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ABSTRACT
POSTERS
ABSTRACTS ARRANGED ALPHABETICALLY BY
FIRST AUTHOR
Paper No.: 3125
FOCUSED CONFERENCE GROUP: P09 - NFLAMMATION AND
IMMUNOPHARMACOLOGY: NEW TOOLS FOR OLD
DISEASES
EFFECT OF CHRONIC MILD STRESS CMS ON CYP1A2
ENZYME ACTIVITY AND INDUCIBILITY IN WISTAR RATS
Ahmed M-D Abdel-tawab(1), AM Hassan(1), AN-E Hassan
Ain Shams University, Faculty of Medicine, Department of
Pharmacology, Cairo, Egypt
Cytochrome P450-1A2 enzyme activity is reported to be affected by
inammatory cytokines and psychological stress. These are also, associated with the pathogenesis of depression. In this work we investigated
the effect of CMS model of depression on CYP1A2 enzyme activity
and inducibility by o-toluidine T. 32 Wistar rats were exposed to CMS
for 4 months then randomly allocated into 3 groups: CMS, CMS-T
(received single injection of i.p. 10 mg/kg o-toluidine 24 hours before
decapitation) and a group left for additional 8 weeks to recover from
CMS, then received T(CMS-RT) Control rats were allocated into two
groups, nave (received vehicle) T groups. CYP1A2 was assessed by
measuring the ratios of caffeine CA and 3 its metabolites; theobromine
TB, paraxanthine PX and theophylline TP in serum samples collected 3 hours after i.p. injection of 20 mg/kg CA. Results showed signicant differences in the behavioral tests assessing induction of
depressive-like behaviors. The PX/CA metabolic ratio was the only metabolic ratio that showed signicant increase in CMS-T compared to C-T
group. This ratio was still higher in CMS-RT group (P < 0.01 in both).
Medians of PX/CA were 0.08, 0.25, 0.17 for T, CMS-T, CMS-RT groups
respectively. Other metabolic ratios medians for these 3 groups were
0.06, 0.09, 0.13 for TB/CA, 0.06, 0.16, 0.1 for TP/CA and 0.2, 0.5, 0.39
for (PX+TB+TP)/CA, respectively. The CMS group showed no signicant difference from nave group. These results suggested that CMS can
be associated with increased CYP1A2 inducibility by o-toluidine.
Paper No.: 1007

FOCUSED CONFERENCE GROUP: PW01 - PHARMACOLOGY
OF ADRENOCEPTORS: EIGHTH SATELLITE MEETING
IN VIVO AND EX VIVO RECEPTOR OCCUPANCY ASSAYS
FOR THE RAT A2A/D RECEPTOR
RS79948 in non-specic binding wells) in 50 mM glycylglycine buffer

for 45 minutes at 4 degrees. Harvesting was carried out using a Brandel
harvester through GF/B lters soaked in 0.5% polyethylimine. For the in
vivo assay, cortex homogenate from rats dosed with RS79948 followed
by intravenous [3H]-RX821002 was ltered as above using a manifold.
Radioactivity was quantied using a scintillation counter. In cortex of
rats (4 per treatment group) dosed with 0.1, 1, and 5 mg/kg RS79948,
31.2, 83.0, and 92.3% occupancy was measured in the ex vivo assay.
These values were consistent with 66 and 93% occupancy measured at
0.3 and 1 mg/kg in the in vivo assay. In conclusion, ex vivo and in vivo
occupancy assays for the measurement of a2 occupancy have been developed. Conducting the assay in the cortex region provides condence that
measurements represent occupancy predominantly at the a2A/D subtype.
Paper No.: 1008

FOCUSED CONFERENCE GROUP:
P11 - G PROTEIN-COUPLED 7TM RECEPTORS: FROM
MOLECULAR TO PHYSIOLOGICAL FUNCTION
DEVELOPMENT OF A P2X7 BINDING AND EX VIVO
OCCUPANCY ASSAY IN RAT BRAIN
Sarah Able, R Fish, L Booth, S Katugampola
Pzer Global Research and Development, Department of Discovery
Biology, Sandwich, UK
The aim of this study was to characterise the binding properties of a
P2X7 antagonist radioligand ([3H]2-cyano-1-[(1S)-1-phenylethyl]-3quinolin-5-ylguanidine) in native tissue, and optimise the binding assay
for use in measuring ex vivo occupancy of P2X7 antagonists. For the in
vitro assay, rat cortex homogenate was incubated with 5nM radioligand
(plus 10 lM of in-house P2X7 antagonist in non-specic binding wells)
in 50 mM Tris/0.1% BSA for 2 hours at 4 degrees. Harvesting was carried out using a Brandel harvester through GF/B lters soaked in 0.5%
polyethylimine. The radioligand concentration was increased (10 nM)
and incubation time reduced (15 minutes) for the ex vivo occupancy
assay. Approximately 60% specic binding was achieved in the cortex
binding assay. The association and dissociation rate of the radioligand
were 0.0021 min-1 nM-1 and0.007 min-1 (half times 45.1 and 88.2 minutes). The Kd derived from kinetic parameters was 3.4 nM, which was
consistent with that derived from saturation experiments of 3.1 nM. In the
brain of rats (4 per treatment group) dosed with3, 10, 30, and 100 mg/kg
of an in-house P2X7 antagonist, 21.5, 27.9, 58.9 and107.4% P2X7 receptor occupancy was measured. In conclusion, an in vitro binding assay
using the P2X7 radioligand has been developed in native tissue and the
ligand properties have been characterised. This assay has been successfully modied for measurement of ex vivo P2X7 receptor occupancy.
Sarah Able, R Fish, C Powell, S Katugampola

The binding of the a2 radioligand [3H]-RX821002 to rat brain has been
well characterised in the literature. The aim of this study was to use this
ligand to develop binding assays to measure ex vivo and in vivo occupancy of a2A/D antagonists in rat brain. Initial autoradiography studies
using subtype selective antagonists conrmed that there was a strong
radioligand binding signal in the cortex region, and that binding to a2A/D
receptors constitued the majority of this signal. For the ex vivo occupancy assay, cortex homogenate from rats dosed with the a2 antagonist
RS79948 was incubated with 1 nM [3H]-RX821002 (plus 100 nM
Paper No.: 1629

FOCUSED CONFERENCE GROUP: P06 - THE HEART GONE
WRONG; STABILIZATION OF CARDIAC FUNCTION - BASIC
EFFECT OF VITAMIN E ON RENAL ISCHEMIC
PRECONDITIONING IN MALE AND FEMALE RATS
Nahid Abotaleb(1), S Aryamanesh(2), SM Ebrahimi(2), M Nobakht(3)
(1) Iran University of Medical Sciences, Department of Physiology,
Tehran, Iran
(2) Tehran University of Medical Sciences, Nursing and Midwifery
Faculty, Tehran, Iran
Journal compilation 2010 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 107 (Suppl. 1), 162692
163
(3) Iran University of Medical Sciences, Department of Histolology and
Neuroscience, Tehran, Iran
Vitamins E play important roles in the protective effect of ischemic preconditioning (IPC). In this study, these antioxidants have been investigated after the induction of ischemia reperfusion (IR) and (IPC) of
kidney. Forty-eight Wistar rats were divided randomly into six groups:
group A (8 male controls), group B (8 female controls), group C (8 male
IR cases) and group D (8 female IR cases). Ischemia was induced by
clamping of left renal arteries for 45 minutes. Rats in group E (8 male
IPC cases) and group F (8 female IPC cases) underwent four cycles of
4-min arterial clamping and 11 min of declamping prior to the nal
45 min ischemia induction. Then, serum was collected to assess the
blood urea nitrogen, creatinine and vitamin E. Also, renal tissues were
obtained for the histological assessments. Vitamin E was signicantly
increased in both male and female rats in IPC group compared with IR
group. Also, its levels showed signicant increase in female IPC group
compared with male IPC group. Histological evaluation showed that in
female rats, injuries induced by IR were signicantly less than male rats
and also protective effects of IPC in female rats were signicantly more
than male rats. Results of the present study show that in female rats,
after renal IPC, an increase of endogenous vitamin E along with a
decrease in tissue injuries is apparent. This might indicate the protective
effects of preconditioning might be due to an increase in vitamin E in
body.
Paper No.: 506

FOCUSED CONFERENCE GROUP: P05 - TRANSLATIONAL
SCIENCE IN THE METABOLIC SYNDROME: BASIC AND
CLINICAL PHARMACOLOGY
THE BLOOD GLUCOSE LEVELS IN HEALTHY VOLUNTEERS
ADMINISTERED WITH REPAGLINIDE AND GENETIC
POLYMORPHISMS OF CYP3A4 AND CYP2C8
Ruzilawati Abu Bakar, MS Ab Wahab, G Siew Hua
University Sains Malaysia, Department of Pharmacology, Kota Bharu,
Kelantan, Malaysia
Repaglinide is a novel prandial glucose regulator (PGR) for the treatment
of type 2 diabetes mellitus. It is mainly metabolized in the liver by
CYP3A4 and CYP2C8. The objective of the present study is to investigate the effects of the CYP3A4 and CYP2C8 genotypes on the repaglinides (Novo Nordisk) blood glucose levels in healthy Malaysian
subjects. Overall, 121 healthy volunteers consisting of 61 men and 60
women enrolled in the study. Each participant received oral 4 mg of repaglinide. Six blood glucose levels (0 min, 30 min, 60 min, 120 min,
180 min and 240 min) per individual were taken according to protocol.
Subjects were genotyped by polymerase chain reaction-restriction fragment length polymorphism for CYP3A4*4,*5 and *18 and by allelesspecic multiplex PCR for CYP2C8*2,*3,*4 and *5. In the group of
121 volunteers, the allele frequency of CYP2C8*1 was 99.17%. The
allele frequency of CYP2C8*2 and CYP2C8*3 were 0.41% respectively.
The allele frequency of CYP2C8*5 was 4.02%. CYP2C8*4 was not
detected in any individual. The frequency of the CYP3A4*18 allele in
the Malaysian population is 2.07%. No mutations were detected for
CYP3A4*4 and CYP3A4*5 alleles. No statistically signicant were
found in the blood glucose response to repaglinide between the genotypes. The present study shows that genetic polymorphisms in CYP3A4
and CYP2C8 are not play a role in the blood glucose response to repaglinide.
Paper No.: 507

FOCUSED CONFERENCE GROUP: P13 - MAXIMISING BENEFITS AND MINIMIZING HARMS FROM DRUGS
POPULATION PHARMACOKINETICS OF REPAGLINIDE IN
HEALTHY MALAYSIAN SUBJECTS AND GENETIC POLYMORPHISMS OF CYP3A4 AND CYP2C8
Ruzilawati Abu Bakar, G Siew Hua, MS Ab Wahab
University Sains Malaysia, Department of Pharmacology, Kota Bharu,
Kelantan, Malaysia
This study investigated the effects of CYP3A4 and CYP2C8 genetic
polymorphisms on repaglinides (Novo Nordisk) pharmacokinetics in
healthy Malaysian subjects (n = 121) who had received oral repaglinide
(4 mg). Blood samplings were done for repaglinides serum concentration determination at 0, 30, 60, 120, 180 and 240 min using highperformance liquid chromatography. Subjects were also genotyped by
PCR-RFLP for CYP3A4*4,*5 and *18 and by an allele-specic multiplex PCR for CYP2C8*2,*3,*4 and *5 alleles. The allele frequencies of
CYP2C8*1, *2, *3, *4 and *5 were 99.17%, 0.41%, 0.41%, 0% and
4.02% respectively. The frequencies of the CYP3A4*4, *5 and *18
alleles were 0%, 0% and 2.07% respectively. CYP2C8 and CYP3A4
genotypes were not signicantly associated with repaglinides blood
glucose lowering effect. On the other hand, the CYP3A4 genotype
signicantly inuenced repaglinides pharmacokinetics. Subjects having
the CYP3A4*1/*18 genotype has a 34% lower mean elimination rate
constant (p = 0.04) and 133% longer elimination half-lives (p = 0.04)
when compared to the normal types (CYP3A4*1/*1). This conrms that
CYP3A4 plays a larger role in metabolizing repaglinide. In conclusion,
genetic polymorphisms of CYP3A4 specically CYP3A4*18 plays a
main role in contributing to the interindividual variability in repaglinides
pharmacokinetics.
Paper No.: 3113

FOCUSED CONFERENCE GROUP: P04 - PHARMACOEPIDEMIOLOGY, CURRENT CONTROVERSIES AND OPPORTUNITIES
THE EFFECT OF ADVERSE DRUG REACTIONS (ADR) ON DRUG
PRESCRIPTIONS DURING ACUTE HOSPITAL ADMISSIONS
Paul Acheampong(1), M Doshi(2), C Doig(2)
(1) North Tyneside General Hospital / Royal Victoria Inrmary, Department ofClinical Pharmacology and General Internal Medicin, Newcastle
Upon Tyne, UK
(2) North Tyneside General Hospital Rake Lane, North Shields, Tyne
and Wear, UK
adr are undesirable effects of drugs beyond their anticipated therapeutic
effects occurring during proper clinical use of drugs (Asscher AW et al,
bmj 1995; 311: 1003-1005). adr are reported to account for 5% of all
hospital admissions, occur in 10-20% of hospital in-patients and are
responsible for 0.1% of medical in-patient deaths (Pirmohamed M et al,
bmj 1998; 316: 1295-1298). adr result in substantial costs directly and
indirectly - to patients and healthcare systems. Data was randomly collected from patients during acute admission to a district general hospital
to assess: 1) the incidence of adr in patients admitted to hospital 2) the
severity of adr using standard denitions [adr resulting in hospitalisation
or prolonged admission, physical disability or malformation to an unborn
foetus (Lazarou J et al, jama 1998; 279: 1200-1205)] and patient perspective, and 3) the effect of previous adr on prescribed medications during acute hospitalisation. In all, 322 acute admissions were screened and
of these 22.4% had adr to previously prescribed medications prior to
admission. Whereas 66.3% of patients considered their adr to be severe,
only 25% of previous adr were severe by standard denition. 18.4% of
adr affected choice of prescribed medications during acute admission to
hospital. The incidence of adr remains high and has enormous effects on
healthcare delivery by way of choice of drugs used during acute hospitalisation. There is an attendant nancial implication in view of the fact that
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most adr are to rst-line drugs and second-line agents are usually more
expensive.
Paper No.: 1134

FOCUSED CONFERENCE GROUP: P15 - ENDOTHELIUM IN
HEALTH AND DISEASE
ANATOMICAL REGIONAL DIFFERENCES IN AORTIC TISSUE
REACTIVITY: THE ROLE OF PERIVASCULAR ADIPOSE
TISSUE IN NORMAL AND DIABETIC RATS
Francis I Achike(1), CB Chua(1), CY Kwan(2)
(1) International Medical University, Department of Clinical Sciences,
Kuala Lumpur, Malaysia
(2) China Medical College, Taichung, Taiwan
The perivascular adipose tissue (PVAT), until recently, was treated as a
non-physiologic structural tissue. Its now known to exert vasorelaxant
and contractile effects. Anatomical regional variations in rat vascular tissue function and in the contribution of human adipose tissue to pathophysiology have been reported. We, therefore, explored possible regional
differences in the effect of PVAT on the contractile responses of windkessel, thoracic and abdominal aortic rings from euglycaemic and streptozotocin-induced diabetic rats. Weighed endothelium-intact or denuded rings
with or without PVAT were dissected from male Sprague-Dawley rats
and mounted in Krebs solution for isometric tension recording. The tissues were contracted with either phenylephrine (PE, 0.1nM - 0.1mM) or
angiontensin-II (Ang-II, 0.01nM - 100nM) under the various experimental conditions. PVAT had no effect on endothelium-intact tissues but it
exerted relaxant, no effect, or contractile (in diabetic tissues) effects,
respectively in the windkessel, thoracic or abdominal endotheliumdenuded tissues. Endothelium-intact or denuded diabetic tissues, respectively, contracted equally or higher than their corresponding euglycaemic
rings. We speculate that PVAT acts like a buffer endothelium with its
relaxant function unmasked once the endothelium is damaged, and like
the damaged endothelium it also promotes vascular contractility in the
diabetic state.
Paper No.: 1135

HEALTH AND DISEASE
POTASSIUM CHANNEL MODULATION OF ACIDOSISINDUCED VASODILATATION OF NORMAL AND DIABETIC
RAT AORTA
dent vasodilatation. 4-aminopyridine or glibenclamide inhibited acidotic

relaxation in diabetic but not euglycaemic ED+ tissues indicating KV
and KATP channel activation in diabetes and their involvement in acidosis-induced endothelium-dependent vasodilatation. In conclusion, acidosis causes endothelium-dependent and -independent vasodilatation in
normal and diabetic tissues via mechanisms differentially modulated by
NO-cGMP and selected K+-channels, some of which, altered in diabetes,
are potential therapeutic targets.
Paper No.: 3248

FOCUSED CONFERENCE GROUP: P02 - TRANSMEMBRANE
TRANSPORT: PERSPECTIVES FOR DISEASE AND DRUG
DISCOVERY
CONSEQUENCES OF CHRONIC POSTNATAL STRESS ON
GLUTAMATERGIC TRANSPORTER AND ON BEHAVIOURAL
TEST IN THE RAT
Gabriela Acosta, MM Odeon, ML Orta, A Salatino
Institute of Pharmacological Research (ININFA), CONICET-UBA, Buenos Aires, Argentina
Early life events have profound consequences in growth and development. It is well known that animals exposed to stressful stimuli during
their early life develop different neurological disorders when they
become adults. We evaluated the consequences of chronic early life environmental manipulations on glutamate transporter (GluT), evaluating the
uptake Glu by synaptosomes isolated from frontal cortex (FC) and hippocampus (Hic) by kinetic parameters, corticosterone levels and GLT-1
expression by western blot. Also we investigated the possiblility of relations between GluT and behaviour. In repeated stress the pups were separated from their mothers and exposed to cold stress (4C) for 1 h at
postnatal days during 20 days. These animals were allowed to a 30 days
recovery period until adulthood. FC and HIC were dissected to study
GluT and trunk blood samples were collected to determinate corticosterone levels. Repeated chronic stress did not change uptake levels in both
areas whereas kinetics parameters were modied. While GLT-1 expression increased on FC and Hic. The levels of corticosterone decreased. In
chronic stress we found an increment in time spent in the illuminated site
of the dark/light transition test. In summary, we have observed regional
changes in GluT produced by chronic stress. These results suggest that a
exposure to postnatal stress at different periods after birth modies GluT,
affects hypothalamic-pituitary-adrenal axis, which could be relevant to
function of GluT in the adult rat brain and induces anxiolytic-like action
in an animal model of anxiety, and alter the glutamatergic neurons.
Francis I Achike, JL Yeo

International Medical University, Department of Clinical Sciences, Kuala
Lumpur, Malaysia
We showed that acidosis-induced vasodilatation does not involve a
cyclooxygenase product, but is via endothelium-dependent and -independent mechanisms, the former modulated by the EDNO-cGMP cascade.
This study explored K+-channel modulation of acidosis-induced vasodilatation of aortic rings from normal male Sprague-Dawley and streptozotocin-induced diabetic rats. Contractile responses to phenylephrine in
endothelium-intact (ED+) and -denuded (ED-) aortic rings in normal (pH
7.40) or acidotic (pH 7.20) Krebs solution were recorded in the presense
or absence of various potassium channel blockers. Barium chloride (Kir
blocker) signicantly inhibited acidotic relaxation in euglycaemic and
diabetic endothelium-intact but not -denuded tissues indicating Kir channel involvement in endothelium-dependent vasodilatation. TEA inhibited
acidotic relaxation in ED+ but not ED- euglycaemic tissues whereas iberiotoxin had no effect in both tissues, indicating involvement of a TEAsensitive (non-BKCa) mechanism in endothelium-dependent relaxation.
In diabetic tissues, however, TEA or iberiotoxin attenuated acidotic
relaxation (ED+ and ED-), although not signicantly in iberiotoxin-treated ED- tissues, indicating BKCa channel activation in diabetes and its
involvement in acidosis-induced endothelium-dependent and -indepen-
Paper No.: 427

FOCUSED CONFERENCE GROUP: P16 - NATURAL
PRODUCTS: PAST AND FUTURE?
MECHANISM OF ANTIPROLIFERATIVE ACTION OF
XYLOPIA AETHIOPICA (ANNONACEAE) DRIED FRUITS
EXTRACT ON HUMAN CERVICAL CANCER CELLS
Oluwatosin Adaramoye(1), N Singh(2), S Meena(2), B Changkija(3),
R Konwar(3), S Sinha(2), J Sarkar(2)
(1) Department of Biochemistry, College of Medicine, University of
Ibadan, Nigeria
(2) Drug Target Discovery and Development Division, Central Drug
Research Institute, Lucknow, India
(3) Endocrinology Division, Central Drug Research Institute, Lucknow,
India
Introduction: African guinea pepper Xylopia aethiopica is commonly
used in traditional medicine for treatment of several diseases. In this
study, we investigated the anti-cancer potential and mechanism of cell
death elicited by Xylopia aethiopica dried fruit extract (XAFE) on human
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cervical cancer cellline, C33A. Materials: Human cancer cell line-C33A
(cervical) was obtained from ATCC. The cell was grown in recommended media supplemented with 10% FBS, 50 lg/ml gentamicin and
2.5 lg/ml amphotericin B in a 5% CO2 humidied atmosphere at 37C.
Results: Our study demonstrated that XAFE treatment led to dose-dependent growth inhibition in various cell lines with selective cytotoxicity
towards cancer cells. Apoptosis was conrmed by nuclear fragmentation
and sub-G0/G1 phase accumulation. Cell cycle was also arrested at G2/
M phase with decreased G0/G1 population. Semi-quantitative gene
expression studies revealed dose-dependent increase of Bax, p53 and
p21 transcripts with decrease in Bcl-2 gene expression. Conclusion:
Taken together, XAFE inhibited cell proliferation, induced apoptosis and
cell cycle arrest in C33A cells, indicating that XAFE may be a potential
therapeutic agent for cancer.
Paper No.: 3230

FOCUSED CONFERENCE GROUP: P19 - GENERAL SESSION
ANALGESIC ACTIVITY OF THE AQUEOUS SEED EXTRACT
OF HUNTERIA UMBELLATA (K. SCHUM.) HALLIER F.
(APOCYNACEAE) IN RODENTS
Olufunmilayo Adeyemi(1), A Adeneye(1,2), T Alabi(1)
(1) University of Lagos, College of Medicine, Department of Pharmacology, Idi-Araba, Lagos State, Nigeria.
(2) Lagos State University College of Medicine, Department of Pharmacology, Ikeja, Lagos State, Nigeria
Different parts of the tropical rainforest tree, Hunteria umbellata
(K. Schum.) Hallier f. (family: Apocynaceae), are highly valued for the
local management of human and veterinary diseases, including genital
infections, diabetes mellitus, obesity, gastric ulcers and pain by African
traditional healers [Falodun A, et al., Pakistani Journal of Pharmaceutical
Sciences 2006; 19(3):256-258]. Recent studies have reported the analgesic and antipyretic effects of the fruit pulp extract of the plant [Igbe I,
et al., Tropical Journal of Pharmaceutical Research 2009;8(4): 331-336].
The present study was designed at investigating the potential analgesic
property and possible mechanism(s) of action 50-200 mg/kg of the aqueous seed extract of Hunteria umbellata (HU) in different experimental
models of algesia using the tail ick, tail immersion, acetic acid-induced
writhing tests and formalin-induced algesia. Results showed that pretreatment with HU produced signicant and dose related (p < 0.05,
P < 0.001) analgesic effect in the treated rats which were mediated via
central and peripheral mechanisms. Overall, results of this study showed
that HU possesses analgesic effect which lends support to its folkloric
use in the local management of pain.
Paper No.: 3392

THE ANALGESIC AND ANTIINFLAMMATORY ACTIVITIES
OF THE AQUEOUS LEAF AND STEM EXTRACT OF
ASYSTASIA GANGETICA
Olufunmilayo O Adeyemi, FR Aigbe, GN Uyaiabasi
in pain threshold comparable to morphine (10mg/kg) in tail ick test and

peak analgesia at 200mg/kg in the hot plate test. The extract (25-200mg/
kg) also produced signicant (p < 0.05) inhibition of oedema comparable
to indomethacin (10mg/kg) in the carrageenan induced paw oedema
model. The extract (200 mg/kg) produced a signicant inhibitory effect
(p < 0.05) comparable to that produced by 1mg/kg dexamethasone in the
xylene induced mouse ear oedema model. Preliminary phytochemical
screening showed that the extract contains alkaloids, tannins, cardiac glycosides and avonoids. It did not produce mortality nor any visible sign
of lethality 24 hours after single oral administration of 10g/kg. The
results show that the extract possesses analgesic and antiinammatory
potentials. Further studies to determine the active component(s) responsible for these activities with the view of isolating a lead compound that
may become a novel drug will be carried out.
Paper No.: 1857

FOCUSED CONFERENCE GROUP: PW23 - APPLYING
PHARMACOGENOMICS (PGX) FROM RESEARCH INTO
CLINICAL PRACTICE: PRESENT AND FUTURE
DPYD POLYMORPHISMS AND ADJUVANT 5-FU/LV
TREATMENT OUTCOME IN COLORECTAL CANCER
PATIENTS
Shoaib Afzal(1), SA Jensen(1), B Vainer(1), U Vogel(2), JB Srensen(1),
HE Poulsen(1)
(1) Copenhagen University Hospital, Rigshospitalet, Laboratory of
Clinical Pharmacology, Copenhagen, Denmark
(2) National Food Institute, Technical University of Denmark,
Copenhagen, Denmark
Dihydropyrimidine Dehydrogenase (DPYD) is the main catabolic
enzyme of 5-Fluorouracil. The objective of this study was to test whether
selected single nucleotide polymorphisms or specic haplotypes in
DPYD inuenced treatment outcome in colorectal cancer patients treated
with adjuvant 5-FU/LV. We studied 302 patients treated with adjuvant 5FU/LV after intended curative resection of stage II-III colorectal cancer.
DNA was extracted from FFPE tumor tissue and analyzed for 4 coding
SNPs in the DPYD gene (DPYD cys29arg, DPYD val166met, ile543val
and the exon 14 deletion variant DPYD 2a) using endpoint reads from
real-time PCR. Furthermore DPYD immunohistochemistry was carried
out. Endpoints were relapse free survival (RFS) and overall survival
(OS). Haplotypes containing 2 polymorphisms on different alleles (40
patients) were associated with improved treatment outcome (OS: HR
0.47, p = 0.02; RFS: HR 0.46, p = 0.01). Furthermore, the presence of
haplotypes containing DPYD val166met polymorphisms were also associated with improved outcome (OS: HR 0.51, p = 0.05; RFS: HR 0.43,
p = 0.01). These associations remained unchanged after adjusting for
known clinical prognostic markers. The two delineated patient groups
contained 40 and 36 patients respectively indicating that 85% of patients
could have a potential for an improved response to 5-FU based treatment. DPYD9a TT and haplotypes containing the DPYD 166 polymorphisms (p = 0.04) were associated with increased protein levels as
determined by immunochemistry. We were able to identify two DPYD
proles that may be useful for allocating colorectal cancer patients to
adjuvant 5-FU based treatment. Our results need conrmation in larger
prospective studies under different treatment regimes.
University of Lagos, Department of Pharmacology, Lagos, Nigeria

The analgesic and antiinammatory activities of the aqueous leaf and
stem extract of Asystasia gangetica (Linn.) T. Anders. [family Acanthaceae] used commonly in sub tropics and tropics for the management of
asthma and pain were evaluated. The analgesic effect of the extract (25,
50, 100 and 200mg/kg) was evaluated in rats and mice using the acetic
acid induced writhing, cold water tail ick and hot plate models. Its antiinammatory effect was evaluated using carragenaan induced rat paw
oedema and xylene induced mouse ear oedema models. The extract (25200mg/kg) signicantly (p < 0.05) reduced the number of writhes in the
acetic acid induced writhing test. At 100 mg/kg, it produced an increase
Paper No.: 2345

MICROPARTICLES FROM PATIENTS WITH METABOLIC
SYNDROME INDUCE VASCULAR HYPO-REACTIVITY VIA
FAS/FAS-LIGAND PATHWAY IN MICE
A Agouni(1), P-H Ducluzeau(2), T Benameur(1), M Sladkova(0), G Leftheriotis(1), O Pechanova(3), Carmen Martinez(1), R Andriantsitohaina(1)
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(1) University of Angers Faculty of Medicine, INSERM, U771, CNRS
UMR, 6214, Angers, France
(2) CHU dAngers, Departement dEndocrinologie et Diabetologie,
Angers, France
(3) Institute of Normal and Pathological Physiology, Slovak Academy of
Sciences, Bratislava, Slovak Republic
Microparticles are small membrane vesicles released during cell activation and apoptosis. Elevated circulating levels of microparticles have
been detected in several cardiovascular diseases including the metabolic
syndrome (MS). The increased number of microparticles was associated
in many of these diseases with vascular dysfunction. In the present study,
we evaluated the effects of in vivo treatment of circulating microparticles
from both patients with MS and healthy subjects on vascular function.
Microparticles obtained from whole blood either from MS patients or
healthy subjects, or a vehicle control were injected intravenously to mice
and vascular reactivity was then evaluated in aorta. Injection of microparticles from MS patients into mice induced vascular hypo-reactivity in
response to serotonin in aorta. Interestingly, hypo-reactivity was reversed
by a nitric oxide (NO)-synthase (NOS) inhibitor, and was associated with
up-regulation of inducible NOS (iNOS) protein expression and an
increased production of NO. The selective cyclo-oxygenase-2 inhibitor
reduced serotonin-induced contraction in vessels from vehicle and
healthy subject microparticles but did not affect response to the same
agonist in MS microparticle-treated mice. MS microparticles also
enhanced prostacyclin production in aorta. In addition, MS microparticles
increased reactive oxygen species production via enhanced expression of
the NADPH oxidase subunits, gp91phox and p47phox. Importantly, the
silencing of the pro-inammatory pathway Fas/Fas-ligand, completely
prevented the vascular hypo-reactivity. These data provide evidence that
circulating microparticles from MS patients induce in vivo vascular dysfunction by increasing both oxidative and nitrosative stresses and by
altering the release of cyclo-oxygenase metabolites through the Fas/FasL
pathway.
Paper No.: 2346

HEALTH AND DISEASE
RED WINE POLYPHENOLS PREVENT METABOLIC AND
CARDIOVASCULAR ALTERATIONS ASSOCIATED WITH
OBESITY IN ZUCKER FATTY RATS (FA/FA)
A Agouni(1), A-H Lagrue-Lak-Hal(1), HA Mostefai(1), A Tesse(1), P
Mulder(2), P Rouet(3,4), F Desmoulins(3,4), C Heymes(5), MC Martinez(1), Ramaroson Andriantsitohaina(1)
(2) INSERM, U644, Rouen, France
(3) INSERM, U858, Toulouse, France
(4) Universite Paul Sabatier, Institut Federatif de Recherche 31,
Toulouse, France
(5) INSERM, U689, Paris, France
Obesity is associated with increased risks for development of cardiovascular diseases. Epidemiological studies report an inverse association
between dietary avonoid consumption and mortality from cardiovascular
diseases. We studied the potential benecial effects of dietary supplementation of red wine polyphenol extract, Provinols, on obesity-associated
alterations with respect to metabolic disturbances and cardiovascular
functions in Zucker fatty (ZF) rats. ZF rats or their lean littermates
received normal diet or supplemented with Provinols for 8 weeks. Provinols improved glucose metabolism by reducing plasma glucose and
fructosamine in ZF rats. Moreover, it reduced circulating triglycerides and
total cholesterol as well as LDL-cholesterol in ZF rats. Echocardiography
measurements demonstrated that ProvinolsTM improved cardiac performance as evidenced by an increase in left ventricular fractional shortening
and cardiac output associated with decreased peripheral arterial resistances in ZF rats. Regarding vascular function, ProvinolsTM corrected
endothelial dysfunction in aortas from ZF rats by improving endothelium-
dependent relaxation in response to acetylcholine (Ach). ProvinolsTM

enhanced NO bioavailability resulting from increased nitric oxide (NO)
production through enhanced endothelial NO-synthase (eNOS) activity
and reduced superoxide anion release via decreased expression of
NADPH oxidase membrane sub-unit, Nox-1. In small mesenteric arteries,
although ProvinolsTM did not affect the endothelium-dependent response
to Ach; it enhanced the endothelial-derived hyperpolarizing factor component of the response. Use of red wine polyphenols may be a potential
mechanism for prevention of cardiovascular and metabolic alterations
associated with obesity.
Paper No.: 744

FOCUSED CONFERENCE GROUP: P01 - CLINICAL
PHARMACOLOGY IN THE EMERGING COUNTRIES
PATTERNS OF INFLUENZA INFECTION IN SYLHET
DIVISION OF BANGLADESH
Salim Ahmed, ATMA Jalil, SS Ahmed, MM Siddika
Jalalabad Ragib-Rabeya Medical College, Department of Pharmacology
& Therapeutics, Sylhet, Bangladesh
The inuenza virus infection varies globally with the genomic diversity
or and clinical variations and regional detection of the patients or and
viral agents is inuenced by the variations of geographic, demographic
condition and patient risk factors. This study is carried out to clarify
about the pattern and seasonality of inuenza infection during two consecutive years from July 2007 to April 2009 in Jalalabad Ragib-Rabeya
Medical College Hospital, Sylhet with active collaboration of ICDDRB
and IEDCR. Three hundred eighty ve (N = 385) patients with acute
respiratory infection were selected as case following WHO selection criteria of avian inuenza A (H5N1) virus infection. In this study throat
swab and anterior nasal swab were collected as clinical specimen from
the cases for detection of Inuenza A (H5N1) by molecular or and
by any other diagnostic methods in BSL-III laboratory of ICDDRB;
Mohakhali, Dhaka. Overall inuenza positivity was 48 (12%), in where
Flu A (n = 27), Flu B (n = 21) and subtypes of Flu A: H1 (n = 16), H3
(n = 11) & H5 (n = 0). This study also suggests that peak seasonality of
inuenza infection is more marked in the month of July, August &
September.
Paper No.: 1851

FOCUSED CONFERENCE GROUP: P11 - G
PROTEIN-COUPLED 7TM RECEPTORS: FROM MOLECULAR
TO PHYSIOLOGICAL FUNCTION
CEREBROVASCULAR CONTRACTILE RECEPTORS
UP-REGULATE VIA A RAF SENSITIVE SIGNALLING PATHWAY
Hilda Ahnstedt, L Edvinsson
Lund University, Division of Clinical Sciences, Department of Experimental Vascular Research, Lund, Sweden
Cerebral ischemia results in a rapid increase in contractile receptors in
the ischemic region, which further impairs the local blood ow and
aggravates the tissue damage. Cerebral ischemia is associated with
enhanced expression of several G-protein coupled receptors (GPCRs)
with strong vasoactive properties such as the endothelin ETA and ETB
receptors, the angiotensin II (Ang II) type 1 receptor (AT1) and the serotonin type 1B receptor (5-HT1B). This receptor up-regulation occurs via
activation of the MEK/ERK1/2 pathway. The present study aimed to
examine the role of Raf, the rst upstream signalling molecule in this
pathway. Cerebral arteries were obtained from patients undergoing neurological surgery (n = 7). The vessels were organ cultured for 48 h in the
absence or presence of Raf inhibitors: SB-386023 or SB-590885. Contractile properties were evaluated in a sensitive myograph by cumulative
addition of 5-carboxamidotryptamine (5-CT, 5-HT1B agonist), Ang II
and endothelin-1 (ET-1). Vessels treated with SB-386023 and
167
SB-590885 showed attenuated contractile responses to 5-CT, and the
effect was most prominent after treatment with SB-590885 (p < 0.05).
The maximum contraction to Ang II was attenuated by SB-590885
(P < 0.01) and only slightly by SB-386023. ET-1 gave a biphasic concentration-dependent response. The high afnity phase corresponding to
the ETB receptor-mediated contraction was decreased in the presence of
SB-590885(p < 0.05). This study shows that Raf plays an important role
in the altered expression of several GPCRs seen after cerebral ischemia
and its inhibition might be a novel approach to reduce tissue damage
after a stroke.
Paper No.: 1480

FOCUSED CONFERENCE GROUP: P17 - NEW APPROACHES
AND TARGETS IN PSYCHIATRY
EFFECTS OF SELECTED GLUTAMATERGIC LIGANDS ON
NOVELTY-INDUCED HYPERACTIVITY OF
GLUA1-DEFICIENT MOUSE LINE
Teemu Aitta-aho, C Procaccini, ER Korpi, AM Linden
Institute of Biomedicine, Department of Pharmacology, Helsinki, Finland
Glutamate AMPA receptors have been implicated in schizophrenia,
which is a major psychiatric illness with unknown etiology. Putative
mouse model of schizophrenia, glutamate AMPA-receptor GluA1-subunit decient mouse line (GluA1-/-mice), displays various symptoms
related to schizophrenia. Among them, a robust spatial novelty-induced
hyperactivity has been shown to be sensitive to a prototypic antipsychotic haloperidol. In our studies, male and female GluA1-/-; mice and
their littermate control wild type mice (GluA1-/-mice & -/- were injected
intraperitoneally with AMPA-receptor antagonist NBQX (10, 30 and
100 mg/kg), AMPA-receptor potentiator CX546 (10, 30 and 80 mg/kg)
or metabotropic glutamate receptor 2/3 agonist LY354740 (15 mg/kg),
placed in a novel cage and thereafter their locomotor activities measured
for 2 h. Vehicle-treated GluA1-/- mice displayed clear hyperactivity in all
experiments. NBQX reduced locomotor activity of both mouse lines at
100 mg/kg dose only. However, the reduction was greater in GluA1-/mice. No effect of CX546 was found in either mouse line. LY354740
reduced locomotor activity in the GluA1-/- males but not in the females,
while the locomotor activity of GluA1-/- mice was unchanged. These
results suggest sensitivity of a tentative antipsychotic LY354740 in a
mouse model of schizophrenia, but in a gender-specic manner.
Paper No.: 1086

DISEASES
INHIBITORY EFFECT OF CARBON MONOXIDE-RELEASING
MOLECULE CORM-2 ON TNF-A RELEASE FROM
ACTIVATED-RBL-2H3 CELLS
Masaaki Akagi(1), Y Yasui(1), M Watanabe(1), A Maruyama(1), N
Matsui(1), N Fukuishi(1), R Akagi(2)
(1) Tokushima University Hospital, Faculty of Pharmaceutical Sciences,
Tokushima,Japan
(2) Yasuda Womens University, Japan
We recently reported that HO-1 inhibited TNF-a release from activatedRBL-2H3 cells, and Zn-protoporphyrin, which was an inhibitor of heme
oxygenase, inverted its inhibition. These results suggest that heme
metabolites can inhibit TNF-a production from the activated-RBL-2H3
cells. In the present study, we examined which heme metabolites had
involved in the inhibition of TNF-a release from activated-RBL-2H3
cells. Heme metabolites (bilirubin, iron, CORM-2 as CO donor) were
co-incubated with anti-ovalbumin(OA) rat serum(IgE) for 2hr, and then

the cells were challenged with OA. Gene expression was analyzed by
RT-PCR. Protein expression was investigated by western blot analysis
and ELISA. TNF-a release was signicantly reduced by CORM-2, not
iron or bilirubin. Inactivated-CORM-2 didnhft reduce TNF-a release.
TNF-a mRNA expression was also signicantly inhibited by CORM-2.
Moreover, CORM-2 reduced phosphorylation of ERK. CORM-2 can
regulate TNF-a production through inhibition of ERK. These ndings
provide new insights into the anti-allergic activities.
Paper No.: 3270

FOCUSED CONFERENCE GROUP: P13 - MAXIMISING
BENEFITS AND MINIMIZING HARMS FROM DRUGS
BIOEQUIVALENCE STUDY OF 1500 MG GLUCOSAMINE
SULFATE IN THAI HEALTHY VOLUNTEERS
Pravit Akarasereenont(1), S Chatsiricharoenkul(1), P Pongnarin(1), K
Sathirakul(2), A Wongkajornsilp(1), S Kongpatanakul(1)
(1) Siriraj Hospital, Faculty of Medicine, Department of Pharmacology,
Bangkok, Thailand
(2) Mahidol University, Bangkok, Thailand
Glucosamine sulfate is widely used to relieve symptoms from osteoarthritis. This study was conducted in order to determine pharmacokinetic
and assessed the in-vivo bioequivalence of two different hard capsule
formulations of glucosamine sulfate when administered as equal dose of
1,500 mg. The two formulations contain different salt form where reference product is NaCl and test product is KCl. A randomized, single dose,
two-treatment, two-period, two-sequence crossover study was conducted.
Twenty-six healthy volunteers were recruited at Siriraj Clinical Research
Unit. Each subject received a dose of 1,500 mg glucosamine sulfate of
both formulations with at least a week washout period. Blood samples
were collected over 24 h after the oral administration. The plasma fractions were analyzed for glucosamine using LC-MS/MS. Twenty-six volunteers enrolled in the present study. Pharmacokinetic parameters were
determined using the non-compartment model. The 90 percent condence intervals of the mean ratios (test/reference) of Cmax (111.19%;
ranged from 93.01%-132.92%) and AUC0-t (107.24; ranged from
87.16%-131.93%) was not contained within the equivalence criteria of
80.00-125.00% (US FDA, 2003). However, this study showed the high
intra-individual CV calculated from ANOVA for Cmax and AUC0-24
(30%). Thus, based on equivalence limits of US FDA (2003), the test
product is not bioequivalent to the reference product in terms of rate and
extent of absorption. However, concerning the wider equivalence criteria
for highly variable drug (EMEA, 2008), the test product is bioequivalent
to the reference formulation in terms of rate and extent of absorption.
Paper No.: 3373

FOCUSED CONFERENCE GROUP: P04
- PHARMACOEPIDEMIOLOGY, CURRENT CONTROVERSIES
AND OPPORTUNITIES
PRESCRIPTION PATTERNS OF CONTROLLED MEDICINES
IN ISTANBUL
Ahmet Akici(1), D Demircan(1), I Topcu(2), H Yilmaz(2), B Donertas(1),
Kemal K Berkman(1)
(1) Marmara University School of Medicine, Department of Pharmacology, Istanbul, turkey
(2) Provincial Health Directorate of Istanbul, Istanbul, Turkey
Objective: Drug utilization study of controlled medicines (CMs) which
are prescribed as red or green color scripts in Turkey, are important to
evaluate rational pharmacotherapy. It was planned to investigate the
details of CMs utilization in Istanbul. Method: A total 12000 prescriptions, containing CMs were collected retrospectively from Provincial
168
Health Directorate of Istanbul medical records between January-March in
2009. CMs use was stratied by some socio-demographic characteristics
of patients, and morbidity patterns among users were investigated.
Results: The average age was 46,6 23,4 and half of them were women.
CMs were prescribed by physicians mainly from psychiatry (34.9%),
internal medicine (10.6%), surgery (9.9%), anesthesia (9.1%), and neurology (7.3%) departments. Only 2.5% of scripts were prescribed by general practitioners and family physicians. The majority of scripts were
prescribed at hospitals (74.4%), mainly contained single drug (94.3%),
and also many of them (77.3%) were green color scripts. The most frequently prescribed drug was alprazolam (27.9%), followed by tramadol
(13.9%), clonazepam (12.5%), biperiden (8.8%), methylfenidate (7.8%),
respectively. The most commonly recorded indications were anxiety
(19.5%), cancer (14.8%), attention deciency-hyperactivity disorders
(12.9%), behavioral disorders (12.5%), depression (10.9%). Conclusions:
This is the rst study in this context which reects CMs utilization in
Istanbul and reveals that CMs were mainly prescribed by specialists in
the treatment of psychiatric disorders in both sexes.
Paper No.: 580

IN VIVO ANTIOXIDANT ACTIVITY OF BYRSOCARPUS
COCCINEUS SCHUM. AND THONN. (CONNARACEAE)
EXTRACT IN CARBON TETRACHLORIDE-INDUCED
HEPATOTOXICITY IN RATS
(1) Kaunas University of Medicine, Kaunas, Lithuania

(2) University of Aarhus, Department of Pharmacology, Aarhus, Denmark
(3) Vilnius University, Institute of Oncology, Vilnius, Lithuania
Photodynamic therapy (PDT) is a treatment method where the combination of laser light and a photosensitizer are combined to produce a
cytotoxic or modifying effect to cancerous tissue by producing reactive oxygen species. Damage of different cellular compartments
induces activation of the cellular DNA damage repair system, which
is important factor modulating tumor sensitivity to this treatment. bGlucans is not present in the membrane of tumor cells and killing of
iC3b-opsonized tumor cells via complement receptor 3 cannot be
induced during PDT. The aim of the present study was to evaluate
the effect of PDT in combination with b-glucans on DNA damage
repair system and cell proliferation and determine whether coadministration of b-glucans produces more effective killing of PDT-treated
tumor cells. C57 Bl/6 female mice bearing Lewis lung carcinoma
were exposed to the treatment with PDT (Photofrin, 10 mg/kg, 24 h
prior to laser irradiation) or/and b-glucans (400 lg/d/mouse, 5 days).
PDT in combination with b-glucans signicantly reduced tumor
growth (P < 0.05, n = 10), proliferating cell nuclear antigen (PCNA)
expression (P < 0.001, n = 9), and increased necrosis in tumor tissue
(P < 0.001, n = 9). Our data demonstrates that b-glucans enhances
efcacy of photodynamic therapy by suppressing activity of DNA
damage repair system and cell proliferation, and inducing more efcient cell killing.
A Akindele(1), K Ezenwanebe(1), C Anunobi(2), Olufunmilayo O Adeyemi(1)

(1) Department of Pharmacology, College of Medicine, University of Lagos, Lagos,Nigeria
(2) Department of Morbid Anatomy, College of Medicine, University of
Lagos, Lagos,Nigeria
The leaf decoction of Byrsocarpus coccineus (Connaraceae) is drunk for
the treatment of jaundice in West African traditional medicine (WATM).
This study was designed to investigate the in vivo antioxidant activity of
B. coccineus aqueous leaf extract (BC) in carbon tetrachloride (CCl4)induced hepatotoxicity in rats. Rats were randomly divided into different
groups (vehicle; CCl4; BC 1000 mg/kg; BC 200, 400 and 1000 mg/kg +
CCl4; and livolin 20 mg/kg + CCl4) and treatment was carried out
accordingly. On the 7th day, rats were sacriced and livers were isolated
to assay for activity of catalase (CAT), superoxide dismutase (SOD),
reduced glutathione (GSH), peroxidase (PX) and level of malondialdehyde (MDA) using standard procedures. Histopathological assessment of
liver samples was also carried out. CCl4 produced signicant (p < 0.05)
reductions in the activity of CAT, SOD, PX and GSH, and conversely
increased MDA level. BC (200, 400 and 1000 mg/kg) produced signicant and dose-dependent reversal of CCl4-diminished activity of the antioxidant enzymes and reduced CCl4-elevated level of MDA. The effect of
livolin on the parameters was generally comparable to those of BC at
the most effective dose of 1000 mg/kg. Histological observation of liver
samples supported the results obtained in this study. BC (+ CCl4) treated
rats generally showed normal hepatocytes with moderate inammation
and livolin treated rats also showed normal hepatocytes but with mild
inammation. The results obtained in this study suggest that BC possesses hepatoprotective in vivo antioxidant activity, thus justifying its use
in WATM for the treatment of liver disease.
Paper No.: 2499

FOCUSED CONFERENCE GROUP: PW04 - ANTIOXIDANTS AS
THERAPEUTIC TARGETS
EFFECT OF B-GLUCANS ON PHOTODYNAMIC THERAPY OF
LEWIS LUNG CARCINOMA
Dalia Akramiene(1), C Aleksandraviciene(3), K Suziedelis(3), U Simonsen(2), E Stankevicius(0)
Paper No.: 751

FOCUSED CONFERENCE GROUP: P14 - ADDICTION AND
DOPING: NEUROBIOLOGICAL AND CLINICAL
NEUROPROTECTIVE EFFECT OF ADENOSINE A1 AND A2A
RECEPTORS IN CHRONIC ALCOHOL WITHDRAWAL
SYNDROME IN MICE
Kiran Kumar Akula, K Chopra, SK Kulkarni
Panjab University, University Institute of Pharmaceutical Sciences,
Chandigarh, India
Alcohol withdrawal following chronic alcohol intake results in moderate to severe withdrawal symptoms. Adenosine, an endogenous neuromodulator, has been reported to reverse the behavioral symptoms of
alcohol withdrawal. The present study was conducted to evaluate the
neuroprotective actions of selective adenosine A1 and A2A receptor
ligands against chronic alcohol withdrawal syndrome in mice. Mice
were made physically dependent on alcohol by chronic intragastric
administration of 35% v/v ethanol (2 g/kg of 10% v/v) twice daily for
10 days. On day 11, animals received only adenosine receptor ligands
twice. After 12 hours of the last adenosine ligand treatment, the withdrawal symptoms were precipitated with RO15-4513 (4 mg/kg, i.p.,
ethanol antagonist) and the mice were tested for withdrawal. Ethanoltreated animals exhibited hyperlocomotor activity and anxiogenic
response. Also, alcohol withdrawal decreased pentylenetetrazol i.v. seizure threshold for the onset of generalized clonus and tonic extensor.
Oxidative-nitrosative stress, caspase-3 activity in cytoplasmic lysate,
and NFjb/p65 unit were signicantly increased in nuclear lysate of the
brain samples of ethanol-treated mice. CHA (0.1 or 0.2 mg/kg, i.p.,
selective A1 agonist), but not CPCA (0.1 or 0.2 mg/kg, i.p., selective
A2A agonist), reduced all the behavioral, biochemical and molecular
changes induced by alcohol withdrawal. On the other hand, the effect
of CHA was reversed by pretreatment with DPCPX (5 mg/kg, i.p.,
selective A1 antagonist). Moreover, A2A antagonist [8-(3-cholorostryl)
caffeine, 4 mg/kg, i.p.] showed neuroprotective action in alcohol withdrawal mice. The results emphasize neuroprotective potential of adenosine A1 receptor agonists or A2A receptor antagonists in attenuating
ethanol withdrawal syndrome.
169
Paper No.: 3380
COMPARATIVE STUDY OF INITIAL AND AQUIRED DRUG
RESISTANCE IN PULMONARY TUBERCULOSIS
Seyed Yousef Agha Alaie(1), SD Mansoori(2), S Khazali(1), AA Hashtroodi(1), S Arami(2)
(1) Artesh University of Medical Sciences, Department of Pharmacology,
Tehran, Iran
(2) National Research Instute of Tuberculosis and Lung Disease, Tehran,
Iran
Resistance to anti-tuberculosis agents particulary multiple drug resistant(MDR) mycobacterium tuberculosis is an important obstacle in the
treatment and control of tuberculosis in the world. Between four years
for 273 smear and culture positive pulmonary tuberculosis patients (both
old = 86/273 and new = 187/273)pretreatment susceptibility tests of isolated bacilli to INH,RIF,EMB and STM were performed by standard proportional method and the result were classied in 3 groups: 1)Newly
diagnosed without any history of treatment 2)patiente with history of
treatment with one course 3)patients with history of treatment for two or
more courses supposed to be MDR cases.The result were collected for
each drug individuall and different combination of two,three and four
medications. Resistance to single drug,two drugs,three drugs and four
drugs wase signicantly increased in group 3 in comparison to group 2
and 1,also signicantly increase in group 2 when compared to group
1.We observed a high rate of primary resistance to INH and STM in
group 1and 2 and a high rate of MDR(INH and RIF resistance)in group
2 and 3. The duration of bacilli exposure to anti tuberculosis agents in
the past is a mager factor in developing resistance .In contrast to WHOs
guideline,due to high rate of primary resistance especially to STM in our
area,we dont recommended addition of STM for treatment of patient
whose initial four drug regimens have been faild (group2).
Paper No.: 3381

FOCUSED CONFERENCE GROUP: P08 - DEVELOPMENTS IN
THE TREATMENT OF SEXUAL DYSFUNCTION AND
DISEASES OF THE LOWER URINARY TRACT
FINASTERIDE AND PRAZOSIN MIXED THERAPY IN
PATIENT WITH BENING PROSTATIC HYPERPLASIA
Paper No.: 2708

FOCUSED CONFERENCE GROUP: P11 - G PROTEINCOUPLED 7TM RECEPTORS: FROM MOLECULAR TO
PHYSIOLOGICAL FUNCTION
PRE-VERSUS POSTSYNAPTIC A2A-ADRENOCEPTORS
FUNCTIONS MEDIATED VIA GAI-ISOFORMS
Julian Albarran-Juarez(1), R Gilsbach(1), R Piekorz(2), K Pexa(2), B
Nurnberg(2,3), L Hein(1)
(1) University of Freiburg, Institute of Experimental and Clinical Pharmacology and Toxicology, Freiburg im Breisgau, Germany
(2) Heinrich-Heine-University, Institute of Biochemistry and Molecular
Biology II, Dusseldorf, Germany
(3) Eberhard-Karls-University, Institute of Experimental and Clinical
Pharmacology and Toxicology, and Interfaculty Center of Pharmacogenomics and PharmaceuticalResearch, Tubingen, Germany
Members of the a2-adrenoceptor family are prototype Gai/o protein-coupled receptors and are involved in the regulation of a number of physiological functions including presynaptic feedback inhibition of transmitter
release, analgesia, sedation, anesthetic-sparing, and locomotor activity. In
the present study we tested whether inhibition of spontaneous locomotor
activity and anesthetic-sparing effect elicited by a2-agonists are mediated
by a2A-adrenoceptors located in adrenergic or non-adrenergic neurons and
whether these responses require specic Gai-isoforms in vivo. We evaluated mice which express a2A-adrenoceptors exclusively on adrenergic cells
under control of the dopamine b-hydroxylase promoter (Adra2AC-/-TG),
mice decient in a2-adrenoceptors (Adra2C-/-, Adra2AC-/-), mice decient
in Gai isoforms or wild-type mice (WT). In Adra2AC-/- animals spontaneous locomotor activity was enhanced compared with WT and Adra2C-/-,
but normalized in Adra2AC-/-TG, thus mediated by a2A-adrenoceptors
located in adrenergic neurons. After administration of the a2-agonist medetomidine locomotor activity was reduced to 9.2 3.8% in WT, 7.9 3.7%
in Gai1-/-, and 4.3 3.3% in Gai3-/- mice; and signicantly attenuated in
Gai2-/- mice to 37.2 8.1%. The anesthetic-sparing effect of medetomidine was ablated in Adra2AC-/- mice and was not rescued by
Adra2AC-/-TG, thus it was mediated by a2A-adrenoceptors present on nonadrenergic cells. In WT, Gai1-/- and Gai3-/- animals medetomidine caused
a leftward shift of the isourane-mediated loss of righting reex curves, but
this effect was completely absent in Gai2-/- mice. Our results suggest that
inhibition of spontaneous locomotor activity and the anesthetic-sparing
effect of a2-agonists may be elicited by a specic coupling of a2A-adrenoceptors via Gai2 proteins.
Seyed Yousef Agha Alaie, SS Kashaf, B Najaan, P Musavi

Artesh University of Medical Sciences, Department of Pharmacology,
Tehran, Iran
Bening prostatic hyperplasia (BPH) is the most common cause of male
urinary obstruction. Medical management of BPH includes anti-androgens and a adrenergic receptor blockers. In this study are compared the
efcacy of mixed therapy of nasteride as a 5-a reductase inhibitor in
combination with prazosin as a specic a 1-adrenergic receptor blocker
(F+P) and nasteride alone (F) on obstructive/stimilative symptom of
BPH.Forty men aging 60 + /-6 (mean+/)SD) years old were divided into
two groups.prazosin 1mg and nasteride 5mg daily and similar dose of
nasteride alone were prescribed to group (F+P) and group (F) for
3 months respectively. afterwhile, mean prostativ volume reduced by
16.5% (p < 0.05) in group (F+P) and 11.3% (p=ns) in group (F).
Obstructive and stimulative sympthom scores reduced by 65.3%
(p < 0.0007). in groups (F+P) and (F), respectively.there was no correlation between change in mean prostatic volume and obstructive/stimulative sympthom scores (r=-0.74). The most complication were impotence
(n = 17), loss of libido (n = 17), reduced ejaculate volume (n = 16) and
dry mouth (n = 15) in which their incidences were not different between
two groups. We concluded that combination of nasteride and prazocin
might accestuate the process of prostatic volume reduction in comparison
to nasteride alone in BPH. This measure did not increase the incidence
of complicatin.So,it may be a suitable alternative for more invasive therapeutic approaches.
Paper No.: 2728

PALMITOYL COA ANTAGONISM OF ADP-INDUCED
RELAXATION OF RAT THORACIC AORTA
Eman Aleshat, S Alexander, V Ralevic
University of Nottingham, Department of Biomedical Science, Nottingham, UK
Palmitoyl CoA has been described to antagonise human P2Y1 receptor
responses in recombinant systems (Coddou C et al. FEBS Lett 2003;
536: 145-150) and in isolated platelets (Manolopoulos P et al. Platelets
2008; 19: 134-145). We have noted that palmitoyl CoA antagonises
P2Y1 receptor-evoked relaxations in rat isolated thoracic aorta (Alexander SPH et al. Proc BPS 2009; 6: 150P). The present study investigated
the reversibility of this interaction in the same tissue from male Wistar
rats (200-250 g), as described previously (Bultmann R et al. Eur J Pharmacol 1998; 359: 95-101). ADP evoked concentration-dependent relaxations of the methoxamine-pre-contracted aorta, 30 lM ADP elicited a
relaxation of 71 4% (n = 8). In the presence of palmitoyl CoA
(10 lM), the response at this concentration of ADP was
18 4%(n = 8). Following washout of palmitoyl CoA for an hour,
170
30 lM ADP evoked a relaxation of 73 4% (n = 8) of the methoxamine contraction. Application of 10 lM palmitoyl CoA in the absence
of ADP elicited a modest relaxation (5 2%), which failed to reach statistical signicance. These results indicate that the antagonism of palmitoyl CoA for the ADP evoked relaxation in rat isolated thoracic aorta
through the P2Y1 receptor is reversible with no signicant direct effect
of pamitoyl CoA on the rat isolated thoracic aorta. Acknowledgement:
We gratefully acknowledge and appreciate the Jordanian Government,
who sponsored this research.
Paper No.: 3104

THERAPEUTIC TARGETS
HYDROGEN SULPHIDE GENERATION IN PORCINE TISSUES
Stephen Alexander(1), M Doe(1), M Garle(1)
University of Nottingham, Department of Biomedical Sciences, Nottingham, UK
Hydrogen sulphide is a gasotransmitter candidate with a suggested physiological role in the regulation of smooth muscle (Li et al Pharmacol Ther
2009 123: 386-400). We have analysed hydrogen sulphide generation in
cell-free extracts using the methylene blue formation method (Stipanuk
MH, Beck PW Biochem J 1982;206: 267-277). In rat liver cytosol, hydrogen sulphide generation in the presence of 5 mM L-cysteine (19 nmol/hr
mg) was inhibited by 95% in the presence of either 10 lM propargylglycine or 100 lM aminooxyacetic acid. In contrast, rat kidney cytosol
hydrogen sulphide generation (4.0 nmol/hr/mg) was not signicantly
inhibited by propargylglycine, but was almost completely inhibited by
aminooxyacetic acid. Porcine liver and kidney cytosolic fractions exhibited much reduced hydrogen sulphide generation (0.6 and 3.9 nmol/hr/
mg, respectively). Cytosolic fractions from porcine smooth muscle
showed a rank order of hydrogen sulphide generation of bronchioles (12)
= splenic artery (10) > bladder (7 nmol/hr/mg). Omission of pyridoxal
phosphate inhibited hydrogen sulphide generation in the bronchioles only.
In the porcine brain, midbrain activity was greater than hippocampus (3.1
and 1.0 nmol/hr/mg, respectively). Bronchiole activity was inhibited by
both propargylglycine and aminooxyacetic acid (20% control), while kidney activity was partly inhibited by propargylglycine (40% control) and
completely by aminooxyacetic acid (3% control). These studies show the
viability of using porcine tissues to assess tissues not readily assayed for
hydrogen sulphide synthesis in rodents. However, the effects of the common inhibitors dont allow identication of the enzymes responsible.
Paper No.: 2246

MUSCARINIC M3ACHR IS REGULATED BY CGMP, VIA A
PKG-II DEPENDENT PHOSPHORYLATION IN PLASMA
MEMBRANES FROM TRACHEAL SMOOTH MUSCLE
Marcelo J Alfonzo, M Alfonzo-Gonzalez, RG Alfonzo, A Misle, IL
Becemberg
Central University of Venezuela Faculty of Medicine, Experimental
MedicineInstitute, Biomembranes Section, Caracas, Venezuela
Muscarinic activation of airway smooth muscle, via m2/3AchR produces
two cGMP signals related to the stimulation of the NO-dependent guanylyl cyclase and the NPR-guanylcyclase. To understand the role of
cGMP on these mAChRs, we decided to evaluate the muscarinic receptor
(3HQNB binding) activity in the presence of ATP and cGMP using
plasma membrane (PM) fractions isolated from bovine tracheal smooth
muscle. We found that cGMP plus ATP change the binding properties of
the mAChRS specially the agonist binding afnity to carbamylcholine
and the antagonist properties for 4-DAMP, which is m3AchR-selective
antagonist, without changing the m2AchRs antagonist binding abilities
of gallamine, AFDX-116DS and methoctramine. These muscarinic binding modications disappeared, after a 4-DAMP alkylation of these PM.
Moreover, activators and inhibitors of GPKG-II affected these binding
activities. This G-kinase was detected by Western blotting in these PM.
Using 32P-ATP, we found that cGMP induced a signicant 32P-phosphorylation in these PM, being immunoprecipated using a m3AchR antibody,
which was obliterated by 4-DAMP, in a dose dependent manner. These
original results indicated that cGMP specically regulated m3AchR, via
phosphorylation by a PKG-II bound to plasma membranes from bovine
tracheal smooth muscle.
This work was supported by grants from FONACIT-Venezuela S12002000411 (ILB) and CDCH-UCV-PG-09-07401-2008/1(RGA).
Paper No.: 2650

FOCUSED CONFERENCE GROUP: PW02 - SYMPOSIUM ON
ADVANCES IN GI PHARMACOLOGY
MECHANISMS UNDERLYING THE MODULATION OF GUT
PERMEABILITY BY CANNABINOIDS
Abdussalam Alhamoruni(1), K Wright(2), A Lee(1), M Larvin(1),
S OSullivan(1)
(1) University of Nottingham, The School of Graduate Entry Medicine
and Health in Derby, Derby, UK
(2) Lancaster University, School of Health and Medicine, Lancaster, UK
The small bowel plays a vital role in preventing ingress of bacteria and
antigens whilst absorbing nutrients. Changes in tight junction proteins
have been reported with agents that modulate gut permeability. We have
previously shown that cannabinoids modulate intestinal permeability, and
the aim of the present study was to examine the underlying mechanisms.
Conuent Caco-2 cells (37oC, 5% CO2) were treated with phytocannabinoids or endocannabinoids (10 lM), with or without AM251 (100 nM,
CB1 receptor antagonist). Transepithelial electrical resistance measurements were made as a measure of permeability. mRNA levels of tight
junction proteins (ZO-1, Occludin and Claudin-1) were determined by
Taqman real time PCR. Signal transduction pathways were investigated
using PD98059 (1 lM, MAPK inhibitor), L-NAME (100 lM, NOS
inhibitor) and KT5720 (1 lM, PKA inhibitor). Over 48 hrs, phytocannabinoids signicantly decreased permeability, while endocannabinoids
increased permeability. Both effects were inhibited by AM251. Both
phyto- and endocannabinoids increased the expression of ZO-1 mRNA.
Claudin-1 was downregulated by endocannabinoids only. Cannabinoids
do not affect Occludin expression. The effects of cannabinoids on tight
junction protein expression were inhibited by AM251. Inhibition of
MAPK signalling blocked the action of phytocannabinoids, but had no
effect on endocannabinoids. Inhibition of NOS signicantly reduced the
effects of endocannabinoids only. Neither phyto- nor endocannabinoid
actions were affected by PKA inhibition. This study demonstrates that
cannabinoids modulate gut permeability and tight junction protein
expression via the CB1 receptor. The effect of phytocannabinoids
appears to be mediated through MAPK signalling pathways, while the
effect of endocannabinoids is NOS-dependent.
Paper No.: 3351

ROLE OF RECEPTOR RECYCLING IN THE RECOVERY OF
CA2 + SIGNALLING BY RECOMBINANTLY EXPRESSED
NEUROMEDIN U 2 RECEPTORS (NMU2)
Khaled Al-Hosaini, RAJ Challiss, GB Willars
University of Leicester, Department of Cell Physiology and Pharmacology, Leicester, UK
Neuromedin U (NmU) regulates many physiological and pathological
events via two family A, G protein-coupled receptors, NMU1 and
171
NMU2. These receptors show ~50% homology and couple to Gaq/11 and
Gai/o leading to increases of intracellular [Ca2 + ]i and inhibition of forskolin-stimulated cyclic AMP accumulation respectively. In HEK293 cells
recombinantly expressing either receptor, NmU binds essentially irreversibly (Brighton et al., 2004). Here, we examined the consequences of irreversible binding on recovery of NmU-mediated Ca2 + signalling
following an initial exposure in HEK-NMU2 cells. Further, we employed
confocal imaging of uorescently labelled NmU (Cy3B-NmU-8) and a
C-terminally eGFP-tagged NMU2 to investigate ligand and receptor trafcking and their relevance to recovery of signalling. We conrmed irreversible ligand binding and demonstrated co-internalization of ligand and
receptor starting within 5min of exposure. Irreversible binding prevented
repetitive Ca2 + signalling when cells were exposed to NmU (10-30nM;
5min), washed and NmU re-applied. Full restoration of the response
required 6h recovery. Inhibition of endosomal acidication by monensin
reduced receptor recycling and slowed re-sensitization, while inhibition
of protein synthesis by cycloheximide had little effect on re-sensitization.
We developed a rapid, low pH wash that removed cell-surface receptorbound NmU, but had no deleterious effects on signal transduction.
Following exposure to NmU, brief acid wash restored an appreciable
NmU-mediated Ca2 + response within 5min with full re-sensitization
within 2-3h. Thus, receptor internalization is required for ligand removal
and cellular re-sensitization to NmU is dependent on receptor internalization and recycling rather than de novo protein synthesis. Brighton P.J.,
et al. (2004) Mol Pharmacol 66, 1544-1556.
Paper No.: 425

FOCUSED CONFERENCE GROUP: P10 - DRUGS FOR HALF
THE WORLD: PAEDIATRIC CLINICAL PHARMACOLOGY
RISK FACTORS FOR ABNORMAL NEWBORN HEARING IN
THE ERA OF EXTENDED DOSE INTERVAL AMIKACIN
ADMINISTRATION
Karel Allegaert, K Kneepkens, K Vandendriessche, F Debruyne, M Rayyan, G Naulaers, C Desloovere
University Hospitals Leuven, Department of Paediatrics, Leuven, Belgium
Introduction: There are conicting reports on the impact of aminoglycoside administration on hearing impairment after NICU admission. We
therefore wanted to assess the impact of amikacin on hearing impairment
following implementation of an extended dose interval regimen.Methods:
Retrospective cohort study. Hearing impairment dened as at least unilateral hearing loss (>40dB), documented by brain stem evoked auditory
response (BERA). Risk factors (amikacin (yes/no, days, through, duration), vancomycin, ventilation, birth weight, bilirubinaemia, CMV, congenital malformations, meningitis, intracranial haemorrhage, convulsions)
were extracted from a prospective collected database and the original
source documents. Chi square test was used to compare risk factors
between neonates with or without hearing impairment. Results: 615 neonates were evaluated. Following re-assessment in 70 (11%), hearing
impairment was documented in 25 (4%). There was no signicant difference in administration, through level, days or cumulative amikacin dose
between impaired and normal hearing neonates. Syndromal/congenital
malformations (10/25) and congenital CMV (4/25) infection were the
most relevant risk factors. Conclusion: Amikacin exposure (through
level, days, cumulative dose) in an extended dose interval approach was
not a risk factor, while syndromal/congenital malformation and CMV
infection were the most relevant risk factors.
Paper No.: 544

DOES THE NEED FOR DOMPERIDONE ADMINISTRATION
SHORTLY AFTER DELIVERY AFFECT THE DURATION
BREASTFEEDING AFTER PRETERM DELIVERY?
Karel Allegaert(1), G Bosmans(2), M Vanhaver(3), V Cossey(1), A Debeer(1), C Vaanhole(1), K Janssens(1)
(1) University Hospitals Leuven, Department of Paediatrics, Leuven,
Belgium
(2) KU Leuven, Department of Clinical Psychology, Leuven, Belgium
(3) KH Limburg, Campus Hasselt, Limburg, Belgium
Introduction: There are several reports (da Silva, CMAJ 2001, Wan, Br J
Clin Pharmacol 2008) on the short term effects of domperidone administration as a galactagogue, but observations on the impact of domperidone
administration on the subsequent duration of breast feeding have not
been reported. Methods: Retrospective study in mothers of preterm neonates (<35 weeks gestational age), admitted in a single NICU in a
6 months time interval. Compare the duration of breast feeding after discharge of the baby and investigate if domperidone was a risk factor for
early failure after discharge (unpaired t test). Results: At delivery, 71/96
mothers of 89/118 newborns had the intention to breastfeed. 58 (81%)
were still breastfeeding at discharge. Mothers who underwent a caesarean
had an increased risk to terminate breastfeeding earlier (6.6 versus
15.5 weeks, p < 0.05) before discharge of the baby. 7 (10%) of the
mothers who were still breastfeeding at discharge received domperidone
shortly after delivery as a galactagogue, but domperione was not a
risk factor for earlier termination (17, SD 7 to 16, SD 11 weeks).
Conclusions: The need to initiate domperidone to facilitate mothers milk
production shortly after delivery does not affect the duration of breastfeeding after discharge in former preterm neonates. The extensive interindividual variability in the duration of breast feeding after discharge
warrants prospective studies.
Paper No.: 2076

P12 - ION CHANNELOPATHIES: NEW WINDOWS ON
COMPLEX DISEASE AND THERAPY
NON-GENOMIC EFFECTS OF OESTROGEN DERIVATIVES ON
BK CHANNELS
M Allen, Jacqueline Maher, A Hunter, J Mabley
University of Brighton, School of Pharmacy & Biomolecular Sciences,
Brighton, UK
There is growing evidence for steroid hormones having non-genomic
actions, including direct actions on the function of ion channels. Oestrogens and xenoestrogens are no exception. Oestrogens activate the BK
channel when the alpha subunit is associated with the beta 1 subunit (De
Wet H et al, Molecular Membrane Biology 2006; 23: 420-429). Interestingly, structurally unrelated antioestrogens such as tamoxifen also activate BK channels with associating beta 1 subunits. We have synthesised
novel steroidal oestrogens incorporating some of the structural motifs of
the non-steroidal antioestrogens. Oestrone analogues were tested for their
ability to activate BK channels in rat aortic rings pre-contracted with
phenylephrine. These analogues were also tested for their ability to activate BK currents in HEK 293 cells expressing BK alpha subunits alone
or in combination with beta 1 subunits. In addition, BK channels were
incorporated in planar lipid bilayers under voltage clamp conditions and
the ability of analogues to directly activate BK channels was determined.
All analogues of oestrone could relax aortic rings, with the following
order of increasing potency quatDME-oestrone, DME-oestrone, oestrone,
Oestrone-oxime. None of the ligands appeared to activate the BK channels in HEK 293 cells expressing alpha subunits alone, but oestrone
could activate BK currents in HEK 293 cells expressing alpha and beta 1
172
subunits. These electrophysiological ndings were mirrored in our single
channel studies conducted in bilayers. Our studies suggest that oestrone
activates the BK channel directly but Oestrone-oxime has additional
endothelium-dependent actions.
Paper No.: 795

EVALUATION OF THE PROULCEROGENIC EFFECT OF
ATORVASTATIN ON INDOMETHACIN INDUCED GASTRIC
INJURY IN RATS
Ilkay Alp(1), O Uyanik(1), H Ozyogurtcu(2), A Gurel(2), P Atukeren(3),
K Gumustas(3), A Kapucu(4), C Demirci(4), O Ozdemir(1), BSU
Dogan(1)
(1) Istanbul University. Faculty of Pharmacy, Department of Pharmacolgy,
Istanbul, Turkey
(2) Istanbul University, Faculty of Veterinary Medicine, Department of
Pathology, Istanbul, Turkey
(3) Istanbul University, Cerrahpasa Medical Faculty, Department of
Biochemistry, Istanbul, Turkey
(4) Istanbul University, Biology Division, Department of Zoology,
Istanbul, Turkey
Statins are suggested to possess healing properties due to their antioxidant
and antiinammatory effects in animal models of inammatory diseases
such as intestinal ulcer and colitis. In contrary, a clinical report indicated
gastric ulcer formation in a patient by use of atorvastatin for 3 months.
We previously observed that acute or subchronic (5 days)administration
of atorvastatin have no favourable inuence on indomethacin-induced
gastric ulcer lesions at 0,5 and 5mg/kg doses in rats. Interestingly, 50 mg/
kg of atorvastatin signicantly aggravated indomethacin-induced ulcer
lesions with mechanisms possibly involving iNOS and increased neutrophil accumulation to gastric mucosa but independently from the inhibition
of mevalonate pathway. Whereas, a direct ulcerogenic effect of atorvastatin was not determined. Herein, we investigated gastric mucosal levels of
TNF-a, myeloperoxidase (MPO), reduced glutathione (GSH), and prostaglandin E2 (PGE2) in order to furtherly enlighten the proulcerogenic effect
of atorvastatin. Gastric ulcer scores obtained in atorvastatin (0.5-50mg/
kg,p.o) administered groups were compared with controls received vehicle plus indomethacin (30mg/kg,i.p). Enhanced gastric mucosal lesions in
rats receiving atorvastatin (50mg/kg) were associated with an increase in
gastric mucosal TNF-a and MPO levels whereas with a decrease in PGE2
and GSH levels. These ndings showed that proulcerogenic effect of atorvastatin is associated with decreased defense mechanisms of gastric
mucosa as well as increased neutrophil inltration and proinamatory
cytokines. Excessive production of NO via iNOS may be correlated with
the generation of TNF-a in the proulcerogenic effect of atorvastatin.
The present work was supported by the Research Fund of Istanbul
University. Project No. T-76 15122006.
Paper No.: 1276

WRONG; STABILIZATION OF CARDIAC FUNCTION
MODULATORY EFFECTS OF ZINC SUPPLEMENTATION ON
CARDIOVASCULAR COMPLICATIONS IN
STREPTOZOTOCIN-INDUCED DIABETES IN RATS
Nouf MI AL-Rasheed, NMI AL-Rasheed, HA Attia, LM Faddah
King Saud University, College of Pharmacy, Department of Pharmacology, Riyadh, Saudi Arabia
vent or delay cardiovascular complications. Diabetes was induced in rats

by a single intraperitoneal injection of streptozotocin (STZ, 40 mg kg-1).
After 45 days, diabetic group showed cardiovascular complications manifested by signicant elevations of troponin-I (0.24 0.03 ng ml-1 Vs.
0.023 0.001, p < 0.001) as well as cardiac enzymes. Total cholesterol,
LDL-C and triglycerides were signicantly increased compared to nondiabetic group. Diabetic group showed impaired oxidative status
manifested by a signicant elevation of lipid peroxides, and reductions
of glutathione and L-ascorbic acid. Serum levels of TNF-a were signicantly increased compared to non-diabetic group (236.5 16.2 ng ml-1
Vs. 169.7 2.9, p < 0.001). Nitric oxide (NO) and vascular endothelial
growth factor (VEGF) were markedly higher indicating endothelial dysfunction and disturbance of angiogenesis. Treatment with glibenclamide
(600 lg kg-1, i.p) resulted in an improvement in most of the deviated
biochemical parameters. However, combination of glibenclamide with
zinc sulfate (20 mg kg-1, i.p) showed more pronounced hypoglycemic
effect and a signicant improvement in cardiac function as compared to
glibenclamide alone treated group. Moreover, LDL-C was signicantly
decreased by the combination (34.46 2.7 mg dl-1 Vs. 49.35 4.1,
p < 0.01). Oxidative stress markers were improved by the combination.
Serum levels of TNF-a, NO and VEGF were signicantly reduced by
the combination. These ndings indicate that combination of zinc with
glibenclamide may potentiate hypoglycemic effect of glibenclamide and
limit the diabetic complications.
Paper No.: 890

ADVERSE EVENTS OF BENZNIDAZOLE IN CHILDREN WITH
CHAGAS DISEASE - A COHORT STUDY
Jaime Altcheh(2), G Moscatelli(2), S Moroni(2), F Garcia-Bournissen(1),
H Freilij(2)
(1) Hospital for Sick Children, Division of Clinical Pharmacology, Toronto, Ontario,Canada
(2) Servicio de Parasitologia, Hospital de Ninos R. Gutierrez, Buenos
Aires,Argentina
Introduction: Chronic Chagas disease (caused by infection with Trypanosome Cruzi) is associated with severe long term complications
such as cardiac disease. Treatment of children with benznidazole can
cure the disease and prevent chronic infection. Benznidazole is associated to a high incidence of adverse drug reactions (ADRs) in adults,
including neuropathy and gastrointestinal symptoms. However, treatment in children seems to be associated with lower incidence and
severity of ADRs. We aimed to describe ADRs in a cohort of children
treated with benznidazole. Materials/Patients: A prospective study of
children with Chagas disease treated with benznidazole in a tertiary
pediatric centre in Buenos Aires, Argentina. Results and Conclusion:
107 children with Chagas were enrolled between 2004 and 2007. Mean
age of the patients was 6.9 years (CI 95%: 10 days - 19 years). 62
ADRs (in 44 children) were observed, most (77.3%) in children over
7 years of age. ADRs could be classied as mild (50; 80.6%), moderate (10; 16%) and severe (2; 3.2%). 75% (32) of the ADRs occurred
in the rst 10 days of treatment. Skin was the most commonly affected
organ (50%), followed by the central nervous system (22%), and gastrointestinal tract (21%). 7 patients did not complete treatment with
benznidazole due to ADRs. Conclusion: Treatment of Chagas disease
with benznidazole is well tolerated in children. Incidence of ADRs is
strongly correlated with age (most ADRs occurred in children over
7 years). Severe ADRs frequently reported in adults were not observed
in our cohort.
Diabetes is one of most prevalent diseases that is now regarded as the

strongest risk factor for cardiovascular diseases. This study aimed to
investigate whether combination of zinc sulfate with glibenclamide preJournal compilation 2010 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 107 (Suppl. 1), 162692
173
Paper No.: 951
POPULATION PHARMACOKINETICS STUDY OF
BENZNIDAZOLE IN CHILDREN WITH CHAGAS DISEASE
Jaime Altcheh(1), G Moscatelli(1), G Mastrantonio(3), S Moroni(1), N
Giglio(1), G Koren(2), H Freilij(1), F Garcia-Bournissen(2)
(1) Hospital de Ninos R Gutierrez de Buenos Aires, Servicio de
Parasitologia y Chagas, Buenos Aires, Argentina
(2) University of Toronto, Hospital for Sick Children, Division of
Clinical Pharmacology & Toxicology, Toronto, Ontario, Canada
(3) LASEICIC, Departamento de Quimica, Universidad de La Plata,
Argentina
Introduction: Chagas disease is caused by Trypanosoma cruzi, and leads
to long term cardiac and gastrointestinal complications. Treatment of
children with benznidazole is effective, but no information on its pharmacokinetics and no pediatric formulation are available. Patients and
Methods: Population pharmacokinetics study in children with Chagas
disease, aged 2-12 years (clinical trials.gov #NCT00699387). Enrolled
children (target N = 50) were treated with oral benznidazole 58 mg/kg/
day BID for 60 days, as per current pediatric Chagas protocols. At least
3 blood samples were obtained after the rst dose, at steady state or after
the last dose. Benznidazole was measured in blood by HPLC. Results
and Discussion: 25 children were enrolled to date. Mean age was 6 years
(range 2.212 years). Median benznidazole dose was 6.3 mg/kg/day.
Median steady-state (trough) benznidazole concentration was 0.99 mg/L,
and highest observed concentration (Cmax) was 11.07 mg/L. All children treated had a positive response, with negativization of PCR for T.
cruzi DNA, and marked decrease in anti T. cruzi antibody titers.
Observed benznidazole concentrations in children were markedly lower
than those reported in adults (treated with comparable mg/kg doses). In
spite of these lower concentrations children treatment was effective and
well tolerated, with few adverse drug reactions (ADRs). Unlike adults,
ADRs in children are uncommon, and severe ADR are rare. It is possible
that the lower blood concentrations, while still providing therapeutic
effect, may be responsible for this lower incidence of ADRs. If conrmed, our results would suggest that dosing modications in adults may
be benecial.
Paper No.: 2982

DISEASES
5-(4-METHANESULPHONYL-PHENYL)-THYAZOLE
DERIVATIVES: NEW TOOLS FOR CHRONIC
INFLAMMATORY DISEASE BIOTHERAPY
Melchor Alvarez de Mon(1,2), F Ledo(3), E SanAntonio(1), E Sanz(1),
R Corcostegui(3), A Berisa(3), V Rubio(3), ML Lucero(3), A Munoz(3),
A de-la-Hera(1,4)
(1) Alcala University, Madrid, Spain
(2) Principe de Asturias University Hospital, Department of Medicine,
Madrid, Spain
(3) FAES FARMA R&D and Innovation, Spain
(4) Molecular Medicine Institute (IMMPA-CSIC/UAH), Spain
Antagonists of TNF-a binding to its receptor are successful examples of
single-cytokine targeted biotherapies of chronic inammatory diseases
such as reumathoid arthritis (RA). However, a large fraction of those
patients are non-responders to a given mono-biotherapy. It would be
highly desirable to nd small molecule drugs that selectively block not
only TNF-a production but also the production of other pathogenic cytokine(s) to assess new poly-biotherapy strategies with novel single drug
tools. Using multiplex immunoassays of cytokines secreted by healthy
human peripheral blood mononuclear cells (PBMC) stimulated with
either CD3 + CD28 antibodies or LPS, we have discovered that
5-(4-Methanesulphonyl-phenyl)-thiazole derivatives are a new drug family able to selectively inhibit both prototype Th1 cytokine IFN-c and
TNF-a production, as exemplied by FAES compound 12 and other
structurally related drugs. Compound 12 inhibited TNF-a mRNA transcription in puried LPS-stimulated human monocytes in vitro, and
reduced TNF-a plasma levels in mice injected with LPS. It also ameliorated the clinical scores of arthritis in a collagen-induced arthritis mouse
model of human RA. Notably, it selectively reduced TNF-a and INF-c
production by PBMC stimulated with LPS and CD3 + CD28 respectively also when PBMC was from RA patients with active disease that
had been resistant to methotrexate treatment, and were to enter anti-TNFa biotherapies. It is now possible to examine putative advantages of
poly-biotherapy of inammatory diseases with complex pathogenic cytokine deregulation with novel single drug tools.
Paper No.: 1403

CARDIAC AND RENAL EFFECTS OF TITYUS SERRULATUS
VENOM AND A NEW ISOLATED PEPTIDE
Renata S Alves(1), ARC Jorge(2), RM Ximenes(2), A Havt(2), NRF
Nascimento(3), MGR Queiroz(1), AMC Martins(1), MH Toyama(4), HSA
Monteiro(2)
(1) Federal University of Ceara, Department of Clinical and
Toxicological Analysis, Ceara, Brazil
(2) Federal University of Ceara, Department of Physiology and
Pharmacology, Ceara, Brazil
(3) Ceara State University, Superior Institute of Biomedical Sciences,
Ceara, Brazil
(4) Paulista State University (UNESP), Sao Paulo, Brazil
Tityus serrulatus is a very dangerous scorpion species accounting for
fatal stings, especially among children in Brazil. The aim of the present
work was to determine the effects of Tityus serrulatus venom and a new
peptide, recently isolated from scorpion venom, weighing 3KDa on cardiac function in vivo. The effects of the crude venom (TsV; 0,6lg/min)
or toxin (PNTsV; 0,2lg/min; n = 6) were studied on mean arterial pressure (MAP), heart rate (HR), urinary ow (UF), glomerular ltration rate
(GFR) and renal cortex blood ux (RCBF), estimated by the clearance of
p-aminohippurate by, in vivo experiments. Data were analyzed by ANOVA and Student t-test using *p < 0.05. Both TsV and PNTsV increased
MAP (C = 102.5 3.5 vs TsV = 134.2 6.3 and PNTsV = 116.0
4.2 mmHg*) but only PNTsV increased HR (C = 239.8 29.35 vs
PNTsV = 355.0 25.30 bpm*). The peptide also increased UF with a
maximum effect at 90 minutes (C = 8.9 1.8 vs PNTsV = 33.8
6.2 lL/min*). While TsV was found to reduce GFR, PNTsV actually
increased GFR (C = 0.18 0.08 vs TsV = 0.03 0.01* vs PNTsV =
0.32 0.05 lL/min/100g) and the renal cortex blood ux was reduced
after the crude venom treatment. In conclusion, In vivo experiments
showed how the crude venom and its isolated components act increasing
arterial blood pressure, heartbeat frequency and altered renal function.
Paper No.: 1140

EVALUATION OF THE ANTICONVULSANT ACTIVITY OF
ZANTHOXYLUM CAPENSE (THUNB.) HARV. (RUTACEAE) LEAF
METHANOL EXTRACT IN MICE
George Amabeoku, CG Kinyua
University of the Western Cape, School of Pharmacy, Cape Town, South
Africa
Zanthoxylum capense (Thunb.) Harv. (Rutaceae) is used in South Africa
by traditional medicine practitioners to treat various ailments including
174
epilepsy. However, the claims of therapeutic success of the plant species
in therapy have not been subjected to any scientic scrutiny. The main
aim of the study, therefore, was to investigate the anticonvulsant activity
of the leaf methanol extract of Z. capense in mice. Chemically-induced
convulsion tests were used to assess the anticonvulsant activity of Z.
capense in mice. The acute toxicity and HPLC studies and the phytochemical analysis of the plant species were also carried out using well
established protocols. Leaf methanol extract of Z. capense and not methanol alone, signicantly antagonized seizures induced by pentylenetrazole (PTZ), bicuculline, picrotoxin and strychnine while only signicantly
delaying the onset of N-methyl-DL-aspartic acid (NMDLA)-induced seizures. Phenobarbitone, diazepam and not phenytoin, signicantly antagonized seizures induced by PTZ, bicuculline and picrotoxin but all three
drugs did not alter NMDLA-induced seizures. Phenobarbitone signicantly attenuated strychnine-induced seizures. The LD50 value obtained
following oral administration of the leaf methanol extract of Z. capense
was above 3200 mg/kg. The phytochemical analysis of the plant species
revealed the presence of alkaloids, triterpene steroids, reducing sugars,
saponins, tannins and quinones while the HPLC study revealed distinct
characteristic peaks .The data obtained indicate that the leaf methanol
extract of Z. capense has anticonvulsant activity which may probably
involve both Gabaergic, glutaminergic and glycinergic mechanisms. The
relatively high LD50 value obtained following oral administration shows
that the plant extract is non-toxic in mice.
Paper No.: 2310

PROTECTIVE EFFECT OF LAFUTIDINE AGAINST
LOXOPROFEN-INDUCED SMALL INTESTINAL LESIONS IN
RATS
Kikuko Amagase, R Kawahara, S Takayama, T Sugihara, A Ochi, S
Kato, K Takeuchi
Kyoto Pharmaceutical University, Division of Pathological Sciences,
Department of Pharmacology & Experimental Therapeutics, Kyoto,
Japan
The effect of lafutidine, a histamine H2-receptor antagonist with the
mucosal protective action mediated by sensory neurons, on loxoprofeninduced smalli ntestinal lesions was examined in rats, in comparison with
other H2-receptor antagonists and an proton pump inhibitor. Male SD
rats were administered loxoprofen Na and killed 24 hr later. Lafutidine,
cimetidine, famotidine or 16,16-dimethyl prostaglandin E2 (dmPGE2)
was given, 0.5 hr before and 6 hr after the administration of loxoprofen,
while ampicillin was given 24 hr and 0.5 hr before. Omeprazole was
given 0.5 hr before loxoprofen treatment. Loxoprofen dose-dependently
caused severe lesions in the small intestine, with the up-regulation of
inducible nitric oxide synthase (iNOS) expression, and an increase of
myeloperoxidase (MPO) activity as well as enterobacterial invasion in
the mucosa. These events were all prevented by dmPGE2 and ampicillin.
Likewise, lafutidine also prevented these lesions with the concomitant
suppression of up-regulaton in iNOS expression, MPO activity as well as
bacterial invasion. However, neither cimetidine, famotidine nor omeprazole had any effect on these lesions. The protective effect of lafutidine
was signicantly attenuated by chemical ablation of capsaicin-sensitive
sensoryneurons. This agent increased by itself intestinal mucus secretion
and signicantly suppressed the decreased mucus response to loxoprofen.
These results suggest that lafutidine protects the small intestine against
loxoprofen-induced lesions mediated by capsaicin-sensitive sensory neurons. This effect may be essentially attributable to the increase of mucus
secretion, resulting in the inhibition of bacterial invasion and iNOS
expression in the intestinal mucosa.
Paper No.: 2332

THERAPEUTIC TARGETS
AGGRAVATION BY SELECTIVE SEROTONIN RE-UPTAKE
INHIBITOR OF ANTRAL ULCERS INDUCED BY
INDOMETHACIN IN RATS: PATHOGENIC IMPORTANCE OF
IMPAIRED ANTI-OXIDATIVE SYSTEM
Kikuko Amagase, A Kojo, C Izuhara, K Nukui, A Tanaka, K Takeuchi
Japan
Clinical studies suggested a risk of gastric adverse reactions on the concomitant use of selective serotonin re-uptake inhibitors (SSRIs) with nonsteroidal anti-inammatory drugs (NSAIDs). We demonstrated the
adverse effect of paroxetine, one of SSRIs, in the rat stomachs when
co-administered with indomethacin, and investigated the mechanism of
this adverse effect. We also examined the effects of various agents on the
antral ulcers induced by indomethacin plus paroxetine. The animals were
fed chow for 1 h after 24 h fasting, then given indomethacin and killed
6 h later. SSRIs were given 30 min before indomethacin. Indomethacin
caused antral ulcers in refed rats. Paroxetine dose-dependently aggravated the severity of these ulcers. The worsening effect of paroxetine
was attenuated by ondansetron, a selective 5HT3 antagonist. The combined treatment with indomethacin and paroxetine caused a signicant
decrease in mucosal superoxide dismutase activity as well as gluthathione content in the antral mucosa. The development of these antral ulcers
was prevented by omeprazole, pepstatin as well as rebamipide. These
results suggest that SSRIs exert a harmful inuence on the antral mucosa
through activation of 5HT3 receptors when given with NSAIDs, resulting in aggravation of antral ulcers, and the process of aggravation may
be modied by the corrosive action of acid/pepsin as well as the
decreased anti-oxidative system. Rebamipide, a mucosal protective drug
developed in Japan, is useful for preventing the antral ulcers caused by
the combined administration of NSAID and SSRI, probably by its
scavenging action as well as its anti-inammatory effect.
Paper No.: 2333
DISEASES
ROLES OF COX/PGE/EP4 RECEPTORS IN HEALING OF
INDOMETHACIN- INDUCED SMALL INTESTINAL LESIONS
IN RATS: RELATION TO VEGF EXPRESSION
Kikuko Amagase, A Kojo, N Abe, R Hatazawa, M Tanigami, K Takeuchi
Japan
Endogenous prostaglandins (PGs) play an important role in the healing
of small intestinal lesions. We investigated the mechanism by which PGs
promote the healing of intestinal lesions in rats, including the determination of EP receptor subtype(s) involved in this action and the relation
with VEGF expression. Intestinal lesions of male SD rats were induced
by indomethacin (10 mg/kg). Indomethacin (2 mg/kg) was given for
6 days, starting 1 day after ulceration. AE1-329 (EP4 agonist) was given
for 6 days in the presence of indomethacin. For studying VEGF expression, indomethacin and AE3-208 were given for 2 days, while AE1-329
was given together with indomethacin. Indomethacin caused severe
lesions in the small intestine, and the lesions healed within 7 days. The
healing of these lesions was signicantly impaired by the repeated treatment of indomethacin. The healing impairment effect of indomethacin
was mimicked by the EP4 antagonist AE3-208 given for 6 days and
reversed by the co-administration of the EP4 agonist. The COX-2 mRNA
expression in the small intestine was up-regulated after ulceration with
increase of PGE2 content. VEGF expression was also increased after
ulceration. The expression of VEGF and the number of microvessels in
175
the mucosa was down-regulated by indomethacin and AE1-208, while
these effects of indomethaicn were reversed by the co-treatment with
AE1-329. These results conrmed an important role of endogenous
PGE2 in the healing of small intestinal lesions and further demonstrated
that this action is associated with the up-regulation of VEGF expression
mediated by the activation of EP4 receptors.
Paper No.: 1358

HEALTH AND DISEASE
SPECIES DIFFERENCE IN CALCIUM DEPENDENCY OF
CROMAKALIM INDUCED RELAXATION IN PORCINE AND
HUMAN CORONARY ARTERIES
Nora Ambrus(1), J Pataricza(1), I Krassoi(2), Z Marton(1), M Bitay(3),
A Varro(1,2), GJ Papp(1,2)
(1) University of Szeged, Department of Pharmacology and Pharmacotherapy, Szeged Hungary
(2) Hungarian Academy of Sciences, University of Szeged, Division of
Cardiovascular Pharmacology, Szeged, Hungary
(3) University of Szeged, 2nd Department of Internal Medicine and Cardiological Center Department of Cardiosurgery, Szeged, Hungary
Introduction: We have shown that relaxation induced by levosimendan, a
potassium channel opener inodilator drug, is decreased with the elevation
of extracellular calcium concentration in porcine and human coronary
arteries (Krassoi I et al, Cardiovasc. Drug. Ther. 2000; 14:691-693). Our
aim was to study the effect of cromakalim, another potassium channel
opener, in isolated human and porcine coronary arteries, and the possible
modulation of cromakalim-induced vasorelaxation at low and high extracellular calcium concentrations. Materials: Isometric tensions of porcine
and human coronary arteries were measured in isolated organ baths.
Contractions were induced by potassium chloride (30 mM) and the prostaglandin analogue, U46619 (0.7 lM). Relaxations by cromakalim
(0.0125-10.0 lM) and 1-EBIO (1-80 lM), an opener of calcium activated potassium channels, at low (1.5 mM) and high (7.5 mM) extracellular calcium concentrations were investigated. Endothelium intact and
deprived arterial preparations were used, and their functions were controlled by bradykinin (1 lM, porcine) and acetylcholine (0.1 lM,
human). Results: High extracellular calcium ([Ca2 + ]o) concentration signicantly enhanced the relaxing effect and the potency of cromakalim
both in porcine and human coronary arteries. In porcine coronary artery,
high [Ca2 + ]o potentiated the effect of cromakalim and 1-EBIO only in
the presence of endothelium. In human coronary artery, the elevation of
[Ca2 + ]o enhanced the relaxation of cromakalim both in the presence and
in the absence of functional endothelium. Conclusion: Results indicate
that the coronary dilating effect of cromakalim largely depends on the
species and is modulated by extracellular calcium concentration with a
partly endothelium dependent manner.
Paper No.: 502

IMPROVING THE ANTIBACTERIAL ACTIVITY OF
LYSOZYME BY CONJUGATION WITH POLYSACCHARIDES
Mahmoud Aminlari
Shiraz University, Department of Biochemistry, Shiraz, Iran
A widespread trend in biomedical sciences is replacing chemical antimicrobials with naturally occurring agents. Lysozyme is an enzyme that
destroys bacteria by lysis of the bacterial cell wall. Lysozyme is found
abundantly in nature and is effect against G-positive bacteria but not
against G-negatives due to the presence of the LPS layer in outer membrane
of the latter. The purposes of this research was to glycosylate lysozyme
with polysaccharides and to study its effect on G-positive and G-negative
bacteria in vitro. Lysozyme and polysaccharides were allowed to react
under mild conditions (60 0C for one week), the glycosylated products
were separated by ion-exchange and gel ltration chromatography and
the identity of the products was conrmed by electrophoresis. The antimicrobial properties of lysozyme-polysaccharide conjugates against test
microorganisms in culture media was evaluated. Under optimum conditions, 3.0 moles dextran, 1.2 moles dextran sulfate and 3.6 mmoles b-glucan were coupled to 1 mole lysozyme. Glycosylated lysozymes exhibited
increased solubility at different temperatures and pHs. Evaluation of the
lysozyme-polysaccharide conjugates against test microorganisms (S. aureus
and E. coli) in culture media indicated an increase in antimicrobial
activity in a concentration-dependent manner such that at 400 lg/ml,
Lysozyme-dextran conjugate decreased S. aureus and E. coli by 2 and 4
log cycle, respectively. Corresponding values for dextran sulfate were 0.6
and 1.1 and for b-glucan were 1 and 3. These results might increase the
applicability of lysozyme as a natural antimicrobial ingredient against a
broader spectrum of bacteria in different pharmaceutical preparations.
Paper No.: 2478

DISCOVERY
CYCLOSPORINE A, BUT NOT TACROLIMUS, SHOW
RELEVANT INHIBITION OF OATP1B1 MEDIATED
TRANSPORT OF ATORVASTATIN
Rune Amundsen(1), H Christensen(1), B Zabihyan(1), A Asberg(1)
School of Pharmacy, University of Oslo, Department of Pharmaceutical
Biosciences, Oslo, Norway
The aim of this study was to investigate and compare the potential of CsA
and Tac to inhibit cellular uptake of atorvastatin mediated by the liver specic organic anion transporting polypeptide 1B1 (OATP1B1) in vitro.
Due to a high incidence of dyslipidaemia following solid organ transplantation, HMG-CoA reductase inhibitors (statins) are now frequently used.
The calcineurin inhibitor cyclosporine A (CsA) however increases systemic exposure of atorvastatin several-fold, an effect not observed with
tacrolimus (Tac), another calcineurin inhibitor. The inhibition of atorvastatin transport via OATP1B1 by CsA and Tac was investigated in
transfected HEK293 cells. An in vitro-in vivo extrapolation (IVIVE) was
performed to estimate the potential for CsA and Tac to inhibit OATP1B1
mediated transport in vivo. CsA inhibited OATP1B1 mediated uptake of
atorvastatin about 90-fold more efciently than Tac, with inhibition constants (Ki) of 0.014 0.003 lM and 1.30 0.27 lM, respectively. The
IVIVE showed that clinically obtainable concentrations of CsA, but not
Tac, inhibit OATP1B1 transport of atorvastatin to a relevant degree. The
estimated degree of inhibition by CsA ranged from 28-77% during a dosing interval while it was below 1% for Tac when considering free concentrations and assuming competitive inhibition. This does however not fully
explain the clinically observed interaction with CsA, suggesting a more
complex inhibitory mechanism may be present. This study provides
evidence that OATP1B1 inhibition is a relevant mechanism for the interaction observed between CsA and atorvastatin. Tac on the other hand does
not have the properties to induce a similar interaction in vivo.
Paper No.: 2809

IN-VITRO CHARACTERISATION OF THE CLOPIDOGREL
BIOACTIVATION PATHWAYS BY CYTOCHROMES P450 AND
POTENTIAL DRUG-DRUG INTERACTIONS
Virginie Ancrenaz(1), Y Daali(1), C Samer(1), M Besson(1),
P Fontana(2), P Dayer(1), J Desmeules(1)
(1) Geneva University Hospitals, Department of Clinical Pharmacology
and Toxicology, Geneva, Switzerland
(2) Geneva University Hospitals, Division of Angiology & Hemostasis,
Geneva, Switzerland
176
Aim: Clopidogrel is a thienopyridine widely used to prevent thrombosis
in patients with acute coronary syndrome or after a stent implantation,
often used in association with aspirin. It is a prodrug which needs to be
activated by CYP450s in two steps to form a pharmacologically active
metabolite. The aim of our work was to characterize the cytochrome
P450 isoforms involved in the two steps of the bioactivation of clopidogrel as well as the importance of each cytochrome in the complete bioactivation mechanism. Methods: Human liver microsomes with selective
CYP450s inhibitors and cDNA expressed human CYP isoforms were
used for clopidogrel metabolism assessment. The intermediate (2oxoclopidogrel) and the active metabolites were analyzed with an HPLC-MS
method. A derivatization method was needed to avoid a rapid degradation of the active metabolite before its determination. Results: The results
obtained with microsomes and human CYP isoforms indicated that
CYP2C9, CYP1A2 and CYP2C19 were the most involved in the production of 2oxo-clopidogrel. CYP3A4, CYP2B6 and CYP2C19 contributed to the formation of the pharmacologically active metabolite from
2oxo-clopidogrel. In the inhibition studies with specic chemical inhibitors, the formation of the active metabolite from clopidogrel was in
accordance with the results obtained in the separate two steps. Conclusion: CYP2C19 is involved in both steps required in the formation of
clopidogrel active metabolite. CYP1A2 and CYP2C9 contributed
substantially to the rst step while CYP3A4 was the most important in
the second step. Hence, genetic polymorphisms or drug-drug interactions
involving these pathways should be considered during clopidogrel
therapy.
Paper No.: 2071

P04 - PHARMACOEPIDEMIOLOGY, CURRENT
CONTROVERSIES AND OPPORTUNITIES
ASSOCIATION BETWEEN FDA DRUG CLASSIFICATION OF
FETAL RISKS AND THE RISK OF MAJORCONGENITAL
MALFORMATIONS: A REGISTER-BASED NATIONWIDE
COHORT STUDY
Jon T Andersen(1,2), M Petersen(1,2), NL Andersen(3), S Afzal(1,2), K
Broedbaek(1,2), BR Hjelvang(1,2), C Torp-Pedersen(4,5), HE Poulsen(1,2,5)
ClinicalPharmacology, Copenhagen, Denmark
(2) Bispebjerg Hospital, Department of Clinical Pharmacology, Copenhagen, Denmark
(3) Psychiatric Center Bispebjerg, Copenhagen, Denmark
(4) Gentofte University Hospital, Department of Cardiology, Gentofte,
Copenhagen,Denmark
(5) University of Copenhagen, Faculty of Health Science,, Copenhagen,
Denmark
Introduction: Since 1979 United States Food and Drug Administration
(FDA) has classied drugs according to their fetal risk when used during
pregnancy. The categories are A, B, C, D, and X; X dening the highest
evidence of risk. The aim of the study was to investigate whether rst trimester dispensing of one or more drugs within each of the categories
was associated with an increased risk of major malformations. Materials:
We conducted a nationwide cohort study using the Danish Fertility Database to identify every mother of children born in Denmark 1997-2005.
All prescriptions dispensed during pregnancy were identied through the
National Prescription Register and categorized according to FDAs classication. We included pregnant women with drugs dispensed only in the
rst trimester and drug-use designated to only one of the FDA-categories
or a not classied category. All diagnoses of major congenital malformations were identied through the National Hospital Register. All
groups where compared to women with no dispensing of drugs during
pregnancy. Results: The preliminary results did not show any signicantly increased risk of major malformations associated with dispensing
of drugs in the six categories. The odds ratio adjusted for age and education of having a child with a major malformation when redeeming a prescription in the respective FDA categories in the rst trimester where
A:1.12(CI95% 0.76-1.66), B:1.06(CI95% 0.96-1.16),

0.92-1.20), D:1.13(CI95% 0.97-1.31), X:1.01(CI95%
classied:1.14(CI95% 0.99-1.32). Conclusion: FDAs
fetal risks due to pharmaceuticals does not seem useful
risk of major malformations.
C: 1.05(CI95%
0.76-1.35), not
classication of
in predicting the
Paper No.: 2072

DRUG USE DURING PREGNANCY: A REGISTER-BASED
NATIONWIDE COHORT STUDY OF FDAS DRUG
CLASSIFICATION OF FETAL RISKS
Jon Andersen(1,2), M Petersen(1,2), N Andersen(3), S Afzal(1,2), K
Broedbaek(1,2), B Hjelvang(1,2), C Torp-Pedersen(4,5),
H Poulsen(1,2,5)
(1) Copenhagen University Hospital, Rigshospitalet, Laboratory of ClinicalPharmacology, Copenhagen, Denmark
(3) Psychiatric Center Bispebjerg, Copenhagen, Denmark
(4) Gentofte University Hospital, Department of Cardiology, Gentofte,
Copenhagen,Denmark
(5) University of Copenhagen, Faculty of Health Science, Copenhagen,
Denmark
Introduction: The knowledge of drugs used during pregnancy is limited.
The aim of the study was to describe the use of drugs during pregnancy
according to the United States Food and Drug Administration (FDA)
classication of fetal risks. Materials: We conducted a nationwide cohort
study using the Danish Fertility Database to identify every mother of
children born in Denmark 1997-2005. All prescriptions dispensed during
pregnancy were identied through the National Prescription Register and
categorized according to FDAs classication of fetal risk. The categories
are A, B, C, D, and X; X dening the highest evidence of risk. All drugs
not classied were grouped as one. Results: We identied 588,832 children born in the study period. During pregnancy 63.6% (374,654) of the
women redeemed a prescription on any drug; 3.1% (18,438) were dispensed a prescription on an X-classied drug during the entire pregnancy
and 3.0% (17,805) during the rst trimester. D-classied drugs were
redeemed by 10.8% (63,687) of the women during pregnancy and by
8.1% (47,729) during the rst trimester whereas C-classied drugs were
dispensed to 30.5% (179,423) during the pregnancy. 38.4% (226,082)
redeemed a prescription on a drug classied as a potential fetal risk (category C, D or X) during the entire pregnancy and 20.4% (123,070) during
the rst trimester. Conclusion: The majority of the pregnant women
redeemed prescriptions on drugs during pregnancy. Furthermore, a large
proportion of the women were dispensed drugs with potentially fetal risk
throughout the pregnancy and during the rst trimester.
Paper No.: 1240

THE ANTIPSYCHOTIC-LIKE EFFECT OF MYRICITRIN, A
PKC INHIBITOR IN MICE
Roberto Andreatini(1), M Pereira(1), L Pereira(1), MABF Vital(1), ARS
dos Santos(2), MG Pizzolatti(3)
(1) Universidade Federal do Parana, Department of Pharmacology
(2) Universidade Federal de Santa Catarina, Department of Physiological
Sciences
(3) Universidade Federal de Santa Catarina, Department of Chemistry
Protein kinase C (PKC) has been associated with bipolar disorder, since
manic patients show elevated platelet membrane-bound/cytosol PKC
177
ratio and antimanic drugs (lithium, valproate and tamoxifen) inhibit PKC
activity (Zarate and Manji, CNS Drugs 2009; 23:569-582). Moreover,
these antimanic drugs blocked amphetamine-induced hyperlocomotion,
an animal model of mania (Einat H et al, J Psychopharm 2006;20:714722; Sabioni et al, Prog Neuropsychopharmacol Biol Psychiatry
2008;32:1927-31). However, this animal model is also employed in the
study of antipsychotic drugs and psychotic symptoms can be seen in
manic patients. Thus, the present study evaluates the effect of mirycitrin
(a avonoid naturally occurring in several plants, e.g. genus Eugenia), a
PKC inhibitor (Meotti et al, J Pharmarcol Exp Ther 2006;316:789-96),
in animal models for antipsychotic-like action. Male Swiss mice treated
with mirycitrin (5 and 10 mg/kg), olanzapine (10 mg/kg; positive control) or their vehicles were tested in: (a) apomorphine-induced climbing;
(b)apomorphine-induced stereotypy; (c) bar catalepsy test (with and without haloperidol co-administration); (d) spontaneous locomotor activity.
Different groups of mice were used in each model. Mirycitrin (10 mg/
kg) and olanzapine blocked stereotypy and climbing induced by apomorphine (1.0 mg/kg). When administered alone, mirycitrin did not produce
catalepsy (up to 30 mg/kg), but it increased the catalepsy induced by
haloperidol (1.0 mg/kg). At the doses tested, mirycitrin did not alter
locomotor activity. These results suggest that mirycitrin has an antipsychotic-like effect at a dose that does not induce extra-pyramidal
side-effects and that PKC inhibition could be a new strategy for the
search of new antipsychotic drugs.
Paper No.: 1274

MAGNESIUM SULPHATE AND SODIUM VALPROATE BLOCK
METHYLPHENIDATE-INDUCED HYPERLOCOMOTION, AN
ANIMAL MODEL OF MANIC STATE
Roberto Andreatini(1), FJ Barbosa(1,2), B Hesse(1), RB de Almeida(1),
IP Baretta(1,3), R Boerngen-Lacerda(1)
(1) Universidade Federal do Parana, Department of Pharmacology, Curitiba, Brazil
(2) Universidade Federal do Parana, Department of Forensic Medicine
and Psychiatry, Curitiba, Brazil
(3) Universidade Paranaense, Instituto de Ciencias Biologicas, Medicas e
da Saude Farmacologia, Umuarama, Brazil
Magnesium sulphate (MgSO4) is used to treat and prevent eclamptic
seizure and several anticonvulsants (e.g. sodium valproate) are clinically
effective antimanic drugs. Some small sample-size, open and/or add-on
clinical studies with MgSO4 found improvement of manic symptoms
(Heiden A et al, Psychiatry Res, 1999;89:239-246; Giannini AJ et al.,
Psychiatry Res 2000; 93:83-7). Thus, these studies suggest a potential
antimanic effect of MgSO4. Psychostimulant-induced hyperlocomotion
is proposed as an animal model for the study of antimanic drugs (Einat H
et al, J Psychopharm 2006;20:714-722; Sabioni et al, Prog Neuropsychopharmacol Biol Psychiatry 2008; 32:1927-31). Therefore, we
evaluated the effect of MgSO4 and sodium valproate (positive control)
on methylphenidate-induced hyperlocomotion in mice exposed to the
open-eld. Acute MgSO4 (300-400 mg/kg), but not valproate (100300 mg/kg), prevents increase locomotor activity induced by methylphenidate (2.5 mg/kg). On the other hand, repeated (14 days) valproate
(300 mg/kg), but not MgSO4 (400 mg/kg), blocked methylphenidateinduced hyperlocomotion. These effects were seen at doses of valproate
or MgSO4 that did not change locomotor activity per se. In conclusion,
acute magnesium sulphate shows antimanic-like effect in this model,
which ts with add-on small clinical studies that employed a short-term
treatment. These results support further studies for investigating MgSO4
as a potential treatment for acute mania. Possible mechanisms of this
action are: NMDA non-competitive antagonism, PKC inhibition and/or
change in calcium effects (Murck H, Nutr Neurosci 2002;5:375-389).
However, this antimanic-like effect of MgSO4 disappears after repeated
treatment, which could be specic for the model used or a side-effect
of MgSO4.
Paper No.: 3114

FRET-BASED EVIDENCE FOR LIGAND-INDEPENDENT
PREASSOCIATION OF 5-HT7 SEROTONIN RECEPTORS WITH
GS
Kjetil W Andressen(1), L Hommers(2), AH Ulsund(1), MJ Lohse(2),
KA Krobert(1), M Bunemann(2), FO Levy(1)
(1) University of Oslo, Department of Pharmacology and Center for
Heart Failure Research, Oslo, Norway
(2) University of Wurzburg, Institute of Pharmacology and Toxicology,
Wurzburg, Germany
In classic pharmacology, G protein-coupled receptors (GPCRs), G proteins and effectors interact sequentially by random collision. Signalling
through GPCRs is both specic and very rapid, indicating that the proteins are efciently organized. Currently, there are two prevailing views
on how receptors, G proteins and effectors are organized; either preassembled in one complex (preassociated/precoupled) or interacting after
receptor-activation (collision-coupling) in discrete microdomains. Our
previous comparison of the Gs-coupled 5-HT4 and 5-HT7 serotonin
receptors revealed fundamental differences in G protein activation.
Whereas 5-HT4 receptor function is consistent with collision-coupling,
the unusual pharmacological properties exhibited by 5-HT7 receptors can
best t a model with receptor and Gs preassociated in the absence of
ligand. To test if 5-HT7 receptors preassociate with Gs, we used Fluorescence Resonance Energy Transfer (FRET) to compare the interaction
between YFP-labeled 5-HT4, 5-HT7 or b1-adrenergic receptors with
CFP-labeled G proteins in HEK293 cells. Agonist-activation of 5-HT4
or b1-adrenergic receptors increased FRET (indicating recruitment of
Gs). In contrast, 5-HT7 receptor activation decreased FRET in a concentration-dependent manner. The dissociation of G protein from 5-HT7
receptors had kinetics identical to G protein activation (dissociation
between Ga and Gbc determined by FRET), but slower than recruitment
of G protein to 5-HT4 and b1-adrenergic receptors. In addition, results
from uorescence recovery after acceptor photobleaching were consistent
with a stable complex between 5-HT7 and G protein. Taken together,
these data provide strong direct support that 5-HT7 receptors preassociate
with G protein, a property that likely accounts for the atypical signalling
characteristics of 5-HT7 receptors.
Paper No.: 3275

SHIKONIN, A NATURAL NAPHTOQUINONE, PROMOTES
WOUND HEALING IN INTESTINAL EPITHELIAL CELLS-18
Isabel Andujar, MC Recio, JL Ros
University of Valencia, Departament of Pharmacology, Burjassot, Valencia, Spain
The intestinal epithelial cell (IEC) barrier plays an important role in
maintaining mucosal immune homeostasis. Dysregulated activation of
the intestinal mucosal immune system eases the penetration of endogenous luminal bacterial ora through tight junction defects in the intestinal
epithelial barrier and this appears to initiate and perpetuate inammation
in inammatory bowel diseases.(1) In our search for anti-inammatory
agents from natural products we found that shikonin, a naphthoquinone
found in the root of Lithospermum erythrorhyzon Sieb. et Zucc., is able
to ameliorate dextran sulphate sodium (DSS)-induced acute colitis due to
the inhibition of the pro-inammatory nuclear transcription factors
NF-jB and STAT3.(2) Moreover, shikonin signicantly reduced the
extent and severity of the colon injury as shown by scores of macroscopic damage, and prevented body weight loss observed in DSS-treated
mice.(3) To further investigate the possible wound healing properties in
178
the damaged intestinal epithelial barrier, we examined effect of shikonin
on IEC-18 cells. IEC-18 monolayers were wounded with a razor blade.
Cells were incubated with or without shikonin 1lM and migration was
blocked with treatment with dexamethasone. After 5h, migration was
assessed by counting cells across the wound edge. Shikonin signicantly
induced epithelial cell migration and wound healing in IEC-18 cells compared to untreated cells or to dexamethasone-treated cells.
(1.) Cario E (2008) Mucosal Immunol 1 (Suppl 1), S62S66
(2.) Ros JL et al. (2009) Meth. Find. Exp. Clin. Pharmacol. 31 (suppl
A) 92
(3.) Andujar I et al. (2008) Meth. Find. Exp. Clin. Pharmacol. 30 (suppl
2) 86
Paper No.: 2862

FOCUSED CONFERENCE GROUP: P03 - ION CHANNELS IN
ANALGESIA & ANAESTHESIA
ROCURONIUM INTERFERES WITH CYTOCHROME P450 IN
VITRO
cytes (NHEK) and to investigate the preventive effect of EPC-K1: a

phosphate diester of vitamin C and vitamin E on UV-radiation-induced
NHEK injury. NHEK were cultured in EpiLife medium supplemented
with Human Keratinocyte Growth Supplement Kit. NHEK viability was
determined by crystal violet (CV) stain 48 hr after UV irradiation. The
mRNA of a transcription factor C/EBP homologous protein (Chop) and
an ER resident molecular chaperone Bip were determined by RT-PCR
analyses. UV was especially effective in the wavelength region 250280 nm in killing NHEK. The minimum exposure dose required to kill
50% of cells (LD50) was 1.64 mJ/cm2 at 269 nm. At 235 and 310 nm,
LD50 in NHEK were 6.62 and 293 mJ/cm2, respectively. After 660 mJ/
cm2 irradiation at 310 nm, the cell viability was signicantly decreased
to 30% of control (without irradiation). The addition of 100 lM EPCK1 after irradiation returned the cell viability to 118%. Six hr after
660 mJ/cm2 irradiation at 310 nm, Chop and Bip mRNA levels in
NHEK were increased 215% and 370% of control and those were
decreased by EPC-K1 treatment. Chop and Bip are responsive to endoplasmic reticulum(ER) stress. These results suggest that EPC-K1 exerts
a protective effect against UV-induced NHEK injury, in part through
the decreased ER stress.
Pavel Anzenbacher(1), E Anzenbacherova(2), A Tunkova(1), M Adamus(3)

(1) Palacky University of Olomouc, Faculty of Medicine and Dentistry,
Department of Pharmacology, Olomouc, Czech Republic
(2) Palacky University of Olomouc, Faculty of Medicine, Department of
Medicinal Chemistry and Biochemistry, Olomouc, Czech Republic
(3) Palacky University of Olomouc Hospital, Department of Anaesthesiology and Resuscitation, Olomouc, Czech Republic
Rocuronium is a relatively new neuromuscular blocking agent (NMBA)
acting as a competitive antagonist of acetylcholine on the postsynaptic
nicotinic receptor of neuromuscular junction with the most quick onset
among currently used non-depolarizing NMBA, and with an intermediate
duration of its action. It is predominantly used to facilitate endotracheal
intubation, to provide skeletal muscle relaxation during surgery, or to
facilitate mechanical ventilation in intubated, critically ill patients. Rocuronium, however, exhibits a great variability in the effect and in the
course of neuromuscular block as in the time of recovery. Variability of
response to rocuronium is according to the literature primarily caused by
pharmacokinetics of this drug. In the literature there are reports indicating that an interaction of rocuronium with liver monooxygenase system
of cytochromes P450 may contribute to these differences. We have found
an inhibition of human cytochrome P450 (form CYP3A4) enzyme activity (6-b testosterone hydroxylation) down to one half of the original
value. The mechanism of this inhibition was evaluated; the results indicate a partially noncompetitive inhibition mechanism. Interactions of this
kind may however inuence the pharmacokinetics of concomitantly
administered drugs.
The nancial support through the MSM6198959216 project is gratefully
acknowledged.
Paper No.: 1732

FOCUSED CONFERENCE GROUP: P19 - GENERAL
SESSION - TOXICOLOGY
ULTRAVIOLET ACTION SPECTRUM AND EFFECT OF
EPC-K1 ON ULTRAVIOLET-RADIATION-INDUCED INJURY IN
CULTURED NHEK
Kayo Aoki(1), T Nakanishi-Ueda(1), M Tsuji(1), T Okuno(2), K Oguchi(1), H Yasuhara(1)
Paper No.: 2994

HEPATOPROTECTIVE EFFECT OF PISTACIA LENTISCUS
AGAINST ISONIAZIDE INDUCED LIVER TOXICITY IN RATS
M Aouichri(1,2,4), M Haouari(1), L Sfaxi(1), R Zermani(3), M Ben-Attia(2), M Lakhal(4), Anis Klouz(4)
(1) Ecole Superieure des Sciences et Techniques de la Sante de Tunis,
Tunisia
(2) Laboratoire de Biosurveillance de lEnvironnement, Faculte des Sciences de Bizerte, Tunisia
(3) Laboratoire dAnatomie et Cytopathologie, Hopital Charles Nicolle,
Tunisia
(4) Centre National de Pharmacovigilance, Service de Pharmacologie
Clinique, Tunisia
Isoniazid (INH) is a widely used drug in association with other antibiotics for the tuberculosis treatment. INH can cause a severe hepatotoxicity in 1% of patients by increasing the production of free
radicals in liver. Plant extracts, especially certain vegetable oils appear
to have benecial antioxidant effects. The aim of this study is to
evaluate the protective effect of Pistacia lentiscus oil (PLO) against
INH hepatotoxicity. Male Wistar Rats were subacutely treated with
INH (25 mg/kg, i.p.) and grouped into the INH group and the INHPLO group for 1 month. The diets fed to the two groups of rats were
identical except the second group (INH-PLO), which received PLO
added to the standard diet. The diet contains 15 percent of PLO by
weight. Animals were sacriced 1 day after the last drug administration and the livers were rapidly removed. While one portion was used
for histopathology, other pieces were stored with plasma samples
immediately at -80C. PLO prevented INH-induced hepatotoxicity,
indicated by both diagnostic indicators of liver damage (alanine aminotransferase and aspartate aminotransferase) and plasma oxidative
stress as well as histopathological analysis. PLO markedly attenuated
the INH effects on the plasma enzymatic antioxidant defence system
(SOD and GPx). Altogether, these results suggest that PLO exerts its
hepatoprotective effects against INH hepatotoxicity by inhibiting the
production of free radicals in addition to its role as a scavenger. Conclusively, PLO might be credited with benecial health aspects and
could also be an important nutritional source rich in natural antioxidants.
(1) Showa University School of Medicne, Department of Pharmacology,

Tokyo, Japan
(2) National Institute of Occupational Safety and Health, Japan
The aims of this study are to determine the ultraviolet (UV:235310 nm) action spectrum for killing normal human epidermal keratinoJournal compilation 2010 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 107 (Suppl. 1), 162692
179
Paper No. 1651
FOCUS GROUP: FC13 - MAXIMISING BENEFITS AND
MINIMIZING HARMS FROM DRUGS
CYNODON DACTYLON A POTENT, ECONOMIC, NATURAL
ANTI-DIARRHOEAL HERB IN DEVELOPING COUNTRIES
Manoj Amrutkar, V Undale, D Bharti, A Bhosale
SGRS College of Pharmacy, Department of Pharmacology, Saswad,
Pune, India
The aqueous extract of aerial parts Cynodon dactylon (poaceae) was
investigated for its anti-diarrhoeal property in term of reduction in the
rate of defecation and consistency of faeces in castor oil induced diarrhea
in Wistar rats to substantiate folkloric claim. To understand the mechanism, the effect was further evaluated on intestinal transit and castor oil
induced intestinal uid accumulation (enteropooling). At various doses
(250 & 500mg/kg body weight) the extract showed a remarkable antidiarrhoeal activity evidenced by the reduction of the rate of defection
and consistency of faecas. Results are comparable to that of standard
drug loparamide (50 mg/kg body weight).A single oral dose of Cynodon
dactylon extract of 250mg/kg body weight produced a signicant
decrease in the severity of diarrhea. Extract produced profound decrease
in intestinal transit (39.66%) also signicantly inhibited castor oil
induced enteropooling comparable to that of intraperitoneal injection of
standard drug atropine sulphate at a doses of 0.1mg/kg body weight and
3 mg/kg body weight respectively. Experimental ndings showed that
aqueous extract of aerial parts of Cynodon dactylon possess signicant
anti-diarrhoeal activity and may be a potent anti-diarrhoeal drug in
future. Furthermore the extract formulated as a syrup (85% simple syrup
& 15% extract) also produced signicant anti-diarrhoeal activity.
Paper No.: 2917

A NEW ANTICOCCIDIAL PRODUCT DEVELOPED FROM
ARTEMISIA SIEBERI PLANT
Hossein Ali Arab(1), A Rasouli(1), J Kaboutari(2), S Rahbari(3)
(1) University of Tehran, Faculty of Veterinary Medicine, Depatment of
Pharmacology, Tehran, Iran
(2) Faculty of Veterinary Medicine, University of Tehran,
(3) University of Shahrekord, Faulty of Veterinary Medicine, Department
of Basic Sciences, Iran
(4) University of Tehran, Faculty of Veterinary Medicine, Department of
Parasitology, Tehran, Iran
Coccidiosis is an important parasitic disease of the chickens causing high
loss in the poultry industry. The emergence of resistance against convention anticoccidial drugs and the concerns of their tissue residues enforced
the attention toward the development of the new drugs. Artemisinin is
one the potential candidate which its anticoccidial effects have been
shown by some studies. This study aimed to develop a new anticoccidial
product from Artemisia sieberi. Artemisinin was extracted from Artemisia
sieberi and the amount of artemisin was determined by HPLC. A formulation of 1% feed additive was produced from Artemisia extract. Coccidiosis was experimentally induced in chickens by oral administration of
Eimeria tenella oocysts. The anticoccidial activity of the new product was
tested by administration of the compound with different doses of 1, 2.5 &
5mg/kg for 5 successive days. A group of infected birds was treated with
1mg/kg pure artemisinin, and the fth group remained as negative control
group. The feces of each group were collected at the end of each day, and
the amount of oocyst per gram (OPG) was determined as the index of the
anticoccidial effects of the compounds. It was found that the new compound was signicantly able to decease the OPG (P < 0.001) in a doseindependent manner. There was not any signicant difference between
the effects of the new compound with the pure artemisinin. The results of
this study suggest that the artemisinin granule formulated form artemisia
sieberi can be introduced, potentially as a new anticoccidial drug.
Paper No.: 1803

HEALTH AND DISEASE
VARIOUS TYPES OF POTASSIUM CHANNELS ARE
ACTIVATED BY THE NEW NITRIC OXIDE (NO) DONOR
[RU(TERPY)(BDQ)NO+]3 + (TERPY) IN MESENTERIC
RESISTANCE ARTERIES OF RENAL HYPERTENSIVE RATS
Alice Araujo(1), J Vercesi(2), J Biazzotto(2), R da Silva(2), L Bendhack(2)
(1) University of Sao Paulo, School of Medicine of Ribeirao Preto,
Department of Pharmacology, Sao Paulo, Brazil
(2) University of Sao Paulo, Faculty of Pharmaceutical Sciences of
Ribeirao Preto, Sao Paulo, Brazil
Nitrosyl ruthenium compounds such as Terpy are very attractive as NO
donors. We previously reported that NO released from Terpy does not
activate K+ channels in aortas from 2K-1C. The aim of this study was to
verify if Terpy relaxes the isolated third branch of rat mesenteric artery
isolated from 2K-1C and normotensive (2K) rats and the cellular mechanisms involved in the smooth muscle mol/L-0.01mmol/L) werel relaxation. Concentration-effect curves for Terpy (0.01 constructed in
endothelium-denuded mesenteric resistance arteries pre-contracted with
phenylephrine. We have analyzed its maximum effect (ME) and potency
(pD2). Terpy induced relaxation of mesenteric arteries from normotensive (ME: 93.9 1.2%; pD2:4.45 0.32; n = 7) and hypertensive (ME:
93.7 1.4%; pD2: 4.51 0.36; n = 8) rats in a similar way, which was
abolished by the soluble guanylyl-cyclase inhibitor, ODQ. The relaxation
was inhibited by the following K+ channel blockers: tetraethylammonium
(TEA, 1mmol/L), glibenclamide (3umol/L), apamin (1umol/L) and 4-aminopyridine (1mmol/L). Taken together, our results show that the new
NO donor Terpy induces vascular relaxation via activation of soluble
guanylyl-cyclase and various types of K+ channels such as ATP-sensitive, small conductance calcium-activated and voltage-activated K+ channels in isolated mesenteric resistance arteries from normotensive and
hypertensive rats.
This study was approved by Ethical Committee of the University of Sao
Paulo (CETEA-FMRP, protocol number: 044/2008).
Supported by FAPESP and CNPq.
Paper No.: 1369

DISCOVERY
UPTAKE OF FOLIC ACID AND METHYLTETRAFOLIC ACID
BY RAT AND HUMAN BLOOD-BRAIN BARRIER
ENDOTHELIAL CELL LINES
Joao R Araujo, P Goncalves, F Martel
Porto University Faculty of Medicine, Department of Biochemistry,
Porto,Portugal
The bloodbrain barrier (BBB) is a selective permeability tissue essential
for central nervous system homeostasis. Folates are essential for the biosynthesis of purines, thymidylate and methionine. Our aim was to characterize 3H-folic acid (3H-FA) and 14C-methyltetrahydrofolic acid (14CMTHF) uptake by rat (RBE4) and human (hCMEC/D3) BBB endothelial
cell lines. Uptake of 3H-FA and 14C-MTHF by RBE4 cells was timedependent and linear for the rst 2 min of incubation; uptake by
hCMEC/D3 cells showed a greater experimental variability. So, further
experiments were performed in RBE4 cells. Uptake of 3H-FA was found
to be stimulated at acidic and alkaline pH, Na+-dependent; stimulated
when F- substituted for Cl-, energy-independent, inhibited by premetrexed (PTX), stimulated by cytochalasin D (cyt D) and not affected by
MTHF, DIDS, SITS, methotrexate (MTX), monensin (MON) and FA.
Uptake of 14C-MTHF was found to be pH-, Na+-, Cl-- and energy-independent, inhibited by PTX and MTX, stimulated by cyt D and not
180
affected by FA, DIDS, SITS, MON and MTHF. RT-PCR analysis demonstrated mRNA expression of RFC (reduced folate carrier) but neither
of FRa (folate receptor a) nor of PCFT (proton-coupled folate transporter) in RBE4 cells, and mRNA expression of both RFC and PCFT,
but not of FRa, in hCMEC/D3 cells. In conclusion, both human and rat
BBB endothelial cells show little capacity for folates uptake.
Paper No.: 2958

EVALUATION OF THE GASTRIC ANTIULCER ACTIVITY OF
A PROTEOLYTIC FRACTION FROM CARICA
CANDAMARCENSIS LATEX
Ana Candida Araujo e Silva(1), FO Lemos(1), C Figueiredo(1), CTR
Viana(1), CM Souza(2), GD Cassali(2), CE Salas(3), MTP Lopes(1)
(1) Federal University of Minas Gerais, Department of Pharmacology,
Belo Horizonte, Brazil
(2) Federal University of Minas Gerais, Department of Pathology, Belo
Horizonte, Brazil
(3) Federal University of Minas Gerais, Department of Biochemistry and
Immunology, Belo Horizonte, Brazil
P1G10 is a proteolytic fraction, rich in cysteine proteases, obtained from
Carica candamarcensis (South America species). Previously, we have
found that these proteases display mitogenic effect, angiogenic and skin
healing properties on mammalian systems. Furthermore, we veried the
gastric protective and healing activities of P1G10 on gastric lesions
induced by indomethacin and acetic acid. In this study, we have investigated the antiulcer activity of P1G10 on other models of lesions, as well
as some pathways involved in this effect. In acute lesions produced by
ethanol, stress and pyloric ligation, P1G10 (10 mg/kg) showed a signicant gastroprotective activity, reducing the number/severity of lesions by
40%, 88% and 64%, respectively. The treatment with P1G10 promoted a
signicant decrease on gastric acidity and on peptic activity, with reductions of 56% (1 mg/kg) and 69% (10 mg/kg). We also demonstrated that
P1G10 increases the gastric levels of glutathione (42%) and that its proteolytic activity is not important for the gastroprotective action. In
chronic lesions induced by acetic acid, P1G10 showed an interesting
healing activity, reducing the ulcer size by 58%. This property is dependent on its proteolytic activity. Furthermore, we showed that P1G10
stimulates about 4-fold the epithelial cell proliferation at the ulcer margins. Thus, the gastroprotective activity plus the ability to stimulate cell
proliferation, makes of P1G10 a potential antiulcer agent.
Financial support: CNPq and Fapemig.
Paper No.: 2084

COMPARISON OF THE NEUROCHEMICAL CHANGES
INDUCED BY ACUTE AND REPEATED ADMINISTRATION OF
MORPHINE, COCAINE AND AMPHETAMINE IN THE RAT
LATERAL SEPTUM
Katherine A Araya, MR Ibanez, V Noches, R Sotomayor-Zarate, K
Gysling,
Ponticia Universidad Catolica de Chile, Department of Cellular and
Molecular Biology, Santiago, Chile
Addictive drugs activate dopaminergic neurons in the ventral tegmental
area. These neurons project to various limbic nuclei such as nucleus
accumbens, prefrontal cortex and lateral septum (LS). We have shown
that acute intravenous administration of morphine increases LS dopamine
(DA) release in a dose-dependent manner. However, the effect of
repeated administration of morphine or other addictive drugs in LS has
not been determined. Thus, we studied the neurochemical changes in LS
induced by acute and repeated administration of morphine (10 mg/kg

i.p.), cocaine (15 mg/kg i.p) and amphetamine (1,5 mg/kg i.p.). We analyzed DA, Glutamate and GABA extracellular levels using microdialysis
in LS of control and repeatedly treated rats with morphine, cocaine and
amphetamine. Addictive state of treated rats was evaluated through the
experimental paradigm of conditioned place preference. Our results
showed that acute administration of morphine and cocaine increased
extracellular DA levels in LS, without changing extracellular Glutamate
and GABA levels. By the contrary, acute administration of amphetamine
increased extracellular GABA levels without any signicant changes in
extracellular levels of DA and Glutamate. Although, the repeated administration of morphine and cocaine produced a signicant increase in DA
extracellular levels in LS, the amount of DA released is signicantly
lower compared to acute administration. These results suggest that LS is
involved as a relay nucleus modulating the response of other limbic
nuclei involved in addictive processes. We will evaluate the inactivation
of LS and its effects on place conditioning induced by morphine and
cocaine.
Funding: MSI P06/008-F and FONDECYT 107-0340
Paper No.: 1155

PUBLICATION TRENDS OF THE CYTOCHROMES P450
RESEARCH: 1977-2008
Charles-Daniel Arreto(1), C Wilson(2), C Robert(1)
(1) Universite Paris Descartes, Laboratoire dAnatomie Fonctionnelle,
Montrouge, France
(2) The University of New South Wales, Sydney, NSW, Australia
This study analyzes trends in cytochrome P450s research publications
from 1977 to 2008. Research articles and review papers with title keywords related to cytochrome-P450 were downloaded from the Web of
Science for four periods: 1977-1978, 1987-1988, 1997-1998, and 20072008. Overall the literature on P450s increased nearly seven-fold (6.9)
from 342 papers in 1977-1978 to 2357 in 2007-2008. For the same literature, the number of countries involved in P450s-related research
increased from over three-fold from 23 in 1977-1978 to 76 in 20072008. Early on in 1977-1978, the leading producers of P450s research
publications consisted of a mixture of western countries (USA, 151
papers; Japan, 42; France, 21and Federal Republic of Germany, 18) and
socialist countries (Germanic Democratic Republic, 23 and the USSR,
23). However, by 2007-2008 the USA with 810 was followed by a mixture of western countries and emerging Asian countries. During the four
time periods, the number of scientic journals publishing P450s papers
increased more than seven-fold (7.7): from 94 journals in 1977-1978 to
724 in 2007-2008. With the exception of two journals (The Journal of
Biological Chemistry and Archives of Biochemistry and Biophysics),
there was a complete turnover among the top-10 most productive journals. While journals focusing on biological chemistry dominated the topranks in 1977-1978, journals focusing on pharmacological approaches
were prominent in the 2007-2008 ranking led by the journal, Drug Disposition and Metabolism. The transition of the P450s literature from biological chemistry to pharmacology shows the importance of translational
approaches for progressing in P450s research.
Paper No.: 2423

ANTINOCICEPTIVE ACTIVITY OF FRUITS OF METHANOLIC
EXTRACT OF CAPPARIS OVATA IN MICE
Rana Arslan(1), N Bektas(1)
Anadolu University, Faculty of Pharmacy, Department of Pharmacology,
Eskisehir, Turkey
181
Aim of the study: In this study, we attempted to identify the possible
antinociceptive action of methanol extract obtained from fruits of
Capparis ovata. Materials and Methods: The antinociceptive effect of
the methanol extract of fruits of Capparis ovata (MEC) was assessed
by intraperitoneal administration in tail immersion, hot plate and writhing tests in mice. Morphine sulfate (5 mg/kg; i.p.) and diclofenac
(10 mg/kg; i.p.) were used as reference analgesic agents. Naloxone
(5 mg/kg; i.p.) was also tested. Results: MEC was studied at the doses
of 50, 100, and 200 mg/kg (i.p.) and showed signicantly antinociceptive activity in all assays. The extract at given dose range reduced the
writing by 32.21, 55.70, and 68.36% respectively. MPE% were
increased by 7.27, 12.07, 14.60% in the tail immersion, and 7.88,
11.71, 16.73% in the hot plate test at the tested doses respectively. Naloxone antagonized antinociceptive effect at the doses of 100 and
200mg/kg whereas partially antagonized the effect of MEC at the dose
of 50 mg/kg. Conclusions: Based on the results obtained, it can be
concluded that MEC has antinociceptive effects both at the peripheral
and central levels.
Keywords: Capparis ovata, fruits extract, antinociception
Paper No.: 2245

CARDIOPROTECTIVE MECHANISM OF TELMISARTAN
THROUGH THE PPAR-C PATHWAY AGAINST
ISOPROTERENOL-INDUCED MYOCARDIAL INFARCTION IN
EXPERIMENTAL DIABETES
Dharamvir Singh Arya, S Goyal
All India Institute of Medical Sciences, Department of Pharmacology,
New Delhi, India
Introduction: Telmisartan is a partial agonist of the PPAR-c. We investigated whether telmisartan improved the pathophysiology of myocardial
infarction in diabetes partially through the PPAR-c pathway by assessing
a variety of indices, e.g., hemodynamic, biochemical, histoarchitectural
changes, and apoptosis. Materials and methods: Diabetes was induced by
a single dose of streptozotocin (70 mg/kg, IP). Diabetic rats received
either telmisartan (10 mg/kg/day, orally), the PPAR-c antagonist
GW9662 (1 mg/kg/day, IP), or both for 14 days with concurrent administration of isoproterenol (85 mg/kg, SC) on days 13 and 14. Results:
Compared with diabetic controls, diabetic rats with myocardial infarction
exhibited altered hemodynamic proles and reduction in the activities of
creatine kinase-MB isoenzyme, lactate dehydrogenase, superoxide
dismutase, catalase, and glutathione level along with increased level of
malondialdehyde in the heart. Further, diabetic animals with myocardial
infarction exhibited increased myonecrosis, edema, and apoptotic cell
death. Treatment with telmisartan signicantly improved the redox status
of the myocardium with subsequent cardiac functional recovery. However, signicant effects were lowered in animals treated with telmisartan
plus GW9662. Telmisartan markedly inhibited Bax expression, TUNELpositive cells, myonecrosis, and edema. On the other hand, administration of telmisartan plus GW9662 did not elicit the same effects, nor did
they increase Bcl-2 protein expression in isoproterenol-induced myocardially infarcted diabetic rats when administered concomitantly or individually. Moreover, down-regulated PPAR-c expression in myocardially
infarcted diabetic hearts was increased by telmisartan treatment. Conclusion: In addition to class effects of ARBs, telmisartan reduces oxidative
stress and apoptosis and improves cardiac function via the PPAR-c pathway.
Paper No.: 2046

DISCOVERY
INTERACTIONS BETWEEN VERAPAMIL AND DIGOXIN IN
LANGENDORFF - PERFUSED RAT HEARTS: THE ROLE OF
INHIBITION OF P-GLYCOPROTEIN IN HEART
M Aylin Arycy(1), E Kilinc(2), O Demir(1), M Ates(3), A Yesilyurt(2), A
Gelal(1)
(1) Dokuz Eylul University School of Medicine, Izmir, Turkey
(2) Ege University, Faculty of Pharmacy, Izmir, Turkey
(3) Dokuz Eylul University, Advanced Professional School of Health
Sciences, Izmir, Turkey
Introduction: P-glycoprotein (P-gp) is expressed in tumor cells as well as
normal tissues including heart. Modulation of P-gp transport in vivo may
lead to increased drug penetrance to tissues with resulting increases in
toxicity. We aimed to investigate the effects of P-gp on the isolated heart
by digoxin infusion in the absence and presence of verapamil. Materials
and Methods: Study was performed in Langendorff isolated perfused rat
hearts. After 20 minute stabilization period with Tyrode Buffer, digoxin
(125 lg/5mL) was infused for 10 minutes in control group (n = 7). The
same dose of digoxin was infused during perfusion with verapamil (1
nM) containing Tyrode buffer (n = 8) in the study group. Outow concentration and cardiac parameters were measured at frequent intervals for
40 minutes. Results: AUEC(0-40 min) for left ventricular developed
pressure (LVDP) was signicantly increased in the presence of verapamil
(4260 39.37 vs 4607 98.09; %95 CI -587.7 to -105.8; p = 0.0083).
The signicant increases in LVDP were at 20, 25, 30, 35 and 40 minutes.
AUC(0-40 min) values for digoxin was lower signicantly in the presence of verapamil. Conclusion: Verapamil increased the positive inotropic effect of digoxin which could be related to the accumulation of
digoxin in the rat heart.
Keywords: P-glycoprotein, drug interaction, heart, digoxin, verapamil
This paper was sent to Basic and Clinical Pharmacology and Toxicology
to be evaluate for publication
Paper No. 1519

FOCUS GROUP: P02 - TRANSMEMBRANE TRANSPORT:
PERSPECTIVES FOR DISEASE AND DRUG DISCOVERY
IMPACTS OF EGFR-ANTISENSE AND CARBENOXOLONE ON
CANCER CELL LINES
Masoud Asadi(1,2), H Hamzeiy(0), J Barar(2), Y Omidi(2), S Mohammadi(1)
(1) Tabriz University of Medical Sciences, Department of Pharmacology
and Toxicology, Tabriz, Iran
(2) Research Center for Pharmaceutical Nanotechnology, Iran
Gap junctions play an important role in the cell proliferation in mammalian cells. However, there are controversial issues about cell communications in neoplastic states. This study was designed to evaluate the
cytotoxic effects of Carbenoxolone as a prototype of inter cellular gap
junction blocker in association with epidermal growth factor receptor
(EGFR) gene silencing using EGFR-antisense therapy. On the basis of
specic and general roles of EGFR in different kinds of cells, three cancerous cell lines(A549, BT20 and MCF7 cells) were cultivated, and at
designated conuency cell monolayers were treated with carbenoxolone
(150 lM) and anti EGFR antisense oligodeoxyneucleotide (ODN). Cellular cytotoxicity were examined using standard colorimetric assay associated with cell viability tests. MCF7 and BT20 cells were signicantly
affected by carbenoxolone and EGFR antisense, but not A549 cells.
Treated MCF7 and BT20 cells with anti-EGFR ODN and carbenoxolone
(alone or as combination) showed signicant toxicity. Our preliminary
examinations with DNA microarray technology conrmed such ndings.
Based upon this investigation, it can be concluded that there exists a
182
novel relationship between cell-cell communications and intercellular
cross talking, perhaps via gap junctions, at which we propose interestingly orchestrated biochemical machinery of cells may be related to
EGFR downstream cell signalling which may play a key role in development of cancer.
Keywords: gap junctions, EGFR-antisense, Carbenoxolone, cancer cell
line
Paper No.: 713

HEALTH AND DISEASE
THE VARIATIONS IN MEASUREMENTS OF BLOOD PRESSUREC VALUES OBTAINED BY THE CONVENTIONAL METHOD
AND THE AMBULATORY BLOOD PRESSURE MONITORING
Mensura Asceric(1), N Mulabegovic(2), E Kapic(2), F Becic(2),
S Loga-Zec(2), J Kusturica(2)
(1) University of Tuzla, Medical Faculty, Department of Pharmacology
and Toxicology, Tuzla, Bosnia & Herzegovina
(2) University of Sarajevo, Medical Faculty, Department of Pharmacology and Toxicology, Sarajevo, Bosnia & Herzegovina
Introduction: More frequent and longer blood pressure (BP) oscillations,
in other words variability, can lead to faster and severe damage of certain
organs and hypertrophy of left venticle and presence of increased risk
factors can lead to exponential growth of risks severe cardiovascular
events. The aim of this paper was to dene and analyze variations in
measurements of systolic and diastolic BP values in treated hypertensive
patients obtained by conventional method during ambulatory monitoring
blood pressure (AMBP). Patients: A total of 72 treated patients have
been observed in 2 groups. The rst group of patients without of echocardiography parameters of HLV and second group of patients with echocardiography diagnosed HLV. Results: It conformed that there were no
signicant gender differences but that there was a signicant growth considering age and artery hypertension length as well as in body mass
index (BMI) values of cholesterol and triglycerides, a genetic predisposition to hypertension, fundus and left ventricular mass index change in
group with HLV. Systolic and diastolic BP values do not show signicant
differences in the groups during the day and they are signicantly higher
than values measured with AMBP method. The systolic and diastolic BP
variability is more noticeable in patients with HLV.Conclusion: The
results of this research have shown that 24-hours AMBP is better indicator of BP variability than use the conventional method.
Paper No.: 575

EVALUATION OF HEPATOPROTECTIVE ACTIVITY OF
GARCINIA INDICA IN RODENTS: EVIDENCE OF
ANTIOXIDANT ACTIVITY
Hardik D Ashar, VS Panda
Principal K. M. Kundnani College of Pharmacy, Department of Pharmacology & Toxicology, Mumbai, India
Garcinia indica is an Indian spice, the fruit of which is widely used in
cooking and its syrup is consumed as a soft drink. The protective effects
of aqueous extracts of the fruit rind of G. indica (GIE) on carbon tetrachloride (CCl4)-induced hepatotoxicity and the probable mechanisms
involved in this protection were investigated in rats. Liver damage was
induced in Wistar rats by administering a 1:1 (v/v) mixture of CCl4 and
olive oil (1ml/kg, i.p.) once daily for 7 days. GIE at 400 mg/kg and
800 mg/kg and the reference drug silymarin (100 mg/kg) were administered orally for 10 days to CCl4 treated rats, this treatment beginning
3 days prior to the commencement of CCl4 administration. Levels of
marker enzymes (AST and ALT), albumin (Alb) and total protein (TP)
were estimated in serum. Antioxidant parameters {reduced glutathione
(GSH), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) and glutathione reductase (GR)} and the lipid peroxidation
marker malondialdehyde (MDA) were determined in liver. GIE and silymarin elicited signicant hepatoprotective activity by attenuating the
CCl4-elevated levels of AST, ALT and MDA and restored the CCl4depleted levels of GSH, SOD, CAT, GPX, GR, Alb and TP. GIE
800 mg/kg demonstrated greater hepatoprotection than GIE 400 mg/kg.
The present ndings indicate that hepatoprotective effects of GIE in
CCl4-induced oxidative damage may be due to an augmentation of the
endogenous antioxidants and inhibition of lipid peroxidation in liver.
(Devasagayam TPA, Mishra A, Bapat MM et al, Curr Sci 2006; 91(1):
90-93).
Paper No.: 2227

EFFECTS OF FREE D-AMINO ACIDS PRESENT IN FOODS ON
HUMAN DERMAL FIBROBLASTS
Yutaka Ashida(1), S Shimada(1), C Okamura(1), M Mita(1),
K Hamase(2)
(1) Shiseido Research Center, Yokohama, Japan
(2) Kyushu UniversityGraduate School of Pharmaceutical Sciences,
Japan
The role of D-amino acids in mammals has been neglected due to their
ultra-low concentrations in tissues. Recent analytical progress has conrmed their existence and demonstrated their involvement in physiological functions, such as neurotransmission and hormone secretion. We have
shown that their contents in skin vary with age. Free D-amino acids in
mammalian tissues may derive from ingested foods, as well as enterobacteria. In this work, we examined their contents in foods and in stratum
corneum, and investigated their physiological functions in human dermal
broblasts. Amino acids were extracted with aqueous methanol from stratum corneum collected by tape stripping. Derivatized D-amino acids in
the extracts were determined by means of two-dimensional HPLC
(NANOSPACE, Shiseido), as reported previously. In human stratum corneum, D-serine, D-alanine and D-aspartate were detected. D-Aspartate
enhanced type I collagen production in broblasts, whereas L-aspartate
did not. D-Aspartate, but not L-aspartate, also showed antioxidative
effects in cells exposed to hydroperoxide or free radicals. D-Aspartate
content in different kinds of foods varied tremendously. Food processing
procedures, including fermentation, may also alter the content of
D-amino acids, and thereby inuence physiological functions. In conclusion, D-amino acids derived from foods may have physiological roles in
humans, and therefore may have potential as pharmacological agents.
Paper No.: 1510

FOCUSED CONFERENCE GROUP: P07 - SIMULATION AND
DATA MODELLING IN DRUG DEVELOPMENT.
PROTECTIVE EFFECT OF AQUEOUS SAFFRON EXTRACT
(CROCUS SATIVUS L.) ON CARBON TETRACHLORIDEINDUCED HEPATOTOXICITY IN RATS
Amir Ashna, H Najafzadeh
Shahid Chamran University, Department of Pharmacology & Toxicology,
Ahvaz, Iran
Crocus sativus L., commonly known as saffron is used for different purposes such as an antispasmodic and expectorant. Recent studies have
demonstrated that saffron extracts have antitumour, radical scavenger,
hypolipaemic, anticonvulsant effects and improve activity on learning
and memory. The aim of the present study was to assess the effect of
183
aqueous saffron extract on carbon tetrachloride-induced hepatotoxicity in
rats. Four groups of rats were used as group1: control; group2: Ccl4;
group3: Ccl4 + vitamin E; group4: Ccl4 + saffron extract. Drugs were
administrated for 7 days. The serum of rats was collected and analyzed
biochemically. The mean of ALT, AST, LDH, GGT, conjugated and total
billirubin, total protein and albumin was compared between groups. The
results show the mean of ALT and AST was increased by ccl4. This elevation was prevented by saffron extract; but vitamin E prevented AST
elevation. The mean of conjugated and total billirubin was increased by
ccl4. This elevation also was prevented by saffron extract. Thus saffron
has protective effect on carbon tetrachloride-induced hepatotoxicity probably by antioxidant action.
Key words: Ccl4, Hepatotoxicity, Rats, Vitamin E, Saffron
Paper No.: 1468

FOCUSED CONFERENCE GROUP: P12 - ION
CHANNELOPATHIES: NEW WINDOWS ON COMPLEX
DISEASE AND THERAPY
THE ROLE OF POTASSIUM CHANNELS IN EXPERIMENTAL
SEPSIS
Jamil Assreuy(1), R Sordi(1), D Fernandes(2)
(1) Universidade Federal de Santa Catarina, Department of Pharmacology, Florianopolis, Brazil
(2) Universidade Estadual de Ponta Grossa, Brazil
This study investigates the involvement of potassium channel (KC) subtypes on sepsis-induced vascular hyporesponsiveness to phenylephrine
(VHP). Wistar rats were submitted to cecal ligation and puncture (CLP)
surgery. Six and 24 hours later, responses to phenylephrine were determined before and 30 min after injection of KC blockers. Additionally,
TEA and GLB were injected 4 hours after CLP and 24 hours after surgery several parameters were evaluated. The KC blockers were tetraethylammonium (TEA; a non-selective KC blocker), glibenclamide (GLB; an
ATP-dependent KC blocker), 4-aminopyridine (4-AP; a voltage-dependent KC blocker) and apamin (a calcium-dependent KC blocker). Results
show that: (1) CLP induced a severe VPH; (2) injection of TEA 30 minutes before phenylephrine injection completely restored the vascular
response to it, 24 hours after CLP; (3) apamin reversed VHP after 6, but
not after 24 hours after CLP; (4) GLB restored phenylephrine response
only 24 hours after CLP and (5) 4-AP was ineffective in all periods. In
addition, the early treatment with TEA reduced plasma NOx, lung MPO,
urea, creatinine and lactate levels, prevented the development of VPH
and reduced the mortality by 50%. Early treatment with GLB was without effect. Thus, at least part of the sepsis-induced vascular dysfunction
is maintained by the activity of distinct KC subtypes, calcium-dependent
KC in the rst hours and ATP-dependent KC in later phases of sepsis. In
addition, blockade of TEA-sensitive KC early after the onset of sepsis
has several benecial consequences reducing organ damage and mortality in experimental sepsis.
Financial support: CNPQ, CAPES, FAPESC
Paper No.: 434

ANTIMITOGENIC AND APOPTOTIC EFFECTS OF
SELECTIVE 5HT3 RECEPTOR ANTAGONIST ON HT29
COLORECTAL CANCER CELL LINE
Ramin Ataee(1), M Zarrindast(2), S Ajdary(1), S Sharyari(1)
(1) Institute Pasteur of Iran, Department of Immunology, Tehran, Iran
(2) Tehran University of Medical Sciences, Department of Pharmacology,
Tehran, Iran
Background: Serotonin has been known to be a mitogen factor in severalmalignancies. It elicits its mitogenic effect through a wide range of
5-HTreceptor subtypes and several internal cellular transcription pathways.According to wide distribution of 5HT3 and 5HT4 receptors in GI
tracts, the mainaim of this study was to investigate effect of these receptors agonists andantagonists in colorectal cell line. Methodology: In cell
culture, weinvestigated the effects of Serotonin (5-HT), 5-HT3 and 5-HT4
receptors agonists and antagonists on proliferation of HT29 cells. We also
testedapoptosis for receptor antagonists on HT29 cells with TUNEL apoptosis test. With ow cytometery we assayed effects of 5HT receptors
antagonist on cell cyclekinetics and with western blot we assayed the protein expression of 5HTreceptors Results: MTT proliferation assay revealed
that Phenylbiguanide (a 5HT3receptor selective agonist) increased proliferation of HT29 cells signicantly and Y25130 Hydrochloride (a 5HT3
receptor antagonist) had the opposite effect; but for 5HT4 receptor antagonist, theses effects were not signicant. Also potent apoptotic and cell
cycle arresting effect was found for selective 5HT3receptor antagonist but
as well we have not seen any signicant effect for 5HT4receptor antagonist Also western blot study showed high protein expression for5HT3
receptor. The ndings of this study provide strong evidence for the potential role of 5-HT3 receptor in colorectal cancer.
Paper No.: 1810

NEUROPROTECTIVE EFFECTS OF THE POLYPHENOLIC
ANTIOXIDANT AGENT, CURCUMIN, AGAINST
HOMOCYSTEINE-INDUCED COGNITIVE IMPAIRMENT AND
OXIDATIVE STRESS IN THE RAT
Amin Ataie, M Sabetkasaei, A Haghparast
Shahid Beheshti Medical University, Department of Pharmacology, Tehran, Iran
Aging is the major risk factor for neurodegenerative diseases and oxidative stress is involved in the pathophysiology of these diseases. In this
study, we investigated the possible antioxidant and neuroprotective properties of the polyphenolic antioxidant compound, Curcumin against homocysteine (Hcy)neurotoxicity. Curcumin (5, 15 and 45 mg/kg) was
injected intraperitonealy once daily for a period of 10 days beginning
5 days prior to Hcy (0.2 lmol/ll) intrahippocampal injection in rats.
Biochemical and behavioral studies, including passive avoidance learning
and locomotor activity tests were studied. We detected Malondialdehyde
(MDA) and Super oxide anion (SOA) in rats hippocampi. Also Immunohistochemical studies were done on hippocampus cells. Results indicated that Hcy could induce lipid peroxidation and increase MDA and
SOA levels in rats hippocampi. Additionally, Hcy impaired memory
retention inpassive avoidance learning test. However, Curcumin treatment decreased MDA and SOA levels signicantly as well as improved
learning and memory in rats. Histopathological analysis also indicated
that Hcy could induce cell death and apoptosis in rats hippocampi and
Curcumin inhibited these toxic effects. These results suggest that Hcy
may induce lipid peroxidation and cell death in rats hippocampi and
polyphenol treatment (Curcumin) has the ability to improve learning and
memory decits by protecting rats hippocampi against Hcy toxicity.
Paper No.: 2828

FOCUSED CONFERENCE GROUP: P18 - NUCLEAR
RECEPTOR TARGETS FOR TREATMENT OF DISEASES
STUDY OF 5HT3 AND HT4 RECEPTORS EXPRESSION IN
HT29 CELL LINE AND HUMAN COLON ADENOCARCINOMA
TISSUES
Ramin Ataee(1), S Ajdary(1), M Zarrindast(2), M Rezayat(2), MA Shokrgozar(5)
(1) Institute Pasteur of Iran, Department of Immunology, Tehran, Iran
(2) Terhran University of Medical Sciences, Department of Pharmacology, Tehran, Iran
(3) Institute Pasteur of Iran, Cell Bank, Tehran, Iran
184
Background: Serotonin (5HT) has been shown to be a mitogenic factor
in several carcinomas. It elicits its mitogenic effect through a wide range
of 5HT receptor subtypes. In this study, the effect of 5-HT, 5HT3 (1-Phenylbiguanide hydrochloride) and 5HT4 (Cisapride) agonists in promoting
the growth of HT29 cell line and the growth-inhibition effect of 5HT3
receptor antagonist (Y-25130 hydrochloride) and 5HT4 receptor antagonist (RS 23597-190) were investigated. The expression of 5HT3 and
5HT4 receptors in human colon cancer tissues and cell line was studied.
Materials and methods: The growth-promoting and growth-inhibition
effect of 5-HT, 5HT3 and 5HT4 agonists and antagonists on HT29 cell
line was studied using MTT assay. Receptor expression has been demonstrated by western blotting. Results: The results showed 5HT, 5HT3, and
5HT4 agonists caused signicant proliferation of HT29 cells. 5HT3 and
5HT4 receptor antagonists had inhibitory effect on the growth of these
cells. Western blot analysis gave bands from colon tissue extracts and
HT29 cell line. Conclusion: The results indicate 5HT3 and 5HT4 receptors express in colon cancer tissue and cell line signicantly and the
expression for 5HT3 receptor is more potent. Furthermore, 5HT plays a
mitogenic role in colon cancer cells and antagonists of 5HT3, and 5HT4
receptors can inhibit cancer cell growth. Key Words: Colon adenocarcinoma, HT29 cell, Serotonin, 5HT3 and 5HT4 receptors.
Paper No.: 634

SINGLE-DOSE PHARMACOKINETICS OF CEFEPIME IN
HEALTHY EGYPTIAN BUFFALO CALVES
Taha A Attia(1), MA Tohamy(2), AAM El-Gendy(2)
(1) Menoa University Faculty of Veterinary Medicine, Department of
Pharmacology, Egypt
(2) Beni-Suef University, Faculty of Veterinary Medicine, Department of
Pharmacology, Egypt
Pharmacokinetics of cefepime was studied after single dose intravenous,
intramuscular and subcutaneous administration. Five apparently healthy
buffalo calves were used in a crossover design with 15-day washout period. After intravenous (i.v) injection of cefepime, the half-lives of distribution and elimination (t0.5(a) and t0.5(b)), volume of distribution at
steady state (Vdss), mean residence time (MRT) and total body clearance
(ClB) were 0.113 and 2.32 h., 0.192 L kg-1., 3.12 h and 0.061 L kg-1 h1
., respectively. Following intramuscular (i.m) and subcutaneous (s.c)
administration of cefepime, the maximum concentration (Cmax) 29.094
and 27.271 ug ml-1 were achieved at a maximum time (tmax) 1.611 and
1.578 h., respectively. The mean values for absorption and elimination
half-lives (t0.5(ab) and t0.5(el)) and MRT were 0.549, 2.422 and 2.242,
4.286 and 4.025 h., respectively. The intramuscular and subcutaneous
bioavailabilities were 95.81 and 85.663%, respectively. The result of invitro protein-binding study indicated that 11.423% of cefepime was
bound to the serum proteins of buffalo calves.
Paper No.: 1517

SEVERAL FRUIT AND BERRY JUICES AND PUREES ARE
POTENT INDUCERS OF ENDOTHELIUM-DEPENDENT
RELAXATIONS IN THE PORCINE CORONARY ARTERY VIA
ACTIVATION OF NITRIC OXIDE AND ENDOTHELIUMDERIVED HYPERPOLARIZING FACTOR PATHWAYS
C Auger, S Trinh, J-H Kim, T Chataigneau, Valerie B Schini-Kerth
University of Strasbourg, Faculty of Pharmacy, UMR CNRS 7213, Illkirch Graffenstaden, France
Polyphenol-rich sources such as red wine have been shown to protect
the vascular system by enhancing the endothelial formation of nitric
oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF),

two major vasoprotective factors. This study evaluated the ability of
several non-alcoholic sources of polyphenols and in particular selected
pure fruit juices and purees to cause endothelium-dependent relaxations
of isolated arteries and identied the mechanism involved. Vascular
reactivity was assessed using porcine coronary artery rings in the presence of indomethacin to prevent the formation of vasoactive prostanoids. The 13 fruit juices and purees tested caused variable
concentration-dependent relaxations in coronary artery rings with endothelium and only small relaxations in rings without endothelium. The
strongest inducers of endothelium-dependent relaxations were blackcurrant, aronia and cranberry followed by lingonberry, blueberry and San
Gabriele grape whereas only small relaxations were observed with
strawberry, apple, raspberry, blackberry, boysenberry, and acerola; elderberry was without effect. Relaxations to aronia, blackcurrant, and lingonberry were not affected by the combination of charybdotoxin plus
apamin (inhibitors of EDHF-mediated responses), slightly reduced by
NG-nitro-L-arginine (an NO synthase inhibitor) and markedly reduced
by the combination of NG-nitro-L-arginine and charybdotoxin plus
apamin. Thus, the present ndings indicate that several fruit juices and
purees are potent inducers of endothelium-dependent relaxations in coronary arteries by stimulating the endothelial formation of NO, and also
EDHF. They further suggest that regular intake of these active rich
sources of polyphenols may increase the protective effect of endothelial
cells on the vascular system.
Paper No.: 2249

DISEASES
WHOLE-BODY DISTRIBUTION AND PHARMACOKINETICS
OF RADIOIODINATED FULLY HUMANIZED ANTI-VAP-1
ANTIBODY A PET IMAGING STUDY OF RABBITS
Anu Autio(1), PJ Vainio(2), A Mali(3), J Vainio(2), S Suilamo(1), V
Oikonen(1), P Luoto(1), M Teras(1), T Karhi(4), A Roivainen(1,4)
(1) Turku University Hospital, Turku PET Centre, Turku, Finland
(2) Biotie Therapies Corp, Turku, Finland
(3) MAP Medical Technologies Oy, Tikkakoski, Finland
(4) University of Turku, Turku Center for Disease Modeling, Turku, Finland
We assessed the usefulness of positron emission tomography (PET) in
evaluation of iodine-124 labeled antibody pharmacokinetics and distribution in rabbits. PET is powerful, non-invasive method particularly
suitable for drug development because of its high sensitivity and ability
to provide quantitative and kinetic data without sacricing the animal.
The antibody (BTT-1023) is rst-in-class, fully human monoclonal antibody to vascular adhesion protein-1 (VAP-1). VAP-1 is an inammation inducible endothelial glycoprotein. It plays a key role in leukocyte
trafcking and is a potential target for anti-inammatory therapy. BTT1020 is potentially useful for the treatment of inammatory diseases
and in vivo imaging of inammation. BTT-1023 was labeled with
[124I] using Chloramine-T method. Its immunoreactivity was retained
after labeling as conrmed in hVAP-1 transfected cells and by timeresolved immunouorometric assay using human recombinant VAP-1.
Ten rabbits were intravenously injected with [124I]BTT-1023. PET/CT
was obtained over the rst 2 h after dosing and at 24, 48 and 72 h
post-injection. Blood samples were collected during PET study to clarify in vivo stability and pharmacokinetics. In rabbits, the radioactivity
was distributed especially to liver and thyroid. Also heart and lungs
showed some uptake whereas brain uptake was very low. The plasma
half-life of [124I]BTT-1023 was 58.3 10.5 h and clearance
7.8 1.8 mL/h/kg. [124I]BTT-1023 showed good in vivo stability
(80% of signal from intact antibody 72 h after injection) and uptake in
VAP-1 positive liver. In conclusion, PET study of [124I]-labeled VAP-1
targeting BTT-1023 further elucidated distribution and kinetics of the
therapeutical antibody.
185
Paper No.: 1095
EFFECT OF THE ANTHRAQUINONE COMPOUND RHEIN ON
HUMAN COLON ADENOCARCINOMA CELL
PROLIFERATION
Gabriella Aviello(1), I Rowland(2), C Gill(3), AM Acquaviva(4),
R Capasso(1), AA Izzo(1), F Capasso(1), F Borrelli(1)
(1) University of Naples Federico II, Department of Experimental
Pharmacology,Naples, Italy
(2) University of Reading, Department of Food Biosciences, Reading,
UK
(3) University of Ulster, Northern Ireland Centre for Food and Health
(NICHE), Department of Biomedical Sciences, Belfast, Northern Ireland,
UK
(4) University of Naples Federico II, Department of Biology and Cellular
and Molecular Pathology, Naples, Italy
Background/Aim: Anthraquinones are the oldest drugs used as laxatives
in clinical practice and as self-medication. Recently the use of anthraquinone laxatives, such as senna (Cassia spp), has been associated with
damage of the intestinal epithelium and increased risk of developing
colorectal cancer. However, human and animal data are inconsistent. In
the present study we evaluated the cytotoxicity and genotoxicity of rhein,
the active metabolite of senna, on human colon adenocarcinoma cells
(Caco-2) and its effect on cell proliferation. Methods: Cytotoxicity studies were performed using MTT and TEER assays; cell proliferation was
investigated using 3H-thymidine incorporation and western blot analysis.
Genotoxicity studies were performed by Comet assay. Results: Rhein
had no signicant cytotoxic effect on proliferating and differentiated
Caco-2 cells. Rhein, at low concentrations (0.1 and 1 lg/ml), signicantly reduced cell proliferation and ERKs expression; by contrast, at
higher concentrations (10 lg/ml), it signicantly increased cell proliferation and MAP kinase activation. Moreover, rhein did not adversely affect
the integrity of tight junctions - and hence the epithelial barrier function
as well as it did not induce DNA damage. Finally, rhein signicantly
inhibited the increase in malondialdehyde and ROS levels induced by
Fentons reagent. Conclusions: Rhein, was devoid of cytotoxic and genotoxic effects in Caco-2 cells. Moreover, at concentrations which are
expected to be yielded in the colon lumen after a human therapeutic dose
of senna, rhein (i) inhibited cell proliferation via a mechanism likely
involving the MAP kinase pathway and (ii) prevented the DNA damage
probably via an anti-oxidant mechanism.
Paper No.: 917

HEALTH AND DISEASE
FUNCTIONAL DISTRIBUTION OF NITRERGIC NERVE
DERIVED FROM PTERYGOPARATINE GANGLION IN
MONKEY CEREBRAL ARTERY
Kazuhide Ayajiki(1), S Kobuchi(1), N Toda(2), M Hanamitsu(3),
T Okamura(4)
(1) Department of Pharmacy, School of Pharmacy, Hyogo University of
Health Sciences, Kobe, Japan
(2) Toyama Institute for Cardiovascular Pharmacology Research,
Toyama, Japan
(3) Department of Otoraryngology, Shiga University of Medical Science,
Shiga, Japan
(4) Department of Pharmacology, Shiga University of Medical Science,
Shiga, Japan
Functional roles of nitrergic nerve innervating monkey cerebral artery
were evaluated by measuring mechanical responses of isolated preparations in vitro and by cerebral angiography in vivo. Nicotine, which stimulates nerve terminals innervating cerebral arteries, produced relaxations
in isolated monkey arteries irrigating the cerebrum and cerebellum. The
relaxations induced by nicotine were abolished by NG-nitro-L-arginine.

We demonstrated, by using arterial angiography, that electrical stimulation of one side of the greater petrosal nerve connecting to the ipsilateral
pterygopalatine ganglion (PPG) with synapse, a parasympathetic ganglion, produced vasodilatation of anterior, middle and posterior cerebral
arteries only in the stimulated side, but failed to produce responses of
superior and anterior inferior cerebellar arteries in the stimulated side and
basilar artery of anesthetized monkeys. It is concluded that nitrergic
nerves innervate all arteries tested and that stimulation of nitrergic nerve
derived from the PPG dilates cerebral arteries, but not cerebellar arteries
and basilar artery. Nitrergic nerve derived from the PPG appears to regulate cerebral vasomotor function whereas nitrergic nerve from unknown
ganglion(s) may regulate the circulation in the cerebellum and brainstem.
Paper No.: 2126

INHIBITORY ACTIONS OF CARVACROL 1,4-CINEOLE BUT
NOT 1,8-CINEOLE ON FIELD STIMULATION OF ISOLATED
RAT VAS DEFERENS: ROLE OF ISOPROPYL GROUP AS A
PHARMACOPHORE
Suleyman Aydin, Y Cakmakci
Anadolu University Faculty of Pharmacy, Department of Pharmacology,
Eskisehir, Turkey
Carvacrol and cineole are volatile and fragrant natural compounds found
in various plants which have medicinal properties in folk medicine. Carvacrol, 1,8-cineole and 1,4-cineole have different chemical structures but
almost similar actions were reported. Actions of 1,4-cineole, 1,8-cineole
and carvacrol were studied on electrical eld stimulated isolated rat vas
deferens in this study. Contractions of vas deferens were inhibited by
Carvacrol (10-4 M) and by 1,4-cineole (10-3 M) whereas 1,8-cineole
was inactive. Based on pharmacological actions and structural properties
of the carvacrol, 1,4-cineole and 1,8-cineole on isolated vas deferens, isopropyl group was proposed to play important role on the inhibitory
actions. Since isopropyl group is widely found in volatile constituents of
natural compounds, isopropyl group of these compounds is proposed to
play role as a pharmacophore.
Paper No.: 2111

THE EFFECTS OF CARVACROL THYMOL AND PROPOFOL
ON ISOLATED RAT ATRIUM
Yasemin Aydin(1), S Arkan(1), S Aydin(2)
(1) Eskisehir Osmangazi University, Faculty of Medicine, Department of
Physiology, Eskisehir, Turkey
(2) Anadolu University, Faculty of Pharmacy, Department of Pharmacology, Eskisehir, Turkey
Thymol and carvacrol are natural compounds widely found in nature but
propofol is a synthetic chemical used as general anaesthetic drug which
are all have chemical similarity being phenolic substances with aliphatic
side chains. Propofol is well known for its cardiovascular effects but
there is no sufcient information on the cardiovascular actions of thymol
and carvacrol. The effects of thymol, carvacrol and propofol were investigated on the contractions of electrical eld stimulation induced isolated
rat atria in the present study. All the test compounds inhibited contraction
of eld stimulated isolated atria. Thymol, which has more structural
similarity to the carvacrol exhibited inhibition only at the 10-4 M but
propofol and carvacrol were observed to inhibit contractions at 10-5 and
10-4 M doses. Although thymol has more structural similarity to carvacrol
than propofol, carvacrol and propofol exhibited similar actions compared
186
to thymol. Thus mechanism of action of carvacrol on myocardium is suggested similar to the propofol including calcium and potassium channels.
Paper No.: 1102

PHARMACOLOGICAL SCREENING OF MEDICINAL PLANTS
IN SWAZILAND
ADP-stimulation with C50~0.5 mM or its abolishment by SU. So

[SU]~Kd(SU) can eliminate >50% of diabetogenic currents in patients
with either A- or B-type KATP inhibited with submillimolar IC50(ATP)
in inside-out membrane fragments. Deciphered mechanisms of HA and
SU-inhibition of diabetogenic KATP tested so far argue that the majority
of HA-KATP patients need SU doses exceeding those currently recommended for treatment of T2DM and illuminate the clinical importance of
the NIDDK/NIH-funded basic analysis of mutant/polymorphic ABCC8/
KCNJ11 identied in humans.
Peter Aziba
Olabisi Onabanjo University, Department of Pharmacology, Ago-Iwoye,
Nigeria
The search for drugs in Africa has been intensied on the potential of
medicinal activities of plants in Traditional medical practice (TMP). In
Swaziland, a tiny kingdom within South Africa, traditional practice is
well accepted by the people due to poverty, cultural afnity and unaffordability of conventional drugs. Problems associated with this practice
bothers on dosage, use, efcacy & side effects of the plant decortion used
in therapy.The institute of medicinal plants and food products research
has focused on the possibility of fostering integration of orthodox and
traditional practice.We have been screening, some plants for pharmacological activities, using basic experimental in vitro technique for receptor
studies (via the organ bath method), analgesic activities (using the Hot
plate method) and inammatory screening of the crude plant extracts (Ledebouria ovaltifolia & mormodica involucrata methanolic extracts).In the
Isolated rat stomach strip preparation, Acetylcholine (2x10-9 - 4x10-8M)
induced contractile responses were inhibited in non- competitive antagonism by extracts at concentration between 1.25.5mg/ml - 2.5mg/ml.shifting dose effect curve to the right. This pattern of inhibition excludes the
possibility of the extract interacting with muscarinic receptor. The result
in analgesic study, the extracts dose dependently evoked a prolonged
reaction time of 85%(p < 0.05) over a period of 20min observation time,
with no overt signs of toxicity. The results in these studies suggest the
extracts contained spasmodic and analgesic activities. Thus the use of
unknown agents may help to assess or evaluate the pharmacological
activities of this plant could be a prelude to drug discovery.
Paper No.: 1597

DISEASE AND THERAPY
DIABETOGENIC ABCC8/KCNJ11: MECHANISMS TO
PERSONALIZED TREATMENT
Andrey Babenko
Paper No.: 2784

ANTIOXIDANT ACTIVITY OF PHENOLIC EXTRACTS OF
TUNISIAN PLANTS AND THEIR EFFECT ON
MITOCHONDRIAL RESPIRATION IN VITRO
S Bacha(1), H Ferchichi(1), I Salouage(1), L Ouanes(2), M Boussad(3),
M Lakhal(1), Anis Klouz(1)
(1) National Center of Pharmacovigilance, Laboratory of Clinical Pharmacology, Tunis, Tunisia
(2) University of Faculty of Medicine, Animal Experiment Unit, Tunis,
Tunisia
(3) National Institute of Applied and Technology, Tunis, Tunisia
Based on the popular knowledge on the therapeutic uses of some Tunisian
plants, we have selected four specimens (Myrtus communis, Hypericum
humifusum, Romarinus ofcinalis and Crataegus azarolus). The aim of
this study is to evaluate the antioxidant effect of these plants by: the determination of total polyphenols content, the Diphenyl Pycril Hydrazil
(DPPH) test and monitoring of malondialdheyde (MDA) used for the
determination of the lipoperoxidation inhibition percentage. We evaluate
the effect of different extracts on a mitochondrial respiration model in vitro.
The concentration of different plants extracts used was 20mg/ml. Our
study shown an antioxidant potential with scavenging activity >85% for
Myrtus communis extracts and <25% for Hypericum humifusum, Romarinus ofcinalis and Crataegus azarolus. The total polyphenols concentration was 103lg/ml for Myrtus communis, and varied from 59.91 to
30.91lg/ml for the other plants. Lipoperoxidation inhibition percentage
was 100% for Myrtus communis and Romarinus ofcinalis, 60% and 35%
for Hypericum humifusum and Crataegus azarolus respectively. The mitochondrial respiration study shows an amelioration of 19.44% for Crataegus azarolus, 13.84% for Hypericum humifusum and 3.75% for
Rosmarinus ofcinalis, compared to no treated mitochondria. This study
we permitted to evaluate the antioxidant activity for a screening of different Tunisian plants. In the other hand, the combination of different tests
used in our model seems to be the best guarantee to select plant extracts
with high potential to be protective against ischemia reperfusion process.
University of Washington, Pacic Northwest Research Institute, Department of Molecular Physiology and Biophysics, Seattle, WA, USA
Sulfonylureas (SU) differentially inhibit different diabetogenic mutant
ABCC8(Sulfonylurea Receptor 1)/KCNJ11(Kir6.2) channels (KATP)
(Babenko et al, NEJM 2006;355:456-466 and Pearson et al, NEJM
2006; 355:467-477). Babenko (JBC 2008; 283:8778-8782) established
the rst, or A-type, mechanism of diabetogenic hyperactivity (HA) of
KATP, its MgATP/ADP-hyperstimulation (HS), claried why even a small
number of mutant channels in a heterozygous KATP population can
hyperpolarize insulin-secreting cells, and uncovered that some A-type
mutations can attenuate SU-inhibition of KATP in MgATP/ADP, thus
explaining in part why therapeutic doses of SU for HA-KATP carriers
often exceed those recommended by the FDA for treatment of type 2
diabetes mellitus (T2DM). Now, I show that insulin release-compromising mutations in domains partnering in SUR1/Kir6.2 inhibitory coupling
(Babenko et al, FEBS Letters 1999; 459:367-76) hyperactivate KATP by
changing the rates of channel transitions to and from its long-lived closed
state with the lowest Kd for inhibitory ATP, thus establishing the second,
or B-type, diabetogenic mechanism of KATP HA. B-type mutations alter
the coupling of SU-binding with the closed channel, but not the MgATP/
Paper No.: 2261

STRIATAL DELIVERY OF CDNF BY AAV2 VECTOR IS
NEUROPROTECTIVE IN A RAT PARTIAL LESION MODEL
OF PARKINSONS DISEASE
Susanne Back(1), J Peranen(2), L Lonka(2), T Tamminen(1), MH Voutilainen(1), A Raasmaja(1), M Saarma(2), PT Mannisto(1), RK Tuominen(1),
(1) University of Helsinki, Faculty of Pharmacy, Division of Pharmacology and Toxicology, Helsinki, Finland
(2) University of Helsinki, Institute of Biotechnology, Helsinki, Finland
Cerebral dopamine neurotrophic factor (CDNF) is a novel neurotrophic
factor that, together with vertebrate and invertebrate mesencephalic
187
astrocyte-derived neurotrophic factor (MANF), belongs to a family of
evolutionarily conserved neurotrophic factors. In our previous work, we
proved that a single injection of MANF or CDNF into rat striatum is able
to protect and restore midbrain dopaminergic neurons in partial lesion
model of Parkinsons disease (PD) in rat (Voutilainen MH et al, J Neurosci 2009; 29: 9651-9659, Lindholm P et al, Nature 2007; 448: 73-77).
According to these results, CDNF and MANF could potentially be used
as treatments in PD. In this study, we constructed adeno-associated viral
serotype 2 (AAV2) vectors encoding CDNF and GDNF genes and
injected them into rat striatum. Two weeks later, rat nigrostriatal dopaminergic tract was lesioned by intrastriatal injection of 6-hydroxydopamine
(6-OHDA, 2x10 lg). To evaluate the progression of the neurodegeneration of the rat nigrostriatal dopamine tract, we measured amphetamineinduced rotational behaviour and tyrosine hydroxylase (TH)-immunoreactivity in rat striatum and SN. Results implies that over-expression of
CDNF in rat striatum is able to protect rat nigrostriatal dopamine neurons
from 6-OHDA-induced nerve cell toxicity with similar efciency as
over-expression of GDNF. This study provides further evidence that
CDNF could be a potential drug candidate for the treatment of PD.
Paper No.: 514

EFFICACY AND SAFETY OF ADD-ON MIRTAZAPINE IN
DEPRESSION
Dinesh Kumar Badyal(1), P Matreja(2), R Deswal(3)
(1) Christian Medical College and Hospital, Department of
Pharmacology, Ludhiana, India
(2) Department Pharmacology, Gain Sagar Medical College and
Hospital, Banur, Patiala, India
(3) Department Of Psychiatry, Christian Medical College and Hospital,
Ludhiana, India
Major depressive disorder (MDD) continues to be a considerable problem, both for clinician and at the public health level. Although antidepressant medications are effective in approximately 70% of patients with
major depressive disorder, they have a delayed onset of therapeutic
effect, problems in remission and adverse effects. Mirtazapine, an atypical antidepressant has certain advantages which makes it an important
option for the treatment of major depression in patients who require additional therapy. The study was conducted in sixty patients suffering from
major depressive disorder. The study was approved by institutional ethics
committee. The duration of study was 1 year. Patients were randomized
to two groups and were given conventional selective serotonin reuptake
inhibitors (SSRI) alone or conventional SSRIs with mirtazapine 7.5 mg
once a day for 6 weeks. Efcacy was evaluated based Hamilton depression rating scale (HDRS), Montgomery and Asberg depression rating
scale (MADRS) and Amritsar Depressive Inventory (ADI) scores. Scores
were recorded at baseline and then at 1, 2, 3, 4, 5 and 6 weeks after the
treatment. The HDRS, MADRS and ADI score reduced signicantly at
each visit as compared to baseline in both the groups. The response rate
and number of remitters were signicantly more with add-on mirtazapine
therapy as compared to conventional therapy at the end of study. The
HDRS-17 subset scores indicate that the add-on mirtazapine therapy was
more effective in improving anxiety and somatic symptoms as compared
to conventional therapy. There was no signicant difference in adverse
effects in the two groups.
Paper No.: 1575

PHARMACODYNAMIC EFFECTS OF
3,4-METHYLENEDIOXYAMPHETAMINE (MDA)
MJ Baggott, J Siegrist, JR Coyle, K Flower, GP Galloway, John
Mendelson
California Pacic Medical Center Research Institute, Addiction and Pharmacology Research Laboratory, San Francisco, CA, USA
Background: MDA, an analog of MDMA (3,4-methylenedioxymethamphetamine, Ecstasy), is an illicitly used drug. In vivo MDMA is
N-demethylated to MDA. This study measured MDA effects in humans
in a controlled setting. Methods: In a placebo-controlled, double-blind,
within-subjects study, 12 individuals received a single 98 mg/70 kg bw
dose of MDA. This is the molar equivalent of 105 mg/ 70 kg bw
MDMA, a well-studied dose. Hormonal (cortisol, prolactin), physiological (HR, BP), and self-report VAS measures of typical MDMA and
hallucinogen effects were obtained. Results: MDA increased cortisol by
16.39 ug/dL (95%CI: 13.03-19.74, P < 1e-3) and prolactin by
18.37 ng/mL (95%CI: 7.39-29.35, P < 1e-3). These hormonal changes
are comparable to those seen after MDMA. Heart rate increased by
9.05 bpm (95%CI: 6.10-11.99, P < 1e-5) and blood pressure increased
by 18.98 / 12.73 mm Hg (Systolic 95%CI: 16.47 - 21.49, P < 1e-7;
Diastolic 95%CI: 10.82 - 14.63, P < 1e-4). Heart rate and systolic
changes were signicantly less than and greater than seen in a previous
study of MDMA (N = 16), respectively (P < 1e-5 and P = 2.42e-7,
respectively). There were robust self-report VAS changes in both
MDMA-like (e.g., closeness to others) and hallucinogen-like (e.g.,
familiar things seem unfamiliar, time distortions, closed-eye visuals)
effects that were generally similar to those seen after MDMA. Conclusions: MDA is a psychoactive sympathomimetic phenethylamine with
effects similar to MDMA. Although differences may exist in the magnitude of physiological effects, the overall proles appear remarkably
similar.
Supported by DA 016776
Paper No.: 931

BIOAVAILABILITY AND BIOEQUIVALENCE OF THREE DIFFERENT SIRUP FORMULATIONS CEFELEKSINE AT CHILDREN IN THERAPY OF SEVERE TONSILLITIS
Adnan Bajraktarevic(1), J Musabegovic(2), T Frankic(2), H Sladic(2), D
Abduzaimovic(3)
(1) Public Health Institution of Canton Sarajevo, Department of Paediatrics,Sarajevo, Bosnia Herzegovina
(2) Pharmaceutical Faculty, Department for Clinical Pharmacology,
Sarajevo, BosniaHerzegovina
(3) Private Biochemical Laboratory, Tesanj, Bosnia Herzegovina
Introduction: Effective criteria for evaluating pharmaceutical antibiotics
drug products saves time for the Health institutions and Pediatric hospitals and ensures that drugs purchased are not associated with hidden
expenses related to drug use. The determination of bioavailability is
dependent on the reliable, precise, and accurate measurement of the
active ingredient, or its metabolites, as a function of time. Methods:
Three same formulations of cefeleksine were compared, a randomized
three-period, three-sequence crossover study were considered the therapy of choice in children with severe bacterial tonsillitis and elevated
CRP. Bioequivalence is usually established by measuring the drug or
its metabolites or both in plasma and serum at 60 children. Results:
There was no correlation between the drug levels and side effects or
initial and subsequent results of curing severe tonsillitis. The 90% condence interval of the relative mean measured Cmax of the test to reference of all three cefaleksine formulation should be between 94% and
98%. There were no signicant baseline demographic or clinical differences between the ethnics, sexual and ages groups. Conclusions: The
change in absorption of same antibiotics of three differenet European
companies was small and unlikely to give signicant differences in
clinical effect in children. The drug product is, on the basis of scientic
evidence before submitted in this sirup cefeleksine application, shown
to meet an in vitro test that has been excellent correlated with in vivo
our data.
188
Paper No.: 932
VITAMINS AS ANTIOXIDANTS HAVE IMPORTANT ROLE OF
ASTHMATIC ADDITIONAL THERAPY IN CHILDREN AND
PROTECTION OF PASSIVE SMOKING
Adnan Bajraktarevic(1), M Miokovic(1), D Abduzaimovic(2), J Ceman
Saric(3), T Frankic(4), J Musabegovic(4), I Tahmiscija(5), A Selimovic(5), JA Maglajlic(6), B Begovic(7)
(1) Public Health Institution of Canton Sarajevo, Department of Paediatrics,Sarajevo, Bosnia Herzegovina
(2) Private Biochemistry and Immunology Laboratory,Tesanj, Bosnia
Herzegovina
(3) Medical Faculty Sarajevo, Department for Biochemistry and Immunology, Sarajevo, Bosnia Herzegovina
(4) Pharmaceutical Faculty, Sarajevo, Bosnia Herzegovina
(5) Pediatrics Clinic, Sarajevo, Bosnia Herzegovina
(6) Pulmology Clinic, Department for Microbiology, Sarajevo, Bosnia
Herzegovina
(7) University Medical Center, Department for Clinical Pharmacology,
Sarajevo, Bosnia Herzegovina
Background: Oxygen is a highly reactive molecule that damages living
organisms by producing reactive oxygen species. Cigarette smoke is a
complex mixture of less than ve thousand chemical compounds of
which free radicals and other oxidants are present in high concentrations.
Methods: Oxidation of proteins caused introduction of carbonyl groups
into the side chains of the protein, providing a convenient and relatively
specic marker of oxidative damage. Authors studied the effects of two
volatile components of cigarette smoke, acetaldehyde and acrolein,
which are present at high concentrations in cigarette smoke on lungs of
children. Vitamins A, C, and E as antioxidants led to the realization of
the importance of antioxidants in the biochemistry of living organisms.
Results: These antioxidant defence elements offer promising chemoprevention targets that have the potential to reduce the burden of asthma.
We have endeavoured to diminish the generation of oxygen free radicals
by decreasing the oxygen concentration in the resuscitating gas. The fall
in antioxidant capacity also correlates with the increased release of oxygen radicals from circulating neutrophils in patients with exacerbations
of asthma bronchale in children. Our study showed that some tocotrienol,
retinol and ascorbic acid have signicant anti-oxidant properties. Conclusions: The use of antioxidants with good bioavailability or molecules that
have antioxidant enzyme activity may be treatments that not only protect
against the direct injurious effects of oxidants. Vitamins antioxidants as
therapeutic targets have important role in obstructive pulmonary disease
in children.
Paper No.: 1237

CUTANEOUS ADVERSE DRUG REACTIONS: ANALYSIS OF
SPONTANEOUS REPORTS FROM HEALTHCARE
PROFESSIONALS IN CROATIA
ed. Overall, most commonly reported reactions were rashes, eruptions

and exanthemas (16.7%,), urticarias (15.9%), pruritus (15.8%,) erythemas (15.1%) and angioedemas (7.0%). Antimicrobial agents (25.0%)
and vaccines (19.6%) were most frequently implicated; together with cardiovasculars and drugs for nervous system, they accounted for more than
70% of reported drugs. There were a total of 107 (9.8%) serious reactions, most common being angioedemas (72.0%), exfoliative conditions
(8.4%), photosensitive reactions (6.5%) and bullous conditions (5.6%)
with drugs implicated being antibacterials (27.1%), cardiovasculars
(19%), drugs for nervous system (13.8%) and antineoplastics and immunosuppressants (9.35%). Conclusion: Antibacterials were the most commonly implicated drugs causing both non-serious and serious cutaneous
reactions reported in Croatia. The reporting rate of serious cutaneous
reactions is not satisfying since spontaneous reporting remains the main
source of safety information in cases of rare severe drug reactions which
are unlikely to be identied in premarketing clinical trials.
Paper No.: 1238

SERIOUS PSYCHIATRIC ADVERSE DRUG REACTIONS
SPONTANEOUSLY REPORTED IN CROATIA FROM 2006 TILL
2009
Ana Balazin, V Macolic-Sarinic, S Arapovic, D Krnic, M Banovac,
S Tomic
Agency for Medicinal Products and Medical Devices, Zagreb, Croatia
Introduction: Recognition of psychiatric adverse drug reactions (ADRs) is
important because they may interfere with the treatment of the primary
illness and be disturbing for patients, families and medical professionals.
Materials: National pharmacovigilance database of spontaneously
reported ADRs was searched. Reports containing psychiatric reactions
that were serious according to Critical Term List of the WHO were
considered for analysis that was done by descriptive methods (SPSS ver.
17.0). Results: 182 spontaneous reports with at least one psychiatric ADR
were identied. One third of them contained at least one serious ADR
(33.0%), which comprised 70 serious spontaneously reported psychiatric
ADRs. Restlessness (37.1%), suicidal behavior (20%) and anxiety
(12.9%) were most commonly reported serious psychiatric ADRs. Antidepressants (24.3%), antipsychotic (17.1%) and vaccines (17.1%) were most
commonly suspected. More female patients (61.4%) experienced serious
psychiatric ADRs. No gender disbalances were found regarding reactions
except for hallucinations which were reported exclusively in female
patients (6 cases; antiepileptic implicated in 50%). Half of patients experiencing serious reactions were adults; elderly patients and infants were
equally represented by 20%. Out of 12 serious psychiatric ADRs reported
in infants, restlessness was most common (91.7%); implicated was either
vaccine (84.3%) or montelukast (16.7%). 11 cases of suicidal behavior
were reported; implicated were antipsychotic (6 cases), antidepressants (4
cases) and antiparkinsonians (1 fatal case). Conclusion: Serious psychiatric ADRs can be challenging to diagnose in everyday practice. When
evaluating psychiatric symptom in any patient, it is important to check
concomitant medication and bear in mind possible psychiatric ADR.
Ana Balazin, V Macolic-Sarinic, S Arapovic, D Krnic, M Banovac,

S Tomic
Agency for Medicinal Products and Medical Devices, Zagreb, Croatia
Introduction: The aim was to explore spontaneously reported serious and
non-serious cutaneous adverse drug reactions (ADRs) and nd classes of
implicated drugs. Materials: We preformed a search within national database of spontaneous ADR reports submitted between January 2006 and
December 2009. Cutaneous reactions categorized as certain, probable,
possible and unlikely, based on WHO causality denitions, were
included in the analysis that was done using descriptive methods (SPSS
ver. 17.0). Seriousness was determined based on Critical Term List of the
WHO. Results: 628 unique cases comprising 1093 reactions were identi-
Paper No.: 1815

HEALTH AND DISEASE
MAST CELL ACTIVATION ALTERS NEURONAL NITRIC
OXIDE RELEASE IN RAT MESENTERIC ARTERY
Gloria Balfagon, J Blanco-Rivero
Universidad Autonoma de Madrid, Madrid, Spain
This study examines how mast cell activation inuences neuronal nitric
oxide (NO) release. Mesenteric arteries from 6-month-old Wistar rats
189
were incubated in the presence of mast cell stabilizers ketotifen and tranilast and mast cell degranulator compound 48/80 (C 48/80). Basal and
electrical eld stimulated (EFS) NO release was measured by uorescence, neuronal NO synthase (nNOS) expression by Western blot and
histamine release by ELISA. NO release was reduced after preincubation
with 0.1 mmol/L tranilast or with 0.1 or 1 umol/L ketotifen for 1, 2 and
3 hours, or with 10 nmol/L ketotifen for 2 and 3 hours, release was not
modied by 10nmol/L ketotifen for 1 hour or 10 umol/L tranilast, and
increased by preincubation with 15 ug/mL C 48/80 for 1, 2 and 3 hours.
nNOS expression was decreased by 3 hours incubation with 0.1 umol/L
ketotifen or with 0.1 mmol/L tranilast, and was increased by 1-hour incubation with 15 ug/mL C 48/80. 3 hours preincubation with 0.1 mmol/L
tranilast or 0.1 umol/L ketotifen decreased histamine release, while the
preincubation with 15 ug/mL C 48/80 increased it. These results indicate
the existence of an interaction between perivascular nitrergic nerve endings and mast cell activation that would inuence neuronal NO release
through a modication in nNOS expression.
This study was supported by Ministerio de Ciencia e Innovacion
(DEP2006-56187-C04-04; SAF2009-10374)
Paper No.: 2069

ARECA NUT DEPENDENCE AMONG CHEWERS IN A SOUTH
INDIAN COMMUNITY WHO DO NOT ALSO USE TOBACCO
(1) University of Sao Paulo - Medical School/ LIM 51, Sao Paulo, Brazil
Albumin is the most prevalent protein in blood. Analbuminemia is associated with increased risk of cardiovascular disease and endothelial dysfunction. Inammatory stimuli such lipopolysaccharide (LPS) is related
to reduced cardiovascular contractile response. Adult (300350g) male
rats Sprague-Dawley (SD) and Nagase Analbuminemic Rats (NAR) were
studied: both groups received LPS (E coli serotype 026:B6) 10 mg/kg,
ip, or saline i.p. Animals were sacriced 4h after injection. Intact thoracic
aortic ring (5-6 mm) of each animal was kept in organ baths (37C 95% O2, 5% CO2) with Krebs solution. Cardiovascular function was performed by catheter in left ventricule (LV) and carotid (dP/dt and Mean
Arterial Pressure, respectively). Under basal condition NAR has an
enhanced response (1.88 fold) to noradrenaline (NE) and lower heart
contractile response (+dP/dt=1.4 fold). LPS stimuli enhanced mortality
(25%); TNF (19.3 fold) and IL10 production (101 fold) on NAR; IL6
levels were enhanced in SD and NAR (526 fold, 914 fold respectively);
LV measurement showed an increase on +dP/dt (mmHg/s) in SD (1.32
fold) whereas a decrease in NAR (1.53 fold); we found a decreased of
-dP/dt (mmHg/s) in SD (1.46 fold), no differences was observed on vascular reactivity to NE, hemodynamic parameters by carotid measures and
serum lactate levels. Prevalence of higher response to NE and elevated
+dP/dt on physiological conditions are essential to NAR survival.
Robert L Balster(1), SJS Bhat(2), MD Blank(1), M Nichter(3),

M Nichter(3)
Paper No.: 883

HEALTH AND DISEASE
TEMPORAL EFFECTS OF ATP/ADP IN A MURINE SEPSIS
MODEL
(1) Virginia Commonwealth University, Richmond, Virgnia, USA

(2) Care Management International, Pune, India
(3) University of Arizona, Tucson, Arizona, USA
Hermes Vieira Barbeiro(1), DF Barbeiro(1), FR Laurindo(2),

HP Souza(1), IT Velasco(1), FG Soriano(1)
The goal of this study was to examine evidence of areca dependence in a

large, representative sample of areca-only (i.e., no tobacco) chewers
using established measurement scales. Information was also gathered on
use patterns in this population. Daily chewers (N = 59) from Karnataka
State, India were surveyed in 2005. Questionnaires assessed chewing history, patterns of use, and dependence features. Additionally, the relationship between topography and dependence scores was evaluated.
Approximately half of respondents reported 1-3 chews/day (mean = 1.9).
The average number of chewing episodes/day was 4.4 and the average
number of nuts/day was 1.2. Users typical chew lasts up to 20 minutes
and includes spitting out the juices and rinsing the mouth with water.
Overall, the levels of reported dependence symptoms were low, but
approximately 44% of chewers endorsed at least one of the following
items: continued use despite illness or wounds, difculty refraining from
chewing in forbidden places, or craving during periods of abstinence.
Approximately 15% of chewers reported at least one quit attempt, and
13.6% had scores indicative of dependence on the modied Cigarette
Dependence Scale (Score >16). Dependence scores were positively correlated with frequency of use. High levels of dependence were not
observed in this sample of regular betel-only users, but many users
reported at least one symptom and a few had several symptoms. The levels of dependence observed in a subset of informants warrant further
investigation as evidence for possible betel dependence
(1) University of Sao Paulo - Medical School, Sao Paulo, Brazil

(2) University of Sao Paulo - INCOR, Sao Paulo, Brazil
Sepsis is one of the main causes of death in non-coronary intensive care
units. One potentially important source on inammatory vascular injury
is lipopolysaccharide (LPS). Adults (255 15g) male Wistar rats
received LPS (E coli 026:B6) 10 mg/kg, ip or saline (control). Animals
were sacriced 8h, 16h and 24h after stimuli. Serum cytokine level
(TNF) was measured by ELISA. Thoracic aortic ring (5-6 mm) was
homogenized in liquid nitrogen and reconstitute to measure: iNOS and
TLR4 by RT-PCR and nitrate by NO-analyser (Sievers Instruments).
Some thoracic ring was kept intact to analyze superoxide (O2-) by chemiluminescence and vasodilation in organ baths (37C - 95% O2, 5% CO2)
with Krebs solution. Studies of O2- and vasodilation were performed on
presence of ATP/ADP. LPS injection enhanced: TNF at 8h (8.2 fold),
16h (7.3 fold) and 24h (7.7 fold); TLR4 at 8h (3.4 fold) and 24h (2.5
fold); iNOS at 8h (11.7 fold); nitrate at 16h (2.8 fold); O2- at 16h (7.8
fold) and 24h (4.7 fold). At 16h of endotoxaemia, ATP provide higher
vasodilation when compared to control (106.72%2.3; 100.15%0.9,
respectively) and reduced O2- levels (1.9 fold); ADP provide higher
vasodilation vasodilation (EC50) sensitivity on endotoxaemic when compared to control (2.0 and 2.6 10-8M, respectively) at the same time. In
spite of inammation maintenance in all periods, ATP/ADP presents a
relevant effect on aorta relaxation only at 16h.
Paper No.: 882

DISEASES
ANALBUMINEMIA: CARDIOVASCULAR FUNCTION OF
ENDOTOXEMIC RATS
Paper No.: 884

HEALTH AND DISEASE
VASCULAR REACTIVITY ON NAGASE ANALBUMINEMIC
RATS CHALLENGED BY LPS
Hermes Vieira Barbeiro(1), CB Lorigados(1), DF Barbeiro(1),

S Catanozi(2), E Nakarandake(2), IT Velasco(1), FB Fusco(2), A dos
Santos Filho(1), FG Soriano(1)
Hermes Vieira Barbeiro(1), CB Lorigados(1), DF Barbeiro(1), S Catanozi(2), E Nakarandake(2), IT Velasco(1), FB Fusco(2), A dos Santos
Filho(1), FG Soriano(1)
190
Hypoalbuminemia is associated with endothelial dysfunction. It is
unclear whether this dysfunction is a direct result of the decreased levels of albumin or whether it is caused by factors as chronic inammation. Adult (300350g) male Nagase analbuminemic rats (NAR) rats
and Sprague-Dawley (SD) were studied before (control) and 4h after
received LPS (E coli serotype 026:B6) 10 mg/kg, ip. Thoracic aortic
ring (5-6 mm) with or without endothelium of each animal was kept in
organ baths (37C - 95% O2, 5% CO2) with Krebs solution. Dose
response curve to noradrenaline (NE) was performed. Serum cholesterol, triglycerides and nitrite was evaluated. Under physiological condition NAR has enhanced levels of cholesterol (2.8 fold), triglycerides
(3.8 fold) and nitrite (3.21 fold). LPS stimuli decreased cholesterol levels (1.6 fold) on NAR and increase triglycerides levels on SD (4.09
fold). Under basal conditions, aortic ring of NAR has elevated response
to NE when compared to SD with endothelium (4.3 0.41g;
2.29 0.29g, respectively) and without endothelium (6.16 0.33g;
3.89 0.25g, respectively); this response was abolished by indomethacin. Endotoxaemia keep higher response to NE in NAR when compared
to SD only in aorta without endothelium (4.82 0.35g; 2.95 0.25g,
respectively) and indomethacin unchanged this response. Higher
response to NE observed on NAR under physiological conditions has
involvement of cicloxygenase and the inammation abolish this difference on aorta with endothelium.
Paper No.: 885

HEALTH AND DISEASE
VOLEMIC REPOSITION ON VASCULAR REACTIVITY OF
ENDOTOXEMIC RATS
Hermes Vieira Barbeiro, RC Petroni, TSL Garcia, DF Barbeiro,
CB Lorigados, FG Soriano, IT Velasco
University of Sao Paulo - Medical School/ LIM 51, Sao Paulo, Brazil
Volume and vasopressor therapy are among the primary goals in the
early management of septic shock and it has been shown to reduce
morbidity and mortality. Adult (220270g) male Wistar rats received
saline i.p. (Control) or LPS (E coli serotype 026:B6) 10 mg/kg, i.p.,
LPS group were separated in 3 groups: LPS; LPS+Saline (Sal) 0.9%
and LPS+Hypertonic 7.5%. Treatment was performed 15 min (i.v.)
and the rats were sacried 24h after LPS stimuli. Serum cytokine
(TNF) level was measured by ELISA. Thoracic aortic ring (5-6 mm)
with and without endothelium was kept in organ baths (37C - 95%
O2, 5% CO2) with Krebs solution for noradrenaline (NE) study (10 to
3000 nM). TNF was enhanced (7.6 fold) after LPS and volemic reposition do not altered this magnication. LPS stimuli decreased maximal response on aorta with and without endothelium when compared
with Control (1.6 fold twice). Absence of endothelium modulated
maximal response on control (1.2 fold) and sensitivity (EC50) on LPS
(2.2 fold). Volemic reposition proportionate higher maximal response
on LPS+Saline (1.4 fold) and higher sensibility (EC50) to
LPS+Hypertonic (2.9 fold) on aorta with endothelium and higher sensibility to LPS+Hypertonic (3.3 fold) on aorta without endothelium
when compare to control. Saline and hypertonic solution enhances
maximal response or sensibility to noradrenaline on isolated aorta of
endotoxemic rats, smooth muscle and endothelium maybe involved in
this nding.
Paper No.: 623

EFFECTS OF SURFACEN IN LOW AND MAINTAINED DOSE
IN THE TREATMENT OF ARDS. PRELIMINARY RESULTS
Yinet Barrese Perez(1), Y Avila Albuerne(1), E Daz Casanas(2),
O Fernandez Lima(2), R Uranga Pina(1), CM Ballagas Flores(1)
(1) National Clinical Trials Coordinating Center, Havana, Cuba
(2) National Center for Animal and Plant Health, Havana, Cuba
The acute respiratory distress syndrome (ARDS) is caused by a pulmonary inammation and destruction of pulmonary surfactant; the application of exogenous surfactant improves oxygenation. Our objective was to
evaluate the effect of SURFACEN in low dose and sustained for three
days on the oxygenation. To this end we conducted a controlled randomized, multicentric clinical trial, in 21 adult patients treated with 100 mg
SURFACEN 3 times for 3 days and 18 remained only with mechanical
ventilation. The response was assessed as positive when the ratio PaO2/
FiO2 200 at the end of treatment. The results showed a better response
indicator PaO2/FiO2 in the treated group (p = 0.0411). There was no difference in the clinical manifestations observed among groups or between
groups (p = 0.308) in the occurrence of adverse events. There were no
differences in mortality (p = 0.296) among groups, 11 patients in
SURFACEN group and 6 in the control group, however the causes of
death were not by ARDS but because the basic pathology that led to the
pulmonary inammation. The treated group showed a decrease
(p = 0.0341) of 7.23 in the days of endotracheal intubation, there was
not difference in the days of mechanical ventilation (p = 0.132) or stay
in the intensive care unit (p = 0.108) although there was a tendency to
be better in the group treated with SURFACEN. We conclude that the
use of surfactant in ARDS improved oxygenation in patients with lower
FiO2 requirements, reduced the time of endotracheal intubation and had
a good safety.
Paper No.: 2842

INTERACTION OF PROTEIN KINASE C AND ALLOSTERIC
MODULATION AT EXTRASYNAPTIC GABA-A RECEPTORS
FOLLOWING CHRONIC ETHANOL TREATMENT
I Barrett, Stephen Kelley
Kent University, Medway School of Pharmacy, Chatham Maritime, UK
Previous studies have shown that extrasynaptic GABA-A receptors are
sensitive to low ethanol doses (Wallner, M.et al., Proc Natl Acad Sci U S
A 2003, 100, 15218-23) However, others have not reproduced this effect
(Borghese, C.M. et al., J Pharmacol Exp Ther., 2006, 316, 1360-68).
This inconsistency may be due to activity of PKC isozymes regulating
GABA-A receptor sensitivity to ethanol. We attempted to address this
question by examining the effects of the PKC inhibitor, calphostin-C
upon receptor sensitivity to low ethanol doses in mouse broblast Ltkcells stably expressing human recombinant a4b3d GABA-A receptors.
Ethanol (0.3, 3, 10, 30 and 100 mM) was co-applied with EC20 GABA
concentrations. The lower doses of ethanol did not potentiate GABAevoked currents in cells expressing GABA-A a4b3d receptors, however
we did nd a signicant potentiation of GABA-induced currents by
100 mM ethanol (p < 0.05). Intracellular application of calphostin-C
(400 nM) resulted in a signicant ethanol potentiation at 30 mM and
100 mM (p < 0.05). We also tested the effects of calphostin-C upon
3a5a-THP neurosteroid potentiation of GABA-induced currents. Calphostin-C reduced 3a5a-THP potentiated currents. Chronic ethanol administration (14 days, 20 mM) had no signicant effect upon 3a5a-THP
potentiation; however calphostin-C application signicantly attenuated
the 3a5a-THP potentiation in cells chronically treated with ethanol
(p < 0.05). These results indicate a complex interaction between PKC
activity and the allosteric modulation of GABA-A receptors.
191
Paper No.: 2879
EFFECTS OF THE ENDEMIC PLANT, ERYNGIUM KOTSCHYI,
ON RAT ISOLATED ILEUM AND DETRUSSOR MUSCLE
E Baydan(1), M Kartal(2), S Aslan(2), S Ynce(3), Begum Yurdakok(1),
H Ekici(1), H Alp(7)
(1) Ankara University Faculty of Veterinary Medicine, Department of
Pharmacology and Toxicology, Ankara, Turkey
(2) Ankara University Faculty of Pharmacy, Ankara, Turkey
(3) Afyon University Faculty of Veterinary Medicine, Turkey
(4) Harran University Faculty of Veterinary Medicine, Turkey
Erngium kotschyi, an endemic plant, have been used in folk medicine in
Turkey, to treat gastrointestinal disorders and for its diuretic, antiinammatory and antinociceptive effects. However no pharmacological evidence for its effectiveness on ileum and bladder has been reported. This
study, was aimed to determine the contractile effect of the lyophilized
extracts of the aereal parts of E. kotschyi on isolated rat ileum and
detrussor muscle (DM) and nancialized by Ankara University (AUBAP). For this purpose, ileal and detrussor muscle strips (n:40) were
taken from 20 healthy male Wistar rats weighting around 250-300 g and
suspended under a load of 1 g. The cumulative concentrations of acethylcholine (ACh) (0.5 log 10-8-10-4 M) and the plant extract (0.0783.125 mg/ml ileum, 3.125-100 mg/ml DM) were applied. The plant
showed contractility on both ileum and DM. EC50 of ACh were determined as (2.5X10-7). The potency of the plant extract with respect to
ACh (pD2: 6.6) was found as 0.37 in ileum (Emax: 0.032 mg/ml, pD2:
2.49) and 0.045 in bladder (Emax: 4.7 mg/ml, pD2: 0.3) in cumulative
applications; suggesting the extract has greater potency in ileum than
DM. It was seen that pretreatment with the plant extract (0.3 mg/ml to
ileum and 4.7 mg/ml to DM) potentiated the cumulative contractions of
ACh and the cumulative concentrations of the plant extract pretreated
with oxybutinin (10 nM) did not have much effect on the contractility.
Further analyzes are currently been undergone in our laboratory to determine the mechanism of its action and complete pharmacodynamic effect.
Paper No. 3415

HEALTH AND DISEASE
THE PECULIARITIES OF ACTION OF PENTOXIFYLLINE ON
RAT ISOLATED AORTA IN EXPERIMENTAL HYPOKINESIA
Aram Baykov, A Manukyan
Yerevan State Medical University M. Heratsi, Department of Pharmacology, Yerevan, Armenia
It is shown by the number of publications that restriction of movement
activity (hypokinesia) is a factor, modifying sensitivity to different vasoactive substances. The aim of this study was to reveal the inuence of
hypokinesia on sensitivity isolated aortic rings (IAR) to pentoxifylline.
The experiments were performed on male mongrel albino rats. The control group of rats was kept in ordinary vivarium conditions and the
experimental group in individual narrow cages for simulation of hypokinesia during 15 days. The ability of pentoxifylline (10-6M) to decrease
isotonic contractions amplitude of IAR contracted by phenylephrine (107M) and P PGF2a - (4, 10-6M) was estimated. Cyclooxygenase (COX)
inhibition was done by diclofenac-sodium (3, 10-6M), and NO-synthase
(NOS) inhibition by 7-nitroindazole (10-4M) to dene changes of
PG- and NO-dependent components of action of pentoxifylline. The
prominent decrease of sensitivity of IAR to pentoxifylline in case of
phenylephrine-induced contraction was observed in hypokinesia with no
changes in case of PGF2a-induced contraction. In COX-inhibition there
was an increase of dilatory effect of pentoxifylline in case of both constrictors in comparison to the corresponding index of control group. In
NOS-inhibition there was an authentic decrease of dilator response of
pentoxifylline in phenylephrine-induced contraction in hypokinesia,
whereas signicant increase of dilatory response was noted in case of

PGF2a -induced contraction in the same conditions. Thus, hypokinesia
decreases sensitivity of IAR to pentoxifylline in case of phenylephrineinduced contraction with no change in case of PGF2a -induced one.
Changes of PG- and NO-dependent components of pentoxifylline action
in hypokinesia have different directions.
Paper No.: 1446

DISCOVERY
RHO KINASE INHIBITOR FASUDIL MODIFIES THE ACTIN
CYTOSKELETON OF ASTROCYTES UP-REGULATING
GLUTAMATE TRANSPORT AND ANTI-INFLAMMATORY
MECHANISMS
Philip Beart(1), C Lau(1), V Perreau(1), N Cheung(2), M Chen(2), L
Bischof(3), R OShea(1)
(1) University of Melbourne, Florey Neuroscience Institutes, Melbourne,
Australia
(2) University of Tasmania, Menzies Institute, Hobart, Australia
(3) CSIRO Mathematical & Information Sciences, North Ryde, Australia
Inhibition of Rho kinase has benets in a number of brain injuries by
reducing glial scarring and thus allowing neuronal plasticity. Astrocytes
exhibit good-bad responses during reactive astrogliosis and glutamate
transporters (EAATs) may be an integral part of these homeostatic
responses. EAAT2 may play key roles in astrocyte biology as an endogenous sensor due to its high abundance in astrocytes. We explored
responses of cultured murine astrocytes to Fasudil (HA-1077) by monitoring cytochemistry (GFAP, F- & G-actin), EAAT function (activity,
cell-surface expression, localization) and the transcriptome by microarray
(Illumina Sentrix MouseRef-8 v2.0). Fasudil (100 lM, 24 h) altered
astrocytic morphology to a physiological stellate phenotype by 1h
with loss of cobble-stoned morphology. Image analyses suggested a
decreased abundance of phalloidin-positive actin stress bres since there
was a signicant conversion of F-actin to monomeric G-actin. Kinetic
studies of glutamate transport revealed a doubling of Vmax and biotinylation with Western blotting showed cell surface expression of EAAT2,
but not EAAT1, was doubled at 24h. Bioinformatics of microarray analyses of RNA from Fasudil- and vehicle-treated astrocytes (2 - 24h)
revealed extensive changes to genes involved in actin cytoskeleton,
TGFb-signalling and anti-oxidant mechanisms. Quantitative RT-PCR
conrmed microarray analyses. Together these data reveal a remarkable
spectrum of responses induced in astrocytes by Fasudil. Not only were
there elevations of anti-inammatory astrocytic responses (BDNF, antioxidant and metabolic responses) and a switch to a physiological stellate phenotype, but the substantial elevation of EAAT2 would imply an
availability to efciently scavenge toxic extracellular L-glutamate.
Paper No.: 1953

WRONG; STABILIZATION OF CARDIAC
FUNCTION -CLINICAL
MECHANICAL AND STRUCTURAL CHARACTERISTICS OF
CAROTID PLAQUES: ANALYSIS BY MULTI-ARRAY
ECHOTRACKING SYSTEM AND MRI
Hele`ne Beaussier, O Naggara, D Calvet, R Joannides, B Laloux,
E Bozec, E Guegan-Massardier, E Gerardin, I Masson, C Oppenheim,
P Boutouyrie, S Laurent
(1) Hopital Europeen Georges Pompidou, Department of Pharmacology,
Paris, France
(2) Centre Hospitalier Sainte-Anne, Departments of Neurology and
Neuroradiology, Paris, France
192
(3) CHU Rouen, Departments of Pharmacology, Neurology and Neuroradiology, Rouen,France
Introduction: Combining functional and structural approaches may
improve the predictive value for plaque rupture and ischemic events.
Two distinct patterns of bending strain (BS) were previously determined
along the common carotid artery (CCA) (Paini et al. Stroke 2007;
38:117-23): Pattern A (i.e. outward BS) and its opposite, Pattern B (i.e.
inward BS). Our aim is to correlate arterial mechanics and composition
of an atherosclerotic plaque at the site of the CCA. Materials/Patients: 27
patients with carotid stenosis and an atherosclerotic plaque on the ipsilateral CCA were included: 18 asymptomatics (AS) and 9 symptomatics (S,
i.e. with previous ischemic stroke). Mechanical parameters were measured at 128 sites on a 4 cm long CCA segment by a novel non-invasive
echotracking system (ArtLab) and plaque composition was determined
by non invasive magnetic resonance imaging (MRI). Results: Pattern A
plaques (n = 21) were more often associated with simple plaque (i.e.
AHA stage I-III) than complex plaque (AHA stage IV-VII), by contrast
to pattern B plaques (n = 25) (chi square P = 0.03). No signicant difference in BS pattern was observed between S and AS carotids. Among S
carotids, plaques were characterized by an outward reModelling
(increased external diameter and no change in internal diameter) whereas
among AS carotids, plaques grew according to an inward remodeling.
Conclusion: Longitudinal mechanics of complex plaques follows a
specic pattern of inward BS and suggest that Pattern B, associated with
an outer remodeling, is a feature of vulnerable plaques.
Paper No.: 1513

RHO-KINASE INHIBITION IMPACTS NEUROGENIC
DETRUSOR OVERACTIVITY IN CHRONIC SPINALIZED
RATS
Delphine Behr-Roussel(1), D Broque`res-You(1), S Oger(1), S Compagnie(1), A Caisey(1), P Denys(2), E Chartier-Kastler(3), F Giuliano(2)
(1) PELVIPHARM, Orsay, France
(2) Raymond Poincare Hospital, Garches, France
(3) La Pitie-Salpetrie`re Hospital, Paris, France
Spinal cord injury (SCI) severely disrupts normal bladder function by
inducing neurogenic detrusor overactivity (NDO). First line treatments
i.e. antimuscarinics often associated with intermittent catheterization are
limited by a moderate clinical efcacy and a signicant incidence of side
effects. Thus, the development of new drugs for the treatment of NDO is
of crucial importance. Rho-kinase has a central role in the regulation of
detrusor smooth muscle contraction since this signalling pathway is
involved in the Ca2 + -sensitization of the smooth muscle. Thus, we
aimed to evaluate the effects of a rho-kinase inhibitor (Y-27632) on urodynamic parameters in SCI rats. Complete T7-T8 spinal cord transection
was performed in 17 female adult Sprague-Dawley rats. At 3-4 weeks
post-SCI, cystometry was performed in conscious rats to determine the
effects of Y-27632 (150 lg/kg, intravenous injection, iv, n = 7) and vehicle (saline, iv, n = 10) on the following urodynamic parameters: maximal
amplitude of micturition pressure (MP); baseline intravesical pressure
(BP); pressure threshold for inducing micturition (PT); intercontraction
interval, (ICI); voided volume; amplitude of non-voiding contractions
(NVC) and volume threshold necessary to initiate NVC. Y-27632 signicantly increased voided volume whereas it did not modify MP, BP, d PT
and ICI. The amplitude of NVC was signicantly decreased and the volume threshold of NVC signicantly increase after Y-27632 administration when compared to vehicle (P < 0.001). Inhibition of rho-kinase
enables a better bladder emptying in the rat model of SCI-induced NDO.
This supports the potential development of rho-kinase inhibitors for the
treatment of NDO.
Paper No.: 1514

RHO-KINASE INHIBITION RELAXES DETRUSOR FROM
NEUROGENIC PATIENTS
Delphine Behr-Roussel(1), S Oger(1), D Gorny(1), J Bernabe(1), E
Chartier-Kastler(2), P Denys(3), F Giuliano(3)
(1) PELVIPHARM, Orsay, France
(2) La Pitie-Salpetrie`re Hospital, Paris, France
(3) Raymond Poincare Hospital, Garches, France
Rho-kinases have a central role in the regulation of bladder smooth muscle. In vitro or in vivo data from animal models of overactive bladder
(OAB) indicate that rho-kinases could be involved in the pathophysiology of OAB but the role of rho-kinases in patients with neurogenic
detrusor overactivity has not yet been explored. We evaluated the effect
of rho-kinase inhibition on detrusor from neurogenic patients pre-contracted with either carbachol or KCl. Bladder samples were obtained
from 13 neurogenic patients who underwent partial or total cystectomy.
Detrusor strips with or without urothelium were mounted isometrically
(resting tension 500 mg) in organ baths lled with Krebs-HEPES maintained at 37C bubbled with 95%O2-5% CO2. After equilibration,
concentration-response curves (CRC) for the rho-kinase inhibitor,
Y-27632, (from 10-8M to 3.10-5M) or vehicle were generated on either
carbachol (1lM)- or KCl (50 mM)-precontracted detrusor (N = 7 in each
condition). Y-27632 induced a signicant concentration-dependent
inhibition of KCl-induced (pD2 = 5.4 0.1;Emax=-31.0 4.3%) and
carbachol-induced contraction of human detrusor strips without urothelium
(pD2 = 5.3 0.1;Emax=-56.1 8.0%). The presence of urothelium did
not modify these inhibitory effects. Thus, rho-kinase inhibition decreases
detrusor contractions from neurogenic patients. The mechanisms responsible for this inhibition are different depending on whether the contraction is induced by carbachol or KCl, involving different signalling
pathways. This supports further investigations regarding the development
of rho-kinase inhibitors for the treatment of OAB.
Paper No.: 534

DISEASES
THE EFFECT OF ACUTE AND CHRONIC ADMINISTRATION
OF NEVIRAPINE ON INTERLEUKIN-2 IN A RAT MODEL
Zanelle Bekker, A Walubo, JB Du Plessis
University of the Free State, Department of Pharmacology, Bloemfontein, South Africa
Nevirapine (NVP) induced hepatotoxicity is regarded as partly due to
hypersensitivity reactions, implying activation of the immune system.
Therefore the aim of this study was to determine the effect of acute and
chronic NVP administration on interleukin-2 (IL-2), a cytokine implicated in cell-mediated toxic-immune reactions, after subclinical immune
stimulation with bacterial lipopolysaccharide (LPS). Male Sprague-Dawley rats were used and the study was approved by the animal ethics
commitee. During acute phase, 2 groups of 10 rats received NVP
(200mg/kg) after pre-treatment with either normal-saline (control) or LPS
(test-group). Five animals were sacriced at 6 and 24 hours after NVP
administration. For the chronic phase, 2 groups of 15 rats each received
NVP (200mg/kg) daily over 3 weeks. After every 7 days, 5 rats from
each group were pre-treated with either normal-saline (control) or LPS
(test-group) and sacriced 24 hours later. Serum IL-2 was measured by
ELISA. Although not statistically signicant, during acute phase, IL-2
concentration (pg/ml: meansd) was higher in the LPS-treated-group than
in the control (421.6 65.7 vs. 386.1 33.4 at 6 hrs; and 398.6 42
vs. 368.9 16.6 at 24 hrs), but during chronic administration, IL-2
193
concentration (pg/ml) was lower in the LPS-treated-group than in the
control at week 1 (298.8 10.8 vs. 359.7 62.9), week 2 (351.9 41.4
vs. 378.9 60.3) and week 3 (313.1 23.2 vs. 469.4 143.1). These
results imply that NVP is a slow-onset IL-2 stimulant as was observed
during chronic administration, and that this was augmented in the acute
phase by LPS. Further studies are needed on the toxicologic signicancy
of these observations.
Paper No.: 2780

HEALTH AND DISEASE
THE ENDOTHELIAL PATHWAY FOR CURRENT SPREAD IS
MODULATED BY SMOOTH MUSCLE K+-CHANNELS
Timea Z Beleznai, KA Dora
University of Oxford, Department of Pharmacology, Oxford, UK
The endothelium forms the conduit for distal spread of hyperpolarizing
signals along the longitudinal axis of resistance arteries. Any agonist that
stimulates smooth muscle cell hyperpolarization has the potential to be
associated with homo- and heterocellular intercellular electrical coupling
and spreading dilatation, and thereby inuence blood ow. We have a
model to study spreading dilatation by isolating and triple-cannulating rat
mesenteric arteries. Arteries are mounted in a gravity-fed pressure myograph and kept at 37C, with one branch of the bifurcation connected to a
syringe pump containing agonist and a uorescent dye. Constant downstream ow through the feed artery allows luminal perfusion of agonists
into the sidebranch without reaching the feed artery, and using confocal
microscopy we can simultaneously measure local dilatation in the branch
and spreading dilatation into the feed artery. Agonists acting selectively
at endothelial (acetylcholine) and smooth muscle (isoprenaline) K+-channels each evoked spreading dilatation with similar mechanical length
constants near 1 mm. The ATP-sensitive K+-channel blocker glibenclamide doubled the length constant for acetylcholine and effectively abolished that for isoprenaline. Combined blockade of large conductance
Ca2 + -activated K+-channels and voltage-gated K+-channels with iberiotoxin or tetraethylammonium together with 4-aminopyridine markedly
and consistently increased the magnitude of spreading dilatation to both
agonists, such that the length constant approached innity in the 2 mm
length of artery. Changes in endothelial cell calcium could not account
for the apparently regenerating current. These data show that activated
smooth muscle cell K+-channels not only serve to modulate contraction,
but also reduce the effectiveness of spreading dilatation.
Paper No.: 1147

GLUTATHIONE S-TRANSFERASE M1 AND GLUTATHIONE
S-TRANSFERASE T1 POLYMORPHISMS AND LUNG CANCER
RISK IN TUNISIAN MEN
Majda-Noura Belkhiria(1), MA Jebali(1), I Harrabi(2), J Knani(3)
(1) Faculty of Medicine of Monastir, Laboratory of Pharmacology,
Monastir, Tunisia
(2) CHU Farhat Hached, Department of Epidemiology, Sousse, Tunisia
(3) CHU Tahar Sfar, Department of Pneumology, Mahdia, Tunisia
Glutathione S-transferases (GST) detoxify polycyclic aromatic hydrocarbons found in tobacco smoke by glutathione conjugaison. Polymorphisms within the GSTM1 and GSTT1 genes, coding for enzymes with
decient or reduced activity, have been studied as potential modiers of
lung cancer risk. In this study, we evaluated the association between the
GSTM1 and GSTT1 polymorphisms and lung cancer risk, and their
interaction with cigarette smoke. The GSTM1 and GSTT1 polymorphisms were determined by multiplex PCR in 80 lung cancer patients
and 86 controls. Associations between specic genotypes and the development of lung cancer were examined by use of logistic regression to
calculate odds ratios (OR) and 95% condence intervals (CI). The frequencies of GSTM1 and GSTT1 null genotypes were 57.5 and 32.5% in
lung cancer patients and 52.3 and 17.4% in controls, respectively. The
GSTM1 homozyous null genotype was not associated with an increase
risk of developping lung cancer. There was a marginally signicant association between lung cancer and GSTT1 null genotype (OR : 2.22, 95%
CI : 1.10-4.61). After grouping according to smoking status, GSTT1 null
genotype was associated with an increase lung cancer risk for smokers
(OR = 2.99, 95% CI = 1.01-8.80). Our ndings suggest that the GSTT1
null genotype may be associated with an increased susceptibility to lung
cancer in Tunisian men.
Paper No.: 1372

A LINEAL MATHEMATICAL MODEL BASED ON HBA1C
LEVELS FOLLOW UP MAY PREDICT THE YEARS OF
EVOLUTION OF TYPE 2 DIABETES MELLITUS STARTING
FROM A SINGLE HBA1C LEVEL QUANTIFICATION
Inmaculada Bellido, AE Santos, L Garca-Carrascal, JJ Sanchez-Luque,
E-I Marquez, E Blanco, A Gomez-Luque
University of Malaga School of Medicine, Department of Pharmacology
and Clinical Therapeutics, Malaga, Spain
Background: Glycosylated haemoglobin (HbA1c) is the best diabetes
control and complication risk predictor. Aim: To design a mathematical
model to predict the type 2 diabetes mellitus (T2DM) evolution time
based in patient HbA1c levels. Methods: T2DM patients were prospectively studied (5 years). All the physiopathological characteristics and
antidiabetics drugs treatments were recorded. The population statistical
analyses was done by ANOVA test, correlations, polynomic interpolation,
regression toward the mean, uni-bivariate linear, non-linear (including
Levenberg-Marquardt algorithm) and logistic regression analysis. Results:
346 patients with T2DM, aged 67.2 10.9 years, male 50.4%, weighed
79.8 1.3, mean body mass index 31.1 0.5, were enrolled. The main
population characteristics were: basal glycaemia 137.5 7.3 mgr/dL,
HbA1c 6.75 0.59, conrmed-diabetes evolution 7.16 0.68 years,
hypertension 6.45%, dyslipemic diseases 6.48%, obesity 6.3%, and
treated with insulin 17.2%, oral antidiabetics 29.8%, anti-hypertensive
agents 33.4%, normolipemiants 53.2%, platelet antiaggregants 43.2%.
The mathematical model which really shown a good adjust to the majority of the patients data was a linear differential equation with a negative
constant of proportionality which may predict the diabetes years of
evolution starting from a single HbA1c level quantication with an
r-squared adjusted for DF of 63.9% and with a standard error of 0.124.
Conclusion: This mathematical model is able to adjust predict the years
of evolution of type 2 diabetes mellitus starting from a single glycosylated haemoglobin level quantication in patients with glycosylated
haemoglobin levels between 5.5% and 9.5%. It is not reliable in case of
very low and very high HbA1c levels, and more than 60 years of diabetes
evolution.
Paper No.: 1373

FOCUSED CONFERENCE GROUP: PW19 - INFLUENCE OF
DEGENERATION AND REPAIR IN THE CNS AND PERIPHERY
THE USE OF E-LEARNING IMPROVED THE
PHARMACOLOGY LEARNING IN CHIROPODISTS
PHARMACOLOGY TRAINING
Inmaculada Bellido(1), E-I Marquez(1), JA Garcia-Arnes(1),
E Blanco(1), A Gomez-Luque(2)
(1) University of Malaga School of Medicine, Department of
Pharmacology and Clinical Therapeutics, Malaga, Spain
194
(2) University Hospital Virgen Victoria, Department of Anaesthesia,
Malaga, Spain
Background: The e-learning prepared teaching material may awake our
students abilities to think, to learn and to develop clinical skills for their
professional lives. Aim: To quantify the efcacy of a PC-supported programme (Chiropharm) added to a face-to-face teaching of Pharmacology
training for Chiropody students. Methods: Students of Pharmacology in
Chiropody training taught by the face-to-face (FtF) (2006-07) were
compared with student taught with FtF complemented with Chiropharm
(ChiropharmP) (2008-09) methodology. ChiropharmP is an interactive
PC-supported programme design using Windows XP, UMAs Moodle
platform, Word, Power Point, Front Page, Hot Potatoes, specialized
medical webs free material and Explorer/Mozilla software, including
texts and slides presentation with iterative buttons linked to hyper-text to
questions, references, gures, lms, iterative auto-evaluations, clinical
cases and high delity simulators. A quality and quantity evaluation of
the students knowledges, and an anonymous satisfaction questionnaire
were analyzed (unpaired-groups student t test). Results: 23 (51% of
n = 45) students were initially trained in ChiropharmP handling during
3 h. They spent a mean of 33 5.7 h using ChiropharmP during the
course development (2.9 0.7h/week/x13 weeks). We founded some
differences between the two groups (FtF vs. ChiropharmP) (p < 0.05):
time of study 6.1 1.7h/week vs. 3.3 0.9h/week; correct resolved
clinical cases 54.5%-91%; approved student 68.1% (46.6% with A-B)
vs. 95.6% (73.9% with A-B); students which prepared themselves for the
honours qualication special exam 5 vs. 16; percentage which consider
this teaching-learning system better than the classic methodology 95%.
Conclusion: The Pc-supported programme ChiropharmP improved the
pharmacology learning and was more pleasant for Pharmacology student
of Chiropody.
Paper No.: 2384

FOCUSED CONFERENCE GROUP: P19 - GENERAL SESSION ONCOLOGY
AN INCREASED PRECRYOPRESERVATION TIME REDUCES
THE FEASIBILITY AND THE RECOUNT OF THE CD34(+)
CELLS OF THE UMBILICAL CORD BLOOD
Inmaculada Bellido(3), L Ponce(1), MC Hernandez(2), M Barrios(1),
I Prat(2), AI Heiniger(1)
(1) Carlos Haya Universitary Regional Hospital. Haematology Service.
Malaga
(2) Regional Center for Blood Transfusion-Umbilical Cord Unit. Malaga.
Spain
(3) University of Malaga School of Medicine, Department of Pharmacology and Clinical Therapeutics, Malaga, Spain
Background: Cryopreservation protocols for umbilical cord bloods (UC)
haematopoietic cells try to approach an acceptable level of recovery
CD34(+) by optimizing the extraction, transport, processed and cryopreservation methods of the UC. It is generally accepted that UC units can
be safely cryopreserved more than 24 h after harvest, but a delay in
freezing can inuence the nal quality of the UC-unit. Aim: To determine if the total time from extraction to cryopreservation, which mainly
dependent on shipment variables, may modify the feasibility and the
recount of the CD34(+) cells in the umbilical cords units. Methods: The
feasibility and number of CD34(+) cells in the UC-units from 5400 UC
samples analyzed in Malagas UC-bank were analyzed. The samples proceeded from 27 Andalusia hospitals (processed from 2009 January to
September (female aged 30.7 0.2 years, gestation weeks 39.8
0.05 weeks, type of delivery eutocic 71.5%, instrumental 16.5%, caesarea 7.6%, male neonate proportion 52.8%). Mother and neonates clinical
data, procedure for UC collection, time of collection, shipment-phases
and cryopreservation-phases, and feasibility and CD34(+) cells were
recorded. Results: Feasible samples shown a lower precryopreservation
time than non-feasible samples (-11.9%, 24.4 0.3h vs. 27.7 0.5h,
P < 0.01). Both feasibility and recount of CD34(+) cells showed an
inverse correlation with precryopreservation time duration with Pearson

correlation coefcient of -0.214 (p = 0.001) and -0.088 (p = 0.049).
Conclusion: The transport, processes and storage time previous to cryopreservation must be reduced as much as possible to minimized the
reduction in the CD34(+) cells of umbilical cord blood feasibility related
with the increment of the precryopreservation time.
Paper No.: 2410

GRAPHIC AND PICTORIAL TABLES EXPLAINING DRUGS
ROUTES ADMINISTRATION REDUCE THE ANXIETY OF THE
HOSPITALIZED CHILDREN IN THE INTENSIVE CARE UNIT
MV Bellido(1), Inmaculada Bellido(2), A Gomez-Luque(3)
(1) Carlos Haya Universitary Hospital, S. Intensive Care Unit, Malaga,
Spain
(2) University of Malaga, School of Medicine, D. Pharmacology and
Clinical Therapeutics, Malaga, Spain
(3) Virgen de la Victoria Universitary Hospital, S. Anaesthesiology,
Malaga, Spain
Background: Art communication techniques are usefully tool to communicate with the children following the narrative-based medicine. Aim:
To evaluate the impact of graphic/pictorial tables explaining drugs routes
administration on pain and anxiety development by the children during
drugs administration in the intensive care unit (ICU). Methods: Childrens hospitalized in the ICU having elective major surgery and oncology therapy were collected prospectively. Clinical stage, surgery and
anaesthesia procedures, oncologic, analgesics, anxiolytics treatments and
therapy-related complications were recorded. Pain and anxiety were measured by a visual analogy scale (VAS), children faces pain scales, and
STAIC questionnaire after the use of a DRA-table to explain the drugs
routes administration and were compared with a group with DRA-tables
explication. esults: 125 children aged 3-10 years (6.5 1.5 years old,
65% male) were enrolled. The procedures included cardiac (51%), digestive (6%), neurosurgery (20%) and oncologic diseases (23%). The drugs
routes administration were oral/nasogastric tube (1%), intranasal (3.1%),
inhalatory (30.9%), intramuscle (1.3%), intravenous (50.2%), intrathecal
(13.5%). Both VAS-pain and VAS-anxiety quantication were higher
(p < 0.05) in control than in DRA-table group (5.6 0.9 vs. 5.1 0.5,
and 7.8 1.1 vs. 5.5 0.6, respectively). The reduction of anxiety in
the DRA-table group with respect to the control group was age and gender-related (higher in children up to 8 years than under 5 years, higher in
female than in male). The control group needs much more analgesics,
anxiolytics and sedans than DRA-table group. Conclusion: Graphic and
pictorial tables explaining drugs routes administration reduce the anxiety
of the hospitalized children in the intensive care unit.
Paper No.: 1897

HEALTH AND DISEASE
ENDOTHELIUM-DERIVED HYPERPOLARIZING FACTOR
AND NITRIC OXIDE REGULATE ARTERIAL WALL
VISCOSITY IN VIVO IN HUMANS
Jeremy Bellien, M Lacob, C Thuillez, R Joannides
Xxxxx
Although the viscoelasticity of conduit arteries has been extensively
investigated, few studies have focused on arterial wall viscosity (AWV)
itself and its regulation by the endothelium in vivo. This is of particular
importance since AWV is a major source of energy dissipation through
the vascular system reducing cardiovascular coupling efciency. We
simultaneously measured radial artery diameter and arterial pressure
195
(NIUS02) in healthy volunteers before and during local infusion of pharmacological inhibitors of endothelial pathways. L-NMMA (8 mmol/min)
was used as NO-synthase inhibitor, tetraethylammonium (TEA: 9 mmol/
min), as blocker of calcium-activated potassium channels, the target of
endothelium-derived hyperpolarizing factors (EDHF) and uconazole
(0.4 mmol/min), as inhibitor of cytochrome epoxygenases which promote the synthesis of epoxyeicosatrienoic acids synthesis, identied as
EDHF in conduit arteries. AWV was estimated from the ratio of the area
of the hysteresis loop of the pressure-diameter relationship to the area
representing the whole energy exchanged during each cardiac cycle.
L-NMMA paradoxically reduced AWV (n = 5: 27.6 0.7 to
23.4 0.7%, P = 0.053). Conversely, AWV was increased by TEA
(n = 6: 25.5 0.5 to 31.3 0.7%, P = 0.040) and uconazole (n = 5:
26.6 0.6 to 30.6 0.6%, P = 0.047). This increase was more marked
with the association of L-NMMA+TEA (n = 6: 27.6 0.9 to
41.0 0.7%, P = 0.002) and L-NMMA+uconazole (n = 6: 26.1 0.7
to 36.3 0.3%, P = 0.001) showing a synergistic effect of both combinations on AWV. These results demonstrate that the endothelium contributes in vivo in humans to the regulation of AWV through an interaction
between NO and a cytochrome-related EDHF.
Paper No.: 2874

ADHERENCE TO ANTIHYPERTENSIVE MEDICATIONS AND
ALL-CAUSE MORTALITY AND CARDIOVASCULAR EVENTS:
A POPULATION-BASED STUDY BASED ON
ADMINISTRATIVE DATA
Silvia Benemei(1), S Saragoni(2), P Batacchi(3), P Geppetti(1),
M Di Bari(4), N Marchionni(4), S Buda(2), ED Esposti(5), LD
Esposti(2)
(1) University of Florence, CIFF, Department of Preclinical and Clinical
Pharmacology, Florence, Italy
(2) CliCon S.r.l., Health, Economics & Outcomes Research, Ravenna,
Italy
(3) University of Florence, CIFF, Pharmaceutical Department ASL10,
Florence, Italy
(4) University of Florence, CIFF, Department of Critical Care Medicine
and Surgery, Florence, Italy
(5) Umberto I Teaching Hospital, Rome, Italy
Adherence to antihypertensive treatment (AT) is limited, ranging from 30
to 70% of treated patients, depending on its denition. The association
between poor adherence to AT and cardiovascular (CV) morbidity/mortality remains largely unexplored. Using administrative databases of citizens living in Florence, Italy, a retrospective cohort study was conducted
with inclusion of records of subjects (>18years), receiving a rst prescription of AT between January 1st, 2004 and December 31st, 2006.
Adherence to AT was dened as the proportion of days (PD) on which a
patient had pills available in the interval from the rst purchase of AT
until all-cause death, non-fatal stroke or myocardial infarction (composite
outcome) or leaving database and classied as poor (PD?40%), moderate
(PD:41%-60%), good (PD:61%-80%), or excellent (PD>80%). Subjects
prescribed AT for secondary CV prevention, occasional users, and those
who suffered a major CV event within 6 months of enrollment were
excluded. Cox regression model was used to determine the association
between adherence and composite outcome. 31,306 records (mean age of
60.2 14.5 years, 52.0% women) were included. Adherence was poor
in 25.7%, moderate in 14.8%, good in 18.1%, and excellent in 41.5% of
subjects. Compared to poorly adherent subjects, the risk of the composite
outcome was progressively lower in those with moderate (HR=0.87),
good (HR=0.69), and excellent adherence (HR=0.53). Sub-optimal
adherence to AT is associated with substantial numbers of avoidable
deaths and CV events in primary prevention. In a public health perspective, this evidence underlines the need for improving adherence to medications in clinical practice.
Paper No.: 492

HONEY: THE OLDEST MODERN REMEDIES FOR MASTITIS
Mokhtar El-Bachir Benhania, L Boukraa
University of Ibn Khaldoun, Department of Agro-Veterinary Sciences,
Tiaret,Algeria
Honey has been used in medicine against bacterial infections for thousands of years, the objective of this study was to evaluate the efcacy of
intramammary honey administered during lactating period, to eliminate
Staphylococcus, Aureus and Pseudomonas aeruginosa infection. Infected
mammary quarters of three cows were treated once daily with 5 ml of
multioral honey per quarter for 2 consecutive days. Milk samples were
taken from affected quarters immediately prior to treatment on d 0, 7, 14,
21, 52 and 60 for microbiological examination. The cure following
administraton of honey was 66% (2/3 cows) at days 7 and 14; 100% (3/3)
at days 21, 52 and 60 for Staphylococcus aureus infection and 66% (2/3)
at days 21, 52 and 60 for Pseudomonas aeruginosa. This preliminary
study has shown the efcacy of honey as natural therapeutic product
against S. aureus and P. aeruginosa sub clinical mastitis. Further study
may elucidate the suseptibility of other pathogens microorganisms implicated in acute clinical and subclinical bovine mastitis (number of cases,
herd size.), the dynamic of honey (distribution, diffusion.) in mammary gland and apropriate intramammary formulation of honey for mastitis prevention and therapy in cows. Finally, honey could be an
alternative for mastitis treatment in both conventional and organic farms.
This will lead to a huge economic and health benet worldwide.
Paper No.: 2943

TOXICITY OF CYTOSTATIC AGENTS TO GRANULOCYTEMACROPHAGE PROGENITORS (CFU-GM) INCREASED IN
ZUCKER OBESE RATS
I Benko(1), Krisztina Geresi(1), K Benko(2), B Szabo(3), A Megyeri(1),
E Ungvari(1), B Peitl(1), Z Szilvassy(1)
(1) University of Debrecen, Medical and Health Science Center, Department of Pharmacology and Pharmacotherapy, Debrecen, Hungary
(2) University of Debrecen, Medical and Health Science Center, Euromedi cDiagnostics, Debrecen, Hungary
(3) Semmelweis University, Heart Center, Budapest, Hungary
Increased risk of anticancer chemotherapy in seriously obese patients is
well-known especially in children with acute myeloid leukemia. Obesity
maybe one of the factors that predict treatment-related toxicity during
chemotherapy. We studied whether the functions of granulopoietic cells
do not be altered by obesity. At the rst sight - granulopoiesis as measured by cellularity, frequency of granulocyte-macrophage progenitors
(CFU-GM) and total CFU-GM content of the femoral bone marrow
did not differ in obese, insulin resistant Zucker rats compared with control, Wistar rats. Nevertheless we tested the vulnerability of their CFUGM cells by culturing them in vitro in the presence of carboplatin and
5-uorouracil, two cytotoxic drugs with different mechanisms of action.
Both drugs were more toxic on CFU-GM cells of insulin resistant Zucker
obese animals than on CFU-GM cells of the control rats. This based on
metabolic disorders at least in partly, because we could demonstrate similar increased toxicity to CFU-GM progenitors originated from the nonobese but insulin resistant Goto-Kakizaki rats in the same dose ranges.
After in vivo administration of rosiglitazone, an insulin sensitizer, the
toxicity of the studied cytostatic drugs decreased proportionally with the
improvement of the insulin sensitivities both in Zucker obese and GotoKakizaki rats. However the increased treatment-related myelotoxicity and
mortality is well-known among obese patients with malignant diseases,
196
only the altered half-lives, volumes of distribution and clearances of the
cytostatic drugs have been taught to be the underlying reasons. Our
results are the rst observation about an impaired granulopoiesis in obese
animals.
Paper No. 3253

ANGIOTENSIN-(1-7) PREVENTS DIABETES-INDUCED
ATTENUATION IN PPAR- C AND CATALASE ACTIVITIES
Ibrahim Benter(1), G Dhaunsi(1), M Yousif(1), S Akhtar(1),
M Chappell(2), D Diz(2)
(1) Kuwait University Faculty of Medicine, Department of Pharmacology, Safat, Kuwait
(2) Wake Forest University. School of Medicine, Winston-Salem, NC,
USA
Angiotensin-(1-7) is a vasodilator peptide that exhibits antihypertensive,
antithrombotic and antiproliferative properties. The mechanisms by
which angiotensin-(1-7) exerts its benecial effects on end-organ damage
associated with diabetes and hypertension are not well characterized.
This study was designed to compare the effects of apocynin with angiotensin-(1-7) on renal vascular dysfunction and NADPH oxidase activity
in a combined model of diabetes and hypertension and to further determine whether chronic treatment with angiotensin-(1-7) can modulate
renal catalase, and peroxisome proliferator activated receptor- c (PPARc) levels in streptozotocin induced-diabetes in both normotensive Wistar
Kyoto rats (WKY) and in spontaneously hypertensive rats (SHR).
Apocynin or angiotensin-(1-7) treatment for one month starting attenuated elevation of renal NADPH oxidase activity in the diabetic SHR kidney and reduced the degree of proteinuria and hyperglycemia, but had
little or modest effect on reducing mean arterial pressure. Both drugs also
attenuated the diabetes-induced increase in renal vascular reactivity to
endothelin-1. Induction of diabetes in WKY and SHR animals resulted in
signicantly reduced renal catalase activity and in PPAR-c mRNA and
protein levels. Treatment with angiotensin-(1-7) signicantly prevented
diabetes-induced reduction in catalase activity and the reduction in
PPAR-c mRNA and protein levels in both animal models. These data
suggest that activation of angiotensin-(1-7)-mediated signalling could be
an effective way to prevent the elevation of NADPH oxidase activity and
inhibition of PPAR-c and catalase activities.
Paper No.: 1287

DISEASES
APPARENT DIFFERENCES IN PHARMACOKINETIC
PERFORMANCE BETWEEN GENERIC MYCOPHENOLATE
MOFETIL TABLET FORMULATIONS
Darren Bentley(1), L Banken(2), R Robson(3)
(1) Roche Products Ltd, Department of Clinical Pharmacology, Welwyn
Garden City, UK
(2) F. Hoffmann-La Roche Ltd, Welwyn Garden City, UK
(3) Christchurch Clinical Studies Trust, Christchurch, New Zealnd
CellCept, mycophenolate mofetil (MMF), is an integral component of
immunosuppressive regimens for the prophylaxis of acute rejection in
patients receiving allogeneic renal, cardiac or hepatic transplants. Several
generic formulations have been approved based on bioequivalence to the
innovator product. However, differences in dissolution rates between
generic formulations observed in vitro may lead to disparities in pharmacokinetic proles. We compared pharmacokinetic proles of three gen-
eric MMF formulations (A] Renodapt, Biocon Ltd; B] Mycept, Panacea

Biotec Ltd; and C] Cellmune, Cipla Ltd) in a randomized, open label,
four-way crossover study that also included CellCept. In each treatment
period, subjects received a single 500 mg MMF tablet with a high fat,
high calorie meal. Plasma concentrations of mycophenolic acid (MPA),
the major active metabolite of MMF, were measured up to 48 hours after
intake. Global statistical comparisons were made between the three generic formulations for Cmax and AUCinf and mean treatment ratios were
calculated. Thirty two healthy male volunteers were enrolled and 31
completed the study. Although global tests could not reject the null
hypothesis, there were apparent differences in the peak exposures to
MPA between the generic formulations. Mean Cmax for B was 23%
lower than C, and that for A was 15% lower than C. However, total
MPA exposures were similar across the generic formulations and CellCept. In conclusion, some apparent differences in pharmacokinetic performance exist between generic formulations. The clinical consequences
(e.g. organ rejection) of these differences when switching between generic MMF formulations remain to be investigated.
Paper No.: 1092

DISEASE AND THERAPY
THE KV11.1 CHANNEL OPENER NS1643 SHORTENS THE QT
IN TRANSGENIC RABBITS WITH LONG QT-SYNDROME 1 IN
VIVO AND SHORTENS THE APD90 EX VIVO, BUT IS
ASSOCIATED WITH INCREASED RISK OF ARRHYTHMIAS
Bo Bentzen(1), S Bahrke(2), K Wu(2), AP Larsen(1), J Biermann(2),
X Peng(3), G Koren(4), M Zehender(2), C Bode(2), M Grunnet(5),
M Brunner(2)
(1) University of Copenhagen, Faculty of Health Sciences, Danish
Arrhythmia Research Centre, Copenhagen, Denmark
(2) Universitatsklinik Freiburg, Innere Medizin III, Freiburg, Germany
(3) Penn State University, Department of Comparative Medicine,
Philadelphia, PA, USA
(4) Brown University Alpert Medical School, Cardiovascular Research
Center, Rhode Island Hospital, Providence, RI, USA
(5) NeuroSearch A/S, Ballerup, Denmark
Aim: Transgenic rabbits expressing pore mutants of hKv7.1(Y315S) display a Long QT-Syndrome-1(LQT1) phenotype. Recently, the compound
NS1643 has been described to increase IKr/Kv11.1 current. We hypothesized that NS1643 would hasten cardiac repolarization and shorten the
action potential duration(APD90) in LQT1 rabbits, thereby normalizing
the QT-interval. Methods: Female transgenic LQT1 rabbits aged
4-6 months were compared to littermate controls(LMC) in vivo and
ex vivo. In vivo ECG studies(n = 9 vs 6/group) in sedated animals were
performed at baseline and during 30min i.v. infusion of NS1643(1.5mg/
kg/min) or vehicle(PEG400/glucose) in a crossover design with 1 week
between treatments. Ex vivo 4 left ventricular monophasic action potentials were recorded from Langendorff-perfused hearts (n = 13 vs 10/
group) at baseline and during 45min perfusion with NS1643(5lM) at
atrial pacing (RR= 400ms). Left ventricular refractory periods(VERP)
were assessed before and after 45min NS1643 infusion. Results: In vivo
NS1643 shortened the QTc after 30min signicantly in LQT1(NS1643:
8.5%0.7; vehicle:2.5%1.1(P < 0.001)), whereas NS1643 did not
shorten the QTc signicantly in LMC. In Langendorff experiments
APD90 was shortened by NS1643(LQT1: 32.0ms 4.3(P < 0.01);
LMC:21.0ms5.0(P < 0.01)). Concomitantly, the VERP was signicantly shortened by NS1643 (LMC:22.6ms9.9; LQT1:23.7ms8.3).
Before NS1643 2 LMC and 5 LQT1 had ventricular brillation either
spontaneously or during VERP-testing, whereas after NS1643 9/10 LMC
and 13/13 LQT1 animals had VF. Additionally, dp/dt was signicantly
decreased(LMC:63%4; LQT1:49%3) after NS1643. Conclusions: We
demonstrate that NS1643 shortens the QT-intervals in LQT1 signicantly
in vivo. Ex vivo NS1643 shortens the APD90 and VERP in both genotypes, but is associated with increased arrhythmia susceptibility and
negative inotropy.
197
Paper No. 3305
FOCUSED CONFERENCE GROUP: FC13 - MAXIMISING
THE STUDY OF RESPONSE TO BRONCHODILATORS
ACCORDING TO POLYMORPHISM B2-ADRENERGIC
RECEPTORS
Nadyia Berdnikova
Moscow Medical Academy, Moscow City Hospital #23, Moscow,
Russian Federation
Aim: to study the inuence of polymorphism 2-adrenergic receptors
(gene ADRB2) on changing of FEV1 after taken ipratropium bromide
and salbutamol in the patients with asthma in dependence of severity of
asthma. Methods: 62 patients (48.6 11.9 ages; 33 men, 29 women) were
included in the study. The genotype (Arg, Gly) was examined by polymerase chain reaction (PCR). All the patients were divided into 2 groups:
I group -FEV1-60%, II group -FEV1-60% pred. FEV1 was measured in
all the patients before and after taken ipratropium bromide 80 mcg
(40 min.) and salbutamol 400 mcg (30 min.). Results: 26 patients have
16ArgGly: FEV1-79.8% (14), FEV1-39.6% (12). Heterosigous showed
increase FEV1 (%) after taken 2 bronchodilators: I- 8.38% and 13% and
II- 10% and 17% (p < 0.05). 16 patients have 16ArgArg: FEV1-74.8%
(10), FEV1-37.6% (6). Homosigous Arg showed increase FEV1 after
taken 2 bronchodilators: I- 1.35% and 16.9% and II- 7% and 13.4%
(p < 0.05). 18 patients have 16GlyGly: FEV1-76.4% (9), FEV1-49.8%
(9). Homosigous Gly showed increase FEV1 after taken 2 bronchodilators: I- 11.7% and 18% and II- 13.3% and 15.6%. Conclusion: only the
60% did not show increase FEV1 after
16GlyGly patients with FEV1U
taken salbutamol. All the other patients showed.
Paper No. 3428

FOCUSED CONFERENCE GROUP: FC16 - NATURAL
STUDY OF INFLUENCY INDOL-3-CARBINOL,
EPIGALLOCATECHIN-3-GALLATE AND SOY ON
PROLIFERATION DISEASES OF THE REPRODUCTIVE
SYSTEM IN WOMEN
Nadyia Berdnikova, L Zorina, S Serebrova
Moscow Medical Academy, Moscow City Hospital #23, Moscow,
Russian Federation
I3C/EGCG/soy are natural products used for treatment. To conrm the
inuence of I3C/EGCG/soy on proliferation diseases of reproductive system in women we searched 30 patients (41.3 4.28 age): with benign breast
diseases-6 patients (20%), myoma of body of uterus and adnexitis-4
(13.3%), adenomyosis and polyp of cervix of uterus-5 (16.6%), myoma
of body of uterus and adenomyosis-8 (26.6%), myoma of body of uterus
and adenomyosis and chronic cystic mastitis-7 (23.3%). Nobody from
patients took the hormonal treatment. Diagnoses were conrmed by sonographic and mammographic. All the patients took I3C/ EGCG/soy per os
for 6 months. The women with benign breast diseases estimated their pain
according questionnaire for pain. Patients (n = 13) showed changes of
scores for pain in mammary glands: before I3C/EGCG/soy with severe
pain-46.15%, moderate-23.07%, mild-30.77%, none-0%; after 3 months:
0%, 23.07%, 15.3% (p < 0.05), 61.63% (p < 0.05) accordingly; after
6 months: 0%, 15.3%, 15.3%, 69.33% accordingly. The size of cysts in
mammary glands changed by sonographic: when before taking I3C/
EGCG/soy it was 1.1 0.3sm then in 3 months it decreased to 0.60 0.22sm
(p = 0.0001). Cysts with size 0,5sm and less werent found in 3 months
by sonographic. Patients (n = 19) showed changes of uterine volume/
myoma: before I3C/EGCG/soy-184.1 4.1ml/80.1 3.6ml; after 3 months168.3 3.5ml (p < 0.05)/72.4 2.4 ml (p < 0.05); after 6 months: 162.2
3.7ml/70.4 2.1ml (p < 0.05). After 3 month taking I3C/EGCG/soy the
decrease in pain, oedema and size of cysts in women with benign breast
diseases was conrmed. We did not nd extra changes after 6 months.
The decrease in uterine volume and myoma was statistically signicant.
Paper No.: 1352

PUBLICATION RATE AMONG INVESTIGATOR-INITIATED
CLINICAL DRUG TRIALS IN DENMARK
Louise Berendt(1,2), KF Bach(1), T Callreus(2), LG Petersen(2),
HE Poulsen(3), K Dalhoff(3)
(1) Bispebjerg University Hospital, GCP Unit, Copenhagen, Denmark
(2) Danish Medicines Agency, Copenhagen, Denmark
(3) Bispebjerg University Hospital, Department of Clinical Pharmacological, Copenhagen, Denmark
Publication of clinical drug trials (CDTs) is an important way to disseminate new knowledge. However, not all CDTs are published. Based on
approved CDT applications, a systematical search in PubMed demonstrated a publication rate of 30% among Danish investigator-initiated
CDTs (Berendt. BMJ 2008; 336 : 33). In order to verify this, we conducted a similar search in which the ndings - negative as well as positive were veried by the investigators. We included all approved CDT
applications submitted to the Danish Medicines Agency in 1999, 2001
and 2003 describing an investigator-initiated trial. Corresponding publications were identied by systematically searching PubMed May-Sept
2009. Only limited data such as investigator, protocol title, drug names
and sometimes a brief description of the study were available. Each submitting investigator was asked to conrm the ndings (trial published/
not published, correct publications). A total of 245 eligible approved trial
applications were identied. Response was obtained from 174 investigators. Of these, PubMed search resulted in 84 published vs. 90 non-published trials, whereas response from investigators resulted in 94
published vs. 80 non-published trials (j statistic 0.771, good agreement).
The overall publication rate based on PubMed search was 46% (112/
245). Dividing trials in +/) response from investigators, the publication
rates were 48% and 39%, respectively. Average number of publications
per trial was 1.8 (median: 5, range: 1-9). Combination of PubMed search
with verication by investigators resulted in a publication rate higher
than PubMed search alone. Reasons are discussed.
Paper No.: 1923

PREVENTION OF URINARY TRACT INFECTIONS IN
NORWEGIAN NURSING HOMES
Jenny Bergman(1), H Salvesen Blix(2), J Schjtt(3,4)
(1) Haukeland University Hospital, Regional Drug Information Centre,
Bergen, Norway
(2) Norwegian Institute of Public Health, Department of Pharmacoepidemiology, Oslo, Norway
(3) Haukeland University Hospital, Section of Clinical Pharmacology,
Laboratory of Clinical Biochemistry, Bergen, Norway
(4) University of Bergen, Institute of Internal Medicine, Section for Pharmacology, Bergen, Norway
Urinary tract infections (UTIs) are the most common infections in nursing homes and prevention may reduce antibiotic use and patient suffering. The spectre of agents used in preventing UTIs in nursing homes is
scarcely documented. The aim of this study was to describe all agents
used to prevent UTIs in Norwegian nursing homes. We carried out a
one-day point-prevalence study in 44 Norwegian nursing homes during
May-April 2006, with registration of all agents prescribed as UTI prophylaxis. The results included 1473 residents. 18% (n = 269) of the residents
had at least one agent recorded as prophylaxis of UTI, varying between
0-50% among the nursing homes. Methenamine was used by 8.8% of all
residents, vitamin C by 5.9%, vaginal and systemic estrogens by 5.5%
and cranberry products by 1.9%. Trimethoprim and nitrofurantoin were
198
used as prophylaxis by 0.9% and 0.7% respectively. The dosage of
vitamin C varied between 60-2000mg a day, cranberry products were
given as stated by the manufacturer and the dosages of other drugs were
within the recommended doses stated in the summery of product characteristics. In conclusion the use of UTI prophylaxis is common but
variable in Norwegian nursing homes. Interestingly, methenamine and
vitamin C, which lack documented effectiveness, are the most frequently
used agents. In contrast, trimethoprim, nitrofurantoin and local vaginal
estrogens have been shown to be effective, but are less frequently used.
We conclude that prescribing habits for effective UTI prophylaxis are
controversial in Norwegian nursing homes.
Paper No.: 1449

SESSION - ONCOLOGY
IMPACT OF ABCB1 VARIANTS ON NEUTROPHIL
DEPRESSION: A PROSPECTIVE STUDY
Troels K Bergmann(1), C Brasch-Andersen(0), H Green(2), M Mirza(3),
K Skougaard(4), J Wihl(5), N Keldsen(6), P Damkier(3), C Peterson(2),
W Vach(7), K Brsen(1)
(1) University of Southern Denmark, Department of Clinical Pharmacology, Odense, Denmark
(2) Linkoping University, Sweden
(3) Odense University Hospital, Denmark
(4) Herlev Hospital, Denmark
(5) Lund Hospital, Sweden
(6) Herning Hospital, Denmark
(7) University of Freiburg, Germany
The standard treatment for ovarian cancer in advanced stages is surgery
followed by taxane-platin therapy. Despite an initial high response rate
most patients eventually relapse. The dose limiting toxicities of paclitaxel
are neutropenia and neuropathy but the inter-individual variability is
large. The purpose of this study was to prospectively investigate the
impact of genetic variants in key drug metabolizing/transporter genes on
paclitaxel toxicity. CYP2C8*3 and three ABCB1 variants were picked
for primary analysis and a host of other candidate genes were assessed in
92 prospectively recruited Scandinavian Caucasian patients with primary
ovarian cancer who were treated with paclitaxel (175 mg/m2) and carboplatin (AUC 5-6) after cytoreductive surgery. Clinical toxicity using
Common Toxicity Criteria for Adverse Events was assessed by a single
investigator for 97% of the patients and hematologic toxicity was registered. Patients carrying one or two variant alleles of ABCB1 C3435T
had progressively more pronounced neutrophil decrease at nadir (P-value
0.03). The same association was found for ABCB1 C1236T and
G2677T/A with P-values of 0.06 and 0.02. No statistically signicant
correlations were found for paclitaxel compliance and sensoric neuropathy. In conclusion: variants in the drug transporter ABCB1 gene impact
on the neutrophil suppressing effect of paclitaxel in patients with ovarian
cancer. To our knowledge this has never been shown before directly in a
prospective study; but a similar result was reported in 18 patients by Sissung et al.(Eur J Cancer 2006;42:2893-6). This novelty has implications
for the understanding of myelosuppression in particular and for tailored
chemotherapy in general.
(1) Karolinska Institute, Neonatal Research Unit Q2:07, Woman and

Child Health, Stockholm, Sweden
rebro University, School of Health and Medical Sciences, O
rebro,
(2) O
Sweden
(3) University of Tennessee, Department of Pediatrics, Memphis, TN,
USA
(4) University of Leicester, Department of Health Sciences, Leicester,
UK
(5) Hospital for Children and Adolescents, Helsinki, Finland
(6) University of Edinburgh, Royal Inrmary of Edinburgh, Neonatal
Unit, Edinburgh, UK
(7) University Hospital Antwerp, Antwerp, Belgium
(8) Friedrich-Alexander Universitdt Erlangen-Nurnberg, Erlangen,
Germany
(9) Uppsala University, Uppsala, Sweden
(10) Lund University, Lund, Sweden
(11) Erasmus universitair Medish Centrum, Rotterdam, The Netherlands
(12) European Paediatric Medicine Company, Brussel, Belgium
(13) Karolinska Institutet, Clinical Pharmacology, Stockholm, Sweden
(14) Hopital Trousseau, Paris, France
Alleviation of pain is a basic and human right regardless of age. Awareness that newborn infants experience pain during surgery, mechanical
ventilation and invasive procedures has increased over the last 20 years.
Despite the accumulating research and various international and national
guidelines for neonatal pain management, there is no recent information
about how this knowledge translates into clinical practice. The NeoOpioid Consortium is a research network with 12 partners supported by
European Commissions Framework Programme 7, Better Use of Medicines. That aims to improve the safety and efcacy of opioid treatment
in critically ill newborns. The EUROPAIN survey, part of the NeoOpioid-project, designed to collect data of neonatal pain management in different countries. Neonatal units in all European countries will be invited
to participate. For ventilated infants, data will be collected about administration of analgesic and sedative medications during a 2-week period.
Documented and reported strategies of pain relief measures will be compared with local and national guidelines. Computerized tutoring material
and data entry systems will facilitate high quality data collection. EUROPain survey will provide units with unique opportunities to compare their
analgesic practices with other units nationally and European basis.
Results will provide a scientic foundation for the development of evidence-based European neonatal pain management guidelines, estimate
the eligible neonatal populations for randomized trials, explore correlations between the burden of neonatal pain and long-term cognitive outcomes, and stimulate drug development specically targeted for
analgesia in children. Research funding from the European Communitys
Seventh Framework Programme, grant agreement no. 223767.
Paper No.: 1672

DISEASES
ANTI-INFLAMMATORY AND ANTIOXIDANT ACTIVITIES OF
AN INDIAN MEDICINAL PLANT, EMBLICA OFFICINALIS, IN
RODENT MODELS OF INFLAMMATION
Jagriti Bhatia, M Golechha, DS Arya, F Tabassum
New Delhi, India
Paper No.: 2081

EUROPAIN EUROPEAN PAIN AUDIT IN
NEONATES NEOOPIOID CONSORTIUM
Lena Bergqvist(1), M Eriksson(2), KJS Anand(3), EM Boyle(4),
H Lagercrantz(1), S Andersson(5), G Menon(6), B van Overmeire(7),
M Keller(8), L Hellstrom-Westas(3) V Fellman(4) RHN van Schaik(11),
J-P Osselaere(12), A Rane(13), J Schollin(2), R Carbajal(14)
Introduction: Emblica ofcinalis has been reported to possess high antioxidant activity. However, the anti-inammatory activity of its fruits has
not been studied earlier. Hence, in the present study the anti-inammatory activity of the standardized hydroalcoholic extract of the fruits of
Emblica ofcinalis (HAEEO) in acute and chronic models of inammation was evaluated. Methods: The acute inammation in rats was induced
by subplantar injection of carrageenan, histamine, serotonin and prostaglandin E2 respectively. Further, the histopathological examination and
assessment of the biochemical markers of oxidative stress was done in
the carrageenan group. Cotton pellet granuloma model was used to
199
evaluate the effect of the HAEEO on chronic inammation. Results: In
the acute model of inammation it was observed that the administration
of HAEEO (300, 500, and 700 mg/kg, i.p.) signicantly and dose-dependently inhibited paw edema induced by all the phlogistic agents. The
HAEEO also signicantly increased glutathione, superoxide dismutase
and catalase activity and reduced the levels of malondialdehyde. Also,
HAEEO at the dose of 500 and 700 mg/kg produced a signicant
decrease in the amount of cellular inltrate and subcutaneous edema
induced by carrageenan. In the cotton pellet granuloma model HAEEO
at all the doses tested signicantly (P < 0.001) reduced the granuloma
formation. Conclusion: The results obtained indicate the HAEEO
possesses signicant anti-inammatory activity in all the models. Thus,
HAEEO may hold therapeutic promise in the management of inammatory conditions.
Paper No.: 1673

STUDY OF THE ANTIHYPERTENSIVE POTENTIAL OF
INDIAN MEDICINAL PLANT, EMBLICA OFFICINALIS, IN A
RAT MODEL OF HYPERTENSION
Jagriti Bhatia(1), F Tabassum(1), DS Arya(1), S Joshi(1), AK Srivastava(2)
We have previously shown that serum estradiol concentrations are

halved in smoking as compared to non-smoking postmenopausal
women during estrogen-progestogen therapy. These studies included
unselected smoking women. We now wished to evaluate if light smoking inuences estradiol kinetics. In addition, we aimed to study acute
effects of tablet intake in smokers versus non-smokers. We used a posthoc strategy in two double-blind randomized trials of estrogen-progestogen treatments in postmenopausal women. Study I: In a 2-year study of
129 women (94 completed) who received either 1mg estradiol continuously combined with 125lg trimegestone or placebo, we compared
smokers (18) to non-smokers (76). Smoking more than 10 cigarettes
per day precluded participation. Serum estradiol and estrone were measured at 0, 3, 6, 12, 18 and 24 months. Multiple linear regression (xed
effects model) showed that smoking women had signicantly lower
serum estradiol (p = 0.0041) and estrone (p = 0.0093) concentrations
compared to non-smokers. Study II: In a subset from a study comparing
combinations of gestodene and 1 or 2mg estradiol in 278 postmenopausal women (153 completed) unselected for smoking status, we studied 2-hour serum estradiol and estrone at 1, 6 and 12 months. We
found that smoking reduced hormone concentrations on both regimens,
for example at 12 months, serum estrone was 157 + /-18 vs 98 + /17 pg/ml in non-smoking versus smoking women at 2 hours (p = 0.04).
In conclusion, we have shown that both heavy and light smoking
reduces serum estradiol and estrone in postmenopausal women undergoing estrogen-progestogen therapy. The effect is seen already within
2 hours after tablet intake.
(1) All India Institute of Medical Sciences, Department of Pharmacology,

New Delhi, India
(2) All India Institute of Medical Sciences, Department of Laboratory
Medicine, New Delhi, India
Introduction: Emblica ofcinalis (EO) has been reported to possess good
antidiabetic, hypolipidemic, cardioprotective and antioxidant activity.
However, the potential of EO in preventing the development of hypertension has not been studied so far. Hence, the present study was designed
to evaluate whether EO has antihypertensive potential in the DOCA-salt
induced model of hypertension in rats. Methods: The DOCA-salt
(20 mg/kg, s.c.) twice weekly, along with 1% NaCl solution substituted
for drinking water, was administered for 5 weeks to induce hypertension
in rats. EO (75, 150 and 300 mg/kg, orally) was concurrently administered with DOCA-salt in rats for 5 weeks. The effect of EO on haemodynamic and biochemical parameters as well as serum nitric oxide and
serum electrolyte (sodium and potassium) levels were determined. The
blood pressure was recorded non-invasively by the tail-cuff method.
Results: EO signicantly and dose-dependently prevented the rise in systolic, diastolic and mean arterial blood pressure and also the heart rate as
compared to the DOCA-salt group. A signicant reduction in malondialdehyde and increased content of glutathione and superoxide dismutase
were observed in the rat heart. Furthermore, EO maintained serum nitric
oxide, sodium and potassium levels as compared to DOCA-salt group.
The maximal signicant effect on all the study parameters was observed
at the dose of 300 mg/kg. Conclusion: EO prevents the development of
hypertension in the DOCA-salt model. This benecial effect may be due
to its antioxidant property.
Paper No.: 1010

BOTH HEAVY AND LIGHT SMOKING REDUCES SERUM
ESTRADIOL AND ESTRONE IN POSTMENOPAUSAL WOMEN
DURING ESTROGEN-PROGESTOGEN THERAPY
Nina Hannover Bjarnason(1), H Jorgensen(2), C Christiansen(3)
(1) Institute for Rational Pharmacotherapy, Danish Medicines Agency,
Copenhagen, Denmark
(2) Department of Clinical Biochemistry, Bispebjerg Hospital, Copenhagen, Denmark
(3) Center for Clinical & Basic Research, Ballerup, Denmark
Paper No.: 3245

CONSUMPTION OF DRUGS AGAINST ADHD IN DENMARK
MORE THAN TEN DOUBLED IN TEN YEARS
Nina Hannover Bjarnason(1), H Jorgensen(2), C Christiansen(3)
(1) Institute for Rational Pharmacotherapy, Danish Medicines Agency,
Copenhagen, Denmark
(2) Department of Clinical Biochemistry, Bispebjerg Hospital, Copenhagen, Denmark
(3) Center for Clinical & Basic Research, Ballerup, Denmark
We have previously shown that serum estradiol concentrations are
halved in smoking as compared to non-smoking postmenopausal
women during estrogen-progestogen therapy. These studies included
unselected smoking women. We now wished to evaluate if light smoking inuences estradiol kinetics. In addition, we aimed to study acute
effects of tablet intake in smokers versus non-smokers. We used a posthoc strategy in two double-blind randomized trials of estrogen-progestogen treatments in postmenopausal women. Study I: In a 2-year study of
129 women (94 completed) who received either 1mg estradiol continuously combined with 125lg trimegestone or placebo, we compared
smokers (18) to non-smokers (76). Smoking more than 10 cigarettes
per day precluded participation. Serum estradiol and estrone were measured at 0, 3, 6, 12, 18 and 24 months. Multiple linear regression (xed
effects model) showed that smoking women had signicantly lower
serum estradiol (p = 0.0041) and estrone (p = 0.0093) concentrations
compared to non-smokers. Study II: In a subset from a study comparing
combinations of gestodene and 1 or 2mg estradiol in 278 postmenopausal women (153 completed) unselected for smoking status, we studied 2-hour serum estradiol and estrone at 1, 6 and 12 months. We
found that smoking reduced hormone concentrations on both regimens,
for example at 12 months, serum estrone was 157 + /-18 vs 98 + /17 pg/ml in non-smoking versus smoking women at 2 hours (p = 0.04).
In conclusion, we have shown that both heavy and light smoking
reduces serum estradiol and estrone in postmenopausal women undergoing estrogen-progestogen therapy. The effect is seen already within
2 hours after tablet intake.
200
Paper No.: 2811
REEP FAMILY MEMBERS MODULATE A2C-ADRENOCEPTOR
EXPRESSION BY AN ER INTERACTION
Susann Bjork(1,2), C Hurt(3), B Kobilka(2), T Angelotti(3)
(1) University of Turku, Department of Pharmacology, Drug Development and Therapeutics, Turku, Finland
(2) Stanford University, Department of Molecular and Cellular Physiology and Medicine, Stanford School of Medicine, Palo Alto, CA, USA
(3) Stanford University, Department of Anesthesia, Stanford University
Medical Center, Palo Alto, CA, USA
As regulators of sympathetic neurotransmitter release, a2A- and a2Cadrenoceptors (ARs) exhibit differential neuronal localization. In nonneuronal cell lines, a2A-ARs target to the plasma membrane (PM) as
opposed to a2C-ARs; but in neuronal cells, also a2C-ARs target to the
PM. We investigated whether accessory Receptor Expression Enhancing
Proteins (REEPs) affect subcellular localization to modulate a2A- and
a2C-AR expression. RT-PCR analysis of cell lysates revealed REEP1,
REEP2 and REEP6 mRNA expression in neuronal but not non-neuronal
cells. A developmental pattern of REEP expression was seen in cultured
sympathetic ganglion neurons. Immunocytochemical staining of surface
and total expression in HEK293 kidney cells revealed co-localization of
a2C-ARs with transfected REEPs in the endoplasmic reticulum (ER).
Transfected REEPs appeared to enhance cell surface expression, but
because of an increase in total a2C-AR expression and not by a selective
increase in PM trafcking (shown by FACS). A direct interaction of
REEPs and receptors was suggested as HA-tagged a2C-ARs
co-expressed with either Flag-REEP1, Flag-REEP2 or Flag-REEP6 were
co-immunoprecipitated with an anti-Flag antibody. Sucrose gradient analysis of REEP expression demonstrated ER fraction localization, without
expression in the Golgi or PM fractions. Finally, glycosylation analysis
revealed that the increase in a2C-AR surface expression was not associated with an increase in mature glycosylation. Thus, enhanced cell
surface expression of a2C-ARs in neuronal cells may be due to effects of
REEP proteins. However, REEP co-expression does not enhance trafcking to the PM. Instead, REEPs and receptors interact in the ER, REEPs
possibly acting as chaperones.
Paper No.: 2720

FOCUSED CONFERENCE GROUP: P19 - GENERAL SESSION HEPATOLOGY
LOVASTATIN PREVENTS CARCINOGENESIS IN A RAT
MODEL FOR LIVER CANCER. EFFECTS OF UBIQUINONE
SUPPLEMENTATION
Linda Bjorkhem-Bergman(1), J Acimovic(2), U-B Torndal(3), P Parini(4),
L Eriksson(3)
(1) Karolinska Institute & Karolinska University Hospital Huddinge,
Division of Clinical Pharmacology, Department of Laboratory Medicine,
Stockholm, Sweden
(2) University of Ljubljana Faculty of Medicine, Institute of Biochemistry, Ljubljana, Slovenia
Division of Clinical Pathology, Department of Laboratory Medicine,
Stockholm, Sweden
Division of Clinical Chemistry, Department of Laboratory Medicine,
Stockholm, Sweden
Statins (HMGCoA reductaste inhibitors) inhibit the mevalonate pathway and efciently reduce cholesterol synthesis. Several studies have
shown that statins may inhibit carcinogenesis although the mechanisms
are not fully understood. Ubiquinone, an antioxidant synthesized in the

mevalonate pathway, is known to be important for cell proliferation
and is increased in preneoplastic liver tissue. The synthesis of ubiquinone is also inhibited by statins and we tested the hypothesis that a
statin-induced effect on carcinogenesis may be mediated by the inhibition of ubiquinone synthesis. We studied the effects of lovastatin treatment given orally with and without addition of ubiquinone in a rat
model for chemically induced hepatocarcinogenesis. The volume fraction of preneoplastic liver tissue was determined, the proliferation of
the cells within the liver tissue was measured and different components
of the mevalonate pathway were monitored. Lovastatin treatment
reduced the volume fraction of liver nodules by about 50% and the
proliferation of the cells within the liver nodules was reduced to one
third. Ubiquinone treatment reversed the statin-induced inhibition of
cell proliferation but did not affect the volume fraction of the liver
nodules. The lathosterol levels were signicantly reduced in the statin
treated rats, indicating inhibition of the mevalonate pathway. In spite
of this, the cholesterol levels were not affected. In conclusion, lovastatin prevents carcinogenesis in a rat model for liver cancer, despite
no change in cholesterol levels. The statin-induced inhibition of cell
proliferation might, at least in part, be explained by the inhibition of
ubiquinone synthesis.
Paper No.: 2721

STATIN TREATMENT REDUCES MORTALITY IN SEVERE
INFECTIONS A SYSTEMATIC REVIEW AND
META-ANALYSIS
Linda Bjorkhem-Bergman(1), P Bergman(2), J Andersson(3), J Lindh(1)
Division of Clinical Pharmacology, Department of Laboratory Medicine,
Stockholm, Sweden
Division of Clinical Microbiology, Department of Laboratory Medicine,
Stockholm, Sweden
Division of Infectious Diseases, Department of Medicine, Stockholm,
Sweden
Several studies have reported improved survival in severe bacterial
infections among statin treated patients. In addition, statins have been
ascribed benecial anti-inammatory effects. The aim of this study was
to evaluate the effect of statin-treatment on mortality in patients with
severe bacterial infections, by means of a systematic review and a
meta-analysis. Studies investigating the association between statin use
and mortality in patients with severe bacterial disease were identied in
a systematic literature review and a meta-analysis was performed to
calculate the overall OR of mortality in statin users. The literature
search identied 552 citations from which 35 relevant studies were
extracted. In all, 17 studies comprising 114 572 patients were included
in the nal analysis. Statin use was associated with a signicantly
(p < 0.0001) reduced mortality in patients suffering from severe bacterial infections (OR 0.53, 95% CI 0.43-0.66). However, all studies
included were of observational design and funnel plot analyses indicated a possible inuence of publication bias (Eggers bias test
p < 0.05). When a precision estimate test was used to adjust for publication bias, the overall OR was reduced to 0.76 (95% c.i. 0.58-1.01).
In conclusion, patients that suffer from severe bacterial infections have
a better outcome if they are on current statin treatment, but the magnitude of this proposed statin effect may have been inated by publication bias. Nevertheless, our data indicate that statin treatment safely can
be continued during severe infections. There is a need for randomised
controlled trials investigating the possible benecial effect of statintreatment in bacterial infections.
201
Paper No.: 1512
HEALTH AND DISEASE
ORAL SILDENAFIL INCREASES SODIUM NITROPRUSSIDE
IONTOPHORESIS INDUCED SKIN HYPERAEMIA IN
HEALTHY VOLUNTEERS
S Blaise, M Hellmann, M Roustit, S Isnard, Jean-Luc Cracowski
Grenoble University Hospital, Inserm CIC3, Grenoble Clinical Research
Center, Grenoble, France
Sildenal, a specic PDE5A inhibitor, is currently tested as a treatment
for severe Raynauds phenomenon. The main objective was to test
whether sildenal, alone or associated with local sodium nitroprusside
(SNP) delivered through skin iontophoresis, increases forearm cutaneous
blood conductance in healthy volunteers. Ten healthy volunteers were
enrolled. Variations in cutaneous vascular conductance following oral
administration of 50 mg or 100 mg of sildenal associated or not with
sodium nitroprusside iontophoresis were expressed as a percentage of
maximal cutaneous vascular conductance and were monitored using laser
Doppler imaging. SNP iontophoresis was performed on the ventral face
of the forearm, one hour after application of lidocaine/prilocaine cream.
Results. Sildenal at 100 mg, but not 50 mg, increased SNP iontophoresis area under the curve (+ 44%, P = 0.03 versus without sildenal) and
increased peak SNP iontophoresis (+29%, P = 0.05). Sildenal at
100 mg, but not 50 mg, increased baseline cutaneous vascular conductance (+ 75%, P = 0.03). Incidence of headache was not different when
SNP iontophoresis was associated with sildenal. One symptomatic arterial hypotension occurred in one volunteer with 50 mg sildenal. Conclusions. Oral sildenal at 100mg potentiates local skin SNP
iontophoresis-induced hyperaemia, with no increased incidence of headaches. The association of oral specic PDE5A inhibitor and nitrates
administered through skin iontophoresis should be further investigated in
diseases such as severe Raynauds phenomenon, with particular attention
to the incidence of arterial hypotension.
Clinicaltrials.gov (NCT00710099)
Paper No.: 2798

DRUGS USE IN PAEDIATRIC POPULATION. EVALUATION IN
TWO AREAS: NEONATOLOGY AND INTENSIVE CARE UNITS
neonatology unit the most frequent reason for the classication of UL

was the use of preparations manufactured (home-label medications) or
modied by the hospital pharmacy (caffeine, dexamethasone or spironolactone). Conclusion: This study shows a high rate of UL and OL drug
use in pediatrics. Regulatory authorities and the pharmaceutical industry
could be the focus for improving the availability of appropriate authorized medicines for children.
Paper No.: 2799

OFF-LABEL VS COMPASSIONATE DRUG USE: LEVEL OF
EVIDENCE FOR APPLICATIONS
Encarnacion Blanco(1), A Munoz(2), I Bellido(1), M Salazar(2),
EI Marquez(1)
(2) Virgen de las Nieves General Hospital of Granada, Spain
Introduction: Some times compassionate drug use can also very confusing: permission to treat a single patient with an unapproved drug outside
of a trial versus the off label utilization. Methods: A cross-sectional study
was undertaken for the compassionate use applications in a general hospital of Granada (Spain). Results: The most commonly prescribed classes
of drug were (% of drugs): L group (50%), B group (13.1%), J group
(8%), and H group (8%), meanwhile B group dominated the number of
applications (45.5%) followed by L group (36.8%). The reasons for compassionate use requests were: access to an unapproved drug or real compassionate use (6 new drugs, 18% of medicinal products) and the
utilization of un authorised medicinal product for an indication, population or route of administration different from the one mentioned in the
SmPC or off label use (82% of drugs). The 46% of petitions was based
on somewhere randomized clinical trial meanwhile the rest had a low
level of evidence (open studies, case reports). Finally, after two years,
half the investigational new drugs are authorized (bevacizumab, trabectedine, tipranavir), while only the 21% of different indications and the
8.3% of different use forms has been approved. There are ten compassionate use petitions based on a high level of clinical evidence without
SmPC variations. Conclusions: Though the drugs selection was rational
in most of the cases, some incongruities were observed. In order to
favour a common approach, better treatment availability and more
dynamic processing, local legal basis has been recently revised.
Encarnacion Blanco(1), I Bellido(1), EI Marquez(1), A Medina(2),

JA Gonzalez-Correa(1)
(2) San Cecilio Clinical Hospital of Granada, Spain
Introduction: Paediatric insufcient drug evaluation and the lack of
adapted pharmaceutical formulations explain the importance of unlicensed (UL) and off label (OL) prescriptions. Moreover, the quality of
information available on formulation and stability is limited, thus preparation of childrens oral medicines is subjected to much variation
between hospitals. Aim: To determine the extent of use of drugs that are
either UL or OL. Patients and Methods: A cross-sectional study including 143 children hospitalised (intensive care unit: 81, neonatology unit:
62). Results: We analysed 601 prescriptions of 91 different drugs. In
intensive care a 22.3% of children received at least one UL prescription:
half not licensed for paediatrics (amiodarone), and half by modication
to a licensed drug (nifedipine). A 87.7% children received at least one
OL prescription; the most common categorie was outside dose recommendations (dexamethasone), followed by used for non-licensed indication (alimemazine for irritability), outside licensed age range
(voriconazole for < 2 years old), and used by unlicensed route. Of the
601 prescriptions a 35% were for drugs without SPC, a 28% for drugs
prescribed in agreement with the SPC, and a 37% were UL/OL. In
Paper No.: 3150

IMMUNOPHARMACOLOGY: NEW TOOLS FOR OLD DISEASES
AGE-DEPENDENT EFFECTS OF DEXAMETHASONE ON
HYPOXIC-ISCHEMIC BRAIN EDEMA
Gabor Blazso(1), M Pasztor(2), A Balogh(1), A Marki(1), G Dombi(2),
G Falkay(1)
(1) University of Szeged, Department of Pharmacodynamics and Biopharmacy, Szeged, Hungary
(2) University of Szeged, Department of Pharmaceutical Analysis, Szeged, Hungary
Objective: Therapeutic value of glucocorticoids in the prevention of hypoxic-ischemic brain edema is still a subject of debate. The aim of the
present study was to examine whether the neuroprotective effect of dexamethasone depends on the degree of maturity of the brain. Method: 1-,
2-, 4- and 12-week-old rats were subjected to unilateral carotid artery
occlusion plus hypoxia in 8% oxygen. The effects of dexamethasone, the
antiglucocorticoid mifepristone and their combination on the cytotoxic
and vasogenic phases of hypoxic-ischemic brain edema were measured
202
in neonate and adult rats. The effects of unarisine and verapamil on the
survival of the animals was also examined alone or in combination with
dexamethason. All the drugs were administered prior to hypoxia-ischemia. Results: Dexamethasone inhibited both the cytotoxic and the vasogenic phases; this inhibition could be antagonized with mifepristone in
neonate rats. Opposite results were observed in 12-week-old animals,
where the glucocorticoid increase of these phases could not be prevented
with mifepristone. In adult rats, dexamethasone increased the Ca2 + content of the hypoxic-ischemic brain hemisphere and the death of the animals. This toxic effect could be antagonized with unarisine and
verapamil. Tritiated dexamethasone was able to penetrate into the hemispheres of 1- or 2-week-old rats, but signicantly less into the brain of 4or 12-week-old animals. This effect lasts until postnatal day 24, when
the brain blood barrier is fully developed. Conclusion: The present ndings support the hypothesis that there is a strong dependence on age corresponding to dexamethasones penetration into the brain.
Paper No.: 3105

CRIP1A REGULATES CB1 CANNABINOID RECEPTOR
INTERNALIZATION AND SIGNAL TRANSDUCTION
Lawrence C Blume(1), CC Bass(1), GD Dalton(1), DE Selley(2), A
Howlett(1)
(1) Wake Forest University Health Sciences, Department of Physiology
and Pharmacology, Winston-Salem, USA
(2) Virginia Commonwealth University, Richmond, USA
CRIP1a (Cannabinoid Receptor Interacting Protein 1a) is an accessory
protein for the CB1 cannabinoid receptor, whose function is as yet
unclear (Neihaus JL et al.,Mol Pharmacol 2008;72:1557-1566). We prepared clones of the N18TG2 neuroblastoma cell that stably overexpress
CRIP1a at 3- to 5-fold greater mRNA levels than WT cells. In CRIP1aoverexpressing clones, CB1 receptor density on the plasma membrane is
modestly reduced compared with WT cells, when examined using immunostaining for surface receptors using an N-terminally-directed CB1 antibody with quantitative detection using the LI-COR Odyssey. In response
to the agonist (WIN55212-2)-stimulation, internalization of CB1 receptors occurs in WT cells, but not in CRIP1a-overexpressing cells. Upon
continued exposure to agonist, cell surface CB1 receptor density was
recovered within minutes in the WT perhaps by externalization, a process
that is also observed in CRIP1a-overexpressing clones. Upon exposure
to the CB1 antagonist rimonabant, the density of cell-surface CB1 receptors increased in both WT as well as CRIP1a-overexpressing cells. Under
conditions in which 2-AG synthesis is blocked, CB1-mediated ERK
phosphorylation in WT N18TG2 cells could be reduced by rimonabant,
consistent with constitutive activation of signal transduction. In the
CRIP1a-overexpressing clones, basal pERK levels were low compared
with WT, and rimonabant had no effect, suggesting the absence of the
constitutive activation. Agonist-stimulated ERK phosphorylation
occurred to the same extent in both WT and CRIP1a-overexpressing
clones. These ndings suggest that the function of CRIP1a may be to
regulate internalization-associated signal transduction events.
Supported by R21-DA025321, R01-DA03690, K01-DA024763, T32DA007246, K01-DA24763
Paper No.: 667

HEALTH AND DISEASE
VASCULAR AT1/AT2 RECEPTOR RATIO IMPACT ON
VASOCONSTRICTION IN EXPERIMENTAL GESTATIONAL
DIABETES
Pregnancy and diabetes mellitus are conditions that separately modify

the vascular response to Angiotensin II(Ang II) by changing AT1/AT2
receptor (R) ratio of expression. We reach to establish the impact of
gestational diabetes (GDM) on AT1/AT2 R and Ang II induced vasoconstriction. Using a conventional isolated-organ system, concentration
response curves to Ang II were constructed using thoracic aortas with
(e+) and without (e-) endothelium from term pregnant (day 20) and
non pregnant rats. Animals received streptozotocin 60 mg/kg ip, an
hypercaloric diet or none. Both pregnancy and experimental diabetes
reduced Ang II vasoconstriction mainly on e+ vessels. The response
was signicantly increased in bboth models of GDM. Losartan
(10-8M) reduced the response to Ang II of the gestational diabetes
e- group, but did not affect e+ aorta rings. AT1R expression increased
in the GDM group. In contrast, increased levels of AT2R were found
in the pregnant and in the diabetic groups, while no change was
observed in the GDM group. These results suggest that pregnancy
and short term exposure to diabetes increase endothelium derived
vasodilators release probably through AT2R, which effects are masked
by an increased AT1R/AT2R ratio when the two conditions are present. This imbalance becomes evident mainly in endothelium intact
vessels.
Paper No.: 2919

DISEASES
THE DIFFERENCES OF ANTIULCER ACTIVITY OF PROTON
PUMP INHIBITOR (PPI) AND H2-ANTAGONIST IN DIABETIC
RATS
Teresa Bobkiewicz-Kozlowska(1), R Forjasz(1), M Nowaczyk(0)
(1) Poznan University of Medical Sciences, Department of Pharmacology, Poznan, Poland
(2) Poznan University of Medical Sciences, Department of Pharmaceutical Technology, Poznan, Poland
Diabetic patients develop a variety of gastrointestinal symptoms
(abdominal pain, vomiting, diarrhea, constipation, delayed gastric emptying). Little attention has been paid to the incidence and healing rate
of peptic ulcer in diabetics, however recent studies indicate that ulcers
are more severe and often associated with complications such as gastrointestinal bleeding. Previous studies documented that streptozotocin
(STZ)-induced diabetes in rats is an accepted model of insulin-dependent diabetes. Experiments were performed on rats with experimental
diabetes. Acute gastric stress ulcers were developed using waterimmersion restraint stress. Ranitidine or pantoprazol (40 mg/kg i.g. or
3 mg/kg i.g., respectively) were administered 30 min before stress.
Severity of gastric mucosa injury was revealed as an ulcer index (UI).
Chronic gastric ulcers were induced using Okabe and Pfeiffer method
with the Konturek modication. Starting on the ulcer induction day,
ranitidine (40 mg/kg i.g., twice a day) or pantoprazol (3 mg/kg i.g.,
twice a day) were administered for 7 days. Tissue concentration of
cytokines TNF-a and IL-1b in gastric mucosa was measured using
ELISA. We concluded that: 1. gastric mucosa is more sensitive to
ulcerogenic stress, but chronic ulcer healing is not impaired in experimental diabetes, however the increase of proinammatory cytokines
tissue level is observed. 2. H2-antagonist and PPI (H2-antagonist
>PPI) protect the gastric mucosa against acute stress ulcer but do not
inuence on self-healing chronic ulcers in diabetic rats. 3. in gastroprotective effect of H2-antagonist lowering TNF-a and IL-1b tissue
concentration can be involved.
Rosa A Bobadilla, C Touno, E Fernandez-Vallin, P Lopez

ESM, Instituto Politecnico Nacional, Mexico City, Mexico
203
Paper No.: 730
RATIONAL DRUG CORRECTION OF THE SYSTEMIC
INFLAMMATORY RESPONSE SYNDROME IN PATIENTS
WITH CONGESTIVE HEART FAILURE
Tamar T Bochorishvili, MA Rogava, ET Tsiwtsiwadze
N. Kipshidze National Centre of Therapy, Department of Cardiomypathy,
Tblisi, Georgia
Introduction: The SIRS which accompained severe form of CHF are
associated with activation of a number of complex and interrelated pathways that include the endotoxemia, tissue hyperoxia and activity of the
intrinsic defense system. The purpose of the present study was to test the
hypothesis that inotropic drug with pronounced antihypoxic/antiischemic
and antioxidant properties, adenocin, would signicantly reduce inammatory signalling and cardiac dysfunction. Materials/Patients: Plasma
levels of cytokines (interleukine-6, interleukine -8, TNF-a and soluble
adhesion molecules, redox potential NAD/NADH, superoxide anion production, activities of superoxide dismutase and catalase were determined
in samples obtained from 78 patients with CHF NYHA classes II-IV,
caused by ischemic heart disease in 65 and dilated cardiomyopathy in 13
patients. All patients including in the study were randomized into 2
groups, control received standard therapy and the main additionally
received adenocin. Results: All biological markers of the intensity of
endotoxemia and oxidative stress, immune inammatory status, redoxpotential, considerably improved in patients of the main group. The signs
of SIRS disappeared in main group, but persisted in patients of control
group. The improvement of the symptoms of SIRS in main group was
coupled with the increasing of ejection fraction by 12%, and NYHA
functional class by the 37% (only 10% in the control group). Conclusion:
The treatment with adenocin cessates the systemic hypoxic and inammatory syndrome, improves cardiac hemodynamics and symptoms of
congestion in patients with manifested form of CHF.
Paper No.: 1934

THE STUDY OF ABCB1 POLYMORPHISMS IN ROMANIAN
EPILEPTIC PATIENTS
Paper No.: 3252

GENDER DIFFERENCE IN THE IMPACT OF LONG-TERM
PSYCHOACTIVE DRUG USE ON COGNITIVE FUNCTIONING:
THE VISAT COHORT
O Boeuf-Cazou(1), B Bongue(2), D Ansiau(3), J-C Marquie(4), Maryse
Lapeyre-Mestre(1)
(1) Universite de Toulouse; UPS, Faculte de Medecine Purpan, Unite de
Pharmacoepidemiologie EA3696, Toulouse, France
(2) CETAF, Centre technique dAppui et de Formation des Centres
dExamens de Sante, Saint-Etienne, France
(3) International University of Monaco, Principality of Monaco, Monte
Carlo
(4) Universite de Toulouse II, Laboratoire Travail et Cognition, UMR
5263 CNRS5, Toulouse, France
Introduction: Few studies have investigated long-term effects of psychoactive drug use among a population aged less than 50. The aim of this
study was to underline the impact of psychoactive drug use on cognitive
functions in a population of workers according to gender. Materials/
Patients: This study included 1,251 French workers from the VISAT
(Aging, Health and Work) cohort whose the objective was to underline
the long-term impact of working conditions on health and aging. VISAT
study included initially 3,237 subjects (current or recently retired workers) born in 1934, 1944, 1954 or 1964 at baseline. Data were collected
through interviews during the annual medical examination by occupational physicians in 1996, 2001 and 2006. Cognitive function was
assessed through ve cognitive tests (immediate free recall test, delayed
free recall test, recognition test, Digit Symbol Substitution Subtest and
visual search speed test). Cognitive scores obtained after a 10-year follow-up were investigated among three categories of psychoactive drug
(hypnotic, anxiolytic and antidepressant) users: none (82.1%), long-term
(9.5%) and occasional (8.4%) with analysis of covariance models
adjusted for several potential confounders. Results: In men, we found an
alteration in delayed recall and recognition tests among anxiolytic users,
and an alteration in the recognition test and the digit symbol substitution
subtest among antidepressants users. In women, we observed an alteration in the selective attention test among hypnotic users. Conclusion:
Long-term use of psychoactive medication could impair different cognitive functions depending on gender and type of drugs.
Ioana Corina Bocsan, AD Buzoianu, FC Militaru, R Popp, A Trifa,

LP Dumbrava, E Brusturean
University of Medicine and Pharmacy, Department of Clinical Pharmacology, Cluj Napoca, Romania
Introduction. Epilepsy is one of the most common, chronic neurological
disorders, affecting approximately 42 million patients worldwide. Even
with more than 15 antiepileptic drugs (AEDs), 30% of patients are still
resistant to AEDs. The pharmacokinetic hypothesis for the refractory epilepsy, postulates that resistance is caused by over expression of multidrug
transporter proteins at the blood brain barrier level, possibly as a result
of active efux mediated by locally over expressed drug transporter proteins. Objective. The aim of this study is to determine the genotype distribution and the allele frequency of the ABCB1 variants in a Romanian
epileptic patients group and compare it with the control group. Methods.
Based on the PCR-RFLP technique, 4 variants of the ABCB1 gene were
studied in 64 epileptic patients and in 200 healthy Romanian volunteers.
Results and conclusions: Our results show low frequency of ABCB1 T
129C and ABCB1 G2677A polymorphisms, only 6.2% of patients being
heterozygous for each of them, the rest of the patients are homozygous
for normal allele. The other two ABCB1 polymorphisms, respectively
2677G>T and 3435C>T, have been frequently found in the patients
group. The distribution of genotypes for ABCB1 3435C>T polymorphism is similar to that of the general population (genotypes CC, CT and
TT had a frequency of 30.8%, 50.8% and 18.4% in the group of patients
compared to 24.5%, 54% and 21.5% in the general population).
Paper No.: 3124

PRO-APOPTOTIC AND ANTI-PROLIFERATIVE MECHANISMS
OF STW 5
G Bonaterra(1,2), S Zugel(2), W Hildebrandt(2), Olaf Kelber(3), D Weiser(3), J Metz(4), R Kinscherf(2)
(1) Section Macroscopic Anatomy, Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany
(2) Department of Medical Cell Biology, University Marburg, Marburg,
Germany
(3) Scientic Department, Steigerwald Arzneimittelwerk GmbH, Darmstadt, Germany
(4) Anatomy and Cell Biology III, University of Heidelberg, Heidelberg,
Germany
Introduction: STW 5 is widely used in the treatment of gastrointestinal
disorders, including functional dyspepsia and colon irritabile, diseases for
which an inammatory etiology is discussed. Our objective was to determine anti-proliferative and pro-apoptotic effects of this combination of
nine plant extracts on colon adenocarcinoma cells (HT-29) in comparison
with aspirin (ASA) or diclofenac (Diclo), for which a reduction of colon
carcinoma risk is known. Methods: HT-29 cells were treated with Diclo,
204
ASA, STW 5 or its components. Anti-proliferative effects and markers
of apoptosis were measured after 72 h. Results: STW 5 (100 `g/ml) and
some of its components induced an inhibition of proliferation by 50-60%
(ASA 0.1 mM or Diclo 2,5 mM 45-50%), a 3 to 4- fold increase in
apoptosis (as well as ASA or Diclo), a 20% to 30% induction of Caspase-3 or BAX expression (ASA or Diclo revealed inhibitory effects), an
inhibition in Bcl2 and p53 mRNA expression, but no change in GDF-15
level (qPCR). Conclusion: STW 5 and some of its components might
have antiproliferative and pro-apoptotic effects on HT-29 cells in vitro.
The STW 5-induced apoptosis pathway is different from the pathway initiated by ASA or Diclo.
Paper No.: 3348

DISCOVERY
A FAST AND SENSITIVE LC-MS/MS ASSAY FOR
QUANTIFICATION OF NORTRIPTYLINE AND ITS ACTIVE
METABOLITES E- AND Z-10-HYDROXYNORTRIPTYLINE IN
HUMAN PLASMA
Gitte Bonke(1,2)*, BP Jensen(2)
(1) * Current address: University of Southern Denmark, Department of
Forensic Toxicology, Odense, Denmark
(2) University of Otago, Department of Medicine, Christchurch, New
Zealand
A fast and sensitive validated liquid chromatography-tandem mass spectrometry assay for quantication of nortriptyline and its active metabolites, E-10-hydroxynortriptyline and Z-10-hydroxynortriptyline in human
plasma following a single oral dose of 25 mg nortriptyline was needed
to support a clinical study (Paper No. 2117). Plasma samples were prepared by protein precipitation with cold methanol:acetonitrile (1:9) and
mean recoveries of 90%, 100%, and 99% were achieved for nortriptyline, E-10-hydroxynortriptyline and Z-10-hydroxynortriptyline. The compounds were separated using a Gemini-NX C18 column, 50 x 2.00 mm
i.d., 3 lm with a mobile phase consisting of 0.1% formic acid in acetonitrile with a gradient of 5-100% acetonitrile over 6 minutes and detected
by ESI+-MS/MS. The standard curves were linear in the range LLOQ
40 ng/ml (r2 0.997) with lower limits of quantication (LLOQ) at
0.2 ng/ml for nortriptyline and 0.5 ng/ml for the metabolites. Intraday
precision C.V. was 6.4% and 16% at LLOQ, interday precision C.V.
was 7.1% and 11% at LLOQ and the accuracy of the method was
within 92-114%. The matrix effect (mean S.D.) for nortriptyline was
54% 6% but the internal standard, nortriptyline-D3 corrected for this.
For the two metabolites it was 87% 3%. The assay has been applied
successfully to a clinical study (as reported in Paper No. 2117).
Paper No.: 898

WRONG; STABILIZATION OF CARDIAC FUNCTION CLINICAL
RELEVANCE OF FREE MPA MONITORING IN EARLY
PERIOD FOLLOWING HEART TRANSPLANTATION
Roselyne Boulieu(1,2), X Cussonneau(1,2), M Larger(1,2), O Bastien(2,3)
(1) University Lyon1- Faculty of Pharmacy, Department of Clinical Pharmacokinetics, Lyon, France
(2) Inserm ERI 22, Lyon, France
(3) University Lyon1, Cardiology Unit, Lyon, France
Mycophenolic acid (MPA), the active metabolite of the pro-drug mycophenolate mofetil, and the phenolic glucuronide (MPAG) are highly
bound to albumin. Scarce data are available on free MPA pharmacokinetics in heart transplantation. The aim of this study was to investigate free
MPA and free MPAG in 16 patients in the early period after heart trans-
plantation and to assess the relation between the free forms and biological data. MPA and MPAG were analyzed by HPLC. Free MPA and free
MPAG were isolated by ultraltration. AUC0-12, C0, free fraction and
Cmax were determined from full pharmacokinetic prole. Creatinine
clearance, blood cell count, hematocrit and hemoglobin were collected.
A wide interindividual variability in free MPA and free MPAG concentrations was observed. Mean absolute AUC0-12 were 0.484 mg.h/L and
691.5 mg.h/L for free MPA and free MPAG respectively. MPAG free
fraction was positively related to MPA free fraction (r = 0.814,
p = 0.001). Free MPA and free MPAG pharmacokinetic parameters were
negatively correlated to creatinine clearance. Moreover, absolute free
MPA C0 was negatively related to red blood cell count, haemoglobin
and hematocrit but not free MPA AUC0-12. This observation which
needs to be conrmed suggests that free MPA C0 could be a relevant
marker related to the myelotoxic effects of the drug and emphasize the
relevance of free MPA and MPAG monitoring in the early period following heart transplantation.
Paper No.: 899

DISEASES
MONITORING OF AZATHIOPRINE METABOLITES IN
PEDIATRIC PATIENTS WITH AUTOIMMUNE HEPATITIS
Roselyne Boulieu(1,2), TMH Nguyen(0), C Le Gall(3), A Lachaux(3)
(1) University Lyon1- Faculty of Pharmacy, Department of Clinical
Pharmacokinetics,Lyon, France
(2) Inserm ERI 22, Lyon, France
(3) University Lyon1, Cardiology Unit, Lyon, France
Azathioprine, an immunosuppressive drug commonly used in the treatment of autoimmune hepatitis (AIH) is converted to 6-thioguanine nucleotides (6-TGN), the active metabolites and to 6-methylmercaptopurine
nucleotides (6-MeMPN) via Thiopurine Methyl Transferase (TPMT).
Few data are available on drug monitoring of azathioprine metabolites in
pediatric patients. The purpose of this study was to investigate thiopurine
metabolites in children with AIH and the relevance of drug monitoring
in regard to efcacy and toxicity. Data from 28 patients with AIH treated
by azathioprine for at least 3 months were recorded. 6-TGN and 6MeMPN concentrations and TPMT activity were determined by HPLC.
Blood cell counts, liver function tests were also collected as well as clinical outcome. A wide interindividual variability in 6-TGN and 6-MeMPN
concentrations was observed in pediatric AIH patients with range values
of 51-1966 and 42-8189 pmol/8x108 RBC for 6-TGN for 6-MeMPN
respectively. 61.4% of the patients achieved remission and only 32.6%
of these children had 6-TGN values within the target range proposed for
IBD (250 450 pmol/8x108 RBC). Azathioprine dosage was signicantly related to 6-TGN and 6-MeMPN levels (r = 0.308, P < 0.001 and
r = 0.405, P < 0.001, respectively). A signicant negative correlation
was observed between 6-TGN concentrations and erythrocyte and lymphocyte counts. Thiopurine metabolite monitoring appears useful in differentiating myelotoxicity and hepatotoxicity due to azathioprine from
disease recurrence and help to identify non-compliant patients. Further
larger study are needed to conrm the optimal recommended target for
thiopurine metabolites to achieve remission in AIH patients.
Paper No.: 3322

ANTI-INFLAMMATORY AND ANTIPROLIFERATIVE
POTENTIAL OF EXTRACTS AND PURIFIED FRACTIONS
FROM MEDITERRANEAN MARINE ALGAE
Abderrahmene Bouraoui(1), W Chaouch(1), L Mhadhebi(1), A Dellai(1),
M Dridi(1), A Laroche(2), F Berrue(3), R kerr(3), J Robert(2)
205
(1) Research Unit URSAM, Laboratory of Pharmacology, Faculty of
Pharmacy of Monastir, Monastir, Tunisia
(2) Institute Bergonie, Laboratory of Pharmacology of Anticancer Drugs,
Bordeaux, France
(3) UPEI, Marine Natural Products Laboratory and Discovery, Charlottetown, Canada
As part of our search for new anti-inammatory or anticancer potential
drugs, organic extracts and puried fractions of marine algae collected
from Mediterranean Tunisia coasts were screened and evaluated for their
pharmacological potential. The present study has established that the
organic extracts of the brown algae of the genus Cystoseira showed a
strong cytotoxic activity against three human cancer cell lines (A-549,
MCF-7, HCT-15), using a MTT cytotoxic assay. At concentrations of 0.1
to 1 mg/ml these extracts suppressed, dose dependently, the proliferation
of the three cell lines by more than 75%. The IC50 values ranged from
40 to 80 lg/ml. We also established that the organic extracts and puried
fractions of the brown algae of the genus Dictyopteris, cystoseira and of
the red algae of the genus Hypnea at different doses (25, 50, 100 mg/kg)
showed a signicant anti-inammatory activity, using the carrageenan
paw edema test in male Wistar rats and in comparison to reference drugs:
dexamethasone (1 mg/kg) and aspirin (300 mg/Kg). The inhibitory effect
on the rats paw oedema is dose dependently and the % inhibition of
oedema 3 h after carrageenan injection ranged from 60 to 85%. In addition to these pharmacological activities, some of puried fractions
showed a strong inhibitory activity against PTP1B: the IC50 ranged
from 5 to 10 lg/ml. In order to isolate and identify the active substances
responsible for these pharmacodiversities of marine algae extracts
and fractions, chemical studies (HPLC, LC /MS and NMR) are under
investigation.
Paper No.: 2349

THE HPA AXIS, CARDIO-VASCULAR SYSTEM AND
BEHAVIOR IN RAT CMS MODEL OF DEPRESSION
Elena V Bouzinova(1), S Christiansen(1), K Henningsen(1), R Palme(2),
O Wiborg(1)
(1) Aarhus University Hospital Risskov, Center for Psychiatric Research,
Aarhus, Denmark
(2) University of Veterinary Medicine, Department of BiomedicalSciences/Biochemistry, Vienna, Austria
Introduction: The Chronic Mild Stress is a well-established and validated
model to develop depression-like symptoms in rodents. In the current
study we have used a rat CMS model to associate the function of the
stress axis with behavioral and physiological parameters. Methods: Rats
are exposed to unpredictable, mild stressors for 8 weeks. Stress
responses, like depression associated anhedonia-like symptoms, are measured by intake of palatable sucrose solution. Already after one week of
stress exposure rats divide in two groups: stress-susceptible, i.e. anhedonic-like, are declining in sucrose intake compared with baseline level
and stress-resilient rats. The activity of HPA axis was measured as faecal
corticosterone metabolites (FCM) in diurnal samples. Different kinds of
behaviors have been screened in open eld, elevated O-maze, shuttle
box, and Y-maze. The tail occlusion method has been used to measure
the blood pressure and heart rate in CMS rats. Results: Signicantly
higher FCM concentrations were observed in CMS animals from rst up
to fourth week of stress. Afterwards FCM returned to the baseline level
and remained unchanged until the end of experiment. The Principal
Component Analysis revealed correlations between anhedonia and some
behavioral parameters. Anhedonic rats have lower anxiety levels in
o-maze and less emotional activity in both o-maze and open eld. No
differences were observed in explicit memory test, but anhedonic rats
made signicantly more mistakes in Y-maze indicating working memory
impairments. Slight increases in heart rate and blood pressure in anhedonic rats were insignicant.
Paper No.: 2669

DISCOVERY
SLCO1B1 VARIANTS AS PHARMACOGENETIC
PREDISPOSITION FOR HEPATOTOXICITY INDUCED BY
HMGCOA REDUCTASE INHIBITORS
Nada Bozina(1), I Merep(2), V Mirosevic(3), T Bozina(1), J Serti(1),
Z Reiner(2)
(1) University Hospital Center, Department of Laboratory Diagnostics,
Zagreb,Croatia
(2) University Hospital Center, Division of Metabolic Diseases,
Department of Internal Medicine, Zagreb, Croatia
(3) Agency for Medicinal Products and Medical Devices, Zagreb,
Croatia
Studies identied a genetic risk factor for myotoxicity induced by HMGCoA reductase inhibitors (statins) (The SEARCH Collaborative Group,
N Engl J Med.;359(8):789-99, Voora et all, J Am Coll Cardiol. 2009;
54(17):1609-16). Researchers observed a strong association between
myotoxicity and variants in the SLCO1B1 gene which encodes
OATP1B1, one of the key hepatocellular uptake transporters providing
extraction of diverse compounds including statins from portal venous
blood into the liver. However, no studies have been done to investigate
genetic risk factors for developing statin induced hepatotoxicity. We performed SLCO1B1 genotyping in patients who developed hepatotoxicity
induced by statins. We analyzed 6 patients (5 male and 1 female, mean
age was 45, range 33-57 years) who developed hepatotoxicity caused by
atorvastatin (20-80 mg/day) in 5 cases and uvastatin in 1 case (80 mg/
day). We genotyped blood of all patients for SLCO1B1 388 A>G and
521T>C and additionally for CYP2C9*2,*,3 in a patient who was taking
uvastatin. Genotyping was performed by real-time PCR methods.
Results. Four patients (one carrier of SLCO1B1 521T/C, another of
521T/C and 388A/G, the 3rd of 388G/G and the 4th of 521C/C and
388G/G) developed hepatic lesion and among them the homozygote carrier for both low activity SLCO1B genotypes had worst clinical presentation. Two other patients developed hepatotoxicity (one patient had
hepatic lesion and another increased transaminases) together with
increased creatin phosphokinase (one was a carrier of CYP2C9*3,
SLCO1B1 388A/G, 521T/C and another of 388G/G). Our preliminary
data indicate associations between hepatotoxicity of statins and both
CYP2C9 and SLCO1B1 polymorphysms.
Paper No.: 2670

THE ROLE OF CYP2D6 AND ABCB1 PHARMACOGENETICS
IN THE CLINICAL EFFICACY OF ORALLY ADMINISTERED
RISPERIDONE
Nada Bozina(1), N Jovanovi(2), M Lovri(1), V Medved(2),
M Kudumija-Slijepe`evi(3), J Grubisin(2)
(1) University Hospital Center, Department of Laboratory Diagnostics,
Zagreb, Croatia
(2) University Hospital Center, Department of Psychiatry, Zagreb,
Croatia
(3) Bjelovar General Hospital, Department of Psychiatry, Croatia
The aim was to evaluate the role of CYP2D6 and ABCB1 variants in the
efcacy and safety of patients treated with oral risperidone. The study
included 85 patients with rst episode of psychosis who were treated
with risperidone and then followed-up for eight weeks. Their psychopathology was assessed using The Positive and Negative Syndrome Scale
(PANSS). The CYP2D6 genotyping was performed by PCR-RFLP (for
*3,*4,*6) and long PCR (for duplications and *5). For ABCB1 G2677T/
A and C3435T, real-time PCR method was applied. Plasma concentrations of risperidone and 9-OH risperidone were measured by HPLC. The
206
distribution of genotypes: CYP2D6 wt/wt, wt/mut, and mut/mut was 44,
32 and 8 respectively; for ABCB1 2677G/G, G/T, T/T was 29, 44 and
12, for 3435CC, C/T and T/T was 25, 39 and 21, respectively. The drug
was prescribed at the mean dose of 3.94 mg 1.47 (range 1-8). The
patients with the CYP2D6 wt/wt genotype had signicantly lower dosecorrected levels of risperidone (C/Ds) (F = 20.46, P < 0.001) and active
moiety (F = 4.496, P = 0.014), while the CYP2D6 mut/mut carriers had
the lowest levels of 9-OH risperidone C/Ds (F = 3.22, P = 0.046). The
ABCB1 genotypes did not signicantly inuence these parameters. A
total of 23 participants (27.4%) were responders (dened as 50%
improvement from baseline in total PANSS) and the ABCB1 3435T
allele increased the likelihood of being a responder for more than seven
times (OR = 7.306, 95% CI = 2.73819.493, P = 0.006). Conclusion:
The CYP2D6 and ABCB1 genotyping may serve for personalizing therapy with risperidone.
Paper No.: 1489

SIMVASTATIN DECREASES MALONDIALDEHYDE LEVEL IN
PLASMA AND TISSUES OF RATS
Vlasta Bradamante(1), M Marija(1), L Jasna(2), K Marijan(1),
K Pasko(3)
(1) University of Zagreb School of Medicine, Department of Pharmacology, Zagreb, Croatia
(2) University of Zagreb School of Medicine, Department of Chemistry
and Biochemistry, Zagreb, Croatia
(3) NMR Center, Zagreb, Croatia
Introduction. It is considered that benecial effect of simvastatin treatment on cardiovascular morbidity is due to its antioxidant activity. Simvastatin also shows antioxidant properties in cisplatin-induced
nephrotoxicity and hepatotoxicity. The aim of this study was to compare
the effect of simvastatin on the malondialdehyde level in plasma and different tissues of rats and to establish whether antioxidant effects exist
after discontinuation of its administration. Material and methods. Two
groups of male rats were under simvastatin treatment which was given
orally (10 mg/kg/day for 3 weeks). At the end of the drug treatment, the
rst group was sacriced and blood and tissue (heart, brain, liver, kidney) samples were taken. Other group was without the for the next
9 days (recovery period) and than sacriced. The malondialdehyde level
was measured in plasma (`mol/l) and tissue (`mol/g tissue) by spectrophotometric method. The results are expressed as the percentage of the
decrease of malondialdehyde level and are compared to the control
group. Data were analyzed by t-test. Results. Simvastatin decreased malondialdehyde level in plasma and all tissue by 20-40%. The same
decrease of malondialdehyde was established in the recovery period, i.e.
9 day after the last dose. Conclusion. Our results have shown that simvastatin exhibits the signicant antioxidant effectiveness which persists
after therapy discontinuation.
Paper No.: 1003

PROTEIN KINASE C (PKC) REGULATION OF TYPE 5
METABOTROPIC GLUTAMATE (MGLU5)
RECEPTOR-STIMULATED CA2 + OSCILLATIONS IN RAT
CEREBROCORTICAL ASTROGLIA
Sophie J Bradley(1), JM Watson(2), RAJ Challiss(1)
(1) University of Leicester, Department of Cell Physiology and Pharmacology, Leicester, UK
(2) GlaxoSmithKline, UK
Astrocytes full vital homeostatic and neuromodulatory roles in the

CNS. Glutamate stimulation of astrocytes generates oscillations in cytosolic Ca2 + concentration via a PKC-dependent mechanism, which has
been shown to involve the rapid and reversible phosphorylation of
mGlu5 receptor in a process referred to as dynamic uncoupling (Nash,
MS et al. J Biol Chem 2002, 277; 35947-35960). Stimulation of rat cortical astrocytes with glutamate induced robust oscillatory changes in [Ca2 +
]i at a single-cell level which could be prevented by pre-addition of the
mGlu5 receptor-selective negative allosteric modulator MPEP. Inhibition
of PKC with staurosporine (3 lM) converted glutamate-stimulated
Ca2 + oscillations into a peak-plateau response. Acute stimulation of protein kinase C with low concentrations of phorbol dibutyrate (PDBu)
caused a decrease in glutamate (100 lM)-stimulated Ca2 + oscillation frequency, with complete inhibition of this response at higher (100 nM)
PDBu. In cell populations, staurosporine or Ro31-8220 caused 2-fold
increases in the maximal agonist-stimulated [3H]-IP accumulation without signicant change in EC50 value. In contrast, pre-incubation with the
Ca2 + -dependent PKC inhibitors Go6976 or PKC20-28 did not affect
glutamate-stimulated Ca2 + -oscillation frequency, or agonist-stimulated
[3H]-IP accumulation. Furthermore, loading astrocytes with BAPTA-AM
(100 lM) had no signicant effect on agonist-stimulated [3H]-IP accumulations. These data provide further evidence for the involvement of
Ca2 + -independent PKCs in the rapid cycles of receptor phosphorylation
and dephosphorylation that likely determine mGlu5 receptor regulation
of Ca2 + oscillation frequency in rat cortical astrocytes and pinpoint postreceptor mechanisms that can shape receptor-driven Ca2 + oscillatory
behaviours.
Paper No.: 1648

CHOLINERGIC AND GLUTAMATE-NITRIC OXIDE
SIGNALLING PATHWAYS IN A GENETIC ANIMAL MODEL
OF DEPRESSION, THE FLINDERS SENSITIVE LINE (FSL) RAT
Linda Brand(1), EL Minnaar(1), J van Zyl(1), G Wegener(2),
BH Harvey(1)
(1) North-West University, Division of Pharmacology, Potchefstroom,
South Africa
(2) University of Aarhus, Centre for Psychiatric Research, Aarhus, Denmark
Depression presents with evidence for degenerative pathology that relates
to disturbances in GABA and glutamatergic pathways, while there is also
evidence for an involvement of cholinergic and nitrergic pathways. The
FSL rat is a well-validated genetic animal model of depression. This
study aimed to determine whether depressive-like behavior is accompanied by altered GABA, NMDA-NO/cGMP and cholinergic signalling in
the hippocampus and frontal cortex of the FSL rat. Total regional brain
nitrogen oxides, GABA, L-citrulline and acetylcholine (ACh) were analysed by different chromatographic methods. NMDA and muscarinic M1
receptor characteristics were determined by radioligand binding assays
and cGMP levels by a radio-immunoassay kit. Behavioural assessments
included open eld and social interaction tests, with results suggestive of
increased anxiogenic behavior in the FSL rat. Signicantly lower basal
cGMP levels were noted in frontal cortex, but not hippocampus, of FSL
versus control rats, although no other differences with respect to indices
of NOS activity, receptor characteristics or GABA levels were apparent
in either brain area. Signicantly elevated frontal cortical, but signicantly reduced hippocampal levels, of ACh were observed in FSL rats.
Results suggest the presence of bidirectional cholinergic dysfunction in
frontal cortex and hippocampus of the FSL rat. Reduced fronto-cortical
cGMP levels in absence of altered NO signalling supports an NO-independent mechanism of cGMP regulation in FSL rats, possibly involving
a muscarinic cholinergic-NOS directed pathway. This will have important
implications for our understanding of ACh-NO interactions in the depressive-like behaviour of the FSL rat and, indeed, in the neurobiology and
treatment of depression.
207
Paper No.: 1848
FOCUSED CONFERENCE GROUP: PW12 - EXPERIMENTAL
PAIN METHODS IN DEVELOPMENT AND VALIDATION OF
ANALGESICS
THE PHARMACOGENOMICS OF ESCITALOPRAM IN THE
TREATMENT OF NEUROPATHIC PAIN
Charlotte Brasch-Andersen(1,2), MU Mller(1,2), SH Sindrup(3),
K Brsen(1), M Otto(3)
(1) Odense University Hospital, Department of Clinical Pharmacology,
Odense, Denmark
(2) Odense University Hospital, Department of Clinical Genetics,
Odense, Denmark
(3) Odense University Hospital, Department of Neurology, Odense,
Denmark
Painful polyneuropathy is a common neuropathic pain condition with
painful diabetic neuropathy being a prominent example. Numerous pathophysiological mechanisms are involved in the generation and maintenance
of neuropathic pain. In spite of increasing knowledge about the mechanisms, the treatment of neuropathic pain is still unsatisfactory. The effects
of a selective serotonin reuptake inhibitor (SSRI), escitalopram, on pain in
polyneuropathy was tested in an earlier randomized, placebo-controlled,
double- blinded cross-over trial including 41 patients who were treated
with 20 mg escitalopram and placebo. The clinical study showed a moderate, good or complete pain relief for escitalopram in 11 patients (responders) but at only a slight or no relief in 30 patients (non-responders).
The difference in response to escitalopram may be caused by genetic differences between the responders and the non-responders, which led to a
pharmacogenomic study investigating genetic differences between the two
groups for functional variants in genes involved in the signalling pathway
for serotonin. We tested the serotonin receptor subunits 5-HTR2C G68C
(rs6318) and 5-HTR2A C1354T (rs6314) both giving rise to amino acid
changes. We furthermore tested a missense variant in the gene coding for
P-glycoprotein, ABCB1 G2677T (rs2032582) and a promoter polymorphism (5-HTTLPR) in the serotonin transporter gene. We were not able to
nd any statistical signicant association between the tested genetic polymorphisms and effect of escitalopram when dichotomizing the patients
into responders and non-responders. Using quantitative assessment of pain
might add more information to the association studies.
Paper No.: 2426

THERAPEUTIC TARGETS
NUCLEIC ACID OXIDATION IS PREDOMINANTLY DETERMINED BY NON-GENETIC FACTORS IN ELDERLY DANISH
TWINS
Kasper Brdbk(1,2), R Ribel-Madsen(3), T Henriksen(1,2),
A Weimann(1,2), M Petersen(1,2), J T Andersen(1,2), S Afzal(1,2),
B Hjelvang(1,2), A Vaag(3), P Poulsen(3,4), H E Poulsen(1,2,5)
(1) Laboratory of Clinical Pharmacology, Rigshospitalet, Copenhagen,
Denmark
(2) Department of Clinical Pharmacology, Bispebjerg Hospital, Denmark
(3) Steno Diabetes Center, Gentofte, Denmark
(4) Novo Nordisk A/S, Soeborg, Denmark
(5) Faculty of Health Sciences, University of Copenhagen, Copenhagen,
Denmark
DNA oxidation is thought to be important in both cancers and in many
non-cancerous conditions. To what extend nucleic acid oxidation is determined by genetic and environmental factors is unknown. The aim of this
study was to examine the contribution of genetic versus environmental
factors to the rate of nucleic acid oxidation by performing a classical
twin study. The study population included 198 elderly twins (aged 62
83 years), 46 monozygotic (MZ) and 53 dizygotic (DZ) twin pairs. Urine
samples were collected for measurement of markers of nucleic acid oxidation: 8-oxodG and 8-oxoGuo using ultra-performance liquid chroma-
tography and tandem mass spectrometry detection. Classical twin

analyses including intraclass correlation coefcients and heritability estimates were used to determine the relative contributions of genes and
environment to the variation in nucleic acid oxidation. There were no statistically signicant differences in intraclass correlations between MZ
and DZ twins neither for 8-oxodG (rMZ = 0.55, rDZ = 0.47; P = 0.43)
nor 8-oxoGuo (rMZ = 0.45, rDZ = 0.58; P = 0.21). The heritability estimates for 8-oxodG and 8-oxoGuo excretion were h2 = 0.17 and h2 ~ 0,
respectively. In conclusion, we demonstrated that in a large population of
elderly Danish twins the rate of nucleic acid oxidation is predominantly
determined by potentially modiable non-genetic factors. (Workshop reference: SW8, Antioxidants as therapeutic agents)
Paper No.: 2575
AL-TRYPTOPHAN RICH DIET IMPROVES ULCER HEALING
VIA ENDOGENOUS MELATONIN, PROSTAGLANDINS AND
DOWNREGULATION OF HYPOXIA INDUCIBLE FACTOR 1A
(HIF-1)
Thomas Brzozowski(1), A Ptak-Belowska(1), R Pajdo(1), S Kwiecien(1),
M Pawlik(1), I Brzozowska(2), PC Konturek(3), WW Pawlik(1), SJ Konturek(1)
(1) Jagiellonian University Medical College, Department of Physiology,
Cracow, Poland
(2) Jagiellonian University Medical College, Department of Anatomy,
Cracow, Poland
(3) Thuringen Clinic, Department of Medicine, Saalfeld, Germany
Exogenous melatonin exhibits gastroprotective and ulcer healing activities but the role of endogenous melatonin derived from daily dietary
intake of L-tryptophan (L-Try) in the mechanism of ulcer healing has not
been extensively studied. We compared the effects of i.g. administration
of vehicle (saline) or melatonin in rats fed with normal, L-Try-depleted
(0% of L-Try) and L-Try rich-diet (Try+ were determined by RT-PCR.,
0.30% L-Try of total protein) of without or with blockade of melatonin
Mel2 receptors with luzindole (20 mg/kg-d i.p.) on healing of gastric
ulcers induced by acetic acid (ulcer area=28 mm2). At 9 days after ulcer
induction, gastric ulcers were measured by planimetry, gastric blood ow
(GBF) was determined by H2-gas clearance technique and plasma L-Try
and gastric mucosal PGE2 concentrations and expression of mRNA for
COX-1, COX-2 and hypoxia HIF-1 Ulcer healing was signicantly
accelerated in rats fed with L-Try rich diet while raising GBF at ulcer
margin, plasma L-Try, melatonin and gastrin levels and mucosal generation of PGE2. In L-Try free diet the plasma levels of L-Try and melatonin levels were signicantly diminished and ulcer healing was
signicantly delayed. Indomethacin and rofecoxib or luzindole, an antagonist of Mel2 receptors, signicantly attenuated L-Try rich diet-induced
acceleration of ulcer healing. was downregulated but COX-1 and COX-2
mRNAs were upregulated by L-Try rich diet. Gastric mucosal HIF-1 We
conclude that endogenous melatonin synthesized from L-Try accelerates
ulcer healing via interaction with Mel2 receptors, an enhancement of gastric microcirculation, mediated by PGE2 derived from COX-1 and COX2 overexpression and activity.
Paper No.: 1996
PKCB/HUR/VEGF A NEW PATHWAY IN DIABETIC
RETINOPATHY
Claudio Bucolo(1), A Pascale(2), M Amadio(2), S Govoni(2),
F Drago(1)
(1) University of Catania, Department of Experimental and Clinical
Pharmacology, Catania, Italy
(2) University of Pavia, Italy
208
Introduction: To investigate whether PKCbII could control VEGF levels
via the mRNA stabilizing human embryonic lethal abnormal vision
(ELAV)-like protein, HuR, in retina of streptozotocin(STZ)-induced diabetic rat. Methods: Retinal tissues, collected after 10 days from STZinjected rats, were processed for western blotting and preparation of
mRNP (ribonucleoproteic complexes). Anti-PKCbI mouse monoclonal
antibody, anti-PKCbII rabbit polyclonal antibody, anti-HuR mouse
monoclonal antibody, anti-VEGF rabbit polyclonal antibody and antialfa-tubulin rat monoclonal antibody were used for western blot analysis.
Samples were processed for immunoprecipitation using anti-HuR antibody, and VEGF mRNA was detected by real-time quantitative PCR.
Statistical analysis was performed by ANOVA by and an appropriate
post hoc comparison test Results: PKCbI and PKCbII were increased in
the retina of STZ injected rats compared to sham (+160% +113%,
respectively). We showed a PKC-mediated phosphorylation of HuR in
the retina from diabetic rats. HuR protein levels increased in STZ-treated
rats (+62% vs. sham), and this was accompanied by an higher phosphorylation in serine residues (+209% vs. sham). These effects were blunted
by selective PKCb inhibitor treatment. A specic binding between HuR
protein and VEGF mRNA in the retina was shown. Further, the PKCb/
HuR activation is accompanied by enhanced VEGF protein expression
and this effect was attenuated by PKCb inhibitor. Conclusion: These
ndings demonstrated the existence of PKC/HuR/VEGF pathway in
experimental diabetic retinopathy. A better dissection of this cascade in
diabetic conditions may help to disclose new potential pharmacological
targets. Acknowledgments: This work was supported by a grant from
MIUR - PRIN 2007.
Paper No.: 2998

EVALUATION OF GUIDELINE-CONFORMITY OF INITIATION
OF ORAL HYPOGLYCEMIC TREATMENT FOR INCIDENT
DIABETES MELLITUS TYPE 2 IN AUSTRIA
Anna Bucsics(1), M Asslaber(1), R Bauer(1), T Burkhardt(1),
M Pogantsch(2), A Schautzer(2), PR Wieninger(1), WC Winkelmayer(3)
(1) Main Association of Austrian Social Security Institutions, Vienna,
Austria
(2) Styrian Sickness Fund, Austria
(3) Harvard University, Boston, MA, USA
Guidelines recommend metformin as rst line oral hypoglycemic (OH)
treatment in patients with diabetes uncontrolled with diet and lifestyle
modications. It is unknown what proportion of incident patients comply
with this recommendation in Austria. We used claims from 11 sickness
funds that covered >95% of the Austrian general population. We identied new users aged 19-100 years of a rst OH drug, dened as a rst
lled prescription after one year without any antidiabetic drug (including
insulin). Among these incident users, we described the OH drug class
used and correlates of guideline-conforming initiation with metformin
monotherapy using logistic regression. Over 18 months (1/2007-6/2008),
we identied 42882 incident users of an OH drug: 70.1% used metformin, 24.7% used a sulfonylurea, and 4.5% initiated treatment with
another class. Independent correlates of metformin initiation were younger age, female gender, waived copayment (as indicator of low socioeconomic status), more recent year, fewer hospital days in the year prior to
rst OH therapy, and more non-OH prescriptions lled during that baseline period (all P < 0.001). There was signicant heterogeneity across
sickness funds. Approximately 20% of metformin initiators had a second
antidiabetic drug added within 12 months. While we were unable to
ascertain specic contraindications to metformin in this dataset (renal
insufciency, hepatic failure), <5% of the general population is expected
to have these conditions. Conclusion: 70% of new initiators of OH treatment in Austria received metformin as recommended by international
guidelines. At least 25% did not, taking into account possible contraindications, providing an opportunity for systematic intervention.
Paper No.: 3155

TNFA DOES NOT INHIBIT INSULIN-STIMULATED 2-DEOXYGLUCOSE UPTAKE IN IN SITU SKELETAL MUSCLE, BUT
DOES INCREASE LIPOLYSIS AND LOWER TOTAL GLUCOSE
UTILIZATION
Christian Selmer Buhl(1), CN Petersen(1), N Jessen(1), O Schmitz(1), S
Lund(2), ES Buhl(1)
(1) Aarhus University, Department of Pharmacology, Aarhus, Denmark
(2) Aarhus Sygehus, Department of Endocrinology M, Aarhus, Denmark
Elevated plasma levels of the proinammatory cytokine TNFa is
strongly correlated to insulin resistance and obesity. However, the exact
role of TNFa in metabolism is not understood. The aim of the present
study was to investigate the effect of TNFa stimulation on insulin sensitivity in muscle using the isolated and perfused rat hindlimb. This model
allows us to investigate direct effects on different types of muscle bres
without interactions with neuro-endocrine pathways. The hindlimbs of
male wistar-rats were mounted and perfused at a constant ow with a
heated and oxygenized Krebs-Ringer solution. Glucose uptake was measured using 2-deoxy-3H-glucose and 14C-mannitol. At the end of the
experiment muscle biopsies were taken out and analyzed for glycogen
content. Perfusion medium was analyzed for glycerol, FFA, lactate and
total glucose utilization. Insulin and TNFa was respectively added in
concentrations of 0, 75, 500 mU/L and 0, 250, 2500ng/L. Insulin
increased glucose uptake by 1.6-3.2-fold at low and 10.0-14.7-fold at
high concentrations. TNFa did not affect basal or insulin-stimulated
2-deoxy-glucose-uptake. However, TNFa did reduce the rate of total
glucose utilization (up to 33%) and did increase the release of lactate (up
to 43.3%) and the amount of glycogen deposited (up to 23.4%) (all
P < 0,05). Furthermore TNFa signicantly increased the lipolysis with
the release of FFA by up to 2.5-fold along with an increase in total
oxygen uptake. The ndings show that TNFa does not affect 2-deoxyglucose-uptake in muscle. However, TNFa mediates an increase in lipolysis that may be at the expense of an attenuated glucose oxidation.
Paper No.: 2468

INNERVATED MYOFIBROBLASTS IN THE URINARY
BLADDER? FUNCTIONAL AND ULTRASTRUCTURAL
EVIDENCE
Elizabeth Burcher(1), P Sadananda(1), S Sandow(1)
University of New South Wales, Department of Pharmacology, Sydney,
Australia
We have reported that neurokinin A (NKA) can contract isolated mucosal strips from porcine bladder, with dense smooth muscle actin staining
on the suburothelial myobroblasts these may mediate the contraction
to NKA (Sadananda et al., Br J Pharmacol 2008; 153, 1465-73). Our
aim was to examine the ultrastructure of the porcine bladder mucosa and
characterize the relationship of the suburothelial myobroblasts with
adjacent cells. Pig bladders obtained from an abbatoir were processed for
electron microscopy. The suburothelial population of myobroblasts
were morphologically similar to the deep myobroblasts of human detrusor, in that they have prominent rough endoplasmic reticulum and elongated processes. Extensive nerve bundles with varicosities were located
below the urothelium and in close proximity to myobroblasts. These
nerve bundles contained individual varicosities with discrete regions
(bare of Schwann cells) containing synaptic vesicles, facing the potential
neuroeffector cell. This is in accordance with the denition of neuroeffector junctions, as neurotransmitter release sites. These dense-cored synaptic vesicles were 100-250 nm in diameter, consistent with a primarily
209
peptidergic content. In low resolution studies (n = 4), synaptophysin and
tachykinin immunoreactivity localised nerve bres containing tachykinins within the suburothelial nerve plexus and in association with
myobroblasts. In conclusion, the strategic location of suburothelial
myobroblasts between the urothelium and afferent nerve plexus suggests that they are involved in sensory processing and cross talk between
cell layers. We hypothesise that peptides such as NKA are released from
these dense-cored synaptic vesicle-containing varicosities, to act directly
on suburothelial myobroblasts, resulting in contraction of the porcine
bladder mucosa.
Paper No.: 1148

DISEASES
SEARCHING FOR NOVEL ANALGESIC AND
ANTI-INFLAMMATORY ANALOGS OF ANANDAMIDE
Sumner Burstein(1), G Li(2), R Salmonsen(1), C McQuain(1), T Al(2),
V Kattamuri(2)
(1) University of Massachusetts Medical School, Department of Biochemistry & Molecular Pharmacology, Worcester, MA, USA
(2) Texas Tech University, Department of Chemistry & Biochemistry,
Lubbock, TX, USA
Recently, data were presented supporting the possibility that analogs of
anandamide called elmiric acids are potential anti-inammatory agents
[Burstein SH, et al. Bioorganic & medicinal chemistry 2007;15:334555]. We have developed a mechanism-based in vitro assay for screening
compounds for potential anti-inammatory activity based on their stimulatory action on prostaglandin J2 levels and have applied this to the novel
compounds described in this report. We determined a rank order of antiinammatory potencies for a series of ten analogs of the endocannabinoid
anandamide that were synthesized via new asymmetric reactions and
multiple-step protection-deprotection procedures. The parent molecules
are, (5Z,8Z,11Z,14Z)-N-(2-hydroxyacetyl)icosa-5,8,11,14-tetraenamide
(GL-A) and (5Z,8Z,11Z,14Z)-N-((1S,2R)-2,3-dihydroxy-1-phenylpropyl)icosa-5,8,11,14-tetraena mide (GL-B). Here we would like to report
some preliminary results for these new anti-inammatory candidates.
GL-B and its enantiomer showed a potency difference of approximately
20 fold. This degree of stereospecicity suggests that the stimulation of
PGJ levels in RAW264.7 cells is probably receptor mediated. Because of
their similarity to anandamide, the cannabinoid receptors are a possibility.
As with the elmiric acids, palmitoyl derivatives have little or no effect on
prostaglandin production whereas arachidonoyl analogs are all relatively
active. Halogen substituents on the phenyl ring showed a range of
potencies all of which were lower than the parent GL-B.
Support was provided by NIDA (1R03DA026960).
Paper No.: 965

EFFECTS OF GONADECTOMY ON HIPPOCAMPAL
AROMATASE EXPRESSION IN BOTH GENDERS
determine the potencial effects of gonadectomy on central aromatase

expression in hippocampus. Female and/or male Sprague-Dawley rats
(200-250g) were used in experiments. Ovariectomy, castration or sham
surgery was performed in anesthetized rats. At the end of 8-weeks, rats
were killed by decapitation and hippocampal tissue homogenates were
prepared and evaluated for aromatase proteins by western blot. b-actin
was used as internal control to ensure equel protein loading. Students
test was used for statistical analysis and P < 0.05 was considered signicant. Aromatase enzyme levels were increased in ovariectomized rats,
but did not alter in castrated rats when compared with control sham operated groups. According to these results it can be suggested that loss of
endogenous peripheral aromatase through surgical gonadectomy alters
hippocampal local aromatase levels in female but not male rats. The
increased hippocampal levels of aromatase after gonadectomy might be a
compansatory mechanism in female brain and this adaptive mechanism
may not be efcient in male brain.
Experimental procedure was approved by Hacettepe University Animal
Ethics Committee (No:2006/8-6).
Paper No.: 2314

ALTERATIONS IN AROMATASE ENZYME LEVELS OF
OVARY, TESTIS, UTERUS AND VAS DEFERENS TISSUES OF
DIABETIC RATS
Nihan Burul-Bozkurt, C Pekiner, P Kelicen
Hacettepe University Faculty of Pharmacy, Department of Pharmacology,
Ankara, Turkey
Although diabetes mellitus (DM) is associated with increased risk of
reproductive problems in both genders, responsible molecular mechanisms under this pathophysiology are not identied yet. Aromatase catalyzes the biosynthesis of estrogens and these hormones have important
roles in reproductive processes. Insulin regulate the activity of aromatase
however there are few data about the effects of diabetes on this enzyme.
Present study was designed to investigate the effects of experimental diabetes on aromatase expression levels in ovary, testis, uterus and vas deferens tissues of rats. Rats were injected with streptozotocin to induce
diabetes. Insulin treatment was also applied. At the end of 4- and
12-weeks, rats were killed by decapitation and ovary, testis, uterus and
vas deferens tissue homogenates were prepared and evaluated for aromatase proteins by western blot. Students t test or one-way ANOVA was
used for statistical analysis. Aromatase expression levels in ovary were
signicantly decreased both in short and long-terms of diabetes. In testis,
enzyme levels were not altered in short-term, but signicantly decreased
in long-term of diabetes. In uterus and vas deferens tissues no signicant
differences were observed at aromatase immunoreactivity. Insulin treatment prevented diabetes-induced changes. These results indicated for the
rst time that, DM altered the expression levels of aromatase both in
ovary and testis tissues but did not affect the enzyme levels in uterus and
vas deferens tissues. According to our results, aromatase might be an
important target molecule for treatment of sexual dysfunction seen in DM.
Approved by Hacettepe University Animal Ethics Committee (No:2006/
8-6).
Nihan Burul-Bozkurt, P Kelicen, C Pekiner

Ankara, Turkey
Aromatase catalyzes the biosynthesis of estrogens from androgens.
Although the primary sources of estrogens are gonads they were
expressed in a variety of tissues including brain. In brain estrogens is
known to play an important role in many physiological processes and
have numerous reproductive and non-reproductive functions. They regulate gene expression, neuronal survial, neuronal and glial differantiation,
synaptic transmission in many central nervous system areas and have
neurotrophic and neuroprotective properties. This study was designed to
Paper No.: 1918

HEALTH AND DISEASE
ANALYSIS OF NITRIC OXIDE SENSITIVE GUANYLYL
CYCLASE ACTIVATION BY CIGUATES BASED ON
MANT-GTP FLUORESCENCE
Mareike Busker, JR Kraehling, M Seeanner, S Behrends
Technical University Braunschweig, Institute of Pharmacology,
Toxicology and Clinical Pharmacy, Braunschweig, Germany
210
Cinaciguat and ataciguat activate nitric oxide sensitive guanylyl cyclase
(NOsGC) independently of heme. It has previously been demonstrated
that MANT-nucleotides ((N-methyl)anthraniloyl-substituted nucleotides)
inhibit nucleotide cyclases by competitive interaction with the substrate
binding site of adenylyl and guanylyl cyclases [Gille et al., J Biol Chem
2004;279:19955-19969]. In the current work we use MANT-GTP derivatives and Foerster resonance energy transfer (FRET) to probe the conformation of the active site of puried NOsGC. In direct uorescence
experiments 2-MANT-3-dGTP was excited at 350 nm and uorescence
was measured at 445 nm: Addition of the a1/b1 isoform of NOsGC
(3 lM) to 2-MANT-3-dGTP (3 lM) led to a small but signicant
increase in uorescence. A non-heme containing NOsGC form (a1/
b1H105A) led to a more pronounced increase in uorescence. Addition
of the novel NOsGC activators cinaciguat or ataciguat increased 2MANT-3-dGTP uorescence for the heme containing and even more for
the non-heme containing form. In a second set of experiments tyrosine
(Y) or tryptophane (W) residues of NOsGC were excited at 280 nm or
295 nm, respectively resulting in uorescence with a maximum at
335 nm. Combination of 2-MANT-3-dGTP and NOsGC led to an additional peak with a maximum at 430 nm indicating the occurrence of
FRET. Preliminary evidence indicates that W669 of the a1 subunit acts
as FRET-donor. Addition of ataciguat further enhanced FRET. In summary, we show that MANT-GTP uorescence can be used in direct
experiments and FRET experiments to detect conformational changes in
the catalytic region of NOsGC induced by novel activator drugs.
Paper No.: 1963

THE STUDY OF CYP2C19 POLYMORPHISMS IN ROMANIAN
EPILEPTIC PATIENTS
Anca Dana Buzoianu, IC Bocsan, FC Militaru, R Popp, A Trifa,
L Perju-Dumbrava
University of Medicine and Pharmacy, Department of Clinical
Pharmacology, Cluj Napoca, Romania
Introduction. Genetic polymorphism of drug metabolizing enzymes is
dened as ability of individuals to metabolize drugs in different degrees,
due to differences in the enzyme capacity and function. CYP2C19, a
member of the cytochrome P450 enzymes family, metabolizes a range of
clinically signicant drugs. Its homologous gene has been shown to be
polymorphic, two major alleles, CYP2C19*2 and *3 being responsible
for the poor metabolizer (PM) phenotype and CYP2C19*4 being associate with ultra-rapid metabolizer phenotype (pharmaco-resistant phenotype). Objective. The aim of this study is to determine the allele
frequency of CYP2C19 variants in a group of epileptic patients and to
compare it with the Romanian general population. Methods. Based on the
PCR-RFLP technique, CYP2C19*2, CYP2 C19 *3 and *4 variants were
studied in 64 epileptic patients and also in 200 healthy, unrelated Romanian volunteers. Results and conclusions: The data obtained shows that
the homozygous genotype for CYP2C19 * 2 allele, responsible for a
poor-metabolizer phenotype for CYP2C19 was met at 7.7% of patients,
compared to the general prevalence of 1.5% of the Romanian general
population. Heterozygous genotype CYP2C19 * 1 / * 2 was met in 21.5%
of patients, a frequency similar to what met in the Romanian general population (24%). CYP2C19 * 3 allele has not been seen in any patient.
Paper No.: 3201

DOCUMENTATION OF RECENT DRUG HISTORY IN
CHILDREN VISITING A PEDIATRIC EMERGENCY CLINIC
Ylva Bottiger(1), E Kimland(1), S Lindemalm(2)
(1) Karolinska Institute, Department of Laboratory Medicine, Division of

Clinical Pharmacology, Stockholm, Sweden
(2) Karolinska Institute, Department of Womens and Childrens Health,
Stockholm, Sweden
Introduction: There is a generally acknowledged lack of evidence in relation to pediatric drug treatment, and off-label treatment has been reported
to be higher in this age group, than in adults. Therefore, it should be
important to document previous use of medicines in children seeking
medical care. The aim of this study was to perform a systematic analysis
of which drugs, prescribed or OTC, and/or natural remedies children had
used prior to visiting a pediatric emergency clinic, and compare this
information with the documentation of drug use in their medical records.
Methods: A questionnaire based study was performed at a paediatric
emergency clinic during three weeks. Very urgent cases and non-Swedish
speaking patients/parents were excluded. The questionnaire was validated
through an interview with a subgroup of participants. Information concerning drug use in the medical records was later compared with the
information from the questionnaires. Results: Out of a total of 963 children who visited the clinic within the time frame, 274 children, aged
2 weeks to 18 years, mean age 3.85 years, were enrolled. Forty percent
had used prescribed drugs, and 65% of the children had used OTC drugs.
Natural remedies were used by 8.4%. In more than half of the medical
records, one or more drugs were not documented. Conclusion: Information concerning the intake of medicines or natural remedies is often missing in the medical record among paediatric patients at an emergency
clinic, which could possibly inuence the safety of the patient in a negative way.
Paper No.: 1486

DISCOVERY
NEW NAPHTHALENE DERIVATIVES OF CITALOPRAM
DISCRIMINATE BETWEEN THE PRIMARY AND THE
ALLOSTERIC BINDING SITES AT THE HUMAN SEROTONIN
TRANSPORTER
Klaus Bges(1), P Bregnedal(1), K Juhl(1), H Zhong(2), O Wiborg(3)
(1) H. Lundbeck A/S, Lundbeck Research, Valby, Copenhagen,
Denmark
(2) H. Lundbeck A/S - Paramus, New Jersey, USA
(3) Aarhus University, Centre for Psychiatric Research, Risskov,
Denmark
The human serotonin transporter (hSERT) has primary and allosteric
binding sites for escitalopram and R-citalopram. While only escitalopram
binds with nanomolar high afnity to the primary binding site the interaction of both compounds with the low afnity allosteric binding site
affect the dissociation half life of [3H]-escitalopram from hSERT. We
synthesized a series of naphthalene derivatives to explore the spatial
requirements of the binding sites and tested them for afnity to the primary binding site and for allosteric modulation of [3H]-escitalopram dissociation. The three possible derivatives with a benzene ring fused to the
isobenzofurane moiety of citalopram all showed high afnity to the primary binding site (Kis 4-12 nM) while their allosteric modulation were
4-11 times weaker than citalopram. The citalopram derivative with a
2-naphthylgroup replacing the 4-uorophenylring had similar prole, and
its enantiomers showed a high stereoselectivity for the primary binding
site (Kis 4.1 and 113 nM, respectively) and a low stereoselectivity for
allosteric modulation (EC50s 41 and 55 microM). The derivative with a
1-naphthylgroup was a selective allosteric modulator. The enantiomers
showed low stereoselectivity for allosteric modulation (EC50s 14 and
19 microM, respectively). Afnities (Kis) for the primary site were 213
and 97 nM, respectively.
211
Paper No.: 1293
A STUDY ON THE PROTECTIVE MECHANISM OF TIBETAN
MEDICINE POTENTILLA ANSERINA ON EXPERIMENTAL
LIVER INJURY
Guangming Cai, Z Zhi, X Fu
Paper No.: 2900

INHIBITORY EFFECTS OF ORIGANUM ONITES ESSENTIAL
OIL AGAINST CONTRACTIONS OF ISOLATED RAT VAS DEFERENS INDUCED BY ANDROCTONUS CRASSICAUDA
VENOM
A Cakmak(1), F Caliskan(2), Suleyman Aydin(1)
China Military Institute of Chinese Materia Medica, 302 Military Hospital of China, Beijing, PR China
Objective: To observe the protection effects of JMS (the active ingredient
of Potentilla anserina) on the hepatic injury in mice and research the possible mechanism of the effects. Methods: Using models of hepatic injury
induced by CCL4, Con-A and DEX, the levels of AST, ALT, TNF-aIFN-b-IL-4 in blood serum and the contents of MDA and GSH-PX in
hepatic tissue were detected. Results: JMS could protect the hepatic cell
directly and reduce the activity of ALT, AST in blood serum signicantly(P). Conclusion: The mechanism of JMS Protection effects on
liver-injured mice should involve inuencing the livers metabolism,
improving capability of anti-oxidation and adjusting the immunity. JMS
could reduce the relief of free radical, cause the inhibition of apoptosis
and protect the hepatic cell by adjusting the cytokine secretion in microenvironment. It could adjust the immune function by correcting the
imbalance of Th1/Th2, so as to keep the homeostasis.
Paper No.: 1924

HEALTH AND DISEASE
PKG MEDIATES THE TESTOSTERONE VASORELAXANT
EFFECT IN HUMAN UMBILICAL ARTERY WITHOUT
ENDOTHELIUM
Elisa Cairrao(1,2), AJ Santos-Silva(1), I Verde(1)
(1) CICS Centro de Investigacao em Ciencias da Saude, Universidade
da BeiraInterior, Covilha, Portugal
(2) Centro Hospitalar da Cova da Beira E.P.E., Covilha, Portugal
The cyclic nucleotides involvement in the vasorelaxation induced by
testosterone in human umbilical artery (HUA) was investigated. The
effect of this hormone on denuded HUA contracted by serotonin (5HT), histamine or KCl was analysed. Testosterone effect on potassium
current (IK) was also studied in HUA vascular smooth muscle cells
(HUASMC). The relaxant effects of Tes, sodium nitroprusside (SNP; a
soluble GC stimulator) and atrial natriuretic peptide (ANP; a particulate
GC stimulator) are similar. The Tes relaxant effect is not different to
the induced by the conjoint application of ANP and Tes. However, the
effects of SNP and Tes seems additive. The inhibition of PKG signicantly reduced the Tes effect independently of the contractile stimuli.
The IK measured is mainly constituted by potassium exit through voltage sensitive potassium channels (KV) and large-conductance
Ca2 + activated potassium channels (BKCa). Testosterone signicantly
activated the basal IK in HUASMC. SNP does not induce a signicant
modication in basal or testosterone stimulated IK. In contrast, ANP
stimulates the basal IK, but does not increase the testosterone stimulation on IK. The activations of the IK induced by testosterone or by
ANP are not signicantly affected by the PKA inhibitor, but are completely inhibited by the PKG inhibitor. Our results show that testosterone and ANP share the same pathway to increase the cGMP and to
induce vasorelaxation, and suggest that Tes increases cGMP levels by
activating particulate GC.
We thank the Gynaecology-Obstetrics Department staff of CHCB and
FCT which supports the fellowship SFRH/BDE/15532/2004.
(1) Anadolu University Faculty of Pharmacy Department of Pharmacology, Eskisehir, Turkey

(2) Eskisehir Osmangazi University Faculty of Science and Letters,
Department of Biology, Eskisehir, Turkey
Androctonus crassicauda is one of the most toxic venomous scorpion
species of the world and many people die due to the envenomation by
this scorpion. Origanum onites is a a plant used for various medicinal
purposes including as antiseptic, antimicrobial and an antidote for toxic
envonamations. The purpose of this study is to investigate the effect of
O. onites essential oil on the contracting effects of A. crassicauda venom
on isolated rat vas deferens. A.crassicauda venom was obtained from
specimens of South-east Turkey and O.onites essential oil, from the distillation of the plants collected from west Anatolia. Our experiments
showed that the contraction obtained by A. crassicauda venom on the
vas deferens was antagonized and relaxed by the application of 10-6,
10-5 and 10-4 M doses of O. onites essential oil. In our study it was
shown that there is an interesting antagonistic interaction between
Origanum onites essential oil and the venom of Androctonus crassicauda.
Our results indicate the possibility of O.onites as a natural antidote
against Androctonus crassicauda which requires new and detailed in vivo
investigations.
Paper No.: 1778

TODAYS USE OF MEDICINAL NATURAL PRODUCTS BY
ITALIAN WOMEN IN SICILY
Gioacchino Calapai, A Pieratti, D Crupi, A De Sarro
University of Messina, Department of Experimental and Clinical Medicine and Pharmacology, Messina, Italy
Introduction: Women are the most consumers of medicinal natural products They use them for typical women disorders (menopausal symptoms
and menstrual disturbances) and eventually for pregnancy and lactation
problems. We propose a survey on their use by sicilian women. Methods:
Data on use of natural medicinal products (such as food supplements,
herbal medicines, omeopathics) and synthetic drugs in either gender diseases or not are collected through interview by 1000 north east sicilian
women aging 15-85 years. To get information about use of drugs and
medicinal natural products a questionnaire is administered in ten different
consulting health units for women. A second way of acting is through
phone interviews. Obtained data are collected in a data-base, and successively statistically analyzed; data on undesired reactions are also collected. Preliminary results: About one third of the interviews (314) were
collected and they show that principally synthetic drugs is used by
74.5%; principally natural medicinal products by 14.5%; we had no
answer by the remaining group of women. Natural products use increases
with the age with maximum use starting from 40 years of age women
and beyond. The major part of women use them without knowledge of
their side effects. The indications most cited are menopause, water retention, stipsis, sleep disturbances, venous insufciency. Conclusions: Use
of medicinal natural products by women of our region is increased but
safety data (warnings, side effects and contraindications) are still poorly
known by consumers.
212
Paper No.: 1868
THE INVERSE AGONIST EFFECT OF RIMONABANT IN
BRAIN MEMBRANES IS NOT MEDIATED BY THE CB1
RECEPTOR
ing low CNS penetration of darifenacin and 5-HMT (limited by PGP

efux), and trospium (limited by permeability and PGP efux). Consistent with these results in vitro, penetration in vivo in rat brain and CSF
was signicant for oxybutynin, solifenacin and tolterodine while trospium, darifenacin and 5-HMT had no signicant CNS penetration. These
differences in CNS penetration may contribute to different CNS side
effect proles across the OAB agents.
Luis F Callado, AM Erdozain, R Diez-Alarcia, JJ Meana

University of the Basque Country, Department of Pharmacology, and
Centro de Investigacion Biomedica en Red de Salud Mental
(CIBERSAM), Leioa, Spain
Previous data suggest that rimonabant behaves as an inverse agonist in
different biological systems Our aim was to characterize the inhibitory
effects of rimonabant on G-protein activation in postmortem human brain
membranes In [35S]GTPcS binding assays performed in human brain
cortical membranes, increasing concentrations of rimonabant blocked the
effect evoked by WIN55,212-2 (10lM) and inhibited the basal binding
of [35S]GTPcS O-2050 also blocked the effect mediated by WIN55,2122 (1lM) but barely affected basal binding of [35S]GTPcS Neither O2050 (10 and 100lM) nor WIN55,212-2 (10lM) were able to modulate
the inhibition of the basal binding of [35S]GTPcS produced by rimonabant [35S]GTPcS binding assays performed in mouse brain revealed that
rimonabant inhibited the basal binding of [35S]GTPcS in the same manner in wild type and CB1 knock out tissue In binding saturation assays
in postmortem human brain membranes, [3H]rimonabant showed a KD
of 58 11 nM The [3H]rimonabant specic binding at a xed concentration of 5 nM was only reduced by 60% in the presence of O-2050
(10lM) These data suggest that the apparent inverse agonist effect of
rimonabant over G-protein activation is not be mediated by the cannabinoid CB1 receptor In addition, rimonabant recognizes a second binding
site distinct from cannabinoid CB1 receptors
Paper No.: 2730

COMPARISON OF CENTRAL NERVOUS SYSTEM
PENETRATION OF ANTIMUSCARINIC DRUGS FOR
OVERACTIVE BLADDER
E Callegari(1), R Webster(2), K Fenner(2), J LaPerle(1), B Malhotra(3),
Peter Bungay(2)
(1) Pzer Inc, Groton, CT, USA
(2) Pzer Inc, Sandwich, UK
(3) Pzer Inc, New York, NY, USA
Common side effects of antimuscarinics for the treatment of overactive
bladder (OAB) symptoms are peripheral (dry mouth and constipation)
while central side effects like cognitive dysfunction may be seen depending on muscarinic receptor subtype afnity and/or central nervous system
(CNS) penetration of specic agents. CNS penetration was evaluated in
nonclinical models in silico, in vitro and in vivo for common OAB drugs:
oxybutynin, trospium, tolterodine, solifenacin, darifenacin and fesoterodine (including its active metabolite, 5-hydroxymethyl tolterodine
(5-HMT)). The molecular and physicochemical properties, cross-membrane permeability (using RRCK cells from Madin-Darby Canine Kidney cell line) and P-glycoprotein (PGP) transporter interaction of each
agent were evaluated as predictors of CNS penetration. Plasma, brain
and CSF concentrations were determined following subcutaneous administration of each agent to rats. Fesoterodine, a prodrug of 5-HMT, is not
detectable in systemic circulation, and was not included in experiments
in vivo. Oxybutynin, solifenacin and tolterodine were not substrates for
PGP, demonstrated high permeability in RRCK cells, and possessed
physicochemical properties predicting moderate-high CNS penetration.
Trospium, 5-HMT and darifenacin were each PGP substrates and demonstrated low, medium and high RRCK permeability, respectively, predict-
Paper No.: 3367

ANTI-INFLAMMATORY EFFECTS OF THE PENTACYCLIC
TRITERPENES A,B-AMIRYNS ON ANIMAL MODELS OF
IRRITATIVE CONTACT DERMATITIS INDUCED BY
TETRADECANOYLPHORBOL 13-ACETATE
Iana Calou(1), D Goncalves(1), T Melo(1), T Olinda(1), GA Brito(1), V
Rao(1), M Chaves(2), F Santos(1)
(1) Federal University of Ceara, Departement of Physiology and Pharmacology, Fortaleza, Brazil
(2) Federal University of Piau, Departament of Organic Chemistry, Teresina, Brazil
The pentacyclic triterpenes a,b-amiryns (AB), isolated from the resin of
Protium heptaphyllium March (Burseraceae), were evaluated on acute
mouse ear dermatitis induced by a single application of tetradecanoylphorbol 13-acetate (TPA, 2.5 mg / ear). The anti-inammatory actions
were accessed by measurements of ear edema, releasing of myeloperoxidase, tissue levels of TNF-a and histological study. Male Swiss mice,
20-25g (n = 8/group) were treated topically with vehicle (2% Tween 80
or only topical acetone), AB (0.125, 0.25 and 0.5 mg/10 microliters/ear)
or dexametasone (0.05mg/ 10 microliters/ear). The animals received
simultaneously, TPA (2.5 mg in acetone/10 microliters/ear). The ear
edema was recorded by a digital caliper (100.174B/DIGIMESS) before
and 4 hours after administration of TPA. After determining the swelling,
ear tissue was collected for determination of the myeloperoxidases
release, evaluation of TNF-as tissue levels and preparation of slides for
histological study. Tests were considered signicant at p < 0.05. The
group treated with AB (0.125, 0.25 and 0.5 mg) has signicantly
reduced the edema to 0,0920 0,01172, 0,0500 0,005375 and
0,0340 0,005207 respectively, when compared to TPA (0,1110
0,01303 mm). Dexametasone signicantly reduced the edema induced
by TPA (0,0140 0,002211 mm). The AB 0.125, 0.25 and 0.5 mg/ear
signicantly reduced the release of myeloperoxidase in 51,47%, 71,28%
and 81,18% respectively, considering the TPA activity 100%. A considerable reduction (48,43%) in the levels of TNF-a was observed when
animals received a topical dose of AB (0.5 mg /ear). Reductions of dermal edema and inammatory cell inltration was observed on animal
treated with AB (0.5 mg /ear).
Paper No.: 3421

THE ANTI-INFLAMMATORY EFFECT OF THE
PENTACYCLIC TRITERPENES A AND B AMIRYNS ON
ANIMAL MODEL OF DELAYED HYPERSENSITIVITY
INDUCED BY REPEATED APPLICATIONS OF OXAZOLONE
Iana Calou(1), D Goncalves(1), T Olinda(1), T Melo(1), I Figueiredo(1),
GA Brito(1), M Chaves(2), V Rao(1), F Santos(1)
(1) Federal University of Ceara, Department of Physiology and Pharmacology, Fortaleza, Brazil
(2) Federal University of Piaui, Department of Organic Chemistry,
Teresina, Brazil
The pentacyclic triterpenes a and b amiryns (AB), isolated from the gum
of Protium heptaphyllium March, were tested in a model of delayed
213
hypersensitivity induced by repeated applications of oxazolone (OXA)
1% (20 microliter/ear every 3 days for 19 days). The parameters used to
measure the anti-inammatory activity of AB were epidermal hyperplasia, tissue levels of interferon c (INF-G) and histological studies. We
used Swiss mice, 20-25g, male (n = 8). The animals were sensitized with
oxazolone (2% in acetona/50 microliter) topically on the abdomen for
two consecutive days. After six days (considered day 1 of treatment)
solution of 1% oxazolone (20 microliter) in acetone and olive oil (4:1)
was topically applied on the ear every three days for 19 days. AB was
administered topically at doses of 0,125; 0,25 and 0,5 mg/20 microliter/
ear 30 minutes before and three hours after administration of OXA. The
ear thickness was recorded every 3 days before AB application until the
end of the protocol. The hyperplasia was recorded by a digital caliper.
For all tests were considered signicant p < 0.05. On 19th day, AB
(0,125, 0,25 and 0,5) reduced the epidermal hyperplasia in a signicant
way (37,800 1,15300; 34,600 0,80550 and 32,400 1,24900,
respectively) when compared to OXA (44,430 0,99660). At a dose of
0,5 mg/20 microliter/ear, AB reduced in a signicant way tissue levels of
INF-G (19,37 10,23) when compared to OXA (477,2 81,50). Histopathological analysis revealed a marked decrease in epidermal hyperplasia and neutrophil inltration in animals treated with AB (0,5mg/20
microliter/ear).
Paper No.: 718

PIROXICAM PROVIDES ADEQUATE ANALGESIA AFTER
IMPACTED LOWER THIRD MOLAR REMOVAL
REGARDLESS OF THE SURGEONS EXPERIENCE
Adriana Calvo, T Dionsio, F Giglio, P Goncalves, C Santos,
University of Sao Paulo Bauru School of Dentistry, Department of Pharmacology,Sao Paulo, Brazil
The aim of this study was to compare the postoperative pain control in
volunteers medicated with piroxicam who underwent impacted lower
third molar removal performed by three surgeons with distinct experiences (a specialist, a PhD student and a senior dental student of the
Bauru School of Dentistry). Fifty volunteers underwent impacted lower
third molar removal under local anesthesia. Piroxicam (20 mg, once
daily) was prescribed to all patients for four days after the surgical procedure, and paracetamol was provided as rescue medication. The volunteers operated by the senior dental student reported higher pain scores at
3, 4, 7, 8, 10, 12, 18, 24 and 48 h in comparison to the patients operated
by the PhD student (p<.05). The volunteers who underwent surgical procedure performed by the PhD student reported lower pain scores at 8 h
in comparison to those whose surgeries were performed by the specialist
(p<.05). All pain scores were lower than 30 mm, which represents a minimal discomfort for all volunteers. Accordingly, volunteers operated by
the three surgeons used the same small amount of rescue medication
(2,159 192.60mg for the specialist; 2,300 208.3mg for the PhD student and 2,506 228.2mg for the senior dental student). These results
suggest that piroxicam provided adequate pain control after impacted
lower third molar removal regardless of the surgeons experience.
Paper No.: 803
HEALTH AND DISEASE
RAT KIDNEY ANTIOXIDANT ENZYMES IN AN EXPERIMENTAL MODEL OF PREECLAMPSIA
Elsa Camacho, J Silva, MG Matos, M Varela, MR Garrido, A Israel
Central University of Venezuela, Caracas, Venezuela
Preeclampsia (PE) is a multifactorial disorder of pregnancy, characterized
by hypertension, proteinuria, edema, endothelial dysfunction, dyslipidemia and oxidative stress. It has been suggested that nitric oxide (NO) is
the most important mediator for the reduction of peripheral vascular
resistance in normal pregnancy, and it is know that this response is

impaired in preeclampsia. The chronic inhibition of NO synthesis in
pregnant rats causes a syndrome similar to preeclampsia which causes
hypertension, proteinuria, thrombocytopenia and intrauterine growth
retardation. In order to establish the role of oxidative stress in this model
of PE, we assessed the activity of antioxidant enzymes: catalase (CAT),
superoxide dismutase (SOD) and glutathione peroxidase (GPX) in the rat
kidney. Pregnant female Sprague-Dawley rats, with 13 days of gestation
were divided into two groups: control (NaCl 0.9%, 7 days) and
L-NAME (50 mg/kg/day, 7 days). Rats were sacriced by decapitation,
the fetuses and the kidneys were extracted. CAT, SOD, GPX activity was
determined by spectrophotometry and nitric oxide synthase (NOS) by the
convertion of radiolabeled L-arginine to L-citruline. Our results demonstrate that treatment with L-NAME in pregnant rats increased MAP
(+20 mmHg), induced proteinuria and decreased the number and weight
of fetuses. In addition, there was a signicant reduction in renal CAT,
SOD, GPX and NOS activities. These ndings indicate the existence of
an alteration in the renal antioxidant enzymes activity associated with the
chronic inhibition of NOS in experimental preeclampsia, and suggest a
role of oxidative stress in PE.
Paper No.: 1365

MEMANTINE PREVENTS THE COGNITIVE DEFICITS
INDUCED BY METHAMPHETAMINE IN RATS
Jorge Camarasa, T Rodrigo, D Pubill, E Escubedo
University of Barcelona, Department of Pharmacology and Therapeutic
Chemistry, Barcelona, Spain
Methamphetamine (METH) is a street drug abused by young people.
This study seeks to determine whether pre-treatment with memantine
(MEM) counteracts the memory impairment induced by METH in
rodents. In mice, both METH (1 mg/Kg) and MEM (5 mg/Kg) induced
a locomotor stimulant response (AUC saline: 72887 6909, MEM:
280605 25130, P < 0.001, METH: 256680 28431, P < 0.001).
Non-spatial memory was assessed in the Object Recognition Test. MEM
pre-treatment recovered the ability of rats to discriminate between a
familiar and a novel object, which had been abolished by the METH
treatment. METH-treated rats showed impaired learning in the Morris
water maze. The escape latency time was shorter in animals treated with
saline (F2,58 = 5.434, P < 0.01), MEM (F2,123 = 4.402, P < 0.01), or
MEM+METH (F2,213 = 7.263, P < 0.001) as trials progressed. In contrast, METH-treated rats showed a higher escape latency (P < 0.05) than
saline-treated animals. Probe trial demonstrated that animals treated with
saline or MEM spent longer in the target quadrant than in the opposite.
Moreover, MEM alone induced an improvement of learning (time spent
in the target: 25.82 2.44 s and opposite: 7.60 1.30 s). METH- treated
rats showed a cognitive decit (time in the target: 17.40 1.23 s and
opposite: 14.62 2.10 s, not different from random: 15.00 s). MEM pretreatment prevented this decit (23.58 3.22 s, target and 11.14 2.35
s, opposite). MEM constitutes the rst successful approach to prevent the
cognitive decits induced by amphetamine derivatives, irrespective of
their neurochemical prole.
Paper No.: 2209

MODULATION OF LIGAND BINDING TO THE ALPHA1B
ADRENERGIC RECEPTOR: A POTENTIAL ROLE FOR THE
SECOND EXTRACELLULAR LOOP
Adrian Campbell(1), I McDougal(2), R Grifth(0), A Finch(1)
(1) University of New South Wales, School of Medical Sciences, Department of Pharmacology, Kensington, NSW, Australia
214
(2) University of Newcastle, School of Environmental and Life Sciences,
Callaghan, NSW, Australia
Activation of the a1B adrenergic receptor (AR) has previously been attributed to the disruption of an interhelical salt-bridge between an aspartate in
transmembrane helix 3 (TMIII) and a lysine in TMVII (Porter JE et al, J.
Biol. Chem. 1996; 271: 28318-23. Our homology model of the a1B AR
based on the crystal structure of bovine rhodopsin (1U19) identied a
putative salt-bridge between the same lysine (K331) and a second extracellular loop (ECL2) aspartate (D191). We propose that the salt-bridge
between TMVII and ECL2 stabilises an active state of the a1B AR. To test
this hypothesis, we created alanine mutants at positions 191 and 331 to
characterise and compare to wild type (WT) receptor and Porter et als
previously described mutants. Saturation data shows no signicant difference in the binding afnity of 3H-prazosin (WT: 295.9 199.4; D125A:
no binding; K331A: 248.9 117.4; D191A: 482.9 94.5; D191A/
K331A: 372.7 96.7 pM, n = 4). Competition binding experiments
show a signicant increase in logKi for (-)epinephrine for D191 mutants
(WT: -5.624 0.03; K331A: -5.641 0.03; D191A: -4.854 0.06;
D191A/K331A: -4.953 0.12 M, n = 3, P < 0.001). Functionally, no
mutants have a signicantly different logEC50 compared to WT (WT:5.534 0.21; K331A: -5.887 0.15; D191A: -5.534 0.25; D191A/
K331A:-5.627 0.11 M, n = 3). Further studies will determine the
effects of these mutations on antagonist inhibition of receptor activation.
The apparent conict between binding and activation data for (-)epinephrine suggests that in the absence of the charge on D191, the receptor is
biased towards the inactive state, decreasing afnity for (-)epinephrine,
but once bound, the receptor can still adopt the active state.
Paper No.: 519

AN OVERVIEW ON ATTENUATION OF DRUG-INDUCED
NEPHROTOXICITY
Guoying Cao, X Hu
Beijing Hospital, Department of Clinical Pharmacology, Beijing, PR
China
Amino glycoside antibiotics, anti cancer drugs, immunosuppressant,
radio contrasts and TCM are associated with nephrotoxicity. Druginduced renal dysfunction generally induces a lysosomal phospholipidosis which is accompanied by cellular necrosis and postnecrotic cell
regeneration. Drug toxicity remains an important cause of acute kidney
injury that, in many circumstances, can be prevented or at least minimized by early intervention. Hydration therapy and structure optimization are widely used in preventing nephrotoxicity of cisplatin while preor coadministration of inhibitors are still questionable. In this article we
review the most prevalent drugs which induce nephrotoxicity, and from
the point of clinical and laboratory research view, analyze the available
evidence for preventive measures.
Paper No.: 2387

HEALTH AND DISEASE
INCREASED SENSITIVITY OF CEREBRAL AND CORONARY
ARTERIES TO GPCR AGONISTS IN SPONTANEOUSLY
HYPERTENSIVE RATS
Lei Cao(1), Y Zhang(1), Y-X Cao(2), L Edvinsson(1), C-B Xu(1)
(1) Lund University, Institute of Clinical Science, Division of Experimental Vascular Research, Lund, Sweden
(2) Xian Jiaotong University College of Medicine, Department of Pharmacology, Xian, Shaanxi, PR China
Hypertension is a major risk factor closely associated with myocardial

infarct and stroke. However, the underlying molecular mechanisms
describing how hypertension leads to vascular damage are not fully
understood. Vascular G-protein coupled receptors (GPCR) mediate vascular smooth muscle contraction and proliferation, which contribute to
narrowing of vascular lumen. The present study was designed to examine
the hypothesis that in hypertension, there is increased sensitivity of cerebral and coronary arteries to vasoconstrictors that activate GPCR. Cerebral and coronary arteries were removed from spontaneously
hypertensive (SHR, n = 8) and normal tension (n = 14) rats. Contractile
responses to GPCR agonists (U46619, thromboxane A2 receptor agonist;
sarafotoxin 6c, endothelin type B receptor agonist and endothelin-1,
endothelin type A and B receptor agonist) were recorded by a sensitive
myograph. Real-time PCR was used to semi-quantify GPCR mRNA
expression. The results show that the contractile responses to U46619,
sarafotoxin 6c and endothelin-1 in cerebral and coronary arteries were
increased in SHR, compared with the arteries from normotensives. In
parallel, there were increased mRNA expressions for thromboxane A2,
endothelin type A and B receptors, suggesting involvement of transcriptional mechanisms. In conclusion, the present study suggests that
increased vascular GPCR expression in hypertension is a mechanism that
may in part mediate the increased sensitivity of cerebral and coronary
arteries seen in hypertension.
Paper No.: 2388

LONG-TERM CIGARETTE SMOKE EXPOSURE INDUCES
BRONCHIAL HYPERRESPONSIVENESS TO G-PROTEIN
COUPLED RECEPTOR AGONISTS
Lei Cao(1), Y Zhang(1), Y-X Cao(2), L Edvinsson(1), C-B Xu(1)
(1) Lund University, Institute of Clinical Science, Division of Experimental Vascular Research, Lund, Sweden
(2) Xian Jiaotong University College of Medicine, Department of Pharmacology, Xian, Shaanxi, PR China
Cigarette smoke exposure strongly associates with airway hyperreactivity (AHR) and inammation. The present study was designed to examine if G-protein coupled receptors (GPCR), inammatory mediators and
Raf-1 activity are involved in the smoke-induced airway pathogenesis.
SD rats (200-300g) were divided into three groups (control, smoke
exposure and GW5074 treatment; n = 10). In smoke exposure group,
rats were exposed to cigarette smoke generated from 10 cigarettes/day
for 8 weeks. Control rats received fresh air only. GW5074 (a Raf-1
inhibitor) treatment group was exposed to the same amount of smoke
and treated by intraperitioneal administration of GW5074 (0.5mg/kg)
every day for 8 weeks. At the end of the experiments, bronchi were
isolated from all groups and cut into ring segments. Contractile
responses of bronchial segments were recorded by a sensitive myogragh. The mRNA expression for GPCR and inammatory mediators
were semi-quantied by real-time PCR. The results show that after
8 weeks of smoke exposure, the airway exhibited increased contractile
responses to carbachol, 5-HT, sarofotoxin 6c and endothelin-1, which
was parallel with enhanced mRNA expression for 5-HT2A, endothelin
type A and B receptors. In addition, there was increase in mRNA
expression of inammatory mediators (IL-1beta, TNF-a and ICAM-1)
in smoke group. Treatment with GW5074 throughout the smoke exposure period abrogated the smoke-induced AHR and inammation. In
conclusion, long-term cigarette smoke exposure results in airway
inammation and AHR to GPCR agonists. Inhibition of Raf-1 activity
might provide new strategies for the treatment.
215
Paper No. 1222
FOCUS GROUP: P09 - NFLAMMATION AND
MITIGATION EFFECT OF ESCULENTIC ACID ON
RADIATION-INDUCED PNEUMONIA AND PULMONARY
FIBROSIS
Yong-Bing Cao(1), S-M Yang(2), Y-P Tian(1), M Zhang(2), L Zhang(3),
K-Z Zhang(2), L-J Yin(2), B-R Zhang(2), R Howell(2), Y-Y Jiang(1) P
Okunieff(2) L Zhang(2)
(1) Second Military Medical University School of Pharmacy, Department
of Pharmacology, Shanghai, PR China
(2) University of Rochester Medical Center, Department of Radiation
Oncology, Rochester, NY, USA
(3) West China Hospital of Sichuan University, Chengdu, Sichuan, PR
China
The effect of Esculentic acid (EA) on radiation-induced pneumonia and
pulmonary brosis and its possible molecular mechanisms were investigated in lung irradiated mouse model. C57BL/6 mice (5-10/group) were
received 15 Gy ionizing radiation (IR) on whole lung. Thirty minutes
late, the mice treated with vehicle alone (as control group) or 10 mg/kg
Esculentic Acid (as test group) orally every other day for days (pneumonia group) or up to 3 months (brosis group). At the different time points
post IR (2.5, 17 days for pneumonia group, and 7 months for brosis
group), the mice were sacriced, the BAL (bronchial alveolar lavage),
plasma and lung tissue were collected and assess for the alterations in
BAL cell counts and differentiation, cytokines and lung brosis with
means of microscope, ELISA or pathological staining. The lung function
was assessed by the breath rate and compliance. The results showed that:
1) EA inhibited neutrophil in BAL 17 days post-radiation; 2) EA
reduced IR-induced P-selectin, PF-4, sTNFR-1, Lymphotaxin on day 2.5
and 17; the IR-induced IL-1b,TNF-a and VCAM-1 were also reduced
on day 2.5; 3) the EA inhibit lung acute and sub-acute inammatory
response induced by radiation; 4) EA reduced the breath rate and
increased the lung compliance 7 months post-radiation; and 5) EA
decreased the amounts of collagen deposited in lung tissue as compared
to the vehicle alone treated mice. In conclusion, the EA mitigates
IR-induced pneumonia and pulmonary brosis via inhibition of the productions of several pro-inammatory cytokines and inammatory mediators.
Paper No.: 823

REMICADE ALLEVIATES AIRWAY INFLAMMATION AND
HYPERREACTIVITY IN ASTHMATIC E3 RATS
epithelial damage. Serum levels of IgE increased, and so did the levels
of NO, iNOS, TNF-a, and IL-4, IL-5, IL-13 in lung homogenate and
serum. Further, the contractile responses in bronchi induced by endothelin-1, sarafotoxin 6c and bradykinin increased, and isoprenalineinduced relaxations decreased. All these changes induced by the sensitization procedure were reduced by the remicade treatment. The results
suggested that Remicade prevented the development of local airway
inammation and antagonized changes of the bronchial smooth muscle
receptor phenotype, thereby blocking the development of airway hyperreactivity of asthmatic rats.
Paper No.: 2668

MMLDL UPREGULATES ENDOTHELIN TYPE A AND TYPE B
RECEPTORS IN RAT BASILAR ARTERY
Yong-Xiao Cao(1), J Li(1), ZY Yuan(1), L Cao(2), CB Xu(2)
(1) Xian Jiaotong University College of Medicine, Department of
Pharmacology, Xian, PR China
(2) Lund University, University Hospital of Lund, Institute of Clinical
Science, Division of Experimental Vascular Research, Lund, Sweden
Mminimally modied low density lipoprotein (mmLDL) can induce an
inammatory response and enhance plaque formation in arteries. Vascular endothelin type A (ETA) and type B (ETB) receptors are implicated
in the pathogenesis of cardiovascular diseases. The present study was
designed to test if mmLDL upregulates ET receptors in rat basilar artery.
Rat basilar artery ring segments were cultured with mmLDL 10 g/ml for
24 h. Tension of the ring segments was recorded by a sensitive myogragh. The mRNA expressions and proteins of ETA and ETB receotors
were semi-quantied by real-time PCR and western blot techniques,
respectively. The results showed that organ culture with mmLDL
increased the contractions induced by sarafotoxin 6c and endothelin-1
and enhanced the expression of mRNA and proteins of ETB and ETA
receptors in in rat basilar artery, suggesting mmLDL up-regulated ETB
and ETA receptors. The general PKC inhibitors, staurosporine, prevented
the upregulation. MAPK inhibitors for extracellular signal related kinases
1 and 2 (ERK1/2), SB386023 and U0126, p38 MAPK, SB203580 inhibited the upregulation of ETB and ETA receptors. C-jun terminal kinase
(JNK), SP600125 and NF-jB, wedelolactone inhibited the upregulation
of ETB. In conclusion, mmLDL up-regulates ETB and ETA receptors in
rat basilar artery. PKC, MAPK and NF-jB signalling pathways seem
involved in the up-regulations.
Yong-Xiao Cao(1), Y Cai(1), SM Lu(1), CB Xu(2), LO Cardell(3)

(1) Xian Jiaotong University College of Medicine, Xian, Shaanxi, PR
China
(2) MedDepartment of Experimental Vascular Research, Institution of
Medicine, Lund University, Sweden
(3) Department of Otorhinolaryngology, Malmo University Hospital,
Malmo, Sweden
Tumour necrosis factor-a (TNF-a) has been implicated in pathogenesis
of asthma, and neutralization of TNF-a is an effective therapy for inammatory diseases. The present study tested the idea that a TNF-a antibody,
remicade, may be useful in the management of asthma. E3 rats were sensitized to OVA/alum and received remicade intraperitoneally. Two weeks
later OVA-PBS was intranasally instilled daily for 7 days. Bronchoalveolar lavage uids (BALF), serum and lung homogenates were collected
for analysis of cells and inammatory mediators. Contractile responses
of lobar-bronchus segments to agonists were tested. Pulmonary tissues
were investigated using histological examination. The results showed that
the sensitized model E3 rats exhibited an increase of the total amount
of cells, primarily eosinophils in BALF and pulmonary tissue, and
Paper No.: 420

INVESTIGATION OF THE RELATIONSHIP BETWEEN
PLASMA L-CARNITINE CONCENTRATION AND ITS
ANTIOXIDANT ACTIVITY AFTER SINGLE ORAL
ADMINISTRATION OF L-CARNITINE IN HEALTHY
VOLUNTEERS
Yu Cao(1), Z-W Han(1), HJ Qu(1), C-B Wang(2)
(1) The Afliated Hospital of Medical College Qingdao University,
PharmacyDepartment, Qingdao, PR China
(2) Department of Pharmacology, Medical college Qingdao University,
Qingdao, PRChina
Introduction: This study was to investigate the plasma concentration
of L-carnitine (LC) after a single oral administration of LC solution in
healthy volunteers and its antioxidant activity. The plasma antioxidant
enzymes and total antioxidant were evaluated through the measure-
216
ment of SOD, GSH-Px, CAT and T-AOC activities in plasma, and
evaluate the relationship between concentration and antioxidant index
in plasma of healthy humans. Materials/Patients: Liquid LC (2.0g)
was administered orally as a single dose in 12 healthy subjects.
Plasma concentration of LC was detected by HPLC in 24h, and the
plasmas were subjected to the measurement of SOD, GSH-Px, CAT
and T-AOC activities by spectrophotometric methods to evaluate the
relationship between concentration and antioxidant activity of LC in
plasma. Results: Antioxidant enzymes,total antioxidant and plasma
concentration increased gradually from 0h to 3.5h point and reached
the peak at 3.5h,then go down gradually, and were as the same level
as the beginning(0h point) at 24h point. Correlation analysis was
studied between plasma concentration and antioxidant activity through
linear regression. The regressive equations were Y = 0.5319X+3.2429
(r = 0.9922), Y = 0.1606X+103.63(r = 0.9316), Y = 0.019X+0.3845
(r = 0.9720),Y = 0.1346X+5.4452 (r = 0.9338) respectively between
LC plasma concentration and antioxidant index (SOD, GSH-Px, CAT,
T-AOC). The antioxidant activity of LC was signicantly positive
correlation with plasma concentrations of LC after single oral administration of L-carnitine in healthy volunteers. Conclusion: Administration
of liquid LC could raise the activities of antioxidant enzymes and total
antioxidant capacity in a concentration-dependent manner of LC in
plasma.
Paper No.: 2373

RESCUE OF TGF-B1 SIGNALLING AS A NEW
NEUROPROTECTIVE TOOL IN ALZHEIMERS DISEASE
Filippo Caraci(1), G Battaglia(2), ML Giuffrida(1), V Bruno(2),
P Bosco(3), MA Sortino(4), F Drago(4), F Nicoletti(2), A Copani(1)
(1) University of Catania, Department of Pharmaceutical Sciences, Catania, Italy
(2) I.N.M. Neuromed, Pozzilli, Italy
(3) IRCCS Oasi Maria S.S, Troina, Italy
(4) University of Catania, Department of Experimental and Clinical Pharmacology,Catania, Italy
Introduction: Transforming growth factor-b1 (TGF-b1) is a neurotrophic
factor that exerts neuroprotective effects in different models of neurodegeneration, including Ab toxicity (Vivien D & Ali C. Cytokine &
Growth Factor Reviews 2006; 17:121-28). Nevertheless the molecular
mechanisms underlying the neuroprotective effects of TGF-1 against
Ab-induced neurodegeneration are presently unknown. Materials and
results: We examined the neuroprotective activity of TGF-b1 in pure cultures of rat cortical neurons challenged with synthetic Ab. Ab induced
the apoptosis of about 30-40% of the total neuronal population. TGF-b1
was equally protective when added either in combination with, or
6 hours after Aba. Co-added TGF-b1 prevented Ab-induced cell cycle
reactivation, whereas lately added TGF-b1 rescued late b-catenin degradation and s hyperphosphorylation. Interestingly, lithium, a mood stabilizer that protects against Ab-induced neurodegeneration, induced both
the synthesis and the release of TGF-b1 from cultured astrocytes. We
also assessed the neuroprotective activity of endogenous TGF-b1 in vivo.
Ab injection into the rat dorsal hippocampus had a small neurotoxic
effect that was amplied by i.c.v. injection of SB431542, a selective
inhibitor of TGF-b1 receptor, thus suggesting that endogenous TGF-b1
acts as a factor limiting Ab toxicity. Conclusions: Our data demonstrate
that TGF-b1 is neuroprotective in vitro and in vivo against Ab-induced
neurodegeneration, and suggest that drugs able to increase TGF-bb1,
such as lithium, might represent a new neuroprotective tool for the treatment of AD.
Paper No.: 2359

HEALTH AND DISEASE
CORRELATION BETWEEN AMINE OXIDASES KINETIC
PARAMETERS AND NITRIC OXIDE PRODUCTION IN
HUMAN MESENTERIC ARTERIES
Maria Margarida Caramona(1), S Nunes(1), J Pereira(2), C Lopes(1), I
Figueiredo(1)
(1) University of Coimbra Faculty of Pharmacy, Laboratory of Pharmacology, Coimbra, Portugal
(2) Portuguese Oncology Institute, Coimbra, Portugal
There are evidences suggesting that the enzymes monoamine oxidase
(MAO, type A and B), semicarbazide-sensitive amine oxidase (SSAO)
and nitric oxide sintase (NOS) play a broad role in the diabetes1,2. However, it is not known the correlation between NO production and the
activity of the enzymes MAO and SSAO. Therefore, the aim of this
work was to study the correlation between the nitrite and nitrate (NOx)
levels and the kinetic parameters of these enzymes in human inferior
mesenteric arteries (n = 12). The NOx concentrations were determined
by Griess reaction and the kinetic parameters were evaluated by radiochemical assay. We found that the NOx levels (57.24 19.60lmol/l))
were positively correlated with MAO-B parameters: Km (r = 0.612,
p = 0.034) and Vmax (r = 0.593, p = 0.042), but it was negatively correlated with SSAO parameters: Km (r= - 0.625, p = 0.029) and Vmax (r=
-0.754, p = 0.0046). In conclusion, our results demonstrate that the correlation between NOx levels with MAO B and SSAO could be a nding
with relevance in some diseases, such as type 2 diabetes, and they are
the rst step for therapeutic strategies using specic inhibitors of these
enzymes.
1 Irer SV et al, Neurotoxicology 2007; 114:811-815.
2 Nunes SF et al, Acta Diabetol 2009; 46: 135-140.
Supported by grant SFRH/BD/23606/2005 from FCT-Portugal.
Paper No.: 2360

PHARMACOLOGICAL TREATMENT OF POSTSTROKE
DEPRESSION
Maria Margarida Caramona(1), E Silva(2), B Oliveiros(3),
E Ponciano(3)
(1) University of Coimbra Faculty of Pharmacy, Laboratory of Pharmacology, Coimbra, Portugal
(2) Comunity Pharmacy, Coimbra, Portugal
(3) University of Coimbra Faculty of Medicine, Laboratory of Pharmacology, Coimbra, Portugal
The issue of poststroke depression has gained worldwide interest in the
past 10 years. There has been a general agreement about the prevalence
of both major and minor depression after stroke. Depression is one of the
major impediments to full physical and mental recovery from stroke. The
aim of this study was to dene the prevalence of depression in stroke
survivors and assess the treatment as well as the impact on their quality
of life. It was designed a prospective study. Patients under antihypertensive therapy visiting community pharmacies all over Portugal were
recruited. Patients medication data, gender, age, smoking history, alcohol
intake, medical conditions, quality of life, were collected in a validated
self administered instrument. The sample included 155 patients (mean
age =66,91 + /- 10 years, 52,9% female) recruited during 2007 that had
a previous stroke and gave informed consent for a direct interview. 30%
of those assessed were depressed. 16% were taking an antidepressant
medication. Poststroke depression and low quality-of-life seem to be
more common among women. Women have less favourable functional
outcome because of higher age at stroke onset and more severe strokes.
Psychiatric disorders, mostly depression, are fairly common in stroke
217
survivors. Although being depressed, few patients were taking antidepressants. We consider that the prevalence of depression is relevant and
that the risk factors as well as the early diagnosis are important for the
best management and prognosis of stroke patients. Further exploration of
this low level of treatment is warranted.
Paper No.: 2931

SESSION - ONCOLOGY
IONIZING RADIATION ALTERS THE BONE MARROW
MICROENVIRONMENT BY INDUCING SENESCENCE OF
MESENCHYMAL STROMAL CELLS
Cynthia Carbonneau(1,2), G Despars(1,2), A Fortin(1,2), O Le(1,2),
T Hoang(1,3), CM Beausejour(1,2)
(1) Universite de Montreal, Department of Pharmacology, Montreal,
Canada
(2) CHU Ste-Justine, Montreal, Canada
(3) IRIC, Montreal, Canada
The extent to which the integrity of the bone marrow (BM) stroma contributes to the success of hematopoietic stem cell (HSC) transplantation
is not well known. Alterations of the BM microenvironment, which has
been shown to occur during aging or following genetic manipulations of
telomere length, impair HSC functions and engraftment. These results
suggest that increased number of senescent marrow stromal cells could
affect the niche functions. Exposure to ionizing radiation (IR), by inhibiting marrow stromal cells colony forming unit (CFU) potential, is
believed to severely impair the BM microenvironment. Here, we demonstrate that IR alters the niche by inducing a p16INK4a-dependent senescence phenotype in marrow stromal cells. Intriguingly and in contrast to
p21 expression, p16INK4a expression is delayed post-IR. In accordance
with this observation, IR-induced long-term loss of BM stromal cell
CFUs potential was partially rescued in p16INK4a decient mice. Moreover, while the absence of p16INK4a expression did not prevent
IR-induced short-term homing impairment, it improved homing potential
long-term post-IR. In addition, IR could contribute to alter BM microenvironment by inducing a p16-independent increase in G-CSF expression.
Indeed, increased levels of G-CSF, a cytokine known to induce HSC
mobilization, were detected in the bone cavity shortly after IR. Co-culture experiments of stromal cells with hematopoietic progenitors further
conrmed that irradiated stromal cells secrete greater levels of G-CSF
than control cells. Together, our results provide a mechanistic link as to
how irradiation, by inducing senescence of the stroma and G-CSF
expression, could impair the function of the hematopoietic niche.
Paper No.: 754

THE EFFECT OF CHEMICAL ABSENCE MODEL ON THE
PROGRESSION OF KINDLING IN WISTAR RATS
Nihan Carcak(1), M Sahiner(2), E Sakally(3), K Tezcan(3), R Gulhan(3),
F Onat(3)
(1) Istanbul University Faculty of Pharmacy Department of Pharmacology, Istanbul, Turkey
(2) Acybadem University Faculty of Medicine, Department of Physiology, Istanbul, Turkey
(3) Marmara University Faculty of Medicine Department of Pharmacology and Clinical Pharmacology, Istanbul, Turkey
Thalamo-limbic circuits are thought to regulate limbic seizure activity
whereas thalamo-cortical circuits are involved in the expression and generation of spike-and-wave discharges (SWDs) in the absence epilepsy
models. Kindling as a model of temporal lobe epilepsies is a well known
model which causes secondary generalized seizures in rats. Recent studies showed that there is resistance in relation with SWD activity to the
kindling progress in animal models of genetic absence epilepsy. c-Butyrolactone (GBL), the prodrug of GHB (c-Hydroxybutyric acid), represent a chemical experimental model of generalized absence seizures. In
this study we aimed to determine whether the resistance to the development of kindling in absence epilepsy can be independent of the genetic
background. Electrodes were stereotaxically implanted into the basolateral amygdala. After a recovery period, the animals were stimulated at their
afterdischarge thresholds twice daily. GBL+KI animals were stimulated
20 min after the intraperitoneal (i.p) administration of GBL twice daily
until they reached stage 5 seizure state. KI animals were stimulated without GBL administration. The seizure severity was evaluated by Racines
scale. The KI animals had stage 5 seizures by 15 stimulations. However,
GBL-KI group reached stage 5 by 30 stimulations. Our data show a
delay in the development of kindling as well as a relation of SWD activity to the kindling progress after GBL administration. The resistance to
limbic epilepsy in absence epilepsy rats seems to be partially inuenced
by the absence epilepsy itself and possibly also be the genetic background.
Paper No.: 1389

FOCUSED CONFERENCE GROUP: P19 - GENERAL 1
SESSION - ONCOLOGY
TEMOZOLOMIDE INDUCES APOPTOSIS AND AUTOPHAGY
IN GLIOMA CELLS
A Carmo(1,2), H Carvalheiro(1), Maria Celeste Lopes(1,2,3)
(1) Center for Neuroscience and Cell Biology, Department of Cellular
Immunology and Oncobiology, Coimbra, Portugal
(2) CIMAGO, University of Coimbra, Coimbra, Portugal
(3) University of Coimbra, Faculty of Pharmacy, Coimbra, Portugal
Glioblastoma is the most malignant primary brain tumor, comprising
approximately 50% of the cerebral gliomas. Despite all the efforts to
improve the efcacy of the treatment, the median survival time for GBM
patients remains approximately 12-14 months. This relative lack of success in GBM therapy is probably due to the genetic and cellular heterogeneity of these tumours and to the development of chemotherapeutic
resistance. The chemotherapeutic treatment of glioma is based on the use
of temozolamide (TMZ). Nevertheless, the median survival of glioma
patients treated with TMZ is 14.6 months. In order to understand the
effect of TMZ in glioma cells, we studied apoptosis and autophagy
induction in TMZ-treated glioma cells. For that, U-118 glioma cells were
incubated with different concentrations of TMZ for different periods of
time. Cell viability was assessed by the MTT assay and cell proliferation
was determined using BrdUrd incorporation. Apoptosis was addressed
by confocal microscopy using hoescht and propidium iodide and by ow
cytometry. Autophagy was evaluated through the LC3 expression which
was determined by western blot. The results indicated that TMZ reduced
the U-118 cell viability and proliferation. Regarding the survival, the
results showed that TMZ induced apoptosis and simultaneously autophagy. Since authophagy is a survival pathway, the low efcacy of TMZ
in the treatment of gliomas may be related with the activation of
autophagy.
Paper No.: 2992

HEALTH AND DISEASE
CORONARY ARTERY BYPASS GRAFTING SURGERY
WORSENS OXIDATIVE STRESS AMONG PATIENTS WITH
CORONARY DISEASE
Manuel Carnero(1), J Munoz-Marin(2), JA Gonzalez Correa(2),
F Reguillo(1), J Navarro-Dorado(3), M Ramajo(3), JP de la Cruz(2),
T Tejerina(3)
218
(1) Hospital Clnico San Carlos, Service of Cardiac Surgery, Madrid,
Spain
(2) Universidad de Malaga, School of Medicine, Department of Pharmacology, Malaga,Spain
(3) University Complutense of Madrid, School of Medicine, Department
of Pharmacology, Madrid, Spain
Introduction: Oxireduction enzymatic mechanisms in human beings can
be physiologically altered due to inammatory or infectious stimuli to
the organism. Inammatory response to heart surgery has been widely
studied, but, up to date, little is known about its possible inuence over
oxidative stress. We studied the variations of multiple oxidative stress
related products and enzymes in a cohort of patients who underwent a
coronary artery bypass grafting surgery (CABG). Methods: We measured
2 hours before and 24 after CABG the concentration of malondialdehidic
acid (MDA), nitrates, and peroxynitrites (all of them considered to be
prooxidative markers); and reducted glutation and mithocondrial superoxide dismutase (SOD-Mn) (antioxidative markers) in a cohort of
patients with coronary disease in a single center. Results: 119 patients
were included in the present study. Statistically signicant differences
were detected in the mean plasmatic MDA concentration before (0.148
mmoL/L (SD 0.12)) and after (0.283 mmoL/L /SD 0.16) surgery
(P < 0.001). Higher concentrations of peroxynitrates (p = 0.443) and
nitrates (p = 0.078) were also detected, though differences did not reach
statistical signicance. On the other hand, lower levels of reducted glutation and SOD-Mn were detected after surgery (p = 0.94 and p = 0.070),
though differences were not signicant. Conclusions: CABG surgery
worsens the oxidative stress in patients with coronary disease higher oxidation activity and greater concentrations of its products and a lower
antioxidant activity.
Paper No.: 3143

ORAL PHARMACOKINETICS OF THREE
FLUOROQUINOLONES IN MEXICANS: EVIDENCE FOR
INTERETHNIC DIFFERENCES
Miriam Carrasco-Portugal(1), G Lozoya-Moreno(2),
S Patino-Camacho(2), F Flores-Murrieta(1,2)
(1) Instituto Nacional de Enfermedade Respiratorias, Mexico City,
Mexico
(2) Escuela Superior de Medicina del Instituto Politecnico Nacional,
Mexico City, Mexico
Interethnic differences in the oral pharmacokinetics of drugs metabolized
by CYP3A4, i.e. nifedipine, cyclosporine, midazolam, sildenal, meloxicam, have been reported, Mexicans reaching higher plasma levels than
Caucasians. It is assumed that such differences are due to a reduced
metabolism by CYP3A4 in Mexicans. However, very frequently, drugs
metabolized by CYP3A4 are also substrates of p-Glycoprotein (p-GP)
and, therefore, participation of this p-GP may contribute to such differences. In this study, we evaluated the oral pharmacokinetics of three
uoroquinolones (ciprooxacin, levooxacin and gatioxacin) that are
p-GP substrates and they are not metabolized by CYP3A4. Seventyseven male healthy subjects were enrolled in this study. The volunteers
were divided in three groups. The rst group received an oral dose of
250 mg of ciprooxacin, the second 500 mg of levooxacin and the
third 400 mg of gatioxacin. Blood samples were drawn at selected
times and plasma was obtained and stored frozen until analyzed by selective high-performance liquid chromatographic methods. Pharmacokinetic
parameters were obtained by non-compartmental analysis and compared
with those reported in other populations. After administration of the
drugs, plasma levels increased rapidly reaching the maximal concentration (Cmax) in about 60 to 90 minutes. Cmax and AUC values observed
in Mexicans were higher than the values described in studies conducted
in Caucausians, however, no important changes in half-life were
observed. These results suggest increased bioavailability of ciprooxacin,
levooxacin and gatioxacin in Mexican subjects, maybe due to

increased absorption of these drugs, by a lower activity of p-GP.
Paper No.: 2395

THIOPURINE METHYLTRANSFERASE
PHENOTYPE-GENOTYPE CONCORDANCE IN CHILDREN
WITH ACUTE LYMPHOBLASTIC LEUKAEMIA
Cher Cartwright(1), C Mitchell(2), N Goulden(3), A Vora(1),
L Lennard(1)
(1) University of Shefeld, Department of Clinical Pharmacology,
Shefeld, UK
(2) University of Oxford, Oxford, UK
(3) Great Ormond Street Hospital, London, UK
The clinical importance of the thiopurine methyltransferase (TPMT)
genetic polymorphism has been illustrated by many studies which have
linked the inheritance of lower TPMT activities with thiopurine sensitivity, specically bone marrow toxicity. The aim of this multi-centre study
was to assess TPMT phenotype-genotype concordance in children with
acute lymphoblastic leukaemia (ALL) treated according to the UKALL2003 trial. Blood samples were requested at 100% protocol mercaptopurine (75mg/sqm), or the maximum tolerated, dose. Red cell TPMT
activity and mercaptopurine derived cytotoxic thioguanine nucleotide
(TGN) metabolites were measured; samples were screened for TPMT*2
and the TPMT*3 family of low-activity variant alleles. Ranges were
compared by the Mann-Whitney test. Samples were forwarded from 800
children (median dose 75mg/sqm, range 7 to 122). TPMT activities ranged from 0 to 26.4 units, median 14.1; 733 children were wild-type, 56
TPMT*1/*3A, 7 TPMT*1/*3C, two TPMT*3A/*3C, one TPMT*3A/
*3A and one TPMT*1/*2. The sensitivity for detecting the variant allele
at 9.5units TPMT was 84% (specicity 94%), at 10.5units 95% (specicity 90%) and at 11.5units 98% (specicity 84%). At 10.5 units the overall concordance was 91%, but only 46% within the heterozygous
phenotype group <10.5units TPMT. Median TGN concentrations in children <10.5 units were 545pmol and >10.5 units 323pmol. Within the
<10.5unit cohort children with a variant allele (n = 61) had 737pmol
compared to 361pmol in wild-type children (n = 72), median difference
354pmol P < 0.001 95% CI 263 to 451. Wildtype children with TPMT
activity less than 10.5units did not exhibit the same metabolite prole as
heterozygous variant allele children.
Paper No.: 1569

PATIENTS ACCESS TO ANTIRETROVIRAL MEDICINES:
HOW DO THEY VIEW THEMSELVES?
DM Carvalho, MH Magalhaes, AMR Felix, ECA Costa, WA Souza,
OMGC Vilas-Boas, Marcia HMC Podesta
Federal University of Alfenas, Department of Pharmacy, Alfenas, Minas
Gerais, Brazil
The Acquired Immune Deciency Syndrome (AIDS) shows a life impact
on the individuals, although the offer of costless treatment to the patient.
The meaning of this research is to analyze the perceptions of the quality
of life on those patients who receive medicines from a public health
unity of Alfenas, Brazil. The patients answered a questionnaire, between
September and October 2009, about specic questions of their disease
followed by the investigation SF-36 (de Soarez, PC et al Pan Am J Public Health 2009; 25: 69-76)This study was approved by the Ethic Committee in Human Research (nordm; 23087003008/2009). From the 92
patients that are in medicament treatment for about 5 years, 49% are
219
female. The most of them are singles (40%) with age predominantly
between 30 to 45 years old (52%), primary school instruction uncompleted (51%) and family earnings approximately US$ 375 by month
(51%). When inquired about AIDS knowledge, patients reported sexual
contamination (75%) and said that they understand, but hide this condition (65%). Concerning about life quality, the most preoccupants factors
for them are physical aspects (56%), vitality (57,8%) and emotional
aspects (60,1%), on media averages between 0 to 100. We concluded that
the AIDS treatment has interference on the self-perceptions of these individuals, already affected by social and cultural conditions.
Paper No.: 2890

MOLECULAR MECHANISMS OF PISTACIA LENTISCUS
ESSENTIAL OIL ANTIPROLIFERATIVE ACTIVITY
from mRNA extracted from 12 pooled samples of internal mammary

artery obtained with written informed consent from seven individuals
undergoing coronary artery bypass graft surgery. The study was
approved by the local research ethics committee. Pooled tissue samples
(4.3 g, sample weight 355 302 mg SD, n = 12) yielded 608 ll total
RNA (sample RNA 51 25 ll SD). A 167 lg sample of this total RNA
gave 2.48 0.008 lg mRNA (SD, n = 2) and was used to prepare a
cDNA library using the OrientExpress Oligo d(T) Primer cDNA kit from
(Novagen #69992-3) from which the phage display library was prepared
using the T7 Select Phage Display System (Novagen #70550-3). Simvastatin was immobilised on a pre-prepared Magic Tag 96-well plate
and a preliminary screen against the new library revealed 25 sequences,
ve of which are of interest for further study. This method has the potential to be scaled to an individual subjecs sample, to allow Magic Tag
analysis of rare diseases and uncommon serious adverse events as well
as to improve personalising treatment to increase efcacy and reduce toxicity in more common, serious disorders.
Elisabetta Casarin(1), B Barocco(1), A Buriani(2), M Carrara(1)

(1) Padova University, Department of Pharmacology and Anesthesiology,
Padova, Italy
(2) Data Medica Research Labs, Padova, Italy
The phytocomplex from Pistacia lentiscus, a shrub of the Anacardiaceae
family, is an essential oil obtained by hydrodistillation of leaves, fruits or
from a trunk exudate (mastic gum). The mastic gum has been known to
be effective in several gastric diseases, against Helicobacter Pylori and
for its antibacterial and antifungine activities. Furthermore, Pistacia oils
major chemical constituents are monoterpenes with chemiopreventive
and chemiotherapic properties. We investigated the antiproliferative properties of the volatile oil from Pistacia lentiscus twigs and leaves using
human cell lines from ovarian (2008 and C13*) and colon (LoVo) adenocarcinoma, and human stable broblast line (HFFF2) as in vitro models.
The MTT test showed that, after 3 hour treatment, phytocomplex (about
150 lg\ml) was able to inhibit the growth of all adenocarcinoma cell
lines. After 24 hour treatment the IC50 on 2008 and LoVo cells resulted
3 times lower. On broblast line the phytocomplex was active only after
72 hour treatment. Western blot analysis conrmed the oil capability to
reduce carcinoma cell growth by decreasing the expression of p-ERK,
MAPKs induced by mitogenic stimuli. Using AnnexinV with propidium
iodide we observed that oil was able to stimulate apoptosis in a dosedependent manner. Analysis of mitochondrial membrane potential, with
Rhodamine123, and ROS generation, with H2DCF-DA, showed the oil
capability to activate mitochondrial apoptotic pathway. Our data also
indicate that Pistacia lentiscus oil caused programmed cell death via a
caspase-independent pathway. We also performed a ow cytometrybased cell cycle analysis, observing that the phytocomplex induced dosedependent arrest in G2M phase.
Paper No.: 1526

A CHEMICAL GENOMICS APPROACH TO
CARDIOVASCULAR THERAPEUTICS
Paper No.: 1527

PERSONALISED THERAPIES USING A CHEMICAL
GENOMICS APPROACH
Katherine Casey-Green(1), A Marsh(1), P Taylor(1), S Ladwa(2), W
Dimitri(3), D Singer(4)
(1) University of Warwick, Coventry, UK
(2) Tangent Reproling Ltd, Bicester, UK
(3) University Hospital, Coventry, UK
(4) Clinical Sciences Research Institute, Coventry, UK
Therapeutic agents may interact with the body in many ways apart from
the major identied mechanism, and often the range of targets through
which a clinical effect is brought about remain unclear. It is desirable to
characterise more fully the action of a pleiotropic therapeutic upon a system, identifying each of the proteins with which it interacts in the cell. In
previous studies, we have developed and tested proof of concept that the
new Magic Tag chemical genomics (Dilly, S. J., et al., Chem. Commun. 2007; 2808-2810) can enable a drug to be covalently immobilised
so that it can be probed by phage display libraries and interacting partners identied. (Ladwa, S. R., et al., ChemMedChem 2008; 3: 742-744).
In order to elucidate structure-function relationships between the immobilised drug molecule and the captured protein, it would also be useful to
be able to remove the covalently bound drug molecule from the resin in
order to observe its conguration. We present here the preparation of a
new cleavable linker immobilisation system based on Wang methodology (Wang, S. S., J. Am. Chem. Soc. 1973; 95: 1328-1333) to allow separation of the immobilised drug - tag moiety, which will allow structural
determination and hence allow for development of further structure-activity assays to investigate new efcacy and toxicity targets. This new
approach thus has the potential to identify important new classes of therapeutic action. It may also unmask new mechanisms for toxicity thereby
allowing development of new structural chemistry approaches to reducing risk drug toxicity.
Katherine Casey-Green(1), A Marsh(1), P Taylor(1), S Ladwa(2), W

Dimitri(3), J Zhang(4), D Singer(4)
(1) University of Warwick, Coventry, UK
(2) Tangent Reproling Ltd, Bicester, UK
(3) University Hospital, Coventry, UK
(4) Clinical Sciences Research Institute, Coventry, UK
The Magic Tag chemical genomics approach (Dilly, S. J., et al., Chem.
Commun. 2007; 2808-2810) has identied novel drug-target interactions
(Ladwa et al. Chem Med Chem 2008;3:742-744). We now report its use
to uncover pathology-specic therapeutic targets for the pleiotropic
HMG-CoA reductase inhibitor simvastatin in patients with severe ischaemic heart disease. A new vascular phage display library was prepared
Paper No.: 1995

FOCUSED CONFERENCE GROUP: P01 - CLINICAL PHARMACOLOGY IN THE EMERGING COUNTRIES
INEQUIVALENCE OF DOXORUBICIN LIPOSOMAL FORMULATIONS COMMERCIALIZED IN LATIN AMERICA
Gilberto Castaneda-Hernandez(1), L Villafana-Godnez(1),
MA Valdes(2), L Gonzalez-Flores(1)
(1) CINVESTAV-IPN, Department of Pharmacology, Mexico DF,
Mexico
(2) Universidad de Sonora, Hermosillo, Mexico
220
It is well established that liposomal formulations signicantly increase
the safety of doxorubicin without compromising the antitumor efcacy
of this agent. The innovator liposomal doxorubicin is Caelyx (Doxil in
USA). A generic formulation, Doxopeg, is presently commercialized in
Latin America, although no information is available regarding its biopharmaceutical properties. Hence, we decided to comparatively study
Doxopeg and Caelyx. Six vials were studied per formulation. Each vial
contained 20 mg doxorubicin in 10 ml, according to the label. Doxorubicin content in Caelyx and Doxopeg was within the 95-105% range of the
labeled dose and no impurities were detected. In the two products, at
least 99.5% of doxorubicin content was encapsulated in liposomes. Both
formulations were similar when examined by electron microscopy and
liposomes exhibited a mean diameter of 100 nm, as determined by light
scattering. However, atomic force microscopy revealed a more stable
liposomal membrane in Caelyx, suggesting differences in lipid composition between formulations. Doxorubicin release rate in human plasma at
37 degrees in 24 h was about 1% of the liposomal content of Caelyx,
being consistent with previous reports. On the other hand, doxorubicin
release from Doxopeg was three times faster, likely due to differences in
the liposomal membrane. Since doxorubicin release rate is critical for
antitumor efcacy, it is concluded that Caelyx and Doxopeg are not
equivalent formulations. In the absence of data on the impact of faster
liposomal doxorubicin release on clinical efcacy and safety, the therapeutic use of Doxopeg is not recommended.
Paper No.: 1033

MANAGEMENT OF CARDIOVASCULAR MEDICATION IN
SURGICAL PATIENTS: INDICATIONS IN SUMMARY OF
PRODUCT CHARACTERISTICS AND SUBSTITUTION FOR
INTRAVENOUS FORMS
Liliana Castanheira(1), P Fresco(2), AF Macedo(1)
(1) University of Beira Interior Faculty of Health Sciences, Health Sciences Research Centre, Covilha, Portugal
(2) Oporto University Pharmacy Faculty, Oporto, Portugal
Introduction: Each year, millions of patients around the world undergo
surgical procedures. The majority of Portuguese surgical patients
(71.5%) were taking regular medication, namely for cardiovascular disorders (53%). Recommendations for management of regular medication in
perioperative period and the availability of intravenous forms of drugs
are critically needed for an efcient iatrogenic risk management. The
purpose of this study was to identify information in summary of product
characteristics for management of cardiovascular medication in perioperative period and the availability of these medications in intravenous
form. Materials/Patients: Summary of product characteristics of cardiovascular medication was screened for information about drug management in the perioperative period. Additionally, it was veried, for the
same drugs, if an intravenous form was available to use in the perioperative period. Results: Of 46 summary of product characteristics screened,
33% had information about its use in the perioperative period and 13%
had intravenous forms available. Conclusions: For the majority of cardiovascular medications, the summary of product characteristics is not
enough to support the management of these medications in the perioperative period. Proper pharmaceutical formulations for cardiovascular drugs,
namely intravenous, are lacking to use in surgical patients.
Paper No.: 2673

NEUROPLASTIC CHANGES PRECEDE CHRONIC
ANTIDEPRESSANT RESPONSES OF THE 5-HT4 RECEPTOR
AGONIST RS67333
Elena Castro, A Daz, EM Valdizan, R Vidal, F Pilar-Cuellar,
J Pascual-Brazo, A Pazos
Institute of Beiomedicine and Biotechnolgy- IBBTEC (UC-CSIC-IDICAN) Department of Physiology and Pharmacology, Santander, Spain
It has been recently suggested that activation of 5-HT4 receptors might
possess antidepressant properties in rats after a 3 days treatment, indicating a new strategy for developing faster-acting antidepressants. In this
regard, here we have evaluated in rat brain the expression of proteins
related to neuroplasticity after a subacute RS67333 treatment and their
correlate on behavioural paradigms. A 3-days treatment with RS67333
(1.5 mg/kg/day), previously shown to increase neurogenesis in dentate
gyrus, induced an upregulation in the expression of BDNF (% increase =
64%; p < 0.05) and pCREB/CREB ratio (% increase = 93%; p < 0.01)
in the hippocampus. A signicant reduction in the forced swimming test
(immobility time) was also observed after 3 (% red= 27%; p < 0.001)
and 7 (% red=29%; P < 0.001) days of treatment. However, in the novelty-feeding suppressed test, a validated paradigm to predict chronic antidepressant efcacy, a signicant reduction in the latency to feed (% red=
49%; p < 0.03) was observed only after 7 days of treatment. Short-term
treatment with RS67333 also failed to downregulate 5-HT4 receptor-coupled adenylate cyclase activity. Our data suggest that neural proliferation-related changes induced by antidepressants precede clinical
improvement. Furthermore, when compared to data regarding classical
antidepressants, our results show that the activation of 5-HT4 receptors
could represent a good strategy for developing antidepressants with a
minor onset of action.
Supported by: Ministerio de Ciencia e Innovacion (SAF07-61862) and
Fundacion Alicia Koplowitz
Paper No.: 1844

CENTRAL INJECTION OF CDP-CHOLINE SUPPRESSES
SERUM GHRELIN LEVELS WHILE IT INCREASES SERUM
LEPTIN LEVELS IN RATS
Sinan Cavun(1), S Kyyycy(2), NF Basaran(1), V Savci(1),
S Imamoglu(2)
(1) Uludag University Faculty of Medicine Department of Medical Pharmacology, Bursa,Turkey
(2) Uludag University Faculty of Medicine Department of Endocrinology, Bursa, Turkey
Introduction: Ghrelin and leptin are two important peptides which control food intake. Recently it was shown that CDP-choline has suppressive effect on appetite. In present study we aimed to test
intracerebroventricular (icv) administration of CDP-choline on serum
ghrelin, leptin, glucose and corticosterone levels in rats. Material and
Methods: Male Wistar rats were used in all experiments. For repeated
blood withdrawal a cannula was inserted into the left carotid artery and
also a cannula was placed into the right lateral ventricule for icv injections. Rats were randomized to three treatment groups and icv CDP-choline was administered at three different doses to the groups as follows:
0.5 (n = 8), 1.0 (n = 8) and 2.0 (n = 8) lmol. Saline injected rats were
used as controls. Blood samples were withdrawn before and 5th, 15th,
30th, 60th and 120th min after icv injections. Results: Baseline serum
ghrelin and leptin levels were not different between groups. Intragroup
analysis revealed that serum ghrelin levels were suppressed signicantly
at 60th min with 1lmol CDP-choline injection (p = 0.025) whereas
serum leptin levels were increased at 60th min with 0.5, 1.0 and
2.0 lmol doses of CDP-choline (p = 0.036, p = 0.012, p = 0.043,
respectively). Serum leptin levels were signicantly higher at 120th min
with 1.0 and 2.0 lmol doses of CDP-choline compared with baseline
values (p = 0.017, p = 0.046, respectively). Central CDP-choline injections also induced a dose- and time dependent increase in serum glucose
and corticosterone levels. Conclusions: These results suggest that central
injection of CDP-choline suppress serum ghrelin levels while it increases
serum leptin levels.
221
Paper No.: 590
CALCIUM ANTAGONISTIC EFFECTS OF SOME
ANTIMIGRAINE DRUGS IN RAT ISOLATED BASILAR
ARTERY
Edip Guvenc Cekic, M Tuncer
Hacettepe University Faculty of Medicine, Department of Pharmacology,
Ankara, Turkey
Vasodilator substances such as CGRP released form central perivascular
nerve endings as a result of activation of calcium channels play a role in
the pathogenesis of migraine. Propranolol, a b-adrenergic receptor
blocker, is used for chronic prophylaxis of the disease. Propranolol has
been reported to block calcium channels in the rat mesenteric artery and
aorta (Priviero FB, et al. 2006; 33: 448-455), however such kind of effect
of the drug has not yet been shown in cerebral arteries. Therefore, we
aimed to investigate calcium antagonistic effects of propranolol and two
drugs, namely pizotifen and methysergide, which are also used for
chronic prophylaxis of migraine, in the rat basilar artery. Both sexes of
Wistar rats (250-350 g) were used. After decapitation, isolated basilar
arteries were mounted in myograph system (Danish Myograph 610M)
and isometric responses were recorded. Propranolol (10 nM-30 lM) and
pizotifen (10 nM-30 lM) caused endothelium-independent relaxations in
the precontracted arteries. Methysergide (10 nM-30 lM) did not induce
any relaxation. In order to examine a calcium antagonistic activity, concentration-response curves to calcium chloride were obtained in a depolarizing Ca-free solution, in the absence and presence of propranolol (130 lM), pizotifen (1-10 lM) and methysergide (1-10 lM). Propranolol
and pizotifen but not methysergide, shifted the calcium concentration response curves to the right. The results suggest that calcium channel
blocking activities of propranolol and pizotifen may contribute to their
antimigraine effect by preventing the release of vasodilator substances
from perivascular nerve endings.
Paper No.: 3237

EFFECT OF CHROMIUM ENRICHED FERMENTATION
PRODUCT OF BARELEY AND BEER YEAST AND ITS
COMBINATION WITH ROSIGLITAZONE ON
EXPERIMENTALLY INDUCED HYPERGLYCEMIA IN MICE
Vlada Cekic(1), V Vasovic(2), V Jakovljevic(2), D Lalosevic(3), M Mikov(1), A Sabo(1)
(1) Hemofarm AD, Medical Department, Belgrade, Republic of Serbia
(2) University of Novi Sad, Faculty of Medicine, Department of Pharmacology, Toxicology and Clinical Pharmacology, Novi Sad, Republic of
Serbia
(3) University of Novi Sad, Faculty of Medicine, Department of Histology and Embryology, Novi Sad, Republic of Serbia
To examine the action of BBCr, rosiglitazone (R) and their combination
(BBCr + R) on glycemia in mice on an empty stomach and hyperglycemia induced with glucose, adrenaline and alloxan. The BBCr preparation
is an auxiliary medicine registered as a diabetic supplement in the treatment of insulin-independent diabetes mellitus. It is fermentation product
of barley and beer yeast enriched with organically bound chromium.
Active ingredient of this product is Glut 4 plus insulin sensitizer with
the percentage of tablet composition of 76.92%. Animals were divided in
three groups, depending of the way of inducing hyperglycemia: Iglucose, single dose 500 mg/kg, p.o.; IIadrenaline, single dose 0.2 mg/kg,
s.c.; IIIalloxan, multiple dose, 100 mg/kg, i.p. Groups were divided into
subgroups according to the preparation (BBCr, R, BBCr+R). In the rst
two groups, hyperglycemia was measured on an empty stomach, 30min
after glucose load, and 45min after adrenaline injection, and at the same
time intervals after a 7-day treatment. In Group III hyperglycemia was

measured both before and after the diabetes-inducing treatment with
alloxan and tested substances (after 12 days). Histological analyses of
sections of the liver and pancreas were carried out in mice of Group III.
In the glucose load test, BBCr and BBCr+R decreased signicantly glycemia value compared to control (7.4 0.6mmol/l : 7.7 0.7mmol/l :
8.9 1.3mmol/l; p < 0.05). In the adrenaline test, R and BBCr+R
decreased signicantly glycemia with respect to control (8.6 1.8mmol/l
: 9.6 2.4mmol/l : 15.2 3.3mmol/l; P < 0.01). After treatment with
alloxan the glycemia was signicantly lower in the groups treated with R
and BBCr compared to control (8.4 0.8mmol/l : 8.6 3.2mmol/l :
18.8 5.5mmol/l, P < 0.01). The least damage to pancreas tissue caused
by alloxan was observed in the subgroups treated with BBCr and
BBCr+R. In the liver tissue, there was more or less pronounced leukocyte inltrate in subgroups treated with BBCr and R.
Paper No.: 1411

DISEASES
DOSAGE REGIMEN OF VANCOMYCIN IN NICU/PICU
PATIENTS
O Cerna(1), Pavla Pokorna(1), J Sedivy(2)
(1) General Teaching Hospital, Department of Paediatrics, Prague, Czech
Republic
(2) General Teaching Hospital, Department of Clinical Biochemistry and
Laboratory Diagnostics, Prague, Czech Republic
Introduction: The value of vancomycin therapeutic drug monitoring and
therapeutic range of vancomycin is discussed for routine monitoring in
pediatric population. Vancomycin is a glycopeptide used in neonates and
children with staphylococcal sepsis. Methods: The goal of this prospective study is to predict a dosage regimen of vancomycin. This presented
study (2004-2009) enrolls n = 57 neonates (gestational age 25-41 weeks,birth weight 750 - 4000 g) during rst month of life and n = 40 children (actual age 6 months -18 years, actual weight 12 - 40 kg) with
blood stream infection (BSI) indentied in n = 53/97 patients. Neonates
received 30 mg/kg/day i.v. divided in two or three doses according to
postnatal age, children received 20 mg/kg/dose i.v. administered by
60 minutes infusion. Trough (Ctrough) or peak (Cpeak) of vancomycin
serum concetrations were determinedbefore and after third dose of
vancomycin or later using Abbott Diagnostics Laboratories Method. Targets were Cmin concentrations between 5 and 15 mg/L and Cmax below
40 mg/L. Vancomycin data were analysed according to one- compartment model using PC- MW Pharm -version 3,30 Programme and various
dosing schemes were simulated. Results: Detected Ctrough concentrations were within targets in 27/97 patients, Cpeak levels were higher than
40 mg/l in 3/97 patients. Vancomycin dosage regimen was individualized
in 31/97 and individual prediciton revisedin 20/31 of patients. Conclusions: The use of individual therapeutic vancomycin monitoring help to
optimize target levels in NICU/PICU patients.
Paper No.: 907

INHIBITION OF CATECHOLAMINE SECRETION BY A
TERPENOID EXTRACTED FROM SYNEILESIS ACONITIFOLIA
(BUNGE) MAXIM IN CULTURED BOVINE ADRENAL
CHROMAFFIN CELLS
L Chai, H Mao, Y Su, Han Zhang, H Guo, Y Jin, Z Zang
Tianjin University of Traditional Chinese Medicine, Tianjin, PR China
Evidences indicated a critical relationship between activation of sympathetic nervous system by stress and chronic joint pain or inammation.
222
Catecholaminergic system was activated as a part of the sympathetic nervous system (CNS) response to stress. The goal of this study was to
examine the effects of a terpenoid, (6b,8b)-6-hydroxyeremophil-7(11)en-12,8-olide, extracted from Syneilesis aconitifolia (Bunge) Maxim on
CA secretion in cultured bovine adrenal chromafn cells, a Chinese herb
which was used to treat rheumatism, arthralgia and some other diseases.
Our results showed that the terpenoid (1lM-100lM) signicantly inhibited CA secretion induced by 300lM acetylcholine (ACh) and 56 mM
K+ solution in a concentration-dependent manner, which are activators
of the nicotinic acetylcholine receptor and voltage-dependent Ca2 + channels, respectively. Raise of intracellular Ca2 + concentration ([Ca2 + ]i) is
a requisite for CA secretion. We next investigated the effects of the terpenoid(1lM-100lM) on various activators induced elevation of [Ca2 + ]i.
The results indicated the terpenoid also inhibited elevation of [Ca2 + ]i
caused by ACh and high K+ in a concentration-dependent manner, which
was consistent with the inhibitory effects on CA secretion. These ndings suggested that the terpenoid inhibited CA secretion in cultured
bovine adrenal Chromafn Cells through suppression on the nicotinic
acetylcholine receptor as well as voltage-dependent Ca2 + channels,
thereby inhibition on the raise of [Ca2 + ]i.
Keywords: catecholamine secretion; calcium; Syneilesis aconitifolia
(Bunge) Maxim; joint pain; inammation; terpenoid
Paper No.: 2344

HEALTH AND DISEASE
ESTROGEN RECEPTOR A AS A KEY TARGET OF RED WINE
POLYPHENOLS ACTION ON THE ENDOTHELIUM
M Chalopin(1), A Tesse(1), MC Martinez(1), D Rognan(2), JF Arnal(3),
Ramaroson Andriantsitohaina(1)
(2) University Louis Pasteur-Strasbourg I, Department of Bioinformatics
of Drugs, UMR 7175 CNRS-ULP, Illkirch, France
(3) University of Toulouse III Paul Sabatier, CHU - Centre HospitalierUniversitaire, INSERM U858, Toulouse, France
A reduction in cardiovascular risk and vascular protection associated
with diet rich in polyphenols are generally accepted; however, molecular
targets for polyphenols effects remain unknown. Meanwhile evidences in
literature have enlightened, structural similarities between estrogens and
polyphenols known as phytoestrogens, and also in their vascular effects.
We hypothesized that estrogen receptor a isoform (ERa) could be
involved in transduction of the vascular benets of polyphenols. Here,
we used ERa decient mice to show that endothelium-dependent vasorelaxation induced either by red wine polyphenol extract, ProvinolsTM, or
delphinidin, an anthocyanin possessing similar pharmacological prole,
is mediated by ERa. Indeed, ProvinolsTM, delphinidin and ERa agonists, 17-b-estradiol and PPT, are able to induce endothelial vasodilatation
in aorta from ERa Wild-Type but not from Knock-Out mice, by activation of nitric oxide (NO) pathway in endothelial cells. Besides, silencing
the effects of ERa completely prevented effects of ProvinolsTM and delphinidin to activate NO pathway (Src, ERK 1/2, eNOS, caveolin-1) leading to NO production. Furthermore, direct interaction between
delphinidin and ERa activator site is demonstrated using binding assay
and docking. Most interestingly, the ability of short term oral administration of ProvinolsTM to decrease response to serotonin and to enhance
sensitivity of the endothelium-dependent relaxation to acetylcholine,
associated with concomitant increased NO production and decreased
superoxide anions, was completely blunted in ERa decient mice. This
study provides evidence that red wine polyphenols, especially delphinidin, exert their endothelial benets via ERa activation. It is a major
breakthrough bringing new insights of the potential therapeutic of
polyphenols against cardiovascular pathologies.
Paper No.: 2470

HEALTH AND DISEASE
THE HYPOCHOLESTEROLEMIC EFFECT OF A NOVEL
SERIES OF COMPOUNDS WITH STRUCTURAL PROPERTIES
OF ALPHA-ASARONE AND FIBRATES IN TYLOXAPOL
TREATED MICE
German Chamorro(1), C Zuniga(1), L Garduno(1), MdC Cruz(1),
R Perez(1), F Labarrios(1), J Tamariz(1)
Escuela Nacional de Ciencias Biologicas, IPN, Mexico D.F., Mexico
Because of the putative link between serum cholesterol and incidence of
atherosclerosis, a novel series of ten analogs of a-asarone related with
brates has been previously prepared. Most of them are phenoxyacetic
acids or their methyl and ethyl esters which was substituted by a single
side chain of the different positions of the benzene ring. They were evaluated in a model of hyperlipidaemia induced in ICR male mice by a single
400 mg/kg intraperitoneal injection of tyloxapol. Mice were treated with
the drugs by gavage 1 h before and 22 and 48 h after the tyloxapol injection at doses of 0, 25, 50 or 100mg/kg. The derivatives exhibited potent
hypocholesterolemic activity, lowering the mice serum cholesterol up to
64.6%, low-density protein cholesterol up to33.8% and triacylglycerols
up to 70.2%. These ndings have led identication of a class of compounds that represent a promising new class of hypolipidaemic drugs.
Paper No.: 2224

HEALTH AND DISEASE
SECRETONEURIN INDUCES ENDOTHELIUM-DEPENDENT
RELAXATION OF THE PORCINE CORONARY ARTERY
Calvin KY Chan(1), JCW Mak(0), PM Vanhoutte(1)
(1) University of Hong Kong, Department of Pharmacology and
Pharmacy, Hong Kong, PR China
(2) University of Hong Kong, Department of Medicine, Hong Kong, PR
China
Secretoneurin is a part of the peptide encoded by the Secretogranin II
gene. Secretoneurin enhances the adhesion and transendothelial migration properties of monocytes. In addition, in regenerated endothelium,
the expression of the Secretogranin II gene is up-regulated. The objective
of the present study was to examine the effects of Secretoneurin on the
vascular reactivity. Isometric tension of rings with or without endothelium from porcine coronary arteries was measured in conventional organ
chambers. Secretoneurin did not induce contraction, but in preparations
contracted by the TP-receptor agonist U46619 it caused relaxation. This
relaxation was endothelium-dependent and reduced by the nitric oxide
synthase inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME). It
was abolished by combined incubation with L-NAME and the cyclooxygenase inhibitor indomethacin. By contrast the relaxation to secretoneurin was not affected by TRAM 34 and UCL 1684. These results suggest
that secretoneurin induces relaxation by activation of both endothelial
nitric oxide synthase and cyclooxygenase, with the nitric oxide pathway
playing a more dominant role.
Paper No.: 981

PROSTACYCLIN IP RECEPTOR ACTIVATION,
TRANSFORMING GROWTH FACTOR SIGNALLING AND
CARDIAC FIBROSIS IN HYPERLIPIDEMIC MICE
Elsa Chan(1), G Dusting (1), N Guo(1), H Peshavariya(1), R Dilley(1),
S Narumiya(2), F Jiang(3)
223
(1) OBrien OBrien Institute and University of Melbourne, Department
of Cytorpotection Pharmacology, Fitzroy, Victoria, Australia
(2) Kyoto University, Faculty of Medicine, Department of Pharmacology,
Kyoto, Japan
(3) Chinese Ministry of Education and Chinese Ministry of Health, Key
Laboratory of Cardiovascular ReModelling and Function Research, Qilu
Hospital, Shandong University, Jinan, Shandong Province, PR China
Prostacyclin IP receptor activation has been shown to protect against cardiac brosis but its molecular mechanisms in the heart remain unknown.
Using mouse cardiac broblasts, we examined whether IP activation with
cicaprost affects collagen expression by interfering with the signalling of
a key brotic factor transforming growth factor (TGF)-b. In broblasts,
TGF-b receptor inhibition suppressed collagen I mRNA by 25 3%
(n = 3-4; P < 0.05) and the phosphorylation of TGF-b signalling protein
Smad2 was detected, suggesting that TGF-b regulates basal collagen
expression. Cicaprost reduced the basal expression of TGF-b responsive
genes including collagen I and plasminogen-activator inhibitor I (PAI-1)
indicating an inhibitory effect of IP receptor activation on TGF-b signalling. cAMP-elevating agents including cicaprost induce phosphorylation
of cAMP response element binding protein (CREB), which competes
with Smad for transcription coactivators such as CBP (CREB binding
protein)/p300 to reduce collagen synthesis. In broblasts with a CREB
mutant protein, cicaprost did not reduce expression of collagen and PAI1. Furthermore, double knockout of apolipoprotein E and IP receptor
[ApoE(-/-)/IP(-/-); n = 4-9; P < 0.05] in mice showed greater collagen
deposition (3-fold) and mRNA (5-fold) in the heart compared to controls
[ApoE(-/-)/IP(+/+)] following two-week angiotensin II infusion (1000 ng/
kg/min, SC). Angiotensin II-induced TGF-b mRNA in the heart was similar in both genotypes, suggesting that IP receptor activation did not affect
TGF-b gene expression, but its downstream signalling mechanism. Our
data suggest the protective effect of IP signalling operates at least partly
by antagonizing TGF-b mediated probrotic effects in cardiac broblasts.
Paper No.: 1554

HEALTH AND DISEASE
NADPH OXIDASE AND TGF-BETA SIGNALLING IN
EXTRACELLULAR MATRIX PRODUCTION IN CARDIAC
FIBROBLASTS
Elsa Chan(1), H Peshavariya(1), G Nancy(1), F Jiang(2), G Dusting(1)
(1) OBrien Institute and University of Melbourne, Department of Cytorpotection Pharmacology, Fitzroy, Victoria, Australia
(2) Key Laboratory of Cardiovascular ReModelling and Function
Research, Chinese Ministry of Education and Chinese Ministry of
Health, Qilu Hospital, ShandongUniversity, Jinan, Shandong Province,
PR China
Oxidative stress, especially activation of the superoxide generating Noxcontaining NADPH oxidase is involved in the development of cardiac
brosis. We hypothesized that a probrotic factor transforming growth
factor (TGF)-b acts via NADPH oxidase to induce collagen production
in mouse cardiac broblasts. TGF-b (10 ng/mL; n = 3) stimulated gene
expression of collagen I (by 2.1 0.4 fold) and the broblast differentiation marker smooth muscle a-actin in mouse cardiac broblasts (by
2.8 0.6 fold). EUK-134 a scavenger of superoxide and hydrogen peroxide suppressed the TGF-b-induced collagen gene expression (by
46 13%; n = 4). By expressing a dominant negative form of NADPH
oxidase in broblasts, TGF-b-induced collagen gene expression was
inhibited, suggesting that NADPH oxidase is important to TGF b signalling in extracellular matrix production. Nox2 and Nox4 are the two
major isoforms found in cardiac broblasts. TGF-b upregulated Nox4
expression (2.9 0.8 fold; n = 3) without affecting Nox2 in mouse cardiac broblasts. On the other hand, Nox1 was undetected suggesting that
Nox4 regulates TGF-b mediated responses. The effect of siRNA against
Nox4 gene expression on TGF-b-induced matrix synthesis is under
investigation. In conclusion, Nox4 may act as an effector molecule in
cardiac broblasts mediating brotic reactions by acting as an intermediary in the signalling of TGF-b.
Paper No.: 1502

HEALTH AND DISEASE
CHARACTERIZATION OF VIP AND PACAP RECEPTORS IN
THE HUMAN CORONARY AND MENINGEAL ARTERY
Kayi Chan(1), M Baun(2), J Danser(1), I Jansen-Olesen(2), AM van
denBrink(1), S Gupta(2)
(1) Erasmus Medical Center, Department of Internal Medicine, Rotterdam, The Netherlands
(2) Glostrup University Hospital, Copenhagen, Denmark
Vasoactive intestinal peptide (VIP) and the pituitary adenylate cyclase
activating polypeptides (PACAPs) are involved in various processes,
including dilation of cephalic arteries. PACAP induces migraine-like
headache in migraine patients. Interestingly, PACAPs do not only stimulate VPAC receptors, like VIP, but also PAC receptors. We characterized
responses to PACAPs and VIP, as well as their VPAC/PAC receptors in
human coronary and meningeal arteries to obtain more knowledge about
their role in migraine. Concentration response curves to the VPAC/PAC
receptor agonists PACAP38 and PACAP27, as well as the VPAC receptor agonist VIP, were constructed in human coronary and meningeal
arteries in the absence or presence of the PAC receptor antagonist
PACAP6-38 or the VPAC receptor antagonist, PG-97269. mRNA
expression was measured using RT-PCR. PACAP38 and VIP induced
relaxations, which was higher in the coronary than in the meningeal
artery. The pEC50 of PACAP38 was smaller than the pEC50 of VIP in
all arteries. The antagonist did not block vasodilation, except for
PG97269, which blocked VIP-induced vasodilation in the coronary
artery. The mRNA expression of VPAC receptors was higher than that of
PAC receptors in coronary and meningeal arteries. PACAP38 and VIP
have a low potency and efcacy in meningeal arteries. PACAP38 is less
potent than VIP in human arteries. Except for VIP-induced vasodilation
in the coronary artery, our antagonist experiments do not conrm
involvement of either VPAC or PAC receptors, although mRNA of these
receptors is present. We suggest that PACAP38 may induce migraine-like
headaches via a mechanism not involving meningeal vasodilation.
Paper No.: 1503

CHARACTERIZATION OF THE CGRP RECEPTOR
ANTAGONIST TELCAGEPANT (MK0974) IN HUMAN
ISOLATED CORONARY ARTERIES
Kayi Chan(1), L Edvinsson(2), S Eftekhari(2), R de Vries(1), S Kane(3),
R Hargreaves(3), J Danser(1), AM van denBrink(1)
(1) Erasmus Medical Center, Department of Internal Medicine, Rotterdam, The Netherlands
(2) Lund University Hospital, Malmo, Sweden
(3) Merck Research Laboratories
Calcitonin gene-related peptide (CGRP) plays a role in primary headaches and CGRP antagonists are effective in migraine treatment. Since
CGRP is also a potent vasodilator in coronary arteries, antagonism of its
receptor might cause coronary side effects. Therefore, we investigated
the effect of telcagepant (MK-0974) on human coronary arteries with different internal diameter. Human alfaCGRP induced concentration-dependent relaxations which was larger in distal (id: 600-1000lm,
Emax=83 7%) than in proximal coronary arteries (id: 2-3mm,
Emax=23 9%) and in coronary arterioles (id: 200-300lm, Emax=15
7%). Pre-treatment with telcagepant (10 nmol/L to 1 lmol/L) antagonized the alfaCGRP-induced relaxation in a competitive manner with a
pA2 value of 8.43 0.24 in the distal coronary arteries. In proximal coronary arteries and coronary arterioles, telcagepant (1 lmol/L) antagonized the alfaCGRP-induced relaxations with a pKB of 7.89 0.13 and
7.78 0.16 respectively. Telcagepant did not induce contraction or relaxation per se. alfaCGRP signicantly increased cAMP levels and this was
224
abolished by pretreatment with telcagepant in distal coronary arteries.
Immunohistochemistry revealed the expression and co-localization of the
receptor elements calcitonin like receptor (CLR) and receptor activity
modifying protein 1 (RAMP1) in the smooth muscle cells in the media
layer of human coronary arteries. The ndings expand our understanding
of on the peripheral vascular properties of telcagepant. When extrapolated to the clinical situation, telcagepant is unlikely to induce coronary
side effects under normal conditions in cardiovascular healthy patients.
Paper No.: 1440

CALCIUM CHANNEL IS INVOLVED IN NEOMYCIN-INDUCED
HAIR CELL TOXICITY IN THE ZEBRAFISH LATERAL LINE
Ming-Huan Chan, C-F Liu, H-H Chen
Tzu Chi University, Department of Pharmacology, Hualien, Taiwan
Aminoglycosides are commonly and clinically used antibiotic agents that
cause severe side effects, such as hearing loss. The extracellular divalent
cations can modulate aminoglycoside-induced sensory hair cell death.
However, the role of cytoplasmic membrane calcium channel in aminoglycoside ototoxicity is not clear. In this study, the lateral lines of zebrash were used to investigate the effects of verapamil, a calcium channel
blocker, and Bay K8644, a calcium channel activator, on neomycininduced hair cell toxicity, which was detected in vivo by the dye FM143FX, a reliable indicator to assess hair cell viability. Results showed
that hair cells from neomycin toxicity were protected under high calcium
(3.3 mM) conditions. On the contrary, lower calcium (33 microM)
enhanced hair cell death mediated by neomycin treated for 30 minutes.
Importantly, verapamil was observed to attenuate neomycin-induced
toxic response in calcium concentrations at 33 and 330 microM. Bay
K8644, in contrast, signicantly reversed the protective action of high
calcium on neomycin-induced hair cell loss. These ndings indicate that
both extracellular calcium concentration and calcium channel activity
inuence hair cell loss from neomycin toxicity.
Paper No.: 671

GASTROPROTECTIVE ACTIVITY OF ANNONA MURICATA L.
ON ETHANOL INDUCED GASTRIC LESIONS IN RODENTS
Pit Foong Chan, R Abdul Hamid
University Putra Malaysia, Faculty of Medicine and Health Sciences,
Department of Biomediacal Science, Selangor, Malaysia
Annona muricata L (AML), locally known as durian belanda belonging to family Annonaceae, is a plant been known to have various pharmacological and nutritional properties in folklore medicine (Dos Santos
et al, 2001; 8: 115-120). The antiulcerogenic effect of a crude hydroalcoholic (80%) extract obtained from AML leaves was evaluated in rodents
by employing absolute ethanol induced ulcer model. AML was administered orally (p.o.) 1h prior to ulcer induction in the dose range of 10, 30,
100 and 300mg/kg. Results of the study revealed that AML at the given
dose range signicantly inhibited the ulcerative lesion in a dose-dependent manner by 22.44, 51.77, 75.43 and 92.80% respectively. Oral
administration of AML (300mg/kg) to L-NAME (a NO-synthase inhibitor)-pre-treated animals, did not reduce the antiulcerogenic activity of
AML, suggesting that the mechanism of pharmacological activity has no
relationship with the nitric oxide (NO). The pre-treatment with N-ethymaleimide, a thiol blocker, including mucosal nonprotein sulfhydryl
groups, increased the gastric lesions in comparison for control groups
(186.33 18 mm2 with saline versus 379.67 15.47 mm2 with NEM).
The rats treated with AML (300mg/kg) inhibited the gastric lesions as
82.02% (pre-treated with saline) and 58.21% (pre-treated with NEM).

This signicant attenuation of gastric lesions showed that the crude
hydroalcoholic extract of AML has active substances that increase the
mucosal nonprotein sulfhydryl groups contents for the gastroprotection
effects.
Paper No.: 1202

BIOEQUIVALENCE STUDY OF TWO FORMULATIONS OF
35MG TRIMETAZIDINE MODIFIED RELEASE TABLETS IN
THAI HEALTHY VOLUNTEERS UNDER FASTING AND FED
CONDITIONS
Weerawadee Chandranipapongse, T Ruangnapa, S Ngokpol,
K Sathirakul, P Pongnarin, S Kongpattanakul
Mahidol University Faculty of Medicine & Siriraj Hospital, Department
of Pharmacology, Bangkok, Thailand
The generic product of trimetazidine, an anti-anginal agent, is now available in Thailand. Bioequivalence study is used to demonstrate that the
generic and the original drug products have comparable rate and extent
of absorption. Thus, the objective of this study was to compare the bioavailability of two modied release tablets of 35mg trimetazidine dihydrochloride (Matenol MR; generic product and Vastarel MR;
innovators product). This study was a single-dose, two-treatment, twoperiod, two-sequence randomized crossover design under fasting and fed
conditions with a minimum of 7 days washout period. Twenty-four
healthy Thai male and female volunteers were enrolled. For both conditions, the volunteers were received a 35mg trimetazidine modied release
tablet of both formulations. Each volunteers were obtained blood samples
16 times over a period of 24 hours after each oral administration. The trimetazidine plasma concentrations were quantied using a validated
method employing liquid chromatography with tandem mass spectrometry with the lower limit of quantication of 0.25 ng/mL. All of the pharmacokinetic parameters were investigated using non-compartmental
analysis model. The results demonstrated that the 90% CI of the geometric mean ratio of Cmax, AUC0-t and AUC0- were within the equivalence criteria (80.00-125.00%) which were 105.53% (95.71%-116.36%),
104.28% (96.24%-112.98%, and 105.26% (96.61%-114.67%) under fasting condition and 110.21% (102.72%-118.25%), 101.95% (94.33%119.19%), and 99.7% (91.18%-109.02%) under fed condition, respectively. No statistical differences of median Tmax (p > 0.05) between two
formulations in both conditions were observed. Furthermore, both preparations were well tolerated and had a few non-serious adverse events. In
conclusion, these two trimetazidine products were bioequivalent.
Paper No.: 1253

THE EFFECT OF C825T POLYMORPHISM OF THE GNB3
GENE ON VALPROATE-INDUCED METABOLIC
ABNORMALITIES IN BIPOLAR DISORDER PATIENTS
Hui Hua Chang(1), PW Gean(2), YK Yang(3), HC Huang(3),
HC Wang(3), RB Lu(0), PS Chen(0)
(1) National Cheng Kung University, College of Medicine, Institute of
Biopharmaceutical Sciences, Tainan, Taiwan
(2) National Cheng Kung University, College of Medicine, Department
of Pharmacology, Tainan, Taiwan
(3) National Cheng Kung University, Hospital and College of Medicine,
Department of Psychiatry, Tainan, Taiwan
(4) National Cheng Kung University, College of Medicine, Institute of
Behavioral Medicine, Tainan, Taiwan
(5) National Cheng Kung University Hospital, Department of Psychiatry,
Dou-liou Branch, Taiwan
225
Valproate (VPA) is a mood stabilizer for treating patients with bipolar
disorder. It may cause metabolic abnormalities in certain bipolar patients.
However, the genetic factors that inucence the susceptibility remain
unclear. Genetic polymorphism of the G-protein 3 Subunit (GNB3) is
reported to be associated with metabolic phenotypes. In the current study,
we investigated the possible associations between the GNB3 variation
and VPA-induced metabolic abnormalities. Subjects (n = 96) who met
the DSM-IV criteria for bipolar disorder were recruited from the National
Cheng Kung University Hospital. Their metabolic indexes were measured. The variation of GNB3 825C/T showed an association with higher
plasma total cholesterol (p = 0.037), triglyceride (p = 0.014), and leptin
(p < 0.001) levels in bipolar disorder patients treated with VPA. After
adjusting for age, gender, types of bipolar disorders, serum concentration
of VPA, the variation of GNB3 825C/T remained signicantly associated
with the levels of serum leptin and BMI (p < 0.001 and p = 0.025,
respectively). In addition, the GNB3 825C/T showed signicant
drug*SNP interactions with TG levels (p = 0.013), leptin levels
(p = 0.013), and BMI (p = 0.018). These results indicated that the T
allele may be associated with lower serum leptin level and BMI in BD
patients treated with VPA.
Paper No.: 1292

SESSION - ONCOLOGY
INDOLE-3-CARBINOL ATTENUATES MIGRATION AND
INVASION OF BREAST CANCER CELLS BY BLOCKING THE
ERK/SP1-MEDIATED MMP-2 GENE TRANSCRIPTION
Hui-Chiu Chang
Kaohsiung Medical University, Graduate Institute of Medicine, Department of Medical Laboratory Science and Biotechnology, Kaohsiung,
Taiwan
Indole-3-carbinol (I3C) is the major bioactive food component of cruciferous vegetables and has been shown to exhibit inhibition of invasion
activity on various cancer cell lines. This study addressed the effect of
I3C on the expression of matrix metalloproteinases (MMPs) and claried
the underlying mechanism. Migration, invasion, MMP-2 activity, and
MMP-2 mRNA level of MCF-7 breast cancer cells were inhibited by
I3C in a dose-dependent manner. Promoter deletion and mutation analysis suggested that I3C inhibited MMP-2 gene transcription via the -85/7 bp promoter region and the Sp1 transcription factor binding site
located within the -72/-64 bp promoter region was important for the inhibition. Chromatin immunoprecipitation assay conrmed that Sp1 proteins
constitutively bound to this consensus sequence in vivo and that the binding was attenuated by I3C. In addition, I3C inhibited the extracellular
signal-regulated kinase (ERK) signalling pathway in MCF-7 cells. The
results suggest that I3C attenuates MMP-2 expression by blocking the
ERK/Sp1-mediated gene transcription to reduce migration and invasion
of breast cancer cells.
Paper No.: 1201

BIOEQUIVALENCE STUDY OF 10 MG RAMIPRIL TABLETS IN
HEALTHY THAI VOLUNTEERS
Somruedee Chatsiricharoenkul, W Thangboonjit, K Sathirakul,
P Pongnarin, K Konhan, S Kongpattanakul
Mahidol University Faculty of Medicine & Siriraj Hospital, Department
The objective of this study was to determine the bioequivalence of
10 mg dose of ramipril tablets between the test product (Ramtace
10 mg, Unison Laboratories, Thailand) and the reference product (Tritace 10 mg, Aventis Pharma S.P.A., Italy). The study was carried out
with a single dose, 2-treatment, 2- period, 2-sequence randomized cross-
over design under fasting condition with a minimum of 14 days washout

period in 24 healthy Thai male and female volunteers. Plasma samples
for determination of ramipril and ramiprilat were obtained pre-dose and
at frequent intervals for up to 72 h post dose. Ramipril and ramiprilat
plasma concentrations were quantied by a validated method employing
high performance liquid chromatography with tandem mass spectrometry
(HPLC-MS/MS) method with the lower limit of quantication (LLOQ)
of 0.05 ng/mL and 0.25 ng/mL for ramipril and ramiprilat, respectively.
All of the pharmacokinetic parameters were investigated using non-compartmental analysis. The result demonstrated the 90% condence interval
(90%CI) of the geometric mean ratio (test/reference) of Cmax, AUC0-72
and AUC0- of ramipril was 97.26% (84.50%-111.93%), 100.70%
(89.47%-113.34%) and 100.29% (88.90%-113.15%), respectively which
were within the equivalent criteria (80.00%-125.00%). For ramiprilat, the
90% CI for Cmax, AUC0-72 and AUC0- was entirely within the equivalence criteria which were 108.87% (103.00%-115.07%), 104.93%
(100.50%-109.55%) and 103.30% (98.03%-108.85%) for Cmax, AUC0-72
and AUC0-, respectively. In conclusion: the 90% condence intervals
for log-transformed geometric mean test/reference formulation ratios of
primary parameters were entirely within 80.00%-125.00%. Thus, it can
be concluded that the test formulation was bioequivalent to the reference
formulation.
Paper No.: 1492

A-LINOLENIC ACID PROTECTS RENAL CELLS AGAINST
PALMITIC ACID LIPOTOXICITY VIA REDUCTION OF
ENDOPLASMIC RETICULUM STRESS
Prabal Chatterjee, E Katsoulieris, J Mabley, M Samai, I Green
University of Brighton, Centre for Biomedical and Health Science
Research, Brighton, UK
Elevated levels of saturated fatty acids (FA) are associated with diabetes/
obesity and with development of diabetic nephropathy. The aim of this
study was to investigate the effects of palmitic acid (PA), a saturated FA,
on renal cells and to evaluate the potential protection afforded by a-linolenic acid (a-LA), a polyunsaturated FA. Cultures of NRK-52E cells, a
rat proximal tubular cell-line, were incubated with PA for 24 h in the
absence or presence of a-LA. Apoptotic cell death was measured using
Hoechst-propidium iodide staining. The expression of CHOP and phosphorylated eIF2a (p-eIF2a) were determined using Western blotting and
used as indicators of endoplasmic reticulum (ER) stress. PA (300 lmol/
L) increased apoptosis from 1.2 + /- 0.2% to 17.1 + /- 2.6% (n = 4,
P < 0.05). PA increased CHOP expression from 0.19 + /- 0.04 to
0.88 + /- 0.14 arbitrary units (au) (n = 6, P < 0.05) and p-eIF2a from
0.32 + /- 0.03 to 0.70 + /- 0.03 au (n = 5, P < 0.05). In the presence of
a-LA (100-300 lmol/L), PA-mediated apoptosis was reduced from
17.1 + /- 2.6% to 4.2 + /- 0.7% (n = 4, P < 0.05), CHOP expression
was reduced from 0.88 + /- 0.14 to 0.22 + /- 0.06 au (n = 6, P < 0.05)
and p-eIF2a expression from 0.70 + /- 0.03 to 0.42 + /- 0.02 au (n = 5,
P < 0.05). In conclusion, these results suggest that a-LA can reduce the
apoptosis of renal cells caused by PA and that this protection involves
reduction of ER stress.
Paper No.: 967

FOCUSED CONFERENCE GROUP: P16 - NATURAL PRODUCTS:
PAST AND FUTURE?
PHARMACOLOGICAL EXPLORATION OF HYPERICUM
PERFORATUM GROWING IN INDIA USING RODENT MODELS
Shyam Sunder Chatterjee(1), V Kumar(2)
(1) Herbal Consultant, Karlsruhe, Germany
(2) Banaras Hindu University, Institute of Technology, Pharmacology
Laboratory,Department of Pharmaceutics, Varanasi, India
226
All therapeutic trials with Hypericum perforatum extracts reported during
past decades have concentrated on its antidepressants like efcacy only.
Preclinical studies have revealed though, that such extracts possess
diverse other therapeutically interesting pharmacological activities as
well. Critical analyses of available preclinical information on such
extracts and their bio-active constituents lead speculate that they could as
well be medicinally used as adaptogens suitable for coping with, or ameliorating, health problems caused by environmental and/or metabolic
stress. Efforts made to experimentally verify this working hypothesis
revealed clear dose dependant anti-aggressive activity of one such extract
in a battery of ve rodent behavioral models commonly used to test antistress activities of psycho-active drugs and agents. Similar was the case
also in rodent metabolic disorders models for hyperglycemia and hypercholesterolemia; two conditions often associated with environmental
stress. Effective oral daily doses of the extract in these models were similar to those needed to observe its anti-depressant like efcacy in rodent
models. Although analogous was also the case for hyperforin, quantitatively the observed effects of the extract was higher than expected by its
hyperforin content. These observations lend further experimental evidences to our conviction that pharmacologically the tested extract cannot
be dened as an antidepressant only, and that hyperforin is not its only
bio-active component.
Rasayan drugs act inside the human body by modulating the neuroendocrino-immunesystems and have been found to be a rich source of
possible therapeutic measure has become a subject of active scientic
investigations. Vajikaran is one of the eight branches that deal with
improving male sexual potency and thereby ensuring a supraja, or better
progeny. The main aim of Vajikaran besides achieving successful copulation for healthy reproduction, with sexual pleasureis an additional benet.
The plant Curculigo orchioides, Astercanthalongifolia, Mucuna pruriens
are well known vajikaran rasayan herbs. The studywas therefore performed to effect of these plants on reproductive parameters.Following
parameters were evaluated the effect of extract on body and organweights, change in histoarchitecture of testis, fructose level in seminalvesicles and hormonal level was studied (Chauhan et al., Fitoterapia 2007;
78:530-534). Administration of ethanolic extract had pronounced anabolic andspermatogenic effect in treated animals as evidenced by weight
gains in the bodyand reproductive organs. Increase in spermatogenesis
was shown in all treatedgroup. The level of follicular stimulating hormone, leutinizing hormone andtestosterone level is signicantly
increased in extract treated group andfructose content in seminal vesicles
was signicantly increases in treatedgroups. Thus it was concluded that
drug was justifying the use in thetraditional system of medicine as a
vajikaran rasayana.
Paper No.: 1004

STRUCTURE ACTIVITY RELATIONSHIP (SAR) OF
HYPERFORIN FOR ITS ANTIDEPRESSANT-LIKE ACTIVITY
PROFILE
Paper No.: 2241

EFFECT OF PRE-ISCHAEMIC CONDITIONING ON ANOXIC
DOPAMINE EFFLUX IN THE MOUSE CAUDATE
Shyam Sunder Chatterjee(1), O Kristhal(2)

(1) Herbal Consultant, Karlsruhe, Germany
(2) Bogomoletz Institute of Physiology, Kiev, Ukraine
Hyperforin is a labile, but quantitatively the major, bio-active secondary
metabolite of Hypericum perforatum with a broad spectrum of therapeutically interesting pharmacological activity prole. Initial SAR studies
have indicated that the phloroglucinol moiety of this structurally complex
molecule could be its essential pharmacophore, and that mono-acylated
hyperforin derivatives could lead to pharmaceutically stable molecules
with antidepressant-like activity proles. Our more recent SAR studies
revealed that such molecules possess also a broad spectrum of ion channels related functions modulating activity prole of major ionotropic
mechanisms. These efforts were made to identify structurally simpler and
pharmaceutically stable molecules with activity proles analogous to hyperforin suitable for drug development purposes. They led not only to a
library of phloroglucinol derivatives with diverse spectra of therapeutically interesting ion channels modulating activities, but also to several
symmetrically di-acylated phloroglucinols with hyperforin-like activity
proles in a battery of in vitro pharmacological screening models. However, in animal models none of these di-acylated phloroglucinols
revealed anti-depressant-like activities. Surprisingly though, a few other
phloroglucinol derivatives with no similarity to the activity prole of hyperforin observed in vitro, were almost as active as hyperforin in
behavioural models of depression. Therefore, we conclude that observed
ion channel functions and biogenic amine reuptake modulating effects of
hyperforin are not involved in its antidepressant like efcacy.
Paper No.: 416

ROLE OF RASAYAN HERBS ON REPRODUCTIVE
PARAMETERS OF MALE RATS
Nikky Kanti Chauhan, AMJ Young, CL Gibson, C Davidson

University of Leicester, School of Psychology, Leicester, UK
Pre-ischaemic conditioning (PIC) with brief periods of ischaemia has been
shown to be neuroprotective against a subsequent ischaemic event. Here
we examined whether short periods of PIC can affect hypoxia induced
dopamine (DA) release in the mouse caudate. Adult male mice (C57Bl/6
~30 g) were killed by cervical dislocation and their brains rapidly
removed. Brain slices (400 lm) were kept in oxygenated articial cerebrospinal uid (aCSF) at room temperature (22C). After at least 45 min
equilibration, slices were transferred to a brain slice chamber and perfused
with oxygenated aCSF at 33C. After a further 45 min equilibration they
were subject to PIC (0, 5 10 or 15 min) comprising hypoxia (N2/CO2)
and reduced glucose (10 to 2 mM) aCSF, then, 60 min later, a 15 min
hypoxic episode. DA efux was measured using fast cyclic voltammetry
at carbon bre electrodes. We recorded a) time from start of hypoxia to
start of DA release; b) time from start of DA release to peak DA release;
c) peak DA release and d) initial rate of DA release (dDA/dt). Data were
analyzed with 1-way ANOVA. The main result was that 10 and 15 min
PIC increased the time to onset of DA efux during the second hypoxic
event (F(3, 18) = 10.7, P < 0.001) values are 349, 561, 684 and 745s for
0, 5, 10 & 15 min PIC respectively. These data suggest that PIC of 10 and
15, but not 5 min are neuroprotective in the mouse caudate.
Paper No.: 3231

DISEASE AND THERAPY
GRP-78 IS A POSSIBLE TARGET IN
GABAA-CHANNELOPATHIES
Ksenia Chekina
Nagendra Chauhan, VK Dixit
Research Institute of Pharmacology, RAMS, Moscow, Russian

Federation
Natural Product Research Laboratory, Department of Pharmaceutical

Sciences, Dr. Hari Singh Gour Vishwavidyalaya, Sagar, India
The pharmacologic analysis of several nontypical anxiolytics (Afobazol,

Ladasten, Noopept, GB-115) revealed that the analyzed compounds have
227
number of properties in common: antioxidant activity, Ca+2-current stabilisation, antiapoptosis effect, and ability to prevent a stress induced
decrease in benzodiazepine binding. Furthermore, the literature analysis
revealed interactions between GABAAcomplex, glucose metabolism and
Ca+2-current regulation. In additional, there are literature data that some
of the mud stabilisation drugs direct effect on GRP78 expression. GRP78
(BIP) is a glucose-regulated chaperon which has binding domains for
Ca+2, caspase, nucleotides, and glucose. For their structure, GRP78 proteins function in neurons as adjusters of Ca+2-current, anti-apoptosis regulators, and starters of antioxidant cascades. It is not clear is GRP78
involved in GABAAcomplex regulation or not, but background of this
assumption consists of many substituted data. In the present paper we
bring out an indirect evidence of interactability between GRP78 and
GABAAcomplex. Yohimbine is a standard-like anxiogenic compound
which, as it shown in literature, leads to decrease in benzodiazepine binding in intact brains and breaks normal Ca+2 current by blocking Na+-channel. If GRP-78 is involved in GABAAcomplex regulation, the
yohimbine-induced decrease in benzodiazepine binding should be levelled
by excess of glucose. In the present study we demonstrate a dose-independent yohimbine-induced decrease in benzodiazepine binding in sinaptosomas and control level of benzodiazepine binding in sinaptosomas after
glucose-enrich incubation medium either with or without yohimbine in it.
So, our present results would be a basis of availability of further research
on GRP-78 as one of possible regulators of GABAA-Cl- channel.
Paper No.: 1761

IN VITRO ANTIPLASMODIAL ACTIVITY OF
8-HYDROXYQUINOLINE DERIVATIVES
National Health Research Institutes, Division of Biotechnology and Pharmaceutical Research, Zhunan, Taiwan
Type II diabetes is one of the common diseases worldwide and
accounted for 90% of the total population of patients with diabetes mellitus. It is a chronic disease status with reduced insulin effectiveness and
glucose tolerability. Normal glucose homeostasis is maintained by gastrointestinal hormones such as the insulinotrpoic incretins family of two
principal members glucose-dependent insulinotropic polypeptide and glucagon-like peptide-1. Both incretins have a short circulation half-life due
to rapid inactivation by the proteolytic enzyme dipeptidyl peptidase IV
(DPP-IV). One therapeutic strategy is, therefore, to maintain the blood
levels of incretins by the inhibition of DPP-IV activities. BPR1G462 is a
novel synthetic small compound that inhibits the DPP-IV activity. We
demonstrated that BPR1G462 is orally active and dose-dependently
inhibits the plasma DPP-IV activities in Sprague-Dawley rats and in dietinduced obese (DIO) mice. Oral administration of BPR1G462 caused a
signicant increase in the plasma levels of active glucagon-like peptide-1
and insulin activities in rats. BPR1G462 also increased the oral glucose
tolerability in lean and DIO mice. BPR1G462 is now a candidate for further preclinical and clinical developments for curing Type II diabetes.
Paper No.: 1695

SESSION - ONCOLOGY
PEPTIDE-CONJUGATED BIODEGRADABLE
NANOPARTICLES AS A CARRIER TO TARGET PACLITAXEL
TO TUMOR NEOVASCULATURE
Hong-Zhuan Chen, D-H Yu, Q Lu, J Xie, C Fang,
Chien-Teng Chen, R van Zyl
Shanghai JiaoTong University School of Medicine, Institute of Medical

Sciences, Department of Pharmacology, Shanghai, PR China
University of the Witwatersrand, Department of Pharmacy and Pharmacology, Johannesburg, South Africa
In present study, we developed a novel drug delivery system (DDS),

nanoparticles conjugated with K237-(HTMYYHHYQHHL) peptides for
tumor neovasculature targeting drug delivery. Paclitaxel, a chemotherapeutic agent with potent antiangiogenic activity, was used as a prototype
drug. We synthesized the aldehyde poly(ethylene glycol)poly(lactide)
(aldehyde-PEG-PLA) block copolymer by ring opening polymerization.
The nanoparticles loading paclitaxel (PTX-NP) were fabricated using the
O/W emulsion and evaporation technique. K237 ligand, a peptide that can
bind to the KDR receptors predominantly expressed on the surface of
tumor neovasculature endothelial cells with high afnity and specicity
and inhibit the VEGF-KDR angiogenic signal pathway, was conjugated
to the aldehyde group of PEG chain using the N-terminal PEGylation
technique. The K237 conjugated paclitaxel-loaded nanoparticles (K237PTX-NP) had a hydrodynamic diameter of 150 nm. The K237 density on
nanoparticle surface was 474. The K237 conjugated nanoparticles could
be signicantly internalized by human umbilical vein endothelial cells
(HUVEC) through K237-KDR interaction, and this facilitated uptake led
to the expected enhanced antiangiogenic activity shown by HUVEC proliferation, migration and tube formation compared to cells treated with
Taxol?and PTX-NP. The long-circulating property and K237 ligand of
K237-PTX-NP warranted rapid, long-term, and accurate in vivo tumor
neovasculature targeting, and thereafter the signicant apoptosis of tumor
neovasculature endothelial cells and necrosis of MDA-MB-231 breast
tumors implanted in female BLAB/c nude mice. This nanoparticulate
DDS offers a new strategy for paclitaxel chemotherapy application and it
could also be used to carry other chemotherapeutic drugs, genes, and proteins with antiangiogenic activity for antiangiogenic cancer therapy.
8-Hydroxyquinoline is a derivative of heterocycle quinoline with reported

antiseptic, disinfectant, antifungal, antiprotozoal, metal chelating and anticancer properties. It is structurally related to antimalarial compounds such
as quinine and chloroquine and derivatives could inhibit the in vitro
growth of the malaria parasite, as well as the formation of the malaria pigment, haemozoin. The antimalarial properties of the 8-hydroxyquinoline
and 14 derivatives were screened using the radiolabelled hypoxanthine
uptake method over a period of 48 hours. Haemozoin formation was
observed by incubating the compounds with haemozoin crystal precursors
under acidic conditions for 24 hours, while toxicity of the compounds
was screened against human kidney epithelial (Graham) cells using the
MTTcell viability assay. 8-Hydroxyquinoline was the most effective of
all the compounds screened (0.432 lM) as compared to quinine
(0.099 lM) against malaria. The antimalarial activity was not related to
the inhibition of haemozoin formation at a molar ratio of 5:1 (IC50:
959 lM) to haematin compared to quinine (0.2:1; IC50: 45 lM). At
1 lM, 8-hydroxquinoline exhibited 25% haemolysis compared to 1% for
quinine. 8-Hydroxyquinoline was 75 times more cytotoxic than quinine
against the kidney epithelial cells. A dichloro- substitution was more
active than dibromo-, dimethyl- and diiodide- substitutions (IC50 value:
1.709 > 4.879 > 6.083 > 7.771 lM, respectively). An amino substitution
increased the antimalarial activity compared to a nitro substitute (0.893
and 4.396 lM, respectively). Elucidating the mechanism of action of
8-hydroxyquinoline as an antimalarial is needed.
Paper No.: 2192

DISCOVERY OF A NOVEL DIPEPTIDYL PEPTIDASE-IV
INHIBITOR BPR1G462
Chiung-Tong Chen, K-C Yeh, C-Y Huang, W-T Jiaang, C-B Hu,
Y-W Huang, T Hsu, J-S Song, T-K Yeh, C-H Chien, S Chen, Y-S Chao
Paper No.: 2044

HEALTH AND DISEASE
ACUTE EFFECT OF SULFAPHENAZOLE ON ISOLATED
MESENTERIC SMALL ARTERIES FROM DB/DB MICE
Hua Chen, U Simonsen, C Aalkjaer
University of Aarhus, Department of Physiology, Aarhus, Denmark
228
db/db mice are fat and hyperglycemic, and an established model for
human type 2 diabetes. Experiments were designed to investigate
whether treatment with sulfaphenazole, a specic cytochrome P450 2C9
inhibitor, could improve endothelial dysfunction in mesenteric small
arteries of db/db mice in vitro. Obese db/db mice and lean db/+ littermates, aged 9-12 weeks, were sacriced after cervical dislocation. First
or second-order mesenteric small arteries were dissected and mounted
for isobaric force measurements. Intracellular calcium ([Ca2 + ]i) transients and production of reactive oxygen species (ROS) were measured
by a confocal laser scanning microscope. Impaired relaxations to acetylcholine (ACh) were observed in mesenteric resistance arteries from db/
db mice. Incubation with 10 M sulfaphenazole for 30 min signicantly
improved ACh-induced relaxation in these mice. The endothelial cell
[Ca2 + ]i transients induced by ACh were signicantly lower and cytosolic ROS signals signicantly more pronounced in arteries from db/db
mice. Both abnormalities were normalized after sulfaphenazole incubation. The present study shows that sulfaphenazole enhances endothelial
dysfunction by decreasing ROS production and increasing [Ca2 + ]i
within endothelial cells. These observations may suggest an effect of
ROS on endothelial cell Ca2 + handling and consequently for endothelial
function in diabetes.
Paper No.: 495

DISEASES
THE ADJUVANT EFFECTS OF SPECIFIC
PHYTOCOMPOUNDS ON TUMOR CELL LYSATE-PULSED
DENDRITIC CELL-BASED VACCINE VIA INDUCTION OF
IMMUNOGENIC TUMOR CELL DEATH
Hui-Ming Chen(1,2), S-S Chen(3), N-S Yang(1)
(1) Department and Institute of Pharmacology, National Yang Ming
University, Taipei, Taiwan
(2) Agricultural Biotechnology Research Center, Academia Sinica,
Taipei, Taiwan
(3) IgE Therapeutics, Inc., Department of Allergy and Immunology, San
Diego, California, USA
An emerging concept of a key-lock paradigm between the immunogenic cell death and dendrite cell (DC) is recently proposed. Immunogenic cell death is characterized by immunogenic factors (HMGB1,
calreticulin, heat shock proteins), which are helpful for the maturation,
antigen uptake and presentation of DC and serve as powerful immunological adjuvants. Whether some specic phytocompounds claimed
to have anti-tumor effects can deliver the immunogenic signals and
keep expending the tumor-recognition factors is a challenging question. To address this possibility, we rst determined the expression
proles of immunogenic factors in tumor cell lysate (TCL) obtained
from B16 cells treated with test phytocompounds (emodin, epigallocatechin-3-gallate, curcumin, shikonin and it derivatives) and chemotherapeutic drugs (doxorubicin and pacilitaxol) at varying
concentrations. Anthracyclines-Dorxorubicin is reported to confer a
higher capacity in eliciting immunogenic cell death. Shikonin, similar
to doxorubicin, can induce tumor cell death and specic immunogenic
factors expression (Hsp70 and Hsp90) in TCL in a dose-dependent
manner, but decrease the survivin expression. The cytotoxic effects of
splenocytes on target cancer cells were much higher in TCL-shikoninDC vaccinated mice than in TCL-DMSO-DC vaccinated mice. TCLshikonin-DC vaccination can substantially decrease the tumor growth
and maintain over 70% of survival rate of tumor-bearing mice after
35 days of tumor cell injection. In conclusion, shikonin is shown here
to sensitize tumor cells to the lytic effects of DC-activated immune
effector cells, which may warrant future evaluation as adjuvant of
tumor lysate-pulsed DC vaccine.
Paper No.: 957

DISEASES
THEOPHYLLINE-SUBSTITUTED KMUP-1 INHIBITS
ALLERGEN-INDUCED PULMONARY VASCULAR AND
BRONCHIAL INFLAMMATION: ENOS-ENHANCEMENT AND
MMP-9-SUPPRESSION
Ing-Jun Chen
Kaohsiung Medical University, Department of Pharmacology,
Kaohsiung, Taiwan
KMUP-1 has been proposed to be a potential treatment for vascular and
bronchial inammation characterized by chronic asthma and obstructive
pulmonary disease. Theophylline has been recognized as a bronchodilator in the treatment of asthmatic attacks and bronchitis related to an immunoresponse. However, a low therapeutic margin of safety has led it
hard to be used. We here investigated the effects of inhaled KMUP-1 in
the non-asthmatic mice and the asthmatic model of ovabumin (OVA)sensitized and challenged mice. Mice were allocated to either: 1) a short
term inhalation protocol, to receive KMUP-1 (1-5 mM) for 30 min in
mice; or 2) a 28-day inhalation protocol to treat asthmatic mice, sensitized with intra-peritoneal OVA on day 1 and day 8 and challenged with
OVA nebulization and treatment with KMUP-1 nebulization (5 mM/30
mins/day) on day 21-27. In both protocols, expression of endothelial
nitric oxide synthase (eNOS) was increased and matrix metalloproteinases-9 (MMP-9) was decreased in lung tissue. Pretreatment of L-NAME
(12 mM, 15 min) reversed these expressions in the short-term experiment. In 28-day experiment, OVA-sensitized decrease of sGCa1 and
PKG and increase of iNOS, ICAM-1, and VCAM-1 expression were
reversed by KMUP-1. Cell count, NO metabolite (NOx) and MMP-9activity in bronchoalveolar lavage uid (BALF) were decreased. KMUP1 reduced OVA-sensitized vascular and bronchial wall thickening,
eNOS-immunostaining at the alveolar septa and MMP-9-immunostaining
in the bronchioles and inltrated inammatory cells in the peri-vascular
and peri-bronchiolar region. KMUP-1 can be useful in inhibiting pulmonary vascular and bronchial inammation in chronic asthma and obstruction pulmonary disease.
Paper No.: 2286

A NOVEL ANTITUSSIVE BOTANICAL DRUG PDC-1427
Jui-Ching Chen, C-Y Lin, Y-L Ku
Medical and Pharmaceutical Industry Technology and Development Center, Natural Product Research Department, Taipei, Taiwan
Chronic cough is a common symptom with signicant morbidity. This
study was aimed at evaluating the antitussive activity of PDC-1427, an
extract generated from natural plant, in citric acid-induced cough reex
assay in guinea pigs. PDC-1427 at doses of 2000, 1000, 500, 250 and
125 mg/kg were given orally twice a day for one day. It is observed that
PDC-1427 (500~2000 mg/kg) demonstrated signicant inhibitory effect
in citric acid-induced cough reex. PDC-1427 at 2000 mg/kg possessed
cough suppression effect comparable to opioid agonist codeine (100 mg/
kg). In addition, PDC-1427 also caused signicant reduction in cough
induced by citric acid challenge at 1, 2, 4 and 6 hours after nal dosing.
A broad spectrum activities study on central nervous system, autonomic
system, cardiovascular function, respiratory function, gastrointestinal system, renal function, allergy, inammation and metabolism as well as in
vitro tissue functions was also conducted for PDC-1427. In toxicological
studies, we found no obvious adverse effects in genotoxicity tests, an
acute oral toxicity study, and a repeated dose 28-day subacute oral toxicity study in rats and dogs. The NOAEL of each study are estimated to be
5000, 1500~4500 and 1500 mg/kg/day, respectively. This study provides
229
novel insights in the therapeutic effect of PDC-1427 against citric acidand capsaicin-induced cough, and then opens scope for further studies on
identication of chemical constituents and pharmacological mechanisms
of PDC-1427.
Paper No.: 1740

SESSION - ONCOLOGY
CRYPTOTANSHINONE HAS OPPOSITE CELL
CYCLE-ARRESTING EFFECTS ON MELANOMA CELLS OF
DIFFERENT METASTATIC CAPACITY
L Chen, S-Z Zheng, A-Y Wang, Lu Yin
(1) Nanjing University of Chinese Medicine, College of Pharmacy, Nanjing, PR China
Introduction: Cryptotanshinone (CTs) is a main active component of
Radix Salviae Miltiorrhizae, which is often used to therapy cancer as traditional Chinese herb.In this study, the effect of CTs on two melanoma
cells(B16BL6/B16) of high/low metastatic capacity has been systematically compared.Methods: We employed the effect of CTs on cell apoptosis or proliferation by Annexin V, TUNEL assay or BrdU assay etc.
Assayed the cell distributions of cell cycle by ow cytometry. The checkpoint integrity was determined by mutational analyses of B-RAF and
N-RAS, and the effect of CTs on the expression of cell cycle associated
proteins was performed by Western blotting. Results:In B16BL6 we
could found that CTs could not detect any obvious apoptosis but inhibited cell proliferation,also CTs induced cell cycle arrest via induction the
expression of checkpoint enforced by p53, Chk1 and Chk2. CTs-induced
G1 arrest of B16BL6 cells was correlated to an increase in p21. By contrast, in B16 cells, the G2/M arrest is the consequence of the induction
of Cdc25c, and the regulation of Cyclin A1, Cyclin B1 and Cdk1/cdc2
expression could be a major contributor .Conclusions: CTs, which has
opposite cell cycle-arresting effect on melanoma cells of different metastatic capacity, may offer opportunities to investigate the reasons for this
different pattern of malignancy.It will be used as a leading compound for
further studies.
Paper No.: 993

SESSION - ONCOLOGY
PULMONARY TOXICITY OF BLEOMYCIN INVOLVED IN
UPREGULATION OF LYSYL OXIDASE ACTIVITY IN
CULTURED HUMAN FETAL LUNG FIBROBLASTS
Lijun Chen, S Li, X Su, G Lin
Sun Yat-sen University Zhongshan Medical School, Department of Pharmaclogy, Guangzhou, PR China
Bleomycin (BLM) has been used as a cancer chemotherapy for many
years. Although pulmonary toxicity and pulmonary brosis of BLM
often occur in clinic, its mechanism is poorly understood. Lysyl oxidase
(LO), a copper (Cu)-dependent amino oxidase, plays a critical role in
morphogenesis and tissue repair of lung and other organs by catalyzing
the crosslinking of elastin and collagen and stabilizing extracellular
matrix (ECM). To better understand mechanisms of BLM-induced pulmonary brosis, we studied changes in LO mRNA, protein, and catalytic
activity levels in cultured human fetal lung broblasts (HLF) exposed to
BLM. Exposure of HLF cells to BLM at 10 lg/ml and 30 lg/ml
increased obviously LO catalytic activity in the conditioned media. The
expression of LO mRNA was enhanced signicantly in HLF cells exposure to BLM at 3 lg/ml. BLM at 3 lg/ml also increased the expression
of 46 kDa preproLO, 50 kDa proLO and 32 kDa mature LO. To probe
mechanisms of upregulation of LO activity, we tested the Cu concentration in the medium of HLF cells exposed to BLM. Consistently, the Cu
concentrations in conditioned media of cultured HLF cells exposed to
BLM were increased signicantly with increases of BLM ranging from

10 to 30 lg/ml. These results suggest that BLM enhanced LO activity in
association of increasing copper concentration in conditioned media of
cultured HLF cells, which may be involved in pulmonary toxicity and
pulmonary brosis of BLM.
Key words: bleomycin (BLM); pulmonary brosis; lysyl oxidase (LO);
copper (Cu).
Paper No.: 2466

DISEASES
OXYTOCIN INJECTED ON OVARIECTOMIZED FEMALE
RATS CAN AMELIORATE HEATSTROKE VIA INCREASING
CIRCULATING ENDOTHELIAL PROGENITOR CELLS
Sheng-Hsien Chen(1), W-T Huang(2), CY Kang(1)
(1) Chi Mei Medical Center, Southern Taiwain University, Department
of Gynecology & obstetrics, Department of Biotechnology, Tainan,
Taiwan
(2) Chia Nan University of Pharmacy Science, Taiwan
Background: The number of circulating endothelial progenitor cells
(EPCs) is decreased in postmenopausal women, and increased in those
on hormone replacement therapy (HRT). Some researchers have indicated that oxytocin (OT) controls differentiation of human mesenchymal
stem cell to osteoblasts (OBs) and reverse osteoporosis. Whether OT can
resuscitate heatstroke rats via increase circulating EPCs is unclear. Aim:
Our current study was designed to investigate the therapeutic effects of
OT on 2 weeks after ovariectomized (OVX) female SD rats subjected to
heatstroke insults. Measurements and Results: Female rats post OVX
2 weeks later were randomly assigned into three groups: a) vehicle 1 cc
subcutaneous injection (sc) only for 8 weeks. b) OT 1mg/kg/day sc for
8 weeks. c) then following normothermia (Urethane-anesthetized rats
exposed to 26 C or 480 min) or heatstroke (Urethane-anesthetized rats
were exposed to an ambient temperature of 43 C to induce heatstrokemean arterial pressure (MAP) decreasing more than 25 mmHg). OT pretreatment on OVX female rats signicantly increased circulating EPCs
before heat stress compared with saline controls via ow cytometry
assay. After the onset of heatstroke, animals treated with saline displayed
hyperthermia, hypotension, bradycardia, hypothalamic neuronal apoptosis and degeneration, and up-regulation of systemic inammatory
response molecules including serum tumor necrosis factor-a, soluble
intercellular adhesion molecule-1 and E-selectin. Heatstroke-induced
hypotension, bradycardia, hypothalamic neuronal apoptosis and degeneration, and increased systemic inammatory response molecules were signicantly attenuated by OT pretreatment. Conclusion: Increased
circulating EPCs produced by OT administered to OVX rats can protect
against heatstroke via attenuation of systemic inammation, neuronal
damage and vascular endothelial damage.
Paper No.: 1459

HEALTH AND DISEASE
ROLE OF RHOA/ROK IN VASCULAR HYPOREACTIVITY
INDUCED BY ENDOTOXIN IN EARLY AND LATE SEPSIS
Shiu-Jen Chen(1), M-H Liao(2), C-C Wu(2)
(1) Kang-Ning Junior College of Medical Care and Management,
Department of Nursing, Taipei, Taiwan
(2) National Defence Medical Centre, Taipei, Taiwan
Blood vessel tone plays an important role in the determination of tissue
perfusion and blood pressure. In severe sepsis, the vascular reactivities to
vasconstrictor and vasodilator are greatly impaired. However, the mechanism of development of vascular hyporeactivity remains unclear. Calcium
230
sensitization and desensitization are involved in mechanisms regulating
vascular tone. Myosin phosphatase (MYPT) and the RhoA-associated
Rho-kinase (RhoA/ROK) pathway are two major cellular targets for regulating Ca2 + sensitivity of agonist-induced contraction. In this study, we
investigated the role of RhoA/ROK in endotoxin (lipopolysaccharide,
LPS)-induced vascular hyporeactivity in early (at 1 h and 2 h after LPS)
and late (at 4 h and 6 h after LPS) sepsis. The sepsis-induced vascular hyporeactivity was performed by intravenous infusion of LPS (10 mg/kg,
10 min) to male Wistar rats. In this study, the changes of hemodynamics,
blood glucose, hepatic (GPT, GOT) and renal (CRE, BUN) function,
plasma nitrate (an indicator of NO), and the vascular reactivities to norepinephrine (NE) and acetylcholine (Ach) of thoracic aorta in vivo and ex
vivo were measured. In addition, the expression of RhoA in the thoracic
aorta was analyzed by immunohistological chemistry and.Western blot,
whereas pathological studies of lungs, livers, kidneys and thoracic aorta
were also examined. Our preliminary results showed that LPS induced
not only dysfunction of liver and kidney but also vascular hyporeactivity
at 1 h, 2 h, 4h, and 6 h in vivo and this hyporeactivity was varied ex vivo
and associated with RhoA expression in early and late sepsis.
Paper No.: 1599

INVOLVEMENT OF AUTOPHAGY IN THE SEX-DIFFERENT
BRAIN INJURY CAUSED BY IRON OVERLOAD
Tzu-Yin Chen, S-I Lue, C Hsu
Kaohsiung Medical University, Department of Physiology, Kaohsiung,
Taiwan
Overproduction of iron, a degradation product of hemoglobin after hemorrhage, leads to free radical formation and oxidative stress in brain tissue. Oxidative stress and subsequent organelle damage simultaneously
induces autophagy that is an intracellular recycling protein degradation
system, while over activation of autophagy may be harmful for the acute
brain injury such as iron overload caused by hemorrhage. Our previous
study showed that infusion of ferrous citrate (FC) in caudate nucleus
(CN) caused a severer brain injury in male rats than that in females and
the estradiol pretreatment diminished the FC-induced brain injury. We
propose autophagy participates in the sex-different FC-induced brain
injury. Microtubule-associated protein 1 light chain 3 was detected to
estimate autophagy. The cleavage of a II-spectrin was quantied to estimate the severity of brain injury. The results showed that iron-induced
autophagy and DNA damage with intact nuclei implying autophagic cell
death were observed. In addition, mice of Atg5(+/)), an autophagyrelated gene, showed a less FC-induced brain decit and injury compare
to Atg5(+/+) mice. The levels of constitutive and iron-induced autophagy
in CN were lower in female rats than in males. Levels of iron-induced
autophagy correlate with the severity of brain injury in both male and
female rats. Moreover, pretreatment of estradiol decreased both the level
of iron-induced autophagy and cleavage of a II-spectrin in females. Since
ER-a expression is higher in females than in males, the inhibition of
iron-induced autophagy by estradiol may mediate the ER-dependent neuroprotection in females from brain injury caused by iron overload.
Paper No.: 1059

MODULATORY EFFECTS OF THE EXTRACT OF BURDOCK
TEA ON HYPERLIPIDEMIA RAT AND RICE MODELS
Xiangguo Chen(1), Y Xu(2), Y Cao(2), Z Han(2), P Li(2), C Wang(3)
(1) Qingdao Food and Drug Administration, Department of Pharmacy,
Qingdao, PR China
Qingdao, PR China
(3) Qingdao University, Medical College, Qingdao, PR China
Arctium lappa Linne (AL; burdock) is a biennial member of the Asteraceae (Compositae) family, and its carrot-like root is commonly cooked
and eaten as a vegetable in parts of Asia. In traditional Brazilian and
Asian folk medicine, AL is used for the treatment of different diseases.
In the present study the effects of the extract of burdock tea on lipid prole were observed in hyperlipemic rats and mice. Seven-week-old male
Wistar rats were fed a high-fat diet to induce hyperlipemia with doses of
extract of burdock tea at 0.83 (low), 1.67 (medium), and 5.0 (high) g/kg
of body weight. The hyperlipidemia mice models were induced by intraperitoneal injection of 75% solution of egg yolk, and the Kunming mice
were intragastric administered with extract of burdock tea (25g/kg, 50g/
kg of body weight). The body weight and the concentrations of total cholesterin (TC), triglyceride (TG) and high density lipoprotein cholesterol
(HDL-C) were detected. The results showed that TG and HDL-C in rats
were decreased signicantly in the two burdock tea groups (1.67g/kg.bw,
5.00g/kg.bw) and the body weight of rats was reduce remarkably. The
TG and HDL-C in mice were remarkably decreased in the burdock tea
groups (50g/kg of body weight). Taken together, these results indicated
that the extract of burdock tea has modulatory effect on blood lipid in
hyperlipidemia model rats and mice.
(Supported by the Shandong Natural Science Foundation of China, No.
Q2008C04 and Z2007c09).
Paper No.: 761

THE MECHANISM OF MNNG-INDUCED-APOPTOSIS: AMPK
COULD BE A TRANSMITTER FROM PARP ACTIVATION TO
MITOCHONDRIAL DYSFUNCTION
Yi-Cheng Chen, J-H Guh
National Taiwan University, Pharmacy Department, Taipei, Taiwan
N-Methyl-N-Nitro-N-Nitrosoguanidine (MNNG), a DNA-alkylating
agent, was well known to exert its cytotoxicity by activating poly (ADPribose) polymerase (PARP) and mitochondrial dysfunction is one of the
mechanisms to induce apoptotic cell death. However, how PARP, a
nuclear protein, conveys the death signal to mitochondria was still
unclear. Here we try to gure out what signal is activated and could be
regarded as an inducer of mitochondrial dysfunction after MNNG treatment. We found that AMP-activated protein kinase (AMPK) could be a
candidate. In PC-3 prostate cancer cells, MNNG can phosphorylate
AMPK on Thr172 and Ser385, indicating the activation. Furthermore,
among the MNNG-treated cells, we separately collected the detached
and attached parts, and observed that AMPK phosphorylation is more
obvious in detached fraction, which are judged dying or to die by ow
cytometry. Besides, MNNG can arrest cells on s/G2 phase at 6 hr, G2/M
phase at 12 hr and nally result in subG1 increase. The combination with
compound C (a specic AMPK inhibitor) can reverse cell cycle arrest
and subG1 increase induced by MNNG. These data suggest the involvement of AMPK in MNNG-induced apoptosis. So we propose that
AMPK could be a transmitter role which accepts the death message from
nucleus to mitochondria in MNNG-induced apoptosis.
Paper No.: 2298

APPROACH OF ACTION MECHANISM OF TCM-98 AND ITS
ACTIVE COMPONENT ON NEURO- INFLAMMATION IN IN
VIVO AND IN VITRO MODELS
Yuh-Fung Chen(1,2), Y-W Wang(2), H-Y Tsai(3)
(1) China Medical University, Department of Pharmacolgy, Taichung,
PR China
231
(2) China Medical University, Graduate Institute of Chinese Pharmaceutical Sciences, Taichung, PR China
(3) China Medical University Hospital, Department of Pharmay,
Taichung, PR China
Cerebral vascular diseases are the third leading cause of death in Taiwan in recent years. Neuro-inammation and neuronal cell death occur
in the early stage of stroke. Our preliminary data showed that TCM-98
and TCM-9811 signicantly reduced the infract volume of the stroke
rat brain. Furthermore, TCM-98 crude and TCM-9811 obviously
decreased both productions of NO and protein expression of iNOS and
COX2 induced by LPS, while increasing p53 protein expression. Specially, the results of TCM-98 crude and TCM-9811 reversing p53 protein attenuated by LPS is interesting for us and remain for further
investigation because p53 traditionally is thought to be involved in
apoptosis, cell cycle arrest, and DNA repair. Although previous studies
have demonstrated p53 is a suppressor of inammatory response in different experimental types, the underlying mechanisms of anti-inammation are poorly understood in microglia cells. In the other hand, TCM9811 was able to inhibit the effects that LPS enhanced the nuclear
translocation of p65 and p50 subunits, an effect preceded by the cytosolic decrease in IjBa and promote in phosphorylation of IjBa. Meanwhile, TCM-9811 also reversed the cytosolic p65 and p50 subunits
expression repressed by LPS. Results from our data showed that TCM98 and TCM-9811 have anti-inammatory actions against inammation
caused by LPS. The possible action mechanism of TCM-98 and TCM9811 involved in BV-2 cell stimulated by LPS model is investigated in
this study.
Paper No.: 3236

SESSION - ONCOLOGY
THE RELATIONSHIP BETWEEN MEKK1 AND
CARCINOGENESIS & DEVELOPMENT OF PANCREATIC
CANCER
Xiaoguang Cheng(1), F Su(1), K Li(1), Y Zhang(1), C Yan(2)
(1) Chinese Academy of Medical Sciences & Peking Union Medical
College, Institute of Materia Medica, Beijing, PR China
(2) Harbin Medical University, The Second Afliated Hospital, Beijing,
PR China
To observe whether the MEKK1 expression is related to pancreatic cancer in vitro, we examined the MEKK1 mRNA and protein levels in different pancreatic cancer cell lines (BxPC3, Capan2, SW1990 and
PANC1). The data showed that MEKK1 mRNA level remained highly
abundant in BxPC3 and Capan2 cell lines while it had weak expression
in the SW1990 and PANC1 cell lines. A Western blot analysis and immunouence assay further conrmed the MEKK1 protein expression in
a similar pattern in the four observed pancreatic cell lines. To identify the
detailed roles of MEKK1 in the regulation of malignant characteristics of
human pancreatic adenocarcinoma in vivo. We analyzed MEKK1 expression in patients samples by immunohistochemistry assay. The results
demonstrated that MEKK1 positive staining was noted in 32 samples
among the 41 pancreatic adenocarcinomas (78.1%). All results suggested
that MEKK1 expression was signicantly associated with differentiation,
TNM staining and lymph node metastasis in the observed pancreatic cancer patients (P < 0.05). The MEKK1 staining did not show signicantly
different among the patients of different gender, age, tumor location and
tumor size (P > 0.05). In addition, there is a mainly founding that
MEKK1 cell location veried with cancer differentiation grade. The
amounts of nuclear staining is 100% in poorly differentiated cancers,
while the amount of nuclear staining in moderate and well differentiated
cancers were 72.2% and 22.2%.In conclusion, this study demonstrates
that MEKK1 plays a major role in the metastasis of pancreatic cancer.
Our ndings suggest that MEKK1 may be a potential molecule target for
pancreatic cancer therapy.
Paper No.: 562

EXPRESSION OF VGLUTS IN THE PROCESS OF
DEGENERATION AND ACQUISITION OF LEARNING AND
MEMORY
Xiaorui Cheng, Y Zhao, W Zhou, Y Zhang
Beijing Institute of Pharmacology and Toxicology, Department of Neuroimmunopharmacology, Beijing, PR China
Vesicular glutamate transporters (VGLUTs) include VGLUT1, VGLUT2
and VGLUT3, and are responsible for the uploading of L-glutamate into
synaptic vesicles. The expression of VGLUTs determines the level of synaptic vesicle lling and glutamate quantal size, indicates the glutamatergic
neurons and synapse number, thus directly inuences glutamate receptors
and glutamatergic synaptic transmission. In order to investigate whether
VGLUTs has correlation with the process of degeneration and acquisition
of learning and memory, we choose senescence-accelerated mouse/prone
8 (SAMP8) as the animal model of degeneration of learning and memory,
mouse through the shuttle-box test as animal model of acquisition of
learning and memory, the expression of VGLUTs and synaptophysin
(Syp) mRNA and protein in the cerebral cortex and hippocampus of the
tested subjects was investigated using QPCR and western-blot technique
respectively. Results showed that VGLUT1, VGLUT2 and Syp in the
cerebral cortex, VGLUT1 and Syp in the hippocampus of SAMP8
decreased with aging and aged senescence-accelerated mouse resistant
strains 1 (SAMR1), while VGLUT2 in the hippocampus decreased in
SAMR1 and increased in SAMP8 with aging. VGLUT3 appeared nonregular pattern. The expression of VGLUT1 mRNA and protein increased
in the process of acquisition of learning and memory in the shuttle box
test. This indicates the complementary expression of VGLUT1 and
VGLUT2 in the hippocampus of SAMP8 and VGLUTs related with learning and memory tightly. These results suggested the involvement of the
brain glutamatergic system in the degeneration of learning and memory in
SAMP8 and glutamateric neuron loss in the brain of the SAMP8.
Paper No.: 2220
ANALGESICS
SINGLE DOSE OF TRAMADOL ON NEUROTRANSMITTER
SYSTEMS IN TARGET AND NON-TARGET AREAS OF RAT
BRAIN WITHOUT NOCICEPTION
Pandanaboina Chetan(1), S Rajbanshi(2), R Wudayagiri(3)
(1) Arkansas State University, Department of Biological Sciences, Jonesboro, AR, USA
(2) Sri. Venkateswara University, Department of Zoology, Tirupati, India
(3) Mahila University, Department of Pharmacy, Tirupati, India
In the present investigation the changes in the levels of Monoamines
such as epinephrine (E), norepinephrine (NE), dopamine (DA), and serotonin (5-HT) and also levels of the major metabolites Homovanillic acid
(HVA) and Hydroxyindole acetic acid (HYAA) were estimated in different areas of the rat brain during the administration of the synthetic opioid
analgesic drug Tramadol (Ultram) without induction of pain. Following
administration of single dose of tramadol (31 mg/kg body weight), the
levels of DA, 5-HT were elevated in all the regions of brain, while NE,
EP, HIAA levels and MAO activity recorded increase in some areas and
decrease in other areas by 3 or 6 hrs. The levels of biogenic amines
returned to the respective control levels by 24 hrs after tramadol administration. The monoamine oxidase activity levels were elevated in all the
brain regions except in hypothalamus after treatment with a single dose
of tramadol. The region specic alterations in biogenic amines and their
metabolites in different areas of rat brain following administration of single dose of tramadol suggest that the analgesic actions of tramadol might
be implicated to the enhanced monoaminergic neurotransmission similar
to other opiods as well as other classes of analgesics.
232
Paper No.: 1707
A GENETIC VARIANT IN THE GENE ENCODING
ADRENOMEDULLIN PREDICTS THE DEVELOPMENT OF
DYSGLYCAEMIA OVER 6.4 YEARS IN CHINESE
Bernard MY Cheung(1), AWK Tso(1,2), KL Ong(1), RYH Leung(1),
SS Cherny(3), PC Sham(3), TH Lam(4), KSL Lam(1,2)
China
(2) University of Hong Kong, Research Centre of Heart, Brain, Hormone
and Healthy Aging, Hong Kong, PR China
(3) University of Hong Kong, Department of Psychiatry and Genome
Research Centre, Hong Kong, PR China
(4) University of Hong Kong, Department of Community Medicine,
Hong Kong, PR China
Adrenomedullin (AM), a vasodilatory peptide, facilitates the differentiation of pre-adipocytes and affects lipolysis and glucose uptake. We investigated the association of common single nucleotide polymorphisms
(SNPs) in the gene encoding adrenomedullin (ADM) with dysglycaemia
in the Hong Kong Chinese population. Four SNPs (rs3814700,
rs11042725, rs34354539 and rs4910118) were genotyped in 1391 subjects without dysglycaemia at baseline from the Hong Kong Cardiovascular Risk Factor Prevalence Study-2 (CRISPS-2), which had a median
follow-up time of 6.4 years. Dysglycaemia included impaired fasting
glucose, impaired glucose tolerance and diabetes according to the WHO
1998 criteria. At follow-up, 382 subjects had developed dysglycaemia.
In stepwise logistic regression, the SNP rs11042725 (minor allele frequency = 28.7%) was a signicant independent predictor of the development of dysglycaemia at 6.4 years (OR=1.31, P = 0.014), together with
age (P < 0.001), plasma triglycerides (P < 0.001), body mass index
(P < 0.001), 2-h glucose after oral glucose tolerance test (P < 0.001),
change in body mass index (P = 0.001) and follow-up duration
(P = 0.006). The association was more signicant in women (OR=1.65,
P = 0.002) and in subjects without regular exercise (OR=1.56,
P = 0.001). In women without regular exercise, the odds ratio (95% CI)
for developing dysglycaemia was 1.93 (1.30-2.85) (P = 0.001). The other
two SNPs, rs3814700 and rs34354539 also showed nominal associations
with the development of dysglycaemia (P = 0.034 and 0.021 respectively). Our study suggests a potential role of genetic variants in the
ADM gene in the development of dysglycaemia. Further studies on the
role of adrenomedullin in glucose metabolism are warranted.
Paper No.: 1708

RELATIONSHIP OF PLASMA INTERLEUKIN-6 AND ITS
GENETIC VARIANTS WITH HYPERTENSION IN HONG KONG
CHINESE
Bernard MY Cheung(1), AWK Tso(1,2), KL Ong(1), RYH Leung(1),
SS Cherny(3), PC Sham(3), GN Thomas(4), TH Lam(5), KSL Lam(1,2)
China
(2) University of Hong Kong, Research Centre of Heart, Brain, Hormone
and Healthy Aging, Hong Kong, PR China
(3) University of Hong Kong,Department of Psychiatry and Genome
Research Centre, Hong Kong, PR China
(4) University of Birmingham, Department of Public Health and Epidemiology, Birmingham, UK
(5) University of Hong Kong, Department of Community Medicine,
Hong Kong, PR China
Interleukin-6 (IL-6) plays a central role in inammation and insulin resistance as well as atherogenesis. We investigated if IL-6 is associated with
hypertension in a sample of the general population, and whether genetic

variants that might inuence plasma IL-6 level could be associated with
hypertension. Plasma IL-6 was measured using a high-sensitivity
enzyme-linked immunosorbent assay in 648 normotensive and 294
hypertensive subjects from the Hong Kong Cardiovascular Risk Factor
Prevalence Study-2 and three tagging single nucleotide polymorphisms
(SNPs) (rs17147230, rs1800796 and rs2069837) in the IL-6 gene were
genotyped. Plasma IL-6 level correlated with age (r = 0.171, P < 0.001),
body mass index (r = 0.109, P < 0.001), systolic blood pressure
(r = 0.083, P < 0.001), pulse pressure (r = 0.092, P = 0.010), C-reactive
protein (r = 0.225, P < 0.001) and high-density lipoprotein cholesterol
(r=-0.100, P = 0.002). Hypertensive subjects have signicantly higher
plasma IL-6 level after adjusting for age and sex (geometric mean (95%
CI) = 0.60 [0.54-0.65] vs 0.47 [0.44-0.50] pg/ml, P = 0.024).In multivariate logistic regression analysis, plasma IL-6 was independently associated with hypertension in women (P = 0.004), but not in men. The SNP
rs1800796 was independently associated with plasma IL-6 level (b=0.098, P = 0.002) in stepwise multiple linear regression. The association
was signicant in both men (P = 0.042) and women (P = 0.016).However, this SNP was not associated with hypertension. Elevated plasma
IL-6 level is associated with hypertension, especially in women. Plasma
IL-6 level is inuenced by the SNP rs1800796. However, this SNP is
not associated with hypertension, suggesting that hypertension is caused
by other factors that elevate plasma IL-6 level.
Paper No.: 1895

RESVERATROL INHIBITS 8-ISO-PROSTAGLANDIN F2ALPHA
PRODUCTION IN YOUNG AND AGED RAT BRAIN
Annalisa Chiavaroli, L Brunetti, G Orlando, L Menghini, L Recinella,
S Leone, C Ferrante, C Di Nisio, R Shohreh, P Di Michele, M Vacca
G. dAnnunzio University, Department of Drug Sciences, Chieti, Italy
Oxidative stress plays a role in the pathogenesis of neurodegeneration
typical of aging and 8-iso-prostaglandin F2a (8-iso-PGF2a), an isoprostane generated from oxygen radical peroxidation of arachidonic acid,
may represent a reliable marker of cellular oxidative damage. Resveratrol
is a naturally occurring phytoalexin that is produced in response to
injury, such as mechanical trauma, ultraviolet light and infection by pathogenic microorganisms, especially fungi, providing means for defense.
Resveratrol has been associated with numerous health benets from
chemoprevention to cardioprotection and might prevent or reverse 8-isoPGF2a production. We have tested the possible antioxidant effects of resveratrol in rat brain synaptosomes obtained from young (3 months old)
and aged (15 months old) male Wistar rats that were perfused, in vitro,
with graded concentration of resveratrol (0.01-50 microM), both in the
basal state and after hydrogen peroxide-induced oxidative stress, evaluating 8-iso-PGF2a levels in the perfusate by radioimmunoassay. In young
rats, we observed a concentration-dependent inhibitory effect of resveratrol on brain production only after hydrogen peroxide-induced oxidative
stimulus. In aged rats, a concentration dependent antioxidant effect of
resveratrol was evidenced both in the basal state and after hydrogen peroxide-induced oxidative stimulus. In conclusion, resveratrol could be
effective in preventing brain oxidative damage both in young and aged
rats, showing a greater inhibition of isoprostane production in the latter.
Paper No.: 1321

DISEASES
INHIBITORY EFFECT OF AS1517499, A NOVEL STAT6
INHIBITOR, ON ANTIGEN-INDUCED AIRWAY
HYPERRESPONSIVENESS IN MICE
Yoshihiko Chiba, M Todoroki, Y Nishida, M Tanabe, M Misawa
233
Hoshi University School of Pharmacy, Department of Pharmacology,
Tokyo, Japan
Interleukin-13 (IL-13) is one of the central mediators for development of
airway hyperresponsiveness in asthma. The signal transducer and activation of transcription 6 (STAT6) is one of the major signal transducers
activated by IL-13, and a possible involvement of IL-13/STAT6 pathway
in the augmented bronchial smooth muscle (BSM) contraction has been
suggested. In the present study, the effect of a novel STAT6 inhibitor,
AS1517499, on the development of antigen-induced BSM hyperresponsiveness was investigated. In cultured human BSM cells, IL-13 (100 ng/
mL) caused a phosphorylation of STAT6 and an upregulation of RhoA, a
monomeric GTPase responsible for calcium sensitization of smooth muscle contraction: both events were inhibited by co-incubation with
AS1517499 (100 nM). In BALB/c mice that were actively sensitized and
repeatedly challenged with ovalbumin antigen, an increased IL-13 level
in bronchoalveolar lavage uids and a phosphorylation of STAT6 in
bronchial tissues were observed after the last antigen challenge. These
mice had an augmented BSM contractility to acetylcholine together with
an upregulation of RhoA in bronchial tissues. Intraperitoneal injections
of AS1517499 (10 mg/kg) 1 h before each ovalbumin exposure inhibited
both the antigen-induced upregulation of RhoA and BSM hyperresponsiveness, almost completely. A partial but signicant inhibition of antigen-induced production of IL-13 was also found. These ndings suggest
that the inhibitory effects of STAT6 inhibitory agents, such as
AS1517499, both on RhoA and IL-13 upregulations might be useful for
asthma treatment.
Paper No.: 1299

IMPAIRMENT OF HEME OXYGENASE-1 EXPRESSION BY
THE DEFECT OF PARKINSON-RELATED PINK1 PROTEIN
Paper No.: 3298

MELAMINE MIGRATION FROM MELAMINE FOOD
CONTACT ARTICLES AVAILABLE ON THE MALAYSIAN
MARKET
Zamri Chik, DEM Haron, MA Mohamad, ED Ahmad, H Taha
University of Malaya, Faculty of Medicine, Department of Pharmacology, Kuala Lumpur, Malaysia
The migration of melamine from melamine-ware products retail in
Malaysia has been determined from 41 products. This study has been
conducted in the midst of public anxiety on the possibility of melamine
leaching into foods that come into contact with the melamine-wares.
Samples of melamine table-wares included cups and plates, forks and
spoons, tumblers, bowls, etc. were collected from various retail outlets.
The samples were exposed to the food stimulant, 3% acetic acid and also
distilled water at room temperature, 70C and 100C for 30 minutes.
The melamine detection was done using Liquid Chromatography-Tandem Mass Spectrometry (LC/MS/MS) method. The column used was a
HILIC C18 with a mobile phase consisting of a mixture of ammonium
acetate / formic acid (0.05%) in water and a mixture of ammonium acetate/ formic acid (0.05%) in acetonitrile with 95/5 (v/v). The limit of
quantitation (LOQ) was 1 ppb. The presence of melamine migration was
detected in all samples. For the articles tested with water, melamine
migration were (median (IQR)) 22.2 (32.6), 49.3 (50.9), 84.9 (89.9) ppb
at room temperature, 70 C, and 100 C, respectively. In acetic acid, melamine migration were 31.5 (35.7), 81.5 (76.2), 122.0 (126.7) ppb at
room temperature, 70 C, and 100 C, respectively. This study suggests
that excessive heat and acidity may directly affect melamine migration
from melamine-ware products. However the results showed that melamine migration in the tested items were well below the specic migration
limit at 30 mg/kg (30,000 ppb) in accordance to The European Commission Directive 2002/72/EC.
Wei-Lin Chien(1), J-R Li(1), M-J Li(3), W-M Fu(0)

(1) National Taiwan University College of Medicine, Pharmacological
Institute,Taipei, Taiwan
(2) National Taiwan University, Neurobiology and Cognition Center,
taipei, Taiwan
(3) National Taiwan University Hospital, Department of Neurology,
Taipei, Taiwan
Parkinsons disease (PD) is one of the most common neurodegenerative
diseases. PINK1 (PTEN induced putative kinase 1) is a mitochondria
serine/threonine protein kinase. Mutation in the PINK1 gene causes an
autosomal recessive form of PD. Although PINK1 is thought to protect
cells from stress-induced mitochondria dysfunction, the etiology related
to PINK1 mutation is still not clear. We have established stable clones
of PINK1 kinase domain mutant G309D in SH-SY5Y. It was found
that ROS and apoptosis increased in G309D mutant cells following
H2O2 treatment. Heme oxygenase 1 (HO-1) increases and exerts its cell
protection when the cells are under oxidative stress. It was found here
that HO-1 increased in wilt-type cells following H2O2 treatment.
Although PINK1 mutant stable clones had a higher basal level of
HO-1, however, HO-1 induction in response to H2O2 and MPP+ treatment was impaired by PINK1 mutation. Using adenovirus infection to
overexpress HO-1 can antagonize the H2O2-induced apoptosis in
PINK1 mutant stable clones. In addition, small hairpin RNA-mediated
knockdown of TNF receptor-associated protein 1 (TRAP1), which is
the substrate of PINK1 kinase, in SH-SY5Y cells also inhibited the
expression of HO-1 in response to H2O2 treatment. TRAP1 can
increase following H2O2 treatment, whereas, the expression of TRAP1
in response to H2O2 was also impaired by PINK1 G309D mutation.
Our ndings suggest a novel pathway by which the defect of PINK1
and its downstream effector TRAP1 inhibits the oxidative stressinduced HO-1 upregulation, the impairment may enhance the dopaminergic neurodegeneration in Parkinson.
Paper No.: 2244

ACTIVATION OF OX1, BUT NOT OX2, RECEPTORS IN THE
PERIAQUEDUCTAL GRAY PRODUCES ANTINOCICEPTION
VIA RETROGRADE ENDOCANNABINOID-INDUCED
DISINHIBITION
Lih-Chu Chiou(1,2,3), Y-C Ho(1), H-J Lee(2), Y-Y Liao(1), S-Y Fu(1),
S-F Teng(3), H-T Liao(3)
(1) National Taiwan University, College of Medicine, Graduate Institute
of Pharmacology, Department of Pharmacology, Taipei, Taiwan
(2) National Taiwan University, Graduate Institute of Zoology, Taipei,
Taiwan
(3) National Taiwan University, Neurobiology and Congnitive Science
Center, Taipei,Taiwan
Orexin A and B are endogenous agonists of a family of orphan Gq-protein coupled receptors, OX1 and OX2. The orexin system has been
implicated in arousal, metabolic, rewarding and pain regulations, and
their action mechanisms have been intensively explored except that in
pain regulation. In this study, we found orexin A and [Ala11,
D-Leu15]orexin B (AL-orexin B), an orexin B analogue selective to
OX2 receptors, increased mouse hot-plate latency when microinjected
into the ventrolateral periaqueductal gray (vlPAG), a midbrain region
mediating pain inhibition. The antinociceptive effect of orexin A, but not
AL-orexin B, was primarily mediated through OX1 receptors, opioidindependent, reversed by AM 251 (a CB1 antagonist), and mimicked by
WIN 55,212-2 (a CB1 agonist). The antinociceptive effect of AL-orexin
B was via OX2 receptors and also opioid-independent. In vlPAG slices,
234
orexin A, via OX1 receptors, caused membrane depolarization and neuronal ring, and depressed GABA-mediated evoked inhibitory postsynaptic currents (eIPSCs) by a presynaptic mechanism. Orexin A-induced
eIPSC depression was reversed by AM 251, mimicked by WIN 55,2122, prevented by U73122 and tetrahydrolipstatin, phospholipase C (PLC)
and diacylglycerol lipase (DAGL) inhibitors, respectively, and enhanced
by URB 602, which inhibits enzymatic degradation of 2-arachidonoylglycerol (2-AG). AL-orexin B also caused membrane depolarization via
OX2 receptors. These results suggest that activation of OX1, but not
OX2, receptors, in the vlPAG induces antinociception through the Gqprotein-PLC-DAGL cascade, yielding 2-AG, an endocannabinoid which
produces retrograde inhibition of GABA release (disinhibition). OX2
receptor activation in the vlPAG also induces antinociception by increasing neuronal activity via membrane depolarization.
Paper No.: 1116

HEALTH AND DISEASE
HIGH-SODIUM LOADED IN RATS IMPAIRS CENTRAL
BAROREFLEX AND ACH-INDUCED VASODILATION
Akiko Chisyaki(1), M Tsuruhisa(1), M Miyakawa(1), T Masuda(1),
Y Hirabara(2), K Honda(1), R Saito(1), Y Takano(1)
(1) Fukuoka University, Department of Pharmacology, Fukuoka City,
Japan
(2) University Miyazaki Hospital, Department of Pharmacy, Japan
Recently, the enzyme 11 b-hydroxysteroid dehydrogenase type 2
(HSD2) and the mineralocorticoid receptors (MR) were identied in the
nucleus tractus solitarius (NTS), in addition to common neurotransmitters; such neurons are termed healdosterone-sensitive HSD2 neuronshf
(J. Comp. Neurol. 2006, 497: 223). Furthermore, a number of studies
have shown that the aldosterone-sensitive HSD2 neurons in the NTS are
involved in sodium appetite or in regulation of body uid circulation as
they are selectively activated by sodium deciency. In the present study,
we tested the hypothesis that the aldosterone-sensitive neurons in the
NTS regulate arterial baroreceptor reex (baroreex) function. To
address this hypothesis, we examined the sensitivity of baroreex
induced by phenylephrine in high-sodium loaded rats. Experiments were
performed by our previous method (J. Pharmacol. Sci. 2007, 104, 402,).
(1) The baroreex sensitivity was signicantly reduced in the high
sodium-loaded rats. (2) The baroreex sensitivity was reversed by microinjection of MR antagonist eplerenone into the nucleus tractus solitarius
(NTS). (3) HSD2 neurons and MRs were identied in the NTS. The
ndings suggest that the aldosterone-sensitive neurons in the NTS may
play important roles of baroreex functions. In addition, we examined
EDHF-mediated relaxation on mesenteric arteries in high sodium loaded
rats by using the type 2 diabetic model, Goto-Kakizaki rats (GK rat).
The endothelium-dependent relaxation to acetylcholine (ACh) ACh in
the high sodium-loaded GK rats signicantly attenuated compared with
that in non-sodium loaded GK rats.
Paper No.: 2483

A NOVEL PROTOCOL FOR THE DISCOVERY OF
ANGIOGENESIS-MODULATORS
Chin-wen C Cho(1), J-C Yeh(1), T-P Fan(1)
University of Cambridge, Department of Pharmacology, Cambridge, UK
Current drug discovery regime employs cell models for screening synthetic compounds or natural products. However, the selection of experimental culture condition (e. g. foetal bovine serum and growth factors)
may mask potential therapeutic properties of the compounds tested. To
overcome this problem, we have developed a novel protocol for identify-
ing angiogenesis modulators, using two endothelial cell (EC) culture

media (EGM-2 and a modied medium, MM) in two in-vitro angiogenesis models (proliferation and tube formation assays). Our data show that
vascular-targetting agents (VTAs) taxol and combretastatin at nM range
produce similar anti-angiogenic effects under both conditions. Conversely,
phytosteriod ginsenosides Rh2 and PPD at lM range exhibit distinct
effects under these two conditions. At 20-40 lM, PPD causes cytotoxicity
in EGM-2, while promoting cellular survival in MM. Western blot analysis demonstrates differential expression of proteins involved in pro-apoptotic or pro-survival pathways in ECs treated with ginsenosides. However,
VTAs result in similar expression of proteins in both conditions. Taxol
and combretastatin are known to target microtubules whereas ginsenoside
Rh2 has been linked to inhibition of matrix metalloproteinases and
expression of junction adhesion molecules in tumour vasculature. Thus,
this novel protocol cannot only detect angiogenesis modulators but also
allow preliminary identication of their underlying mechanisms.
Acknowledgement: supported by Cambridge Overseas Trust.
Paper No.: 2149

ASSOCIATION OF THE ABCB1 3435C>T POLYMORPHISM
AND ADVERSE EVENTS OF CILOSTAZOLE IN HEALTHY
SUBJECTS
Sung Kweon Cho(1,2), LA Lim(1,2), SB Jang(1,2), YJ Lee(1,2),
DH Lee(1,2), HK Son(1,2), JY Chung(1,2), K Park(1,2)
(1) Yonsei University College of Medicine, Department of Pharmacology, Seoul, South Korea
(2) Yonsei University, Brain Korea 21 Project for Medical Science,
Seoul, South Korea
Cilostazole, a type III phosphodiesterase inhibitor, has been widely used
to control symptoms of lower extremity peripheral arterial disease and
intermittent claudication. Adverse events of Central Nervous System
(CNS) such as headache, nausea and dizziness are frequently reported
during the treament. This study was aimed to propose the role of ABCB1
polymorphism in the case of CNS adverse events of cilostazol. We
examined the the relationship between the genotypes and the adverse
events of cilostazol after single oral administration of Pletalc(Otsuka) in
84 korean healthy volunteers who participated in clinical studies to
investigate safety and pharmacokinetics of newly developed cilostazol
formulations. Fifty-eight subjects were genotyped for ABCB1. Adverse
events were classied into 2 categories, headache or the other CNS
symptoms such as nausea, vomiting and dizziness. The relationship
between the adverse events and genotype was assessed by the chi-square
test by using the SPSS 17.0. The reported headache cases in 3435CC,
CT and TT group are 3, 12 and 4 out of 24, 24 and 10, respectively. A
signicant difference was observed in the incidence of headache by a 2.5
fold increase (p = 0.02) in the subjects carrying 3435T allele compared
to those who do not carry that allele. All other symptoms were not signicant. Our data suggest that ABCB1 3435T allele is associated with
the headache after taking cilostazole. Further study is required to establish and validate the role of ABCB1 polymorphism in the incidence of
headche during cilostazole treatment.
Paper No.: 2166

EFFECTS OF CYP2D6*10 ALLELE ON THE
PHARMACOKINETICS OF ATOMOXETINE IN HEALTHY
KOREANS
Chang-Ik Choi, J-W Bae, M-J Kim, C-G Jang, S-Y Lee
Sungkyunkwan University, College of Pharmacy, Laboratory of Pharmacology, Suwon, South Korea
235
Atomoxetine is a selective norepinephrine receptor inhibitor (SNRI), and
is usedfor the treatment of Atomoxetine is a selective norepinephrine
receptor inhibitor (SNRI), and is used for the treatment of attention-decit hyperactivity disorder (ADHD) in children, adolescents and adults.
Atomoxetine is metabolized via aromatic ring-hydroxylation, benzylic
hydroxylation and N-demethylation in human. Among these, aromatic
ring-hydroxylation is the major metabolic pathway and is primarily mediated by CYP2D6, the highly polymorphic drug metabolizing enzyme.
CYP2D6*10 allele, causing the decreased CYP2D6 enzymatic activitiy,
is specically and frequently distributed in Asians, including Korea. We
investigated effects of CYP2D6*10 allele on the pharmacokinetics of
atomoxetine. After genotyping for CYP2D6 with volunteers, 32 healthy
Korean subjects were selected for this study. They were grouped according to CYP2D6 genotype, group 1 (n = 9, CYP2D6*wt/*wt), group 2
(n = 13, CYP2D6*wt/*10), and group 3 (n = 10, CYP2D6*10/*10). A
single oral dose of 40 mg of atomoxetine was administered to each subject and plasma concentration of atomoxetine was determined by using
high-performance liquid chromatography-tandem mass spectrometry system. Mean AUC values of atomoxetine in group 2 (1903 1289 nghr/
mL) and group 3 (2640 742 nghr/mL) were signicantly higher than
those in group 1 (768 125 nghr/mL) (P < 0.05 and P < 0.001,
respectively). Elimination half-life (t1/2) of atomoxetine in group 3 was
signicantly longer than in group 1 and group 2 (P < 0.001 and
P < 0.05, respectively). Oral clearance (CL/F) of atomoxetine in group 2
(26.8 10.4 L/hr) and group 3 (16.2 4.2 L/hr) was signicantly lower
than in group 1 (53.2 7.4 L/hr) (both P < 0.0001). In conclusion,
CYP2D6*10 allele is found to affect the pharmacokinetics of atomoxetine in Koreans.
Paper No.: 3314

DOSE ADJUSTMENT OF OMEPRAZOLE BASED ON CYP2C19
GENOTYPE
Chang-Ik Choi, Sung-Min Moon, Jung-Woo Bae, Mi-Jeong Kim,
Choon-Gon Jang, Seok-Yong Lee
Sungkyunkwan University, School of Pharmacy, Suwon, South Korea
Omeprazole is a proton pump inhibitors (PPIs) that suppresses gastric
acid secretion by specic inhibition of the H+/K+-ATPase in the gastric
parietal cell, and is used for the treatment of gastroesophageal reux disease (GERD), the healing of erosive esophagitis, and Helicobacter pylori
(H. pylori) eradication. Omeprazole is extensively metabolized by cytochrome P450 enzymes, and CYP2C19 is the main metabolizing enzyme
which is known to be highly polymorphic. It is reported that CYP2C19
genetic polymorphism can affects the pharmacokinetic and pharmacodynamic changes of several PPIs, including omeprazole. So we evaluated
the optimal dose adjustment of omeprazole in relation to CYP2C19
genotype. Forty-two healthy Korean volunteers were selected and were
divided into three groups according to CYP2C19 genotype,
CYP2C19EM (CYP2C19*1/*1, n = 15), CYP2C19IM (CYP2C19*1/*2
and CYP2C19*1/*3, n = 15), and CYP2C19PM (CYP2C19*2/*2,
CYP2C19*2/*3, and CYP2C19*3/*3, n = 12). Each group was received
a single oral dose of 75 mg, 60 mg and 20 mg omeprazole, respectively,
and the plasma concentration of omeprazole was measured by using
LC-MS/MS system. AUC0- of omeprazole in each genotype group
(CYP2C19EM, CYP2C19IM and CYP2C19PM group) was
3239 1997 ng/mLhr, 4000 1173 ng/mLhr and 3690 1179 ng/
mLhr, respectively. These differences were not statistically signicant,
indicating similar plasma concentration of omeprazole between three
genotype groups. Doses of omeprazole used in this study can be applied
to the CYP2C19 genotype-based omeprazole dose determination in
Koreans.
Paper No.: 3315

DISCOVERY
PHARMACOKINETIC INTERACTIONS OF CIMETIDINE AND
PRAMIPEXOLE IN RELATION TO OCT2
Chang-Ik Choi(1), S-M Moon(1), J-W Bae(1), M-J Kim(1), C-G Jang(1),
S-Y Lee(1)
Sungkyunkwan University, School of Pharmacy, Su-Won, South Korea
Pramipexole is in a class of non-ergoline dopamine agonist and is
approved for the treatment of Parkinsons disease and restless legs syndrome (RLS). After administration, pramipexole is well absorbed and
undergoes little presystemic biotransformation. Approximately 90% was
excreted in the urine as unchanged form, with possible involvement with
organic cation transporters (OCTs) in the renal tubules, especially OCT2.
So we investigated the effects of cimetidine, known as an OCT2 inhibitor, on the pharmacokinetics of pramipexole. Eighteen healthy Korean
subjects with OCT2 808GG genotype enrolled an open-labeled, twophase parallel clinical study. In the rst phase (control phase), each subject received a single oral dose of 0.25 mg pramipexole. In the second
phase (cimetidine phase), the subjects administered cimetidine 400 mg
twice daily for ve days. On the morning of day 6, the subjects received
a single oral dose of 0.25 mg pramipexole 2 hours later the administration of a 400-mg single oral dose of cimetidine. Plasma concentration of
pramipexole was determined by using LC-MS/MS system. In cimetidine
phase, Cmax and AUC0- of pramipexole was 21% and 43% higher than
those in control phase (P < 0.001 and P < 0.0001, respectively). Oral
clearance of pramipexole in cimetidine phase was 30% lower than that in
control phase (P < 0.0001). Other pharmacokinetic parameters were not
statistically signicant. From these results, OCT2 is found to be associated with the renal excretion of pramipexole in vivo. Further studies on
the relationship between OCT2 genetic variants and pharmacokinetics of
pramipexole will be meaningful.
Paper No.: 2881

PHARMACOGENETIC STUDY ON METABOLIC SYNDROME
INDUCED BY ATYPICAL ANTIPSYCHOTICS AND MOOD
STABILIZERS
Eva Choong(1), M Etter(2), B Oneda(1), H Tayebi(3), G Bondol(2),
CB Eap(1,3)
(1) CHUV - Unit of Biochemistry and Clinical Psychopharmacology,
Centre for Psychiatric Neuroscience, Department of Psychiatry, Lausanne-Prilly, Switzerland
(2) University of Geneva, and University Hospital of Geneva, Department of Psychiatry, Switzerland
(3) University of Geneva and University of Lausanne, School of PharmaceuticalSciences, Geneva, Switzerland
Metabolic syndrome (weight gain, alteration of lipid and glycaemia proles) induced by atypical antipsychotics and/or mood stabilizers are of
high clinical concern for the long term patient morbidity and mortality.
The major aim of this trial is to identify pharmacogenetic susceptibility
factors for metabolic syndrome. For this purpose, a transversal study was
performed in a Swiss out-patient psychiatric division, with the inclusion
of 196 psychiatric patients. 69% of patients were receiving atypical antipsychotics, and 31% lithium or valproate (two mood stabilizers also
known to induce weight gain). Genotyping of pharmacodynamic candidate genes was performed using rtPCR and allelic discrimination assays,
after validation of the method by direct sequencing. Uncoupling protein
2 (UCP2), a mitochondria membrane transporter involved in the release
of stored energy, leptin receptor (LEPR) and fat mass and obesity
236
associated gene (FTO), 2 genes playing a role in satiety, were chosen as
candidate genes. Result: UCP2 rs660339 polymorphism is associated
with differences in HDL-cholesterol levels (p = 0.002), and with obesity,
with CC and CT carriers presenting a 3.1-fold increased risk of obesity
(95%CI 1.2-9.9) as compared to TT carriers. Signicant association
between BMI change and LEPR polymorphism were found in female
patients treated with all studied drugs (p = 0.039), and between BMI
change and FTO polymorphism in patients treated with risperidone or
olanzapine (p = 0.003). Predicting metabolic syndrome side effects
remains complex and requires further investigations. However, each relevant gene showing an association with this important side-effect could
assist in the choice of the appropriate treatment for each individual
patient.
Paper No.: 2262

QUANTITATIVE PHOSPHOPROTEOMICS DISSECTION OF
7TM RECEPTOR SIGNALLING USING FULL AND BIASED
AGONISTS
Gitte Lund Christensen(1), CD Kelstrup(2), C Lyngse(3), U Sarwar(1),
R Bgebo(1), SP Sheikh(4), S Gammeltoft(1), JV Olsen(2),
JL Hansen(3),
(1) Glostrup Hospital, Department of Clinical Biochemistry, Glostrup,
Copenhagen, Denmark
(2) University of Copenhagen, Novo Nordisk Foundation Center for
Protein Research, Faculty of Health Sciences, Copenhagen, Denmark
(3) University of Copenhagen, Danish National Research Foundation
Centre for Cardiac Arrhythmia, Laboratory for Molecular Cardiology,
Department of Department ofBiomedical Sciences, Copenhagen,
Denmark
(4) University of Southern Denmark, Odense University Hospital,
Department for Biochemistry, Pharmacology & Genetics, Odense,
Denmark
Seven-transmembrane receptors (7TMRs), signal through heterotrimeric
G proteins, but can also activate G protein-independent signalling pathways of which the impact and complexity are less understood. The
Angiotensin II type 1 receptor (AT1R) is a prototypical 7TMR an important drug target in cardiovascular medicine. Biased agonists which
blocks Gaq protein activity, and simultaneously activitate G protein independent pathways, confer important perspectives in treatment of cardiovascular diseases. In this study we performed a global quantitative
phosphoproteomics analysis of the AT1R signalling network. Applying
high-resolution mass spectrometry (LTQ Orbitrap MS) we compared the
phosphoproteome of the AT1R agonist Angiotensin II with the biased
agonist SII Angiotensin II. We quantied more than elleven thousand
phosphorylation sites of which 879 were regulated by Angiotensin II.
45% of the Angiotensin II regulated phosphorylations were also regulated by SII Angiotensin II. Analysis of phosphorylation site patterns displays a striking distinction between protein kinases activated by Gaq
protein-dependent and independent mechanisms. This study provides
substantial novel insight into Angiotensin II signal transduction and is
the rst study dissecting the differences between a full and a biased agonist from a 7TMR on a systems-wide scale. Importantly, it reveals a previously unappreciated diversity and quantity of Gaq protein-independent
signalling and uncovers novel signalling pathways. We foresee that the
amount and diversity of G protein independent signalling may be more
pronounced than previously recognized for other 7TMRs as well. Quantitative mass spectrometry is a promising tool for evaluation of signalling
properties of biased agonists to other receptors in the future.
Paper No.: 2317

CHANGING DOCTORS PRESCRIPTION BEHAVIOUR. A
DRUG COMMITTEE PERSPECTIVE: TNF INHIBITORS
Hanne R Christensen, H Thomsen
Bispebjerg Hospital, Department of Clinical Pharmacology, Copenahgen,
Denmark
Introduction: The introduction of TNF inhibitors in rheumatoid arthritis
has resulted in improved treatment options but also a marked increase in
drug expenses. In 2009 adalimumab, iniximab and etanercept were
responsible for the highest drug expenditure in Denmark; 75 mil. Euro.
Of the ve regions in Denmark, the Capital Region is the only one with
specic guidelines in this area. The TNF inhibitors adalimumab, etanercept and iniximab were considered to have equal clinical efcacy in the
treatment of rheumatoid arthritis, but use of iniximab was estimated as
the cheapest TNF inhibitor, and hence was chosen as 1. Line treatment
when a TNF inhibitor was needed. Methods: The drug committee did a
joint venture with the specialists in rheumatology in completing a guideline for the use of TNF inhibitors for treating patients with rheumatoid
arthritis. A goal was set, that 80% of all new patients diagnosed with
rheumatoid arthritis and approved for TNF inhibitor treatment, should
receive iniximab. Results: After implementation of the guideline all
new patients were monitored in the period January October 2009, and
out of 136 patients, 111 received iniximab corresponding to 82%,
which was a marked change in prescription behaviour. In the four other
regions, no such change was observed. Conclusion: Even though it is
difcult to change doctors prescription behaviour, guidelines for the initial treatment of patients with rheumatoid arthritis can be complied with,
when implemented on rheumatology wards.
Paper No.: 1696

DISCOVERY
PHARMACOGENETICS OF METFORMIN
Mette Marie Hougaard Christensen(1), CB Andersen(2), P Damkier(2),
HB Nielsen(3), K Brsen(1)
(1) University of Southern Denmark, Institute of Public Health & Odense
UniversityHospital, Department of Biochemistry and Pharmacology,
Odense, Denmark
(2) Odense University Hospital, Department of Biochemistry and Pharmacology, Odense, Denmark
(3) Odense University Hospital, Department of Endocrinology & University of Southern Denmark, Faculty of Health Sciences, Institute of Clinical Research, Odense, Denmark
Introduction: Genetic polymorphisms in metformin transporters and their
impact at the steady state metformin plasma concentrations is evaluated
in a large well characterized type 2 diabetes cohort given by South Danish Diabetes Study. SNPs in PMAT, OCT1-2 and MATE 1-2 have been
linked to reduced pharmacodynaminc response and large interindividual
pharmacokinetic variation. Materials/Patients: South Danish Diabetes
Study was designed as a 2 x 2 x 2 factorial prospective, randomized,
double-blinded, placebo-controlled, multi-centre study comprising 386 in
8 parallel groups. It was initialized to investigate the optimal pharmacological combination of metformin, rosiglitazon and insulin Asp/NPH in a
cohort of type 2 diabetics. One hundred and eighty-six patients were
allocated to metformin, and repeated measurements of steady state concentrations were collected. The nal results will be adjusted for gender,
age, BMI, duration of disease, metformin dose, co-medication, smoking,
creatinine clearance, HbA1c-pretreatment and polymorphisms in PMAT,
OCT 1-2 and MATE 1-2. Results: The preliminary unadjusted result
for 456 repeated measurements of metformin conrm the large inter-
237
individual variation in steady state metformin concentration:
769 649ng/ml (SD), range: 9.7 to 4133ng/ml. In the cohort 369 samples could be genotyped. In the renal transporter OCT2, the A270S genotype frequencies were determined: Heterozygous 19%, homozygous
0.5% and wild-type 80.5%, which corresponds well with the databases at
NCBI. Conclusion: The preliminary results strengthen the thesis that
Danish type 2 diabetics have the same allele frequencies in transporter
SNP as healthy Caucasian, and it indeed underscores the enormous interindividual variation in metformin steady state concentration.
Paper No.: 493

IMPROVEMENT OF MEMORY IN MICE AND INCREASE OF
HIPPOCAMPAL EXCITABILITY IN RATS BY GINSENOSIDE
RG1S METABOLITES GINSENOSIDE RH1 AND
PROTOPANAXATRIOL
Shifeng Chu, J Chen, Y Wang, N Chen, J Zhang
Institute of Materia Medica, Chinese Academy of Medical Sciences &
PekingUniversity, Department of Pharmacology, Beijing, PR China
Abstract: The aim of present study is to observe the effect of ginsenoside
Rg1s metabolites (primary metabolit ginsenoside Rh1 and end metabolite protopanaxatriol) on learning and memory function. We employed
the step through test and electrophysiological study to investigate the
effects of Rh1 and Ppton learning and memory as well as hippocampal
excitability. The behavioral study showed that both ginsenoside Rh1 and
Ppt signicantly ameliorated memory-impaired model induced by scopolamine in mice. Consistently, electrophysiological work revealed that
Rh1 and Ppt as well as their precursor Rg1 all increased hippocampal
excitability in the dentate gyrus of anesthetized rats. The metabolism of
Rg1 in cerebrospinal uid was detected by HPLC-MS, indicating that
Rg1 could not convert into Rh1 or Ppt. These results demonstrated that
both Rh1 and Ppt have similar effect on improving memory and hippocampal excitability, suggesting that the role of ginsenosides sugarmoeties in biological activities is not as necessary as traditionally
considered.
Paper No.: 1563

FRACTIONATION OF SAW PALMETTO EXTRACTS AND
BIOASSAY FRACTIONS IN ISOLATED RAT PROSTATE
Thiam Chua, J Simpson, S Ventura
tude to the contractions produced by the crude extracts. These responses

were attenuated in the presence of cocaine (3 lM) (P < 0.001, n = 6,
repeated measures ANOVA) or prazosin (0.3 lM) (P = 0.001, n = 6,
repeated measures ANOVA). Analysis of NMR and mass spectra and
comparison with authentic material conrmed that this bioactivity was
due to tyramine in the active fraction. In conclusion, tyramine in saw palmetto extract causes indirect a-adrenoceptor mediated contractions via
the release of noradrenaline from sympathetic neurons.
Paper No.: 956

HPLC METHOD DEVELOPMENT & VALIDATION FOR THE
DETERMINATION OF WARFARIN IN HUMAN PLASMA
Yung An Chua, WZW Abdullah, SH Gan
University Sains Malaysia, Human Genome Center, Kelantan, Malaysia
Introduction: In many third world countries, not all laboratories have
sophisticated equipment such as Liquid chromatography Mass Spectrometry (LCMS) for fast analysis of serum drug levels. Objective: The aim
of our study is to develop a simple High Performance Liquid Chromatography (HPLC) method coupled with a UV detector for the determination of racemic serum concentrations of warfarin. Method: Warfarin and
phenylbutazone (internal standard) were spiked into 1 ml of human
serum before extraction using a liquid-liquid extraction (LLE) method
consisting of 3 ml ethyl-acetate. The samples were dried and reconstituted in 100 ml of acetonitrile before HPLC injection (10 ml). The
mobile phase comprised of 30% acetonitrile and 70% potassium dihydrogen orthophosphate buffer (0.01 M) with pH adjusted to 6.5. Separation
was performed on an endcapped C18 (250 X 4.6 mm I.D., 5 mm)
reversed phase column. The ow rate was set at 1.0ml/min with a total
run time of 14 min. The Photodiode Array (PDA) absorbance range for
warfarin was set as 210-350 nm. Result: The extraction recovery for warfarin was excellent (98%). Our limit of detection was 50 ng/ml. The
method was linear over the concentration range between 100 ng/ml and
6.0 mg/ml. The precision as well as accuracy meets the guideline laid by
the FDA.Conclusion: The developed HPLC method was found to be
suitable for the determination of serum warfarin concentrations for a clinical study.
Paper No.: 1997

PHARMAPOLY, GAME AS A TEACHING STRATEGY IN
PHARMACOLOGY CLASSES
Luis Cifuentes
Monash University, Monash Institute of Pharmaceutical Sciences, Parkville, VIC, Australia
University of the Andes, Faculty of Medicine, Bogota, Colombia
Extracts of saw palmetto berries (Serenoa repens) have been widely used
as a herbal remedy for benign prostatic hyperplasia (BPH). However, little is known about the benecial physiological role of the bioactives and
which of the active phytochemicals are involved in improving the symptoms of BPH. The purpose of this study was to isolate and investigate
the bioactive compounds from saw palmetto extract that affect prostatic
smooth muscle. A commercially available saw palmetto extract (MediHerb Pty Ltd, Warwick, Queensland) was lyophilized and subjected to
fractionation using column chromatography. The composition of fractions was assessed by proton nuclear magnetic resonance spectroscopy
(1H NMR) and mass spectrometry (MS). Non-polar fractions were found
to contain fatty acids and their esters as the major constituents. In contrast, polar fractions revealed carbohydrate components (mono- and disaccharides) and alkaloids. In addition, the polar fractions were found to
produce contractions of isolated rat prostates that were similar in magni-
Introduction. Pharmacology teaching is an ongoing challenge for the teacher and students, in order to achieve that knowledge and motivation to
persist. The aim of this paper is the incorporation of games as a teaching
strategy (Ulrich D., et al. J of Nursing Education 2005;44:338-339) in
pharmacology classes, through the development of a material called
Pharmapoly. Materials. Using the principles of the game called Monopoly, I have developed a board with eighteen portraits of famous pharmacologists due to their discoveries. Cards and dices to play are related to
the area of pharmacology. Each pharmacologist has assigned several
questions related to the topics of research and development, pharmacokinetics and pharmacodynamics, which must be answered correctly by students to advance and nish with the highest number of pharmacologists
in their possession. Results. During the 1st half of 2009 it was made a
pilot with 43 students of the Faculty of Medicine, University of Los
Andes in Colombia, with the module of pharmacodynamics. The
238
comments at the end of the module by students were: 1. It was fun, 2.
We have learned a lot, and 3. We want to continue playing and learning.
Conclusion. The teaching of pharmacology can be facilitated by the incorporation of methodologies such as educational games, which complement
any teaching activity and generate continuous motivation in students.
Paper No.: 1998

SUMMARY OF PK AND PD IN A PRACTICAL WAY
Luis Cifuentes
University of the Andes, Faculty of Medicine, Bogota, Colombia
Introduction. One of the great barriers of medical students who are in
training of pharmacology and even the medical graduates is to understand and internalize the basic principles of pharmacokinetics (PK) and
pharmacodynamics (PD). The aim of this paper is to present an approach
of a summary of PK and PD in only one sheet for each concept. Materials: We have reviewed in 10 of pharmacology reference texts, the basic
principles of PK and PD, which has been supplemented with the teachers teaching experience in pharmacology. The result was an integration of all key concepts for PK and PD, in only one sheet, respectively.
Results. Each of the two sheets previously have been delivered to undergraduate medical students in teaching modules of PK and PD. Their conclusions after students have seen these sheets before a class of PK and
PD, were how easy the approach was, and after the module they mentioned that facilitates the recall the concepts, interaction with reference
texts and as continuous reference material. Conclusion. This tool has the
advantage of being able to access through the Internet via public domain
to help medical students and health professionals on the need to have
continued incorporation on PK and PD in the study and prescription of
drugs.
Paper No.: 1869

CROTOXIN: A TOXIN FROM RATTLESNAKE VENOM THAT
INDUCES A LONG-LASTING INHIBITORY EFFECT ON THE
PHAGOCYTIC ACTIVITY OF NEUTROPHILS
Maria Cristina Cirillo(1), TS Lima(1), SC Cataneo(1), SC Sampaio(1),
MC Della-Casa(2)
(1) Butantan Institute, Laboratory of Pathophysiology, Sao Paulo, Brazil
(2) Butantan Institute, Laboratory of Immunopathology, Sao Paulo,
Brazil
Crotalus durissus terricus snake venom (CdtV) and crotoxin, its main
component, have long-lasting anti-inammatory properties and inhibit
the phagocytic activity of macrophages. Recently, we demonstrated that
CdtV inhibits phagocytosis by neutrophils. Despite these ndings, the
component of CdtV responsible for phagocytosis inhibition is unknow.
This study investigated the effect of crotoxin and other fractions (peak I
and III), obtained during crotoxin purication, on C3b-receptor-mediated
phagocytosis, by neutrophils. Neutrophils were obtained from peritoneal
cavity of Wistar rats, 4h after intraperitoneal administration of carrageenan (4.5 mg/kg) and phagocytosis of opsonizaded zymosan was evaluated after different treatments. In vitro, cells (1.2x106cells/mL) were
incubated with crotoxin (peak II), peak I or III, at different concentrations
(0.02, 0.04, 0.08, 0.16 or 0.32 lg/mL) or RPMI1640 (control). In vivo,
crotoxin (0.11 mg/kg) or saline (control) was administered subcutaneously to rats at different time periods: 2h, 1, 4 or 14 days before or 1h
after the administration of carrageenan. In vitro, crotoxin signicantly
reduced the phagocytic activity of neutrophils (0.02 lg/mL: 29%,
0.04 lg/mL: 24%, 0.08 lg/mL: 28%, 0.16 lg/mL: 26% and 0.32 lg/
mL: 24%). A single dose of crotoxin also reduced the percentage of
phagocytosis (2h: 24%, 1 day: 31%, 4 days: 25%, 14 days: 18% and 1h
later: 35%). These results show that crotoxin is the component responsible for the inhibitory effect of CdtV on phagocytosis by neutrophils. Taking into account the role of neutrophils in inammation, these data
reinforce that crotoxin is a potential natural product in controlling inammatory diseases.
Support: FAPESP and CNPq/INCTTOX.
Paper No.: 1870

CROTOXIN: A TOXIN FROM RATTLESNAKE VENOM WITH
POTENTIAL ANTI-INFLAMMATORY PROPERTIES
Maria Cristina Cirillo(1), FPB Nunes(1), BC Zychar(1),
MS Della-Casa(2), SC Sampaio(1), LRC Goncalves(1)
Brazil
Crotalus durissus terricus (CdtV) snake venom exhibit a long-lasting
anti-inammatory effect, inhibiting paw edema and cell migration to the
peritoneal cavity induced by carrageenan. However, the contribution of
puried components of CdtV on this effect has not been evaluated yet;
thus the effect of crotoxin (CTX), the main toxin of CdtV, and other
components were investigated on paw edema, cellular migration and leukocyte-endothelium interaction induced by carrageenan. A single dose of
CTX (0.89lg/50lL s.c.), other components (1.0lg/50lL s.c.) or saline
(50lL) was injected in mice, 7 days before intraplantar injection of carrageenan (300lg/50lL) or saline (contralateral paw) and edema was evaluated. Total and differential cell counts were determined 4h after
intraperitoneal injection of carrageenan (300lg/200lL) in animals treated
with CTX, saline or other components 7, 14 or 21 days earlier. Leukocyte-endothelium interaction was induced 1h after injection of carrageenan or saline into the scrotum of animals, 7 or 14 days before the
injection of CTX, saline or other components. Adherent, or in rolling
leukocytes were counted by intravital microscopy 1h after carrageenan or
saline injection. The results showed that a single dose of CTX, but not
other components, reduced edema (30%), cell migration (30%) and
altered leukocyte-endothelium interaction, increasing the rolling (38%)
and reducing the adherent cells (63%) induced by carrageenan compared
with controls. Thus, CTX is the component involved in the long-lasting
anti-inammatory effect of CdtV, indicating that it might be used as a
new approach for treating inammatory diseases.
Supported by FAPESP, CAPES and CNPQ/INCTTOX.
Paper No.: 1871

INVOLVEMENT OF FORMYL PEPTIDE RECEPTORS ON THE
LONG-LASTING ANTI-INFLAMMATORY EFFECT OF
CROTOXIN, A TOXIN FROM RATTLESNAKE VENOM
Maria Cristina Cirillo(1), FPB Nunes(1), BC Zychar(1),
MS Della-Casa(2), SC Sampaio(1), LRC Goncalves(1)
Brazil
Crotalus durissus terricus snake venom (CdtV) and crotoxin, the main
component of this venom, present long-lasting anti-inammatory proprieties on paw edema and cell migration induced by carrageenan. Other
investigation showed that CdtV and crotoxin inhibit phagocytosis by neutrophil and macrophages. Moreover, crotoxin increases the production of
lipoxin A4, by macrophages. Lipoxins, anti-inammatory mediators generated by lipoxygenase pathway exert their biological actions by binding
239
to specic receptors FPR2/ALX that belongs to formyl peptide receptors
family. In this study we investigated the involvement of formyl peptide
receptors on the long-lasting inhibitory effect of crotoxin on cell migration induced by carrageenan. The Boc-2 (10 lg/200 lL i.p.), a selective
antagonist of formyl peptide receptors was administered 30 minutes prior
to the subcutaneous injection of crotoxin (0.89 lg/50 lL) or saline. The
treatment with crotoxin (single dose) or saline was undertaken 1 hour, 7
or 14 days before intraperitoneal injection of carrageenan (300 lg/
200 lL). After 4 hours of carrageenan injection, cellular migration to the
peritoneal cavity was evaluated. Pretreatment of animals with crotoxin,
signicantly diminished carrageenan-induced cell inux into the peritoneal cavity, when compared with the control group. These decreases
were 47% (1 hour), 44% (7 days) and 33% (14 days). Pretreating animals with Boc-2 abolished the decrease in cell migration induced by crotoxin in all time evaluated. In conclusion, results suggest that formyl
peptide receptors seem to play a role in the anti-inammatory effect of
crotoxin and also indicate the involvement of the lipoxin in this effect.
Supported by FAPESP, CAPES, CNPQ/INCTTOX.
Paper No.: 3118

TYPE 2 DIABETES-ASSOCIATED NON-SYNONYMOUS
MUTATIONS IN THE GENE CODING FOR THE MELATONIN
MT2 RECEPTOR IMPAIR MELATONIN SIGNALLING
Nathalie Clement(1), A Bonnefond(2), K Fawcett(3), E Vaillant(2),
J-L Guillaume(1), B Balkau(4), M Marre(5), G Charpentier(6),
M Vaxillaire(2), I Barroso(3), R Jockers(1), P Froguel(2,7)
(on behalf of the MAGIC consortium)
(1) Institut Cochin, Inserm U567, CNRS-UMR8104, Paris, France
(2) CNRS-UMR8090, Pasteur Institute, Lille, France
(3) Wellcome Trust Sanger Institute, Metabolic Disease Group, Hinxton,
UK
(4) INSERM-U780, Villejuif, France
(5) INSERM-U695, Paris, France
(6) Corbeil-Essonnes Hospital, Endocrinology-Diabetology Unit,
Corbeil-Essonnes,France
(7) Imperial College, Department of Genomic Medecine, London, UK
Using genome-wide association study data, we have recently identied a
single nucleotide polymorphism (SNP) located within the MTNR1B gene,
encoding the MT2 melatonin receptor, associated with an increase of
both fasting plasma glucose (FPG) levels and risk of type 2 diabetes
(T2D). Melatonin is a neurohormone, which regulates the circadian
rhythm and may contribute to glucose homeostasis. Melatonin signalling
is mainly mediated by its G protein-coupled MT1 and MT2 receptors.
Though the association signal of the MTNR1B SNP rs10830963 is
strongly signicant (P < 10-30), its effect on FPG and T2D risk is modest. We hypothesized that rare non-synonymous mutations in MTNR1B
may have a larger effect on T2D risk and may contribute to the haplotype association signal. We sequenced MTNR1B exons in 6,878 European samples including 2,271 individuals with T2D. All identied
mutations were generated and expressed as Myc-tagged fusion proteins.
Ligand binding properties, surface expression and signalling (cAMP,
ERK1/2) of mutant receptors was studied in HEK 293 cells. We identied a total of 40 non-synonymous variants including 35 rare mutations
(minor allele frequency < 0.001). Preliminary statistical analyses showed
that these mutations tend to be more prevalent in individuals with T2D,
compared to nondiabetic controls. So far we have found that three mutations are devoid of 125I-MLT binding, all present in individuals with elevated FPG. Subsequent analyses are in progress. Our present ndings
show that MTNR1B coding regions are variable with putative loss of
function mutations being identied only in individuals with T2D or
impaired fasting glucose. Given that MTNR1B may mediate melatonininduced inhibition of insulin secretion, wouldnt we expect loss-of-function mutations to decrease FPG/decrease risk of T2D.
Paper No.: 2665

HEALTH AND DISEASE
CONTRIBUTION OF SMOOTH MUSCLE BKCA CURRENTS
TO OUTWARD CURRENTS RECORDED FROM
ENDOTHELIAL CELLS IN SITU
Belen Climent(1), P de la Villa(2), U Simonsen(3), A Garca-Sacristan(1), L Rivera(1)
(1) University Complutense of Madrid Faculty of Pharmacy, Department
of Physiology, Madrid, Spain
(2) University of Alcala, Department of Physiology, Madrid, Spain
In rat mesenteric arteries large-conductance calcium-activated K channels
(BKCa) have been reported to be restricted to vascular smooth muscle
cells (VSMC) (Mistry & Garland, J Membr Biol 164:125 38, 1998). The
present study hypothesized that changes in K current in the vascular
smooth muscle layer will result in changes in endothel cell (EC) K currents. Simultaneous recordings of vascular tone and ionic currents by use
of in situ patch clamp were performed on rat mesenteric arterial segments
mounted in a microvascular myograph. Families of depolarizing voltage
pulses (from -60 to +140mV; holding potential -60mV) were applied
before and after: phenylephrine (Phe), acetylcholine (ACh)+Phe; and iberiotoxin (IbTx)+ACh+Phe or carbenoxolone+ACh+Phe. In mesenteric
arteries incubated with carbenoxolone (45-60 minutes), the same protocol was applied in the presence of Phe+ACh and after IbTx+Phe+ACh.
Outward potassium currents were recorded. ACh induced potassium-current amplitude was decreased both by IbTx (30 3 to 11 3 pA/pF;
n = 5; P < 0.01) and carbenoxolone (35 5 to 19 7 pA/pF; n = 5;
p < 0.05). Incubation with carbenoxolone abolished the effect of IbTx
on the potassium currents (n = 7, p < 0.05). IbTx-induced reversion of
the ACh induced arterial relaxation with or without carbenoxolone was
similar (87%, n = 7; 91%, n = 5, respectively, p > 0.05). Gap junctions
are active in EC in situ and the blockade with IbTx suggests that BKCa
channels contribute to these currents. These data suggest that the currents
recorded from EC in situ are modulated by smooth muscle cell BKCa
currents, an effect mediated most likely through gap junctions.
Supported by M.E.C., Spain, Grant No. BFU2007-67732/BFI.
Paper No.: 2666

HEALTH AND DISEASE
INCREASED OUTWARD CURRENTS RECORDED IN SITU
FROM ENDOTHELIAL CELLS OF RABBIT CORONARY
ARTERIES AFTER ISCHEMIA REPERFUSION
Belen Climent(1), AL Garca-Villalon(2), N Fernandez(2), L Monge(2),
G Dieguez(2), A Garca Sacristan(1), L Rivera(1)
(1) University Complutense of Madrid Faculty of Pharmacy, Department
of Physiology, Madrid, Spain
(2) University Autonoma of Madrid Faculty of Pharmacy, Department of
Physiology, Madrid, Spain
Ischemiareperfusion is a clinical and experimental event that can produce dysfunction of the coronary vasculature, and evidences suggest that
endothelium dysfunction is involved. Rabbit hearts were perfused at constant ow and then exposed to 60 min global zero-ow ischemia followed by 30 min reperfusion. Segments of the descendent coronary
artery were mounted in a microvascular myograph. Simultaneous recordings of vascular tone and ionic currents by use of in situ patch clamp
were performed both on control and ischemic arteries. Families of depolarizing voltage pulses (from -60 to +140mV; Vh= -60mV) were applied
before and after endothelin 1(ET-1), and ET-1 + acetylcholine (ACh).
Outward potassium currents were recorded. The peak current value was
similar in ischemic and control segments (353 59, n = 10, 281 47
pA, n = 9; respectively; p = 0.167). However, the current density was
240
higher in ischemic vessels (48 6, and 25 5 pA/pF, respectively;
p < 0.05). In contrast, membrane capacitance values were lower in ischemic arteries (7 1 vs 12 1 pF; P < 0.01). ET-1 simultaneously
increased isometric tension and decreased outward currents in both arteries (14.9 3 pA, n = 5, P < 0.05 and 7,3 3, n = 4, P < 0.01, respectively). After ET-1, ACh induced relaxation and increased outward
currents in control but not in ischemic segments (23 4, P < 0.01; and
10 4 pA/pF p = 0.089, respectively). These preliminary data may suggest that increased current density along with capacitance reduction after
ischemia reperfusion injury might be due to an increase in K+ current
expression and to an impaired gap junctional communication. Further
experiments are needed to clarify this hypothesis.
Supported by M.E.C., Grants No. BFU2007-67732/BFI, and F.I.S., No
PS09/00394, Spain.
Paper No.: 2804

HEALTH AND DISEASE
CERAMIDE IS INVOLVED IN THE HYPOXIC
VASOCONSTRICTION OF CHICKEN PULMONARY AND
CHORIALLANTOIC ARTERIES
Angel Cogolludo(1), E Villamor(2), J Moral-Sanz(1), R Scheepers(1), B
Barreira (1), C Menendez(1), L Escolano(1), L Moreno(1), F Perez-Vizcaino(1),
(1) University Complutense of Madrid School of Medicine, Department
of Pharmacology,Madrid, Spain
(2) Univertsity of Maastricht, The Netherlands
Ceramide is a sphingolipid-derived second messenger that plays an
important role in numerous physiological processes such as cell growth,
apoptosis and differentiation. We have recently reported that neutral
sphingomyelinase (nSMase)-derived ceramide is involved in hypoxic
pulmonary vasoconstriction in rats (Cogolludo et al, Cardiovasc Res.
2009; 82: 296-302). We hypothesized that ceramide may play a role in
other species and in other vessels exhibiting a hypoxic vasoconstrictor
response. Thus, in the present study we have studied if ceramide mediates the hypoxic responses in chicken pulmonary (PA) and choriallantoic
arteries (CA). Experiments were performed in CA and PA isolated from
chicken fetuses (15-day incubation) and newborns (1-3days), respectively, and in myocytes isolated from these tissues. Exposure to hypoxia
induced a contraction in both PA and CA. Similarly, hypoxia increased
the ceramide content measured by immunocytochemistry. The nSMase
inhibitor GW4869 (10 lM) reduced the contractile responses induced by
hypoxia but not those elicited by endothelin-1. Hypoxic responses were
also attenuated by an anticeramide antibody. Moreover, C6-ceramide
(10 lM) and bacillus cereus SMase (100 mU/ml) mimicked the effects
of hypoxia, i.e. they produced a sustained contractile response. Finally,
the mRNAs of nSMase2 was expressed in PA and CA as measured by
real-time RT-PCR. In conclusion, nSMase-derived ceramide plays a key
role in the hypoxic responses found in two specialized oxygen sensing
vessels from chickens.
Supported by Fundacion de Investigacion Medica Mutua Madrilena.
Paper No.: 1034

INHIBITION OF THE 26S PROTEASOME COMPLEX BY
CHEMICAL CONSTITUENTS OF SMILAX REGELII AND ITS
POSSIBLE CHEMOTHERAPEUTIC CAPABILITIES ON
MAMMARY TUMOURS IN SPRAGUE DAWLEY RATS
Damian Cohall(1), PDA Singh(2)
The folklore anti-neoplastic claim of Smilax regelii was investigated on

mammary tumour models in female Sprague Dawley rats by probing
the plants ability to inhibit the 26S Proteasome complex (Adams J,
Drug Discovery Today 2003; 8: 307-315). Bioactivity of the plants
extract was assayed in vitro with the 26S proteasome. Rats were treated
with the aqueous extract (35.7 mg plant material/Kg i.p.) in a spontaneous tumour model. A possible bioactive constituent of the extract and
MG-132, a known 26S proteasome inhibitor (Adams J, Drug Discovery
Today 2003; 8: 307-315), were administered (1.0 mg/Kg i.p.) to respective groups of animals in a 7, 12-Dimethylbenzanthracene induced
tumour model. Tumour sizes were estimated using the formula, 1/
6p[(d1 d2)3/2] where d1 and d2 are the two perpendicular diameters
(Vigushin DM et al, Clinical Cancer Research 2001; 7: 971-976). Histopathology was done on tumour tissue samples. The extract inhibited the
26S Proteasome dose dependently. Chemical analysis of the extract
indicated that a possible bioactive constituent is Dioctyl Adipate.
Tumour growth within the groups treated with the aqueous extract
(p > 0.05, n = 12, Wilcoxon Signed Ranked test) and Dioctyl Adipate
(p > 0.05, n = 6, Students T test) was not signicant compared to the
control groups. The tumours in both models were broadenomas and
remission of the neoplasia in the test groups was due to induced apoptosis. Dioctyl Adipate and its analogues are potential anti-neoplastic
agents. Further investigation of these agents as drug leads in the drug
discovery pipeline is recommended.
Paper No.: 3291

ACTIVATION OF PEROXISOME PROLIFERATOR-ACTIVATED
RECEPTOR B/D (PPARB/D) BY GW501516 PREVENTS FATTY
ACID-INDUCED NF-JB ACTIVATION AND INSULIN
RESISTANCE IN SKELETAL MUSCLE CELLS
T Coll, AM Gomez-Foix, Manuel Vazquez-Carrera
At present it is unknown whether Peroxisome Proliferator-Activated
Receptor (PPAR)b/d activation prevents fatty acid-induced inammation and insulin resistance in skeletal muscle cells. In C2C12 skeletal
muscle cells, the PPARb/d agonist GW501516 prevented phosphorylation of IRS-1 at Ser307 and the inhibition of insulin-stimulated Akt
phosphorylation caused by exposure to the saturated fatty acid palmitate. This latter effect was reversed by the PPARb/d nantagonist
GSK0660. Treatment with the PPARb/d agonist enhanced the expression of two-well known PPARb/d-target genes involved in fatty acid
oxidation, carnitine palmitoyltransferase-1 (CPT-1) and pyruvate dehydrogenase kinase 4 and increased the phosphorylation of AMPK, preventing the reduction in fatty acid oxidation caused by palmitate
exposure. In agreement with these changes, GW501516 treatment
reversed the increase in DAG and PKC activation caused by palmitate. These effects were abolished in the presence of the CPT-1 inhibitor etomoxir, thereby indicating that increased fatty acid oxidation was
involved in the changes observed. Consistent with these ndings,
PPARb/d activation by GW501516 blocked palmitate-induced NF-jB
DNA-binding activity. Likewise, drug treatment inhibited the increase
in interleukin 6 expression caused by palmitate in C2C12 and human
skeletal muscle cells as well as the protein secretion of this cytokine.
These ndings indicate that PPARb/d attenuates fatty acid-induced
NF-jB activation and the subsequent development of insulin resistance
in skeletal muscle cells by reducing DAG accumulation. Our results
point to PPARb/d activation as a pharmacological target to prevent
insulin resistance.
(1) University of the West Indies, Faculty of Medical Sciences, Cave Hill
Campus, Black Rock, St Michael, Barbados
(2) University of the West Indies, Mona Campus, Jamaica
241
Paper No.: 729
EFFECT OF ENVIRONMENTAL TEMPERATURE ON
NOREPINEPHRINE-INDUCED CONTRACTION IN ISOLATED
CHICKEN CUBITAL VEIN: GENDER VARIATION
Simon Comerma-Steffensen(1), J Rojas(1), A Risso(1), M Rossini(1),
V De Basilio(2), I Oliveros(3), H Zerpa(1)
(1) Central University of Venezuela, Veterinary Faculty, Department of
Biomedical Sciences, Maracay, Venezuela
(2) Central University of Venezuela, Agronomy Faculty, Maracay, Venezuela
(3) National Institution of Agropecuary Research, Venezuela
The rise of global environmental temperature might impact thermoregulation of several organisms. To assess this issue in chickens (Ross linebroilers), segments of the wing cubital vein were harvested from 5 weeks
old individuals (n = 5-9) killed for human consumption. Isolated vessels
were obtained from male and female chickens, maintained at two breeding temperatures (30C warm and25C cool) and prepared for isometric tension recording in organ baths. Endothelium integrity was
established by the relaxing response to acethylcolinein phenylephrinecontracted vessels. The response to a depolarising Krebs solution (DKS
118 mM KCl) was assessed followed by the construction of concentration response curves (CRC) to norepinephrine (10-9-10-4M). The Emax
and pD2 values were compared by unpaired Students tests. The DKS
peak height contraction was similar between males and females at both
breeding temperatures. Norepinephrine-induced Emax was also similar
between gender and temperatures; however, the norepinephrine-induced
Emax was higher (p<0.05) in vessels from females bred at cool
(1.85 0.4g/mg) vs. warm (0.88 0.1g/mg) temperatures.These
results might suggest a reduced efcacy to norepinephrine in cutaneous
veins from females chickens under warming conditions, in contrast to the
response in males. The potential better physiological adaptability of
supercial veins from females chickens might contribute to overcome
higher environmental temperatures.
Paper No.: 1153

RECEPTOR MECHANISMS OF THE LONG-LASTING AND
PERSISTENT ARTERIAL CONTRACTILE EFFECTS OF
ENDOTHELIN-1
Matthijs Compeer(1), M Meens(1), C Holtke(2), W Neugebauer(3), J De
Mey(1)
(1) Maastricht University, Department of Pharmacology & Toxicology,
Maastricht, The Netherlands
(2) The University Hospital of Munster, Munster, Germany
(3) Sherbrooke University, Sherbrooke, QC, Canada
Introduction: Endothelin-1 (ET-1) acts as a high afnity, irreversible
polyvalent ligand on ETA-receptors. Relations between these characteristics have not been established. Materials: We used rat mesenteric resistance arteries, rhodamine-labeled ET-1 (to monitor ligand-binding by
2-photon laser scanning microscopy focusing on smooth muscle) and
native ET-1 (monitoring contractions by wire-myography in conditions
minimizing endothelial and sensory-nervous effects). Results: ET-1 (1 16nM) caused strong contractions, persisting in the absence of free agonist. The ETB-antagonist BQ788 (1uM) partly prevented binding, but
did not modify contractile effects of ET-1. The peptidergic ETA-antagonist BQ123 (1uM) prevented but not reversed binding persisting in presence of BQ788. The potent non-peptidergic ETA-antagonist PD156707
(1 - 100nM) and BQ123 (0.1 - 3uM) reduced sensitivity to ET-1-induced
contractions. They partly and reversibly reduced contractions in the pres-
ence and after exposure to ET-1. Preventive effects of Cy5.5-PD156707

and FITC-BQ123 were comparable to their unlabeled analogues. However, the bulky labeled antagonists were less effective in reducing contractions in presence and after exposure to ET-1. Conclusion: These
ndings indicate sequential bitopic binding of ET-1 to ETA-receptors;
one agonist part interacts dynamically with an antagonist-sensitive binding-site (site H). Thereafter, another agonist part binds irreversibly to
another part of the receptor (site L). Signalling is triggered by agonistoccupied site L and can be enhanced by site H. Differences between
large versus small antagonists suggest charnie`re (hinge) behaviour of
the agonist molecule, irreversibly bound to one of its binding sites. This
study was performed within the framework of TI Pharma projects T2301 and T2-108.
Paper No.: 1013

JNK SIGNALLING PATHWAY INVOLVES IN AB1-40-INDUCED
NEURONAL DEATH AND PROTECTIVE EFFECT OF
EXTRACTS OF GINSENG AND GINKGO BILOBA
Weihong Cong(1), J Liu(1), L Sheng(1), C Lin(2)
(1) China Academy of Chinese Medical Sciences, Xiyuan Hospital,
Research Center, Beijing, PR China
(2) Shandong University of Traditional Chinese Medicine, Shandong, PR
China
b-amyloid peptide (Ab) has been implicated as a key molecule in the
neurodegenerative cascades of Alzheimers disease (AD). Meanwhile,
studies on the c-Jun N-terminal kinase (JNK) signalling pathway strongly
suggested that it plays an important role in mediating the neuronal death
induced by Ab. Therefore, we aimed to investigate the protective effects
of a combination of extracts of ginseng and Ginkgo biloba (NWK) on
neuronal death induced by Ab 1-40 in rats and the candidate molecular
mechanism. A 4-week NWK (150 mg/kg and 15mg/kg, respectively)
administration to rats was performed daily after bilateral injection of Ab
1-40 (4 lg/lL for each side) into hippocampus. Nissl staining, TUNEL
staining and phosphorylated JNK (p-JNK) immunohistochemical staining
were adopted to observe the changes of hippocampal neurons. Western
blot was used to detect the phosphorylated proteins of c-Jun and JNK.
The results showed that NWK (150 mg/kg) could prevent the loss of
Nissl staining neurons, reduce the TUNEL-positive and p-JNK-positive
neurons, and inhibit c-Jun and JNK phosphorylation in hippocampus.
Our study suggested that JNK signal transduction pathway involve in the
Ab 1-40-induced neuronal death and the combination of extracts of ginseng and Ginkgo biloba might attenuate the neuronal toxicity ofAb 1-40
by blocking JNK signalling cascades.
Paper No.: 1382

VASCULAR INSULIN RESISTANCE IN PENILE ARTERIES
FROM A RAT MODEL OF METABOLIC SYNDROME
C Contreras, A Sanchez, B Climent, A Garca-Sacristan, S Benedito,
Dolores Prieto
University Complutense of Madrid, Faculty of Pharmacy, Department of
Metabolic and cardiovascular abnormalities accompanying metabolic
syndrome, i.e. obesity, insulin resistance (IR) and hypertension, are all
associated to endothelial dysfunction and act as independent risk factors
242
for erectile dysfunction (ED). Our purpose was to investigate the vascular effects of insulin in penile arteries (PA) and whether these effects are
impaired in a rat model of IR/metabolic syndrome. PA from Obese
Zucker rats (OZR) and from Lean Zucker rats (LZR), were mounted on
microvascular myographs and the effects of insulin were assessed in the
absence and presence of endothelium and of specic inhibitors of nitric
oxide (NO) synthase (NOS), phosphatidil-inositol-3-kinase (PI3K), mitogen-activated protein kinase (MAPK), endothelin 1 (ET-1) receptors and
cyclooxygenase. The insulin-induced changes in the intracellular
Ca2 + concentration [Ca2 + ]i were also examined. OZR exhibited mild
hyperglycemia, hypercholesterolemia, hypertryglyceridemia and hyperinsulinemia. Insulin induced endothelium and NO-dependent relaxations in
LZR that were impaired in OZR. Inhibition of PI3K reduced the insulinand the b-adrenoceptor agonist isoproterenol-induced vasodilatation
mainly in arteries from LZR. Antagonism of ET-1 receptors did not alter
insulin-induced relaxation in either LZR or OZR, but MAPK blockade
increased this vasodilatation in OZR, which was inhibited by cyclooxygenase blockade. Insulin induced a decrease [Ca2 + ]i which was
impaired in OZR. In conclusion, insulin-induced vasodilatation is
impaired in PA of OZR due to altered NO release through the PI3K pathway and unmasking of a MAPK pathway-mediated vasoconstriction.
This vascular IR is likely to contribute to the endothelial dysfunction and
to the ED reported in IR states.
Supported by grant SAF2006-09191 from MICINN
Paper No.: 2077

HIGH LEVEL OF BLOOD ZOLPIDEM CONCENTRATIONS IN
THE MORNING AND IN THE EARLY AFTERNOON AFTER A
SINGLE DOSE OF A HYPNOTIC DRUG FOLLOWED BY A
NIGHT OF SLEEP IN HEALTHY ELDERLY SUBJECTS :
POTENTIAL LINK WITH IMPAIRED DRIVING
Antoine Coquerel(1,2), M Loilier(1,2), S Marie(1,2), F Bertran(3),
V Lelong-Boulouard(1,2), P Denise(2,3), M-L Bocca(2,4)
(1) University Hospital, Department of Pharmacology, Caen, France
(2) Caen University Research Team, ERI 27 Inserm - EA 3917, Caen,
France
(3) Caen University Hospital, Department of Pysiology, Caen, France
(4) Paris XI, University, Paris, France
Old people (OP) are drivers and may be hypnotic drug consumers.
Recently European community promoted research on psychotropic drugs
consumption to prevent trafc accidents. Usually the hypnotic tolerance
tests like driving ability are achieved with young healthy patients. We
tried to determine whether in OP - 55 to 65 - a single dose of a hypnotic
like Zolpidem (Zp), Zopiclone (Zc) or Flunitrazepam (FNZ) induce
residual effects versus placebo (PCB). Drugs were assayed with Gas and
Liquid Chromatograhy coupled to Mass Spectrometry to measure concentrations 10 and 16 h after drug intake. Methods: 16 OP. None consumed psychotropic medication. The night before a session, they took a
standard dose of either Zp (10 mg), Zc (7.5 mg), FLN (1 mg) or PCB at
23 h. The study was conducted double-blind, random order of treatments, each session being separated by >8 days. Results: 6/16 subjects
had residual effects and increased trough levels of Zc or Zp until midday.
Particularly 11/16 Zp had signicant concentrations >15 mg/L as threshold. mean SEM at 9 h and 15 h were respectively 69 17 and
41 10 mg/L. The mean terminal half life was 4.0 h. Conclusion and
discussion: the incidence of high residual concentration of Zp is in contrast with our previous ndings in young subjects (Bocca &al., Psychopharmacology 1999; 143: 373-9).The Zp residual effects were related to
pharmacokinetic prole. This suggests revisions of Zp dosage for people
over 55.
Paper No.: 1533

DIAGNOSIS AND TREATMENT OF SEIZURES IN THE
NEONATAL INTENSIVE CARE UNIT
N Cosic-Cerovac(1), Milca Prostran-Veselinovic(2), N Jovic(1),
R Stojanovic(2), Z Stojanovic(2)
(1) Belgrade University School of Medicine, Department for Neurology
and Psychiatr yfor Children & Youth, Belgrade, Republic of Serbia
(2) Belgrade University School of Medicine, Department of Clinical
Pharmacology, Pharmacology and Toxicology, Belgrade, Republic of
Serbia
Neonatal seizures are heterogeneous in origin and are the most common
neurological emergency in newborn infants. We studied a group of 22
infants with clinically observed seizures. Gestation ranged from 34 to
41 weeks and time of rst seizure ranged from 2 hours to 25 days of
age. Initial investigations included results of tests for blood sugar and
serum electrolytes, blood cultures, neurological examinations, and appropriate specialized studies (EEG and ultrasongraphy of infant brain). Subtle (70%) and multifocal clonic seizures (15%) were the most common
types of seizures. Status epilepticus was observed in 1 patient. Etiology
was determined in 92% of infants and the commonet was asphyxia. Interictal EEG discharges were noted in 25% of infants. AED treatment was
necessary in 15% of infants (phenobarbital, phenytoin and diazepam).
Continuous AED therapy was instituted in 10%. real time ultrasonography and EEG are very reliable methods of diagnosing these conditions
and should be part of the routine screening of all infants with seizures.
Paper No.: 2729

DISEASES
EFFECT OF CROTOXIN, THE MAIN TOXIN OF THE
RATTLESNAKE C.D.TERRIFICUS VENOM, ON SECRETORY
ACTIVITY OF PERITONEAL MACROPHAGES DURING
TUMOR PROGRESSION
ES Costa, AMSC Francisco, OJ Faiad, Y Cury, Sandra C Sampaio
Butantan Institute, Laboratory of Pathophysiology, Sao Paulo, Brazil
Crotoxin (CTX) inhibits tumor growth and modulates the function of
macrophages. Despite these evidence, the contributiuon of macrophage
inhibition to the decrease in tumor growth, caused by CTX, was not
determined yet. Macrophages provide a defense mechanism against
tumor cells and two distinct polarization states, M1 and M2, have been
described for these cells. In the beginning of tumor progression, M1
macrophages release reactive nitrogen/oxygen intermediates and the
cytokines TNF-a, IL-1b and IL-6. In contrast, during tumor development, the release of these mediators by tumor-associated macrophages
(M2 cells) is inhibited, contributing to tumor development. In the present
study, the effect of CTX on the activity (H2O2, nitric oxide-NO and cytokines release) of macrophages obtained from peritoneal cavity of Walker
256 tumor-bearing rats, was investigated. Male Wistar rats were inoculated (2x107, s.c.) with tumor cells and treated with CTX (18lg/300ll/
rat, s.c.) on day 1 (M1 macrophage) or 5 (M2 macrophage) after cell
injection. Macrophages were obtained on day 14 after cell inoculation.
CTX (day 1 and 5, respectively) stimulates H2O2 (149% and 162%) and
NO (77% and 94%) production and the release of the cytokines IL-1 b
(day 1: 71%) and TNF-a (day 5: 1.59 fold). These results indicate that
CTX modies the secretory activity of M2 cells, which may contribute
to the inhibitory action of the toxin on tumor growth. These data reinforce the actions of CTX on defence mechanisms and bring new perspectives for the development of a new substance with therapeutic properties.
Supported by FAPESP(08/53840-8,09/52330-9), CNPq/PIBIC, INCTTOX.
243
Paper No.: 3278
DISEASES
POSSIBLE ROLE OF THE ENDOCANNABINOID SYSTEM IN
THE ANALGESIC EFFECT OF PARACETAMOL-IBUPROFEN
ASSOCIATION - AN EXPERIMENTAL STUDY
Mihnea Costescu, H Paunescu, OA Coman, I Fulga
University Carol Davila School of Medicine and Pharmacy, Department
of Pharmacology and Pharmacotherapy, Bucharest, Romania
Aim: Evaluation of a potentiation of the analgesic effect between paracetamol and ibuprofen and implication of endocannabinod system. Materials and methods: 10 male albino mice groups were used. The
experiments were supervised by an institutional ethic board. The tested
substances were: paracetamol orally administered; ibuprofen and AM281
(CB1 receptor antagonist) intraperitoneally administered. Two analgesia
test were used: acetic acid 0,75%(v/v) writhing test, using as parameter
the antinociceptive percent; hot plate test at 55C, parameters used - time
until paw liking and until jump. Statistic analysis used ANOVA or nonparametric tests. Results: In writhing test a dose-analgesic effect relationship was obtained, with ED50 = 400 mg/kg bw for paracetamol and
75 mg/kg bw for ibuprofen. Using different proportion of these two substances the analgesic effect was additive. Association of 80 mg/kg bw
for paracetamol and 60 mg/kg bw for ibuprofen with AM281 decrease
the association effect. In hot plate test doses up to 1000 mg/kg bw of
paracetamol and up to 240 mg/kg bw ibuprofen had no analgesic effects,
the association of 600 mg/kg bw paracetamol and 30 mg/kg bw ibuprofen was analgesic and this effect was antagonized by AM281. Conclusions: Paracetamol and ibuprofen had an additive analgesic effect in
writhing test and reciprocal potentiation in hot plate test. Adding AM281
to the association paracetamol-ibuprofen decrease the analgesic effect in
the writhing test and eliminated the effect in the hot plate test. Inuencing endocannabinoid system might be responsible for a part of analgesic
effect of paracetamol-ibuprofen associations. Key words: ibuprofen, paracetamol, endocannabinoids, analgesia.
Paper No.: 1989

OXALIS PES-CAPRAE INHIBIT NA
CONCENTRATION-RESPONSE CURVES IN HUMAN
INTERNAL MAMMARY ARTERY
Maria Cotrim(1), M Campos(1), M Gaspar(1), D Fonseca(1),
C Frigerio(1), N Gomes(1), M Antunes(2)
(1) University of Coimbra Faculty of Pharmacy, Center for Pharmaceutical Studies(CEF), Laboratory of Pharmacology, Coimbra, Portugal
(2) University Hospital of Coimbra (HUC), Department of Cardiothoracic Surgery, Coimbra, Portugal
Introduction: After a meta-analysis of the published papers in natural
sources for neurological bioactivity we veried that many are the natural
sources and the structures that can be utilised on this purpose (Gomes et
al., 2009). Duarte et al. (1993) found that Oxalis pes-caprae L. relax the
contractions induced by NA, KCl or phorbol 12-myristate-13-acetate in
rat aorta strips. Abdalla et al. (1994) reported a concentration-dependent
relaxation of the tone of ileum, epinephrine- precontracted pulmonary
artery and mild relaxation of acetylcholine- precontracted trachea for this
compound. We have study the effects of Oxalis pes-caprea leaves extract
on NA concentration-response curves in human internal mammary artery.
Material and methods: The avonoid/phenolic acid prole was carried
out as been proposed by Campos (1997). The structural identications
were made according to Campos & Markham (2007). Vascular rings of
the human internal mammary artery were cut into four segments. After
an equilibration period of 2 hours at a resting tension of 1.5 g, changes
in isometric tension were measured using the PowerLab data acquisition

package. A volume of 200 ll of the 0,2 mg/ml Oxalis pes-caprae L.
leaves extract, was used to test the effect on the NA concentrationresponse curves (10-7-10-4M) in human internal mammary artery.
Results: Oxalis pes-caprae L. inhibited NA concentration-response
curves on human internal mammary artery.
Results: Oxalis pes-caprae L. inhibited NA concentration-response
curves on human internal mammary artery.
Abdalla et al., 1994, Phytoth. Res., 8: 265-70; Campos M.G., Mitchel
K., Cunha A., Markham K., 1997, Phytochem. Anal., 8, 181-185; Campos M.G. and Markham K. R., Ed. Imprensa da Universidade de Coimbra, Portugal 2007; Duarte et al., 1993, Rev. Cienc. Biomed., Sao Paulo,
14:89-96 Gomes et al., 2009, Prog. Neuro-Psychopharmacol. & Biol.Psych, 33, 1372-89
Paper No.: 2404

BEHAVIORAL EFFECTS OF 5-GNTI AND 6-GNTI IN MICE
Alan Cowan(1), S Inan(1)
Temple University School of Medicine, Department of Pharmacology,
We are comparing the in vivo pharmacology of 5-GNTI (5-guanidinonaltrindole), a selective j opioid receptor antagonist, and its 6-regioisomer, commonly described as a selective agonist for the j-d receptor
heteromer. 5-GNTI (0.03-3 mg/kg) precipitated frenzied scratching after
s.c. injection behind the neck of male, S.W. mice (25-30 g; n = 8-10), as
did 6-GNTI (0.30-30 mg/kg). The A50 values were 0.17 and 2.28 mg/
kg, respectively. The nature of the spectacular behavioral arousal was
studied in the hexobarbital sleeping time test. Three groups of mice
(n = 9-10) received hexobarbital (100 mg/kg, i.p.), followed 3 min later
by s.c. saline, 5-GNTI (0.30 mg/kg) or 6-GNTI (4 mg/kg). The three
mean latencies (min) to recovery of the righting reex (71 + /- 10,
s.e.m., 69 + /- 12 and 64 + /- 10, respectively) were not signicantly different after Kruskal-Wallis nonparametric ranking analysis. We conclude
that, despite 6-GNTI being heralded as a tissue-selective analgesic with
potentially reduced side effects, it provokes the same compulsive scratching as does 5-GNTI, and this persistent behavior may be viewed either
positively as a useful preclinical model for drug discovery or negatively
as a portent of ultimate dermatological woe.
Paper No.: 3307

DISCOVERY
THE ROLE OF P-GLYCOPROTEIN IN BACTERIAL
ATTACHMENT TO INTESTINAL CELLS
Andrew Crowe(1,2)
(1) Curtin University, School of Pharmacy, Perth, WA, Australia
(2) Curtin Health Innovation Research Instutute, Perth, WA, Australia
This study investigated the effect of changes to P-glycoprotein function
and expression on bacterial attachment to Caco2 and RKO gastrointestinal cells using 6 species of bacteria (E. coli, Staph. aureus, Salmonella
typhimurium, Klebsiella aerogenes, Clostridium sporogenes or Pseudomonas aeruginosa). The RKO cell line was chosen to provide a cell line
with minimal P-gp expression. Following incubation of gastrointestinal
cells with the P-gp inhibitors, bacteria incubated with a stable uorescent
dye (BacLight Green) were added and incubated at 37C for various
times between 30 and 240 min. Fluorescence intensity corrected for
background was used to compare bacterial attachment to these cell lines
244
with either the P-gp inhibited, induced (using rifampicin) or normal
expression. It was found that P-gp inhibition resulted in a signicant
increase of all bacterial attachment to Caco2 cells. PSC-833 incubation
resulted in Pseudomonas attachment increasing by 65%, while E. coli
resulted in a doubling of attached bacteria. Salmonella, Klebsiella, Staphlococcus, and Clostridium had up to a 3 fold increased attachment in
Caco-2 cells. RKO cells did not alter their bacterial attachment with
PSC-833. Western blotting conrmed the presence of P-gp in Caco-2
cells, and absence in RKO cells. In addition, rifampicin, a P-gp inducer,
resulted in a reduction in Salmonella and Klebsiella attachment of up to
50%. These results suggest P-gp expression may contribute to the resistance of potential bacterial toxicity in the gut, by preventing them accumulating, which in turn reduces the risk of gastrointestinal disorders.
Paper No.: 1534

POTENTIATING EFFECT OF DILTIAZEM ON PENTOBARBITAL HYPNOSIS MAY BE RELATED TO THE 5-HT1A AND
5-HT2A/2C RECEPTORS: THE DRN, TMN AND VLPO AS KEY
ELEMENTS IN THE PATHWAY
Su-Ying Cui, Y-H Zhang, X Zhao, X-Q Zhang
Peking University, School of Basic Medical Science, Department of
Pharmacology, Beijing, PR China
Our previous researches indicated that the augmentative effect of diltiazem on p entobarbital sleep may be mediated by the serotonergic system, and the VLPO-TMN neuronal circuit may play a key role. This
study was undertaken to investigate t he role of dorsal raphe nucleus
(DRN) in rats and two subtypes of serotonergic r eceptors (5-HT1A and
5-HT2A/2C) on the augmentative effect of diltiazem on pento barbital
sleep in mice and rats. The results showed that diltiazem obviously pro
longed the duration of pentobarbital-induced sleep with signicant
increase in NREM sleep time including light sleep time and deep sleep
time. These effects we re signicantly inhibited by the 5-HT1A agonist
8-OH-DPAT and 5-HT2A/2C agonist DOI and potentiated by atagonists
both of 5-HT1A receptor (p-MPPI) and 5-HT2A/ 2C receptor (ritanserin). Perfusion of diltiazem into DRN also dose-dependently increased
non-rapid eye movement (NREM) sleep including light sleep (LS) and
SWS. On the other hand, diltiazem signicantly increased c-Fos expression which is suppressed by pentobarbital in rat DRN. From these
results, it should be presum ed that the augmentative effect of diltiazem
on pentobarbital-induced sleep may be inuenced by 5-HT1A and 5HT2A/2C receptors and the DRN-VLPO-TMN neuronal ci rcuit may
play a key role.
Acknowledgement: This work was funded by grants from the National
Natural Science Foundation of China (no. 30640070 and 30772556) and
the 985 project from Peking University.
Paper No.: 3193

SELECTIVE MODULATION BY SINGLET OXYGEN OF
CCK1R AND OTHER G PROTEIN-COUPLED RECEPTORS IN
LIVE CELLS
Zong Jie Cui, Y Wang, TT Li, SF Zhang, B Nashun, HY Liang, ZQ Han
Beijing Normal University, Institute of Cell Biology, Beijing, PR China
respiratory burst. We have previously found that plasma membranelocalized singlet oxygen produced by photodynamic action permanently
activates CCK1 receptors in rat pancreatic acini. Materials: In the present work we used isolated rat pancreatic acinar cells, hepatocytes, cultured AR4-2J cells, to examine triggering by plasma membranelocalized singlet oxygen after brief photosensitization of sulphonated
aluminium phthalocyanine, of calcium oscillations in Fura-2-loaded
cells. Results: It was found that permanent calcium oscillations were
induced in pancreatic acinar cells, due to activation of CCK1 but not
M3 receptors. In hepatocytes singlet oxygen desensitized a1 adrenergic
and V1a vasopressin receptors. In AR4-2J cells, photodynamicallygenerated singlet oxygen activated CCK1 but desensitized NK1 receptors. Effects of membrane impermeant methionine / cysteine oxidant
chloramine T and cysteine oxidants PCMB, MTSEA, DTNB were
investigated. It was found that methionine / cysteine oxidation produced
reversible calcium oscillations via CCK1 receptor activation in pancreatic acinar cells. In AR4-2J cells, oxidation of extracellularly accessible
methionine residues induced permanent calcium oscillations due to
CCK1 receptor activation. In a mouse model of acute pancreatitis, pancreatic acini isolated during the initial but not later stages of pancreatitis
showed spontaneous but persistent calcium oscillations blockable by
CCK1 receptor antagonist FK480. Conclusion: Such data indicate that
oxidation of extracellular methionine residues in CCK1 receptor results
in its permanent activation, reversed only by methionine sulfoxide
reductase (MSR). This has important implications for GPCR modulation
in pancreatitis and other inammatory diseases.
Paper No.: 2967

HEALTH AND DISEASE
EPAC PROTEINS AS A DETERMINANT FOR
FORSKOLIN-INDUCED VASORELAXANT EFFECT AND
CAMP-INHIBITION OF STORE OPERATED CA2 + ENTRY IN
VASCULAR MYOCYTES
Andrea Cunas, J Elies, M Campos-Toimil
University of Santiago de Compostela, Department of Pharmacology,
Santiago de Compostela, Spain
The mechanism of the cAMP-induced vasorelaxation is poorly understood, although it may be due partially to interferences in the regulation
of cytosolic Ca2 + concentration ([Ca2 + ]i). The discovery of the cAMP
target Epac (exchange proteins directly activated by cAMP), have
explained previously unrecognized roles for cAMP that are independent
of its traditional effector cAMP-dependent protein kinase (PKA). We
have studied the effects of forskolin, an adenylylcyclase inhibitor, and
the role of the two main cAMP effectors in isolated endotheliumdenuded rat aorta. In rat aortic smooth muscle cells (RASMC), we have
measured [Ca2 + ]i with fura-2 and intracellular cAMP levels by uorescence resonance energy transfer (FRET) using epac-cAMPs dyes. Phenylephrine-induced contractions were concentration-dependently inhibited
by forskolin, both in the presence or in the absence of extra cellular
Ca2 + . Forskolin also reduced the contractions elicited by reintroduction
of external Ca2 + after a phenylephrine-induced contraction in a Ca2 + free solution. Forskolin effects were partially reduced by PKA inhibitors (KT5720, Rp-cAMPs) and they were reproduced by the Epac activator 8-pCPT-2-O-Me-cAMP. In RASMC, forskolin dose-dependently
inhibited increases in [Ca2 + ]i induced by arginine-vasopressin(AVP)
and the store-operated Ca2 + entry (SOCE) activated after depletion of
intracellular Ca2 + stores with AVP. FRET experiments shown that the
effects of forskolin on [Ca2 + ]i correlate well with the increases of
cAMP levels induced by this agent. In conclusion, forskolin-induced
increases of cAMP in RASMC inhibit intracellular Ca2 + release and
the subsequent transmembrane SOCE. The effects of cAMP are mediated by activation of Epac in these cells, although a role of PKA cannot be excluded.
Introduction: Singlet oxygen is produced by photodynamic action after

UVA irradiation of endogenous photosensitizers, or during neutrophil
245
Paper No.: 2849
ADHERENCE TO TREATMENT: FEASIBILITY OF A SPECIFIC
SELF-REPORTED QUESTIONNAIRE TO ASSESS
MEDICATION TAKING BEHAVIOUR
Josip Culig(1), M Leppee(1), J Boskovic(2)
(1) Andrija Stampar Institute of Public Health, Department of Pharmacoepidemiology, Zagreb, Croatia
(2) Pharmacy Simac, Zagreb, Croatia
Non-adherence to medication behaviour is observed in all situations
where self- administration is required. There is strong evidence that
many patients with chronic illnesses have difculty adhering to their
treatment regimens. These results show poor management and control of
the illness, which reduces the patients quality of life and wastes health
care resources. Adherence is simultaneously inuenced by various factors. The adherence assessment was done among the patients with
chronic diseases in the city of Zagreb. They were interviewed in different
community pharmacies by standardized self-reported questionnaires
offered by pharmacist. The questions concerned personal data, history,
health status, relationships with doctors and pharmacist, medication taking behaviour. According to 635 collected questionnaires there were
1357 diagnosed chronic diseases (roughly 2 per patient). The leading disease was essential hypertension (361, as rst diagnosis 239), followed by
dislipidaemia (125, only 15 as rst) and diabetes mellitus (120, as rst
69).The non-adherent behaviour has been found in 58, 3% of patients.
As the main reason emerged forgetfulness (60%), followed by being
away from home (45, 4%), and running out of pills (44, 4%). The dosing
schedule was a problem for 40, 9% of the patients and polypharmacy for
39, 5%. Frequent changes of prescribed therapy were blamed by 26% of
patients for non-adherent behaviour (in hypertensives slightly higher 31,
9%) and avoiding side-effects were reason in 29, 6% (31% in hypertensives). The patients understanding and perception of the disease itself
might improve adherence behaviour signicantly. The intervention strategies to improve the patient-health care provider relationship is crucial.
Paper No.: 2066

HYDROGEN SULFIDE IMPROVES NEUTROPHIL MIGRATION
AND SURIVAL IN SEPSIS VIA K+ATP CHANNEL ACTIVATION
Fernando Cunha, F Spiller, M Orrico, F Souto, J Alves-Filho,
S Ferreira, J Hothersall, A Freitas
University of Sao Paulo, School of Medicine Ribeirao Preto, Department
of Pharmacology, Sao Paulo, Brazil
The recovering of neutrophil migration to infectious focus improves the
survival in severe sepsis. Recently, we demonstrated that the cystathionine c-lyase (CSE)/hydrogen sulde (H2S) pathway increases the neutrophil recruitment to inammatory focus during acute localized
inammation. The present study evaluates if H2S administration
increases the neutrophil migration to infection focus and survival of mice
with sepsis induced by cecal ligation and puncture (CLP). It was
observed that the pre-treatments of mice with H2S donors (NaHS or
Lawessons reagent) improved leukocyte rolling/adhesion in mesenteric
microcirculation as well as the neutrophil migration into the infection
focus. Consequently, bacteremia levels were reduced, hypotension was
prevented and the survival rate enhanced from ~13% to ~80%. Notably,
even when treatment was delayed (6 h post-CLP), a highly signicant
reduction in mortality as compared to untreated mice was still observed.
Moreover, H2S-pretreatment prevented the down-regulation of CXCR2
and L-selectin and the up-regulation of CD11b and GRK2 in neutrophils
during sepsis. H2S also prevented the reduction of ICAM-1 expression
on endothelium of the mesenteric microcirculation in severe sepsis.
Conrming the critical role of H2S on sepsis outcome, pretreatment with

dl-propargylglycine (a CSE inhibitor) inhibited neutrophil migration to
infectious focus and induced high mortality in mice subjected to nonsevere sepsis (from 0% to ~80%). Finally, the benecial effects of H2S
were blocked by glibenclamide (a K+ATP channel blocker). These results
showed that H2S restores the neutrophil migration to infectious focus
and improves survival outcome in severe sepsis by a K+ATP channel
dependent mechanism.
Paper No.: 2727

DISEASES
OPIOID RECEPTOR ACTIVATION AND EXPRESSION IS
REGULATED BY PERIPHERAL INJURY IN BOTH DORSAL
ROOT GANGLIA (DRG) AND NERVE PAW (NP) OF RATS
Yara Cury(1), V Zambelli(1), AC Fernandes(1), V Gutierrez(1),
C Parada(2)
(2) UNICAMP, Department of Physiology and Biophysics, Campinas,
Sao Paulo, Brazil
Previous data demonstrated that, in rats, prostaglandin E2- PGE2 and
chronic constriction injury-CCI of sciatic nerve increase peripheral analgesic efcacy of opioid drugs and also of crotalphine, a novel peptide
that induces analgesia mediated by activation of j- and d-opioid receptors. The aim of this study is to characterize the mechanisms involved in
the increase of the efcacy of CRP caused by tissue injury. For this purpose, the effect of PGE2 (intraplantar route) and CCI on opioid receptor
expression/activation in DRG and NP of rats was evaluated by immunoblotting and ELISA assays. In NP, PGE2 increases the expression of land j-opioid receptors (43 and 71%, respectively) and decreases (30%)
the expression of d-opioid receptors. l- and j-opioid receptor expression
is also increased in the DRG (79 and 168%, respectively). CCI up-regulates l-opioid receptors in NP (27%) and DRG (49%) and d-opioid
receptors in DRG (35%). In contrast, j-opioid receptors are down-regulated by CCI (51% in NP and 21% in DRG). Despite increasing the
expression of opioid receptors, PGE2, per se, did not alter the conformation of opioid receptors. CRP (0.0006lg/paw) or DAMGO (5lg/paw, lopioid agonist) activate, respectively, j- and l-opioid receptors, per se
(23 and 16%, respectively) or in the presence of PGE2 (15 and 50%,
respectively). These results indicate that peripheral opioid receptor
expression and activation are distinctly regulated by the presence of acute
or chronic injury and provide evidence regarding the effectiveness of
peripheral opioids in both acute and chronic pain.
Support: FAPESP (07/03404-4), INCTTOX Program, CNPq
Paper No.: 960

DISEASES
PD98059, A SPECIFIC MITOGEN-ACTIVATED PROTEIN
KINASE INHIBITOR, ATTENUATES THE DEVELOPMENT OF
BLEOMYCIN-INDUCED LUNG INJURY
Salvatore Cuzzocrea(1,2), M Galuppo(1), E Mazzon(1), E Esposito(1,2),
R Di Paola(1), T Genovese(1), P Bramanti(2)
(1) University of Messina School of Medicine, Department of Clinical &
Experimental Medicine & Pharmacology, Messina, Italy of Medicine,
University of Messina, Italy
(2) IRCCS Centro Neurolesi Bonino-Pulejo, Messina, Italy
Mitogen-activated protein kinase (MAPK) signalling pathways involve
two closely related MAPKs, known as extracellular signal-regulated
kinase (ERK)-1 and -2. The aim of the present study was to evaluate the
246
contribution of MAPK3/MAPK1 in the development of lung inammation and brosis caused by bleomycin-administration (1 mg/kg) in mice.
To this purpose, we used 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059), which is an inhibitor of MAPK3/MAPK1. Mice
subjected to intra-tracheal administration of bleomycin developed signicant lung injury characterized by marked neutrophil inltration and tissue
oedema. An increase in immunoreactivity to nitrotyrosine, iNOS, TNF-a
and IL-1b was also observed in the lungs from bleomycin-treated mice.
PD98059 treatment (10 mg/kg, 10% DMSO, i.p.), shows therapeutic
effects on pulmonary damage, decreasing many inammatory and apoptotic parameters: 1) cytokines production, 2) IkB-a degradation and NFjB nuclear traslocation 3) iNOS expression 4) nitrotyrosine and PARP
localization 5) the degree of apoptosis, evaluated as Bax and Bcl-2 balance, FAS ligand expression and TUNEL staining. Taken together, these
results clearly demonstrate treatment with the MEK1 inhibitor PD98059
reduces the development of lung injury and inammation induced by
bleomycin in mice.
Paper No.: 2749

SEROTONIN IS INVOLVED IN AIRWAY HYPERREACTIVITY
AT BOVINE TRACHEAL SMOOTH MUSCLE
Darwin J Da Costa-Guevara(1), E Trejo(2), M Alfonzo(2)
(1) Central University of Venezuela Faculty of Pharmacy. Department of
Pharmacology, Caracas, Venezuela
(2) Central University of Venezuela Faculty of Medicine, Caracas,
Venezuel
Serotonin or 5-hydroxytryptamine (5-HT) has different effects in the
smooth muscle due to a great variety of receptors. The 5-HT is a common
regulator of the arterial pressure, gastrointestinal motility, etc. In addition,
serotonin role in the modulation of the smooth muscle tone of the upper
airways is a key factor of controversy in the pathophysiology of the bronchial hyperreactivity. In this sense, little is known about the receptors and
the mechanism that exactly are involved in bronchial hyperreactivity.
Some subtypes of 5-HT receptors are claimed to be associated with this
function are: 5-HT1, 5-HT3, 5-HT7 and more frequently 5-HT2 receptors.
Two pharmacological mechanisms have been proposed to explain the 5HT induced contraction of the upper airway smooth muscle: a) Acting
directly via binding to specic 5HT receptors localized on the smooth
muscle, or b) indirectly when 5HT facilitates the acetylcholine (ACh)
release from of neurons or other deposits by means of 5-HT receptors and
this release ACh contracts the airway smooth muscle. The aim of this
study was evaluated using isolated bovine trachea strips in organ bath the
effect of muscarinic inhibitors such as atropine (1lM) and 5-HT antagonists (1lM) on contraction of upper airway smooth muscle in the presence
of 5-HT. The atropine was capable of block the contractile effect of 5-HT
1lM in about 75% and the ritanserina almost completely. These result
suggest that the 5-HT2 receptors are important in this contractile effect of
the 5-HT and that the ACh participate in this action.
Paper No.: 2813

FOCUSED CONFERENCE GROUP: P11 - G PROTEIN-COUPLED 7TM RECEPTORS: FROM MOLECULAR TO PHYSIOLOGICAL FUNCTION
LACK OF KININ RECEPTORS AFFECTED ACE EXPRESSION
IN MICE ABDOMINAL AORTA
Eliete da Silva Rodrigues, RP Martin, JB Pesquero, SI Shimuta
UNIFESP, Department of Biophysics, Sao Paulo, Brazil
Reduced responsiveness of abdominal aorta to bradykinin (BK), desArg9
BK and to angiotensin II (AngII) was described in mice decient in kinin
B1 (B1KO) and B2 (B2KO) receptors. The expression levels of the kinin
receptors and of AngII AT1 receptor were low in these transgenic mice.
Then the role of angiotensin-converting enzyme (ACE), a link between
kallikrein-kinin and renin-angiotensin systems, was evaluated. Contractile responses to angiotensin I (AngI) and to BK were recorded in the
presence and absence of ACE inhibitor, lisinopril, in rings of aorta of the
transgenic animals. The efcacy of AngI induced responses was reduced
in B1KO and B2KO, suggesting that ACE activity was lower than in
WT mice. However BK-induced responses which were reduced in these
transgenic animals were potentiated in the presence of lisinopril, indicating that the enzyme activity was not inhibited. Noteworthy, AngI
induced contractions in B1KO was inhibited by lisinopril, whereas those
of B2KO were partially affected, suggesting that a role for ECA. The
nding that there was a signicant reduction in the ACE expression level
in B1KO but not in B2KO mice is another fact to be considered. Our
present results suggest that the ACE inhibitor can potentiate BK-induced
effect by inducing cross talk between ACE and BK B2 receptor beyond
blocking BK inactivation. The nding that the efcacy of AngI was
decreased in both transgenic mice but lisinopril exerted partial inhibitory
effect on the B2KO reinforces our hypothesis that a regulation between
ACE, ACE inhibitor and kinin receptors may occur.
Paper No.: 1078

SESSION - ONCOLOGY
NOVEL IMIDAZO[1,2-A]PYRIDINES IN THE INDUCTION OF
COLONIC CELL DIFFERENTIATION AND APOPTOSIS
Nurit Dahan-Farkas(1), C De Koning(2), L Harmse(1), H Davids(1)
(1) University of the Witwatersrand, Department of Pharmacy and Pharmacology, Parktown, South Africa
(2) University of the Witwatersrand, Department of Chemistry, Parktown,
South Africa
Over the past few decades, a major research thrust has been directed
towards the development of anti-cancer agents through both empiric
screening and rational design of new compounds. These attempts are
made to reduce the severe adverse effects associated with existing cancer
chemotherapeutic agents, as well as to reduce the development of drug
resistance. The aim of the study was thus to screen novel imidazo[1,2a]pyridines for cytotoxic activity, as well as to determine the level of differentiation and the induction of apoptosis in the colonic carcinoma cell
lines, viz. HT-29 and Caco-2. All compounds were synthesised using
standard organic chemistry techniques. In vitro cytotoxicity of the synthetic compounds were tested against the HT-29 and Caco-2 cell lines. In
order to determine the cytotoxic effects of these compounds, the MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay
was used. The measurement of cell differentiation was determined by the
alkaline phosphatase assay, and a colorimetric assay was used for the
determination of caspase activity. The percentages of apoptosis were
determined using the Annexin V-FITC apoptosis detection kit and cytochrome c levels were determined by Elisa. Compounds CL 3 and CL 6
showed signicant cytotoxic activity (p < 0.05) against both cell lines,
with IC50 values ranging from 7.38-14.34 lM. The compounds did not
produce a signicant reduction in white blood cell viability in comparison to camptothecin (p > 0.05). CL 3 induced the highest amount of
apoptosis. Maximal caspase 3 expression was observed between 412 hours after addition of the active compounds.
Paper No.: 941

HEALTH AND DISEASE
A COMPARATIVE STUDY OF A-ADRENERGIC RECEPTOR
MEDIATED CA2 + SIGNALS AND CONTRACTION IN INTACT
HUMAN AND MOUSE VASCULAR SMOOTH MUSCLE
Jiazhen Minnie Dai(1,2), H Syyong(1), C van Breemen(1),
J Navarro-Dorado(2), S Redondo(2), M Alonso(3), T Tejerina(2)
247
(1) University of British Columbia, Child and Family Research Institute,
Department of Anesthesiology, Pharmacology and Therapeutics, Richmond. BC, Canada
(2) Universidad Complutense,Department of Pharmacology, School of
Medicine, Madrid, Spain
(3) Hospital Clinico San Carlos, Service of General Surgery. Madrid,
Spain
Paper No.: 1093

HEALTH AND DISEASE
ACTIVATORS OF SMALL CONDUCTANCE
CALCIUM-ACTIVATED POTASSIUM CHANNELS
SELECTIVELY ENHANCE BRADYKININ NO-MEDIATED
VASODILATATION IN PORCINE RETINAL ARTERIOLES
In virtually all experimental animal studies on intact vascular smooth

muscle of a variety of blood vessels, physiological and pharmacological agonists initiate oscillatory uctuations in intracellular Ca2 + to initiate and maintain vasoconstriction. In this study we compared
phenylephrine-mediated Ca2 + signals and contraction in native human
and mouse smooth muscle cells situated in the intact media of small
mesenteric arteries. In addition we employed electron microscopy to
examine the cytoplasmic distribution of the sarcoplasmic reticulum
(SR). Phenylephrine elicited tonic contractions in both vessel types,
asynchronous Ca2 + oscillations in the mouse mesenteric smooth muscle cells, but only single transient Ca2 + signals in the human mesenteric smooth muscle cells. While nifedipine inhibited 90% of the
phenylephrine-induced tonic contraction in mouse mesenteric arteries,
it only slightly attenuated those in human mesenteric arteries. The
remaining large nifedipine-resistant component in human mesenteric
arteries was abolished by the Rho-kinase blocker HA-1077. Signicant
differences were also observed in the membrane ultrastructure of the
two types of arteries. Whereas the supercial SR was abundant in the
mouse vessels and many junctions were observed between the plasmamembrane (PM) and the SR, the smooth muscle of human mesenteric
arteries had far less peripheral SR and was almost devoid of PM-SR
junctions. As we had previously reported that the PM-SR junctions are
essential for maintenance of Ca2 + oscillations, we hypothesized that in
the relatively old human patients the change in Ca2 + signalling pattern
was due to impaired SR relling via areas of close apposition between
the PM and SR.
Thomas Dalsgaard(1), C Kroigaard(1), M Misfeldt(2), T Bek(2),

U Simonsen(1)
Paper No.: 2207

COMPARATIVE STUDIES ON THE ANTIMUTAGENICITY
AND ANTIOXIDANT ACTIVITIES OF THE FRUITING BODY
AND MYCELIUM OF THAI GANODERMA LUCIDUM
Theera Dalodom, A Aramphongphan
Mahidol University, Faculty of Science, Department of Pharmacology,
Bangkok,Thailand
Ganoderma lucidum (GL, Lingzhi) has been suggested as a candidate
for prevention and treatment of different diseases, including cancer.
Thai Ganoderma lucidum (G2), which has been grown in Thailand as
part of the Royal Project since 1988 was studied for its safety and efcacy. The present study aimed at comparing the fruiting body (whole
fruiting) and mycelium of GL hot water extracts with regards to their
in vitro mutagenic and antimutagenic potential by bacterial reverse
mutation assay, antioxidant activity by free radical scavenging activity
(DPPH assay), and iron chelating activity. The results showed that both
GL extracts ranging from 0.15 to 3.0 mg/ml was neither mutagenic nor
antimutagenic in bacterial system. Higher antioxidant activity in term
of DPPH scavenging free radicals was found in fruiting body extract
when compared with the mycelium extract. Moreover, both GL extracts
showed a slight effect of chelating activity on Fe2 + . Signicantly, the
antioxidant capacity correlated with the total phenolic content. While
no tested concentrations of GL extracts were toxic to TA 98 and
TA100 salmonella typhimurium, the highest non-mutagenic concentration was cytotoxic to lung carcinoma cell lines (A549) as determined
by Trypan blue assay. This is the rst comparative study on the antimutagenic and antioxidant activities of Thai Ganoderma fruiting body
and mycelium extracts.

(2) Aarhus University Hospital, Department of Ophthalmology, Aarhus,
Denmark
Introduction: The present study investigated whether the selective activator of small (SKCa) and intermediate (IKCa) conductance calcium-activated potassium channels, NS309 (6,7-dichloro-1H-indole-2,3-dione
3-oxime), or the selective activator of SKCa2 and SKCa3 channels, CyPPA (cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]amine), enhanced endothelium-dependent vasodilatation in porcine retinal
arterioles. Methods: Localisation of SKCa3 and IKCa protein was examined by immunolabelling. Endothelial cell calcium was measured by uorescence imaging with oregon green. For functional studies, arterioles
were mounted in microvascular myographs for isometric tension recordings and concentration-response experiments for bradykinin, NS309, and
CyPPA, were performed. Results: SKCa3 and IKCa protein was localised
in the endothelium. Bradykinin, but neither NS309 nor CyPPA increased
endothelial cell calcium. Pre-incubation with NS309 or CyPPA enhanced
bradykinin relaxation. This enhanced relaxation was abolished by blocking SKCa channels with apamin. In the presence of NS309 or CyPPA,
mainly inhibition of NO synthase with asymmetric dimethylarginine, but
also inhibition of cyclooxygenase with indomethacin, reduced bradykinin
relaxation. Bradykinin relaxation was completely abolished by inhibition
of NO synthase and cyclooxygenase together with a NO scavenger, oxyhaemoglobin. Conclusion: In porcine retinal arterioles, SKCa3 and IKCa
protein is localised to the vascular endothelium. Bradykinin increases
endothelial cell calcium followed by opening of SKCa and IKCa channels.
Without altering endothelial cell calcium, NS309 and CyPPA enhance the
NO-mediated bradykinin relaxation via activation of SKCa channels.
These results implicate that activating SKCa channels improves endothelium-dependent relaxation and make this channel a potential target for
treatments aimed at restoring retinal blood ow.
Paper No.: 1682

HEALTH AND DISEASE
NITRITE AND NITROGLYCERIN PREFERENTIALLY DILATE
LARGE SYSTEMIC ARTERIES DURING HYPOXIA
Thomas Dalsgaard(1), C Kroigaard(1), A Fago(2), U Simonsen(1)
(2) Aarhus University, Department of Biological Sciences, Aarhus, Denmark
Several mechanisms in the wall of large arteries have been proposed to
reduce nitrite to nitric oxide (NO) during hypoxia. This study investigated whether the vasodilating effect of nitrite and nitroglycerin (GTN)
is similar in large and small arteries. The results were compared to other
NO donors. Furthermore, the role of mitochondrial aldehyde dehydrogenase (ALDH2) in nitrite and GTN relaxation was investigated. Rat aorta,
small femoral, and mesenteric arteries (diameter (lm): 2044 8 n = 42,
8 n = 42, and 8 n = 24, respectively) were mounted in microvascular
myographs for isometric tension recordings and concentration-response
curves were performed for nitrite, GTN, NO solution, and S-nitrosogluthathione (GSNO), during normoxia (21% O2) and hypoxia (5% or 1%
O2). Nitrite, NO solution, GSNO, and GTN induced concentrationdependent relaxations in all arterial segments, but these relaxations were
more pronounced in aorta both during normoxia and hypoxia compared
248
to small systemic arteries. In aorta, nitrite and GTN relaxation was
increased during hypoxia compared to normoxia. During hypoxia,
ALDH2 inhibition with cyanamide, reduced nitrite and GTN relaxations
in aorta, whereas in small femoral arteries only GTN relaxation was
reduced. Guanylate cyclase inhibition with 1H-[1,2,4]oxadiazolo-[4,3a]quinoxalin-1-one (ODQ) reduced relaxations to nitrite, GTN and NO
in both aorta and small femoral arteries. These results suggest that nitrite
relaxation is more pronounced in large arteries compared with systemic
small arteries due to lower sensitivity to NO in small arteries. Moreover,
the effect of cyanamide in aorta during hypoxia suggests an involvement
of ALDH2 in the pronounced nitrite and GTN relaxation observed.
Paper No.: 2833

AN ENZYME-LINKED IMMUNOSORBENT ASSAY FOR
THERAPEUTIC DRUG MONITORING OF TRASTUZUMAB
Francois Darrouzain(1), I Teulon(2), T Ternant(3), D David(1),
D Degenne(4), H Watier(4), P Gilles(1)
(1) Tours University Hospital, Department of Pharmacology & Toxicology, Tours,France
(2) INSERM, Centre de Recherche en Cancerologie de Montpellier,
University ofMontpellier, Montpellier, France
(3) Tours University Hospital,Department ofObstetrics and Gynecology,
Tours, France
(4) Tours University Hospital, Department of Immunology, Tours,
France
Trastuzumab is a humanized recombinant monoclonal IgG1 antibody
directed against the extracellular domain of Human Epidermal Growth
Factor Receptor 2 (HER2) and used in the treatment of breast cancers
overexpressing HER2. Trastuzumab has a large inter-individual pharmacokinetic variability. An assay measuring trastuzumab serum concentrations in treated patients is therefore needed.Microtiter plates were
sensitised with three different proteins: a fusionprotein consisting in
recombinant HER2 extracellular domain linked to human immunoglobulin Fc (HER2-Fc) and two single chain variable fragments (SCFV) of an
anti trastuzumab antibody. After incubation with the sample, bound trastuzumab was detected using peroxidase-conjugated goat F(ab)2 fragment
specic for human Fcc (HRP-anti hIgG). The analytical performances of
the three different methods were compared. The HER2-Fc method had
the best analytical performances and the larger standard calibrator range.
The intra-day precision for quality controls were (percent coefcients of
variation, CV%): 10.2%, 3.1% and 14.6% for 0.4 lg/mL, 3 lg/mL and
15 lg/mL, respectively. The corresponding bias measures (percent deviation) were +0.37%, -18.2% and -3.3%, respectively. The between-days
precision was 16.3%, 9.2% and 6.4% for for 0.4 lg/mL, 3 lg/mL and
15 lg/mL, respectively. The corresponding bias were 11.4%, -8.2% and
-2.6%, respectively. The limit of detection was 0.62 lg/mL. Lower and
upper limits of quantitation were 0.4 lg/mL and 15 lg/mL, respectively.
This method is rapid, accurate and reproducible, and can be used for
pharmacokinetic and therapeutic drug monitoring studies of trastuzumab.
Paper No.: 1193

FOCUSED CONFERENCE GROUP: P19 - GENERAL SESSION TOXICOLOGY
TOPOTECAN VITREOUS LEVELS AFTER PERIOCULAR OR
INTRAVITREAL DELIVERY IN HEALTHY ALBINO RABBITS:
AN ALTERNATIVE FOR ADVANCED RETINOBLASTOMA
CHEMOTHERAPY
Denisa Darsova(1), P Pochop(1), E Klapkova(2), D Kodetova(3), H Tesfaye(2), J Uhlik(4), L Vajner(4)
(1) Charles University 2nd Faculty of Medicine and Motol Hospital,
Department of Ophthalmology, Prague, Czech Republic
Department of Clinical Biochemistry and Pathobiochemistry, Prague,

Czech Republic
Department of Pathology and Molecular Medicine, Prague, Czech
Republic
Department of Histology and Embryology, Prague, Czech Republic
Background: Intravenous topotecan (TPT) was proved to be effective in
the treatment of advanced retinoblastoma. The local ocular TPT delivery
might result in higher intraocular chemotherapeutic levels eliminating
exposure to non-target tissues. Methods: Six albino rabbits received single
TPT dose of 2mg/1ml by periocular injection into right eyes (POI group).
Six albino rabbits (TII group) received a single transcorneal intravitreal
injection of TPT (1lg/0.1ml) into right eyes. Left eyes were treated with
the same volume of sterile water. Blood and vitreous samples were collected at 0,1,2,6,24,48,168 and 480 hours. Plasma and vitreous levels of
TPT were determined by HPLC. AUC was calculated using trapezoidal
rule. Clinical evidence of toxicity was classied into four grades according to the anatomical structure. Results: Mean AUC was higher for TII
group (780.365 lg/ml.h) than for POI group (mean: 640.3 lg/ml.h). Relatively high TPT concentration was measured in left eyes (mean: 1.86lg/
ml) and in plasma (mean: 0.79 lg/ml) in POI group. Two rabbits in POI
group developed grade 1 - periocular toxicity, two had grade 3 - ocular
toxicity and two grade 4 systemic toxicity. In TII group, TPT vitreous
levels were observed until 480 hours. Five rabbits in TII group developed
grade 3 toxicity. Conclusion: Reached AUC in POI and TII groups was
higher than expected target AUC of 80-120ng/ml.h. TPT delivered periocularly developed grade 4, while TPT delivered intravitreally developed
grade 3 toxicity. Transcorneal intravitreal delivery of topotecan might be
promising in the treatment of advanced retinoblastoma.
Supported by the grant GAUK Nr. 63008.
Paper No.: 406

COULD VITAMIN K2 GUARD AGAINST EXPERIMENTALLY
INDUCED GASTRIC STRESS ULCERS IN RATS
Inas Darwish(1), A Mohamed(2), H Abdel Aziz(3)
(1) Alexandria University Faculty of Medicine, Department of Clinical
Pharmacology, Alexandria, Egypt
(2) Alexandria University Faculty of Medicine, Department of Physiology, Alexandria, Egypt
(3) Alexandria University Faculty of Medicine, Department of Medical
Biochemistry, Alexandria, Egypt
The present study was planned to evaluate the possible gastro protective
effect of vitamin K2 on water immersion plus restraint stress- induced
gastric ulcers in rats. It was carried out on 40 male albino rats that were
divided into 5 equal groups .Two of them served as control groups; the
rst is normal and the second is subjected to stress ulcer induction but
not pretreated. Water immersion plus restraint stress (WIRS) was the
experimental model for gastric stress ulcer done in four groups. Pretreatment with vitamin K2 in a dose of 15mg/kg/day orally for 7days in the
third group was done. While in the fourth group with alendronate (ALD)
in an oral daily dose of 20mg/kg/day for 7 days, or combination of the
mentioned drugs in the same previous doses for 7 days in the fth one.
Then they were subjected to WIRS-induced gastric ulcers. Ulcer index,
gastric tissue levels of myeloperoxidase(MPO)and serum levels of both
epidermal growth factor (EGF)and interleukin -10(IL-10) were assessed
in all groups. The present study revealed that Vitamin K2 induced significant reduction of the mean values of ulcer index and tissue levels of
MPO. Furthermore, there were signicant increase in the levels of protective cytokines as IL-10 as well as to increase the levels of ulcer healing growth factor; EGF as compared to the control groups. Thus, vitamin
K2 could have a potential clinical benet in preventing gastric ulceration
The Experimental protocol was approved by the Medical Ethical Committee of the Faculty of Medicine, Alexandria University August; 2009.
249
Paper No.: 440
MENATETRONE AND NIGELLA SATIVA: A PROPHYLACTIC
ROLE IN CORTICOSTEROID INDUCED OSTEOPOROSIS IN
THE RAT
have proposed for the rst time that endogenous hydrocortisone may
play a role in ischemic preconditioning phenomena.
Keywords: Hydrocortisone, Infarct size, Ischemia-reperfusion model,
Ischemic preconditioning, serum cortisole.
Suzan A Darwish, HM Khalifa, IE Darwish, EI Anwar
Paper No.: 892

HEALTH AND DISEASE
EXPOSURE TO CONCENTRATED AMBIENT FINE
PARTICLES (PM2.5) OF URBAN SAO PAULO AIR POLLUTION
INDUCES ENDOTHELIAL DYSFUNCTION AND STRUCTURAL
CHANGES IN RAT PULMONARY ARTERIES
Alexandria University Faculty of Medicine, Department of Pharmacology, Alexandria, Egypt

The long term use of corticosteroids adversely causes osteoporosis and
consequently bone fracture. In the presented wok, osteoporosis was
induced by the SC injection of (methyl prednisolone acetate10 mg / kg
daily for 4 weeks) to male albino rats (CIOP) which served as our experimental model. The following drugs were tested: nigella sativa oil (NSO)
800 mg/ kg and menatetrenone 7.5 15, 30 mg / kg (vitamin K2). The
tested drugs were given with calcium (10mg/kg +vitamin D3(8IU/kg)
.The following parameters were assessed after 28days of treatment:
serum osteocalcin (OC), serum osteoprotegrin(OPG) serum total alkaline
phosphatase (ALP), 24 hr fasting urinary hydroxyproline (UHP). Histological examination of lumbar vertebrae was done at the end of the
experiment .Treatment with NSO, nearly demonstrated the classical
structure of the trabicular system. This was evidenced by a signicant
increase in serum OC reduction of UHP increase in serum OPG and
reduction ALP The strong positive correlation between serum OC and
ALP reected a perfect mineralization activity of NSO. Treatment with
menatetrenone (7.5mg / kg) demonstrated normalization of trabicular
architecture and bone markers. The combination of NSO (800 mg/kg)
and menatetrenone(7.5 mg/kg) caused a classical structure of the trabicular system. UHP and total serum ALP approached normal levels. Both
serum OPG and OC exceeded normal levels showing an excessive activity on bone formation. Our results suggest that nigella sativa oil and menatetrenone almost restore normal bone structure of CIOP in rats
especially if used in combination.
Paper No.: 490

CARDIOPROTECTIVE EFFECTS OF EXOGENOUS AND
ENDOGENOUS HYDROCORTISONE IN THE RABBIT MODEL
OF ISCHEMIA-REPERFUSION
Ali Davarian, V Khori, O Shirinbeik Mohajer
Golestan Cardiovascular Research Centre, Faculty of Medicine, GolestanUniversity, Department of Pharmacology, Golestan, Iran
Reducing the infarct size in acute myocardial infarction is one of the
most important goals driving new drug research and development. During the last two decades, many clinical studies have found cardioprotective effects of corticosteroids, but their exact role in ischemic
preconditioning remains questionable. The aim of the present study was
to determine the protective effects of hydrocortisone sodium succinate on
myocardial preconditioning in rabbit hearts. Twenty-four male New Zealand rabbits were divided randomly & equally to four groups: 1) control,
2) Infarct, 3) Ischemic preconditioning (IP) and 4) Hydrocortisone
(HYD). The HYD group received 50mg/kg Hydrocortisone 45min before
major ischemia. Serum levels of cardiac troponin-T(cTNT) and cortisole
were measured before and after the protocols. Triphenyl-tetrazolium
chloride staining was used to determine the infarcted area. In the present
study, exogenous hydrocortisone decreased infarct size by 53% in comparison to the infarct group. Serum level of cortisole was increased in the
IP and HYD groups, and was signicant in the HYD group (P < 0.01).
An increasing trend in cortisole level was associated with a decreasing
trend in infarct size and cTNT in the IP and HYD groups (p > 0.01). In
conclusion, we showed that hydrocortisone has cardioprotective effects
when injected before the onset of myocardial infarction. In addition, we
Ana Paula Davel, M Lemos, L Pastro, L Rossoni, P Saldiva

University of Sao Paulo, Department of Physiology and Biophysics, Sao
Paulo, Brazil
This study was design to investigate the effects of daily inhalation of
urban Sao Paulo PM2.5 in vascular reactivity and structure of pulmonary
arteries, the primary vascular target of inhaled air pollution. For this, rats
were daily exposed to 600lg/m3 concentrated PM2.5 from urban Sao
Paulo (n = 10) or ltered (n = 10) air, during 2 weeks, 5 hours/ day.
After exposure, rats were sacriced by abdominal aorta exsanguination
and the lungs were removed. Extralobar pulmonary arteries were isolated
and cut into rings placed on organ bath containg Krebs-Henseleit solution (37C, pH=7.4). The maximal contraction of rings was tested with
KCl (75 mM). Concentration-response curves to acetylcholine, phenylephrine and sodium nitroprusside were obtained. Morphometric analysis
of pulmonary arterioles was performed, specically targeting the bronchiole alveoli transition region. The results obtained demonstrated that pulmonary arterial rings of PM2.5 exposed animals showed signicant
reduction of maximal relaxation induced by acetylcholine, whilst sodium
nitroprusside-induced relaxation was similar between exposed and control groups. Contractile responses of pulmonary arteries to KCl and phenylephrine did not modify after PM2.5 exposure. Morphometry of
intrapulmonary arteries showed that PM2.5 exposure promoted pulmonary arteriole narrowing, especially on the smaller arterioles. The present
results indicate that short exposure to urban levels of PM2.5 could
induce endothelial dysfunction and adverse structural alterations of pulmonary vascular territory.
Financial support: FAPESP.
Paper No.: 893

ROLE OF B2-ADRENOCEPTOR ON VASCULAR
INFLAMMATORY MEDIATORS AND OXIDATIVE STRESS
INDUCED BY ISOPROTERENOL TREATMENT
Ana Paula Davel, C Wenceslau, G Ceravolo, MH Carvalho, P Brum,
L Rossoni
University of Sao Paulo, Department of Physiology and Biophysics, Sao
Paulo,Brazil
Overactivation of b-adrenoceptors following chronic treatment with isoproterenol leads to hyperreactivity to phenylephrine, oxidative stress and
increases proinammatory cytokines expression on rat aorta. The aim of
the present study was investigate the b-adrenoceptors subtype(s)
involved in these vascular effects of isoproterenol treatment. Male wildtype and b1- or b2-adrenoceptors knockout mice (b1-KO, b2-KO) were
daily treated with isoproterenol (15 lg/g, sc) or vehicle for 7 days. Vascular reactivity and biochemical experiments were conducted on thoracic
aortic rings. Isoproterenol treatment enhanced the vasoconstrictor
response to phenylephrine in aortas from wild-type and b1-KO mice.
L-NAME effect on phenylephrine-induced contraction was impairs and
superoxide dismutase improved vascular reactivity only in wild-type and
250
b1-KO mice treated with isoproterenol. Further, these vessels showed
enhanced uorescence to oxidative products of dihydroethidine. Aforementioned effects of isoproterenol treatment were not observed in
b2-KO. Isoproterenol treatment increased aortic protein expression of interleukins-1b and -6, and p65 subunit of NF-jB in wild-type mice.
Moreover, inhibition of NF-jB activity with sodium salicylate normalised to control values the hyperreactivity to phenylephrine observed on
aortas from isoproterenol-treated wild-type mice. These proinammatory
effects were abolished on aortas from b2-KO treated with isoproterenol.
Immunohistochemistry analysis reveals that isoproterenol treatment did
not modify aortic expression and distribution of b-adrenoceptors. The
results provides evidences suggesting a pivotal role of b2-adrenoceptor
subtype mediating oxidative stress and proinammatory status associated
with the hyperreactivity to phenylephrine induced by persistent b-adrenergic stimulation.
Financial support: FAPESP and CNPq.
Paper No.: 1650

IMPACT OF AMBIENT EXTRACELLULAR CALCIUM ON
ANALYSIS AND INTERPRETATION OF AGONISM AND
ALLOSTERIC MODULATION OF THE EXTRACELLULAR
CALCIUM SENSING RECEPTOR (CASR)
(1) Federal University of Fluminense, Department of Physiology and

Pharmacology, Rio de Janeiro, Brazil
(2) Federal University of Rio de Janeiro, Rio de Janeiro, Brazil
Introduction: Leptin, an adipocyte-derived hormone, is an important regulator of energy metabolism with relevant vascular effects. Leptin may
increase blood pressure through enhanced sympathetic activity and it also
exerts direct depressor effects on vascular tone by stimulating endothelial
production of nitric oxide in a dose-dependent manner (Zanetti M et al,
Atherosclerosis 2004; 175: 253259). The aim of the present work is to
study if neonatal leptin treatment develops vascular alterations. Methods:
Pups were divided into two groups. Leptin group, injected daily with leptin (8lg/100g sc) and Control group injected saline daily, for the rst
10 days of lactation. The animal groups were studied at one, three and
ve months of age. Rings (4mm long) from thoracic aorta artery were
used for assessing vascular reactivity. The maximal contraction of each
ring was determined by cumulative addition of phenylephrine (10-10 to
10-6 mol/l). Acetylcholine (10-9 to 10-5 mol/l) was added cumulatively
during a maximal contraction to phenylephrine. Results: No differences
were observed in phenylephrine dependent aorta contraction at the different ages studied. However, acetylcholine EC50 was statistically different
for Leptin group (1.36x10-7M) compared to Control group (6.53x107M) at 1 month of age. There was no difference at 3 and 5 months of
age. Discussion: The higher potency of acetylcholine in Leptin group at
1 month of age agrees with previous works that shows leptin decreasing
blood pressure via NO production. Possible adaptations can explain the
lost of this activity at the other ages studied.
Financial Support: FAPERJ, CNPq, CAPES, PROPPI/UFF.
Anna E Davey(1), K Leach(1), AD Conigrave(2), PM Sexton(1),

A Christopoulos(1)
(1) Monash University, Monash Institute of Pharmaceutical Sciences &
Department Pharmacology, Parkville, VIC, Australia
(2) University of Sydney, School of Molecular and Microbial
Biosciences, Sydney, Australia
The extracellular calcium sensing receptor (CaSR) is a family C G protein-coupled receptor (GPCR) that differs from most other GPCRs in that
physiological activation by its endogenous ligand, extracellular calcium,
occurs over a very narrow concentration range. Moreover, cell-based
assays used to study GPCRs usually need to be performed in the presence of different amounts of ambient calcium in the buffer media, introducing an additional layer of complexity into the analysis of CaSR
functional data. The current study compared different modes of analysis
of C/R data generated for the interaction between CaCl2 and the human
CaSR, stably expressed in a HEK293 TREx cell line. We nd that a logarithmic parameterisation of the Hill equation that specically incorporates the xed concentration of ambient buffer calcium into the analysis
provides appropriate parameter estimates with minimal need for additional data transformations. Simulations also revealed that the effect of
increasing the concentration of ambient agonist is to decrease the apparent Hill slope and increase agonist potency. This effect on the Hill slope
was also noted experimentally (using intracellular calcium mobilisation
assay) in the presence of the positive allosteric modulator cinacalcet, suggesting that the mechanism underlying the phenomenon is potentiation
by the modulator of ambient as well as added calcium. Collectively,
these results provide new insights into some of the determinants of
experimentally derived CaSR C/R data, and suggest caution in the
(over)interpretation of mechanisms underlying allosteric modulator drug
action at this receptor.
Paper No.: 2698

HEALTH AND DISEASE
CHANGES IN RATS VASCULAR REACTIVITY IN RESPONSE
TO NEONATAL INDUCED HYPERLEPTINAEMIA
Fernanda Carla Ferreira de Brito(1), NAV da Motta(1), EB Marques(1),
CBV Scaramello(1), LL da Silva(2)
Paper No.: 3387

VANCOMYCIN DOSING AND MONITORING IN CHILDREN:
COMPLIANCE WITH GUIDELINES IN A BELGIAN
TEACHING HOSPITAL
Pieter De Cock(1), E Haegeman(1), N Goerlandt(1),
P Vanhaesebrouck(2), V Stove(3), A Verstraete(3), P Schelstraete(4),
H Robays(1)
(1) Ghent University Hospital, Department of Pharmacy Department,
Ghent, Belgium
(2) Ghent University Hospital, Department,of Neonatology, Ghent, Belgium
(3) Ghent University Hospital, Department of Clinical Chemistry, Ghent,
Belgium
(4) Ghent University Hospital, Department of Pediatric Pulmonology and
Infectious Diseases, Ghent, Belgium
Introduction: the aim of this study was to evaluate the compliance of
vancomycin prescribing and therapeutic drug monitoring (TDM) in children, with updated hospital guidelines. Methods/patients: a retrospective,
observational study was conducted on 60 vancomycin therapies in 56
patients. Results: in 17 therapies (28.3%), a wrong dosing regimen was
initiated. From the 40 correctly initiated and evaluable dosing regimens,
37.5% resulted in a therapeutic, 32.5% in a subtherapeutic and 30% in a
potentially toxic trough level; 3 correctly initiated therapies were not
evaluable. Trough level monitoring was minimally needed a 138 times
from which 54.3% was monitored at the appropriate time, 18.1% too late
and 2.9% too soon; in 24.7% of cases no trough level was taken. Peak
level monitoring was inappropriate in 29 cases (100%). A dose adjustment was justied in 69 cases: 36.2% was done at the appropriate time,
29% too late (median of 3 doses, range : 1-22) and 34.8% was not performed at all. In 5 cases, the dose adjustment could not be justied from
the reported trough levels. 38 dose adjustments were evaluable: 50%
resulted in a therapeutic, 36.8% in a subtherapeutic and 13.2% in a
potentially toxic level; 7 dose adjustments were not evaluable. Conclusion: these results demonstrate the stringent need for thorough education
on guidelines to ensure efcacy and minimize toxicity of vancomycin
251
treatments. Moreover, this study highlights the value of TDM in therapy
optimisation, even when correct initiating dosing regimens are used.
Paper No.: 1962

CANNABIDIOL CONTROLS INTESTINAL INFLAMMATION
THROUGHT THE MODULATION OF ENTERIC GLIAL CELL
ACTIVATION
Daniele De Filippis(1), G Esposito(2), M Cipriano(1), C Scuderi(2),
J De Man(3), L Steardo(2), T Iuvone(1)
(1) University of Naples Federico II, Faculty of Pharmacy, Department
of Experimental Pharmacology, Naples, Italy
(2) University of Rome La Sapienza, Vittorio Erspamer Faculty of
Pharmacy, Department of Human Physiology and Pharmacology, Rome,
Italy
(3) University of Antwerp, Faculty of Medicine, Division of Gastroenterology, Antwerp, Belgium
Introduction. Abnormalities of the enteric nervous system (ENS) are
essential elements in the pathogenesis of gastrointestinal disorders. During gut inammation enteric glial cells (EGCs) proliferate and release
neurotrophines, growth factors and pro-inammatory cytokines which, in
turn, amplify the immune response. The discovery of molecules able to
control ECG-activation by this way modulating inammation is therapeutically interesting. Cannabidiol (CBD) represents a good candidate at
this attempt, since several evidence show that cannabinoids control neurogenic inammation, both in CNS and ENS. Methods. Intestinal biopsies from patients suffering from ulcerative rectocolitis (URC) both in
active and remission phase, were stimulated with LPS plus INF-c for
24h in presence or absence of CBD. Moreover, mice with LPS-induced
acute intestinal inammation were treated with CBD. Intestinal tissues
were processed for biochemical, histological and immunohistochemical
analysis. Results. Stimulation of URC biopsies with LPS+INF-c
increased glial cell activation and intestinal damage. CBD reduced
S100B and iNOS protein expression indicating an anti-inammatory
effect of CBD. In in vivo experiments, a signicant increase of S100B
protein in the intestine of mice treated with LPS was found. Moreover,
CBD pre-treatment reduced glial cell activation. Histological, biochemical and immunoistochemical analysis demonstrated that CBD decreased
mast cell and macrophage number in LPS-treated intestine. Finally, CBD
treatment of LPS-mice blunted the pro-inammatory responses, as
revealed by the reduction of TNF-a expression. Conclusion. The presentr
results suggest that CBD, by modulating glial activation, was able to
control the inammatory environment consequently protecting against
intestinal damage.
Paper No.: 1254

P19 - GENERAL SESSION - ONCOLOGY
NEW HPLC-MASS SPECTROMETRY METHOD FOR THE
SIMULTANEOUS QUANTIFICATION IN HUMAN PLASMA OF
THE ANTILEUKEMIA DRUGS IMATINIB, DASATINIB, AND
NILOTINIB
Silvia De Francia(1), A DAvolio(2), F De Martino(1), E Pirro(1),
F Piccione(1), S Racca(1), F Di Carlo(1)
(1) University of Turin, Department of Biological and Clinical Sciences,
Orbassano,Italy
(2) University of Turin, Department of Infectious Diseases, Turin, Italy
resistance may occur. Thus, two second-generation Bcr-Abl inhibitors,

dasatinib and nilotinib, active against most of imatinib resistant Bcr-Abl
mutants, have recently been tested in clinical trials. At present imatinib
pharmacokinetics has been well investigated, while only limited information on dasatinib and nilotinib are available. In support of current clinical
studies suggesting Bcr-Abl inhibitors use in sequential/simultaneous
administration, these knowledges should be achieved. Furthermore BcrAbl inhibitors plasma concentrations measurement has become an essential tool for CML patients management, useful to evaluate compliance,
potential drugs interactions, treatment efcacy, and drug-related adverse
events. Recently many laboratories reported the development of procedures for Bcr-Abl inhibitors quantication, but, no methods for their
simultaneous determination were available. Aim of our study has been
the development and validation of an original HPLC-mass spectrometry
method for simultaneous quantication of imatinib, dasatinib and nilotinib in human plasma. After a simple protein precipitation extraction,
drugs chromatographic separation was achieved on a C18 reverse-phase
column at 1ml/min ow rate. Validation results were all in concordance
with international guidelines (FDA, 2001): mean intra-day precision
4.3%; mean inter-day precision 11.4%; mean accuracy 1.5%; extraction
recovery 95-114%; mean regression coefcient 0.99. Method developed
then can be useful to achieve informations on dasatinib/nilotinib pharmacokinetics and due to its high extraction efciency and to its good reproducibility it can be suitable to perform therapeutic drug monitoring in
clinical routine.
Paper No.: 2878

THERAPEUTIC DRUG MONITORING: HYDROQUINIDINE
ADMINISTRATION IN A NEWBORN INFANT WITH SHORT
QT SYNDROME. A CASE REPORT
Silvia De Francia(1), E Pirro(1), E Banaudi(2), C Riggi(2),
F De Martino(1), F Piccione(1), F Di Carlo(1)
(1) University of Turin, Department of Biological and Clinical Sciences,
Orbassano,Turin, Italy
(2) O.I.R.M. - S. Anna Hospital, Department of Pediatric Cardiology,
Turin, Italy
The rst-choice therapy in adult patients affected by short QT syndrome,
agenetic anomaly which may cause severe arrhythmias and death, is the
implantablecardioverter-debrillator device (ICD). The most effective
pharmacologictherapy is hydroquinidine, indicated for patients who
refuse an ICD andchildren, even if there are very little data about pharmacological therapy inthis age range. On July 2005 cardiologists asked
the laboratory of clinicarmacology to measure hydroquinidine plasma
levels in a newborn with historyof short QT syndrome in her family and
recognized as being affected by amutation in the IKr encoding gene. The
ECG pattern at birth was consistent withthe diagnosis (QTc 310 msec).
Antiarrhythmic prophylaxis with oralhydroquinidine was started in the
rst week of age. The plasma tests wereperformed by HPLC-UV. Separation was achieved with a C18 column. Mobile phasewas 0.1% triethylamine (pH 2.4) - acetonitrile (85:15) at constant ow rate of0.550 ml/
min and the eluate was monitored at 250 nm. Quinine was internalstandard. The starting hydroquinidine dose was 4 mg/kg TID, while some
monthslater dosage was increased up to 10 mg/kg TID in order to reach
a QTc interval >360 msec and basal hydroquinidine plasma levels > 0.6
ug/ml. It was observed astrong correlation between hydroquinidine
plasma levels and prolongation of QTinterval. An increase in drug biotransformation ability reached by liver duringthe rst months of life is
probably responsible for the need of greater dosesof hydroquinidine for
maintaining therapeutic plasma levels.
Chronic myeloid leukemia (CML) is a myeloproliferative disorder. Current frontline therapy is imatinib, a Bcr-Abl kinase inhibitor. Although
most patients show excellent responses to imatinib treatment, clinical
252
Paper No. 2913
DISEASES
INFLUENCE OF PROLONGED-RELEASE ASPIRIN ON
PLATELET FUNCTION IN PATIENTS WITH CHRONIC
CORONARY DISEASE
Jose P de la Cortes(1), A Guerrero(1), E Rueda(2), J Munoz-Marn(1),
JA Lopez-Villodres(1), JA Gonzalez-Correa(1)
(1) University of Malaga, School of Medicine, Department of Pharmacology, Malaga, Spain
(2) University Hospital Virgen de la Victoria, Service of Cardiology,
Malaga, Spain
Aim: to establish the inuence on platelet function of prolonged-release
aspirin in patients with chronic coronary disease. Methods: clinical trial
parallel, double-blind, randomized study on the inuence of two forms of
presentation of aspirin (immediate-release ASA, ASA-IR and prolongedrelease, ASA-PR), on the platelet function in patients with chronic coronary heart disease (Nordm; EudraCT: 2004-000398-76). Patients were
evaluated prior inclusion (V0) to 3 (V1), 6 (V2) and 12 (V3) months. We
collected clinical, analytical and biochemical parameters related to platelet function, endothelial and oxidative status. Statistical analysis was performed uni and bivariate (chi square and t-student). Results: included in
the study were 100 patients, 50 per treatment arm. Characteristics of the
population: 79% men, average age of 64.2 10.1 years. Cardiovascular
disease: IAM (61%), coronary syndrome (57%), stable angina (45%).
CVRF: hypercholesterolemia (88%), hypertension (62%), diabetes
(28%), tobacco (13%), older than 70 years (30%), BMI> 30 (39%). Compliance was over 98%. The inhibition of platelet aggregation was similar
after taking ASA-IR and ASA-PR (1.41 vs. 0.28 . 1.99 0.22 ohms V3-). The levels of thromboxane B2 were signicantly higher in patients
who took ASA-PR (15.17 2.03 vs. 30.90 2.21 pg/mL), although adequate regarding the efcacy antiaggregant. The plasma levels prostacyclin
were signicantly higher in patients who took ASA-PR (1.34 0.08 vs.
0.63 0.06 pg/mL). For none of the above parameters were observed
signicant changes over time. Conclusions: The antiaggregant effect of
aspirin prolonged-release is similar to aspirin immediate-release, but
showed less activity on the synthesis of prostanoids.
Paper No.: 2061

HYDRO-ALCOHOLIC EXTRACT OF AC
AI (AN AMAZON
PLANT) PREVENTS ENDOTHELIAL DYSFUNCTION,
OXIDATIVE STRESS AND DEVELOPMENT OF
RENOVASCULAR HYPERTENSION
Miguel de Lemos Neto, CA Costa, RS de Moura, LCRM Carvalho,
PRB Oliveira, PJC Sousa, AC Resende
Rio de Janeiro State University, Department of Pharmacology. Rio de
Janeiro, Brazil
Hydro-alcoholic extract of aca (an Amazon plant) prevents endothelial
dysfunction, oxidative stress and development of renovascular hypertension. Lemos M; Costa CA; Soares de Moura R; Carvalho LCRM; Oliveira PRB; PJC Sousa; Resende AC. UERJ. Polyphenols possess
antihypertensive, antioxidant and vasodilator activities. The aim of this
study was to investigate the protective effects of aca stone extract (ASE,
200 mg/Kg/day) rich in polyphenols in 2-kidney, 1-clip (2K-1C) renovascular hypertension that is associated with an endothelial dysfunction
and oxidative stress. Young male Wistar rats used to obtain 2K-1C and
control rats (2K) received daily treatment with vehicle or ASE for
40 days, and systolic blood pressure (SBP, mm Hg) was measured by
plethysmography. The vasodilator effect of acetylcholine (ACh, 1-100
pmol) was studied in mesenteric arterial bed. Determination of oxidative
damage was estimated by formation of thiobarbituric acid reaction sub-
stances (TBARS) in mesenteric arteries and superoxide dismutase

(SOD), catalase (CAT) and glutathione peroxidase (GPx) activities (U/
mg protein) by spectrophotometry. SBP was increased in 2K-1C and
treatment with ASE (232 12 vs 137 8) prevented the development
of hypertension. The reduced vasodilator effect (% of relaxation) of ACh
in 2K-1C rats was recovered by the ASE (45 2 vs 62 2). ASE
decreased the levels of TBARS in 2K-1C rats (1.5 0.2 vs 0.3 0.1).
The activities of SOD, CAT and GPx were reduced in 2K-1C rats and
ASE increased these activities (14 1.5 vs 24 1.5; 0.8 0.07 vs
2.1 0.2; 0.06 0.006 vs 0.12 0.008, respectively). Chronic treatment
with ASE prevents the development of hypertension and improves endothelial dysfunction and oxidative stress in 2K-1C rats.
Paper No.: 2123

HEPARIN PREVENT THE CARDIOTOXIC EFFECT OF
BOTHROPSTOXIN I & II
Miguel de Lemos Neto(1), HD Ricardo(1), MM Machado(1),
VV Martins(1), BL Cons(1), A Cintra(2), PA Melo(1)
(1) Rio de Janeiro State University, Department of Pharmacology &
Medicinal Chemistry, Rio de Janeiro, Brazil
(2) University of Sao Paulo Faculty of Clinical Pharmaceutics of Riberao
Preto, Departament of Clinical Diagnostics, Roxicology and Barmatology, Sao Paulo,Brazil
We investigated the cardiotoxic effects of Bothropstoxin I and II (BthTX
I and II) which are toxins isolated from Bothrops jararacussu venom and
we also tested heparin (30-100mcg/mL) in these effects. We performed
the experiments on Langendorff preparation with isolated rat hearts.
These hearts were continuously perfused (2-5mL/min) with a Ringers
solution at 37C and recorded the electrocardiogram (EKG) and the cardiac tension. Both toxins at 10mcg/ml, induced a negative inotropic
effect over time leading to heart stoppage. BthTX I and II decreased the
EKG wave amplitude to 83.6% of the control. BthTX II increased the
perfusion pressure, enlarged the PR interval and decreased the QRS
amplitude on the EKG. Heparin 100mcg/ml abolished the BthTX I and
BthTX II cardiotoxic effects. Images obtained with heart incubated in
1% triphenyl tetrazolium chloride (TTC), showed the more damaged area
with BthTX II than BthTX I that were completely neutralized by heparin.
Heparin was able to antagonize completely the heart stoppage, changes
in the EKG and the damage induced by these toxins in the preparation of
isolated heart.
Support - CAPES, CNPq, PRONEX e FAPERJ.
Paper No.: 783

DISEASES
THE ANTI-INFLAMMATORY AND ANTIHYPERNOCICEPTIVE ACTIVITIES OF LASSBIO-1141: THE
ROLE OF ADENOSINE RECEPTORS
Cleverton KF de Lima, RB Lacerda, LL da Silva, CAM Fraga, E de
Jesus Barreiro, ALP de Miranda
University of Rio de Janeiro, Departament of Pharmacology, Rio de
Janeiro, Brazil
Introduction: Nowadays the therapies for chronic inammatory diseases
are unsafe or inefcacious, so adenosine and its receptors arise as potential targets for the control of the inammation. Materials: It was used
male and female Wistar rats and BALBc mice, weighing 120-180g and
25-35g, respectively. All the studies have been approved by the ethic
committee from UFRJ. Results: LASSBio-1141(100lmol/Kg, p.o.) was
able to inhibit the thermal hypernociception induced by carrageenan (La-
253
vich TR et al., Braz. J. Med. Biolog. Res. 2005, 38: 445-451), and by
capsaicin (Mizushima T et al. Pain 2005, 113:51-60) in 60% and 51%,
respectively. The compound inhibited the paw edema induced by carrageenan (Ferreira SHJ Pharmacy Pharmacol.1979, 31: 648) after 3h of
stimuli with Emax= 64% and ED50 = 14,3lmol/Kg, this effect, such as
antihypernociceptive effect, was totally blocked by the unspecic adenosine receptor antagonist, teophyline (10mg/Kg, i.p.), and by the adenosine deaminase (ADA, 2unit/mL, i.pl.), an enzyme which metabolize the
adenosine. In the end of the evaluation with LASSBio-1141 (100lmol/
Kg, p.o.) in the paw edema (6h after), the neutrophil recruitment was
indirectly measured through assay of myeloperoxidase (MPO) activity
(Posadas I et al. Br. J. Pharmacol. 2004 142: 331-338) and it has reduced
the inltrated in 59%. LASSBio-1141 was able to inhibit the TNF-a production in LPS-stimulated peritoneal macrophage from mice, with Emax=
98% and IC50 = 292nM, this effect was blocked by ADA. Conclusion:
LASSBio-1141 arises as a potential candidate to control the inammation
and its mechanism of action can involve the modulation of extracellular
levels of adenosine.
Paper No.: 1482

HEALTH AND DISEASE
ROLE OF PROTEIN DISULFIDE ISOMERASE IN
ANGIOTENSIN II INDUCED CONTRACTION IN VASCULAR
SMOOTH MUSCLE CELLS FROM SPONTANEOUSLY
HYPERTENSIVE RATS
(1) University of Padova, Department of Pharmacology and Anesthesiology, Padova,I taly

(2) University of Padova, Department of Medical and Surgical Sciences,
Padova, Italy
Fluvoxamine, a selective serotonin reuptake inhibitor, is a rst-choice
antidepressant agent for treatment of depression in elderly subjects and
patients with cardiovascular diseases. Since limited and conicting data
are available regarding age-related modications of uvoxamine pharmacokinetics, this study aims to reassess the effect of age and ascertain the
possible impact of chronic heart failure (CHF) on the oral disposition
kinetics of uvoxamine. To achieve this goal, a single 50 mg-dose of this
drug was administered orally to 10 healthy young adults, 10 healthy
elderly subjects and 10 elderly patients with CHF. Plasma uvoxamine
concentration was measured for up to 96 hours. With the exception of
apparent distribution volume, all main pharmacokinetic parameters of the
drug were signicantly modied in elderly with respect to young subjects: peak concentration was about doubled (31 19 vs 15 9 ng/ml;
P < 0.05); the area under the concentration-time curve was almost three
times higher (885 560 vs 304 84 ngh/ml; P < 0.05); half-life was
prolonged by 63% (21.1 6.2 vs 12.9 6.4 h; P < 0.01), and oral clearance was halved (1.12 0.77 vs 2.25 0.66 l/hkg; P < 0.001). CHF
had no signicant effect on any pharmacokinetic parameters. Thus, aging
causes considerable impairment of uvoxamine disposition, whereas no
signicant pharmacokinetic alterations are produced by CHF. Therefore,
adjustment of initial dose and subsequent dose titrations may be required
in elderly subjects, whereas no further dose reduction is necessary in
patients with CHF.
Livia de Lucca Camargo, ACD Androwiki, GS Ceravolo, AZ Chignalia,

Alexandre Denadai-Souza, MN Muscara, MHC de Carvalho, ZB Fortes,
M Janizewski, LR Lopes
University of Sao Paulo, Institute of Clinical Biomedics, Department of
Pharmacology, Sao Paulo, Brazil
Enzymatic ROS generation is an inherent component of redox signalling.
We demonstrated that the redox chaperone protein disulde isomerase
(PDI) modulates NADPH oxidase dependent ROS generation in VSMCs.
However, the role of PDI in oxidative stress and in vascular alterations
observed in hypertension is unknown. Inhibition of PDI was evaluated in
vascular reactivity to angiotensin II (Ang II) in isolated mesenteric arteriolar bed from SHR and Wistar rats. To investigate the mechanisms
involved, VSMCs from mesenteric arteries were cultured. The expression
of PDI in VSMCs was analysed by WB. Ang II-induced ROS production, c-Src activation and intracellular free calcium concentration ([Ca2 +
]i) were measured by dihydroethidium derived uorescence, WB and
uo-3AM uorescence, respectively. Experiments were done in the presence of the PDI inhibitor, bacitracin (0.5 mM), and the NADPH oxidase
inhibitor, apocynin (30lM). Vasoconstrictor responses to Ang II were
substantially suppressed with bacitracin in Wistar (P < 0.05, n = 6) and
SHR (P < 0.05, n = 5). Additionally, PDI expression was higher in SHR
VSMCs as compared to Wistar and this was concomitant to an increase
in ROS generation (P < 0.05, n = 3). PDI inhibition signicantly
reduced Ang II dependent ROS generation, c-Src phosphorylation and
[Ca2 + ]i responses in both Wistar (P < 0.01, n = 4) and SHR cells
(P < 0.01, n = 3). Therefore, the reduction of Ang II mesenteric vasoconstriction after PDI inhibition could be related to a decrease in ROS
production, c-Src phosphorylation and [Ca2 + ]i in VSMC from Wistar
and SHR. In summary, our data suggest that PDI could be a new player
in the oxidative stress and vascular dysfunction observed in
hypertension.
Paper No.: 1888

FLUVOXAMINE PHARMACOKINETICS IN ELDERLY
SUBJECTS AND PATIENTS WITH CHRONIC HEART FAILURE
Paper No.: 2062

PROTECTIVE ACTION OF HYDRO-ALCOHOLIC EXTRACT
OF AC
AI (AN AMAZON PLANT) ON EXPERIMENTAL
METABOLIC SYNDROME IN C57BL/6 MICE
RS de Moura, PRB Oliveira, CA Costa, LCRM Carvalho, Miguel de Lemos Neto, PJC Sousa, AC Resende
Rio de Janeiro State University, Department of Pharmacology, Rio de
Janeiro, Brazil
Protective action of hydro-alcoholic extract of aca (an Amazon plant)
on experimental metabolic syndrome in C57BL/6 mice. Soares de Moura R, Oliveira PRB, Costa CA, Carvalho LRM, Lemos M, Sousa
PJC, Resende AC. Rio de Janeiro State University.This study was
designed to determine the protective effects of aca stone extract (ASE,
300 mg/Kg/day) in C57BL/6J mice fed a high-fat diet (HFD) that
delineate components of metabolic syndrome. C57BL/6 mice at
6 weeks of age were fed a control diet and received water (CD; 10%
fat) or ASE (CD+ASE) and a HFD (60% fat) that received water
(HFD) or ASE (HFD+ASE) for 12 weeks. The vasodilator effect of
acetylcholine (ACh, 0.1-1000 nmol) was studied in pre-contracted and
perfused mesenteric arterial bed of the mice. Body weight, plasma total
cholesterol, triglyceride, glucose and insulin levels were determined
and the insulin resistance measured by HOMA index. Oral glucose tolerance test (OGTT) was also assessed by oral administration of glucose (2g/Kg) at time 0 and measurement of glucose at different times.
Vasodilator response to ACh was reduced in HFD and ASE restored
the response. Increased body weight, plasma triglyceride, total cholesterol, glucose levels and insulin resistance were observed in HFD and
reduced by ASE. Treatment with ASE also reduced glucose intolerance
observed by OGTT in HFD. The results demonstrate a protective
effect of an extract obtained from aca stone in metabolic syndrome,
since the endothelial dysfunction, obesity, hypercholesterolemia, hyperglicemia and insulin resistance induced by HFD were reduced by oral
treatment with the extract.
Sara De Martin(1), R Orlando(2), M Floreani(1), P Palatini(1)

254
Paper No.: 2124
PREVENTIVE ACTION OF AC
AI (AN AMAZON PLANT)
HYDROALCOHOLIC EXTRACT ON DELETERIOUS EFFECT
OF CIGARETTE SMOKE IN MOUSE LUNGS
Roberto Soares de Moura, AC Resende, M de Lemos Neto, MAS Silva,
ETL Trajano, JN Alves, RT Nesi, PJC Sousa, LC Porto, SS Valenca
Rio de Janeiro State University, Department of Pharmacology, Rio de
Janeiro, Brazil
Cigarette smoke-induced lung damage is frequently associated either
with unbalanced protease, antiprotease production or increased oxidative
status. The present study was undertake in order to nd out if an hydroalcoholic extract obtained from acai fruits can reduce the lung damage
observed in mice that inhales smoke from regular cigarette. C57BL/6
male mice were exposed to 12 commercial full-avor ltered Virginia
cigarettes per day, for 7 days/week during 60 days (CS) or smoke from
cigarette containing acai extract (CS+A). Control group was shamsmoked. Lung histological analysis of CS group showed a typical pattern
of murine emphysema, with alveolar enlargement and areas of initial
brosis while CS+A presented a reduction in the alveolar enlargement
and no areas of initial brosis. The number of leukocytes (macrophages
and neutrophils) in the bronchoalveolar lavage was signicantly higher
in the CS group than in the CS+A and control. GPx activity was reduced
in CS but fully restored in CS+A. The nitrite was reduced in the CS
compared to control group and CS+A group. Myeloperoxidase activity,
lipid peroxidation (4-HNE), metalloelastase (MMP-12) and neutrophil
elastase were elevated in CS but not in control and CS+A. The results
suggest that harm induced by cigarette smoke may be signicantly
reduced by the presence of acai extract inside the cigarette.
Paper No.: 2430

GASTROPROTECTIVE EFFECT OF OLEA EUROPAEA LEAF
EXTRACT IN EXPERIMENTALY INDUCED GASTRIC
LESIONS IN RATS
Dragana Dekanski(1), S Ristic(1), V Piperski(1), N Radonjic(2),
N Petronijevic(2), D Mitrovic(3)
(1) Galenika a.d. R&D Institute, Biomedical Research, Belgrade,
Republic of Serbia
(2) Institute for Medical Biochemistry, School of Medicine, Belgrade
University, Belgrade, Republic of Serbia
(3) Institute for Medical Physiology, School of Medicine, Belgrade
University, Belgrade, Republic of Serbia
In this study we investigated the gastroprotective effect of dry olive leaf
extract (DOLE) in two established preclinical models of gastric damage:
gastric lesions induced by absolute ethanol and by cold restraint stress
(CRS) in Wistar rats. Different doses of standardized olive leaf extract
were applied intragastrically 30 minutes prior to absolute ethanol administration or before CRS induction. Gastric lesions were evaluated and
ulcer index was calculated. Malondialdehyde (MDA) concentration as an
index of lipid peroxidation, was determined in gastric mucosa since lipid
peroxidation has important role in pathogenesis of these conditions. The
effects of applied DOLE were compared with effect of pretreatment of
quercetin as positive control. Absolute ethanol and CRS caused severe
gastric lesions in all non-pretreated animals. Pretreatment with DOLE
(40, 80 and 120 mg/kg), as well as with quercetin (100 mg/kg), signicantly (p < 0,001) attenuated induced gastric lesions. MDA concentration signicantly increased in gastric mucosa after ethanol administration
(593 3.86) and in CRS exposed rats (445.4 43.6) vs. 287.1
15.6 nmol/mg prot. in control, non-pretreated group. Pretreatment with
reference drug, quercetin, restricted rise in MDA concentration, and this
effect was statistically signicant. All doses of DOLE decreased MDA
level in ethanol experimental model signicantly while MDA was

reduced signicantly by pretreatment with two higher doses of DOLE in
CRS. The results obtained indicate that DOLE possesses strong gastroprotective potential in experimentally induced gastric lesions, possibly
related to its antilipid peroxidative activity.
Paper No. 619

FOCUS GROUP: FC16 - NATURAL PRODUCTS: PAST AND
FUTURE?
PHARMACOLOGICAL PROFILE AND THERAPEUTIC
POTENTIALITY OF NATURAL PRODUCT OBTAINED FROM
STEM BARK OF MANGIFERA INDICA L (VIMANG) AND ITS
GLUCOSILXANTONA, MANGIFERIN
Rene Delgado-Hernandez(1), B Garrido Suarez(1), D Garcia-Rivera(2),
GL Pardo-Andreu(3), P Hernandez Casana(4), I Rodeiro-Guerra(5),
I Hernandez-Balmaseda(5), N Merino -Garcia(2), MM GuevaraGarcia(1), A Alvarez-Leon(2), Y Maragino(3), AJ Nunez-Selles(6)
(1) National Center Coordinating of Clinical Trials, Havana, Cuba
(2) Center of Biomolecular Chemistry, Havana, Cuba
(3) Pharmacy Institute, Havana University, Havana, cuba
(4) Center of Molecular Immunology, Havana, Cuba
(5) Center of Marine Bioactive Compound, Havana, Cuba
(6) Department of Science. Ministry of Health, Havana, Cuba
The aqueous extract from stem bark of Mangifera indica L (VIMANG)
has been used in Cuba during several years in ethnomedical practices for
the improvement of quality of life of patients with different pathologies.
A phytochemical characterization of the extract has led to the isolation of
nine phenolic constituents, with the glucosylxanthone mangiferin as a
major component, and different microelements as zinc, copper, and selenium. The extract has demonstrated as the main pharmacological property its antioxidant activity. Others studies have shown that the extract
also possesses others pharmacological activities, such as: anti-inammatory, antiallergic, analgesic and inmunomodulador, with very complex
and multifactorial mechanisms of action involved. These properties are
related to its scavenger capacity of different reactive oxygen species. The
interaction of mangiferin and others component of the extract with iron
represent an important antioxidant mechanism recently characterized in
our studies. On the other hand, mangiferin and Vimang have the property
of modulating different mediators involved into immune response. In
general, the total extract and its xanthone, mangiferin are involved in
several inmunomodulatory processes, properties that confer an important
therapeutic potentiality as adjuvant in the preparation of phytopharmaceuticals products for the treatment of pathologies where oxidative stress
and immunomodulator disorders are related with their etiology. Different
clinical studies are conducted at this moment in order to get new knowledge about its therapeutic potentiality.
Key words: Mangifera indica L, Vimang, mangiferin, antioxidant, antiinammatory, immunomodulation
Paper No.: 2191

MECHANISMS OF THE DILATOR ACTION OF A DANSHEN
AND GEGEN FORMULATION ON RAT BASILAR ARTERY
Shirlly Y Deng(1), FFY Lam(1), ESK Ng(1), JHK Yeung(1),
YW Kwan(1), CBS Lau(2), JCM Koon(2), PC Leung(2), KP Fung(1,2)
(1) The Chinese University of Hong Kong, School of Biomedical
Sciences, Shatin, Hong Kong, China
(2) The Chinese University of Hong Kong, Institute of Chinese
Medicine, Shatin, Hong Kong, China
Danshen (root of Salvia miltiorrhiza) and Gegen (root of Pueraria lobata) are two herbs used in traditional Chinese medicine, most commonly
for their putative cardioprotective and anti-atherosclerotic effects. In this
255
study, the actions of a Danshen and Gegen formulation (DG; ratio 7:3)
were investigated on rat isolated basilar artery rings precontracted with
100nM U46619. DG produced concentration-dependent relaxation of the
artery rings with an IC50 of 0.90 0.12mg/ml. Mechanical removal of
the endothelium or pre-treatment with 10mM of a potassium channel
inhibitor tetraethylammonium (TEA) had no effect on the DG-induced
vasodilator response, but pre-treatment with 100mM TEA suppressed
the maximum response to DG by 18% (P < 0.05). Involvement of
Ca2 + channels was investigated in artery rings incubated with Ca2 + -free
buffer and primed with 100nM U46619 for 5 minutes prior to adding
CaCl2 to elicit contraction. Pre-incubation of the artery rings with DG
for 10 minutes produced concentration-dependent (1, 3 and 7mg/ml) and
total inhibition on the CaCl2-induced vasoconstriction. These ndings
suggest DG relaxes the basilar artery primarily by inhibiting Ca2 + inux
in the vascular smooth muscle cells, and a minor component is mediated
by opening TEA-sensitive K+ channels. DG could be a useful cerebroprotective agent in some patients with occlusive cerebrovascular disease.
Paper No.: 813

PROTECTIVE EFFECT OF VEGF ON ETHANOL-INDUCED
HEMORRHAGIC GASTRIC EROSIONS IN RATS
Xiaoming Deng(1,2), X Xiong(1), Z Sandor(1,2), S Szabo(1,2)
(1) VA Long Beach Healthcare System, Department of Diagnostic &
Molecular Medicine HCG, Long Beach, CA, USA
(2) University of California, Irvine, CA, USA
Treatment with the peptide or gene of vascular endothelial growth factor/
vascular permeability factor (VEGF/VPF) accelerated healing of experimental gastric and duodenal ulcers (Szabo et al., DDS 1998;43:40S-45S;
Jones et al., Gastroenterology 2001;121:1040-7; Deng et al., J Pharmacol
Exp Ther 2004;311:982-988.). Since VEGF/VPF increases vascular permeability, we tested the hypothesis that it might create a perivascular
histodilutional barrier, hence delay the absorption of damaging chemicals that are known to induce vascular injury and hemorrhagic erosions.
Fasted S-D rats were given rat VEGF164 (ProSpec) 0.01, 0.04, or 0.2
ug/100g by gavage at 30 min before 75% ethanol (1 ml/kg) intragastrically. Control rats were pretreated with equal amount saline. All the rats
were euthanized 1 hr after ethanol administration. Hemorrhagic gastric
mucosal lesions were measured by an Imaging Analysis System (MetaMorph) and calculated as % of glandular stomach. The results showed
that VEGF/VPF pretreatment with 0.01, 0.04, or 0.2 ug/100g prevented
gastric mucosal lesions from 10.6 1.7% (controls) to 8.9 1.8,
0.9 0.2, or 2.8 0.7%, respectively. Statistical analysis indicated that
the doses (0.04 and 0.2 ug/100g) of VEGF/VPF signicantly reduced the
gastric mucosal lesions (p < 0.01 and 0.05, respectively) when compared
with controls. Conclusions: 1) This is the rst demonstration that pretreatment of VEGF/VPF offered dose-dependent gastroprotection in rats
exposed to ethanol. 2) The likely mechanisms might be related to a histodilutional barrier induced by VEGF/VPF. 3) Further studies are needed
to elucidate the molecular mechanisms on this new form of gastroprotection by VEGF/VPF.
Paper No.: 2853

THE RELAXANT EFFECT OF HYDROGEN SULFIDE IS
POTENTIATED WHILE THAT OF ACETYLCHOLINE IS NOT
ALTERED IN PULMONARY AND MESENTERIC ARTERIES
ISOLATED FROM STREPTOZOTOCIN-INDUCED DIABETIC
RATS
Merve Denizalti(1), TE Bozkurt(1), I Sahin-Erdemli(1), N Abacioglu(2)
(1) Hacettepe University, Faculty of Pharmacy, Department of Pharmacology, Ankara,Turkey

(2) Gazi University, Faculty of Pharmacy, Ankara, Turkey
Hydrogen sulde (H2S) is an endogenous gas which has potent relaxant
effect in vascular and nonvascular smooth muscles. The involvement of
H2S in several pathological states including diabetes has also been
reported. In the present study, we have investigated the relaxant effect of
H2S in mesenteric and pulmonary arteries isolated from 12 weeks-diabetic rats. Diabetes was induced by tail vein injection of streptozotocin
(STZ) (35 mg/kg) dissolved in 0.1 M citrate buffer. The control group
received citrate buffer alone. Insulin-treatment was applied by using insulin implants. At the end of 12 weeks the mesenteric and pulmonary arteries were isolated and the relaxation response to H2S donor sodium
hydrogen sulde (NaHS) (0.1 mM-3 mM) and acetylcholine (10 nM-30
`M) was elicited. The relaxation responses of the arteries isolated from
12 weeks-diabetic rats were found to be increased when compared to that
obtained from non-diabetic controls. ATP-sensitive potassium channel
(KATP) blocker glibenclamide inhibited the relaxation response to NaHS
in the arteries isolated from either diabetic or non-diabetic group of rats.
Concurrent insulin treatment of STZ-injected rats prevented the potentiation of the relaxant effect of NaHS in the mesenteric and pulmonary
arteries. However, acetylcholine-induced relaxation responses were not
altered in diabetic group of rats. In conclusion, STZ-induced experimental diabetes resulted in enhanced relaxation response to H2S in mesenteric and pulmonary arteries of rats. The potentiation of the relaxation
response to H2S in diabetic arteries may indicate a role against the vascular complications of diabetes.
Paper No.: 1686

HEALTH AND DISEASE
SIGNALLING VIA IRAG REGULATES NO- AND
ANP-MEDIATED SMOOTH MUSCLE RELAXATION
Matthias Desch(1), B Hieke(1), D Bernhard(2), K Hoecherl(3),
F Hofmann(2), J Schlossmann(1)
(1) University of Regensburg, Department of Pharmacology and
Toxicology, Regensburg, Germany
(2) Technical University of Munich, FOR 923, Munich, Germany
(3) University of Regensburg, Department of Physiology, Regensburg,
Germany
NO / ANP / cGMP signalling is important for many physiological functions e.g. vascular tone regulation, vascular smooth muscle proliferation,
gastrointestinal motility or neurotransmission. IRAG (IP3 Receptor Associated cGKI substrate) is highly expressed e.g. in smooth muscle cells
and might play an important role for cGKI downstream signalling. To
elucidate the role of IRAG for NO- and ANP-mediated smooth muscle
tone regulation, cGKI localization and blood pressure adjustment we
generated IRAG knockout mice by targeted deletion of exon 3. Hormone
precontracted aortic tissues of IRAG decient mice showed an impaired
relaxative response to exogenous and endogenous cGKI activating compounds like NO, ANP, Acetylcholine and cGMP. The cGKI dependent
reduction of tonus by high potassium depolarization was not affected in
IRAG decient tissue in comparison to wild type tissue. Reduction of
hormone-induced calcium rise by NO, ANP and cGMP was abolished in
IRAG decient vascular smooth muscle cells (VSMC) in contrast to wild
type VSMC. The localization of cGKIa- and cGKIb-isozymes in cultured aortic smooth muscle cells was not altered in absence of IRAG.
Basal mean arterial blood pressure, heart rate and activity were not changed in IRAG-/- mice. Interestingly, under pathophysiological conditions
like sepsis (induced by E.coli lipopolysaccharide treatment) IRAG decient mice were resistant to blood pressure reduction. These ndings
indicate that blood pressure regulation via cGKI / IRAG under physiological terms might be counterbalanced whereas this signalling pathway
might play an important role for blood pressure modulation under pathophysiological conditions.
256
Paper No.: 1282
ROLE OF CX43 IN THE MAINTENANCE OF SPONTANEOUS
ACTIVITY IN THE GUINEA PIG PROSTATE
Anupa Dey, S Kusljic, RJ Lang, B Exintaris
Monash University, Department of Medicinal Chemistry and Drug
Action, Melbourne, VIC, Australia
Background: Investigate the role of CX43 in the maintenance of spontaneous activity in young and aging guinea-pig prostates. Methods: Conventional intracellular microelectrode and tension recording techniques
were used. Results: Experiments were performed using gap junctional
uncouplers 18b GA (40lM), CBX (50lM) and octanol (0.5mM), on
cells displaying slow wave activity and on spontaneously contracting tissue in young and aging guinea-pig prostates. 40lM 18b GA and 50lM
CBX both abolished slow wave activity in young and aging prostates
and signicantly depolarised the membrane potential by approximately
5mV (n = 5, student paired t-test p < 0.05). 50lM CBX and 40lM 18b
GA abolished all spontaneous contractions in young and aging guineapig prostate tissue (2-way ANOVA, Bonferroni post test, P < 0.01)
(n = 5). Although, lower concentrations of CBX (10lM) and 18b GA
(10lM) signicantly reduced the spontaneous contractions within aging
guinea pig prostates while the young tissue remained unaffected from
control (students paired t-test p > 0.05). Octanol abolished slow wave
activity in both age groups of guinea-pig prostates (paired students-t test,
p < 0.05). Octanol also signicantly depolarised the membrane potential
from the control values in both age groups (paired students-t test,
p < 0.05). Within both age groups of animals octanol inhibited all contractile activity at two different concentrations (0.5mM and 1mM) (2-way
ANOVA, Bonferroni post test, P < 0.01) (n = 5). These effects were
reversed upon washout. Conclusion: Results demonstrated that when gap
junctional communication via CX43 was impaired spontaneous activity
was abolished at a cellular and whole tissue level; CX43 is therefore
essential for the maintenance of activity in the guinea-pig prostate gland.
Paper No.: 2401

OXALIPLATIN-DEPENDENT NEUROPATHY IN THE RAT:
PAIN BEHAVIOUR AND PERIPHERAL NERVOUS SYSTEM
ALTERATIONS
Lorenzo Di Cesare Mannelli(1), L Bonaccini(2), A Pacini(2),
C Ghelardini(1), A Bartolini(1)
(1) University of Florence, Department of Pharmacology, Florence, Italy
(2) University of Florence, Department of Anatomy, Florence, Italy
Oxaliplatin, a platinum-based chemotherapeutic agent, has become a
standard treatment for advanced colorectal cancer and a valid option for
patients in the adjuvant setting. The dose-limiting toxicity of this
compound is the development of a peripheral neuropathy with gloveand-stocking distribution sensory loss, combined with paresthesia, dysesthesia, and pain. In order to delucidate the morphological and molecular
alterations that occur in the peripheral nervous system during neuropathy,
we daily injected rats with 2,4 mgkg-1 oxaliplatin intraperitoneally.
Twenty-one days later, the paw-pressure test evidenced a signicantly
reduced pain threshold consisting in mechanical hyperalgesia. The histochemical study of the peripheral nerve did not highlight variations in
myelin thickness or axonal diameter. On the contrary, the treatment
increased the incidence of eccentric nucleoli and multinucleolated neurons in the lumbar dorsal root ganglia (DRG). Moreover, a signicant
reduction in the somatic area of small and medium neurons in the oxaliplatin groups was observed. The immunohistochemical analysis showed
an oxaliplatin-induced alteration of gene expression prole. The expression of the cellular injury marker ATF3 was enhanced both in axons and
in myelinating Schwann cells of the sciatic nerve, as well as in neurons

and satellite cells of DRG. The expression of the structural protein
NF200 was unaltered in the nerve, whereas the majority of DRG neurons
displayed a dramatic decrease. An understanding of the factors involved
in the development and maintenance of neuropathy and the study of the
neuron vs glia signalling may lead to mechanism based therapies that
prevent/treat neuropathic pain and improve neurorestoration.
Paper No.: 2000

HEALTH AND DISEASE
GENETIC TARGETING OF COX-1 AND COX-2 IN MURINE
AORTIC ENDOTHELIAL CELLS REVEALS THE
ANTIOXIDANT EFFECT OF ENDOTHELIAL COX-2
Andrea Di Francesco(1), L Di Francesco(1), M Dovizio(1), F Seta(2),
C Funk(2), P Patrignani(1)
(1) G. dAnnunzio University, Department of Medicine and Center of
Excellence on Ageing, Chieti, Italy
(2) Queens University, Departments of Physiology and Biochemistry,
Kingston, Canada
Prostacyclin restrains inammation in endothelial cells through the induction of heme oxygenase(HO)-1, an antioxidant enzyme. Using genetic
strategies of selective inhibition of cyclooxygenase (COX)-1 and COX-2
in mice, we explored the contribution of COX-isozymes to prostanoid
generation and HO-1 expression in murine aortic endothelial cells (MAECs) in response to interleukin(IL)-1b(10ng/ml). MAECs were isolated
from wild-type (WT), COX-1 and COX-2 knock-down(KD) mice, and
cultured in EBM-2 supplemented with 10% FBS for 6-24 hours. COX-1,
COX-2, HO-1 and terminal prostaglandin synthases(tPGS) levels were
assessed by Western blot analysis, and released prostanoids were evaluated by specic immunoassays techniques. COX-1, but not COX-2, was
detectable in unstimulated MAEC. IL-1b caused COX-2 induction by 4and 8-fold in WT and COX-1 KD cells, respectively. COX-1 and tPGS
levels were not signicantly modulated. The induction of COX-2 was
associated with signicant (P < 0.05) increase of 6-keto-PGF1a>PGE2 >
PGF2a in a time dependent fashion, both in WT and COX-1 KD cells.
PGD2 was not detected. In contrast, COX-2 KD cells were associated
with complete inhibition of prostanoid generation. COX-1 KD cells
exhibited substantially enhanced (2 fold) expression of COX-2 wich translated into signicant (P = 0.007) higher levels of prostacyclin versus WT
(11611 1678 vs 4342 674.2 pg/ml, respectively). HO-1 expression
was strongly suppressed in COX-2 KD MAEC but unaffected by COX-1
manipulation. In conclusion, COX-2 may counteract cytokine-dependent
activation of endothelial cells through the induction of HO-1. Interestingly
COX-2 expression seems to be under the negative control of COX-1.
Whether this phenomenon is mimed by pharmacological inhibition of
COX-1 remains to be elucidated.
Paper No.: 1774

DISEASES
ANALYSIS OF THE IMMUNOGENICITY OF AN INTERFERON
B1A BIOSIMILAR
Roberto Diez(1), M Kauffman(1), A Sterin-Prync(1), M Papouchado(1),
E Gonzalez(2), A Grossberg(3), S Chuppa(3), B Odoriz(4), C Vrech(5),
H Ferro(1)
(1) Bio Sidus S.A., Buenos Aires, Argentina
(2) CDM, Buenos Aires, Argentina
(3) Medical College of Wisconsin, WI, USA
(4) Hospital Central, UNC, Mendoza, Argentina
(5) Sanatorio Allende, Cordoba, Argentina
257
Introduction: Interferon (IFN) b, like other therapeutic proteins derived
from human genes by recombinant DNA technology, can be immunogenic, potentially inuencing its efcacy and safety. Blastoferon is a
pharmaceutical product of human IFN-b1a currently marketed in Argentina and Latin America as a biosimilar to the innovator IFNb1a product
for the treatment of multiple sclerosis (MS). The aim of this paper is to
present the assessment of Blastoferon immunogenicity. Materials and
patients: Immunological recognition by monoclonal and polyclonal antibodies (with either neutralizing and non-neutralizing activity) was followed by IFN biological activity testing, comparing Blastoferon to other
IFN-b products, including WHO standard. After marketing approval, a
pharmaceutical program was established, including active surveillance at
two sentinel sites in Buenos Aires and Cordoba cities. Serum of their
patients (n = 37) were tested for anti-IFNb antibodies by ELISA and in
positive cases, also for neutralizing activity against the antiviral effect of
IFN-b on Wish cells challenged with Vesicular Stomatitis Virus. Results:
recognition by the panel of antibodies showed that Blastoferon shares
immunological determinants with other human interferon-b products,
especially IFNb1a. At two years of follow-up, IFNb -binding antibodies
have been detected by enzyme immunoassay (EIA).in 72.9% of the
patients, whereas neutralizing antibodies were found in only 8.1% of the
MS patients registered in the ongoing Blastoferon pharmacovigilance
program. Conclusion: Blastoferon appears to generate antibodies at a
rate similar to the innovator IFNb1a product administered by subcutaneous route.
Paper No.: 1775

EMERGENCE OF H274Y MUTATION AND OSELTAMIVIR
RESISTANCE DURING TREATMENT IN TWO PATIENTS IN
THE 2009 EPIDEMICS OF SWINE FLU IN ARGENTINA
Paper No.: 2114

BISPHOSPHONATE-RELATED OSTEONECROSIS OF THE
JAW (BONJ)
G Dimosiari, Konstantinos Imprialos, Xxxxx Mironidou-Tzouveleki
Aristotle University of Thessaloniki, Medical School, A Laboratory ofPharmacology, Thessaloniki, Greece
Background: Bisphosphonates are primary agents in the current pharmacological arsenal against osteoclast-mediated bone loss due to osteoporosis, Paget disease and hypercalcaemia associated with certain malignant
tumors. They are also frequently prescribed for prevention and treatment
of low bone density and osteogenesis imperfecta. They have been shown
to reduce skeletal morbidity in multiple myeloma as well as a wide range
of solid tumors affecting bone. Despite these uses, the current recognition
that bisphosphonate use can be associated with pathologic complications
such as osteonecrosis of the jaw has brought skepticism and has planted a
new eld for research regarding the widespread prescription of these
drugs. Currently the American Dental Association denes bisphosphonate-associated osteonecrosis as an exposed area of necrotic jaw bone
present for at least eight weeks in patients using bisphosphonates. Recent
studies have documented that osteonecrosis, associated with the use of
bisphosphonates, primarily refers to intravenously administered drugs,
nitrogen-containing, on a long-term basis, and few cases have been
reported after short-term or oral delivery. Purprose and Methods : Since
2003, the number of publications on the subject has dramatically
increased. The purpose of this paper is to report on characteristics, mechanisms of action, risk factors and incidence of BONJ by comprehensively
reviewing the literature via pub med research. Conclusion: Though evidence supports clinical decisions in favor of the patient taking oral bisphosphonates, it is ultimately the prescribing physicians duty to weigh
the benets and risks of oral bisphosphonate use individually before determining the call for temporary or permanent termination of medication.
Roberto Diez(1), ML Valinotto(2), P Barrero(2), M Viegas(2), MC Alvarez Lopez(3), E Lopez(3), AS Mistchenko(2)

(1) University of Buenos Aires, School of Medicine, Department of
Pharmacology, Buenos Aires, Argentina
(2) Hospital de Ninos R Gutierrez, Virology Lab, Argentina
(3) Hospital de Ninos R Gutierrez, Department of Medicine, Argentina
Introduction: Oseltamivir was the most commoly used antiviral in the
2009 epidemics of swine-origin inuenza A H1N1 in Argentina and elsewhere. The emergence of resistance is an expected result of exposure to
and use of any anti-infective agent. The most frequent event resulting in
oseltamivir resistance is the substitution of histidine by tyrosine at position 275 of neuraminidase, encoded by the H274Y mutation The aim of
this report is to present genotypic and phenotypic evidence of oseltamivir
resistance emergence, mediated by the H274Y mutation in two cases during the 2009 epidemics in Argentina. Materials and patients: Complementary DNAs including the 274 codon were obtained by RT-PCR using
viral RNAs extracted from nasopharyngeal aspirates. Sequencing and pyrosequencing was performed following the WHO Inuenza A (H1N1)
NA-H274 protocol. Neuraminidase activity in the presence or absence of
oseltamivir was performed by chemiluminescence with comercial
reagents (NA-Star Inuenza Neuraminidase Inhibitor Resistance Detection Kit). Results: Sequential samples of two pediatric patients under
oseltamivir treatment were analyzed. Pre-treatment samples were composed of 100% oseltamivir-sensitive variants. In Case 1, the oseltamivirresistant variant was found eight days after the beginning of treatment. In
Case 2 the viral population became resistant on the second day of
treatment, with 83% of the viral population bearing the mutation and
reaching 100% on the seventh day. Conclusion: We describe the intratreatment emergence of oseltamivir resistance in two pediatric patients.
Pyrosequencing allowed us to detect variant mixtures, showing the
precise moment when the viral population changed from sensitive to
resistant.
Paper No.: 1921

A COMMUNITY PHARMACY MULTINATIONAL PROJECT TO
INVESTIGATE THE PRESCRIPTION DRUG ABUSE
Paola DIncau(1), M Lapeyre-Mestre(2), A Carvajal(3), U Bergman(4),
ER Heerdink(5), RV Stichele(6), D Macias(3), L Pourcel(2),
A Conforti(1),
(1) University of Verona, Department of Medicine and Public Health,
Section of Pharmacology, Verona, Italy
(2) Universite Paul Sabatier, CEIP-A, Unit of Pharmacoepidemiology,
Toulouse, France
(3) University of Valladolid, Institute of Pharmacoepidemiology,
Valladolid, Spain
(4) Karolinska Institute, Department of Physiology & Pharmacology,
Stockholm, Sweden
(5) University of Utrecht, Department of Pharmacoepidemiology,
Utrecht, TheNetherlands
(6) Ghent University Hospital, Department of Pharmacology, Ghent,
Belgium
Prescription drug abuse and diversion have been reported as a critical
concern in terms of patient care and public health, receiving conspicuously targeted consideration from health authorities. The aim of this article was to analyse the feasibility of carrying out the OSIAP system to
detect the potential abuse of marketed drugs in a multinational community pharmacy setting. An average of 2105 community pharmacies took
part in the OSIAP project during 2006 and 2007. They reported a total
of 862 suspect prescriptions concerning 1220 different drugs. The mean
age of the total sample of subjects presenting suspect prescriptions was
45 years (SD, 16.7) and the majority of them were women. The most
258
frequently reported criteria of suspicion was modication of the prescription and most suspect prescriptions regarded the ATC N class. Of
these drugs, 54% were psycholeptics (54% anxiolytics, 40% hypnotics
and sedatives and 6% antipsychotics), 23% analgesics (72% opioids,
23% other analgesics and antipyretics and 5% antimigraine preparations)
and 11% psychoanaleptics (66% antidepressants, 29% psychostimulants,
5% anti-dementia drugs and 1% psycholeptics and psychoanaleptics).
The OSIAP system provided useful information resulting from the
patients everyday life, thus conrming potential role of a pharmacy network in limiting drug diversion. Further projects should be developed
taking into consideration a variety of intervention strategies, from psychiatric intervention to practical law enforcement strategies. They should
entail the collaboration of multidisciplinary efforts involving the abusers
themselves in frontline educational activities.
Boeuf O & Lapeyre-Mestre M. Drug Safety 2007; 30: 265-276.
Paper No.: 1922

GENDER DIFFERENCES OF ADRS RELATED TO
PSYCHOTROPIC DRUG USE: A SURVEY FROM FRANCE,
ITALY AND SPAIN
Paola DIncau(1), M Lapeyre-Mestre(2), M Sainz(3), M Donati(1),
A Carvajal(3)
(1) University of Verona, Department of Medicine and Public Health,
Section of Pharmacology, Verona, Italy
(2) Universite Paul Sabatier, CEIP-A, Unit of Pharmacoepidemiology,
Toulouse, France
(3) University of Valladolid, Institute of Pharmacoepidemiology, Valladolid, Sain
It is well recognized that being female appears to be a risk factor for
developing ADRs. A number of studies clearly suggest that ADRs are
50 to 75% more likely in women than men. At the same time, nervous
system agents represents one of the most frequent drug classes reported
(20%) to elicit adverse events. A female propensity to experience or
report drug-related adverse effects may result from gender-related differences in drug exposure as well as in the number of drugs prescribed, in
the drug pharmacokinetics and pharmacodynamics. Nonetheless, the reasons for this increased risk in female patients are not entirely clear, notably whether adverse drug reactions among women reect an
inappropriate use of psychotropic medicines. The results from the European Study of the Epidemiology of Mental Disorders (ESEMED) and
from the Ordonnances Suspectes Indicateur dAbus Possible (OSIAP)
project suggest that among European countries, France, Italy and Spain
reordered one of the highest percentages of psychotropic drug use. On
the basis of these assumptions, the aim of this study is to compare in
males and females incidence, type, seriousness and drugs involved in
ADRs reported in a regional pharmacovigilance centre of each of these
countries respectively, Midi-Pyrenees, Veneto and Castilla-Leon, using
spontaneously reported cases between 2007 and 2009. This analysis will
also compare the incidence, type, seriousness and psychotropic drugs
involved in ADRs in males and females, as spontaneously reported to
these three regional pharmacovigilance centres.
Van der Heyden JHA et al. Pharmacoepidemiology and Drug Safety
2009;18: 11011110
Paper No.: 3353

P19 - GENERAL SESSION - TOXICOLOGY
ROLE OF SISTER CHROMATID EXCHANGE ANALYSIS IN
ASSESSMENT OF PROGUANIL GENOTOXICITY IN
CULTURED HUMAN LYMPHOCYTES
Domagoj Dinter(1), G Gajski(2), V Garaj-Vrhovac(2)
(1) Pliva Croatia Ltd., Oral Solid Forms, Zagreb, Croatia

(2) Institute for Medical Research and Occupational Health, Mutagenesis
Unit, Zagreb, Croatia
Proguanil hydrochloride is a prodrug of cycloguanil, a dihydrofolate
reductase inhibitor, which in malaria parasites Plasmodium falciparum
blocks DNA synthesis. In this study, evaluation of DNA damage in
human lymphocytes after proguanil treatment in vitro was made by virtue
of measuring frequency of sister chromatid exchanges (SCE) as a sensitive biomarker in assessment of DNA integrity since SCE are known to
increase as a result of the exposure to various genotoxic agents. Different
concentrations of proguanil obtained from the plasma concentrations;
130 ng/ml for prophylactic treatment and 520 ng/ml for the treatment of
malaria were used with and without metabolic activation (S9) in different
lenghts of time. SCE technique allows metaphases in rst, second and
third division to be recognized after culturing. For this reason, the SCE
analysis was also employed in estimation of proliferation kinetics after
the proguanil treatment. Results of the frequencies of SCE in human lymphocytes did not show statistically signicant deviation from the control
samples even though mean SCE per cell in all the exposed samples was
slightly higher than in corresponding controls and was more prominent
with metabolic activation (S9). In addition, the percentage of high frequency cells (HFC) for each sample was estimated using the pooled distribution of all SCE measurements. This parameter also did not show
signicant deviation compared to the control samples. Results revealed
that proguanil did not inhibit lymphocyte proliferation in signicant manner, therefore proguanil can be considered relatively safe from the aspect
of genotoxicity, especially if used for prophylactic treatment.
Paper No.: 2951

ALUMINIUM PHOSPHIDE POISONING - A RETROSPECTIVE
STUDY
Nandlal Disania, L Sharma, S Janani, BM Gupta
SMS Medical College, Department of Forensic Medicine, Jaipur, India
Aluminium Phosphide Pesticide poisoning is an important cause of morbidity and mortality in India, there has been an explosion of reports from
parts of India predominantly being northwest and central India. The
study was aimed to generate a baseline data on the epidemiological factors contributing to the incidence and mortality due to Aluminium Phosphide poisoning, so as to highlight the problem which requires planned
and concentrated efforts in dealing with it on a broader horizon. Since
prevention is the only logical approach there is an urgent need to take
appropriate steps to prevent loss of lives. The analysis of the data
revealed that the cases of Aluminium Phosphide poisoning brought to
the mortuary of S M S Medical College, Jaipur(Rajasthan) for medico
legal autopsy, during ve years period i.e. January 2005 to December
2009, with highest incidence in 2008. The age group ranged between 14
to 65 years, with maximum incidence between 22 to 29 years and males
outnumbering females. The main mode of poisoning was suicidal. The
use of poisoning Aluminium phosphide for suicidal purpose has markedly increased during the last decade contributing 6.2% of the total mortality .The mechanism of action of Aluminium Phosphide is not clearly
known. More basic research is required to nd exact mechanism of
action of phosphine so that a specic antidote can be developed.
Paper No.: 1891

ENGAGEMENT OF IMIDAZOLINE RECEPTORS IN EFFECTS
OF IMIDAZOLINE DRUGS ON ISOLATED RAT HEART ATRIA
Justyna Dlugokecka, A Radwanska, R Kaliszan, A Nasal
Medical University of Gdansk, Department of Biopharmaceutics and
Pharmacodynamics, Gdansk, Poland
259
The main sites of action of imidazoline drugs are located in the brain
and in peripheral vasculature. Recently, attention has been payed to the
role of imidazolines on physiology of the heart. However, no systematic
comparative studies were reported regarding the activity of imidazoline
drugs towards imidazoline receptors in the heart preparations. In this project the effect of I1- (clonidine, rilmenidine, moxonidine and
AGN192403) and I2-imidazoline receptor ligands (2-BFI, BU239) were
studied on isolated rat heart atria. The spontaneously beating right and
electrically driven left atria were treated with cumulative concentrations
of the agents studied. The inotropic and chronotropic responses of imidazolines were measured at the presence of xed concentrations of aadrenergic (phentolamine or yohimbine) or I1/I2-imidazoline (idazoxan)
receptor blockers. These effects were calculated as per cent of changes of
the control value of atria rate or amplitude preceding the administration
of each agent studied. Log EC50 parameters were also calculated. The
positive inotropic effect were evoked with the rank order of potency: clonidine > BU239 > rilmenidine > moxonidine. These effects were diminished by idazoxan. AGN192403 did not change the amplitude of left
atria. 2-BFI weakly diminished the rate ofbeating of atria; moxonidine
and rilmenidine had no effect. In conclusion, imidazoline receptors of I1
subtype may be involved in inotropic reaction of the agents studied, but
this effect depends mainly on the a2/a1 adrenergic receptors. Engagement
of I2 imidazoline receptors, along with the a2 adrenergic ones, in chronotropic activity of isolated right atria of rat has been demonstrated.
Paper No.: 2990

HEALTH AND DISEASE
THALIDOMIDE PRODUCES SMOOTH MUSCLE
RELAXATION
James R Docherty, SW Seto
Royal College of Surgeons in Ireland, Department of Physiology, Dublin,
Ireland
We have found that thalidomide produces hypotension in conscious and
anaesthetised rats, and have investigated the mode of action. Male Wistar
rats (250g) were killed by CO2 overdose, and the tail artery, aorta and
vas deferens was removed and rings were set up in small vessel myographs. Thalidomide (10-100 uM) produced signicant relaxations of
phenylephrine contracted tail artery and inhibited contractions to calcium
restoration in the presence of phenylephrine but not KCl. Hence, thalidomide did not act as an L-type calcium channel blocker. Thalidomide also
inhibited nerve-evoked contractions of epididymal, but not prostatic, portions of rat vas deferens, an action shared with the T-type calcium channel blocker NNC 55-0396. Thalidomide did not reduce the maximum
contraction to noradrenaline in aorta. Relaxant actions in vitro do not
seem to involve a1-adrenoceptors, caffeine sensitive calcium stores, glibenclamide sensitive potassium channels, PKC or P38 MAP kinase. Like
thalidomide, the T-type calcium channel blocker NNC 55-0396 (100
uM) inhibited contractions to calcium restoration in the presence of phenylephrine but not KCl. combination with NNC 55-0396, thalidomide
failed to produce any further inhibition of the contraction to calcium restoration in the presence of phenylephrine. It is concluded that thalidomide produces vascular and non-vascular relaxations by a mechanism
distinct from L-type calcium channel block, but its actions resemble
those of T-type calcium channel blockers.
Paper No.: 2300

CYP2D6 AND CYP2C19: A COMPARISON OF DIRECT VERSUS
PREDICTED PHENOTYPING IN A SOUTH AFRICAN
POPULATION
Tyren Dodgen(1), D Cromarty(1), H Fickl(2), M Pepper(3,4)
(1) University of Pretoria, Department of Pharmacology, Pretoria, South

Africa
(2) University of Pretoria, Unit for Inammation and Immunity and Academic Division NHLS, Department of Immunology, Pretoria, South
Africa
(3) University of Pretoria, Department of Immunology, Pretoria, South
Africa
(4) Geneva University Medical Centre, Department of Genetic Medicine
and Development, Geneva, Switzerland
Knowledge of cytochrome P450 (CYP) isoenzyme activity is used to
predict inter-individual variability in drug metabolism. Several genetic
screening tests are available, including the FDA-approved Roche AmpliChip CYP450 test, which predicts for poor, intermediate, extensive and
ultra-rapid metabolisers (PM, IM, EM and UM respectively). However,
several non-genetic factors inuence drug metabolism and contribute to
the nal phenotype, thereby confounding predicted phenotyping based
on genetic screening alone. Samples collected from subjects treated with
specic probe drugs (dextromethorphan for CYP2D6 and omeprazole for
CYP2C19) at 0, 2, 3 and 4 hours post administration were genotyped
using the AmpliChip CYP450 test. A semi-automated on-line solid phase
extraction liquid chromatograph tandem mass spectrometer (LC-MS/MS)
method was developed and validated in-house for simultaneous phenotyping using plasma concentrations of the drugs and metabolites to determine a metabolic ratio. For CYP2D6, 55% of the sampled population
were EM, 42% were IM, 1% were PM and 2% could not be classied
using the AmpliChip. For CYP2C19, 93% were EM and 7% PM. Phenotyping 3 hours post administration reveals that the sampled population
was 48.1% EM, 44.2% IM and 7.7% PM for CYP2D6 and 45.5% EM,
42.4% IM and 12.1% PM for CYP2C19. A signicant discordance was
thus observed between genotype and phenotype, and the reasons for this
discrepancy are being addressed. Direct rather than predicted phenotyping may prove to be a simple and efcient means of determining interindividual variability in drug metabolism.
Paper No.: 1081

THYROXINE MEDICATION AND CYP 3A4 ACTIVITY AS
MEASURED BY THE URINARY 6B-HYDROXYCORTISOL/
CORTISOL RATIO: A PILOT STUDY
Josef Donnerer(1), J-P Siest(2), E Beubler(1)
(1) Medical University of Graz, Institute of Experimental & Clinical
Pharmacology, Graz, Austria
(2) Stabiligen, Nancy, France
Introduction: The aim of the study was to investigate a possible CYP
3A4- inhibiting activity of long-term oral thyroxine hormone treatment.
An inhibition of the CYP 3A4 enzyme has so far only been demonstrated in vitro for triiodothyronine in cultured hepatocytes. Methods: We
performed a pilot study by using urinary 6b-hydroxycortisol and urinary
6b-hydroxycortisol/cortisol ratio as a non-invasive marker of microsomal
CYP 3A4 activity. Ten individuals, male and female, age 20 64 years
and taking a daily dose of thyroxine between 75 and 160 lg for at least
1 year were compared with 10 age-matched control persons. Morning
spot urine samples collected from 8:00 a.m. to 12:00 p.m. were studied
using ELISA kits from Stabiligen. Results: The mean SEM of the 6bhydroxycortisol/cortisol ratio was 11.19 1.38 in the control group and
16.43 3.52 in the thyroxine group, with no signicant difference
between the 2 groups. The individual values differed by a factor of 4 in
the control group and by a factor of 9 in the thyroxine group. The ratio
of 6-hydroxycortisol [nmol/l] versus creatinine [mmol/l] was
163.5 17.1 in the control group and 179.1 21.9 in the thyroxine
group. Conclusion: The data from our pilot study conrm the marked
inter-individual variation in the morning spot urine 6b-hydroxycortisol/
cortisol ratio. They further show that it is most unlikely of thyroxine
medication being a microsomal CYP 3A4 inhibitor in vivo.
Acknowledgement: This study was supported by the Franz-Lanyar-Stiftung (project 336).
260
Paper No.: 2657
HEALTH AND DISEASE
PROTEIN DISULFIDE ISOMERASE REGULATES REACTIVE
OXYGEN SPECIES GENERATION AND ANGIOTENSIN II
INDUCED CONTRACTION IN AORTA FROM WISTAR RATS
Ana Alice Dos Santos Dias(1), LL Camargo(1), GS Ceravold(1),
ACD Androwiki(1), MHC Carvsalho(1), FRM Laurindo(2),
M Janiszewski(1), LR Lopes(1)
(1) University of Sao Paulo, Department of Pharmacology, Sao Paulo,
Brazil
(2) University of Sao Paulo, Heart Institute (InCor), Sao Paulo, Brazil
Angiotensin II (Ang II) reactive oxygen species (ROS) generation is
involved in the modulation of vascular resistance. We have shown previously that the generation of ROS by Ang II can be regulated by a thiol
oxidoreductase, protein disulde isomerase (PDI). Thus, in the present
study we investigated the role of PDI in ROS generation and Ang IIinduced aorta contraction in Wistar rats. PDI was found in all layers of
the aorta. ROS generation (dihydroethidium uorescence) increased after
stimulation with Ang II (30 min), an effect diminished by the PDI inhibitor, bacitracin (BAC; 1mM, 30 min). The role of PDI and ROS in vasoconstriction was evaluated in concentration-effect curves to Ang II (1nM
to 1 lM) in aortic rings with (E+) and without endothelium (E-) in the
presence and absence of BAC (1mM, 15 min) and antioxidant enzymes
superoxide dismutase (SOD, 150U/ml, 30 min) and catalase (CAT;
300l/ml, 30 min). Ang II increased contraction in both E+ and E- rings.
This effect was reduced in the presence of BAC, (P < 0.01; n = 9),
SOD (p < 0.05; n = 7) and CAT (p < 0.05; n = 7). These results suggest
that PDI modulates ROS production and the contractile response to Ang
II in aortic rings. Therefore, PDI may be a new player in the control of
vascular tone.
Paper No.: 2463

RESULTS ABOUT THE INFLUENCE OF METFORMIN ON
THE CONCENTRATIONS OF MAGNESIUM,ZINC AND
COOPER IN NIDDM PATIENTS
Monica Daniela Dosa(1), LC Petcu(2), C Gales(3), M Nechifor(4)
(1) Constanta University Faculty of Medicine, Department of Pharmacology, Constanta, Romania
(2) Constanta University Faculty of Dentistry, Department of Biophysics
and Medical Informatics, Constanta, Romania
(3) UMF Gr.T.Popa, Department of Histology, Iasi, Romania
(4) UMF Gr.T.Popa, Department of Pharmacology, Iasi, Romania
Introduction: The aim of this study was to investigate the inuence of
the treatment with metformin in NIDDM patients, who didnt received
medication, on the concentrations of magnesium, cooper and zinc. Materials: We studied 30 newly diagnosed adults with NIDDM, in diabetes
clinic of the Faculty of Medicine,Constanta, who received metformin,
1000 mg/day, and 17 control healthy subjects. Methods.We determined
the concentrations for: glicemia, cholesterol, tryglicerides, HDL, LDL,
magnesium, copper and zinc, HbA1c, urine magnesium, copper and zinc
and intraerythrocyte magnesium, before and after 3 months of therapy.
The results were statistically interpreted. Results: Our data showed in
diabetic patients compared with healthy group: lower plasma levels in
magnesium (1.95 0.19, vs 2.2 0.18 mg/dl; p < 0.05) and in zinc
(67.56 6.12 vs 98.41 20.47 lg/dl; p < 0.05), increased levels of cooper (111.91 20.98 vs 96.33 8,56 lg/dl; p < 0..05) increased urinary
magnesium (237.28 34.51 vs 126.25 38.82 mg/24h; p < 0.05)
increased urinary zinc (1347.54 158.24 vs 851.65 209.75 lg/24;
p < 0.05), lower intraerhythrocyte magnesium (5.09 0.63. vs 6.38
0.75 mg/dl; p < 0.05). The treatment with metformin reduced signicant
urinary concentration in magnesium (t = 5,35, df= 23,93; P < 0.001),

increased signicant the intraerythrocyte magnesium (t= - 3,09, df= 45,
p = 0.002), and did not modify signicant the urinary zinc and cooper
concentrations.There was a positive correlation between cooper and glycated haemoglobin (r = 0.517;p < 0.003) and a negative correlation
between zinc and glicemia (r= -0.399;p < 0.029). Conclusions: In diabetic patients plasma magnesium and zinc was decreased, after metformin treatment the ratio of plasma between cooper and zinc, increased in
a positive correlation with the increase of glycated haemoglobin, and the
rate of intraerythrocyte magnesium increased in a positive correlation
with the decrease of glycated haemoglobin.
Paper No.: 2311

DISEASES
NCX4040, A NITRIC OXIDE-DONATING ASPIRIN, EXERTS
ANTI-INFLAMMATORY EFFECTS THROUGH INHIBITION OF
IKB-A DEGRADATION IN HUMAN MONOCYTES
Melania Dovizio(1), E Ricciotti(2), L Di Francesco(1), P Anzellotti(1),
T Salvatore(1), A Di Francesco(1), MG Sciulli(1), G Pistritto(3),
A Monopoli(4), P Patrignani(1)
(1) G. dAnnunzio University and G. dAnnunzio University Foundation, Ce.S.I, Chieti, Italy
(2) University of Pennsylvania, Philadelphia, PA, USA
(3) University of Rome Tor Vergata Medical School, Rome, Italy
(4) NicOx Research Institute, Bresso, Milan, Italy
NO-donating aspirins consist of aspirin to which a NO-donating group is
covalently linked via a spacer molecule. NCX4040 and NCX4016 are
positional isomers with respect to the-CH2ONO2 group (para and meta,
respectively) on the benzene ring of the spacer. Because positional isomerism is critical for antitumor properties of NO-donating aspirins (Kash
et al., JPET, 2005, 312:978-988), we aimed to compare their anti-inammatory effects versus aspirin in vitro. Thus, we assessed their impacts on
cyclooxygenase(COX)-2 activity(by measuring PGE2 levels), protein
expression, and cytokine generation(IL-1b, IL-18, TNF-a, and IL-10) in
human whole blood and isolated human monocytes stimulated with LPS.
Interestingly, we found that micromolar concentrations of NCX4040, but
not NCX4016 or aspirin, affected COX-2 expression and cytokine generation. We compared the effects of NCX4040 versus NCX4016 or aspirin
on IkB-a stabilization and proteasome activity in the LPS-stimulated
human monocytic cell line(THP1). At micromolar range, NCX4040
inhibited IkB-a degradation, as opposed to aspirin and NCX4016. In
fact, NCX4040 caused concentration-dependent accumulation of IkBa
and phospho-IkB-a. This effect was not reversed by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, an inhibitor of guanylyl cyclase, thus excluding the contribution of NO-dependent cGMP generation. In contrast,
IkB-a accumulation by NCX4040 may involve an inhibitory effect on
proteasome functions. Indeed, NCX4040 inhibited 20S proteasome activity when incubated with intact cells but not in the presence of cell lysate
supernatants, thus suggesting an indirect inhibitory effect. In conclusion,
NCX4040 is an inhibitor of IkB-a degradation and proteasome function
and it should be taken into consideration for the development of novel
anti-inammatory and chemopreventive agents.
Paper No.: 1904

STATINS CONSUMPTION IN THE REPUBLIC OF CROATIA IN
2005, 2006, 2007 AND 2008
Pero Draganic, S Zezelic, V Macolic-Sairinic, S Tomic
Agency for Medicinal Products and Medical Devices (ALMP), Zagreb,
Croatia
261
Objectives: Drug usage evaluation is a system of continuous, systematic,
criteria-based drug evaluation that ensures the appropriate use of drugs.
Statins are drugs used to prevent cardiovascular diseases. The importance
of collecting data on statins consumption is in correlation with the
improvement on cardiovascular diseases therapy. Methods: ALMP Croatia collected and processed data on consumption of the medicines, and
the usage between 2005-2008 was processed by the DDD/1000inh/day
and ATC classication, and analyzed according to the nancial indicators. Results: The percentage of statins (C10AA group) within total
nancial resources indicates: in 2005 5.20%, in 2006 6.58%, in 2007
8.17%, and in 2008 8.22%. By processing consumption data as DDD,
between 2005-2008 within C10AA group shows 35.62, 45.77, 67.89 and
68.37 DDD/1000inh/day, respectively. The individual expenditure of statins indicates: simvastatin 20.23, 20.87, 23.5, and 23.77 DDD/1000inh/
day in 2005-2008 periods; atorvastatin 13.03, 20.73, 39.86 and 36.89
DDD/1000inh/day in 2005-2008 periods. Conclusion: Between 20052008 all statins obtained in the Croatian market showed a continuous
increase in DDD/1000inh/day as well as in nancial costs. For the entire
period atorvastatin and simvastatin were the most prescribed drugs. The
overall consumption of statins in Croatia (35-67x DDD/1000inh/day) is
lower than in EU and other Western countries, where a consumption of
statins is about 80-200 DDD/1000inh/day.
Paper No.: 2769

INTERACTION BETWEEN CICLETANINE AND
PRECONDITIONING IN CONCIOUS RABBITS WITH INSULIN
RESISTANCE
Laszlo Drimba, B Peitl, T Patonay, Z Szilvassy
University of Debrecen, Department of Pharmacology and
Pharmacotherapy and Pharmapolis Debrecen Ltd, Debrecen, Hungary
Cicletanine, a cyclic GMP phosphodiesterase inhibitor has been shown
to potentiate the anti-ischaemic effect of preconditioning. It has also been
shown that hypercholesterolemia blocks preconditioning in conscious
rabbits. The aim was therefore to asses if cicletanine restores the rapid
pacing (RP)-induced preconditioning phenomenon in insulin resistant
rabbits. Right ventricular electrode and left ventricular polyethylene catheter-instrumented conscious rabbits were subjected to two consecutive 5min periods of RP with an interpacing period of 5 minutes. The 1st RP
served as an ischemic challenge and to induce preconditioning, the 2nd
one was to test the preconditioning effect. The resulting intracavital STsegment elevation and increase in left ventricular end-diastolic pressure
(LVEDP) were measured after pacing periods. Both variables were signicantly reduced after the 2nd RP compared to those measured after the
1st one. Cicletanine (20 mg/kg) applied intravenously 20 min prior to
the 1st preconditioning attenuated both postpacing ST-segment elevation
and LVEDP-increase and the protection was further amplied by preconditioning. The same animals were then made insulin resistant by partial
hepatic sensory denervation by perineurial treatment with 2% capsaicin
solution around the anterior hepatic plexus. This decreased whole body
insulin sensitivity 2 weeks later. The development of insulin resistance
resulted in the loss of the RP-induced preconditioning effect on both
parameters, nevertheless, cicletanine was found to signicantly reduce
both ST elevation and LVEDP-increase produced by the 1st preconditioning. This protection also was amplied by preconditioning. We conclude that CIC produces an anti-ischemic effect and preserves PC in
conscious rabbits with neurogenic insulin resistance.
Paper No.: 2885

MANAGEMENT OF ACUTE MYOCARDIAL INFARCTION IN
FRANCE: RESULTS FROM INCLUSION OF A NATIONAL
COHORT
C Droz-Perroteau(1), C Dureau-Pournin(1), D Thomas(2),
N Danchin(3), J Tricoire(4), J Benichou(5), F Paillard(6),
P Ducimetie`re(7), S Hercberg(8), H Mazi(1), E Guiard(1),
M-A Bernard(1), P Blin(1), Nicholas Moore(1)
(1) Universite de Bordeaux, INSERM CIC 0005, Bordeaux, France
(2) Hopital Pitie-Salpetrie`re, Paris, France
(3) Hopital Europeen Georges Pompidou, Paris, France
(4) Cardiologist, Toulouse, France
(5) CHU de Rouen-INSEM U657, Rouen, France
(6) CHU de Pontchaillou, Rennes, France
(7) INSERM, Villejuif, France
(8) INSERM U557-INRA-CNAM-CRNH, Bobigny, France
This study was performed at the request of the Health Authorities to
assess the impact of recommended cardiovascular treatments in secondary prevention after acute myocardial infarction (AMI) in real-life practice. A national cohort study was designed to include 5000 recent AMI
(<3 months) recruited by hospital and non-hospital cardiologists. At
inclusion, a standardized questionnaire was used to collect socio-demographic data, AMI characteristics, cardiovascular risk factors, history and
associated cardiovascular diseases, the last biological balance sheet, cardiovascular drugs used, prescription of cardiovascular rehabilitation program. Between May 2006 to June 2009, 5540 patients were included
(3306 by 346 hospital cardiologists and 2234 by 389 non-hospitals);
77.6% were male, mean age was 62.1 years. It was the rst AMI for
86.7%, 70.5% had the 3 AMI criteria (symptomatic, electrical, enzymatic), 8.2% a LVEF <40%. Concerning the cardiovascular risk factors,
9.6% were current smokers, 16.7% had diabetics, 44.6% had history of
hypercholesterolemia, 43.6% history of arterial high blood pressure. The
4 recommended cardiovascular drugs in secondary prevention were prescribed to 70.5% (73.7% of hospital cardiologists and 65.8% of non-hospitals): aspirin or other anti-platelet agents to 99.3% (99.4% of hospital
cardiologists and 99.2% of non-hospitals), statins or others hypolipidemic drugs to 96.1% (96.6%, 95.3% respectively), b-blockers to 88.9%
(90.5%, 86.5% respectively) and IEC or ARA II to 80.7% (82.9%,
77.5% respectively). A cardiovascular rehabilitation program at inclusion
was prescribed for 40.5% (46.6%, 31.5% respectively). This study shows
that the 4 recommended cardiovascular drugs in secondary AMI prevention are now widely used in France.
Paper No.: 2080

EFFECT OF SIRNA-MEDIATED KNOCKDOWN OF ALDH2 ON
GTN-INDUCED CGMP FORMATION IN PK1 CELLS
Yohan DSouza, BM Bennett
Queens University, Department of Pharmacology & Toxicology, Kingston, Ontario, Canada
Recent studies suggest a primary role for aldehyde dehydrogenase 2
(ALDH2) in mediating the biotransformation of organic nitrates, such as
glyceryl trinitrate (GTN), to the proximal activator of soluble guanylyl
cyclase (sGC), resulting in increased cGMP accumulation and vasodilation. In studies using stably transfected porcine renal epithelial cells
(LLC-PK1 cells) overexpressing ALDH2, we found that overexpression
of ALDH2 did not signicantly alter GTN-induced cGMP accumulation
compared to wild type cells. However, it was possible that GTN bioactivation mediated by endogenous ALDH2 was maximal in wild type cells,
and that overexpression of ALDH2 had no further impact on GTN bioac-
262
tivation. Accordingly, we used siRNA-mediated knockdown of ALDH2
to examine the effect of decreased ALDH2 expression on GTN-induced
cGMP formation. PK1 cells treated for 72 hours with 50 nM of two siRNAs directed against ALDH2 resulted in a greater that 95% reduction in
ALDH2 protein. Exposure of cells to 1 uM GTN for 3 minutes resulted
in a 5-6 fold increase in cGMP accumulation, with no differences in the
GTN-induced cGMP response in wild type cells compared to cells treated with the two siRNAs or an siRNA negative control. Preincubation of
cells with 10 lM GTN for 2 hours resulted in a similar inhibition of the
cGMP response on subsequent exposure to GTN (GTN tolerance) in all
treatment groups. Together, these results indicate that ALDH2 does not
mediate the mechanism-based biotransformation of GTN to activators of
sGC, and that GTN tolerance can occur in the absence of ALDH2.
Paper No.: 1675

ANALGESICS
COMPARISON OF THE PD/PK OF ROTUNDINE CRYSTAL
FORM A AND C IN MICE
Lida Du(1), X Li(1), J Ying(2), X Bai(2), S Yang(2), H Zhang(2),
G Wang(1), Y Lu(2)
(1) China Pharmaceutical University, School of Pharmacy, Nanjing, PR
China
(2) Institute of Materia Medica, Chinese Academy of Medical Science,
Nanjing, PR China
Rotundine is isolated from Chinese traditional herb, and have been used
as analgesics clinically for decades. But the absorption of rotundine in
gastrointestinal tract is rare and unstable. We researched the crystal forms
of rotundine and investigated their relationship of PD/PK in mice. The
crystal forms of rotundine were prepared by the physical methods and
tested by X-ray diffraction, DSC and infraredspectrum. The analgesic
effects of rotundine were tested by the methods of hot plate and writhing
induced by acetic acid in mice. The concentrations of rotundine in blood
were measured by the method of HPLC. The results show that the crystal
form C is a new one and has high absorption ratio in mice. The number
of writhing induced by acetic acid in mice from 36.67 + /-10.19
decreased to 27.00 + /- 13.39 and 21.17 + /- 13.73 for form A and C
respectively when administrated 200 mg/kg orally. The Cmax for the
form A, C are 4.8872 (mg/L) and 5.5410 (mg/L), the Tmax(min) for two
different forms are 45.0 min(form A) and 75.0 min (form C). Compared
the results of PD and PK of rotundine in mice, it can be concluded that
the new crystal form C of rotundine is a optimal form for the drug preparation with a good absorption and exert better analgesic effects than other
forms.
Acknowledgment: this work was supported by the National Major project No:2009ZX09302-003 and basic science and techniques research
project: No: 2008IM022200
Paper No.: 1730

EFFECTS OF ULTRA-LOW DOSES OF PERORAL ANTIBODIES
TO ANGIOTENSIN II RECEPTOR IN NON-INVASIVE AND
INVASIVE RAT MODELS OF HEART FAILURE
Julia Dugina, IA Kheyfets, SA Sergeeva, OI Epstein
Materia Medica Holding Company, Department of Research & Development, Moscow,Russian Federation
failure:isoproterenol-induced (ISO) HF and HF induced by permanent

coronary occlusion(CO). In ISO HF male and female Wistar rats
(220-250 g) were administered per osdaily with cardosten (2.5 ml/kg),
losartan (20 mg/kg) for 28 days starting onday 7 after twice repeated sc
injection of isoproterenol (80 mg/kg). In CO HFmodel male Wistar rats
(240-340 g) were treated per os daily with vehicle,cardosten (7.5 ml/kg),
losartan (20 mg/kg) for 30 days starting one day beforecoronary occlusion. In ISO HF cardosten was considerably better than placebo andalmost as effective as losartan in improving exercise performance
(a tendencyin males, and signicant changes by 27.8% and 34.5% vs
baseline respectively infemales), cardiac output, LV function. In CO HF
model cardiac output measuredboth in vivo and in vitro is tended to
increase by cardosten (32.9% and 47.4 vsvehicle respectively), while losartan had no any noticeable effect. Totalperipheral resistance was
decreased by both of losartan and cardosten (p < 0.05).Increase in stroke
volume in cardosten (42% vs vehicle) and aortic ow (60.3% 66.3% vs
vehicle) was registered. Heart rate was not changed in the all groupsas
compared to control. In summary, cardosten had comparable benecial
effectsto that of losartan in two models of CHF.
Paper No.: 758

EFFECT OF ATORVASTATIN ADDITION TO CARDIOPLEGIC
SOLUTION ON MYOCARDIAL PROTECTION IN ISOLATED
RAT HEART
Deniz Kaleli Durman(1), M Agirgol(1), M Kucur(2), F Basinoglu(3),
O Ozdemir(1), BS Uydes-Dogan(1)
(1) Istanbul University, Faculty of Pharmacy, Department of
Pharmacology, Istanbul, Turkey
(2) Istanbul University, Cerrahpasa Medical Faculty, Fikret Biyal Central
Research Laboratory, Istanbul, Turkey
(3) Haseki Training and Research Hospital, Biochemistry Laboratory,
Istanbul, Turkey
Cardioplegia is a selective cardiac arrest technique, which aims to prevent myocardial damage and provide adequate heart hemodynamics in
cardiac surgery. For minimising myocardial damage, various agents
accepted to be cardioprotective are added to cardioplegic solution. Statins
are reported to have protective effects in myocardial ischemia-reperfusion
(I/R) injury independently from their lipid-lowering effects. Herein, we
aimed to investigate the effect of atorvastatin addition to cardioplegic
solution on myocardial protection. Isolated rat hearts perfused in Langendorff system at constant pressure (80mmHg) were subjected to 40min.
ischemia/60min. reperfusion. Cardioplegia was administered 3min.
before ischemia by St.Thomas2 cardioplegic solution (+4C) at a constant ow (10ml/min). Atorvastatin was added to cardioplegic solution at
increasing concentrations (5x10-6M, 10-5M, 5x10-5M). Cardiac function
(left ventricular developed pressure, heart rate, double-product), coronary
hemodynamics (coronary ow, coronary vascular resistance) and myocardial necrosis (creatine kinase, troponin-T, necrotic area) parameters
were evaluated and compared with cardioplegia. For investigating the
mechanism of action, atorvastatin (10-5M) was studied in the presence
of PI3 kinase inhibitor, wortmannin (10-7M). When compared to cardioplegia, addition of atorvastatin to cardioplegic solution signicantly
enhanced postischemic cardiac recovery, whereas no change was
observed in coronary hemodynamics. Furthermore, cardiac enzyme levels and infarct area were also signicantly reduced. Wortmannin inhibited
benecial effects of atorvastatin on cardioplegia. These ndings demonstrated that, atorvastatin strengthens protective effect of cardioplegia via
PI3 kinase related mechanism which may provide an additive clinical
benet in minimising myocardial damage during cardiac surgery.
The present work was supported by the Research Fund of Istanbul University. Project No. T-1892.
Ultra-low doses of antibodies to C-terminal of AT1 receptor to angiotensin II(cardosten) is a novel antibody-based therapeutic for the treatment
of chronicheart failure (CHF). The drug was tested in rat models of heart
263
Paper No.: 2776
CROSSTALK BETWEEN GABAB AND GROUP III MGLU
RECEPTORS FOLLOWING INTRANIGRAL DRUG
ADMINISTRATION IN THE RESERPINE-TREATED RAT
Susan Duty, M Broadstock
Kings College London, Wolfson Centre for Age-Related Diseases,
London, UK
Cross talk between Class C GPCRs has received much attention at the
molecular level, though little is known about this phenomenon in vivo.
Here, we examined whether crosstalk between group III mGlu and
GABAB receptors was seen in vivo using reversal of reserpine-induced
akinesia as our measure. Male rats, cannulated above the substantia nigra
pars reticulata (SNpr) were rendered akinetic with reserpine (5 mg kg-1
s.c.). 18h later, rats received a unilateral injection of either L-SOP (group
III agonist; 750 nmol in 2.5 ll PBS), baclofen (GABAB agonist; 800ng
in 0.5 ll PBS) or vehicle into the SNpr. 4h later rats received a second
injection of either L-SOP or baclofen (doses as above). Contraversive
360o rotations were quantied for up to 60 min following each injection.
Data are expressed as mean s.e.m (n = 6). The rotational responses to
L-SOP and baclofen were reduced following second injection of the
same agonist (P < 0.05; paired t-test between injections). The response
to L-SOP was also signicantly reduced following initial injection with
baclofen (7 3 rotations 30 min-1 versus 30 6 after initial vehicle
injection; P < 0.05; unpaired t-test). In contrast, the response to baclofen
was signicantly enhanced following initial injection with L-SOP
(329 29 rotations 60 min-1 versus 106 4 rotations following initial
vehicle injection; P < 0.05; unpaired t-test). These data conrm that crosstalk occurs in vivo between group III mGlu receptors and GABAB
receptors but that the consequences of this can produce opposite effects,
depending on the order of receptor activation.
Paper No.: 2492

RUSSIAN BIOSIMILAR EPOETINS AND THEIR POSSIBLE
USE IN SPORT
Yuliya I Dykhal, GI Krotov, PV Postnikov, GM Rodchenkov
FGUP Antidoping Centre, Department of Peptide Doping Analysis, Moscow, Russian Federation
Biopharmaceuticals have revolutionized the treatment of many diseases.
The advent of recombinant erythropoietins has greatly beneted patients
with anemia related to chronic kidney disease and cancer. In parallel they
became potential agents for use in sports as doping due to their similarity
to endogenous EPOs. Recent expire of the patent restriction leads to
development of new versions of these products that may stimulate
demand and reduce prices. For now several biosimilar epoetins are available on Russian biopharmaceuticals market: Epocrine, Erythrostime,
Vero-epoetine, Eralphone and some illegal forms. These epoetins were
studed for possible differences in erythropoietin content, potency, possible
immunogenesity and isoform distribution. For those purposes different
methods were used. For isoform distribution isoelectric focusing with
dobble-blotting were performed, and differences in molecular mass were
shown in SDS-electrophoresis study. Total protein was determined by
Bradford assay meantime concentration of EPO was determined by ELISAs and LIA. Some urine samples which contain mentioned epoetines
were analysed and they shown that results sometimes are very complicated and almost impossible to interpret as positive case following identication criteria set in doping control analysis even if its very likely that
athlet abused epoetine. The study shows that epoetin products from different manufacturers differ in composition. Some of the products failed to
meet their own specications, indicating that some of the manufacturers
do not have adequate quality control over their production process. This
could result in undesired clinical effects and even lead to lethal outcome.
Paper No.: 1747

TAURINE AND ITS ANALOGUE TAUROPYRONE:
COMPARATIVE PHARMACOLOGICAL ACTIVITIES
Zane Dzirkale(1), J Pupure(1), J Rumaks(1), M Vanina(1), S Svirskis(1),
R Mezhapuke(2), MA Fernandes(3), G Duburs(2), R Muceniece(1),
V Klusa(1)
(1) University of Latvia, Department of Pharmacology, Riga, Latvia
(2) Latvian Institute of Organic Synthesis, Riga, Latvia
(3) University of Coimbra, Faculty of Sciences, Department of Zoology,
IMAR-CMA, Coimbra, Portugal
Taurine, a sulphur-containing amino acid, is characterized by its hydrophilicity and poor absorption. Tauropyrone, a taurine-containing 1,4-dihydropyridine derivative, is suggested to have a higher activity than that
of taurine due to the lipophylic cyclic 1,4-dihydropyridine (DHP) moiety
that might serve also as a carrier element capable of facilitating the compounds delivery to cell targets. In the present study, we compared the
effects of taurine and tauropyrone in in vitro experiments: binding to
GABA-A receptor, and inuence on mitochondrial processes; and in in
vivo tests: inuence on behavioral effects caused by GABA-A receptor
ligands bicuculline, diazepam and ethanol. Our results demonstrated: 1)
unlike taurine, tauropyrone lacked GABA-A receptor binding afnity,
and did not inuence bicuculline convulsant activity; 2) neither taurine
nor tauropyrone inuenced mitochondrial processes, and did not alter
diazepam myorelaxing action; 3) tauropyrone showed more pronounced
anti-ethanol effect (shortening of ethanol-sleeping time). One may suggest that tauropyrone molecule due to its DHP moiety has a hindered
access to GABA-A receptor GABA site, but the enhanced modulatory
inuence on ethanol-mediated cellular events.
Acknowledgements: ESF Nr.2009/0217/1DP/1.1.1.2.0/09/APIA/VIAA/
031, Latvian Council of Science Grant Nr.05-1418; ESF 2004/3; COST
D34.
Paper No.: 2867

MODELLING OUTCOMES OF ANTIBIOTIC THERAPY BY
DISCRETE EVENT SIMULATION (DES) OR
MULTI-AGENT-BASED SIMULATION (MABS) OF
NOSOCOMIAL PNEUMONIA
Mario Eandi(1), P De Luca(1), L Pradelli(2), S Iannazzo(2)
(1) University of Turin, Department of Anatomy, Pharmacology and
Forensic Sciences, Torino, Italy
(2) Adres srl - Turin, Torino, Italy
Discrete-event-simulation (DES) and multi-agent-based-simulation
(MABS) are two approaches to Modelling complex processes. DES
micro-simulates therapeutic pathways of individuals and is increasingly
used in cost-effectiveness studies. MABS is a new scientic instrument
well-suited to study how interactions of autonomous agents (e.g. microorganisms-host-antibiotic) driven by a simple local rule can generate the
emergence of global outcomes from the bottom up. We developed a
DES and a MABS model to analyze outcomes of nosocomial pneumonia
(NP) treated with carbapenems. Both models predict individual outcomes
by simulating in vivo microbiological efcacy according to PK-PD relationships. To evaluate the clinical results, the DES model integrates the
surrogate parameter time-supra-MIC (%T>MIC) with the baseline risk
of death. The MABS model evaluates outcomes by replicating the
dynamics of pathogen (replication and death rates) as inuenced by host
264
and antibiotic, and of patients (healing or death rate, resistance) as inuenced by pathogen and antibiotic. Outcomes predicted by both models
compare well to those registered in comparative trials in response assessment at end of treatment (EOT), test-of-cure (TOC), and late-follow-up
(LFU). Despite non-inferiority results in cure and death rates, results
obtained from DES model showed a superior effect of doripenem over
comparators in a number of areas including seizures, emerging resistance, length of stay (LOS) in general ward and in ICU on mechanical
ventilator. The analysis performed by MABS model lead to similar conclusions. Yet, MABS Modelling is even more exible and is suitable to
nd the optimal solution to critical drug management by computer-experimenting virtual reality.
Paper No. 3393

FUNCTIONAL EXPRESSION OF CB1 RECEPTORS IN RAT
BRAIN NUCLEAR FRACTIONS
Leire Echeazarrra(1), S Barrondo(1), GG del Cano(2), M Montana(1),
ML de Jesus(1), J Salles(1)
(1) Departamento de Farmacologa. Facultad de Farmacia (UPV/EHU);
Centro de Investigacion Biomedica en Red de Salud Mental. CIBERSAM, Vitoria-Gasteiz, Spain
(2) Departamento de Neurociencias, Facultad de Farmacia (UPV/EHU).
Vitoria-Gasteiz, Spain
The aim of the present work was to study the presence of CB1 receptors
in rat brain nuclear fractions and its functional coupling to Gi/o proteins.
To this aim, inmunohistochemistry, double inmunouorescence, immunoblotting, and [3H]SR141716A and GTPc[35S] binding assays were
performed. Inmunohistochemistry revealed the classical presynaptic prole of CB1 distribution together with a nuclear inmunolabelling. Double
inmunouorescence of CB1 receptors and lamin B1 (a marker of the
nuclear envelope) conrmed the nuclear localization of CB1. Employing
enriched nuclear preparations, the nuclear location of CB1 receptors and
their downstream signalling partners Gi/o was also demonstrated by western blot analysis. The selective CB1 antagonist radioligand,
[3H]SR141716A, recognized a specic population of binding sites in
nuclear membranes, with a density (Bmax of 1,04 0,24 pmol/mg) and
afnity (KD of 1,6 0,5 nM) similar to those obtained in cytoplasmatic
membranes. In order to evaluate the functional coupling of CB1 receptors GTPc[35S] binding assays were performed in nuclear membranes.
The cannabinoid agonist WIN55,212-2 was able to stimulate the
GTPc35S] binding with and efcacy (Emax of 89,5 7.3% over basal)
and a potency (EC50 of 0,63 0,06 lM) in the same order as those
obtained in cytpolasmatic membranes.Our results demonstrate the functional expression of CB1 receptors in nuclear fractions. Due to the role
of these receptors in the regulation of cell proliferation, survival and
apoptosis, our results open and interesting subject for future studies about
the functional responses specically mediated by nuclear CB1 receptors.
Funding: SAIOTEK S-PEO8UN29 and SE-PE07UN21, UPV/EHU
NUPV08/05 and CIBERSAM.
Paper No.: 642

DISCOVERY
EXTRACELLULAR LOOPS 2 AND 4 OF GLYT2 ARE
REQUIRED FOR N-ARACHIDONYL-GLYCINE INHIBITION
OF GLYCINE TRANSPORT
Concentrations of glycine are regulated via the Na+/Cl--dependent glycine transporters, GLYT1 and GLYT2. N-Arachidonyl glycine (NAGly)
is an endogenous inhibitor of GLYT2 with no effect on GLYT1. Understanding the molecular basis of NAGly interactions with GLYT2 may
allow for the development of novel analgesics. We investigated whether
extracellular loops 2 and 4 (EL2/4) are important for NAGly sensitivity
between GLYT1 and GLYT2 and also a series of related N-arachidonylamino acids. Chimeras were constructed between GLYT1 and GLYT2
with their EL2 and/or EL4 regions switched. Point mutations of all
GLYT2 EL4 residues which differed from GLYT1 were mutated to the
corresponding residue in GLYT1. Transporters were tested for their
sensitivity to GLYT2 inhibitors: NAGly, N-arachidonyl-L-alanine (NALAla), N-arachidonyl-D-alanine (NADAla) and N-arachidonyl-c-aminobutyric acid (NAGABA) using electrophysiology (n 5/transporter).
GLYT2 is inhibited (and not GLYT1) by NAGly, NADAla and NAGABA whereas GLYT2(GLYT1EL2) and GLYT2(GLYT1EL4) had
reduced sensitivities. Interestingly, GLYT2 and GLYT2(GLYT1EL2) are
inhibited by NALAla whereas GLYT2(GLYT1EL4) is not. GLYT2R531L
and GLYT2K532G had reduced sensitivity to NAGly (IC30; 13 2lM;
9 1lM, respectively) compared to GLYT2 (IC30: 3.4 0.6lM) while
GLYT2I545L had markedly reduced sensitivity to NAGly (IC30:
>30lM). GLYT2R531L, GLYT2K532G and GLYT2I545L also had
reduced sensitivities to NALAla (IC30: 14 1lM; 12 1lM; and
21 1lM, respectively) compared to GLYT2 (IC30: 5.9 0.7lM). In
conclusion, EL2 and EL4 of GLYT2 are important in the inhibition of
GLYT2 by NAGly, NADAla and NAGABA while only EL4 of GLYT2
is required for NALAla inhibition of transport. Key residues in GLYT2
EL4 required for NAGly and NALAla sensitivity are R531, K532 and
I545.
Paper No.: 1806

HEALTH AND DISEASE
EVIDENCE FOR FUNCTIONAL TWO-PORE DOMAIN
POTASSIUM CHANNELS (K2P) IN RAT MESENTERIC
ARTERIES
Gillian Edwards, E Porter, N Ashton, A Gurney, A Weston, I Johnson
University of Manchester, Faculty of Life Sciences, Manchester, UK
The K2P channels TREK and TRAAK are activated by the polyunsaturated fatty-acid (PUFA) arachidonic acid (AA) and are present in rat mesenteric arteries (Gardener et al., Br J. Pharmacol. 2004; 142:192-202).
Although Blondeau et al., (Circ Res. 2007; 101:176-84) showed PUFAs
activate K2P channels in murine arteries, their functional role in rat arteries is unknown. In our study, isolated Wistar rat (225-250g) mesenteric
arteries were pre-contracted with U46619 and then exposed to AA (1100microM) in the absence and presence of chlorpromazine (TREK/
TRAAK inhibitor; 1microM). No differences were detected between
AA-induced relaxation log EC50 values in the absence and presence of
chlorpromazine (-5.4 0.1 vs -4.9 0.2, n = 4, respectively). However,
in the continuing presence of the nitric oxide synthase inhibitor
L-nitroarginine (L-NOARG, 100microM) and the cyclo-oxygenase
inhibitor indomethacin (INDO, 10microM), the AA log EC50 value was
shifted signicantly by chlorpromazine at 1microM (-5.1 0.05, AA
with L-NOARG/INDO vs. -4.7 0.05, AA + chlorpromazine with
L-NOARG/INDO, p < 0.0001; n = 4) and 10microM (-5.2 0.07, AA
with L-NOARG/INDO vs. -4.6 0.2, AA + chlorpromazine with
L-NOARG/INDO, p = 0.0053; n = 3). These data suggest that in the
absence of relaxing factors such as nitric oxide and endogenous cyclooxygenase products, activation of K2P channels by PUFAs generates
measurable mechano-inhibitory effects in rat mesenteric arteries.
Work funded by the British Heart Foundation.
Amelia Edington, R Ryan, R Vandenberg

University of Sydney, School of Medical Sciences, Discipline of Pharmacology, NSW, Australia
265
Paper No.: 2976
DISPROPORTIONALLY IMPAIRED MICROVASCULAR
STRUCTURE IN PATIENTS WITH VERY MILD ESSENTIAL
HYPERTENSION
Ashkan Eftekhari(1,2), ON Mathiassen(1,2), NH Buus(2,3), O Gotzsche(2), MJ Mulvany(2), KL Christensen(1,2)
(2) Aarhus University Hospital, Department of Medicine and Cardiology
A, Aarhus, Denmark
(3) Aarhus University Hospital, Department of Renal Medicine, Aarhus,Denmark
Essential hypertension (EH) is characterized by reduced vasodilatory
capacity of the resistance vasculature. The purpose of our study was to
investigate if coronary ow reserve (CFR) and minimum forearm vascular resistance (Rmin) are affected proportionally to the increased blood
pressure (BP). 75 previously untreated EH patients (age 48 yr, 60%
males) without previous cardiovascular disease and with 24-h systolic
80 mmHg were
BP (SBP) l 130 mmHg or diastolic BP (DBP) Y
assigned into two groups: either Very Mild EH (125/79 mmHg) or MildModerate EH (145/89 mmHg). Twenty ve healthy normotensive
controls were used . CFR was measured with trans-thoracic echocardiography, forearm Rmin was measured using plethysmography. Systemic
vascular resistance index (SVRI) was calculated from cardiac output
measurements using a gas rebreathing technique. Compared to control,
24-h mean BP was raised 15% in Very Mild EH and 29% in Mild-Moderate EH. CFR was decreased by 23% in Very Mild EH and 29% in
Mild-Moderate EH, respectively. Rmin was elevated by 49% in Very
Mild EH and 75% in Mild-Moderate EH, as compared to control persons. Thus both CFR (P < 0.01) and Rmin (P < 0.01) were altered more
than could be expected from the raised BP level in Very Mild EH. In
contrast, SVRI increased proportionally to the increased BP (19%, Very
Mild EH; 33%, Mild-Moderate EH). In patients with very mild EH,
structural remodelling of the myocardial and the forearm resistance
vasculature seems disproportionally impaired compared to that expected
from the modest rise in BP.
Paper No.: 915

ANALGESICS
NEUROTROPIN RELIEVES THE OXALIPLATIN-INDUCED
PERIPHERAL NEUROPATHY BY INHIBITING
NEURODEGENERATION
Nobuaki Egashira, T Kawashiri, H Watanabe, Y Ikegami, T Yano,
H Ikesue, R Oishi
Kyushu University Hospital, Department of Pharmacy, Fukuoka, Japan
Oxaliplatin is a key drug for colorectal cancer, but it causes acute peripheral neuropathy (triggered by cold) and chronic neuropathy (sensory and
motor dysfunction). Oxaliplatin is metabolized to oxalate and dichloro(1,2-diaminocyclohexane)platinum [Pt(dach)Cl2]. Recently, we demonstrated the involvement of oxalate in oxaliplatin-induced cold
hyperalgesia but not mechanical allodynia, and preventive effect of preadministration of Ca2 + or Mg2 + on the cold hyperalgesia in rats (Sakurai
et al, Pain 2009; 147: 165-174). Neurotropin, a non-protein extract from
the inamed rabbit skin inoculated with vaccinia virus, has been used to
treat various chronic pains. We previously reported that repeated administration of neurotropin reverses the paclitaxel-induced neuropathy without affecting anticancer activity in rats (Kawashiri et al, Eur J Cancer
2009; 45: 154-163). In the present study, we investigated the effect of
neurotropin on the oxaliplatin-induced neuropathy in male Sprague-Dawley rats. Repeated administration of oxaliplatin (4 mg/kg, i.p., twice a
week) caused cold hyperalgesia (acetone test) in the acute phase and
mechanical allodynia (von Frey test) in the chronic phase. Repeated
administration of neurotropin relieved the oxaliplatin-induced mechanical
allodynia but not cold hyperalgesia, and inhibited the oxaliplatin-induced
axonal degeneration in rat sciatic nerve. Neurotropin also inhibited the
oxaliplatin-induced neurite degeneration in cultured PC12 and rat dorsal
root ganglion (DRG) cells. On the other hand, neurotropin did not affect
the oxaliplatin-induced cell injury in rat DRG cells. These results suggest
that repeated administration of neurotropin relieves the oxaliplatininduced mechanical allodynia by inhibiting the axonal degeneration and
it is useful for the treatment of oxaliplatin-induced neuropathy clinically.
Paper No.: 2807

USAGE OF AND ATTITUDES TOWARDS THE DRUG-DRUG
INTERACTION DATABASE SFINX A QUESTIONNAIRE
STUDY WITHIN SWEDEN
Birgit Eiermann(1), M Andersson(2), M-L Ovesjo(2), P Bastholm(1),
A Veg(1), M Edlert(1), Y Bottiger(2)
(1) Stockholm County Council, Department of Drug Management and
Informatics, Stockholm, Sweden
(2) Karolinska Institutet, Division of Clinical Pharmacology, Department
of Laboratory Medicine, Stockholm, Sweden
Introduction: Drug-drug interactions (DDI:s) are a common cause for
adverse drug events or reduced drug effect. Therefore avoidance of
DDI:s through usage of a decision support system for DDI:s can improve
quality of care. The DDI database SFINX, developed and used in Sweden and Finland is available through a web application, presenting colour
coded alerts when entering a drug or substance list. Clicking on the alert
button will lead to a short description of the medical consequence of a
DDI followed by useful clinical recommendations, mechanism and background information. Method: In a questionnaire study 11 763 registered
users of SFINX were asked about their use of SFINX, its inuence on
decision making and the perceived benets. Results: 2707 (23%) answers
from physicians (43%), pharmacists (18%) and other health care personnel (20%) were received. The majority of physicians and pharmacists
used the database during patient contact (60 resp. 50%). 75% of the users
always read the alert messages instead of just reacting to a warning signal. Over 56% of the physicians take actions receiving a DDI alert such
as change of drug therapy and informing the patients. The most important benets using SFINX are that physicians learn about DDIs (77%)
and that they receive conrmation of the patients drug lists regarding
interactions (73%). Conclusion: DDI databases with focus on concise
and short recommendation texts are seen as useful tools to support physicians decision making process during drug prescribing and to increase
their knowledge about DDI:s.
Paper No.: 1878

TESTOSTERONE INHIBITS ENDOTHELIAL NITRIC OXIDE
SYNTHASE MRNA EXPRESSION AND NITRIC OXIDE
PRODUCTION
Lena Ekstrom, C Skogastierna, A Rane
Karolinska Institute, Department of Laboatory Medicine, Stockholm,
Sweden
Background: Accumulating evidence indicates that abuse of anabolic
androgenic steroids (AAS) cause cardio-vascular adverse effects and disease. One early event in cardiovascular diseases is endothelial dysfunction. Several vasoactive mediators are released from the endothelium,
including the vasodilator nitric oxide (NO) which is synthesized by
266
endothelial nitric oxide synthase (eNOS). Surprisingly, there are no studies available on how AAS affect the endothelial metabolism of NO.
Aims: To investigate the effects of testosterone on the endothelial production of NO in vitro and in vivo. Methods: Testosterone (500 mg) was
administrated as a single dose to healthy volunteers and the morning
urine was collected prior to and 2 days after injection. The urinary excretion level of NO was assessed by a colorimetric assay. Moreover, testosterone was added to the medium of a human endothelial cell-line and
eNOS mRNA was quantied by real-time PCR. Results: The in vivo
results show that the urinary NO level signicantly decreased by 25%
two days after testosterone administration. The in vitro studies show that
testosterone inhibits the eNOS mRNA expression after 48 hours. When
the antioxidant selenomethionine was included, the eNOS mRNA
expression was not inhibited by testosterone, indicating that the downregulation of eNOS may partly be induced by oxidative stress. Conclusion: These results show that a supra-physiologic dose of testosterone
decreases the expression of eNOS and consequently the formation of
NO, indicating that testosterone may induce endothelial dysfunction. It is
possible that this effect contributes to the cardiovascular adverse effects
observed in AAS abusers.
Paper No.: 1867

DISEASES
CANNABINOIDS AND CALCIUM MOBILISATION IN
MICROGLIA CELLS
Khalil El Deeb(1), SPH Alexander(2), D Pritchard(3), DA Kendall(2)
(1) University of Nottingham Medical School, School of Biomedical
Sciences, QueensMedical Centre, Nottingham, UK & Al-Azhar University, Faculty of Medicine, Department of Pharmacology, Damietta, Egypt
(2) University of Nottingham Medical School, School of Biomedical
Sciences, Queens Medical Centre, Nottingham, UK
(3) University of Nottingham Medical School, Queens Medical Centre,
Nottingham, UK
(4) University of Nottingham Medical School, School of Pharmacy,
Queens Medical Centre, Nottingham, UK
Introduction Activation of microglial cell is a key feature in a wide range
of neuroinammatory CNS disorders such as stroke and multiple sclerosis. Cannabinoid compounds are known to have an immunomodulatory
effects through their actions on CB2 receptors and, potentially GPR55, a
cannabinoid-related receptor (Kreitzer & Stella, 2009). In the present
study, we investigated the effects of cannabinoid receptor ligands against
LPI and ADP on Ca2 + levels in BV-2 mouse microglial cells. Materials:
Intracellular calcium levels ([Ca2 + ]i) were monitored using Fluo-4
using a FlexStation-96 (Kasorn et al., 2006). Results expression of
GPR55 mRNA in BV-2 cells was indicated by Taqman qRT-PCR analysis. While AEA and 2AG, as well as HU210, CP55940, WIN55212-2
and rimonabant did not alter [Ca2 + ]i (9 1, 8.3 3, 10 1, 9.8
1.3, 8.8 1.3 and 8.4 1.9% ATP response respectively n = 5) cannabidiol and THC produced modest increases in [Ca2 + ]i (15 2 and
16 1.9%, respectively n = 5). Pre-treatment with cannabidiol or THC
(10 lM) produced signicant attenuations of LPI- (9 1, 2 0.5%
respectively n = 3) and ADP-evoked (31 8, 6.3 1.6% respectively
n = 3) [Ca2 + ]i responses. Conclusion these nding are consistent with
a non-selective inhibition of evoked Ca2 + release by cannabinoid
ligands in BV-2 microglial cells. The functional implications of these
effects on microglial calcium mobilization remain to be determined.
(Kasorn A et al. (2006). pA2 online 3: 24P.Kreitzer FR, Stella N (2009).
Pharmacol Ther 122: 83-96).
This work was made possible by a scholarship to Khalil Eldeeb from the
Egyptian government.
Paper No.: 2119

TUMOR NECROSIS FACTOR ALPHA INHIBITION BY
PENTOXIFYLLINE PROTECTS FROM THE IMPAIRMENT IN
VASCULAR REACTIVITY ASSOCIATED WITH INSULIN
RESISTANCE
Hany El-Bassossy(1), M El-Moselhy(2), M Fouad(1)
(1) Zagazig University Faculty of Pharmacy, Department of Pharmacology, Zagazig, Egypt
(2) University of Minia Faculty of Pharmacy, Department of Pharmacology, Minia, Egypt
Alterations of vascular reactivity due to insulin resistance play roles in
diabetic vascular complications. Tumor necrosis factor a (TNF-a) is an
important pro-inammatory cytokine. Here we have investigated the
potentially protective effect of TNF-a inhibition against the changes in
vascular reactivity associated with insulin resistance. Insulin resistance
was induced by including fructose (10%) in drinking water. while TNF-a
was inhibited by pentoxifylline (50mg.kg-1) for 8 weeks. Serum levels
of glucose, insulin, TNF-a was determined. Rings of rat isolated thoracic
aorta were used for histopathological examination and to measure the
responses to receptor (phenylephrine, PE) and depolarization (KCl) vasoconstrictors, and the endothelial dependent (ACh) and independent
(sodium nitroprusside, SNP) relaxants. Fructose drinking induced a signicant increase in insulin level and insulin resistance index. The insulin
resistance was accompanied with a signicant elevation in TNF-a level.
While pentoxifylline administration prevented the elevation in TNF-a
level and inhibited the developed insulin resistance. Insulin resistance
signicantly increased contraction of aortic rings to PE and KCl and
decreased relaxation to ACh and SNP. TNF-a inhibition by pentoxifylline protected against insulin resistance-induced hyperresponsiveness to
PE without affecting responses to KCl while it prevented the hyporesponsiveness to ACh and SNP. Insulin resistance was associated with
marked inltration of leukocytes in the adventitia and endothelial cells
pyknosis. TNF-a inhibition by pentoxifylline prevented the leukocyte
inltration and signicantly inhibited the observed pyknosis. In conclusion, inhibition of TNF-a by pentoxifylline protects from the impairment
in aortic vascular reactivity associated with insulin resistance, by a mechanism involving an inhibition in leukocyte inltration.
Paper No.: 3309

PROTEIN KINASE C INHIBITION PROTECTS FROM THE
IMPAIRMENT IN VASCULAR RESPONSIVENESS
ASSOCIATED WITH INSULIN DEFICIENCY BUT NOT
INSULIN RESISTANCE
Hany El-Bassossy, A Fahmy, W Barakat, N Dsokey
Zagazig University, Faculty of Pharmacy, Department of Pharmacology,
Zagazig, Egypt
Alterations in vascular reactivity play important roles in diabetic vascular
complications. In addition, stimulation of protein kinase C (PKC) has
been reported in diabetes. Here we have investigated the potential protective effect of PKC inhibition against the changes in vascular reactivity
associated with diabetes. Two animal models of diabetes were used.
Insulin deciency was induced by injecting rats with streptozotocin
(50 mg.kg-1) while, insulin resistance was induced by fructose (10%) in
drinking water. Rings of isolated thoracic aorta were used to measure the
responses to receptor (phenylephrine, PE) and depolarization (KCl) vasoconstrictors, and the endothelial dependent (ACh) and independent
(sodium nitroprusside, SNP) relaxants. Chelerythrine, a selective PKC
267
inhibitor, was added to organ bath in a nal concentration of 10 lM.
Streptozotocin injection signicantly decreased serum insulin level while
increasing glucose level. The produced insulin deciency signicantly
increased contraction of aorta to PE and KCl without affecting relaxation
to SNP. PKC inhibition by chelerythrine completely protected against
insulin deciency-induced hyperresponsiveness to PE and KCl. Fructose
drinking induced a signicant increase in insulin level and insulin resistance index. The developed insulin resistance signicantly increased contraction of aorta to PE and KCl and decreased relaxation to ACh without
affecting relaxation to SNP. Despite completely restoring responsiveness
in insulin deciency model, PKC inhibition by chelerythrine had no
effect on the impaired responsiveness to PE and KCl in insulin resistance
model. In conclusion, inhibition of PKC by chelerythrine protects from
the impairment in aortic vascular reactivity associated with insulin defeciency but not insulin resistance.
Paper No.: 2796

DISEASES
PERIPHERAL EFFECTS OF THE MARIJUANA SMOKE IN
THE LUNG OF MICE
Krisztian Elekes(1), K Sandor(1), E Szoke(1), D Toth(3), A Markovics(1),
I Szitter(1), L Jakab(2), B Szakacs(1), L Kereskai(4), J Szolcsanyi(1),
T Molnar(2), Z Helyes(1)
(1) University of Pecs Faculty of Medicine, Department of Pharmacology
and Pharmacotherapy, Pecs, Hungary
(2) University of Pecs Faculty of Medicine, Surgery Clinic, Pecs,
Hungary
(3) University of Pecs Faculty of Medicine, Analgesic Research
Laboratory, Pecs and Gedeon Richter Plc, Budapest, Hungary
(4) University of Pecs Faculty of Medicine, Department of Pathology,
Pecs, Hungary
Although the central nervous system effects of marijuana smoke has
been extensively studied, there are less and contradictory data on its
peripheral actions. Since human case reports described emphysema and
lung inammation in marijuana smokers, the aim of the present study
was to examine these alterations in a predictive mouse model. Male CD1
mice were exposed to smoke of cigarettes containing chopped dried marijuana (0.4 + 0.02w/w%. THC; 2 cigarettes twice a day throughout 1-3months). Experiments with research cigarettes were paralelly performed
for comparison. Airway reactivity to inhaled carbachol was measured
with unrestrained whole body plethysmography and the total cannabinoid
content of the urine was determined every week. Histological examination was performed, myeloperoxidase activity as a marker of granulocyte
accumulation and IL-1b concentration were measurement from the lung
samples. Prominent bronchial hyperreactivity was observed from the second week in the marijuana-exposed group and only from the middle of
the third month in cigarette smoke-exposed mice, the extent was signicantly smaller in the latter group. The histological pictures showed denitive inammatory changes. At 2 and 3 months the number of
inammatory cells and the empysema markedly increased, goblet cell
hyperplasia, perivascular granulocyte accumulation and large number of
eosinophilic giant cells were also observed. In the cigarette smokeexposed group inammation and emphysema appeared later and the histopathological changes were markedly less severe. Myeloperoxidase content and IL-1b concentrations were markedly elevated in both groups.
These data clearly show that marihuana smoke induces more severe
inammation, emphysema and hyperresponsiveness in the mouse airways than the conventional cigarettes.
Paper No.: 1175

EFFECT OF GABA MIMETICS ON THE DURATION OF
IMMOBILITY IN BEHAVIORAL DESPAIR SWIM TEST
Abdalla Elhwuegi, N Zahaf
Alfateh University of Medical Sciences Faculty of Pharmacy, Department of Pharmacology and Clinical Pharmacy, Tripoli, Libya
Introduction: Studies regarding the role of GABA in depression are conicting. Therefore, it was decided to study the antidepressant effect of
different drugs that enhance the GABA system by different mechanisms
against forced swim test (FST). Materials and methods: Adult Albino
mice divided to several groups of 6 animals, each received an intraperitoneal injection of either imipramine (10, 20, or 30 mg/kg), diazepam (0.5,
1 or 2 mg/kg) vigabatrin (100,200, or 300 mg/kg), zolpidem (2.5, 5, or
10 mg/kg), or alprazolam (1, 2.5 or 5 mg/kg). Control groups received
the appropriate vehicle. One hour after injection the duration of immobility for 5 minutes was calculated in the FST. The percentage change in
the duration of immobility from control group was calculated. Statistical
signicance (p < 0.05) between treated and control group was calculated
using unpaired t-test. Results: Imipramine produced a signicant dose
dependent decrease in the duration of immobility (78%, 74% and 56%
respectively). Diazepam, vigabatrin and zolpidem produced a signicant
dose dependent increase in the duration of immobility (119%, 126% and
128%), (116%, 124% and 128%) and (108%, 109% and 119%) respectively. The 2 low doses of alprazolam produced a signicant increase
(115% and 120%), while the high dose produced a signicant decrease
in the duration of immobility (74%) Discussion: Except for the high dose
of alprazolam, all of the selected GABAmimetic drugs had a depressant
like action in FST. This can be the result of a direct and/or an indirect
effect of these drugs on other neurotransmitter systems including GABA.
Paper No.: 2920

NEPHROTIC SYNDROME IN CHILDREN: GENETIC
VARIABILITY OF THE CLINICAL PRESENTATION AND
THERAPEUTIC RESPONSE
Valery Elie(1), M Fakhoury(1), Y Medard(1), A Rousseau(3),
G Deschenes(2), E Jacqz-Aigrain(1)
(1) Hospital Robert Debre, Department of Paediatric Pharmacology and
Pharmacogenetics, Paris, France
(2) Hospital Robert Debre, Department of Paediatric Nephrology, Paris,
France
(3) Clinical Research Unit (URC-EST), Paris, France
Introduction: Nephrotic syndrome is the most common kidney disease in
childhood, of unknown origin. In its steroid sensitive form, remission is
usually achieved within 4 weeks of prednisone but almost 70% of
patients will experience relapses. This ongoing multicentric prospective
case-control study aims to explore the impact of genetic polymorphisms
affecting glucocorticoid disposition and response on the outbreak, outcomes and interindividual variability of response to glucocorticoids in
pediatric nephrotic patients. Materials: Enrolled patients were treated
according to recommendations of the Society of Paediatric Nephrology
and paired unaffected controls selected according to sex and age. Patients
were genotyped for 4 single additional single nucleotide polymorphisms
(SNPs): glucocorticoid receptor (GR) rs6196, rs6198, rs33388 and
FK-binding protein 5 rs1360780. GR individual haplotypes were inferred
using HapMap database, PHASE and Haploview softwares. All patients
were also genotyped using Illumina Innium Whole-Genome Genotyping beadarray for 17826 SNPs (1395 genes). Statistical analyses are performed with Plink 1.07, Thesias and SPSSE Statistics 17. Results: 132
patients with a rst outbreak of NS were included: 78 boys and 54 girls
268
(sex ratio: 1.44/1), aged 5.25 3.26 [0.10-15.10] years. Minor allele frequencies were: rs6196 (13.40%), rs6198 (15.17%), rs33388 (38.99%)
and rs1360780 (31.30%). Preliminary results using a logistic regression
model of relapse predicted 80.6% of overall outcomes (83.3% of relapsers and 76.9% of non relapsers). Conclusion: Further statistical analyses
will be performed to evaluate the impact of genetic background on the
clinical outcome and variability of steroid treatment response in nephrotic syndrome.
Paper No.: 1180

DISCOVERY
THE EFFECT OF LOPERAMIDE AND FENTANYL ON THE
DISTRIBUTION KINETICS OF VERAPAMIL IN THE LUNG
AND BRAIN IN SPRAGUE DAWLEY RATS
Iman El-Kiweri(1), MT van Patot(2), YL Zhang(2), U Christians(2),
T Henthorn(2)
(1) King Saud Bin Abdulazziz University for Health Science, College of
Nursing, Department of Basic Science, Jeddah, Saudi Arabia
(2) University of Colorado, Denver Health Sciences Center, Denver, CO,
USA
Verapamil has been shown to inhibit fentanyl uptake in vitro and is a
potent P-glycoprotein inhibitor. Tissue partitioning of loperamide, a commercially available opioid, is closely controlled by the P-gp efux transporter. The following studies were designed to evaluate the effect of
opioids on verapamil partitioning in the lung and brain, in vivo. Opioid
(fentanyl or loperamide) was administered by intravenous infusion to
Sprague Dawley rats alone or in combination with verapamil and plasma,
with lung and brain tissues were collected at 1, 5, 6, 8, 10 and 60 minutes. Drug dispositions were modeled by recirculatory pharmacokinetic
models. Fentanyl slightly increased the verapamil lung (PL) partition
coefcient yet decreased the brain (PB) partition coefcient. Furthermore, loperamide signicantly increased PL and PB. Fentanyl reduced
the verapamil volume of distribution (V1) and verapamil elimination
clearance (ClE). Fentanyl decreased verapamil brain partitioning, yet
increased verapamil lung partitioning. Also, loperamide increased lung
and brain partitioning in vivo. These results suggest that verapamil and
fentanyl may be substrates of an unidentied inward transporter in brain
tissue and conrm that verapamil and loperamide are substrates of the
efux transporter P-gp.
Paper No.: 972

COMPARATIVE EFFICACY AND BIOAVAILABILITY OF
DIFFERENT PRAZIQUANTEL BRANDS
Naglaa El-Lakkany(1), S Seif el-Din(1), AN Sabra(1), M Ibrahim(2),
S Botros(1)
(1) Theodor Bilharz Research Institute (TBRI), Department of
Pharmacology, Giza, Egypt
(2) Egyptian International Pharmaceutical Industries Company (EPICO),
El-Asher Men, Ramadan City, Egypt
The efcacy, bioavailability and drug metabolizing enzymes mainly
involved in the metabolism of the commercial brands of praziquantel
(PZQ) in Egypt were examined in comparison with the original pure
powder. Mice infected with PZQ-susceptible (CD) or insusceptible (EE2)
S. mansoni isolates were divided each into 7 groups, 6 of them received
PZQ brands (Distocide, Epiquantel, Biltricide, Bilharzid, Praziquantel,
and pure PZQ), while the seventh one was left as infected untreated.
Seven weeks PI, worms were quantied and hepatic CYP450 and cyt b5
were examined. To study PZQ pharmacokinetics, groups of normal mice
were given different PZQ brands and subdivided into subgroups, killed
at 2, 5, 15, 30, 60, 90, 120,150, 180, 240 and 360 minutes post dosing.
Signicant decrease in worm burden was recorded in all PZQ brands
treated mice infected with CD isolate and it was to a less extent in mice
infected with the EE2 isolate. Biltricide and Distocide showed higher
worm reduction with least inhibition of CYP450 and cyt b5 as that of
pure powder. Pharmacokinetic data revealed higher Cmax and AUC0-6h
for both formulations versus that for PZQ in pure powder. PZQ T3A,
Bilharzid and Epiquantel showed in addition to less efcacy, higher Kel
and lower t, Cmax and AUC0-6h. The 32-46% reduction of their bioavailability was shown to reect on their antischistosomal efcacy and
recovery of drug metabolizing enzymes. Conclusion; the quality of generic PZQ should include in addition to examining the physicochemical
characteristics of the brands, biological testing including efcacy and
bioavailability studies.
Paper No.: 973

P19 - GENERAL SESSION - HEPATOLOGY
POTENTIAL ANTIFIBROTIC EFFECTS OF LOSARTAN OR
SILYMARIN COADMINISTRATION WITH PRAZIQUANTEL
AGAINST EXPERIMENTAL SCHISTOSOMA
MANSONI-INDUCED LIVER FIBROSIS
Naglaa El-Lakkany(1), W El-Maadawy(1), A Ain-Shoka(2),
A Badawy(3), F Ebeid(1)
(1) Theodor Bilharz Research Institute (TBRI), Department of Pharmacology, Giza, Egypt
(2) Cairo University Faculty of Pharmacy, Department of Pharmacology,
Cairo, Egypt
(3) Theodor Bilharz Research Institute (TBRI), Department of Pathology,
Giza, Egypt
The main cause of mortality and morbidity in human schistosomiasis is
hepatic brosis, which characterized by excessive accumulation of extracellular matrix (ECM). This study investigates the possible antibrotic
effects of losartan and silymarin at different stages of schistosomal hepatic brosis. Parasitological, biochemical and histological parameters
which reect disease severity and morbidity were examined. Moreover,
the effects of both drugs on ECM turnover were assessed by determination of hydroxyproline (Hyp), serum MMP-2 and TGF-b1. S. mansoniinfected mice were divided into 7 groups: Control untreated (i), treated
with either losartan (ii), or silymarin (iii) alone in doses of 50 or 750 mg/
kg/day for 5 days/wk for 6 wks respectively starting from the 4th, 8th,
12th wks PI, treated with praziquantel (PZQ), 500 mg/kg/day for 2 days
7 wks PI (iv), treated with losartan or silymarin in addition to PZQ (v &
vi) and treated with both losartan and silymarin in addition to PZQ (vii).
Administration of losartan or silymarin alone caused remarkable decrease
in worm burden, hepatic and intestinal tissue egg load with an increase
in dead eggs. Best results were obtained when losartan or silymarin were
co-administered with PZQ in the acute stage of infection as revealed by
normalization of ALAT, Hyp, MMP-2, TGF-b1 and of GSH versus PZQ
treated group. Taken together, these data suggested diminution of liver
brosis with a decrease in the severity of murine schistosomiais and
point to losartan and silymarin as a convenient and promising coadjuvant
therapeutic tool in the therapy of early schistosomal hepatic brosis.
Paper No. 1087

FOCUS GROUP: P04 - PHARMACOEPIDEMIOLOGY,
CURRENT CONTROVERSIES AND OPPORTUNITIES
ANALYSIS OF PHARMACOTHERAPY FOR PREGNANT
WOMEN IN PRIMORYE REGION
Ekaterina Eliseeva, Y Feoktistova, L Lozinskaya
Vladivostok State Medical University, Department of Clinical Pharmacology,Vladivostok, Russian Federation
269
Materials: 703 medical charts of pregnant women were analyzed by continuous sampling method (2004-2007). All the women consulted clinical
pharmacologist or geneticist regarding possible fetotoxic effects in pregnancy age from 3 to 32 weeks. Results: taking medication was carried
mostly in 1-3 and 6-7 weeks of pregnancy age. Average number of drugs
taken by a pregnant woman was 2.95 + 0.22. Total number of medicines
was 2078. Polypharmacy was noted in 85 (12.1%). Indications for pharmacotherapy were pelvic inammatory disease, respiratory infections,
UTI, GI disorders, connective tissue diseases, arterial hypertension and
epilepsy. Prescriptions included antibiotics 423 (60.17%), NSAID 98
(13.9%), CNS-affecting drugs 69 (9.8%), female sex hormones 68
(9.67%), corticosteroids 44 (6.25%), vaccines 37 (5.26%), mucolytics and
antitussives 50, bronchodilators (21). Also were used anticonvulsants
(21), opiate receptor agonists and amphetamines (9), PKKN drugs (32),
nootropics (35). ACE inhibitors were used in 2 patients. Two women took
veroshpiron in I trimester. According to the FDA classication 41.81% of
prescribed medicines is potentially toxic to the fetus (category C, D and
X of the FDA). Only 3,75% of drugs could be considered safe (Category
A). 26,27% prescriptions were relatively safe (category B). In our study
28,15% of the prescribed medications are not included in the FDA classication. Their risk for pregnancy is not known (NR). Conclusions: our
results suggest no sufcient drug safety for the fetus in pregnancy.
Paper No. 1088

MEDICAL PROGNOSIS OF PERIOPERATIVE PROPHYLAXIS
EFFICIENCY IN CHOLECYSTECTOMY
Ekaterina Eliseeva, V Shevtsov
Vladivostok State Medical University, Department of Clinical Pharmacology, Vladivostok, Russian Federation
Materials: 487 medical records of patients in three hospitals of the Primorsky Territory (2007-2008) were analyzed by sample comparing method
according to kea words like: surgical treatment of cholelithiasis, the
absence of infectious diseases, rare complications. Exclusion criteria
were: intolerance to antibacterial drugs in medical history; incomplete
record in medical charts, death during surgery or within 30 days after.
Results: Antibiotic perioperative prophylaxis (APP) has been performed
15.22% of patients, subjected to lap cholecysyectomy (cefazolin, cefuroxime and cefoperazone). APP was used in 48% of open cholecystectomy cases. Cefazolin, cefuroxime combined with metronidazole and
cefoperazone were used in 48% of the open colecystectomies. In laparoscopic cholecystectomy cases the frequency of postoperative infections
(PI) was 4,89% versus open surgical method - 32,77%. Atypical location
of the gall bladder was the very high risk for the development of PI. Relatively possible risks in laparoscopic cholecystectomy were: concrements
in the common bile duct (OR 0,23), recent exacerbations of cholecystitis
(OR 0,16). The relative PI risks after open cholecystectomy were: age
(OR 0,98), of obstruction of common bile duct (OR 0,78), recent episodes
of acute cholecystitis OR (0,36), presence of jaundice before surgery (OR
0 43), duration of surgery (OR 0,99). Conclusion: APP does not reduce
the incidence of PI in laparoscopic cholecystectomies. In open cholecystectomy APP reduces incidence of postoperative infection (OR 9,24). Further study is needed to reduce the routine use of cefoperazone for APP.
Paper No.: 2102

GASTROPROTECTIVE EFFECT OF PEROXISOME
PROLIFERATOR-ACTIVATED RECEPTOR-C (PPARC) AGONIST, ROSIGLITAZONE AGAINST STRESS-INDUCED
GASTRIC MUCOSAL INJURY IN RATS
Mohamed A EL-Moselhy(1), WH Nazmy(2), HM Ibrahim(2)
(1) University of Minia, Department of Pharmacology, Minia, Egypt

(2) University of Minia, Department of Physiology, Minia, Egypt
This study was designed to investigate the protective effects of rosiglitazone against acute gastric mucosal lesions induced by cold-restraint stress
ulcer (CRS) in rats and the mechanisms underlying these effects. Rats
were pylorically ligated and divided randomly into following groups:
control, CRS & CRS pretreated with rosiglitazone (3 mg/kg/day, orally
for7days) groups. Blood samples were collected from the heart; left to
clot and the sera were separated for determination of tumor necrosis factor-a (TNF-a) level. Animals were sacriced and gastric juice was collected for determination of volume, free and total acid outputs, mucin
concentration and pepsin activity. The gastric mucosal lesions were
assessed and the stomachs were scraped and the gastric mucosa was analyzed for determination of lipid peroxides, nitric oxide (NO), and prostaglandin E2 (PGE2) levels. Our results revealed that CRS induced gastric
mucosal ulceration mainly through an increase in gastric acidity, pepsin
activity, lipid peroxides and TNF-a levels with concomitant reduction in
NO, mucin and PGE2 levels compared to the control non-stressed rats.
Rosiglitazone pre-treatment signicantly attenuated the gastric mucosal
lesions induced by CRS, which was accompanied by signicant reduction in lipid peroxides and TNF-a levels and concomitant increase in
mucin concentration. Additionally, rosiglitazone caused a signicant rise
in gastric mucosal nitrite and PGE2 levels. In conclusion: Rosiglitazone
exerts a protective effect against CRS induced gastric ulcer possibly via
NO, PGE2 and mucus production as well as anti-inammatory and antioxidant mechanisms.
Paper No.: 2971

HEALTH AND DISEASE
RHO KINASE-DEPENDENT CALCIUM SENSITIZATION IN
THE INTERPLAY BETWEEN PRESSURE- AND
AGONIST-INDUCED CONSTRICTION OF CEREBRAL
ARTERY
Ahmed El-Yazbi(1), RP Johnson(1), K Takeya(2), EJ Wlash(1),
MP Walsh(2), WC Cole(1)
(1) University of Calgary, Department of Pharmacology and Physiology,
Calgary, ALB, Canada
(2) University of Calgary, Department of Biochemistry, Calgary, ALB,
Canada
Calcium sensitization is the mechanism by which smooth muscle cells
attain an elevated level of myosin regulatory subunit phosphorylation
and contraction with no change in cytosolic calcium concentration. This
process has been implicated in the myogenic response of resistance
arteries and in agonist-induced constriction of conduit arteries. Only
limited information is available concerning the interplay of myogenicand agonist-induced calcium sensitization in resistance arteries due to
technical difculties associated with quantication of phosphoprotein
markers of this mechanism; i.e. myosin light chain phosphatase targeting subunit (MYPT1) and CPI-17. Here, we used a high sensitivity
western blotting technique to detect a role for Rho kinase-mediated calcium sensitization in the myogenic response of cerebral arteries, to
assess the role of this mechanism in serotonin (5-HT)-evoked constriction, and whether there is an interplay between these constrictor stimuli
in the control of calcium sensitization. Pressure in the range of 10100 mmHg increased phospho-MYPT1-T855, but not -T697. 5-HT
evoked calcium sensitization only in the presence of myogenic constriction at 60 and not at 10 mmHg, and increased phosphorylation was
detected at MYPT1-T855 and -T697. Dual phosphorylation may be
required to permit increased force, a view that is supported by phosphorylation of both sites at an extreme pressure of 140 mmHg. No role
for CPI-17 was identied. These ndings provide the rst biochemical
evidence of an interplay myogenic and agonist dependent mechanisms
for control of calcium sensitization.
270
Paper No.: 923
PHARMACOKINETICS OF FLORFENICOL (WATER SOLUBLE
FORMULATION) IN HEALTH AND PASTEURELLA INFECTED
BROILER CHICKENS
Paper No.: 1798

DISEASES
ANTIVIRAL ACTIVITY OF ANAFERON FOR CHILDREN IN
PANDEMIC A/H1N1 IN MICE
Hesham Elzorba, H El-Banna
Oleg Epstein, JL Dugina, SA Tarasov, SA Sergeeva
Cairo University, Department of Pharmacology, Cairo, Egypt
Materia Medica Holding Company, Department of Research & Development, Moscow, Russian Federation
Florfenicol have been approved in the European Union for use in cattle
and pigs as injectable solution for treatment of respiratory diseases in
cattle but now it introduced in some countries as oral solution for the
treatment of several poultry diseases. The aim of the present study is
to describe the Pharmacokinetics of orfenicol (water soluble formulation) in broiler chickens after either a single intravenous and/or oral
administration at a dose of 30 mg/kg body weight. Meanwhile, its disposition in control healthy and Pasteurella-infected broilers was compared. Following the IV administration of the drug in healthy and
diseased birds, the drug plasma concentration declined in a biphasic
pattern. The maximum plasma concentration of orfenicol in control
healthy and diseased was reached one hour after its oral administration,
but the peak level detected in control broilers was higher than that
detected in infected birds. Data of the present study showed that volume of distribution (Vdss, 1.98 L/kg), total body clearance (ClB,
0.55 L/kg/h) in infected birds were higher than that determined in control birds compared to values determined in healthy ones (Vdss, 1.3 L/
kg, ClB, 0.38 L/kg/h). On the other hands, systemic bioavailability
were signicantly lower (F %,55.6%) in diseased broiler compared to
values determined in healthy ones (F %,71.5).
Paper No.: 491

ANTIBIOTIC ACTIVITY AGAINST URINARY PATHOGENS IN
HOSPITALIZED MEDICAL PATIENTS IN EASTERN NIGERIA
Oguamanam Enwere, K Duruewuru, V Okwara
Imo State University Teaching Hospital, Department of Internal Medicine, Orlu, Imo State, Nigeria
Urinary tract infection causes high morbidity and increases cost of care
in clinical practice. Quinolones have been recommended as rst line
drugs for uncomplicated urinary tract infection. Changing antibiotic
sensitivity patterns are becoming a concern in Nigeria due to the high
rate of self medication and substandard drugs in the Nigerian market.
This study, scheduled to last a one year period, assessed sensitivity of
commonly available antibiotics to isolated urinary pathogens in admitted patients who had urine analysis and culture as part of their
workup. 29 positive cultures (20 males and 9 females) were recorded
so far. 9 patients had self medicated with antibiotics prior to admission
(all men). 12 samples grew escherichia coli (41.4%), 15 for staphylococcus aureus (51.7%) and one each for proteus mirabilis and enterococcus spp (3.4% each). There was a high resistance rate against the
quinolones (33%-50%), nitrofurantoin and nalidixic acid (85.7% &
68.8% respectively). Resistance against gentamycin was 67.9%, streptomycin (17.4%) and coamixiclav (68.8%). E.coli did not have any
resistance against levooxacin. Only 82.8% of patients had pyuria
while 79.3% had proteinuria, 44.8% had hematuria and 24.1% had
glycosuria. The rising resistance to antibiotics is alarming; discouraging
self medication is very important at preventing rising resistance to antibiotics and physicians must be notied on the changing antibiotic sensitivity patterns in clinical practice.
Antiviral activity of anaferon for children, ultra-low doses of oral antibodies to interferon c, in the mice models of non-lethal and lethal A/California/07/09 (H1N1) infection was studied. First study included 96
female Balb/c mice (15-17 g) infected intranasally with 2.3 lg EID50/
mouse. Anaferon for children and distilled water (control) were administered intragastrically BID 0.2 mL/mouse starting 5 days prior to and
continuing for 8 days after inoculation. Oseltamivir was given 20 mg/kg/
day BID for 5 days with rst treatment 1hr after the challenge. Virus
titers in lungs on days 2, 4, 6, 8 after inoculation were assessed. Both
anaferon for children and oseltamivir signicantly reduced A/H1N1
titers: by 2.7 and 2.4 times respectively. Second study included 105
female Balb/c mice (16-20 g) infected intranasally with 3xLD50. Anaferon for children and distilled water were administered intragastrically BID
0.2 mL/mouse in combination with free access to drinking water (in
active treatment group anaferon for children serve as drinking water)
starting 5 days prior to and continuing for 13 days after challenge. Oseltamivir was given 20 mg/kg/day BID for 3 days with rst treatment 4hr
before challenge. Virus titers in lung tissue on days 3 and 6 after inoculation were assessed. Anaferon for children signicantly reduced mortality
of mice: in control group survival was 12.5%, in oseltamivir group
10%. The titet measurements in lungs conrmed the survival data. This
study provides evidence of antiviral activity of anaferon for children
against pandemic A/California/07/2009(H1N1) inuenza virus infection
in mice.
Paper No.: 2257

RESISTANCE OF ESCHERICHIA COLI AND KLEBSIELLA
PNEUMONIAE ISOLATES IN THREE TERTIARY INTENSIVE
CARE UNITS: A RESULT OF INSUFFICIENT EMPLOYMENT
OF INFECTION CONTROL MEASURES OR INADEQUATE
ANTIBIOTIC CONSUMPTION?
Viktorija Erdeljic, I Francetic, Z Bosnjak, A Budimir, S Kalenic, L Bielen,
R Likic, K Makar-Ausperger
University Hospital Zagreb, Department of Medicine, Zagreb, Croatia
Introduction: Escherichia coli (E.coli) and Klebsiella pneumoniae
(K.pneumoniae) are among the most important causes of serious nosocomial bacterial infections, and their resistance to antimicrobial agents has
become an increasingly relevant problem. We evaluated reasons (insufcient employment of infection control measures vs. inadequate antibiotic
consumption) for high resistance levels of E.coli and K.pneumoniae isolates to uroquinolones, aminoglycosides and beta-lactams in three tertiary intensive care units. Methods: Data on monthly uroquinolones,
aminoglycosides and beta-lactams consumption and resistance rates of
K.pneumoniae and E.coli isolates to these antibiotics were analyzed for
the period April 2006-July 2007 using distributed lags time series analysis. In addition, pulsed-eld gel electrophoresis (PFGE) was done on a
representative sample of 78 E.coli and 66 K.pneumoniae isolates (17%
and 23% of all isolates, respectively) to evaluate the type of resistance:
clonal or polyclonal. Results: A signicant temporal relationship between
uoroquinolones, aminoglycosides and beta-lactams consumption and
the resistance rates of E.coli and K.pneumoniae isolates to these antibiotics was identied. PFGE typing showed a wide diversity of strains: 57%
271
of E.coli and 61% of K.pneumoniae isolates were considered distinct as
they demonstrated <90% similarity with any other isolate. The remaining
isolates clustered into 16 and 10 clonal groups, respectively. For 10/16
and 8/10 clonal groups, respectively, the isolates belonging to each group
came from the same ICU. Conclusion: We were able to demonstrate antibiotic use as the main reason for the emergence of resistant E.coli and
K.pneumoniae strains, thus conrming the usefulness of antimicrobial
use restrictions as a method of resistance control.
Paper No.: 2460

CLINICAL PHARMACOLOGY OUTPATIENT CLINIC:
OUTCOMES AND MATERNAL CHARACTERISTICS
ASSOCIATED WITH PREGNANCY EXPOSURE TO FDA
CATEGORY X DRUGS
Viktorija Erdeljic(1), I Francetic(1), K Makar-Ausperger(1), R Likic(1),
M Radacic-Aumiler(1), D Juricic(1), L Bielen(1)
University Hospital Zagreb, Department of Medicine, Zagreb, Croatia
Introduction: Risk classication systems have been set up to guide safe
drug use during pregnancy. The most frequently used is the FDA pregnancy risk categorisation which places the drug in one of ve categories
(A, B, C, D, X) according to the level of risk to the infant, FDA X category being the category of contraindicated drugs in pregnancy. However,
drug safety classications give a very crude estimation of risk and should
only be used as general guidelines. Methods/Patients: We retrospectively
evaluated medical records on 130 consultations on the use of FDA X
drugs in pregnancy (2001-2007). Results: Out of 1003 pregnant women
consulted, 130 were taking an FDA X drug during the 1st trimester of
pregnancy. Most women were reffered during the rst trimester of pregnancy (88%). Maternal characteristics were as follows: 25% women had
an university degree, 36% had comorbidity, 26% were cigarette smokers,
for 39% this was the 1st pregnancy, 20% women were prescribed >2
drugs during pregnancy. The most frequently FDA X prescribed drugs
were oral contraceptives (75%), followed by oral anticoagulants, retinoids, methotrexate, etc. Follow-up was available for 50% women: Normal fetal outcomes were reported for 76% pregnancies, spontaneous
abortions for 9%, and articial abortions for 12% pregnancies. One baby
was born with coarctation of the aorta. In more than 2/3 of cases the clinical pharmacologists risk estimation was lower than X. Conclusion: Our
study shows high prevalence of consultations for drug exposures to FDA
X drugs in pregnancy in the clinical pharmacology outpatient clinic.
Paper No.: 2411

SUBSTANCE P INTERACTING PROTEINS: POTENTIAL
MECHANISMS FOR NON-RECEPTOR MEDIATED EFFECTS
Nuray Erin(1), V Bui(1), G Clawson(2)
(1) Akdeniz University, Department of Pharmacology, Antalya, Turkey
(2) Penn State University, Department of Pharmacology, Hershey, USA
Substance P (SP) belongs to the tachykinin family of peptides which is
involved in chronic inammation, cancer, and neurodegenerative disorders. Inammatory and carcinogenic effects of SP are believed to be
mediated by neurokinin 1 receptors (NK1Rs). SP also has anti-inammatory as well as anti-carcinogenic properties, although the mechanisms
underlying these activities are not known. We hypothesized that direct
interaction of SP with key binding partners mediate certain effects of
SP.Biotinylated SP was incubated with skin extracts and binding proteins
were precipitated using streptavidin beads. After PAGE, novel bands that
differed from controls were excised and analyzed using nanocapillary
reverse phase HPLC coupled with mass spectrometry. We found eight
high condence proteins, each of which contained three or more peptide
sequences with high homology scores and good spectra. Three of the
proteins (collagen type 1a1, procollagen type 1a2, and keratan sulfate
proteoglycan lumican) were extracellular matrix components. Another
two (myosin and actin) are part of a specialized membrane structure
involved in cell migration.The nal protein was identied as Histone 3.
Interaction of SP with three of the high condence proteins was veried
in skin extracts by immunoblot analyses. Interaction of SP with keratan
sulfate proteoglycan was also demonstrated in primary murine tumors.
Paper No.: 2751

THE EFFECTS OF SERTRALINE ON BEHAVIORAL
ALTERATIONS CAUSED BY ENVIRONMENTAL
ENRICHMENT
Kevser Erol(1), E Yildirim(1), E Ulupinar(2)
(1) Eskisehir Osmangazi University, School of Medicine, Department of
Pharmacology, Eskisehir, Turkey
(2) Eskisehir Osmangazi University, School of Medicine, Department of
Anatomy, Eskisehir, Turkey
Introduction: Depression, anxiety, motor skills and learning ability could
be affected by rearing environment. Neurotransmitters such as norepinephrine, dopamine and 5-HT may participate in the plasticity of environmental enrichment. In the present study, the effects of enriched
environment and social isolation on motor activity, anxiety, depression,
and also the effects of sertraline on these behaviors were evaluated.
Materials and Methods: After weaning at postnatal day 21, Wistar rats
were divided in different rearing conditions (Standard:SC, Enriched:EC,
Isolated:IC). Animals in SC were housed as group of 4 rats in regular
size cages. Animals in EC were housed as group of 12 rats in three large
cages connected via tunnels, with different toys. Animals in IC were
housed individually in metal cages. Six weeks later activitymeter, elevated plus maze, rota rod, Grip, forced swimming and sucrose preference
tests were applied to all animals. All these tests were repeated after i.p.
injection of sertraline (10 mg/kg/day) for 7 days. Results: Environmental
enrichment reduced stereotypic behavior, improved motor coordination
and facilitated learning skills. There were no signicant changes in anxiety and depression in EC and IC. Sertraline reversed the effects of
enriched environment on the stereotypic behavior; It reduced depression
in SC and EC but did not change it in IC. Sertraline was anxiolytic in
EC. The animals in IC prefered more sucrose than the others, and sertraline increased sucrose consumption. Conclusion: Rearing conditions
affect the behavioral and motor functions. The effects of sertraline
change depend on rearing conditions of animals.
Paper No.: 1174

DEVELOPMENTS IN THE TREATMENT OF SEXUAL
DYSFUNCTION AND DISEASES OF THE LOWER URINARY
TRACT
Saintmoses Eromosele(1), V Eruanga(2), PA Francis(3)
(1) The Pedagogues Educonsult, Benin City, Nigeria
(2) University of Benin, Department of Pharmacology, Benin City, Nigeria
(3) Ostee Trust Services Limited, Nigeria
Objective: To evaluate the relationship between lower urinary tract symptoms and erectile dysfunction in different male populations. Methods:
272
Data sources: PubMed (Medline), clinical evidence, Embase, Cochrane
reviews, and articles from reference lists. Selection criteria: Selection criteria in search databases were lower urinary tract symptoms, LUTS, comorbidity (MeSH), impotence (MeSH), sexual dysfunction, and male.
Studies on these subjects, and concerning men aged 30 years or older,
were eligible for inclusion in this review. Both community-based and
clinical-based studies were included. Results: 30 studies were eligible for
inclusion, representing 72 400 men. These studies showed a signicant
positive relationship between lower urinary tract symptoms and erectile
dysfunction.
Paper No.: 763

PRESCRIPTION OF POTENTIALLY INAPPROPRIATE
MEDICATIONS: COMPARISON BETWEEN INTERNAL MEDICINE SERVICE AND GERIATRIC UNIT
obstructive pulmonary disease (COPD) develop a disproportionate

response leading to pulmonary hypertension. The aim of this study was
to analyze whether polymorphisms in nSMase2 (SMPD3 gene) can make
patients more prone to suffer from pulmonary hypertension. After
informed consent, genomic DNA was isolated from blood samples of 14
patients suffering from COPD or interstitial lung disease, 5 of them without and 9 with associated pulmonary hypertension (group III Who classication), from 3 patients with pulmonary arterial hypertension (PAH,
group I) and from 7 controls. Nine fragments covering the whole gene
sequence were amplied by PCR and sequenced. A missense mutation
(C617Y) in heterozygosis was found in one of the patients with class I
and in one the patients with class III pulmonary hypertension but in none
of the COPD patients without pulmonary hypertension or in controls. In
conclusion, this pilot study suggests that mutations in the cysteine 617 of
the nSMase2 which belongs to the catalytic center of the enzyme might
be associated with the development of pulmonary hypertension. The
sample size does not allow to draw denitive conclusions but the results
guarantee further research.
Leslie Escobar(1), P Jara(1), M Jiron(1), M Rain(1), G Martinez(2),

C Dechent(2)
(1) Universidad de Chile, Facultad de Ciencias Qumicas y Farmaceuticas, Santiago,Chile
(2) Hospital Clinico de la Universidad de Chile, Santiago, Chile
With the exception of specialized geriatric services, in Chile there is a
scarcely knowledge about potentially inappropriate medications (PIM)
prescription to elderly. This study was conducted for comparing frequency of PIM to elderly patients attended in an internal medicine (IM)
service and a geriatric unit(GU), and to assess their outcomes over functionality. A prospective follow-up study was performed in GU and IM
services of a teaching hospital. Patients inclusion criteria were: 65 years
old, both sexes, without terminal disease, >3 days of hospitalization.
PIM was evaluated using 2003 Beers criteria and functionality by Barthel index. Data were analyzed using STATA 10.1. Eighty-one IM
patients and 70 GU patients were enrolled. In total 86(57%) were
females. GU patients were signicantly older than IM patients
(80.5 7.7 versus 76.5 7.3 years) and IM patients had lower mean
number of co-morbidities than GU patients (3.9 1.9 versus 5.3 2.6,
respectively). During hospitalization, 1029 medications were prescribed
in IM versus 684 in GU. 4.5% and 4.7% of them corresponded to PIM,
respectively. These results show that a 44.4% of IM patients received at
least 1 PIM compared with 37.1% in GU patients, mainly NSAIDs concomitantly with anticoagulation therapy and amiodarone. Mean number
of medication/patients was also signicantly higher in IM (12.7 5.3)
compared with GU (9.8 4.5). GU patients lost more than 14 points in
Barthel score during hospitalization, but there were no statistically differences between both services. More data analysis is needed for establishing associations between functionality and PIM. Also, additional studies
with an extended sample are necessary.
Paper No.: 2037

DOES MISSENSE MUTATION IN THE SMPD3 GENE
INCREASE THE RISK OF PULMONARY HYPERTENSION?
Luca Escolano(1), MA Nieto(2), L Moreno(1), A Cogolludo(1), A
Roman(1), Jose Luis Alvarez-Sala(2), F Perez-Vizcaino(1)
Paper No.: 1364

IN VITRO NEUROCHEMICAL PROFILE OF MDMA AND ITS
METABOLITE ALPHA-METHYLDOPAMINE
Elena Escubedo, S Abad, I Torres, J Camarasa, D Pubill,
Several studies suggest that MDMA-induced neurotoxicity is dependent
on its metabolic disposition. In rats, the N-demethylation of MDMA
leads to the formation of a-methyldopamine (MeDA). Both compounds
have different afnity prole for the serotonin transporter (SERT) (Ki =
3.10 1.48 lM for MDMA and 0.12 0.15 mM for MeDA) but similar afnity for the dopamine transporter (DAT) (assessed by [3H]WIN
35428 binding)(Ki = 14.78 5.58 lM and 16.94 8.73 lM respectively). They inhibited [3H]DA uptake with IC50 values: 3.90 2.30 and
0.11 0.02 lM respectively. The DA uptake inhibition by MDMA but
not by MeDA is dependent on NOS, PKC and nicotinic receptor. Conversely, MeDA preincubation followed by washout induces a reduction
in [3H]WIN 35428 binding, but not MDMA, pointing to a direct effect
of the metabolite on DAT. A hypothesis about MDMA neurotoxicity
states that DA enters in serotonergic terminals where it is deaminated by
MAO-B, resulting in free-radical formation and the selective degeneration of the serotonergic axons. The effect of both compounds on MAOB was very different (IC50 values of 1.00 0.23 mM MeDA and
70.10 13.20 lM MDMA). MeDA but not MDMA inhibited DA
uptake via SERT (IC50 about 20 nM at DA 5 nM) in hippocampal synaptosomes. Both MeDA effects, MAO-B and DA uptake inhibition via
SERT, point to the absence of neurotoxic effect of this metabolite.
Paper No.: 2091

HEALTH AND DISEASE
PULMONARY PRESSURE REDUCTION ATTENUATES
EXPRESSION OF PROTEINS IDENTIFIED BY LUNG
PROTEOMIC PROFILING IN PULMONARY HYPERTENSIVE
RATS
(1) University of Complutense, Department of Pharmacology, Madrid,

Spain
(2) Hospital Clinico San Carlos, Madrid, Spain
Yvonne Eskildsen-Helmond(1), L stergaard(1), B Honore(2), L Bech

Thorsen(1), J Baandrup(1), B Elmedal Laursen(1), H Vorum(2),
M Mulvany(1), U Simonsen(1),
Previous results show that neutral sphingomyelinase type 2 (nSMase2) is

involved in the signalling cascade of hypoxic pulmonary vasoconstriction
both in vitro and in vivo. In humans the magnitude of hypoxic vasoconstriction is variable: some patients suffering hypoxia due to chronic

(2) Aarhus University, Department of Medical Biochemistry, Aarhus,
Denmark
273
The present study was designed to analyze protein regulation in the lungs
of chronic hypoxic rats in order to identify novel signalling pathways,
which could provide new targets for the treatment of pulmonary hypertension. This was achieved by proteomic studies in which proteins from
lung homogenates from ve hypoxic rats were compared to those from
ve normoxic rats. In a second study the expression of these proteins
and correlation to alterations in vascular muscularization was also investigated in lungs from hypoxic rats that had received treatment with either
an activator of soluble guanylyl cyclase, BAY 412272, or an inhibitor of
phosphodiesterase type 5, sildenal. The proteomic study revealed upregulation of guanine nucleotide-binding protein beta (Gb-beta), glutathione
S transferase omega 1, cathepsin D (CatD), chloride intracellular channel
subunit 5, Annexin A4, F-actin capping protein CapZ (CapZalpha), and
the translation factor elongation factor 1 delta in lungs from chronic hypoxic rats with pulmonary hypertension. Immunohistochemistry showed
that many of these proteins were expressed in the pulmonary vascular
wall (e.g. CapZalpha, CatD, and annexin A4) and immunoblotting
showed these proteins correlated to alterations in muscularization. Both
treatments inhibited hypoxia-induced increase in right ventricular systolic
pressure and pulmonary arterial muscularization and prevented most of
the protein regulations observed after hypoxia. These ndings suggest
that pulmonary pressure is an important factor for initiating signalling
pathways leading to protein expression and muscularization in the pulmonary vasculature.
Paper No.: 3117

HEALTH AND DISEASE
UNFOLDED PROTEIN RESPONSE INVOLVED IN OXIDIZED
LDL-EVOKED ENDOTHELIAL DYSFUNCTION
Yvonne Eskildsen-Helmond(1), M Jrgensen(1), E Stankevicius(1),
N Christophersen(1), E Falk(2), U Simonsen(1)
(2) Aarhus University Hospital, Department of Cardiological Medicine,
Aarhus, Denmark
Accumulation of cholesterol in the endoplasmatic reticulum (ER) contributes toapoptosis of macrophages and smooth muscle cells in atherosclerotic lesions. Thepresent study was designed to investigate whether
unfolded protein response(UPR) is activated in endothelial cells in atherosclerotic aortae and cellsexposed to oxidized low density lipoprotein
(ox-LDL). Immunohistochemistry wasperformed for glucose-regulated
protein (Grp78) and measurements of nitric oxide(NO) and calcium in
human umbilical vein endothelial cells (HUVEC). Grp78, asmarker for
UPR activation, was extensively expressed in atherosclerotic plaques,in
endothelium and adventitia localised in the ascending part of aorta
(ApoE-/-mice (n = 8)). GRP78 expression was also increased in HUVEC
exposed to acomponent of ox-LDL, lysophosphatidyl choline (LPC), and
a chemical inducer ofER stress tunicamycin (TM). In HUVEC, endothelial NO synthase (eNOS) expressionwas unaltered, while eNOS phosphorylation at serine1177 was unaltered afterincubation with LPC and
increased with tunicamycin. However, histamine (1`M)-induced increases
in NO concentration (dNO, 22.9 5.9 nM) were decreasedafter incubation with LPC (7.8 1.7) and TM (7.9 2.1), and inhibited in thepresence of asymmetric dimetylarginine. Our results indicate activation of
theUPR system associated with reduced NO release, suggesting involvement inendothelial dysfunction caused by Ox-LDL.
Paper No.: 2972

THERAPEUTIC TARGETS
MELATONIN MODULATES OXIDATIVE STRESS AND
PREVENTS CELL DEATH IN HUMAN LEUKOCYTES. EFFECT
OF INTRACELLULAR CALCIUM OVERLOAD AND AGE
Javier Espino(1), I Bejarano(1), SD Paredes(1), D Gonzalez(1),
RJ Reiter(2), JA Pariente(1), AB Rodrguez(1)
(1) University of Extremadura, Faculty of Science, Department of Physiology,Badajoz, Spain

(2) University of Texas Health Science Center, Department of Cellular &
Structural Biology, San Antonio, USA
Ageing is associated with an increased production of free radicals and
alterations in the mechanisms of adaptation to oxidative stress. In fact,
the free radical theory of ageing proposes that deleterious actions of free
radicals are responsible for the functional deterioration associated with
ageing. Moreover, a close relationship exists between calcium homeostasis and oxidative stress. The current work was aimed at proving the
effect of melatonin on the levels of reactive oxygen species (ROS) and
cell viability in human leucocytes collected from young (20-30-year-old)
and elderly (65-75-year-old) individuals under both basal and oxidative
stress-induced conditions. Treatments with 10 nM FMLP and/or 1 uM
thapsigargin induced a transient increase in cytosolic free-calcium concentration ([Ca2 + ]c) in human leucocytes due to calcium release from
internal stores, and led in turn to oxidative stress, as assessed by intracellular ROS measurement. Non-treated leucocytes from aged individuals
exhibited higher ROS levels and lower rates of cell survival when compared to leucocytes from young individuals. Similar results were
obtained in FMLP and/or thapsigargin-treated leucocytes from elderly
individuals when compared to those from the young individuals. Melatonin treatment signicantly reduced both hydrogen peroxide (H2O2) and
superoxide anion levels, likely due to its free-radical scavenging properties, and enhanced leucocyte viability in both age groups. Therefore, melatonin may be a useful tool for the treatment of disease states and
processes where an excessive production of oxidative damage occurs.
Funded by BFU2007-60091 (MEC-DGI) and PRI07-A024 (Junta de Extremadura)
Paper No.: 961

DISEASES
THE ABSENCE OF A FUNCTIONAL PEROXISOME
PROLIFERATOR-ACTIVATED RECEPTOR-ALPHA GENE IN
MICE ENHANCES DOPAMINERGIC NEURON SENSITIVITY
TO 1-METHYL-4-PHENYL-1,2,3,6-TETRAHYDROPYRIDINE
Emanuela Esposito(1,2), E Mazzon(1), I Paterniti(1), T Genovese(1),
S Cuzzocrea(1,2)
(1) University of Messina School of Medicine, Department of Clinical &
Experimental Medicine & Pharmacology, Messina, Italy
The nuclear receptor peroxisome proliferator-activated receptor (PPAR)a plays a prominent role in several physiological processes including the
inammatory response, and its activation mediates a reduced production
of proinammatory factors. Using PPAR-a knockout (KO) mice, we
report a detailed investigation of the role of PPAR-a in cerebral damage,
associated inammatory and antioxidant processes in a subacute model
of Parkinsons disease (PD). N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), is used in rodent and cell models to elicit neurochemical
alterations associated with PD. Immunohistochemistry and Western blotting analysis were performed to detect the expression of neuronal and
inducible nitric oxide synthase (NOS) and cyclooxygenase (COX)-2,
matrix metalloproteinase (MMP)-2 and -9 activity, 3-nitrotyrosine formation, MAPK and NF-jB activation, apoptosis, comparing mice lacking
PPAR-a KO with wild-type (WT) mice. Brain oxidative stress was dramatically enhanced in KO mice, as documented by an increased content
of malondialdehyde, decreased levels of glutathione, a marked increase
of oxidative products, and no induction of uncoupling protein 2. PPAR-a
deciency appears to aggravate the severity of brain injury through an
increase in extracellular matrix formation and inammatory parameters.
These data indicate that PPAR-a plays an important role in integrating
and regulating central inammation, antioxidant mechanisms in the
process of MPTP-induced toxicity, and suggest that the use of PPAR-a
agonists may be of interest for the prevention of PD.
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Paper No.: 1210
PACEMAKER CELLS AND CONTRACTILITY IN THE
PROSTATE GLAND
Betty Exintaris, A Dey, S Kusljic, M Lam, D-T Nguyen, R Lang
Action, Parkville, VIC, Australia
Introduction: Morphologically-distinct, c-Kit immunoreactive prostaticinterstitial cells (PIC) are likely to generate the pacemaker signal thatmanifests as slow wave activity and resultant contractility in the smooth
musclecells of the guinea-pig prostate. Methods: In this study, we have
compared thespontaneous activities and cell types in younger (300-500g)
and older animals(>900g), using tension-recording techniques, intracellular micro-electrodes andimmunohistochemistry. Results: Slow waves
recorded within the guinea pigprostatic stroma had similar frequencies to
that of the spontaneous contractionswithin both age groups of animals.
In addition, older prostates (n = 78) had ahigher basal tension of
6.0 0.3mN than the younger prostates (n = 51) 4.7 0.66mN.Pacemaker activity was recorded in approximately 10% of all electricalrecordings in the younger guinea-pig prostate (n=>100). In the older guineapigprostate, pacemaker activity was not recorded (n = 84), however cells
exhibitinglarge-amplitude spontaneous transient depolarisations (STD)
were recorded in 9%of all electrical recordings. Microscopic examination
revealed a distinct layerof c-Kit and connexin 43-immunoreactive cells
in the region between the smoothand glandular layers in younger and
older guinea-pigs; c Kit positive cells werealso sparsely distributed
within the glandular layer and among smooth musclecells (n = 5). Conclusion: These results suggest that that older prostates have anincreased
level of smooth muscle tone. In addition, with age, there is achange in
the proportion of cells exhibiting pacemaker activity; however c-Kitimmunoreactive cells are present, indicating that the characteristics of the
PICmay change with age.Supported by the NH&MRC
Paper No.: 2454

CAN STEADY STATE PLASMA CONCENTRATION OF
EFAVIRENZ PREDICT VIROLOGIC OUTCOMES IN
TREATMENT NAIVE HIV ETHIOPIAN PATIENTS?
Abiy H Eyakem(1,2), W Amogne(3), G Yimer(1), E Makonnen(1),
G Aderaye(3), A Worku(4), L Lindquist(6), L Bertilsson(2),
J Burhenne(5), E Aklillu(2)
(1) Addis Abeba University, Faculty of Medicine, Department of Pharmacology, Addis Ababa, Ethiopia
(2) Karolinska Institute, Department of Laboratory Medicine, Stockholm,
Sweden
(3) Department of Internal Medicine, Faculty of Medicine, Addis Abeba
University, Addis Abeba, Ethiopia
(4) Addis Abeba University, Faculty of Medicine, School of Public
Health, Addis Abeba, Ethiopia
(5) University of Heidelberg, Department of Clinical Pharmacology and
Pharmacoepidemiology, Heidelberg, Germany
(6) Karolinska Institute, Department of Internal Medicine, Stockholm,
Sweden
Previously, the therapeutic plasma concentration of efavirenz has been
suggested to range between 1 4 lg/mL. Efavirenz plasma concentration < 1 lg/mL is associated with therapeutic failure while > 4lg/mL is
associated with CNS toxicity. This study was designed to investigate the
steady-state plasma concentration of efavirenz and compare its relationship with the treatment outcome measured by virologic success among
treatment nave HIV infected Ethiopian patients. A cohort of HIV
infected adult participants (n = 118), nave for HAART with CD4 counts
less than 200 cells per mm3 were recruited. Participants received 600mg
daily dose of efavirenz. 16 h 1 h post-dose, blood samples were collected on the 4th week after initation of efavirenz based treatment. Steady
state plasma concentrations of efavirenz were determined using LC/MS/
MS and viral loads (VLs) were determined on week 24 using Abbott M2000 RT. The mean plasma efavirenz concentration at week 4 was
1.5 0.12 lg/ mL while the median was 1.1 lg /mL. Proportion of participants in sub-therapeutic (<1 lg/mL), therapeutic (1- 4 lg/mL) and
toxic (< 4 lg/mL) ranges were 41.5%, 55.1% and 3.4% respectively.
The proportion of participants with virologic success among participants
with plasma efavirenz concentration <1, 1-4 and < 4lg/mL were 100%,
91.4% and 100% respectively. Our result indicates no association
between the described therapeutic range (1-4 lg/mL) categorization
based on plasma concentrations of efavirenz and therapeutic success
measured by VL among Ethiopian HIV infected individuals.
Paper No.: 1495

DISEASE AND THERAPY
VARIABLE EXPRESSION OF ABCC8 AND ABCC9
TRANSPORTERS IN PREGNANT RAT MYOMETRIUM:
INFLUENCE OF GESTATION AGE
George Falkay, N Lovasz, E Ducza, R Gaspar
University of Szeged Faculty of Pharmacy, Department of Pharmacodynamics and Biopharmacy, Szeged, Hungary
Potassium channel activation has been shown to decrease uterine tone
which is considered as a target for inhibition of uterine activity in the
treatmet of preterm labour. This study was designed to examine the
expression and role of SUR1 (ABCC8) and SUR2 (ABCC9) subunits of
KATP channels in pregnant rat myometrium specially with regard to the
contractility. RT-PCR was performed to detect the presence of sulphonyurea binding regulatory subunit 1 and 2. The effect of the KATP channel opener pinacidil on the contraction provoked by electric eld
stimulation (EFS) (50Hz) and by oxytocin were investigated on the isolated pregnant uterus. The SUR 1 and SUR 2 were markedly increased
at the early stage of the pregnancy. The highest level was detected at day
6 of the pregnancy while at late stage the levels of the transporters were
signicantly decreased. Pinacidil inhibited the oxytocin and EFS induced
contraction. Glibenclamide as a selective blocker of KATP channel antagonised the pinacidil induced inhibition of contraction. The pharmacological reactivity of KATP channels might be attributed to the levels of SUR1
and SUR2 subunits. This is the rst direct study to dene the potencial
role of SUR subunits in the pregnant rat myometrium. These ndings
may have a potencial therapeutic implication for managing preterm
labour in the future.
Acknowledgement:This work was supported by a Hungarian Otka
Research Grant (K62707).
Paper No.: 561

HEALTH AND DISEASE
TANSHINONE IIA INHIBITS ER-A ACTIVITY IN MCF-7
BREAST CANCER CELL
Guanwei W Fan(1), XM Gao(1), H Wang(1), Y Zhu(1), J Zhang(2),
LM Hu(1)
(1) Tianjin University of Traditional Chinese Medicine, Tianjin, PR
China
(2) Nankai University, Tianjin, PR China
Phytoestrogens are a diverse group of plant-derived compounds that
structurally or functionally mimic mammalian estrogens and show potential benets for human health. An increase in phytoestrogen research
over the past few decades has demonstrated the biological complexity of
275
phytoestrogens. Phytoestrogens can bind to estrogen receptors (ERs) and
mimic some actions of human estrogen through the activation or inactivation of certain genes.Tanshinone IIA (Tan IIA), a major component
extracted from a traditional herbal medicine Salvia miltiorrhiza BUNGE,
has been implicated as a chemopreventive agent against breast cancer.
However, the mechanisms behind it cancer-protective effects are not
clear. The goal of this study was to determine whether the antiproliferative action of this compound on breast cancer cell (MCF-7) is mediated
by estrogen receptors (ERs). The percentage of cell viability was evaluated by MTT assay. It was found that Tan IIA exhibited antiproliferative
effects in MCF-7 cells in the range of 10-6M10-5M. In RT-PCR assay,
Tan IIA(10-5M) was found to inhibit pS2 expression at 6h (0.35 0.27fold), at 24h (0.80 0.32-fold) when compared to untreated control. In
transfection assay, Hela cells were co-transfected with pERE-luc, pTKRen, ER-a or ER-b, Tan IIA(10-6M10-5M) decreased the ER-a transactivation. Our data unveil, that the anticancer action of Tan IIA in ER-a
positive breast cancer cells mediated, in part, by inhibit the activity of
ER-a and the expression of pS2.
Paper No.: 3218

VASORELAXANT EFFECTS OF COPTISINE IN RAT AORTIC
RINGS
Lian-Hua Fang, L-L Gong, J-J Hu, H-L Qin, Y Lv, G-H Du,
Chinese Academy of Medical Science & Peking Union Medical College,
Institute of Materia Medica, National Center for Pharmaceutical Screening, Beijing, PR China
Coptisine is an isoquinoline alkaloid isolated from Coptidis Rhizoma.
Several biological actions of Coptidis Rhizoma have been reported. The
present study was performed to investigate the vasorelaxant effects of
coptisine and its mechanism in isolated rat aorta. Coptisine(1~200?M)
inhibited norepinephrine(NE) and KCl induced sustained contraction
with pEC50 values of 4.49 0.48 and 4.85 0.57, respectively. Pretreatment of coptisine also suppressed the concentration-response curves
to NE and KCl. The vasorelaxant effect of coptisine was endothelium
dependent. In addition, the vasorelaxant effect of coptisine was partially
reduced by pretreatment with Nj-nitro-L-arginine methyl ester, methylene blue and indomethacin, respectively. In endothelium-denuded aortic
rings, the vasorelaxant effect of coptisine was reduced signicantly by 4aminopyridine. However, glibenclamide and tetraethylammonium failed
to do so. Moreover, coptisine inhibited not only the NE-induced transient
contraction caused by cytosolic Ca2 + release from endoplasmic reticulum in Ca2 + -free medium, but also the contractions induced by 60 mM
KCl buffer with different Ca2 + concentrations. This study showed that
coptisine inhibited both endothelium-dependent and -independent contraction in rat aortic rings. These results suggested that NO-cGMP pathway may be involved in the endothelium-dependent relaxation of
coptisine, and activation of voltage-dependent K+ channel contributes in
part to the endothelium-independent relaxation of coptosine. Furthermore, coptisine may inhibit the extracellular Ca2 + inux by blocking
both voltage- and receptor-operated Ca2 + channel.
Acknowledgements: This study was supported by special foundation on
scientic & technological basic work(2007FY130100) and national scientic & technological major special project(2009ZX09302-003,
2009ZX09501-021) provided by China Ministry of Science and Technology. Corresponding authors: dugh@imm.ac.cn (G.-H. Du).
Paper No.: 2956

DISTRIBUTION OF METHYLPHENIDATE AND RITALINIC
ACID IN ORAL FLUID
Magi Farre(1), R Pardo(1), E Marchei(2), O Garcia-Algar(3), M Pellegrini(2), R Pacici(2), S Pichini(2)
(1) IMIM-Hospital del Mar, UAB, Department of Human Pharmacology

and Neurosciences, Barcelona, Spain
(2) Department of Therapeutic Research and Medicines Evaluation, Istituto Superiore di Sanita, Rome, Italy
(3) IMIM-Hospital del Mar, UAB, Department of Pediatrics, Barcelona,
Spain
Introduction: Oral uid as an alternative matrix for monitoring drug use
due to its ease-of-collection and non-invasiveness. The aim of this study
was to evaluate the pharmacokinetic prole of methylphenidate (MPH)
and its metabolite ritalinic acid (RA) in oral uid and in plasma after
controlled MPH administration. Materials/Patients: Eight male healthy
volunteers participated in the study. Subjects received a single dose of
20 mg short-release (n = 5) or extended-release MPH (n = 3) by oral
route. Samples of oral uid and plasma were collected along 24 hours.
Concentrations of MPH and RA in oral uid and plasma were measured
by liquid chromatographyelectrospray ionization mass spectrometry
using a validated method. Apparent pharmacokinetic parameters of MPH
in plasma and oral uid were estimated. Results: MPH and RA were
detected in oral uid. Whereas parent drug concentrations in oral uid
were one order of magnitude higher than those in plasma, the opposite
was observed for RA, which resulted 10 times more concentrated in
plasma. Oral uid concentrations of MPH ranged between 0.5-466.7
mcg/L and peaked at 0.5 h after short release administration and ranged
between 0.7-89.5 mcg/L and peaked at 2 h after extended release administration. Conclusions: The results obtained support the measurement of
MPH in oral uid as a valuable alternative to drug monitoring in plasma
both in clinical and toxicological studies. Acknowledgements.
Supported in part by grants from ISS-Rome, FIS-RTA (RD06/0001/
1009), 2009 SGR 718 and FIS (R. Pardo is the recipient of a fellowship
Rio Hortega, no. CM08/00051).
Paper No.: 2957

MINI-CASES AS A METHOD TO DYNAMIZE LECTURES OF
AUTONOMIC NERVOUS SYSTEM PHARMACOLOGY IN A
MEDICAL SCHOOL
Magi Farre(1), J-E Banos(2)
(1) IMIM-Hospital del Mar, UAB, Dept. Human Pharmacology and Neurosciences, Barcelona, Spain
(2) Universitat Pompeu Fabra, Department of.Experimental and Health
Sciences, Barcelona, Spain
Introduction: We report here a preliminary study using a series of minicases or very short problems in conjunction with traditional lectures for
teaching medical students. Materials: We used ten mini-cases in our lectures on autonomic nervous system pharmacology. Mini-cases consist of
a ve-line text with two questions on the subject of the lecture. They
were delivered just before the lecture, and students were given ve minutes to answer the questions at the end. Teachers evaluated their correctness and were able to award extra points to the overall mark in the
regular exam. At the end of term the experience was evaluated by asking
students to answer ten questions anonymously. Results: Attendance to
lectures increased when mini-cases where used in comparison to other
lectures of different parts of the Pharmacology program. Students handed
in a mean of 7-8 mini-cases and gave a mean score of 7.5 for their usefulness in understanding the subject better and 7 for their interest. When
asked about their reasons for taking part, they scored 8.5 for the possibility of obtaining an extra mark, 7.5 for the idea that they would understand the subjects better. Around 70% reported that mini-cases increased
attendance at lectures and 95% said they would participate even if extra
marks were not available. All participants reported that they would participate in similar experiences in the future. Conclusions: We conclude
that mini-cases may be a cost-effective method for improving students
attendance at lectures, as well as their understanding of the autonomic
nervous system pharmacology.
276
Paper No.: 1026
ANXIOLYTIC-LIKE EFFECT OF SKF-38393 IN COMBINATION
WITH LOW DOSE OF 17B-ESTRADIOL IN
OVARIECTOMIZED RATS
Paper No.: 1048

DISCOVERY
CANNABIDIOL INHIBITS BCRP AND P-GP IN HUMAN
PLACENTAL TROPHOBLAST CELL LINES BEWO AND JAR
Yulia Fedotova
Valeria Feinshtein(1), G Holcberg(2), Z Ben-Zvi(1)
Institute for Experimental Medicine, Department of Neuropharmacology,

St Petersburg, Russian Federation
(1) Ben-Gurion University, Department of Pharmacology, Beer-Sheva,

Israel
(2) Soroka University Medical Center, Beer-Sheva, Israel
Dopamine systems are activated by stress and involved in stress-related

behaviors such as anxiety. On the other hands, estrogens have been
implicated in anxiety behavior. Recent evidence has shown that estrogen
receptors can be activated by a variety of neurochemical compounds,
including the dopaminergic ligands acting on D1-receptor. The aim of
the present study was to explore the antianxiety-like effects of D1-receptor agonist SKF-38393 (0.1 mg/kg, i.p.) or D1-receptor antagonist SCH23390 (0.1 mg/kg, i.p.) alone or in a combination with low dose (0.5
mkg/rat, s.c.). The anxiety-like behavior was assessed in the white model
(BWM) and the open eld test (OFT). In the BWM, SKF-38393 signicantly reduced anxiety behavior in OVX females as compared with control group. SKF-38393 in a combination with low dose of 17b-E2
induced more profound anxiolytic effect in OVX rats as compared to
OVX rats treated with SKF-38393 alone. In the OFT, SKF-38393 alone
or in combination with 17b-E2 increased grooming and rearing without
signicant changes in locomotor activity as compared to OVX and control groups. SKF-38393 simultaneously restored mRNA expression of
D1- and 17b-E2-receptors in the hypothalamus. In conclusion, SKF38393 alone or in combination with low dose of 17b-E2 may exert antianxiety-like effect on OVX females through D1- and 17b-E2-receptors,
via altering the expression of these receptors in the brain. Thus, the
results demonstrate that 17b-E2 and SKF-38393 behaviorally and neurochemically interact to enhance anxiolytic action and can potentiate effects
of each other.
Paper No.: 2274

ROPREN IS A POLYPRENOL PREPARATION FROM
CONIFEROUS PLANTS THAT AMELIORATES COGNITIVE
DEFICIENCY IN A RAT MODEL OF B-AMYLOID
PEPTIDE-(25-35)-INDUCED AMNESIA
Yulia Fedotova(1), V Soultanov(2), V Roschin(3), T Nikitina(4)
(1) IP Pavlov Institute of Physiology RASCi, Department of Neuroendocrinology, St.Petersburg, Russian Federation
(2) Solagran Limited, Melbourne, VIC, Australia
(3) State Forest Technical Academy, Department of Wood Chemistry, St
Petersburg,Russian federation
(4) IM Sechenov Institute of Evolutionary Physiology and Biochemistry,
RussianAcademy of Science, Laboratory of Functional Biochemistry of
Invertebrates, StPetersburg, Russian Federation
This study assesses the efcacy of a xed dose of a novel preparation
Ropren (a plant preparation isolated from the neutral fraction of an
extract of spruce and pine needles) on cognitive impairments in rats with
b-amyloid peptide-(25-35)-induced amnesia. Ropren was administered
at a dose of 8.6 mg/kg for 28 days, per os, to rats with b-amyloid peptide-(25-35)-induced amnesia. Cognitive performance was assessed using
the passive avoidance paradigm and the Morris water maze. The behavior of animals was assessed in the open eld test. After four weeks,
Ropren treatment signicantly improved non-spatial and spatial learning in rats with b-amyloid peptide-(25-35)-induced amnesia. The results
of the present study suggest that Ropren, a novel plant preparation,
ameliorates cognitive deciencies in an animal model relevant to Alzheimers disease.
Cannabis is the most commonly used illegal drug, when approximately

4% of women in the USA report using illicit drugs during pregnancy,
with marijuana being the most popular one (75%). BCRP and P-gp are
efux transporters expressed at the apical membrane of the syncytiotrophoblast and are putatively important protective proteins for the fetus.
To day, very little is known about the inuence of cannabinoids on
BCRP and P-gp in the placental barrier. Our study focused on cannabidiol (CBD) effect on the functionality of BCRP and P-gp in BeWo and Jar
cells, while MCF-7 cells were used as control for BCRP function properties. CBD cytotoxycity was tested rst. Non toxic concentrations of
CBD were then used in Mitoxantrone (MX) (substrate of P-gp and
BCRP) uptake assay. Cells were preincubated with/without CBD for
30min, followed by MX addition and further incubation for 30min.
Nicardipine was used as P-gp inhibitor. CBD 25 and 10 lM signicantly
inhibited BCRP dependant MX efux in MCF-7, BeWo and Jar cells
(for 10uM 40%, 25% and 22%, respectively). Nicardipine inhibition of
MX efux in Jar and BeWo cells was signicantly lower compared to
CBD inhibition of MX efux, indicating that CBD inhibited both BCRP
and P-gp dependant MX efux. We showed that BCRP and P-gp efux
function can be inhibited after only 1h exposure to CBD. These ndings
indicate that marijuana consumption during pregnancy (even at the very
early stages of gestation) could jeopardize fetal wellbeing by inhibiting
active efux placental barrier transporters.
Paper No.: 2701

EFFECT OF ULVA LACTUCA IN LIVER CONSERVATION IN
WISTAR RAT MODEL
H Ferchichi, I Salouage, S Bacha, L Ouanes, M Lakhal, Anis Klouz
(National Center of Pharmacovigilance, Laboratory of Clinical Pharmacology, Tunis, Tunisia
The purpose of our work is to evaluate the extracts stemming from the
green seaweed Ulva lactuca to improve the organs conservation solution
against cold hepatic-ischemia effects in Wistar rats. Rats were randomized between 3 groups: - Sham group (shortly preserved): livers were
washed with 0.9% NaCl, and then placed in a preservation solution
(KrebsHenseleit) for 30 min at 4 C. - For the other two groups (no
treated and treated group), livers were preserved under the same conditions for 24 h. For treated group, Ulva lactuca solution was added to the
preservation solution at a concentration of 40 `g/ml. After liver conservation, we realized an extraction of mitochondria for the dosage of malondialdehyde (MDA) and to evaluate the mitochondrial respiration.
Transaminase levels (AST and ALT) were assessed in the preservation
liquid. A fragment of liver used for histological analysis. The results of
AST and ALT levels decrease signicantly in the treated group (201 and
163 U/I) compared with no treated group (716 and 466 U/I) respectively.
The amelioration percentage of lipoperoxidation inhibition was 15,21%
and the amelioration percentage of mitochondrial activity was 13%,
between treated and no treated groups. The necrotic areas were irreversible in the no treated group. Extensive vacuolar degeneration (reversible
lesion) was found in the treated group. The addition of the Ulva extract
in standard conservation solution permitted us to avoid deleterious effects
277
of the cold ischemia in Wistar rats. The use of this extract will improve
the graft quality before its transplantation.
Paper No.: 1522

ACETALDEHYDE STIMULATES IL-6 MRNA EXPRESSION
AND IL-6 PROTEIN SECRETION IN CULTURED RAT
ASTROCYTES
Polonca Ferk(1), L Sarc(2), B Wraber(3), M Lipnik-Stangelj(3)
(1) University of Maribor Faculty of Medicine, Department of Pharmacology and Toxicology, Maribor, Slovenia
(2) University Medical Centre, Poison Control Centre, Ljubljana,
Slovenia
(3) University of Ljubljana, Faculty of Medicine, Ljubljana, Slovenia
Several disorders in the CNS, related to excessive ethanol consumption,
seem to be associated with altered cytokine production. Astrocytes are
important source of several cytokines, including a pro-inammatory
cytokine IL-6, and signicantly contribute to the immune response in the
brain. Recent studies have shown that some of the ethanol effects are
mediated through acetaldehyde, the primary metabolite of ethanol. We
hypothesized that acetaldehyde could be, at least partly, responsible also
for ethanol immunomodulatory effects in the brain. Specically, we
investigated a possible inuence of acetaldehyde on the IL-6 mRNA
expression and IL-6 protein secretion in cultured rat astrocytes. Primary
cultures of cortical astrocytes were prepared from the brain of neonatal
Wistar rats. The cells were incubated with 1mM acetaldehyde, for
4 hours, 24 hours (acute exposure) or for 7 days (chronic exposure).
Total RNA was isolated and IL-6 mRNA expression assessed by quantitative real-time PCR. IL-6 protein secretion was determined using appropriate ELISA protocol. Acetaldehyde increased the IL-6 mRNA
expression after 4 hours of incubation and IL-6 protein secretion after
acute and chronic exposure. The IL-6 protein secretion was dose-dependent and time-dependent. Short-term exposure to acetaldehyde showed
higher stimulation of IL-6 protein secretion compared to long-term exposure. We conrmed that ethanol immune effects in the brain could be at
least partly due to its major metabolite, acetaldehyde. However, further
studies are needed to quantify the contribution of acetaldehyde to the
overall brain damage after ethanol exposure.
Paper No.: 2837

DISEASES
TRPA1 PLAYS AN IMPORTANT ROLE IN TNFA-INDUCED
MECHANICAL HYPERALGESIA
carried out using mixed ANOVA followed by the Sidak test. P < 0.05
was considered signicant. Animals received TNFa (10 pmol/paw, ipsilateral paw) and Tyrode (50 ll/paw, contralateral paw). TRPA1 involvement was analyzed in WT and TRPA1 knockout (TRPA1KO) mice and
CD1 mice treated with the selective TRPA1 receptor antagonist AP-18
(i.pl., 25 nmol/paw, co-injected with TNFa or intrathecal, prior to
TNFa). TNFa evoked a signicant bilateral hyperalgesia in WT mice
that lasted up to 7 days. In contrast, TRPA1KO mice exhibited signicant less hyperalgesia after TNFa injection. Additionally, both i.pl. and
intrathecal treatment with AP-18 signicantly prevented TNFa-induced
hyperalgesia in CD1 mice. Herein, these results show that TRPA1 is
important to the development of TNFa-induced mechanical hyperalgesia.
Thus, we present novel evidence of TRPA1 participation in pain processing. We suggest TRPA1 plays a role in maintaining inammatory hyperalgesia associated with TNFa. Keeble, J. et al. (2005) Arthritis Rheum.
52: 3248-3256. Russell FA, et al. (2009) Pain. 142: 264-274. This work
was supported by ARC and the BBSRC/Pzer.
Paper No.: 791

DISEASES
MEROPENEM CONTROLLED-RELEASE SYSTEM
Gonzalo Fernandez Govantes, JJT Duran, ST Duran
Universidad Complutense de Madrid, Farmacia y Tecnologa
Farmaceutica, Madrid, Spain
Surgical infections are one of the main problems in hospitals, not only
because they pose a risk for the patients, but also due to their economic
implications. The aim of this project is to obtain controlled-release systems of meropenem for implantation and use them as a local treatment in
surgery, as it is a broad spectrum antibiotic and it can prevent the colonisation of bacteria. The materials used are meropenem and polylacticcoglycolic acid, which is a biodegradable polymer. Initially, meropenem
was validated following the USP method, based on HPLC. Afterwards,
the polymer was atomized by spray drying, obtaining microspheres of
PLGA. Finally, tablets baring a small diameter were obtained by direct
compression (Murakami H et al, J. Controlled Release. 2000, 67:29-36).
Both microspheres and tablets have been assayed. The microsphere
assays included size, morphology and residual solvents by GC; and
weight, porosity, morphology, delivery assay, and other properties for the
tablets. It resulted that microspheres had a diameter of 10 lm, were
roughly spherical and had an insignicant proportion of dichloromethane. On the other hand, the tablets produced had diameter of 7 mm,
weight about 95 mg and provided an optimal delivery of meropenem. In
the future, these tablets will be assayed in laboratory animals. In conclusion, it is possible to obtain local delivery systems of meropenem with
favourable properties, which could provide higher efciency and lower
adverse effects than current therapies.
Elizabeth S Fernandes(1), FA Russell(1), D Spina(2), R Graepel(1),

C Gentry(3), J Keeble(4), S Bevan(3), SD Brain(1)
(1) Kings College London, Cardiovascular Division, London, UK
(2) Sackler Institute of Pulmonary Pharmacology, London, UK
(3) Wolfson Centre for Age-related Diseases, London, UK
(4) Kings College London, Pharmaceutical Sciences Division, London,
UK
The mechanisms linking TNFa and inammatory pain are unclear. Previously, we showed that TRPV1 is involved in TNFa-induced thermal hyperalgesia (Russell et al., 2009) and that TNFa levels are raised in a
model of CFA-induced joint inammation (Keeble et al., 2005). Here,
we investigated the contribution of TRPA1 receptors in inammatory hyperalgesia induced by intraplantar (i.pl.) TNFa injection. Mechanical hyperalgesia was assessed in both hind paws by using an automated Von
Frey system before (baseline) and after treatment. Statistical analysis was
Paper No.: 2714

ASPARAGUS OFFICINALIS REGULATES CHOLESTEROL
METABOLISM AND IMPROVES ANTIOXIDANT STATUS IN
HYPERCHOLESTEROLEMIC RATS
Aangeles Fernandez-Arche, D Garca -Gonzalez, R Saenz-Rodriguez,
R Puerta-Vazquez
University of Sevilla, Department of Pharmacology, Sevilla, Spain
Asparagus ofcinalis L. has a high antioxidant capacity, associated to its
total phenolic content. There are large population studies showing that
vegetables rich in antioxidants are associated with a low incidence of
278
cardiovascular diseases. Objetive: To evaluate the effect of Asparagus ofcinalis on plasma lipids levels in rats fed a cholesterol-rich diet. Methods: The rats in the control group were fed a standard laboratory diet; the
rats in the hypercholesterolemic group were fed a cholesterol-rich diet .
The rats in others groups were treated with freeze-dried asparagus at a
dose of 500, 250, and 125 mg/Kg of body weight /day, while being fed
the cholesterol-rich diet. At the end of treatment animals were sacriced
and the livers were collected and weighed. According to the standard
methods, total cholesterol (TC), triglicerides (TG), and high density lipoprotein cholesterol (HDLc) of the plasma, were analyzed. Low density
lipoprotein cholesterol (LDLc) and atherogenic index was calculated by
using Friedenwalds formula and the equation: TC/HDL-c, respectively.
Results: Asparagus, signicantly decreased the plasma level of TC
(P < 0.01) against the hypercholesterolemic group. LDLc values in the
asparagus groups were lower than the hypercholesterolemic group
(p < 0.05), HDL-c levels, a benecial effect was observed although the
increase was not signicative. TG was slightly reduced but the effect
was not signicant against the reference group. The antioxidant status
(catalase and superoxide dismutase activities) and protection against lipid
peroxidation was also improved.
Paper No.: 1157

CONGENITAL-NEONATAL HYPOTHYROIDISM IMPACTS
HEPATIC LIPID METABOLISM IN ADULTHOOD
Leandro Fernandez-Perez(1), R Santana-Farre(1), L Henrquez-Hernandez(1), C Bocos(2), N Kahlon(3), E Herrera(2), N Gunnar(3), P Parini(4), A Flores-Morales(3),
(1) University of Las Palmas de Gran Canaria, Department of Clinical
Sciences, Molecular and Translational Endocrinology Group, Cancer
Research Institute of The Canary Islands (ICIC), Spain
(2) University of San Pablo-CEU, Department of Biochemistry and
Molecular and Cellular Biology, Faculties of Pharmacy and Medicine,
Spain
(3) Karolinska Institute, Department of Molecular Medicine and Surgery,
Sweden
(4) Karolinska Institute Huddinge, Division of Clinical Chemistry,
Department ofLaboratory Medicine, and Molecular Nutrition Unit,
Center for Nutrition andToxicology, Sweden
Thyroid hormones (TH) are essential for development, growth, and
metabolism. If not treated immediately, congenital hypothyroidism (CH)
has a profound impact on physiology and can imprint neurological and
endocrine systems, which, in turn, led to mental retardation, growth
arrest, and metabolic disturbances. To test if CH may inuence liver gene
expression program in adulthood, pregnant rats were given the antithyroid drug methimazole between gestational day 12 and post-natal day
(PND) 30 to induce CH in male offsprings. Upon weaning at PND30,
exposure to MMI was ceased. On PND80, we analyzed changes in
serum and hepatic lipids and liver transcriptome to explore whether CH
inuences liver physiology in adulthood in comparison with age-matched
rats without CH (INTACT). Body weight of PND80 rats with CH was
reduced; however, serum levels of T3, cholesterol, and triglycerides
showed no signicant differences in relation to INTACT. In contrast,
hepatic content of fatty acids, triglycerides, cholesteryl esters, and phospholipids were reduced in the CH group. Liver transcriptome was signicantly modied by CH. The mRNA levels several genes associated with
metabolism and transport of lipids were signicantly modied and differently-expressed genes were mostly mapped into lipid metabolism GO
categories as well as PPARalfa and complement-coagulation cascades
signalling pathways. In summary, we provide in vivo evidence that CH
causes long-lasting effects on hepatic functions in part through changes
in transcriptome. This underscores the critical role of T3 during neonatal
life for maintaining a normal developmental program of growth and lipid
metabolism.
Paper No.: 1105

DISEASES
MODULATION OF TH2-CYTOKINES PRODUCTION AND
ADHESION MOLECULE EXPRESSION ON BONE MARROW
AND PERIPHERAL BLOOD EOSINOPHILS IN ALLERGIC
MICE BY INDUCIBLE NITRIC OXIDE SYNTHASE
Heloisa HA Ferreira(1), EH Pellaquini(1), LR Benetti(1), LGR Fernandes(2), WMSC Tamashiro(2)
(1) San Francisco University, Laboratory of Inammation Research, Sao
Paulo, Brazil
(2) UNICAMP, Department of Microbiology and Immunology, Campinas, SP, Brazil
We investigated the role of the inducible nitric oxide isoform (iNOS) in
the migration of eosinophils to the lungs and the mechanisms by which
NO exerts these effects. Results demonstrate that the peak of eosinophil
inltration into the lungs of ovalbumin-sensitised control (non-treated)
mice, seen at 48h after antigen challenge, was signicantly reduced by
treatment of allergic mice with iNOS inhibitor (1400W). Flow cytometric
analysis showed that treatment resulted in a signicant increase in Mac-1
expression (85% increases) on bone marrow (BM) eosinophils at 24h, as
compared to control mice. However, at 24h, 1400W decreased Mac-1
and VLA-4 expressions on eosinophils by about 40% and 76%, respectively, in the peripheral blood (PB). In contrast, increases in both Mac-1
and VLA-4 expressions (42% and 64%, respectively) were observed at
48h on PB eosinophils. These results show a temporal effect of NO upon
the expression and function of Mac-1, which is the chief adhesion molecule involved in eosinophil efux from the BM at 24h. In contrast, Mac1 and VLA-4 were involved in eosinophil mobilization from PB to lungs
at 48h after antigen challenge. Furthermore, 1400W signicantly reduced
lung tissue levels of IL-4 at 24 and 48h and IL-5, IL-13 and eotaxin at
48 and 72h after OVA-challenge. In conclusion, results suggest the
involvement of NO in the modulation of Th2-derived cytokines and
eotaxin secretion in the lungs and in the expression of adhesion molecules on BM and PB eosinophils that may inuence cell migration to the
lungs of allergic mice.
Support: FAPESP
Paper No.: 786

ANALGESICS
CHARACTERIZATION OF THE ANTINOCICEPTIVE AND
ANTI-INFLAMMATORY ACTIVITIES OF NICOTINAMIDE
AND ITS ISOMERS IN DIFFERENT EXPERIMENTAL MODELS
Wallace Ferreira, A Godin, L Rocha, R Vieira, E Nascimento, J Seniuk,
M Coelho
Federal University of Minas Gerais, Department of Pharmacology &
Pharmaceutic Products, Belo Horizonte, Brazil
Nicotinamide, member of the vitamin B3 family, presents antinociceptive
activity and there are also evidences of its anti-inammatory property
(CUZZOCREA, S et al., Life Sci., 1999; v. 65: p. 1297,). However, there
is not any information about effects induced by picolinamide or isonicotinamide, isomers of nicotinamide, on the nociceptive or inammatory
responses. To establish a structure-activity relationship, we have investigated the effects induced by the three isomers in models of nociceptive
and inammatory pain and edema. Female Swiss mice were used. Nicotinamide, isonicotinamide (500 or 1000 mg/kg) or picolinamide (62.5 or
125 mg/kg) were administered per os. The nociceptive response was
evaluated in the formalin model. In the model of carrageenan-induced
edema, each isomer was administered 1 h before and 2 h after the
inammatory stimulus. The motor activity was investigated using a
279
rota-rod apparatus. The results were analyzed by one way Anova followed by Newman-Keuls test (p < 0.05). Nicotinamide (1000 mg/kg)
inhibited the rst and second phases of the formalin model. Only the second phase of this response was inhibited by isonicotinamide (500 or
1000 mg/kg) or picolinamide (125 mg/kg). Nicotinamide (1000 mg/kg),
picolinamide (125 mg/kg) and isonicotinamide (500 or 1000 mg/kg)
inhibited the paw edema. None of the substances impaired the motor
activity of mice. This study shows that the antinociceptive and antiinammatory activities of nicotinamide extend to its isomers. Despite
some evidences of potential molecular targets, the mechanisms that contribute to these activities are still uncertain. The substances may prove to
be useful as anti-inammatory and analgesic drugs.
Paper No.: 787

ANALGESICS
CHARACTERIZATION OF THE ANTINOCICEPTIVE AND
ANTI-INFLAMMATORY ACTIVITIES OF NICOTINIC ACID
AND ITS ISOMERS IN DIFFERENT EXPERIMENTAL MODELS
Wallace Ferreira, A Godin, L Rocha, R Vieira, E Nascimento, J Seniuk,
M Coelho
Federal University of Minas Gerais, Department of Pharmacology &
Nicotinic acid is one of the two forms of the B3 vitamin. In addition to
being a nutrient, it is a clinically applied drug. In high doses, it has been
used as a lipid-lowering agent (BODOR, ET et al., Br. J. Pharmacol.,
2008; v. 153: p. 568,). Nicotinamide, the amide derivative of vitamin B3,
exerts a number of anti-inammatory properties (CUZZOCREA, S et al.,
Life Sci., 1999; v. 65: p. 1297,). Thus, we investigated the effects
induced by nicotinic acid and its isomers, picolinic and isonicotinic
acids, in models of nociceptive and inammatory pain and also edema,
as these effects have not been investigated. In the formalin model in
female Swiss mice, nicotinic (250 or 500 mg/kg), picolinic (62.5 or
125 mg/kg) or isonicotinic (250 or 500 mg/kg) acids were administered
per os, 1 h before the injection of the noxious stimulus. The evaluation
of motor activity was evaluated in the rota-rod. When paw edema was
induced by carrageenan, each isomer was administered 1 h before and
2 h after the inammatory stimulus. Results were analyzed by one way
Anova followed by the Newman-Keuls test. The results show the antinociceptive activity of nicotinic and picolinic acids and also the anti-inammatory activity of nicotinic acid. The study shows the structure-activity
relationship of the molecules. Despite the lack of precise information of
its mode of action, nicotinic acid is safe, approved for clinical use, and
represents a potential analgesic and anti-inammatory agent. More
research is needed to elucidate its mechanisms of action.
Paper No.: 1790

BEER POLYPHENOLS AGAINST HUMAN GLIOBLASTOMA:
EFFECTS OF XANTHOHUMOL ON T98G CELL APOPTOSIS
Michela Festa, A Capasso, L Parente, CW DAcunto, C Pizza, S Piacente
(1) University of Salerno, Department of Pharmaceutical Science, Fisciano, Salerno, Italy
Glioblastoma multiforme (GBM) is the most common primary brain
tumor in humans and has a median survival of 6 months from the time
of diagnosis. The lack of effective current therapy, including surgery,
radiation and chemotherapy, has not signicantly improved the prognosis
of patients with GBM (Terzis et al, Opin Biol Ther. 2006; 6:739-749).
Thus, new therapeutic strategies need to be explored. Xanthohumol
(XH), a prenylated chalcone of the hop plant (Humulus lupulus L.), has
shown cancer chemopreventive, antiproliferative and anticancer activities
in several human malignancies (Zanoli & Zavatti, J Ethnopharmacol.
2008; 116:383-96). The aim of this study was to investigate the effect of
XH on human glioblastoma T98G and U87MG cells. Xanthohumol
induced apoptosis in concentration-dependent manner in T98G but not in
U87MG cells. XH induced apoptosis in T98G cells by elevating intracellular oxidative stress generating reactive oxygen species (ROS). XH
treatment resulted in activation of the mitogen-activated protein kinase
(MAPK) ERK1/2, and pretreatment with an ERK inhibitor PD98059 partially reduced XH-induced apoptosis. We found that XH-mediated production of a large amount of ROS caused activation of ERK1/2 and
changes in mitochondrial membrane potential. Pretreatment with the antioxidant N-acetylcysteine (NAC) strongly inhibited the XH-induced generation of ROS, phosphorylation of ERK1/2, and apoptotic cell death.
XH-induced apoptotic cell death was mediated by activation of the effectors caspases and cleavage of PARP-1 and was blocked by pretreatment
with Z-VAD-FMK (a pan-caspase inhibitor). These results suggest the
potential application of xanthohumol as a novel chemiotherapic agent for
the treatment of glioblastoma.
Paper No.: 2979

CENTRAL NERVOUS SYSTEM PATHOLOGY DURING
PREGNANCY. STUDY OF 25 CLINICAL CASES USING AN
EVIDENCE-BASED APPROACH
Isabel Vitoria Figueiredo(1,2), AC Rama(1,2,3), O Isabel(3),
F Tavares(4), T Santos(4), F Fernandez-Llimos(5),
MM Castel-Branco(1,2), A Cabral(1), M Caramona(1,2)
(1) University of Coimbra, Faculty of Pharmacy, Department of Pharmacology, Coimbra, Portugal
(2) University of Coimbra, Center for Pharmaceutical Studies, Coimbra,
Portugal
(3) Coimbra University Hospitals, Pharmacy Department, Coimbra,
Portugal
(4) Coimbra University Hospitals, Department of Maternal Foetal
Medicine, Coimbra, Portugal
(5) University of Lisbon, Faculty of Pharmacy, Department of Social
Pharmacy, Lisbon, Portugal
Central nervous system (CNS) pathology during pregnancy is becoming
more frequent among our pregnant women. Confronted with the complexity in managing foetal risk, efcient literature searching in addition
to the type and accuracy of clinical and medication data collection are
becoming crucial skills for evidence based practice as a support to clinical decision. Study of 25 cases of women with CNS diseases that were
pregnant or planned to be and were exposed to drugs. Assessment of
quality/utility of information provided to support clinical decision. Medical records analysis for pregnant, foetus and newborn follows-up. Evaluation of the parameters related to the likely effects described. Clinical
queries (2005-2009) of 25 cases were examined. 23 pregnants: 6 with 512/weeks and 17 with 13-24/weeks of gestation. Ages ranged from 1639 years. Pathologies occurring more frequently: depression, anxiety and
epilepsy. Associated conditions: hypertension, hypercholesterolaemia,
asthma. They were exposed to 33 CNS drugs, alone or in association
(with higher prevalence of diazepam, paroxetine and alprazolam) and to
22 other drugs. Information provided was considered reliable, accessible,
complete and applicable and used for clinical decision in all cases. There
was the possibility of establishing a causal relationship in 5 cases, where
newborns had abstinence and/or oppy child syndromes. One quarter of
pregnant women came to us in the rst trimester. There were 5 newborns
in 16 presenting withdrawal syndrome and oppy child syndrome. We
propose to create a structured formulary for collecting information oriented to medication, disease and clinical situation of the pregnant, foetus
and newborn.
280
Paper No.: 1633
ADVERSE DRUG REACTIONS IN INFANTS AND CHILDREN
Houda Filali, A Elgharbi, F Hakkou
Ibn Rochd Hospital, Department of Pharmacology, Casablanca, Morocco
The cause of iatrogenic accidents drug preferentially affects extreme
ages. Objective: To analyze the case reports concerning children and
infants (18 years or younger) in Ibn Rochd Hospital Materials and methods: Cases report of adverse drug accidents (ADRs) in patients aged 18
and under are analyzed in a retrospective study of 1 year (Sept.2008Sept.2009). Finding: Eighty-two ADRs reported, representing 23.4% of
all ADRs collected. The main events are 24 toxidermas, 24 digestive disorders, 10 haematological and neurological disorders, 6 disturbances of
general condition, 5 events nephro-urological. We also identied 14
overdose caused by therapeutic errors. The major drug makers are antiinfectives (37.8%), antineoplastics (23.1%), anti-inammatories (14.6%),
drugs of neuro-psychiatric (10%) with antiepileptic (6%). The high number of daily medications promotes the risk of drug interactions responsible for iatrogenic injuries in 14.6% of patients in this study. Off label
prescriptions were identied in 10.9% of cases. Conclusions: Adverse
drug reactions are not common in paediatric patients, and most are mild.
However, due to limitations of clinical trials in children, pharmacoepidemiological studies may be the only source of information on the benetrisk prole of drugs received by these patients, and as such require special attention.
Paper No.: 1634

CAUSES OF THERAPEUTIC ERRORS IN OLDER ADULTS
Houda Filali, H Elkarimi, D Mzah, A Miftah, F Hakkou

Introduction: The Sweet syndrome or acute febrile neutrophilic dermatosis is a table whose toxidermie stereotype is that of fever, leukocytosis with neutrophils, a rash made with painful plaques and dense
dermal inltration with neutrophils histologically. The diagnosis is
based on a series of arguments. The forms of the drugs heal after
stopping the drug responsible. Observations: We report two cases of
Sweets Syndrome Drug: the rst is a woman of 52 years treated for
10 days amoxicillin-clavulanate for furunculosis foot. 2 days after
completion of treatment, she presented an eruption febrile generalized
erythemato-papular. Laboratory studies showed a major inammatory
syndrome with leukocytosis with neutrophils. Histological examination
of a skin biopsy conrmed the diagnosis of Sweets syndrome. Systemic corticosteroids led to the disappearance of the clinical and biological symptoms. The second observation concerns a woman aged
60, also treated with amoxicillin-clavulanate for angina. At days 5 of
the treatment, she developed papulopustular rash erythematous conuent bunched on the hands and feet. Laboratory tests and histology
were similar to the rst case. The evolution was marked regression of
lesions after steroid therapy. Analysis and discussion: In two cases,
Sweets syndrome is well dened criteria of Sweets syndrome drug
proposed by Walker and Cohen. In addition, the study of accountability inherent in accordance with the WHO method has proved plausible
for the two cases, based on chronological criteria C2 and semiotic S2,
while the extrinsic accountability found a score B1 not known for the
amoxicillin-clavulanate.
Paper No.: 1350

THE SCAFFOLD PROTEIN NHERF2 DETERMINES
NEURONAL ION CHANNEL COUPLING OF GLUTAMATE
MGLUR5 AND NUCLEOTIDE P2Y1 RECEPTORS
Alexander K Filippov(1), J Simon(2), EA Barnard(2), DA Brown(1)

Objectives: To evaluate the reasons of unintentional therapeutic errors in
older people, the types of drugs most frequently involved, and medical
outcomes associated with these adverse drug events. Materials and methods: Retrospective analysis of cases reported between 2005 and 2009 at
pharmacovigilance center of hospital Ibn Rochd, involving adults aged
65 and over with a potentially toxic exposure due to inadvertent errors
therapy. Results: Of the 65 notications in older adults who reported
treatment errors, including 27 cases have been followed by a known
medical outcome. A major effect or death occurred in 16 cases. The most
common reasons for therapeutic errors were made inadvertently or medication administered twice, wrong drug given or taken, and the other
wrong dose. The reasons for the higher rate of major impact or deaths
were drug interactions, health professional or iatrogenic errors, and more
than one product containing the same ingredient. Certain classes of drugs
such as analgesics, anticoagulants, psychotherapy and some cardiovascular drugs were associated with high risk factors. Conclusion: interest to
evaluate the therapeutic errors in older adults to determine the causes of
errors related to the most frequently reported, and the reasons and drugs
involved with errors that result in serious consequences. Knowing why
they occur can help Causes of Therapeutic Errors in Older Adults.
Paper No.: 1635

DISEASES
THE SWEET SYNDROME : TWO CASES INDUCED BY
AMOXICILLIN- CLAVULANIC ACID
(1) University College London, Department of Neuroscience, Physiology

and Pharmacology, London, UK
(2) Cambridge University, Department of Pharmacology, Cambridge, UK
Expressed mGluR5 and P2Y1 receptors show promiscuous ion channel
coupling in sympathetic neurons: their stimulation inhibits M-type (Kv7,
K(M)) potassium currents and N-type (CaV2.2) calcium currents
(Kammermeier & Ikeda, Neuron 1999; 22: 819; Brown et al., J.Aut.Nerv.Syst. 2000; 81: 31). These effects are mediated by Gq and Gi/o
G proteins respectively. In contrast, activation of endogenous mGluR5
and P2Y1Rs in hippocampal pyramidal cells inhibited K(M)-current
(Filippov et al., J. Neurosci. 2006; 26: 9340), but not CaV (unpublished).
We ask why the difference? One possibility is that it relates to the differential distribution of the mGluR5/P2Y1R-binding scaffold protein
NHERF2 (Fam et al, PNAS 2005; 102: 8042; Paquet et al., JBC 2006;
281: 29949). Thus, we found that hippocampal neurons showed strong
NHERF2 immunoreactivity whereas sympathetic neurons showed none.
We therefore tested the effect of expressing NHERF2 (by intranuclear
cDNA injection) on the effects of mGluR5 and P2Y1R activation on
CaV2.2 and K(M) currents in sympathetic neurons. NHERF2 co-expression did not affect the amount of M-current inhibition produced by either
receptor but strongly reduced CaV inhibition by mGluR5 activation
(from 18 2.8%, n = 11 to 4 1.2%, n = 10), and by P2Y1R activation
(from 38 4.3%, n = 10 to 10 3.0%, n = 10). NHERF2 expression
had no signicant effect on CaV inhibition by norepinephrine (via a2adrenoceptors, which do not bind NHERF2), or on CaV inhibition produced by an expressed P2Y1 receptor lacking the NHERF2-binding
DTSL motif. Thus, NHERF2 selectively restricts downstream coupling
of mGluR5 and P2Y1Rs in neurons to Gq-mediated responses such as
K(M) inhibition.
Support: Wellcome Trust grant 090370.
Houda Filali, I Rahmoun, A Elgharbi, F Hakkou

281
Paper No.: 687
THE EFFECT OF CENTRALLY INJECTED GLY-L-GLN
(GLYCYL-GLUTAMINE) ON NUCLEUS ACCUMBENS GABA
OUTPUT AFTER ACUTE MORPHINE ADMINISTRATION
Nesrin Filiz Basaran(1), S Cavun(1), RL Buyukuysal(1),
WR Millington(2)
(1) Uludag University, Faculty of Medicine, Department of Medical
Pharmacology, Bursa,Turkey
(2) Albany Collage of Pharmacy, NY, USA
Substance abuse and dependence is growing health problem all around
the world. At present still there is not effective and accurate treatment for
substance addiction. The most important site of action of all addictive
drugs is mesolimbic dopaminergic pathway. We previously showed that
Gly-Gln, a dipeptide synthesized by proteolytic clevage from endogenous b-endorphin, blocked the positive reinforcing and rewarding effects
of morphine, also delayed tolerance to morphine, inhibited morphine
addiction and suppressed withdrawal syndrome symptoms. After this
results we have focused on the possible mechanism of action of Gly-Gln.
We rst studied on dopamine and its metabolite DOPAC. After swowing
inhibitor activity on morphine evoked dopamine release in nucleus
accumbens we tested Gly-Glns effect on GABA release. In this study
we used 200-300g Sprague Dawley male rats. Microdialysis probes were
placed in to Nacc (bregma 0 point, AP:+1.8; L:-1.0; V:-6.8) and intracerebroventricular (ICV) canulla to left lateral ventricul (bregma 0 point,
AP:-1.6; L:+0.9; V:-4.0) for injection Gly-L-Gln. Gly-L-Gln was administered 2 minutes before morphine injection. Morphine injection was performed via intraperitoneal (IP) canulla. Our results showed that after
morphine (2.5 mg/kg, IP) injection, Nacc GABA output start to increase
in 20. minute about 426% of baseline values and goes signicantly high
during rst hour. Gly-L-Gln (100 nmol/5ll ICV) blocked this increased
GABA release signicantly. The effect of Gly-L-Gln on addiction and
rewarding impact of morphine, nicotine and ethanol is known. Gly-LGln is thought to show this effect also by altering GABAergic tonus
besides dopamine in Nacc.
Key-words: Gly-Gln, Morphine, GABA, Nucleus accumbens, Microdialysis,
_
BITAK
Supported by TU
and Uludag University (BAP)
Paper No.: 1530

CONTRIBUTION OF CYP2C8 TO THE METABOLISM OF
MONTELUKAST IN VITRO
Anne Filppula, J Laitila, PJ Neuvonen, JT Backman
University of Helsinki and Helsinki University Central Hospital, Department of Clinical Pharmacology, Helsinki, Finland
The leukotriene receptor antagonist montelukast is extensively metabolised. The majority of its oral dose is excreted in bile, mainly as a secondary metabolite formed by oxidation of metabolite 6 (M6). In vitro
studies with high montelukast concentrations have shown that cytochrome P450 (CYP) 3A4 catalyses montelukast sulfoxidation (M2
formation) and 21-hydroxylation (M5), and CYP2C9 catalyses 36hydroxylation (M6) (Chiba et al. Drug Metab Dispos 1997;25:10221031). We have studied the contributions of different CYP enzymes,
particularly the contribution of CYP2C8, to the hepatic microsomal
metabolism of clinically relevant montelukast concentrations (0.021 lM) in vitro. The effects of CYP isoform inhibitors on the metabolism
of montelukast were examined using pooled human liver microsomes
(HLM). In addition, montelukast metabolism by a range of human
recombinant CYP isoforms was investigated. Both inhibition and recombinant enzyme studies veried the central role of CYP3A4 in the formation of M2 and M5b (the more abundant diastereomer). Recombinant
CYP3A5 also mediated M2 and M5b formation. However, the CYP2C8

inhibitors trimethoprim and gembrozil 1-0-b glucuronide inhibited the
depletion of 0.02 lM montelukast and formation of M6 more potently
than did the CYP2C9 inhibitor sulfaphenazole. Similarly, M6 formation
was more effectively catalysed by recombinant CYP2C8 than by
CYP2C9 at low montelukast concentrations. At <0.1 lM montelukast,
the contribution of CYP2C9 was minimal, less than 10% of that of
CYP2C8. Moreover, recombinant CYP2C8 also mediated the further
metabolism of M6. In conclusion, CYP2C8 plays a major role in the
main metabolic pathway of montelukast at clinically relevant montelukast concentrations in vitro.
Paper No.: 2265

THE CONSERVED PROLINE OF HELIX 6 PLAYS A ROLE IN
THE FOLDING AND EXPRESSION OF A1B AND
B2 ADRENERGIC RECEPTORS
Angela M Finch, SJ Humphrey, AP Campbell
University of New South Wales, School of Medical Sciences, Department of Pharmacology, Sydney, NSW, Australia
It has been proposed that prolines within transmembrane a-helices play a
role in the regulation of protein folding or alternatively have dynamic
structural roles as hinges, pivots or switches. To investigate the role of
proline in the transmembrane a-helices of G-protein coupled receptors
(GPCRs), we focused on the conserved proline of helix 6 (designated
P6.50 according to the Ballesteros-Weinstein numbering scheme) of the
Family A GPCRs. We mutated P6.50 of the a1B and b2 -adrenergic
receptors (AR). Mutation of P6.50 to alanine or glycine in the a1B-AR
resulted in no change in agonist binding or receptor activation; however,
receptor expression was halved. In contrast in the b2-AR these mutations
resulted in a 75-85% loss in receptor binding sites (Bmax wildtype:
25.1 4.5; P6.50A: 3.8 0.7, P6.50G: 6.3 1.2 pmol/mg, n = 4-6).
The b2-AR mutants had unaltered [3H]-DHA antagonist binding afnities
(KD wildtype: 1.78 0.66; P6.50G: 1.03 0.37; P6.50A: 2.35 0.68,
nM, n = 4-6) however they had signicantly increased agonist (epinephrine) afnity (Ki wildtype: 16.72 2.0; P6.50G: 6.90 0.79; P6.50A:
2.80 0.76 lM, n = 4, p < 0.05). These data suggest that the mutant
receptors may favour an active conformation. The use of a pharmacological chaperone (propanolol 100nM) lead to a substantial recovery of surface expression of both b2-AR mutants. The chaperoned b2-AR mutants
displayed wildtype agonist and antagonist binding properties. These ndings support the notion that P6.50 is critically involved in folding and
expression of the b2-AR, however once the receptors are inserted into
the plasma membrane P6.50 is not required for maintaining the structural
integrity of the receptor.
Paper No.: 2266

VIRTUAL SCREENING USING A1ADRENERGIC RECEPTOR
PHARMACOPHORES IDENTIFIES A NOVEL STRUCTURAL
FRAMEWORK FOR A1A/D-ADRENERGIC RECEPTOR
SELECTIVE LIGANDS
Angela M Finch(1), ES Stoddart(1), IJA MacDougall(2), R Grifth(1,2)
(1) University of New South Wales, School of Medical Sciences, Department of Pharmacology, Sydney, NSW, Australia
(2) The University of Newcastle, School of Environmental and Life
Sciences, Australia
Pharmacophores are in silico tools for drug discovery that represent an
exciting opportunity to increase the efciency and reduce the cost of
282
identication of hit compounds. In this study a1- adrenergic receptor
(AR) pharmacophores were used to identify novel a1-AR selective compounds. Using ve-feature pharmacophores for selective antagonists at
each of the a1-AR subtypes the National Cancer Institute (NCI), Tripos
LeadQuest (TDD) and Maybridge 2004 databases were mined. 12 compounds from these databases were selected, based on diversity of structure, predicted high afnity and selectivity at the a1D- subtype compared
to a1A- and a1B-ARs. The selected compounds were subsequently tested
in vitro for afnity and efcacy. 9 out of 12 of the tested compounds displayed afnity at the a1A and a1D -AR subtypes and 6 displayed afnity
at all three a1-AR subtypes, no a1B-AR selective compounds were identied. These results represent a high hit rate, indicating our pharmacophores can successfully screen for ligands with a1-AR afnity. The
efcacy of the compounds that displayed afnity for the a1-AR subtypes,
measured as total inositol phosphate production, indicate 8 of the 9 compounds with a1-AR afnity displayed antagonist efcacy, with one compound (NCI-2) displaying partial agonist characteristics (pEC50
5.26 0.15 M, n = 3). This virtual screen has successfully identied an
a1A/D-AR selective antagonist (NCI-5), with low lM afnity (pKi, a1A
5.75 0.14, a1B 4.96 0.12, a1D 5.82 0.1, n = 3) with a novel structural scaffold of a an isoquinoline ring system with a rigid four ring
structure and good lead-like qualities ideal for further drug development.
Paper No.: 2909

HEALTH AND DISEASE
PROSTAGLANDIN E2 PROMOTES ANGIOGENESIS BY
SYNERGIZING WITH FIBROBLAST GROWTH FACTOR
RECEPTOR-1/FIBROBLAST GROWTH FACTOR-2 SYSTEM
Federica Finetti, S Donnini, L Morbidelli, A Giachetti, M Ziche,
University of Siena, Department of Molecular Biology, Siena, Italy
Prostaglandin E2 (PGE2), the major product of cyclooxygenase, has been
shown to promote angiogenesis, however the functional and molecular
mechanism by which PGE2 induces this process is not completely understood. We investigated the PGE2 effects on the pro-angiogenic broblast
growth factor receptor-1 (FGFR-1)/broblast growth factor-2 (FGF-2)
signalling in cultured microvascular endothelial cells and in ex vivo and
in vivo assays. We demonstrated that PGE2 synergizes with FGF-2
and activates FGF-2 pathway. In particular PGE2 promotes FGFR1 and
FRS2a (FGFR substrate 2a) phosphorylation, endothelial nitric oxide synthase, the MAPK ERK 1/2 and the transcription factor STAT-3 activation
and FGF-2 protein up-regulation. In cells KO for FGF-2, PGE2 loses the
pro-angiogenic activity supporting the key role played by FGFR1/FGF-2
signalling cascade in PGE2 effects. FGF-2 over-expression, in turn, activates an autocrine/paracrine cycle in endothelial cells, which controls cell
survival and proliferation. In vivo, in the rabbit cornea and Matrigel plug
assay, the synergism between PGE2 and FGF-2 promotes a robust angiogenesis. In conclusion we demonstrated that the synergistic interaction of
PGE2 and FGF-2 promotes the angiogenic switch through activation of
the autocrine/paracrine FGF-2/FGFR1 signalling cascade. The synergism
between the PGE2 and FGF-2 signalling pathways here described may
substantiate the use of antitumor drug combinations, the most notable
being cyclooxygenase inhibitors with growth factors or growth factor
receptor inhibitors.
Paper No.: 1017

COMPARISON OF GABA-A IN VIVO OCCUPANCY IN RAT
BRAIN AND SPINAL CORD
GABA-A receptor occupancy has been validated clinically using [11C]

umazenil PET imaging in human brain (Van Laere, Biol Psychiatry
2008; 64: pp153-161) and preclinically using [3H]umazenil binding in
rodents (Atack et al, Neuropsychopharmaology 1999; 20: 255-262).
Technically PET imaging in the spinal cord can be more challenging than
using brain. This aim of this study was to compare GABA-A in vivo
receptor occupancy in rat whole brain vs spinal cord in order to determine if brain occupancy can be used as a surrogate for spinal cord occupancy when the spinal cord is the target tissue. Male CD rats (200-250g,
Charles River) were dosed orally with vehicle, and a range of GABA-A
selective ligands including SL651498 (3-100mg/kg), TPA023B (0.110mg/kg), or TP-13 (0.1-10mg/kg). At 3 min prior to sacrice all rats
received, an iv dose of [3H]umazenil (10lCi/kg). At 60 min rats were
sacriced, brain/spinal cord removed, homogenised and ltered over GF/
B lters by washing with 2x5mls ice cold buffer. Non specic binding
was determined in a separate group of rats pre-treated with bretazenil
(5mg/kg, i.p. for 30 min). Dose - occupancy curves have shown similar
Occ50 values (a dose that produces 50% receptor occupancy) in rat
whole brain versus spinal cord for SL651498 (194 and 165mg/kg respectively), TPA023B (0.09 and 0.1mg/kg respectively), TP-13 (3.7 and
1.9mg/kg respectively). This data from a range of GABA-A ligands provides condence that whole brain PET may be used as a surrogate for
spinal cord occupancy in translatable human studies.
Paper No.: 2281

MULTI-TARGET DRUGS FOR COGNITIVE IMPAIRMENT
THERAPY: IN VITRO INHIBITION OF HISTAMINE H3
RECEPTORS AND CHOLINESTERASE BY ORIGINAL
DONEPEZIL HYBRIDS
L Flammini(1), S Bertoni(1), C Giorgio(1), M Tognolini(1),
Vigilio Ballabeni(1), G Morini(2), M Incerti(2), E Barocelli(1)
(1) University of Parma, Department of Pharmacological, Biological
Sciences and Applied Chemistries, Parma, Italy
(2) University of Parma, Pharmacy Department, Parma, Italy
Introduction: A new paradigm called multi-targetdirected ligand
design is emerging for the development of new successful drugs for the
treatment of neurodegenerative diseases such as Alzheimers disease. A
current treatment of this multifactorial syndrome is based on acetylcholinesterase inhibitors endowed with a poor ability to alter disease progression and coupled with severe adverse side effects. The selective
blockade of histamine H3 receptors, auto- and heteroreceptors negatively modulating the synthesis and release of histamine and other neurotransmitters (such as acetylcholine) in different brain areas, has been
proven to ameliorate learning and memory in experimental models.
Therefore, some hybrid molecules have been developed combining the
structure of Donepezil with that of new H3 antagonists, to meet the goal
for development of original neuroprotective agents with improved tolerability. Materials: Donepezil and ve new hybrids were evaluated in vitro
for their afnity (pKi), antagonistic potency (pKB) at rodent and human
histamine H3 receptors and for their ability to inhibit rat brain cholinesterase activity (pIC50). Results: Donepezil that inhibits cholinesterase
(pIC50 = 7.45) shows a two order of magnitude lower H3 binding afnity
(pKi = 5.30) than hybrids. All the new hybrids were potent H3 antagonists (pKB from 7.47 to 8.10; pKi from 7.34 to 8.18) exhibiting lower
anti-cholinesterase activity (pIC50 from 5.83 to 6.09) than Donepezil. No
species-specic differences were detected between human and rat histamine H3 receptor afnity. Conclusion: From this study the compound
M122, possessing a quite balanced H3 antagonistic/anti-cholinesterase
activity, can be selected for further in vivo studies.
Rebecca Fish, S Able, C Powell, L Booth

283
Paper No.: 750
ACUTE ORAL AND ACUTE DERMAL TOXICITY STUDY OF
DIOSCOREA HISPIDA (NAMI) AQUEOUS PLANT EXTRACT IN
SPRAGUE-DAWLEY RATS
Mona Liza Flores(1,2)
(1) University of the Philippines College of Medicine, Department of
Pharmacology and Toxicology, Manila, The Philippines
(2) Philippine Council for Health Research and Development - Department of Science and Technology, Manila, The Philippines
Dioscorea hispida, popularly known as nami in the Tagalog regions,
belongs to the family Dioscorecae. It has a wide variety of species found
growing in different countries and is widely distributed in the Philippines. Nami is believed to possess therapeutic potentials and is used for
various purposes such as medication to pains, bilious colic, nausea of
pregnancy, for spasmodic conditions, prevention of miscarriage, anodyne
and maturative in cases of tumors and buboes, to reduce swelling due to
insect bites or jelly sh sting, and as foodstuff and was found to have
sedative effects. Tubers of nami contain the alkaloid dioscorine responsible for its toxicity that can result to death. The activity of the Dioscorea
species has been attributed to the presence of and the actions of various
steroidal saponins (diosgenin an aglycone), alkaloids (dioscorine, dioscine), and other alkaloids which are derived from nicotinic acid. In this
study, nami was prepared by maceration and the aqueous extract was
lyophilized. Administration and application of the plant material on sprague-dawley rats follows the OECD guidelines for testing of chemicals Up-and-Down-Procedure Guideline 425 for acute oral test and FixedDose-Procedure Guideline 434 for acute dermal test. Preliminary acute
toxicity test showed depression as the major toxidrome observed for the
live test subjects dosed between 175 mg/kg and 375 mg/kg. Mortality
was seen at 470 mg/kg. Acute dermal toxicity test showed no evident
dermal toxicity at the dose of 200 mg/kg.
Paper No.: 3147

POPULATION PHARMACOKINETICS OF OSELTAMIVIR
CARBOXYLATE IN MEXICAN PATIENTS WITH INFLUENZA
A(H1N1)
Francisco Flores-Murrieta(1,2), M Barranco-Garduno(1),
M Carrasco-Portugal(1), H Leon-Molina(1), A Cervantes-Nevarez(1),
G Castaneda-Hernandez(3)
(1) Instituto Nacional de Enfermedade Respiratorias, Mexico City,
Mexico
(2) Escuela Superior de Medicina del Instituto Politecnico Nacional,
Mexico City, Mexico
(3) CINVESTAV-IPN, Mexico City, Mexico
The orally active neuraminidase inhibitor oseltamivir has shown to be
effective against the 2009 (H1N1) virus, being widely used for both,
treatment and prophylaxis. Oseltamivir is readily absorbed from the gastrointestinal tract and then extensively biotransformed to its active metabolite, oseltamivir carboxylate (OC). OC is minimally bound to plasma
proteins exhibiting a volume of distribution (Vd) equivalent to the extracellular volume of body water. OC is eliminated by renal clearance (CL)
by both, glomerular ltration and tubular secretion. Almost all this information has been generated in healthy volunteers or patients with mild
inuenza, being data on severe ill patients very scarce. The purpose of
this study was to evaluate OC pharmacokinetics in adult hospitalized
patients diagnosed with inuenza A(H1N1) using a population approach.
Plasma samples from 15 healthy volunteers and from 77 patients receiving 75 or 150 mg b.i.d. were obtained and analyzed by HPLC. Vd and
CL of OC were respectively increased and reduced, compared with
healthy volunteers. Notwithstanding, therapeutic plasma concentrations

were achieved with 75 mg bid. This was observed regardless of BMI or
if oseltamivir was given via nasogastric tube. Hence, higher doses are
not required for obese of severely ill patients. Moreover, oseltamivir
combinations with probenecid are not justied, as patients already exhibit
a reduced CL. Our data support current recommendations for oseltamivir
dosing in adult patients with inuenza A(H1N1), and point that higher
doses are not rational from a pharmacokinetic point of view.
Paper No.: 2844

SYNERGISTIC INTERACTION BETWEEN PPAR AGONISTS
AND ALBUTEROL ON HUMAN BRONCHIAL SMOOTH
MUSCLE CELL PROLIFERATION
Stefano Fogli, L Picchianti, F Stefanelli, MC Breschi
University of Pisa, Department of Psychiatry, Neurobiology,
Pharmacology & Biotechnology, Pisa, Italy
An important aim in asthma therapy is to prevent the accelerated growth
of bronchial smooth muscle cells (BMSC) which leads to hyperplasia
and bronchial hyperreactivity. In this study, we investigated the antiproliferative effect of the combination of albuterol with different PPAR-c agonists on human BSMC maintained in an optimized medium containing
epidermal growth factor and basic broblastic growth factor. RT-PCR
and Electrophoretic Mobility Shift Assay were used to assess PPAR-c
gene and protein expression, respectively. Apoptosis and necrosis were
assessed by ELISA. Synergism or antagonism was quantied by the
combination index (CI) method (Chou TC et al, J Natl Cancer Inst 1994;
86: 1517-1524). PPAR-c gene was highly expressed in BSMC and the
protein was identied in cell nuclei. Stimulation of BSMC with growth
factors for 48 h induced a 4-fold increase in cell proliferation which was
signicantly prevented by single-agent albuterol, rosiglitazone or 15deoxy-d 12, 14-prostaglandin J2 with IC50 mean values of 7.37 1.75,
8.52 1.26, and 10.1 1.37 mM, respectively. Selective b2-adrenoceptor (b2-AR) and PPAR-g antagonists dose-dependently block the antiproliferative effect of drugs. Combination of albuterol with PPAR-g agonists
at nanomolar concentrations, allows obtaining the 50% growth inhibition
at levels from 2 to 10-fold lower than those required for each drug alone
(CI <1), without induction of apoptosis or necrosis. These data indicate
the presence of a synergistic interaction between PPAR-g agonists and
bronchodilators on airway smooth muscle cell proliferation, thus supporting the therapeutic potential of this combination in chronic airway
diseases.
Paper No.: 3181

MONTELUKAST REVERSES ALBUTEROL-INDUCED
B2-ADRENOCEPTOR TOLERANCE IN AIRWAY SMOOTH
MUSCLE
Stefano Fogli, F Stefanelli, L Picchianti, MC Breschi
University of Pisa, Department of Psychiatry, Neurobiology, Pharmacology & Biotechnology, Pisa, Italy
The regular use of b2-adrenoceptor (b2-AR) agonists has been associated
with proasthmatic-like changes that limit their efcacy and potentially
increase the risk of asthma exacerbation (Spitzer WO et al, N Engl J
Med 1992; 326: 501-506). We sought to determine whether montelukast
prevents homologous b2-AR desensitization in tracheal muscular tissues
derived from guinea-pigs and in human bronchial smooth muscle cells
(BSMC). The guinea-pig model of albuterol-induced b2-AR tolerance
described in the present study closely resembles the clinical setting since
284
it was characterised by b2-AR agonist hyporesponsiveness (-25%) and
hyperreactivity to cholinergic stimuli (+53%). Montelukast, given intraperitoneally at 5 mg/kg/d for 6 consecutive days, completely reversed
albuterol-induced b2-AR tolerance in homologously desensitised tracheal
smooth muscle, without signicantly affecting the cholinergic constrictor
response. In vitro ndings demonstrated that albuterol-induced cAMP
stimulation was signicantly reduced in homologously desensitised
BSMC (P < 0.001), in the absence of changes in b2-AR mRNA levels.
In this experimental setting, montelukast 30 lM for 24 h was able to
reverse albuterol subsensitivity induced by homologous desensitization
(P < 0.01). No substantial change in b2-AR transcription was observed
in desensitised cells, as compared with those treated with montelukast.
Overall, our data suggest the presence of a positive pharmacodynamic
interaction between montelukast and b2-AR agonists, thus supporting
the use of this combination to improve the therapeutic index of
bronchodilators.
Paper No.: 1406

EXERCISE INFLUENCE ON THE
AMPHETAMINE-DEPENDENT BEHAVIOUR AND DOPAMINE
IN STRIATUM, FRONTAL CORTEX AND HIPPOCAMPUS
Carlos Alberto Fontes-Ribeiro, E Marques, F Pereira, AP Silva,
T Macedo
University of Coimbra Faculty of Medicine, Institute of Pharmacology
and Experimental Therapeutics, Coimbra, Portugal
The dopaminergic mesocorticolimbic and nigrostriatal systems are considered to be involved in drug addiction. However, there is little information about the effect of repeated exercise on the synthesis, metabolism
and action of dopamine (DA) in the central nervous system, in the presence of amphetamines. Thus, our aims were to verify the inuence of
exercise on addiction using an amphetamine (AMPH)-induced conditioned place-preference (CPP) in rats, and to determine DA and metabolites in striatum, frontal cortex and hippocampus. Adult male SpragueDawley rats were trained under a 8 week treadmill running program.
The CPP test was performed in trained and untrained groups. Conditioning was performed for 8 days with i.p. injection of 2 mg.Kg-1 AMPH or
saline. Animals were sacriced 24 h after the last AMPH or saline, and
striatum, hippocampus and frontal cortex were dissected for DA and
metabolites measurement by HPLC-EC. Rats without exercise showed
preference for the compartment associated with AMPH, an effect which
did not occur with the animals with training. Amphetamine decreased
striatal DA content and turnover from both trained and untrained rats.
Training reduced and increased the DA content in frontal cortex and hippocampus, respectively. According to these results, exercise may prevent
the AMPH-seeking behaviour but not changes the impact of AMPH on
striatal DA synthesis. Data also suggest that exercise may correlate with
changes in DA dynamics in the frontal cortex and hippocampus.
Paper No.: 2321

MACRO-STUDY ON-LINE OF SEARCHING IN INTERNET
FOR HEALTH AND DRUG PRODUCTS: A SOCIAL
PHARMACOLOGY APPROACH
Juan Antonio Formigos(1), M Palmero(1), JL Alloza(2)
(1) University of Alicante, Department of Optic, Pharmacology and
Anatomy, San Vicente del Raspeig, Alicante, Spain
(2) University of Alcala, Department of Pharmacology - Social Pharmacology Group, Alcala de Henares, Madrid, Spain
Internet is a new stage of our civilization that provides any kind of information. In recent years searching on health and medical treatments is one
of the most widely used on the Internet. We have developed an epidemiological transversal and observational study, using a self-administrated
questionnaire on the Web, with free access at any time. The target was
the community of The University of Alcala de Henares, Madrid (Students, Lecturers and Researchers, and the administrative staff services),
over 30.000. 45.2% of Internet users have sought information about
medications on occasion. The searchers were characterized by size
of internet use (OR=2.43, 95%CI=1.97-2.99) related with a greater
condence in the contents of the network (OR=1.55, 95%CI=1.30-1.85).
It was more evident for those health related careers (OR=2.07,
95%CI=1.69-2.53), the chronically ill people (OR=1.41, 95%CI=1.091.82). The latter were more concerned about the medicines safety
(OR=1.33, 95%CI=1.09-1.62), and also read the prospectus over the rest
(OR=1.66, 95%CI=1.26-2.18). The knowledge provided by internet was
used positively by 21.3% in a doctors consultancy. Our results on the
internet and health care are applicable to the current European society.
This study supports that new social trends run in a similar way that has
been assessed in the US in recent years. Internet plays a denite role in
health care.
Paper No. 3423

SELF-ASSESSMENT WITH MOODLE FROM ACTIVITIES
PROPOSED BY STUDENTS
Juan Formigos-Bolea, M Palmero, V Maneu, C Garca-Cabanes
University of Alicante, Department of Optics, Pharmacology & Anatomy, Alicante, Spain FARMAGITE
Preliminary results of an experiment in the area of pharmacology. Background: The B-learning improves academic performance [1]. Blended
learning is very popular and well received among students [2]. Students
can learn through taking the role of a teacher [3]. Methods: The experience was performed in the subject of Pharmacology, Nutrition and Dietetics of the second year of the Nursing Degree during the 2009-2010
academic years at the University of Alicante (Spain). A total of 180 students enrolled were invited to perform self-assessment tests (J-click and
hot potatoes) into a Moodle platform. The activities and test questions
were proposed by students themselves. The role of teachers has been
limited to monitor the consistency of the questions asked, respecting the
characteristic form of expression and the students vocabulary. Results:
The interim results show strong trends in favour of this procedure. These
ndings indicate that students who participated by providing questions
and solving quizzes obtain better academic performance than those who
were limited to answering the tests and these, in turn, stand on that have
prevented their participation in the test. Conclusions: The approach of
self-assessment exercises collaboratively by students, under supervision
of faculty, is shown as a useful tool and could be useful in the transition
to new teaching strategies suggested by the European Higher Education
balos C et al . RCCV. 2009; 3:198-207. [2]. Shah IM et
Area [1]. [1]. A
al. Emerg Med J. 2008; 25:354-357. [3]. Yeung MA et al. Australasian
Journal of Peer Learning. 2008; 1: 26-39.
Paper No.: 887

FOCUSED CONFERENCE GROUP: SOCIETY WORKSHOP SW14
A RAPID AND SENSITIVE HIGH-PERFORMANCE LIQUID
CHROMATOGRAPHIC METHOD TO QUANTIFY CARBAMAZEPINE, OXCARBAZEPINE, ESLICARBAZEPINE ACETATE
AND THEIR MAIN METABOLITES IN HUMAN PLASMA
Ana Fortuna(1,2), G Alves(2,3), L Santos(1,2), A Falcao(1,2), P Soaresda-Silva(4)
285
(1) Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal
(2) Centre for Neurosciences and Cell Biology, University of Coimbra,
Coimbra, Portugal
(3) Health Sciences Research Centre, University of Beira Interior,
Covilha, Portugal
(4) Department of Research and Development, BIAL, S. Mamede do
Coronado, Portugal
A simple and fast HPLC-UV method was developed and validated for
simultaneous quantication in human plasma of three structurally related
antiepileptic drugs, carbamazepine, oxcarbazepine and eslicarbazepine
acetate, and their main metabolites carbamazepine-10,11-epoxide, racemic licarbazepine and trans-diol. Aliquots (500ll) of blank plasma from
healthy subjects were spiked with known amounts of all compounds and
internal standard (10,11-dihydrocarbamazepine). The analytes were
extracted from plasma by solid-phase extraction (SPE) and, thereafter,
separated at 40C on the reversed-phase column LiChroCART Purospher Star (C18, 3lm, 55mm x 4mm) using an isocratic elution with a
mixture of water, methanol and acetonitrile (64:30:6, v/v/v) as mobile
phase and detected at 235 nm. Under these chromatographic conditions,
all studied drugs and metabolites were baseline separated in less than
9 minutes and no peaks due to the plasma matrix interfered at their retention times. The assay was linear (r2 > 0.997) in the ranges of 0.05-30,
0.05-20, 0.15-4, 0.1-30, 0.1-60 and 0.1-10lg/ml for carbamazepine,
oxcarbazepine, eslicarbazepine acetate, carbamazepine-10,11-epoxide,
licarbazepine and trans-diol, respectively. The overall intra and interday
precision did not exceed 15% and the accuracy was within 15% considering all compounds. Precision and accuracy at the lower limit of quantication was also demonstrated. In addition, the assay afforded high
recoveries for all analytes. Thus, the selective SPE procedure combined
with the simple and fast chromatographic run make this assay a useful
tool to support the therapeutic drug monitoring of such antiepileptic
drugs and future pharmacokinetic clinical studies.
Paper No.: 888

LACK OF INTERFERENCE OF CARBAMAZEPINE AND ITS
METABOLITES IN THE ENANTIOSELECTIVE THERAPEUTIC
DRUG MONITORING OF ESLICARBAZEPINE ACETATE
Ana Fortuna(1,2), G Alves(2,3), J Sousa(1,2), R Direito(2,3),
M Rocha(2,4), A Falcao(1,2), P Soares-da-Silva(5)
(1) Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal
(2) Center for Neurosciences and Cell Biology, University of Coimbra,
Coimbra, Portugal
(3) Pharmacology Deapratment, Faculty of Pharmacy, University of
Coimbra, Coimbra, Portugal
(4) Pharmaceutical Services, Coimbra University Hospital, Coimbra,
Portugal
(5) Department of Research and Development, BIAL, S. Mamede do
Coronado, Portugal
As add-on therapy, eslicarbazepine acetate (ESL) phase III clinical trials
conducted in patients with refractory partial-onset seizures have evidenced good efcacy, safety and tolerability, even though ~60% of the
enrolled patients were taking carbamazepine (CBZ). Considering the
pharmacological disadvantages of CBZ and the similar spectrum of efcacy of CBZ and ESL, the switching to ESL may be successful in many
patients. As ESL is extensively converted to S-licarbazepine (S-Lic;
~95%), the enantioselective assessment of S-Lic is expected to be useful.
This study investigated the plasma levels of S-Lic and R-licarbazepine
(R-Lic) in patients under CBZ long-term treatment to assess the potential
interference of CBZ or its metabolites in the enantioselective therapeutic
drug monitoring (TDM) of ESL. For that a chiral HPLC-UV assay developed and validated within our research group was used. Twenty-four
patients admitted to the Coimbra University Hospital and receiving CBZ
long-term treatment were identied. Blood samples were collected and
serum CBZ levels were measured by the usual TDM protocol. Aliquots
of plasma were also submitted to a chiral HPLC-UV analysis. The bioan-
alytical data indicated that S-Lic and R-Lic were not present at detectable
levels in plasma samples of the CBZ-treated patients. The chromatograms generated by the analysis of patient plasma samples, when compared with those obtained from blank plasma samples spiked with S-Lic
and R-Lic, showed the absence of interferences. These data support the
usefulness of the chiral HPLC-UV method validated by our group for enantioselective TDM of ESL (mainly S-Lic) during the switching from
CBZ to ESL.
Paper No.: 1759

MORPHOLOGICAL EVALUATION OF RAT LIVER TISSUE
AFTER THE SUBCUTANEOUS INJECTION OF
1,2-DIMETHYLHYDRAZINE
Ana Fortuna(1,2), R Direito(3), G Alves(2,4), J Sousa(1,2),
A Falcao(1,2), A Cabrita(5)
(2) University of Coimbra, Center for Neurosciences and Cell Biology,
Coimbra, Portugal
(3) University of Coimbra, Faculty of Sciences and Technology,
Biochemical Department, Coimbra, Portugal
(4) University of Beira Interior, Health Sciences Research Centre,
Covilha, Portugal
(5) University of Coimbra, Faculty of Medicine, Institute of Experimental Pathology, Coimbra, Portugal
The exposure to 1,2-dimethylhydrazine (DMH) is usually associated to
colorectal cancer induction. However, one can predict that the liver may
be also an important target for DMH toxicity, since it is the primary
organ of metabolism, playing a major role in the bioactivation and detoxication of xenobiotics. Therefore, the present study was carried out to
evaluate the morphological and histopathological modications in liver
tissue following the administration of DMH to rats. Groups of adult male
Wistar rats were treated twice-weekly with subcutaneous injections of
DMH at a dose of 15 mg/kg body weight for 8 weeks. Hematoxylin and
eosin histological analysis using optic microscopy revealed the existence
of hepatic necrosis following the 8-week treatment period with DMH.
Aiming to evaluate the regenerative capacity of liver in the sequence of
the administration of DMH, groups of rats were sacriced at different
time points after the last dose: immediately after and, 4 and 24 weeks
later. The histological examination showed that, in all exposed animals,
the observed stasis, cytoplasmic changes, steatosis, inammatory inltration and necrosis, were not reversible six months after the last administration of DMH. It was also noticeable that at the end of six months no
evidences compatible with development of hepatic neoplasia were found.
Accordingly, these results give enough support for the experimental evidence of DMH-induced hepatotoxicity in rats. In addition, this wellestablished experimental model will allow us to study, in the future, the
signalling pathways involved in such histopathological alterations
induced by DMH in the liver of rat.
Paper No.: 1841

DISCOVERY
GENE EXPRESSION AND COMPARATIVE GENOMIC
HYBRIDIZATION PROFILES OF 6-MERCAPTOPURINE- AND
6-THIOGUANINE-RESISTANT HUMAN LEUKAEMIA CELLS
Alan Fotoohi, J Hashemi, A Moshfegh, C Larsson, F Albertioni
Karolinska Institute, Department of Medicine & Clinical Pharmacology,
Stockholm, Sweden
Treatment of the MOLT4 human T-cell leukemia cells with 6-mercaptopurine (6-MP)and 6-thioguanine (6-TG), resulted in acquired resistance as
286
a result of low expression of concentrative nucleoside transporter 3
(CNT3) and equilibrative nucleoside transporter 2 (ENT2) genes. The
activity of thiopurine methyl transferase enzyme and other enzymes in
metabolism pathway of 6-MP and 6-TG was unchanged. The exception
was >40% reduction in expression of guanine monphosphate synthetase
(GMPS) in both resistant sub-lines. In order to identify other modications at RNA and DNA levels associated with 6-MP and 6-TG resistance, we analyzed the patterns of gene expression and comparative
genomic hybridization proles by resistant sublines and compared with
the wild-type MOLT4 cells. Expression of several nucleoside transporter
genes at mRNA level were down-regulated in the both thiopurine-resistant sub-lines. Besides mRNA expression of genes encoding the purine
de novo synthesis enzymes was reduced equally in both resistant cell
lines; 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase
by 20%, and glycinamide ribonucleotide transformylase by 40%, respectively, which along with a signicant reduction in expression of GMPS
indicate defected purine metabolism in these resistant sublines. Interestingly, reductions observed in some apoptotic genes: caspase 1 and 4
encoding genes were down-regulated in 6-MP resistant cells by 75 and
80%, respectively. While the expression of mRNA of Fas receptor was
reduced by >50% in 6-TG resistant cells, the apoptosis-inducing serinethreonine kinase 17b gene was down-regulated in both 6-MP- and 6-TGresistant sub-lines by 50 and 40%, respectively. Our results suggest contribution of several genes in development of resistance to thiopurines.
Paper No.: 1706

HEALTH AND DISEASE
HIGH SALT INTAKE ABOLISHES AT2-MEDIATED
VASODILATION OF PIAL ARTERIOLES IN RATS
Sebastien Foulquier(1), C Perrin-Sarrado(1), F Dupuis(1), F Soussi(1),
P Liminana(1), Y-W Kwan(2), C Capdeville-Atkinson(1), I Lartaud(1),
J Atkinson(1),
(1) Nancy University Faculty of Pharmacy, EA3452 Drug Targets, Formulation & Preclinical Drug Evaluation, Cardiovascular Pharmacology
Laboratory, Nancy, France
(2) Chinese University of Hong Kong Faculty of Medicine, Basic Medical Sciences, Hong Kong, PR China
Objectives: Angiotensin II (Ang II) has a dual effect on the cerebral circulation, inducing constriction via AT1 and dilatation via AT2 receptors
(VINCENT et al., Stroke 2005, 36, 2691-2695). We hypothesized that
HS specically changes functional Ang II-responses of cerebral arterioles. Methods: Three m.o. male Wistar rats received (drinking water)
Na+ 172 mM or 4 lM (control). Internal diameter of pial arterioles (ID)
was measured (cranial window) following Ang II 10-6M (AT1-mediated
vasoconstriction) and Ang II 10-8M + losartan 10-5M (AT2-mediated
vasodilation) suffusion at day 4 or 30. Vasoactivity was evaluated following serotonin (5-HT) 10-6 M and sodium nitroprusside (SNP) 10-5M.
Results (m+/)sem; n = 5-10; * p < 0.05 vs control same day, 2 way
ANOVA + Bonferroni): HS did not modify mean arterial pressure (control d4; d30 106 + /-5; 109 + /-4; HS 103 + /-3; 110 + /-6 mmHg).
Vasoconstrictor responses to Ang II (d4; d30, control -16 + /-1; -14 + /1; HS -18 + /-2; -14 + /-1% baseline ID) did not change. AT2-mediated
vasodilation (control d4; d30 8 + /-2; 5 + /-2%) was abolished by HS at
d4 (-2 + /-2* %) and even reversed to vasoconstriction at d30 (-7 + /-2*
%). Vasoconstriction to 5-HT (control -15 + /-3; -8 + /-1; HS -12 + /-2;
-10 + /-1%) did not change, nor did vasodilation to SNP (control 84 + /12; 87 + /-8; HS 86 + /-11; 82 + /-8%). Conclusion: HS does not change
arterial pressure in Wistar rats, but specically abolishes AT2-mediated
vasodilation. This effect is immediate (d4) and maintained in time (d30).
Were AT2-mediated vasodilation be protective, its abolition would be
deleterious in case of stroke.
Paper No.: 2854

DISEASES
INHIBITION OF NF-JB ACTIVATION BY CYMBOPOGON
CITRATUS LEAVES AND THEIR POLYPHENOLS IN HUMAN
MACROPHAGES: A POTENTIAL NATURAL SOURCE OF
ANTI-INFLAMMATORY DRUGS
Vera Francisco(1,2,3), A Figueirinha(1,4), BM Neves(2,3),
C Garcia-Rodriguez(5), MC Lopes(2,3), MT Cruz(2,3), MT Batista(1,3)
(1) Centro de Estudos Farmaceuticos, Faculdade de Farmacia-Universidade de Coimbra, Coimbra, Portugal
(2) Centro de Neurociencias e Biologia Celular, Universidade de
Coimbra, Coimbra,Portugal
(3) Faculdade de Farmacia, Universidade de Coimbra, Coimbra, Portugal
(4) Departamento de Ambiente, Instituto Politecnico de Viseu, Viseu,
Portugal
(5) Instituto de Biologia y Genetica Molecular, Universidad de Valladolid
- CSIC,Valladolid, Spain
Introduction: Most of pharmaceutical drugs presently available are
derived from natural products because the bioactivity of their phytoconstituents. Phenolic compounds have important biological properties, being
a potential source of new therapeutic agents for the treatment of inammation and related diseases like cancer and cardiovascular pathologies.
Since nuclear transcription factor (NF)-jB regulates the expression of
many pro-inammatory genes we evaluated the effect of an infusion of
Cymbopogon citratus (Cc) leaves and its polyphenols on lipopolysaccharide (LPS)-induced NF-jB activation, in human macrophages. Materials:
Cc dry leaves were extracted by infusion (5g/150ml H2O). Subsequently,
lipids and essential oils were removed with n-hexane and the extract was
freeze-dried (Cc extract). Different Cc polyphenolic fractions (PFs),
namely phenolic acids, avonoids and tannins, were obtained for Cc
extract fractionation on a reverse phase semipreparative column Lichroprep RP-18 and in a gel chromatography on a Sephadex LH-20 column. As an in vitro model of inammation, human monocyte-derived
macrophages stimulated with LPS from E. coli were used. The effect of
Cc extract and PFs on NF-jB activation was evaluated by Western blot
using anti-inhibitory-jB (IkB) and anti-phospho-IjB antibodies. Results:
We observed that Cc extract inhibited LPS-mediated IjB degradation and
increased the levels of IjB phosphorylation, while PFs inhibited LPSmediated IjB phosphorylation and degradation, consistent with inhibition
of NF-jB activation and anti-inammatory effect. Conclusion: Polyphenolic compounds from Cymbopogon citratus inhibited NF-jB activation.
Therefore, Cc extracts are good candidates for the development of new
drugs to treat diseases with a strong inammatory component.
Supported by FCT (PTDC/SAU-FCF/105429/2008 project and SFRH/
BD/46281/2008.
Paper No.: 3109

DISEASES
ESTROGEN MINIMIZES CELL DEATH CAUSED BY H2O2 IN
CELLS OF RAT GLIOMA
Lucas AM Franco(1), L Yshii(1), E Kawamoto(1), L Lima(1),
C Scavone(1), C Munhoz(1)
Universidade de Sao Paulo (USP), Department of Pharmacology, Sao
Paulo, Brazil
Evidence suggests that glial cells play a major role in neuronal signalling
and inammatory responses in the Central Nervous System (CNS).
Chronic inammatory responses as well as glia activation are associated
with neurodegenerative diseases, such as Parkinsons and Alzheimers.
Estrogen (E2) is well known by its neuroprotective actions and the
estrogen receptors ESR1, ESR2 and GPER are very important for the
287
manifestation of these effects in the brain. This work investigated the
protective roles of E2 in C6 cell line of rat glioma, after damage induced
by hydrogen peroxide (H2O2). PCR, western blot and immunouorecence assays conrmed the presence and functionality of estrogen receptors ESR1 and GPER in C6 cells. Our results also conrmed that H2O2
induced death in C6 cells. And the pre-treatment with E2 (2 hours and
24 hours) decreased the H2O2 toxicity in a dose-dependent manner.
These results highlight the involvement of E2 and its receptors in preventing cellular damage in glial cells. In addition, they also suggest that
the E2 protective effects may be associated with rapid, non-genomic
actions of E2 membrane receptors.
Paper No.: 3375

DISEASES
EFFECT OF RED-WINE POLYPHENOLIC COMPOUNDS ON
ALLERGEN INDUCED HYPERREACTIVITY OF THE
AIRWAYS
Sona Franova(1), M Joskova(1), E Novakova(2), K Adamicova(3),
G Nosalova(1), O Pechanova(4)
(1) Comenius University, Jessenius Faculty of Medicine, Department
of Pharmacology, Martin, Slovakia
(2) Comenius University, Jessenius Faculty of Medicine, Department of
Microbiology, Martin, Slovakia
(3) Comenius University, Jessenius Faculty of Medicine, Department
of Pathologcal Anatomy, Martin, Slovakia
(4) Slovak Academy of Sciences, Department of Normal and Pathological Physiology, Bratislava, Slovakia
The results of some epidemiologic studies have suggested that the consumption of the food containing polyphenolic compounds might reduce
the occurrence of asthma symptoms. The aim of our experiments was to
evaluate the effect of 21 days red-wine polyphenolic compounds (Provinol) administration on allergen (OVA) induced inammation of the airways in experimental conditions. The allergic inammation of the
airways was induced in guinea pigs by 21 days ovalbumin sensitization.
The tracheal smooth muscle reactivity was examined by in vitro method
to bronchoconstricting mediator histamine (10-8-10-3 mol.L-1); the
changes in airways resistance by in vivo method to nebulized histamine
(10-6 mol.L-1). The histological investigation of the tracheal tissue and
BALF levels of inammatory cytokines IL-4, IL-5 were used as parameters of airway inammation. 21 days administration of Provinol caused a
signicant decrease of specic airway resistance after histamine nebulization and decline in tracheal smooth muscle contraction amplitude to this
bronchoconstrictor. Provinol minimized the degree of inammation estimated on the basis of eosinophil calculation and levels of inammatory
cytokines IL-4 and IL5. In conclusion, Provinol administration in ovalbumin-sensitized guinea pigs leads to bronchodilating and antiinammatory
effects, suggesting their possible use in therapy of allergic inammatory
conditions of airways.
Work was supported by Grant VEGA 1/0073/08, Center of Experimental
and Clinical Respirology Project is co-nanced from EC sources
ERDF European Regional Development Fund
Paper No.: 1864

HEALTH AND DISEASE
IMPACT OF WINDOW L-TYPE CA2 + INFLUX ON ACTIVITY
OF CA2 + CHANNEL BLOCKERS IN AORTIC SMOOTH
MUSCLE CELLS OF C57BL6 MICE
Introduction. L-type Ca2 + channel (CC) blockers (CCBs) reduce blood

pressure more effectively in hypertensive than in normotensive subjects
and are more effective in vascular smooth muscle (VSM) than in cardiac
muscle. Several attempts have been made to explain these unique features. The depolarization of the resting potential by hypertension and the
depolarized resting potential of VSM in comparison with the heart not
only favour the inactivated state of the CC, but also increase window
L-type Ca2 + inux. Methods. The present study investigated whether the
CC agonist BAY K8644 and whether nifedipine, verapamil or diltiazem,
representatives of different CCB classes, alone or in combination, modulated window Ca2 + inux (Fura-2 method) and isometric contraction
during depolarisation of C57Bl6 mouse aorta segments with 50 mM K+.
Results. High K+-induced contractions were biphasic. The fast force
component coincided with Ca2 + inux via a population of CC that
exhibited fast activation and then inactivation. The slow, sustained force
component was attributed to voltage-dependent, but time-independent
Ca2 + inux displaying incomplete inactivation (window) and was significantly more sensitive to CCBs than the fast component. Stimulation of
CC Ca2 + inux with 30 nM BAY K8644 decreased, whereas conditions
that favoured window Ca2 + inux increased the efcacy of CCBs to
inhibit isometric contractions. Conclusion. Results suggest that inhibition
of window L-type Ca2 + inux may play a dominant role in the antihypertensive effects of CCBs.
Paper No.: 1865

HEALTH AND DISEASE
SIGNIFICANCE OF WINDOW L-TYPE CALCIUM INFLUX
FOR CONTRACTION OF C57BL6 MOUSE AORTIC
SEGMENTS
Paul Fransen, CE Van Hove, J van Langen, H Bult
University of Antwerp, Division of Pharmacology, Antwerp, Belgium
Recently, differential expression of spliced isoforms of the voltage-gated
calcium channel (VGCC) gene Cav1.2 was demonstrated along the vascular tree. These isoforms display cell-specic electrophysiological properties with respect to the window VGCC Ca2 + inux and sensitivity to
L-type Ca2 + channel blockers. We investigated whether the window
VGCC Ca2 + inux plays a physiological role for blood vessel constriction. Methods. Ca2 + mobilisation (Fura-2 assay) and isometric contraction were studied in aortic segments of C57Bl6 mice. The membrane
potential was clamped at xed potentials by increasing external K+ concentration. Results: Above 20 mM, K+ induced biphasic contractions.
The fast component coincided with Ca2 + inux via a VGCC population
that exhibited fast activation and then inactivation. The slow force component was temporally related to a slow voltage-dependent, but timeindependent Ca2 + inux. The slow contraction was not due to Ca2 +
induced Ca2 + release, as it was not affected by dantrolene, ryanodine or
depletion of sarcoplasmic reticulum Ca2 + stores with cyclopiazonic acid.
By modulating external Ca2 + levels before and after depolarisation with
extracellular K+, we could attribute the sustained contraction to a population of VGCCs displaying incomplete inactivation (window). At normal
extracellular K+ concentration, this window was operative, allowing a
continuous VGCC-mediated Ca2 + inux leading to basal tone. Basal
inux and force were both suppressed upon hyperpolarization by adding
the ATP-dependent K+ channel agonist levcromakalim. It is concluded
that window L-type Ca2 + inux has a profound physiological impact on
basal tension and isometric contraction of C57Bl6 mouse aortic smooth
muscle cells.
Paul Fransen, CE Van Hove, C Michiels, J van Langen, H Bult

University of Antwerp, Division of Pharmacology, Antwerp, Belgium
288
Paper No.: 2112
ACTIVITY ASSESSMENT OF INDIVIDUAL CYP2D6 VARIANTS
USING POPULATION PHARMACOKINETICS OF
DEXTROMETHORPHAN
Uwe Fuhr(1), D Frank(1), A Gaedigk(2), T Klaassen(1),
D Tomalik-Scharte(1), Ar Jetter(1,3), U Jaehde(4), R Suverkrup(4),
J Kirchheiner(5), K Abduljalil(1,6)
(1) University Hospital, University of Cologne, Department of
Pharmacology, Cologne, Germany
(2) The Childrens Mercy Hospital and Clinics, Division of Developmental Pharmacology & Experimental Therapeutics, Kansas City, USA
(3) University Hospital Zurich, Division of Clinical Pharmacology and
Toxicology, Department of Internal Medicine, Zurich, Switzerland
(4) University of Bonn, Institute of Pharmacy, Bonn, Germany
(5) University of Ulm, Department of Pharmacology of Natural Products
& Clinical Pharmacology, Germany
(6) SimCYP Limited, Shefeld, UK
CYP2D6 activity is rate limiting for the formation of dextrorphan from
dextromethorphan. Respective metabolic ratios in urine and plasma are
thus used for CYP2D6 phenotyping. CYP2D6 has an extensive genetic
polymorphism. To enable prediction of phenotype from genotype, currently individual alleles are often categorized in null, reduced, high, and
increased activity allele groups, which are used to derive CYP2D6 activity scores. The aim of this study was to quantify the effect of major
CYP2D6 variants on dextromethorphan metabolism. To this end, plasma
and urine concentrations of dextromethorphan and dextrorphan were
evaluated from 36 healthy Caucasian male volunteers who participated
in three studies with administration of a single oral dose of 30 mg dextromethorphan-HBr. Concentrations were quantied by LC-MS/MS. All
data were modelled simultaneously using the population pharmacokinetic
software NONMEM. A ve-compartment model was able to describe
the data adequately. The clearance fraction attributable to an individual
CYP2D6*1 allele was about 2.5-fold higher than that attributable to a
CYP2D6*2 allele (5010 [95% CI 3579-6441] vs. 2020 [624-3416] L/h),
while the metabolic activity related to a CYP2D6*41 allele was much
lower (85 [63.8-106.2] L/h). Urinary pH was conrmed as a signicant
covariate for dextromethorphan renal clearance. These results clearly
show that important information is lost when CYP2D6*1 and *2 are considered as equal. The lack of differential information on the activity
attributable to individual alleles may be one explanation why there is
considerable interindividual variability in the pharmacokinetics of dextromethorphan and other CYP2D6 substrates within currently used
CYP2D6 activity score levels.
Paper No.: 2146

HYDROGEN SULFIDE PROMOTES FUNCTION OF T-TYPE
CALCIUM CHANNELS AND SECRETION OF PROSTATIC
ACID PHOSPHATASE IN NEUROENDOCRINE-LIKE
PROSTATE CANCER CELLS FROM THE
ANDROGEN-SENSITIVE LNCAP CELL LINE
Kazuki Fukami(1), R Kasamatsu(1), S Yoshida(2), F Sekiguchi(1),
A Kawabata(1)
(1) Kinki University School of Pharmacy, Division of Pharmacology and
Pathophysiology, Osaka, Japan
(2) Kinki University School of Science and Engineering, Department of
Life Science,Osaka, Japan
Prostate cancer is one of the most common malignancies among men. An
increased population of prostate cancer cells acquiring a neuroendocrine
(NE) phenotype is involved in the malignancy and androgen-independent

pathology of prostate tumors. In the process of acquisition of the NE phenotype, Cav3.2 T-type calcium channels might be up-regulated, participating in the enhancement of secretory functions. Given our latest
evidence for activation/sensitization of Cav3.2 by hydrogen sulde
(H2S), a gasotransmitter, in the present study, we asked if H2S regulates
functions of NE-like cells originating from the androgen-sensitive prostatic adenocarcinoma cell line LNCaP. LNCaP cells were differentiated
by stimulation with dibutyryl cyclic AMP into NE-like cells characterized
by neurite outgrowth and expression of T-type calcium channel-dependent membrane currents. The NE-like LNCaP cells exhibited increased
steady-state expression of mRNA for prostate-specic antigen (PSA) and
decreased response to androgen, compared with undifferentiated cells.
NaHS, a donor of H2S, enhanced T-type currents in NE-like LNCaP cells,
an effect abolished by mibefradil, a pan-T-type channel blocker. NaHS
also caused secretion of prostatic acid phosphatase (PAP) in NE-like cells.
Expression of cystathionine-b-synthase (CBS) and cystathionine-b-lyase
(CSE), distinct H2S-forming enzymes, was detected by Western blotting
in both undifferentiated and differentiated LNCaP cells. Our data demonstrate that H2S promotes secretion of PAP in the NE-like LNCaP cells
most probably through activation of T-type calcium channels. We thus
speculate that H2S might function as a general secretagogue for potential
mitogenic factors including neuropeptides in the androgen-independent
stage, participating in the progression of prostate cancer.
Paper No.: 2200

HEALTH AND DISEASE
ROLE OF HIF-1B IN ENDOTHELIAL CELLS IN
MONOCROTALINE-INDUCED PULMONARY HYPERTENSION
IN MICE
Yayoi Fukuhara(1), S Tomita(1), Y Imamura(1), Y Horinouchi(1),
M Imanishi(1), T Sakurada(1), T Kitagawa(2), T Tamaki(1)
(1) University of Tokushima Graduate School, Institute of Health
Biosciences, Department of Pharmacology, Tokushima, Japan
Biosciences, Department of Cardiovascular Surgery, Tokushima, Japan
Pulmonary hypertension (PH) is a life-threatening disease characterized
by signicant increases in pulmonary arterial pressure (PAP) and right
ventricular hypertrophy (RVH). Hypoxia inducible factor (HIF)-1 has
also been shown to be related to pulmonary vascular reModelling in PH.
However, it is unclear the function of concerned factor in each cell type.
Therefore, in this study, we investigated that HIF-1 in pulmonary aortic
endothelial cells would be important mechanisms of PH development
and vascular remodeling. Endothelial cell-specic HIF-1b-decient (HIF1b+/)) mice were generated using a Cre-loxP system. PH was induced
by monocrotaline (MCT). Four weeks after MCT injection, HIF-1b+/)
mice showed considerably less RV hypertrophy than wild type (WT)
mice. The right ventricle to left ventricle plus septum [RV/ (LV+S)] ratio
was 23.4 2.5% in HIF-1b+/) mice, whereas WT mice was
29.2 3.2% (p < 0.05). Medial hypertrophy is a hallmark of pathological vascular reModelling in PH. The medial thickness of PAs was markedly increased in the MCT-treated WT mice compared with untreated
WT mice. However, MCT-treated HIF-1b+/) mice signicantly inhibited
the increase in percent medial thickness (WT mice:23.7 5.9%, HIF1b+/) mice:12.2 4.5%, p < 0.05). MCT-induced PH is attenuated in
endothelial cell-specic HIF-1b decient mice. These results suggest that
the development of PH may be formed via HIF-1b in endothelial cells.
Paper No.: 471

PHARMACOVIGILANCE BETWEEN SCIENCE AND CRAFT
Giovanni Furlan
289
Bracco Imaging SpA, Department of Drug Safety & Pharmacoepidemiology, S. DonatoMilanese, Italy
Nowadays, drug safety is mainly an observational, rather than experimental science. Following collection of case reports, causality assessment
is performed and aggregate case reports are analysed, mainly by looking
for confounding factors and by focusing on pathology. Whenever an
important potential risk is identied, one or more studies may be conducted to conrm or disprove the signal. Often the results of the studies
are confounded and/or contradictory. Drug safety is currently engaged in
detecting a problem once it has already occurred, once the drug is on the
market. Drug safety should be engaged in primary prevention, so to prevent or minimize important risks before the drug is marketed. The aim
should be to identify/raise hypothesis on drug potential risks in pre-clinical stage and to test these hypothesis in subsequent clinical stage so to
have a precise idea of the drug risks once it is launched. Since the drug
primary and secondary mechanisms of action are the root cause of any
adverse reaction, pharmacology is a powerful tool to forecast the drug
safety prole. Despite the ability of pharmacology to forecast type A
adverse reactions in humans, sometimes these reactions are identied
once the drug has been marketed for a relatively long period of time.
This because only subjects having other risk factors for the iatrogenic
disease experience it; furthermore, severity, signs and symptoms of the
disease vary greatly from subject to subject. Just observing the adverse
events/reactions without having an understanding of the drugs pharmacology can be very confusing.
Paper No.: 1410

BIOEQUIVALENCE STUDY OF TWO FORMULATIONS OF
DESMOPRESSIN NASAL SPRAY: A COMPARISON BETWEEN
THE NOVEL KW-8008RTS TO BE STORED AT ROOM
TEMPERATURE AND THE CONVENTIONAL KW-8008 TO BE
STORED IN REFRIGERATION
Hidetoshi Furuie(1), M Yagi(1), T Yajima(2), N Sakoda(2), K Matsumoto(2), H Ando(3), Y Amano(3), T Sakurai(1)
Paper No.: 2787

MOLECULAR DYNAMICS SIMULATIONS OF SEROTONIN
AND S-CITALOPRAM IN SEROTONIN TRANSPORTER
Mari Gabrielsen, AW Ravna, K Kristiansen, I Sylte
University of Troms, Department of Medical Biology, Troms, Norway
Transporters of the monoamines serotonin (SERT), dopamine (DAT) and
noradrenaline (NET) belong to the neurotransmitter:sodium symporter
(NSS) family. These presynaptic transporters remove neurotransmitters
from the synaptic cleft, thus reducing the number of neurotransmitters
available for interaction with postsynaptic receptors. The transporters are
major targets for antidepressant drugs, the selective serotonin reuptake
inhibitors (SSRIs) and the tricyclic antidepressants (TCAs), and are also
the targets for psychostimulants such as cocaine, MDMA (ecstasy), and
d-amphetamine. The three-dimensional structure of SERT has not yet
been determined. However, the rst three-dimensional structure of a NSS
family member, the Aquifex aeolicus leucine transporter, LeuT, was
recently solved (Nature 2005; 437:215-23). Based on a comprehensive
alignment of NSS family members (Mol Pharmacol 2006; 70:1630-42)
and the LeuT crystal structure, a homology model of SERT has been
generated using the ICM program package. Different tryptamine derivatives and the SSRI S-citalopram have been docked into the putative substrate binding site of SERT, yielding two possible binding modes of the
ligands. In order to discriminate between these binding modes and
between the tryptamine derivatives (substrates) and S-citalopram (inhibitor), the SERT model has been embedded in a POPC lipid bilayer, generated by using the CHARMM-GUI membrane builder (J Comput Chem
2008; 29:1859-65), and long molecular dynamics (MD) simulations of
ligand-SERT-membrane complexes have been performed.
Paper No.: 2019

THE EFFECT OF RESVERATROL ON ILEAL SMOOTH
MUSCLE: FUNCTIONAL INVESTIGATION IN A RAT SEPSIS
MODEL
(1) Heishinkai Medical Group Inc., OPHAC Hospital, Osaka, Japan

(2) InCROM, Osaka, Japan
(3) Kyowa Hakko Kirin Co., Ltd, Japan
N Gacar(1), SS Gocmez(2), G Gacar(1), IK Celikyurt(1), Oguz Mutlu(1),

T Utkan(1)
Introduction: Desmopressin is a synthetic analogue of vasopressin and

useful for treatment of nocturnal enuresis. The conventional desmopressin nasal spray KW-8008 is inconvenient to carry because it should be
stored in a refrigerated state. KW-8008RTS is a novel formulation of desmopressin nasal spray under development and intended to be stored at
room temperature. We planned a bioequivalence study of these two formulations. However the study has some limitations, as Joukhadar et al.
suggested that there is a high inter-subject CV of approximately 60% and
intra-subject CV of more than 30% in plasma pharmacokinetics of desmopressin. Objective: The primary objective was to evaluate the bioequivalence between KW-8008RTS and KW-8008. The secondary
objective was to evaluate the safety of these formulations. Method: The
study was carried out in 28 healthy male volunteers in an open, randomized two-way crossover design with a single dose administration of
20µg (10µg per nostril) desmopressin in order to study bioequivalence and safety. Wash out period was at least 1 week. To avoid
large variation in plasma pharmacokinetics, the subjects were placed in a
decubitus position with a nasal clip. Results: The 90% condence intervals of geometric mean ratios of Cmax and AUC0-t were 89.4%-102.2%
and 80.7%-106.5%, respectively. They implement the criteria of bioequivalence. No drug-related adverse events were observed. Conclusion:
The novel KW-8008RTS storable at room temperature and the conventional KW- 8008 to be refrigerated were safe and considered to be bioequivalent.
(1) Kocaeli University Medical Faculty, Department of Pharmacology,

Kocaeli, Turkey
(2) Namyk Kemal University Medical Faculty, Tekirdag, Turkey
Sepsis, a common and signicant complication of acute illness, often
leads to a progressive and catastrophic condition known as multiple
organ dysfunction. The intestine is one of the major organs that are
involved in sepsis. Gastrintestinal stasis during sepsis may be associated
with gastrointestinal smooth muscle dysfunction. Resveratrol, is a natural
phytoalexin present in grapes and red wine, which possesses a variety of
biological activities including anti-inammatory and antioxidative activities. Therefore, we aimed to examine whether the protective effects of
resveratrol on isolated intestinal smooth muscle responses and TNF-a
levels in septic rats. Polymicrobial sepsis was induced by the cecal ligation and perforation (CLP) procedure (24 h model). Rats received resveratrol (100 mg/kg,ip) or saline immeadiately after CLP. At 24 h after
CLP, the animals were anesthetized and contractile and relaxant
responses were measured in strips of ileal smooth muscle. We also measured TNF-a levels in blood samples. Contractile responses to KCl and
carbachol and relaxant responses to EFS were signicantly decreased in
the strips of the sepsis group than control group (p < 0.05). Plasma
TNF-a levels of sepsis group were higher than those of control group
(p < 0.05). The impaired contractions and relaxations of strips were
markedly improved by treatment of resveratrol. Furthermore, increased
TNF-a levels of sepsis group reversed to the controls with pretreatment
290
of resveratrol. The results of the present study indicate that resveratrol
signicantly improved CLP-induced relaxant and contractile responses
and that the benecial effects of resveratrol might be attributed to its antiinammatory activity.
Paper No.: 1032

EFFECTS OF RESVERATROL ON LEARNING AND MEMORY
IN RATS
Nejat Gacar(1), O Mutlu(1), T Utkan(1), I Komsuoglu Celikyurt(1),
SS Gocmez(2), G Ulak(1)
Kocaeli, Turkey
Resveratrol (3,5,4-trihidroksi-trans-stilben) is a polyphenol which is
found especially in grape and red wine and exerts biological activities. In
recent studies resveratrol is shown to protect neuronal cells with its antioxidant activity, improve memory functions in streptozotocin induced
dementia models and reverse acetylcholine esterase activity. The aim of
this study was to investigate the effect of resveratrol on emotional and
spatial memory in naive rats and also on scopolamine induced memory
impairment in passive avoidance and Morris water maze (MWM) tests.
Male Wistar-albino rats and three different doses of resveratrol (12.5, 25
and 50 mg/kg) were used. Two way ANOVA post hoc Dunnett-t test is
used for the statistical analysis. There was no signicant difference
between all groups for the rst day latency while scopolamine signicantly shortened the second day latency compared to control group and
resveratrol reversed this effect in all doses used in the passive avoidance
test. In the MWM test, 50 mg/kg resveratrol signicantly increased the
time spent in the escape platforms quadrant in the probe test while scopolamine decreased this parameter. The effect of scopolamine was
reversed by 50 mg/kg resveratrol. There was no signicant difference for
the total number of movements and the total distance travelled in the
locomotor activity test in all groups. Resveratrol reversed the effect of
scopolamine and even had improving effects in naive animals at higher
doses. The results of this study should be claried by further studies
including different learning and memory tasks.
Paper No.: 2700

ANALYSIS OF N-ACETYLTRANSFERASE 2 PHENOTYPE AND
GENOTYPE IN A TUNISIAN POPULATION
E Gaes, Anis Klouz, I Salouage, S Trabelsi, I Hamza, N Jebabli,
M Aouichri, M Lakhal
National Center of Pharmacovigilance, Laboratory of Clinical
Pharmacology, Tunis, Tunisia
N acetylation polymorphism has been documented as a representative
pharmacogenetic trait. It is involved in the metabolism of a large number
of drugs and carcinogens and has diverse clinical consequences. The aim
of our study is to determine the acetylation phenotype, and to compare
acetylation phenotype with NAT2 genotype in Tunisian tuberculosis
patients. It consists on a prospective study including tuberculosis patients
in 2005 and in 2009. NAT2 phenotype was determined for two populations by calculating acetylation index 3 hours after isoniazid administration, the NAT2 genotype was only analysed by PCR/RFLP for patients
included in 2005. In 2005, we found a bimodal distribution of acetylation
phenotype (75% slow acetylators (SA) and 25% rapid acetylators (RA)).
The distribution of SA and RA genotypes was respectively 56% and
44%. The 341 C-to-T transition was the predominant mutation (37,3%).
Ten NAT2 alleles were found in our population, NAT2*4 was the major
one. Thirty-two different genotypes were found. The major genotype
was NAT2*6B / NAT2*4. The concordance value was 79%. The K statistics indicated moderate agreement between genotype and phenotype with
K = 0,55. In 2009, the distribution of the acetylation phenotype was
55,4% SA and 44,6% RA indicating a good concordance with genotype.
Results obtained in 2005, make us vigilant about the collection of blood
samples by respecting exactly the delay of 3 hours after isoniazid administration. This permit to obtain the results that should be obtained in
2005. Thus we can predict easily acetylation phenotypes with a poor
error risk.
Paper No.: 2785

TUNISIAN CLINICAL PHARMACOLOGY EXPERIENCE
E Gaes, R Sahnoun, D Bensaid, N Jebabli, I Salouage, S Trabelsi,
I Hamza, M Aouichri, Anis Klouz, M lakhal
National Center of Pharmacovigilance, Laboratory of Clinical Pharmacology, Tunis, Tunisia
Clinical pharmacology is the science of drugs and their clinical use; it
connects the gap between medical practice and laboratory science. The
main objective is to promote the safety of prescription, maximise the
drug effects and minimise the side effects. At the beginning of the Clinical Pharmacology department activity on 1976, drug monitoring concerns only few molecules by the determination of correlation between
doses and concentrations. The activity of our department has been
evolved over the years and the number of monitored drugs has signicantly increased. In fact, it increased from 1777 assays in 2000 to 7885
in 2009. By the way, we developed a new approach using classic pharmacokinetic for the determination of pharmacokinetics parameters.
Which was rst determined only for cyclosporine and busulfan. In order
to minimise drug toxicity due to inter and intra-patients pharmacokinetic
variability, we developed drug population pharmacokinetic for several
anti-cancerous and immunosuppressive drugs. This approach proposes a
limited sampling strategy to estimate individual pharmacokinetics parameters. In conclusion, the clinician claim is increasing; this shows that the
activity of the Tunisian Clinical Pharmacology department is important
to guide clinician to promote the efciency of such treatment and to
avoid toxicity.
Paper No.: 3176

ANGIOTENSIN II MODULATES A1D-ADRENOCEPTORS
EXPRESSION IN VASCULAR SMOOTH MUSCLE CELLS
Itzell Gallardo-Ortiz(1), P Lopez-Sanchez(2), JJ Lopez-Guerrero(1),
M Ibarra(1), R Villalobos-Molina(1)
(1) UNAM, FES-Iztacala, Mexico City, Mexico
(2) ESM, I.P.N., Mexico
Angiotensin II (Ang II) plays an important function regulating systemic
arterial pressure; it provokes cardiovascular reModelling and hypertension, as well as increase in a1-adrenoceptors mRNA and protein. The
aim of this work was to evaluate the inuence of Ang II on a1-adrenoceptors in isolated smooth muscle cells of Wistar rat aorta. Smooth muscle cells were incubated with Ang II (100 nM) for different periods of
time (30min-24hrs), and protein was determined by Western blot for a1adrenoceptor subtypes. Data show an important increment of a1D-adrenoceptors after 30 min of Ang II incubation, then a time-dependent diminution in the expression of protein at later measurement points. Whereas
291
a1A-adrenoceptors shown opposite effects, they decreased at short times
and returned to basal. a1B-adrenoceptors were not modied by the peptide. Maximal expression was blocked by losartan and inhibited by
cycloheximide. Our data suggest that stimulation of AT1 receptors by
Ang II increased a1D-adrenoceptors in isolated cells, and this effect is
due to synthesis de novo of that receptor. In addition, data support a
putative crosstalk between Ang II and a1D-adrenergic pathways, and this
effect might be related with hypertension and hypertrophy induced by
Ang II.
Supported in part by grant IN224408 PAPIIT, DGAPA, UNAM.
Paper No.: 2296

DISEASE AND THERAPY
CONTRIBUTION OF TRPC1 TO THE PATHOGENESIS OF
DYSTROPHIC MICE: ANALYSIS OF DOUBLEKNOCK-OUT
TRPC1 -/- DYSTROPHIN
Chiara Gallo(1), OM Dorchies(1), G Shapovalov(1), N Zanou(2),
P Gailly(2), E Roulet(1), UT Ruegg(1)
(1) University of Geneva, School of Pharmaceutical Sciences, Laboratory
ofPharmacology, Geneva, Switzerland
(2) Universite Catholique de Louvain, Institute of Neuroscience, Laboratory of CellPhysiology, Brussels, Belgium
Calcium dysregulation has been shown as one of the triggering events
causing muscular degeneration in Duchenne muscular dystrophy (DMD).
The increased Ca2 + inux could result from transient membrane lesions
or from inux through store operated channels (SOC) or stretch activated
channels (SAC). These inuxes may be due to an over-activation of cationic channels belonging to the TRP family. Since TRPC1 is a candidate
for SOC function and has been shown to be involved in skeletal muscle
function, we have generated a double knock-out TRPC1-/- - dys- (mdx)
mouse to analyze the involvement of TRPC1 in Ca2 + regulation and
muscle function in the dystrophic phenotype. Characterization of
TRPC1-/- - dys- (mdx) was performed by physiological, functional,
behavioral and molecular tests including TRPC1-decient, dystrophindecient and normal mice as controls. SOC activity was analyzed with
Ca2 + imaging experiments (Fura-2 on bers) and 45Ca2 + ux experiments on entire muscle, using the SERCA inhibitor, thapsigargin. SAC
activity was tested using 45Ca2 + inux under hypo-osmotic conditions.
Electrophysiology was performed to characterize TRPC1 current on isolated Flexor Digitorium Brevis muscle bers. Isometric force and fatigue
were measured in vivo. Fatigability was also tested with a grid test. Histology was performed on Tibialis Anterior muscle. Up until now,
although TRPC1 seems to play a role in force and fatigue mechanism of
otherwise normal mice, the contribution of TRPC1 to the pathogenesis
of mdx dystrophic mice is not clear. Further investigations are in
progress.
Paper No.: 1574

A SIMPLE, NOVEL METHOD FOR ASSESSING MEDICATION
ADHERENCE: CAPSULE PHOTOGRAPHS TAKEN WITH
CELLULAR TELEPHONES
for how to best accomplish this goal. In this study, we looked at three
types of adherence measurement methods (capsule count, MEMS and a
novel method- pictures taken by cellular telephones) and compared their
usefulness and accuracy. Overall adherence estimated by capsule count
was 94.9% ( 13.5%), by MEMS 93.6% ( 15.0%), and by photos
76.9% ( 14.6%). Weekly photographs and MEMS agreed with weekly
capsule counts with similar frequency (36% vs. 39%, respectively; OR
1.11, p= .79). When weekly measures disagreed with capsule count,
MEMS overestimated adherence more than photographs (39% vs. 14%;
OR 3.88; P < 0.001) and photographs underestimated adherence more
than MEMS (49% vs. 22%; OR 3.48; P < 0.001). Comparing against
capsule count, the novel method was found to be as useful as MEMS.
Given the ubiquity of cellular telephone use, and the relative ease of this
adherence method, we believe it to be a useful and cost effective
approach to adherence measurement that should be utilized by providers
in the future.
Supported by NIH DA018179.
Paper No.: 953

CORRELATION OF CYTOCHROME P450 3A4*18 GENETIC
POLYMORPHISMS WITH POSTOPERATIVE FENTANYL
REQUIREMENTS
Siew Hua Gan, PCS Tan, SK Hassan, N Abdullah
Interindividual variability in drug responses may be attributable to genetically-determined alteration in enzyme activity. In this study, the association between cytochrome P450 3A4 (CYP3A4) genetic polymorphisms
and postoperative fentanyl requirements was investigated. Patients
(n = 94) scheduled for gynaecological laparotomy received IV fentanyl
infusion (3 lg/kg/hr) after induction of general anaesthesia. Postoperative
fentanyl requirements were quantied by using a patient-controlled analgesia (PCA) and the number of IV fentanyl rescue analgesia required
were recorded. Pain control was assessed using visual analogue scores
(VAS) and fentanyls adverse effects were documented. CYP3A4*4,
CYP3A4*5 and CYP3A4*18 alleles of cytochrome P450 3A4 were identied by polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP). Differences in fentanyl requirements and VAS
among the various genotypes were compared. No mutations were
detected for CYP3A4*4 and CYP3A4*5 alleles. Allelic frequency for
CYP3A4*18 was 2.7% where eighty nine patients (94.7%) were wildtype, ve (5.3%) were heterozygous while none was homozygous. No
signicant difference was demonstrated between the genotype groups in
terms of fentanyl consumptions, pain control and adverse effects. The
result showed that genetic polymorphism of CYP3A4*18 does not play
an important role in inuencing postoperative fentanyl requirements.
Paper No.: 954

HPLC METHOD DEVELOPEMENT FOR THE SIMULTANEOUS
DETERMINATION OF ARABINOSIDE-C AND DOXORUBICIN
CONCENTRATIONS IN HUMAN SERUM
Suew Hua Gan, AF Mistiran, AD Abdullah
GP Galloway, JE Guillen, JR Coyle, K Flower, John Mendelson

California Pacic Medical Center Research Institute, Addiction and Pharmacolog Research Laboratory, San Francisco, CA, USA
Medication non-adherence is an important factor in both everyday
healthcare and research methodology. Though many types of adherence
measuring utilities have been applied, there is as yet no gold standard
Arabinoside-C (Ara-C) and doxorubicin hydrochloride (DOX) are two

anticancer agents commonly used for many types of cancers. In this
study, a new HPLC method was developed for the simultaneous determination of the two drugs in human serum. The effects of 1) using different
buffer types 2) different buffer pHs 3) adding different organic modiers
292
such as triethylamine and acetic acid 4) changing column temperature 5)
changing mobile phase composition 5) varying the injection volume and
6) changing UV detector wavelength were investigated. Chromatographic separation was successfully achieved on a 250 mm X 4.6 mm
(C-18) column using a mobile phase consisting of a mixture of ammonium hydrogen phosphate (0.01M) adjusted to pH 6.2 and acetonitrile
(55:45). The ow rate was 0.5 ml/min and UV detection was done at
275 nm. The column temperature was set at 30C. The proposed HPLC
method was successfully applied to the determination of the investigated
drugs with good precision and accuracy as per recommended by the
FDA.
ischemia, whereas under the conditions of experimental myocardial

infarction its effect obviously decreases and in case of combined cardiocerebral pathology it is fully absent. By contrast, Afobazol (10 mg/kg) in
comparison with intact animals substantially increases cerebral perfusion
during vascular pathology of brain or heart, and it is even more effective
under the conditions of combined cardiac and cerebral vascular pathology. Thus, in conditions of combined ischemic damage of brain and
heart the cerebrovascular effects of Afobazol signicantly increase, while
Nimodipin has no effect. It indicates that under the conditions of cardioneurologic disturbances these two drugs have different mechanisms of
cerebrovascular activity.
Paper No.: 991

EFFECT OF TAURINE, PYRIDOXINE AND A LIPOIC ACID ON
CAFETERIA DIET FED RATS
Paper No.: 1213

DISCOVERY
PHARMACOKINETIC INTERACTION BETWEEN PUERARIN
AND EDARAVONE AND EFFECT OF BORNEOL ON THE
BRAIN DISTRIBUTION KINETICS OF PUERARIN IN RATS
Priya Ganpathy, R Ghosh, S Pitre, V Kadam
C Gao, X Li, T Li, L Wang, Ming Xue

Bharati Vidyapeeths College of Pharmacy, Department of Pharmacology, Navi-Mumbai, India
Metabolic syndrome, a condition which lacks a concise denition and
treatment attracts research for newer therapies. Increased consumption of
westernized diet worldwide has contributed to more individuals being
diagnosed with metabolic syndrome. The cafeteria diet model, which
incorporates high calorie western food items, mimics the features of
human metabolic syndrome well and is a widely used screening model
for metabolic syndrome. In the present study, we have investigated the
effect of a combination of nutritional supplements on the three major
components of metabolic syndrome viz. obesity, insulin resistance and
dyslipidemia. Male Sprague-Dawley rats were fed high fat cafeteria diet
for a period of 5 weeks. The treatment group received a combination of
taurine, pyridoxine and a lipoic acid. The results of this in-vivo study
indicates that the proposed combination shows statistically signicant (p
< 0.05) improvement in the body weight, serum levels of glucose, triglycerides, total cholesterol, free fatty acids and HOMA-IR. The candidate
combination proves to be benecial in the cafeteria diet induced weight
gain, glucose and lipid imbalances and insulin resistance. Hence, from
the results of current in-vivo study we conclude that by combining nutritional supplements based on their individual pharmacologic potentials we
can alleviate the features of metabolic syndrome.
Paper No.: 1511

HEALTH AND DISEASE
THE DIFFERENCE IN CEREBROVASCULAR EFFECTS OF
AFOBAZOL AND NIMODIPIN UNDER THE CONDITIONS OF
COMBINED CARDIAC AND CEREBRAL VASCULAR
PATHOLOGY
Tamara Ganshina, R Mirzoyan, N Khaylov, S Seredenin
Capital Medical University School of Chemical Biology and Pharmaceutical Sciences, Department of Pharmacolgy, Beijing, PR China
The aim was to investigate the pharmacokinetic interaction between
puerarin and edaravone and the effect of borneol on the brain distribution
kinetics of puerarin in rats.A reversed-phase high performance liquid
chromatography method was developed and validated for the simultaneous determination of puerarin and edaravone in rat plasma. The detection method was successfully applied to compare the pharmacokinetic
interaction and brain distribution kinetics of puerarin and edaravone,
using in situ microdialysis sampling, in rats after intravenous administration and co-administration with a single dose. The method gave good linearity and no endogenous material interfered with the two target
compounds and I.S. peaks. The limit of detection of puerarin and edaravone was 0.03 and 0.05 ug/ml, respectively. The precision determined of
testing were all within 10%. The combination of puerarin and edaravone
reduced drug elimination rates, gave a wider distribution and the dispositions of both drugs in rats were optimized. The amount of distribution of
puerarin in brain tissues were increased signicantly and the elimination
of puerarin was obviously slow with borneol pretreated. The results provide an important information for an improved combined use of puerarin
and edaravone with borneol pretreated in the clinical practice.
Keywords: Puerarin; edaravone; borneol; pharmacokinetic interaction;
brain distribution kinetics; microdialysis
Paper No.: 1787

PINOCEMBRIN PREVENTS GLUTAMATE-INDUCED
APOPTOSIS IN SH-SY5Y NEURONAL CELLS VIA DECREASE
OF BAX/BCL-2 RATIO
Mei Gao(1), W-C Zhang(2), J-J Hu(1), G-H Du(1)
Russian Academy of Medical Sciences, V.V.Zakusov Institute of Pharmacology, Moscow, Russian Federation
Combined blood supply disturbances of the two most important organs
brain and heart occur in clinical practice quite often. However we were
unable to nd any information about the inuence of drugs on cerebral
circulation on the experimental models of ischemic cardiac damage or
during combined disturbances of cerebral and coronary circulation. A
well known calcium channel antagonist Nimodipin and selective anxiolytic drug with neuroprotective activity Afobazol were chosen for our
research. The experiments have shown that Nimodipin (0,03 mg/kg)
enhances cerebral perfusion of both intact animals and during cerebral
(1) Chinese Academy of Medical Sciences & Peking Union College,

Institute of Materia Medica, National Center for Pharmaceutical Screening, Beijing, PR China
(2) Beijing Institute of Technology, School of Life Science & Technology, Beijing, PR China
Pinocembrin is the most abundant avonoids in propolis, and has been
proven to have antioxidant, antibacterial and anti-inammatory property.
To assess the protective effects of pinocembrin on neurons, SH-SY5Y
neuronal cells were pretreated with pinocembrin for 2 h followed by
co-treatment with glutamate (2 mM) for 12 h. Cell viability was
293
determined by(3,4,5-dimethylthiazol-2-yl)-2,5-diphenylte-trazolium bromide assay, and apoptosis was conrmed by cell morphology, capillary
zone electrophoresis and ow cytometry assay. Cell morphology was
evaluated with Hoechst33258/PI dye. Treatment with pinocembrin (10-5,
10-6, 10-7mol/l) increased cell viability dose-dependently, inhibited LDH
release and attenuated apoptosis. Intracellular free [Ca2 + ] was increased
after glutamate exposure, and this increase was attenuated in cells treated
with pinocembrin. bax mRNA expression increased remarkably following
glutamate exposure and pinocembrin treatment manifested a reduction
effect. bcl-2 mRNA expression changes were not detected in groups with
or without pinocembrin. Western blotting results indicated that pinocembrin treatment reduced the expression of Bax and had no effect on Bcl-2,
thus decreased the BaxCBcl-2 ratio, which is in consistent with the gene
expression result. Pinocembrin could also down-regulate the expression
of p53 protein, and inhibit the release of cytochrome c from mitochondria
to cytosol. Thus we conclude that pinocembrin exerts its neuroprotective
effects in glutamate injury model partly by inhibiting p53 expression,
thus BaxCBcl-2 ratio, and the release of cytochrome c.
Acknowledgments: This work was supported by National Natural
Science Foundation of China(No. 30630073) and National Science and
Technology Major Projects for Key New Drug Innovation
(No.2009ZX09302-003).
Paper No.: 988

SESSION - HEPATOLOGY
CHARACTERIZATION OF METABOLITES AND
CYTOCHROME P450 ISOFORMS INVOLVED IN THE
MICROSOMAL METABOLISM OF ACONITINE
Yue Gao
Beijing Institute of Radiation Medicine, Department of Pharmacology &
Toxicology, Beijing, PR China
Intoduction: Veratridine is a lipid-soluble alkaloid extracted from Veratrum ofcinale and other species of the family Liliaceae. Veratridine prevents inactivation of Na+ channel via binding the receptor site 2, causes
inux of sodium ion and depolarization and induces apoptosis of neuronal
cells. Materials: In the present study, we investigated the metabolism of
veratridine and the effects of selective cytochrome P450 (CYP) inhibitors
on the metabolism of veratridine in rat liver microsomes. The metabolites
were separated and assayed by iquid-chromatography-electrospesults:
Result showed that four CYP isoforms (CYP1A, CYP2B, CYP2E1,
CYP3A) were involved in the metabolism of veratridine in vitro and
seven metabolites of veratridine were detected incubating with rat liver
microsomes. Conclusion: Some of the metaboliteswere presumed to be
potentialmediates of neurotoxicity via protein binging. Further research in
vivo needs to link the metabolism of veratridine to its toxicity.
Paper No.: 769

POTENTIALLY INAPPROPRIATE MEDICATIONS IN
ELDERLY ATTENDED AT PRIMARY CARE
pharmaco-therapeutically at elderly people attended in a primary care center of Santiago, Chile. Also prevalence of PIM was determined. A crosssectional study including interviews and revision of medical records in a
representative sample of elderly was developed. Adherence to treatment,
health-related quality of life (VAS-HRQoL), and functionality were measured. PIM was dened by 2003 Beers criteria. Data were analyzed using
STATA 10.1. Eighty three patients were included; 62.7% were women,
mean age was 72 6.2 years. Hypertension (91%) and osteo-articular diseases (64%) were the most frequent illness. Within last year 25% of
patients were hospitalized, and 35% suffered at least one fall. The 92%
were functionally independents, and their mean VAS-HRQoL was 62.5
22.5. The 53% received poly-pharmacy, and the 83% was adherents to
treatment. The 30.1% received PIM, and PIM use was the only variable
statistically associated with a greater number of prescriptions (5.7 2,51
versus 4.7 1,70, respectively). Prevalence of PIM found in present study
could indicate that it is necessary to assess the effect of PIM use over
health resources utilization, and HRQoL of older adults. However it is
necessary to do additionally studies including a greater number of
patients, representative of the national older population.
Paper No.: 891

ESTIMATION OF TRANSFER OF NIFURTIMOX, FOR
CHAGAS DISEASE, INTO BREASTMILK. POPULATION
PHARMACOKINETICS AND SIMULATION ANALYSIS
Facundo Garcia-Bournissen(1), J Altcheh(2), A Panchaud(3), S Ito(1)
(1) Hospital for Sick Children, Division of Clinical Pharmacology,
Toronto, Ontario, Canada
(2) Servicio de Parasitologia, Hospital de Ninos R. Gutierrez, Buenos
Aires, Argentina
(3) Swiss Teratogen Information Service, Division of clinical Pharmacology and Toxicology, University Hospital, Lausanne, Switzerland
Introduction: Women with Chagas disease receiving treatment with nifurtimox are discouraged from breastfeeding. Many patients who would
receive treatment with nifurtimox live in extreme poverty, and have limited access to resources such as clean water and baby formula and may
not have safe alternatives to breast milk. We aimed to estimate, using
limited available pharmacokinetics data, potential infant exposure to nifurtimox through breast milk. Methods: Original nifurtimox plasma concentrations were obtained from published studies. Pharmacokinetic
parameters were estimated using nonlinear mixed effect Modelling with
NONMEM version VI. One thousand nifurtimox plasma-concentration
proles were simulated and used to calculate the amount of drug that an
infant would be exposed to, if breastfed 150 ml/kg/day. Results and Conclusion: Estimation of bBreast milk concentrations on the basis of peak
plasma levels (1,361 ng/ml) and milk-plasma ratio were estimated. We
calculated infant nifurtimox exposure of a breastfed infant of a mother
treated with this drug to be below 10% of the maternal weight-adjusted
dose, even if milk plasma ratio were overestimated. Simulation led to
similar estimates. Risk for signicant infant exposure to nifurtimox
through breast milk seems small and below the level of exposure of
infants with Chagas disease receiving nifurtimox treatment. This potential degree of exposure may not justify discontinuation of breastfeeding.
Jose Luis Garcia Fuentes(1), M Jiron(1), I Ruiz(1), M Santibanez(2)

(1) Universidad de Chile, Departamento de Ciencias y Tecnologia
Farmaceutica, Santiago, Chile
(2) Departamento de Salud, Comuna de San Ramon, Santiago, Chile
Quality of drugs prescription in elderly has been estimated applying some
clinical tools. In Chile, older adults are principally attended at the primary
health care system, and until now there are no studies for detecting
the use of potentially inappropriate medicines (PIM) in these subjects.
The purpose of this study was characterized socio-demographic and
Paper No.: 3407

FAVORABLE STUDENT ATTITUDES TOWARDS HOSPITAL
TRAINING IN PHARMACOLOGY IN NURSING DEGREE
Cristina Garca-Cabanes, V Maneu, MM Palmero, J Formigos-Bolea
294
Pharmacology is a crucial discipline for students of nursing. Since
2000, the Area of Pharmacology of the University of Alicante has
been developing clinical training in Pharmacology as a part of the
hospital training. The objective of our study was to assess students
opinion about the hospital training in Pharmacology as a model for
strengthening learning objectives from Pharmacology theoretical classes. The implementation of this study in the subject Pharmacology
of the second year of the Degree in Nursing, began the academic
year 2008-2009 and it has continued during the academic year 20092010. A total of 394 students did the hospital training during the second semester distributed in 9 hospitals. During the hospital training
the students had to report the pharmacological treatment of 3 patients:
prescribed drugs, therapeutic use, dosage prescribed, routes of administration and adverse reactions. A teacher of pharmacology visited
them once a month in order to solve doubts. At the end of the clinical training, evaluation was accomplished with an anonymous survey
through an online questionnaire self-administrated in the intranet. On
a scale of 1 to 10, results showed that students are aware of the
importance of the Pharmacology to nurses activity. Regarding Pharmacology clinical training, students think that acquired knowledge is relevant to their professional work. Respondents think that it should be
maintained in the new EHEA curricula. Student attitude was assessed
by noting their degree of agreement using a Likert scale. The overall
student attitude towards hospital training on pharmacology was positive.
Paper No.: 3277

SESSION - ONCOLOGY
CYTOTOXIC ACTIVITY OF BIOACTIVE FRACTIONS OF
BIOACTIVE FRACTIONS OF ASPARAGUS OFFICINALIS L.
M Dolores Garca-Gimenez, R De la Puerta, MA Fernandez-Arche,
MT Saenz-Rodrguez
University of Sevilla, Faculty of Pharmacy, Department of Pharmacology, Sevilla, Spain
Background: Triguero asparagus from Huetor - Tajar is a popular vegetable consumed in many part of the world but this product also has a
wide spectrum of bioactive compounds as polyphenols, dietary ber
and saponins. Objetive: In this work, we report the cytotoxic activity
of bioactive fractions of Asparagus ofcinalis provided by Huetor-Tajar
(Granada) (avonoids, bers, saponins and freeze-dried asparagus).
Methods: The HT-29 human colon adenocarcinoma cell line was used.
The cytotoxic activity was evaluated using the sulforhodamine B
(SRB) assay following protocols established by the National Cancer
Institute, National Institutes of Health, Bethesda, MD. This colorimetric
assay estimates cell number indirectly by staining total cellular protein
with the dye SRB. HT-29 cells were seeded in 96-well plates (5000
cells/well) and, after 24 h, the cells were treated for 48 h with the fractions. The protein-bound dye was dissolved in 10 mM Tris base solution for optical density determination at 492 nm using a microplate
reader.Cells viability was expressed as porcentage in relation to controls. Data were averaged from al least three independent experiments
and are expressed as means SEM. The working concentrations used
were 1,10,25,50,100 and 1000 lM. Results: Our results show that the
avonoids and saponins fractions inhibit the proliferation of HT-29
human colon adenocarcinoma cells in concentration-dependent manners.
IC50 values were 60 4.68 and 73.80 lM respectively. 5-FU was
used a positive control (IC50:6.35 4.32lM). Cell viability induced
by the avonoids and saponins fractions was not reduced in the presence of the antioxidants NAC and MnTMPyP. This suggests that reactive oxygen species are not involved in the cytotoxic activity of these
fractions.
This sudy was supported by MCYT-AGL 2007-63703/ALI.
Paper No.: 1760

EFFECT OF METHYLENEDIOXYMETHAMPHETAMINE
(MDMA) ON CYTOSOLIC CALCIUM LEVELS IN PC 12
CELLS. ROLE OF NICOTINIC RECEPTORS
Sara Garcia-Rates, E Escubedo, J Camarasa, D Pubill
Previous work from our group pointed to a role of a7 nicotinic acetylcholine receptors (nAChR) in the neurotoxicity induced by methamphetamine (MA)and MDMA because methyllycaconitine (MLA), a specic
a7 nAChR antagonist, attenuated neurotoxic effects such as oxidative
stress and dopaminergic damage. Also, we demostrated that MA and
MDMA displaced specic nicotinic receptor radioligands indicating that
they can directly interact with nAChR. Once demonstrated the afnity
we studied the effect of preincubation with MDMA on nAChR density
and, as nicotinic ligands such as nicotine do, MDMA induced binding
upregulation of both a7 and heteromeric nAChR types after incubation
times ranging from 6 to 48 h. We have now studied the effect of MDMA
on the activation of nAChR subtypes and the consequent calcium mobilization using uorimetry in Fluo-4-loaded PC12 cells. MDMA produced
a rapid and sustained increase in uorescence without reaching AChinduced maximum effect, and inhibited the ACh- induced response.
These results suggest that MDMA acts as a partial agonist on a7 nAChRs as the response was almost completely inhibited by MLA (10 nM)
and a-bungarotoxin (100 nM) but not by the b2 antagonist dihydro-berythroidine. Subsequently, calcium-induced Ca2 + release from endoplasmic reticulum and entry through voltage-operated calcium channels
are also implicated. Also, treatment with MDMA for 24 h signicantly
increased basal Ca2 + levels and induced an increase in a-spectrin breakdown products indicating calpain and caspase 3 activation. Moreover,
pretreatment with MDMA induced functional up-regulation of calcium
responses to specic agonists of both heteromeric and a7 nAChR suggesting a nicotine-like regulatory effect.
Paper No.: 1951

OXYTOCIN ANTAGONIST PREVENTS PRO-COGNITIVE
EFFECT OF ANGIOTENSIN IV
Paul Gard, C Naylor
University of Brighton, Centre for Biomedical and Health Science
Research, Brighton, UK
Angiotensin IV (AIV) is a hexapeptide fragment of angiotensin II which
has been shown to enhance memory consolidation and recall in rat and
mouse models. The mechanism of this effect is unknown. AIV has been
shown to act via insulin-regulated aminopeptidase (IRAP), which previously has been known as placental leucine aminopeptidase and oxytocinase; AIV inhibits the aminopeptidase activity of this enzyyme. Low doses
of oxytocin have similarly been shown to enhance aspects of memory in
animal models. The possibility therefore exists that the effects of AIV on
memory are mediated by an accumulation of endogenous oxytocin consequent to inhibition of oxytocinase. Using novel object recognition, memory was quantied as the proportion of time spent exploring the novel
object compared with the familiar object (D score). The effects of subcutaneous injection of 4.7lg.kg-1 AIV on novel object recognition in male
and female C57/BL6 mice were determined in the presence and absence
of 10 lg.kg-1 d(CH2) 5 [Tyr(Me)2, Orn8-AVT], an oxytocin receptor
antagonist. In comparison to saline control, AIV signicantly enhanced
novel object recognition in both male and female mice. Prior administration of the oxytocin antagonist reduced the effects of the AIV such that
they were not signicantly different from saline control, without having a
deleterious effect on the saline treated animals. Neither the AIV nor the
295
oxytocin antagonist signicantly inuenced mouse locomotor activity.
These results raise the possibility that the pro-cognitive effects of AIV are
mediated by accumulation of endogenous oxytocin although the selectivity of the antagonist actions warrant futher investigation.
Paper No.: 2052

INTRANASAL ADMINISTRATION OF THE DOPAMINE D3
RECEPTOR ANTAGONIST SB-277011A INHIBITS COCAINE
SELF-ADMINISTRATION AND COCAINE-ENHANCED
BRAIN-STIMULATION REWARD IN RATS
Eliot Gardner(1), Xiao-Qing Peng(1), Xia Li(1), Charles Ashby(2),
Zheng-Xiong Xi(1)
(16%) two days after the testosterone injection (p = 0.007). This is the
rst time a perturbation in the lipoprotein prole is observed after only a
single dose of testosterone. The circulatory level of total testosterone was
associated with the total cholesterol levels on day 2, indicating that the
observed effect is directly associated with testosterone and not an articial
effect of the injection i.e. stress. Moreover, HMGCR specic mRNA level
in HepG2 cells and protein expression in blood of the volunteers was
induced by testosterone. Our results may provide a partial molecular
explanation for the vasculatory adverse side effects of AAS abuse.
Paper No.: 3265

NEUROPROTECTIVE EFFECT OF DIMEBON IN RATS WITH
INTRACEREBRAL POST-TRAUMATIC HEMATOMA
(HEMORRHAGIC STROKE)
(1) National Institute on Drug Abuse, Intramural Research Program, Baltimore, MD, USA
(2) Saint Johns University, Jamaica, New York, USA
TL Garibova(1), TA Voronina(1), SA Litvinova(1), VV Grigoriev(2),

SO Bachurin(2), Tatiana S Kalinina(1)
SB-277011A is the most well characterized selective DA D3 receptor

antagonist. Extensive research during the past decade demonstrates that
systemic administration of SB-277011A consistently inhibits the actions
of cocaine or other addictive drugs in most animal models relating to
drug addiction. However, further development of SB-277011A has been
halted by GlaxoSmithKline Pharmaceuticals, due to unexpectedly poor
bioavailability (~2%) and a very short half-life (<20 min) in primates.
Recent research indicates that a wide variety of therapeutic compounds
with poor bioavailability can be delivered intranasally from nose into the
brain. In the present study, we investigated whether intranasal microinjections of SB-277011A inhibit intravenous cocaine self-administration
or cocaine-enhanced brain-stimulation reward (BSR) similar to that produced after intraperitoneal administration. We found that intranasal microinjections of SB-277011A produced a dose-dependent reduction in
break-point level for intravenous cocaine self-administration under progressive-ratio reinforcement conditions. Intranasal administration of SB277011A also produced a signicant attenuation of cocaine-enhanced
BSR in rats. These ndings suggest that intranasal administration of SB277011A is as effective in attenuating cocaines rewarding efcacy as
intraperitoneal SB-277011A administration. Such data support the potential use of intranasal SB-277011A in the treatment of drug addition in
humans.
Supported by NIDA IRP.
(1) Russian Academy of Medical Science, Zakusov Institute of Pharmacology RAMS, Moscow, Russian Federation
(2) Institute of Physiologically Active Compounds RAS, Moscow,
Russian Federation
Paper No.: 1834

TESTOSTERONE ENANTHATE INDUCES THE EXPRESSION
OF HMGCR AND CHOLESTEROL LEVELS
Paper No.: 1023

DISEASES
THE PROTECTIVE EFFECT OF ATORVASTATIN AGAINST
PROLIFERATIVE ALTERATIONS PRODUCED BY CROTON
OIL ON MOUSE EPIDERMIS
Nina Garevik, C Skogastierna, A Rane, L Ekstrom

Karolinska Institute, Division of Clinical Pharmacology, Department of
Laboratory Medicine, Stockholm, Sweden
Background: The rate-limiting step in the cholesterol synthesis is catalysed by HMG-CoA reductase (HMGCR). It is well established that anabolic-andogenic steroids induce deleterious alterations in lipid
metabolism which may increased the risk of coronary artery disease. In
this study we investigated whether a single dose of testosterone enanthate
affect the cholesterol biosynthesis and the expression of the HMGCR
Study Design and Methods: 55 healthy male volunteers were given 500
mg testosterone enanthate as a single intramuscular dose of TestovironDepot. Total cholesterol levels prior to, and two days after testosterone administration were analysed. Protein expression of HMGCR in
whole blood was investigated by Western blotting. In order to study
whether testosterone regulates the mRNA expression of HMGCR, in vitro
studies were performed in a human liver cell-line (HepG2). Results and
Discussion: The total level of cholesterol was signicantly increased
This investigation was carried out with purpose to examine the neuroprotective properties of dimebon (2,3,4,5-tetrahydro-2,8-dimethyl-5-(2(6-methyl-3-pyridyl)ethyl)-1H-pyrido(4,3-b)indole), an agent with neuroprotective activity, a positive modulator of NMDA and AMPA subtype
of glutamatergic receptors. The study used new models of intracerebral
post-traumatic hematoma (hemorrhagic stroke). Hemorrhagic stroke (HS)
was produced in animals by cerebral tissue destruction in the internal
capsule region. Dimebon in dose of 0.1 mg/kg was administered i.p. 3-4
hrs following the operation. It was established that the agent diminished
HS-induced neurological decit, movement co-ordination disturbances.
Dimebon was found to prevent the animal death caused by HS. The animals which were learned the passive avoidance reex prior to HS on
Day 3 after operation demonstrated disturbances in the retrieval of conditioned passive avoidance reex (PAR). With dimebon PAR retrieval
disorders were much less pronounced. The results obtained from these
studies provided fair evidence of neuroprotective and memory-restoring
properties of dimebon in a new model of hemorrhagic.
Alireza Garjani(1), Y Doustar(2), H Rezazadeh(1), S Andalib(1), N

Maleki-Dizaji(1)
(1) Tabriz University of Medical Sciences, School of Pharmacy, Department of Pharmacology, Tabriz, Iran
(2) Islamic Azad University Faculty of Veterinary Medicine, Tabriz, Iran
A growing body of preclinical data indicates that statins may have antineoplastic properties, but some studies raise the possibility that statins
may possess a carcinogenic potential. A single application of croton oil
to mouse skin induces an ordered sequence of ultrastructural changes in
the cells of epidermis, which correlate with changes in thickness, number
of nucleated cell layers, and mitotic index of the interfollicular epidermis.
To evaluate the effect of atorvastain on ultrastructural and histological
alterations produced by croton oil, Swiss albino mice were received atorvastatin (3, 6, 9 mg/kg/day; orally) for a period of 2 weeks prior to single
topical application of croton oil (0.5% in 200ll acetone/mouse) to the
296
shaved backs of the animals. After 24 hour animals were killed under
deep anesthesia and the skin was excised and xed in Stieves solution
and then processed for histological examination. All parameters: disturbance of cell polarity, inammatory response, nucleated cell layer, thickness of epidermal layer, and mitotic index were inhibited dose
dependently (P < 0.001) by pre-treatment with atorvastatin. The number
of cells in mitosis in 2500 lm2 of interfollicular epidermis was reduced
from 3 0.3 in untreated group to 2 0.2, 1 0.2 (P < 0.01), and 0.6
0.2 (P < 0.001) by 3, 6, and 9 mg/kg of atorvastatin, respectively. The
results of this study show strong anti-inammatory and anti-proliferative
effects of atorvastatin and suggest that statins may suppress events associated with carcinogenesis.
Paper No.: 2733

A JERTE VALLEY CHERRY-BASED PRODUCT ACTS AS AN
ENHANCER OF SLEEP, IMPROVES MOOD AND POSSESSES
ANTIOXIDANT PROPERTIES
Maria Garrido(1), D Gonzalez-Gomez(2), M Lozano(2), MT Hernandez(2), MS Salcedo(3), C Barriga(1), SD Paredes(1), AB Rodriguez(1)
(1) University of Extremadura Faculty of Science, Department of Physiology, (Neuroimmunophysiology and Chrononutrition Research Group)
Badajoz, Spain
(2) Technological Institute of Food and Agriculture (Intaex), Badajoz,
Spain
(3) Perpetuo Socorro Hospital, SES, Childrens Psychiatry Unit, Badajoz,
Spain
Tryptophan, serotonin and melatonin are implicated in the regulation of
sleep. Melatonin directly, or indirectly by supplementation with its precursor tryptophan, functions as a powerful antioxidant. Jerte Valley cherries are rich in tryptophan (Cubero J et al, Food Anal. Methods 2009;
doi 10.1007/s12161-009-9084-1), serotonin and melatonin (GonzalezGomez D et al, Eur. Food Res. Technol. 2009; 229:223-229). The study
was aimed at evaluating the effect of Jerte Valley cherry product intake
on the sleep-wake cycle and mood of middle-aged volunteers as well as
determining the participants urine levels of total antioxidant capacity,
cortisol, 5-hydroxyindolacetic acid, and 6-sulfatoxymelatonin. Volunteers
consumed 28 g of this product twice a day for 5 days. Actigraphic monitoring was used to record and display the temporal patterns of the individuals activity and rest during 5 days before the beginning of the assay
(basal), 5 days of assay (assay) and 5 days afterwards (post-assay). Psychological tests were performed in basal, assay and post-assay conditions. Total antioxidant capacity, cortisol, 5-hydroxyindolacetic acid and
6-sulfatoxymelatonin were analyzed using colorimetric or ELISA assay
kits in rst-void morning and 21:00 h urines collected on the third and
fth day in basal, assay and post-assay conditions. The ingestion of this
product improved nocturnal rest and mood, decreased cortisol and
increased total antioxidant capacity, 5-hydroxyindolacetic acid and 6-sulfatoxymelatonin urinary levels. The product may be employed as a
source of antioxidants and be useful in states where sleep and mood disorders have been reported.
Funded by PDT0A008-Junta de Extremadura.
Paper No.: 1370

DISEASES
EFFECT OF AT1 RECEPTOR BLOCKADE ON THE
ANTIOXIDANTS ENZYMES ACTIVITIES IN AN
EXPERIMENTAL MODEL OF PERIODONTAL DISEASE
INDUCED BY LIPOPOLYSACCHARIDE IN THE RAT
In addition to its vasoactive actions, angiotensin II (AngII) has been

implicated in the release of pro-inammatory cytokines, activation of
mitogenic factors, oxidative stress and modulation of nitric oxide synthesis, through stimulation of AT1 receptor (AT1R).AT1R stimulation
increases oxidative stress through activation of NAD(P)H oxidase.
Increased reactive oxygen species (ROS) production leads to tissue damage associated with activation of the inammatory process. Local administration of bacterial endotoxin (lipopolysaccharide, LPS) in the gum of
the rats produces an inammatory response that progress in periodontal
disease. AT1R blockade reverse the AngII mediated pro-inammatory
effects in the vasculature system and in chronic hypertension. The aim of
the present study was to assess the role of AT1R blockade in the inammatory reaction and ROS generation in an experimental model of LPSinduced periodontal disease in the rat. Male Sprague-Dawley rats (220240g) were divided in four groups: Control, LPS, VAL: Valsartan (20
mg/kg, p.o, 7 and 14 days) and LPS+VAL. Rats were anesthetized with
10% ketamine (60 mg7kg) and euthanized by decapitation, the maxilla
dissected and the connective tissue collected in the appropriate buffer.
CAT, SOD, GPx activity was determined by spectrophotometric analysis
and NOS activity was assayed by monitoring the conversion of radiolabelled L-arginine to L-citrulline. LPS signicantly increased tissue CAT,
SOD, GPx and NOS activity. VAL blunted the LPS-increased antioxidant
enzymes and NOS activity. Our results indicate a role AngII and AT1
receptor in the pathogenesis of the LPS-induced periodontal disease.
(Mision Ciencia, Proyecto ECCV No. 2007001585).
Paper No.: 2305

DISEASES
NOVEL SMALL MOLECULAR WEIGHT CCL2 INHIBITORS
REDUCE INFLAMMATORY RESPONSE IN MICE
Beatrice Garrone, A di Matteo, G Mangano, C Bartella, C Apicella,
N Cazzolla, G Furlotti, A Guglielmotti
Angelini Research Center-ACRAF, Department of Pharmacology, Rome,
Italy
Prevention of inammation through blockade of the chemokines/chemokine receptors system is a major target for pharmacological intervention.
Angelini has an ongoing development program on CCL2 inhibition
based on an original small molecule, bindarit, which is a selective inhibitor of CCL2 synthesis. Bindarit has recently completed a phase II clinical
study in patients with diabetic nephropathy and is currently tested in coronary in-stent restenosis. Based on bindarit promising results, a group of
derivatives was selected from in-house library and screened in order to
select new CCL2 synthesis inhibitors. Activity of compounds on CCL2
production was evaluated in vitro on LPS-stimulated MonoMac6 cells.
CCL2 accumulation was quantied by ELISA. In addition, the effect on
specic CCL2 mRNA transcripts was also assessed. In vivo effects were
studied in mice measuring CCL2 plasma levels in LPS-treated animals
as well as thioglycollate-induced peritoneal macrophages recruitment. In
the MonoMac6 model, compounds showed CCL2 inhibition with an
IC50 in the range 20-329 lM. The most active compounds (IC50 lower
than 100 lM) were tested in vivo. Intraperitoneal administration of AF6,
AF11 and AF13 signicantly reduced CCL2plasma levels with 39, 42
and 51% inhibition, respectively. Moreover, AF1, AF13and AF14
showed a signicant effect on thioglycollate-induced peritonitis by
decreasing macrophages recruitment in the range 30-43%. Present results
and potential advantages of CCL2 synthesis inhibitors versus CCL2-specic mAbs and receptor antagonists call for further characterization
of these novel small molecular weight compounds in inammatory
conditions.
Maria del Rosario Garrido, MG Matos, JA Silva, Y Mathison, A Israel

University of Venezuela, Schook of Pharmacy, Laboratory of Neuropeptides, Caracas, Venezuela
297
Paper No.: 2352
QUALITY OF ANTIBIOTIC PRESCRIBING IN INFANT IN
AMBULATORY CARE
Kristina Garuoliene, J Gulbinovic
Department of Pathology, Forensic Medicine & Pharmacology, Vilnius,
Lithuania
Inappropriate antibiotic prescribing in infants increases antimicrobial
resistance, unnecessary health care costs and may inuence infants
health status in the future life. Quality of antibiotic prescribing in infants
in ambulatory care in Lithuania during 2003-2008 was analysed. The
data on antibiotic dispensing was obtained from the population based
electronic database of the Lithuanian Compulsory Health Insurance
Information system. This database provides the complete prescription
medication history of patients, diagnosis according IDC-10, identies
prescriber and dispensing pharmacy. The study included all infants (0-12
months of age), who claimed at least one dispensing for any systemic
antibacterial (ATC code: J01) during 2003-2008. The number of dispensed prescriptions per 1000 infants decresed from 1047 in 2003 to 719
in 2008. The prevalence rate of infants treated with antibiotics decresed
from 56,7% to 43,8% during study period. The percentage of infants
who received multiple courses of antibiotic treatment (more that 3 per
year) decresed from 18,5% in 2003 to 15,2% in 2008. The most common
indications for antibiotic prescribing were acute bronchitis, acute pharyngitis, and common cold which made up 29%, 24% and 13% out of all
antibiotic prescriptions, respectively. Broad spectrum penicillins with or
without b lactamase inhibitors were the most often used antibiotics and
made up 70% out of all prescriptions. Three fold differences in antibiotic
prescribing were found between different regions. Obtained results suggest wide unnecessary prescribing of antibiotics in infants.
Paper No.: 1974

THE EFFECT OF NOCICEPTIN ON UTERINE
CONTRACTIONS IN TERM-PREGNANT RATS IN VITRO
Robert Gaspar(1), A Klukovits(1), K Tekes(2), O Gunduz(3),
S Benyhe(3), A Borsodi(3), BH Deak(2), J Hajagos-Toth(1), J Verli(1),
G Falkay(1)
(1) University of Szeged Faculty of Pharmacy, Department of Pharmacodynamics and Biopharmacy, Szeged, Hungary
(2) Semmelweis University, Department of Pharmacodynamics,
Budapest, Hungary
(3) Hungarian Academy of Sciences, Institue of Biochemistry, Biological
Research Centre, Szeged, Hungary
The actions of the endogenous peptide nociceptin (N/OFQ) on the myometrium have not been investigated previously. Our aim was to study
the presence and functional role of N/OFQ in the modulation of uterine
contractility in pregnant rats at term.The presence of N/OFQ and its
receptors (NOP) in the uterus were detected by radioimmunoassay (RIA)
and radioligand-binding experiments. The N/OFQ-stimulated G-protein
activation was assessed by a [32S]GTPcS-binding technique. The effects
of N/OFQ in uterine rings precontracted with KCl or oxytocin were also
tested in vitro. Uterine levels of cAMP were measured by enzyme immunoassay (EIA). The K+ channel blockers tetraethylammonium and paxilline were used to study the role of K+ channels in mediating the uterine
effects of N/OFQ. Both N/OFQ and NOP were present in the uterus. N/
OFQ revealed a maximum contraction inhibition of ~30%, which was
increased to 40% by naloxone. Naloxone and pertussis toxin signicantly
attenuated the G-protein-stimulating effect of N/OFQ. The uterine cAMP
levels were found to be elevated by N/OFQ and naloxone, and after
preincubation with pertussis toxin. Tetraethylammonium and paxilline
reduced the contraction-inhibiting effect of N/OFQ and naloxone to ~10

and 15%. We presume that N/OFQ plays a role in regulating uterine contractility at term. Its effect is mediated partly by Gs-proteins coupled to
NOP receptors and elevated cAMP levels, and also by Ca2 + -dependent
K+ channels. Our results demonstrate a novel action and signalling pathway for N/OFQ that might be a potential drug target.
Paper No.: 1260

EFFECT OF GRAPEFRUIT JUICE ON DRONEDARONE PHARMACOKINETICS IN HEALTHY SUBJECTS
Christine Gaud(1), A Brunet(2), E Sultan(2), J Galleyrand(3), T Duvauchelle(4)
(1) Sano-Aventis R&D, Department of Clinical and Exploratory Pharmacology,Montpellier, France
(2) Sano-Aventis R&D, Department of Global Metabolism and Pharmacokinetics,Montpellier, France
(3) Sano-Aventis R&D, Department Biostatistics and Programming,
Montpellier,France
(4) Institut ASTER, Paris, France
Dronedarone is a multichannel blocker developed for the treatment of
patients with atrial brillation. It is highly metabolized by CYP3A4 with
a rst-pass metabolism. This study evaluated the effect of multiple ingestions (tid) of 300mL double-strength grapefruit juice (GFJ), on pharmacokinetics (PK) of dronedarone and its active metabolite, SR35021. This
randomized, open-label, nonplacebo-controlled, 2-treatment (dronedarone + water or dronedarone + GFJ), 2-period crossover study was conducted in 24 healthy young men. Each period had 3 phases: Phase 1 and
Phase 2 with single doses of dronedarone in fasted and fed conditions
(data not presented), and Phase 3 with multiple doses of dronedarone
400 mg bid in fed condition for 10 days. GFJ was ingested for 20 days.
Plasma dronedarone/SR35021 concentrations were determined using a
validated liquid chromatography-mass spectrometry method. Non-compartmental PK parameters were calculated. Concomitant oral ingestions
of double-strength GFJ 300 mL tid increased maximal plasma concentration (Cmax) and area under the plasma curve (AUC0-12) of dronedarone
after 400 mg bid multiple doses in fed condition by2.5- and 3.0-fold,
respectively, and did not change SR35021 Cmax and AUC. For electrocardiography (ECG) parameters, mean changes from baseline were
+19.4 msvs. +5.7 ms in PR and +21.5 ms vs. +7.8 ms in QTc during
dronedarone + GFJ and dronedarone + water co-administrations, respectively. In very stringent conditions, GFJ increased dronedarone exposure
by approximately 3-fold, an increase that was associated with consistent
changes of dronedarone in ECGparameters. Patients should, therefore,
avoid GFJ beverages while taking dronedarone.
Paper No.: 1261

EFFECTS OF DRONEDARONE ON THE
PHARMACOKINETICS AND PHARMACODYNAMICS OF
WARFARIN IN HEALTHY SUBJECTS
Christine Gaud(1), J-P Duret(2), A Brunet(2), E Sultan(2),
R Ebrahimi(3)
(1) Sano-Aventis R&D, Department of Clinical and Exploratory Pharmacology, Montpellier, France
(2) Sano-Aventis R&D, Department of Global Metabolism and Pharmacokinetics, Montpellier, France
Montpellier, France
298
patients with atrial brillation (AF). This study assessed the effects of
multiple doses of dronedarone on the single dose pharmacokinetics (PK)
and pharmacodynamics(PD) of warfarin, a frequently co-administered
drug in patients with AF. Following a randomized 2-treatment, 2sequence, crossover design, 17 healthy young men received a single dose
of warfarin 30 mg in Period 1 and a 14-day multiple administration of
dronedarone 600 mg bid co-administered with a single dose of warfarin
30 mg on Day 8 of Period 2. Plasma was analyzed for R- and S-warfarin
(2 chiral forms) using a validated high-performance liquid chromatography/ultraviolet method. Non-compartmental PK parameters and PD
activity (international normalized ratio [INR]) were determined. Ratios of
geometric means (warfarin + dronedarone/warfarin alone) with 90% condence intervals (CI) were: R- warfarin, 1.04 (0.981.11) for maximal
plasma concentration (Cmax) and 1.11 (1.051.18) for area under the
plasma curve (AUClast); S- warfarin, 1.07 (1.001.14) for Cmax and
1.19 (1.131.26) for AUClast. INR ratios of geometric means (warfarin
+ dronedarone/warfarin alone) with 95% CI were 1.06 (0.991.13) at
peak and 1.07 (1.021.12) for AUC. Dronedarone at a higher dose than
the proposed therapeutic one (400 mg bid) had a very limited effect on
PK of S-warfarin and no effect on R-warfarin. There were no clinically
relevant effects on INR, suggesting that monitoring should be done in
accordance with the warfarin label.
Paper No.: 1262

EFFECTS OF DRONEDARONE ON THE
PHARMACOKINETICS OF DIGOXIN IN HEALTHY SUBJECTS
Christine Gaud(1), F Hurbin(2), A Brunet(2), E Sultan(2),
J Galleyrand(3), E Guenole(4)
(1) Sano-Aventis R&D, Department of Clinical and Exploratory
Pharmacology, Montpellier, France
Montpellier, France
(4) Therapharm Recherches, Caen, France
patients with atrial brillation. In vitro, it has a potential to inhibit
digoxin secretion mediated by P-glycoprotein (P-gp). This study assessed
the effects of multiple doses of dronedarone on multiple-dose pharmacokinetics (PK) of digoxin and the tolerability of co-administration. A placebo-controlled, 2-treatment crossover study was conducted in 20
healthy young men receiving digoxin 0.25 mgqd for 10 days (loading
dose, 0.75 mg), alone or co-administered with dronedarone 400 mg bid
for 10 days in fed condition. Plasma was analyzed for digoxin with a validated immunoassay method. Non-compartmental PK parameters were
calculated. ECG and vital signs were regularly performed. Ratios of geometric means (digoxin + dronedarone/digoxin alone) with 90% condence intervals were 1.75 (1.581.93) for maximal plasma concentration
(Cmax), 2.57(2.212.98) for area under the plasma curve (AUC0-24),
0.57 (0.460.69) for renal clearance (ClR0-24), and 1.45 (1.191.78) for
urinary excretion (Ae0-24).The tolerability of the co-administration was
satisfactory. No prolonged QTc were observed. Dronedarone at therapeutic dosing regimen signicantly increased steady state digoxin exposures
by 2.6-fold, due to decreased digoxin renal clearance and increased
absorption through P-gp inhibition at both levels. When digoxin is coadministered with dronedarone, it is recommended that digoxin dose
should be reduced by 50% and serum digoxin levels, clinical signs and
ECG should be closely monitored according to label.
Paper No.: 1263

PHARMACOKINETIC AND PHARMACODYNAMIC
INTERACTION OF DRONEDARONE AND VERAPAMIL IN
HEALTHY SUBJECTS
Christine Gaud(1), S Doroumian(2), A Brunet(2), E Sultan(2),
J Galleyrand(3)
Montpellier France
patientswith atrial brillation. It is highly metabolized by, and has a
potential to inhibit, CYP3A4. This study assessed the pharmacokinetic
(PK) and pharmacodynamic (PD) interactions of multiple doses of dronedarone and the calcium-channel blocker verapamil (CYP3A4 substrate).
A randomized crossover study with 13-day washouts was conducted in
21 healthy young men receiving dronedarone 400 mg bid alone, verapamil 240 mg qd alone and co-administration for 14 days in fed condition.
Plasma was analyzed for dronedarone and its active metabolite,
SR35021, using a validated liquid chromatography mass spectrometry/
mass spectrometry method, and for verapamil/norverapamil using a
validated high-performance liquid chromatography/ultraviolet method.
Non-compartmental PK analyses were performed. Effects on electrocardiography (ECG) parameters were evaluated. Ratio of geometric means
(verapamil +dronedarone/verapamil alone) with 90% condence intervals
(CI) were 1.30(1.141.48) and 1.29 (1.151.44) for verapamil and norverapamil area under the plasma curve (AUC0-24), respectively. Ratio of
geometric means (dronedarone +verapamil/dronedarone alone) with 90%
CI were 1.48 (1.381.58) and 0.98(0.921.04) for dronedarone and
SR35021 AUC0-12, respectively. Co-administration increased the PR
and QTcF (Fridericias correction) greater than each product alone (mean
changes compared with dronedarone alone: +10.2 ms and +11.9 ms,
respectively; compared with verapamil alone: +15.8 ms and +31.9
ms,respectively). Dronedarone increased verapamil exposure 1.3-fold,
and verapamil increased dronedarone exposure 1.5-fold. Due to alterations in ECG values, verapamil should be initiated at low dose and
up-titration should be done only after ECG assessment.
Paper No.: 1264

PHARMACOKINETICS AND TOLERABILITY OF
DRONEDARONE IN PATIENTS WITH MODERATE HEPATIC
IMPAIRMENT
Christine Gaud(1), A Brunet(2), E Sultan(2), J Galleyrand(3),
K Lasseter(4), T Marbury(5)
Montpellier,France
(4) Clinical Pharmacology Associates, Miami, FL, USA
(5) Clinical Research Center, Orlando, FL, USA
patients with atrial brillation. It is highly metabolized by CYP3A4 and
highly bound to plasma protein. This study assessed the pharmacokinetics (PK) of dronedarone and its active metabolite (SR35021), and the
pharmacodynamics (PD) and tolerability of dronedarone in patients with
299
hepatic impairment. Thirteen patients with moderate hepatic impairment
(Child-Pugh class B) were globally matched by sex, age, and weight
with 13 healthy subjects. After determining that PK changes were limited
(<2-fold increase in exposure) and safety results were satisfactory in 4
patients and 4 subjects receiving 400 mg qd for 7 days, the study proceeded with 400 mg bid (therapeutic dose) for 7 days in 8 patients and 8
subjects. Total and unbound plasma concentrations of dronedarone/
SR35021 were assessed using a validated liquid chromatography-mass
spectrometry method, and a non-compartmental analysis was performed.
Steady state was reached within 3 to 6 days in patients and subjects for
dronedarone and SR35021. In patients as compared to subjects, total and
unbound steady state exposures increased by 1.3- and 1.9-fold for dronedarone, respectively, and decreased by ~ 53 % and ~62% for SR35021.
For electrocardiography (ECG) parameters, mean changes from baseline
were +16.77 ms vs. +7.97 ms in PR and +8.58 ms vs. +5.37 ms in QTcF
in patients and subjects, respectively. Some patients experienced mild
gastrointestinal disorders. Based on mild to moderate exposure increases,
ECG effects and tolerability, dronedarone may be used at the therapeutic
dose in patients with moderate hepatic impairment.
Paper No.: 2242

STEREOSELECTIVITY OF THE RECOMBINANT AND NATIVE
HISTAMINE H3 RECEPTOR ISOFORMS: THE
H3-AUTORECEPTOR IS A SHORT ISOFORM
Florence Gbahou, A Rouleau, S Morisset, J-M Arrang
INSERM U894 Centre de Psychiatrie et Neurosciences, Sainte-Anne,
Paris, France
The histamine H3 receptor (H3R) was pharmacologically characterized as
an autoreceptor regulating histamine synthesis and release in the rat
brain. Activation of histaminergic neurons promotes arousal, attention,
improves learning and, has been proposed as a symptomatic therapeutic
approach in human attentional and ageing disorders such as Alzheimers
disease. Since its cloning in 1999, the molecular studies of the receptor
have shown that the H3R belongs to the G-protein-coupled receptor family, displays constitutive activity, and has many isoforms generated by
deletion of a pseudo-intron in the third intracellular loop. In spite of the
length of this loop, only minor pharmacological differences have yet
been reported between H3R isoforms. In this study, we hypothesized that
a deletion in this important loop would promote some modications in
the 3D structure of the H3R. To support our hypothesis, we used stereoenantiomers which are molecules of identical composition but with different three-dimensional structures. Two pairs of R(-) and S(+) agonist
and antagonist enantiomers displaying a high stereoselectivity at native
H3-autoreceptors were studied in various functional tests at native H3Rs
and recombinant isoforms including the non-deleted H3R isoform and a
short isoform common to all species. Our data show strong differences
between the stereoselectivity, i.e. conformations, of the isoforms, and
show that a short isoform plays the role of H3-autoreceptor.
Paper No.: 2988

CHARACTERIZATION OF A LIPID KINASE INVOLVED IN
THE SYNTHESIS OF ALKYL LYSOPHOSPHATIDIC ACID
Amanda Gellett, Y Kharel, K Lynch
to the glycerol backbone via an acyl, an alkyl, or an alkenyl linkage.

Acyl LPA is the most abundant of the three classes, however, alkyl LPA
has been shown to contribute to the aggressiveness of ovarian cancer and
platelet aggregation. There are multiple potential pathways for alkyl LPA
synthesis. Intracellularly, alkyl LPA could be formed by the esterase,
KIAA1363, deacetylating 2-acetyl MAGE (monoalkylglycerol ether) followed by phosphorylation by a heretofore unidentied lipid kinase. An
alternate intracellular pathway would involve phosphorylation of alkyl acylglycerols by DGK (diacylglycerol kinase), with subsequent cleavage
of the acyl fatty acid by phospholipase A2. The alkyl acylglycerol, 2-acetyl-MAGE, is phosphorylated by human ovarian cancer cell (SKOV-3)
lysates and mouse liver homogenates. Conversely, direct phosphorylation
of MAGE to form alkyl LPA could not be demonstrated in these extracts.
Forced expression of various isoforms of mammalian DGK in HEK293T
cells resulted in increased phosphorylation of 2-acetyl-MAGE in cell
extracts. Thus phosphorylation of 2-acetyl-MAGE by DGK followed by
deacetylation is a potential route for the intracellular synthesis of alkyl
LPA. Sphingosine and analogs such as the immunomodulatory prodrug,
FTY720, increase Type I DGK activity in broken cell assays. Ongoing
studies include determining whether stimulation of whole SKOV-3 cells
by sphingosine analogs increases production of alkyl LPA.
This research was supported by NIH grants R01 GM067958 and T32
GM007055.
Paper No.: 3140

DISEASES
DICLOFENAC SODIUM SUPPRESSES RAT CALVARIAL BONE
DEFECT REGENERATION
T Georgopoulou-Karanikola(1), Stergios Tsartsalis(2), H Kalekou(3),
AA Veis(1), B Kokkas(2), AT Tsirlis(1), M Mironidou-Tzouveleki(2)
(1) Aristotle University of Thessaloniki School of Dentistry, Department
of Alveolar Surgery, Implantology and Radiology, Thessaloniki, Greece
(2) Aristotle University of Thessaloniki School of Medicine, A Laboratory of Pharmacology, Thessaloniki, Greece
(3) Aristotle University of Thessaloniki School of Dentistry, Department
of Oral Medicine and Oral Pathology, Thessaloniki, Greece
Introduction: Diclofenac sodium is a non-steroidal anti-inammatory
drug, inhibiting both cyclooxygenase-1 and cyclooxygenase-2, which is
frequently prescribed for analgesic therapy. However, its effect on bone
regeneration has raised doubts about its use after several medical procedures in medicine and dentistry. The purpose of this study was to examine the effect of diclofenac on rat calvarial bone defect regeneration.
Materials: Adult male Wistar rats (250-300g) underwent a surgical procedure in which two articial defects 6-mm in diameter were made on each
of the parietal bones by means of a specic trephine bar. Rats were
divided in two groups, A and B, of 8 rats in each group. The animals of
group A received daily intramuscular injections of diclofenac sodium
(5mg/kg) for 4 weeks, while the non-treated animals of group B served
as control. Calvarial bone samples from the defect area were decalcied
in EDTA in acid buffer and embedded in parafn. H+E stained sections
from the sagittal diameter of the defect area were subjected to histological and histomorphometric analysis. Results: At 4 weeks a thin layer of
brous connective tissue had formed in the defect area closing the defect.
Partial bone regeneration in the defect area was observed in both groups.
Histomorphometric analysis revealed a lower percentage of new bone in
group A (15,55 10,34) compared to group B (29,34 13,47) (p <
0,05). Conclusion: Our results suggest that diclofenac has an inhibitory
effect on the process of bone regeneration in the rat calvarial defect
model.
University of Virginia, Department of Pharmacology, Charlottesville,

VA, USA
Lysophosphatidic acid (LPA) designates a group of phosphoglycerides
divided into three groups based on whether the carbon chain is connected
300
Paper No.: 2438
CARBOPLATIN-INDUCED TOXICITY INCREASED TO
GRANULOCYTE-MACROPHAGE PROGENITORS (CFU-GM)
IN OTSUKA-LONG-EVANS-TOKUSHIMA FATTY (OLETF)
RATS
rats increased the latency time during learning, short and long memory
retention tests. In hot-plate analgesic test and in analgesy-meter test and
ketamine-treated rats did not change the latency of reaction compared to
controls. Our results permitted us to suggest that ketamine improved
learning and memory processes and has weak effect on nociception in
doses applied.
References: 1. Riedel G. et al. Behav. Brain Res, 2003, 140 (1-2):1-47.
2. Furst Z. Neuropsychopharmacol. Hung, 2008, 10:127-30.
Krisztina Geresi(1), K Benko(2), B Szabo(3), A Megyeri(1), B Peitl(1),

Z Szilvassy(1), I Benko(1)
(1) Medical and Health Science Center, University of Debrecen, Departmant of Pharmacology and Pharmacotherapy, Debrecen, Hungary
(2) Euromedic Diagnostics, Medical and Health Science Center, Univesity of Debrecen, Debrecen, Hungary
(3) Heart Center, Semmelweis University, Budapest, Hungary
OLETF rats have a congenital defect in the expression of CCK-1 receptor gene. Because of the increased food intake a metabolic syndrome has
developed early. In our other experiments we found an impaired granulopoiesis in Zucker obese rats, probably by metabolic changes due to insulin resistance. We study the toxicity of carboplatin, an alkylating-related
cytotoxic drug on granulocyte-macrophage progenitors (CFU-GM) in
OLETF and in their healthy counterparts, LETO rats. At rst sight granulopoiesis, as measured by cellularity, the frequency of CFU-GM
and the CFU-GM content of the femoral bone marrow – did not
differ in obese OLETF and LETO rats. However, testing the vulnerability
of CFU-GM cells cultured them in the presence of carboplatin, we
detected an increased damage in the CFU-GM progenitors obtained from
OLETF compared with LETO. After in vivo administration of a CCK-1
selective antagonist, lorglumide, we could establish a similarly increased
toxicity on the progenitors in LETO rats as in CCK-1 decient OLETF
rats. Lorglumide might have a direct effect on progenitor cells, because it
could inhibit colony formation of CFU-GM cells of LETO rats also in vitro. As far as we know, this is the rst nding concerning CCK-1 receptorial effects on CFU-GM hemopoietic progenitors. Obesity may alter
the function of CFU-GM cells by different mechanisms. As obesity has
become pandemic the higher risk for myelotoxicity of anticancer treatment has a growing importance. The CCK-1 receptor deciency maybe
also some of the reason of the increased risk of anticancer chemotherapy
in some obese patients.
Paper No.: 1550

EFFECTS OF KETAMINE ON NOCICEPTION, LEARNING
AND MEMORY IN RATS
Damianka Getova, N Doncheva
Medical University Plovdiv, Department of Pharmacology, Soa, Bulgaria
Ketamine is intravenous anesthetic acting on NMDA-glutamate receptors
(1), which are involved in nociception and memory functions (2). We
study the effects of ketamine using two nociceptive tests and active and
passive avoidance tests. Rats (n = 10) were chronically treated: 1st group
with saline, 2nd, 3rd and 4th groups with ketamine 10, 15 and 20 mg/kg.
Active avoidance test an automatic reex conditioner was done with
learning and memory tasks and the number of avoidances and escapes
were counted. Step-through and step-down passive avoidance tests were
done with learning, short and long memory retention task. It was measured the latency of reaction. The nociceptive tests were done hot-plate
and alangesy-meter and the reaction time were measured. In active
avoidance test the control rats learned the tasks and increased the number
of avoidances on learning session as well as on memory retention test.
Ketamine in all doses used increased the number of avoidances on learning and on memory retention test. The number of escapes was not changed. In both passive avoidance tests the controls and ketamine-treated
Paper No.: 2495

OXALIPLATIN-INDUCED NEUROPATHY: GLIAL
ACTIVATION IN SPINAL CORD AND BRAIN
Carla Ghelardini(1), L Di Cesare Mannelli(1), L Bonaccini(2),
A Pacini(2), A Bartolini(1)
(1) University of Florence, Department of Pharmacology, Florence, Italy
(2) University of Florence, Department of Anatomy, Florence, Italy
Oxaliplatin, unlike other platinum derivatives, does not result in signicant renal impairment or ototoxicity, and it has only mild hematological
and gastrointestinal toxicity. On the other hand the limiting side effect is
its neurotoxicity that acts as bases for a neuropathic syndrome. A tangled
panel of symptoms may be disabling for these patients, adversely affecting activities of daily living and thereby quality of life. Chronic pain as
well as the nding of nerve hyperexcitability suggest an involvement of
the central nervous system. In a rat model of painful oxaliplatin-induced
neuropathy (2,4 mgkg-1 intraperitoneally, daily injected for 21 days), we
investigated the prole of glia activation in the spinal cord and in brain
areas involved in pain sensation. In the central nervous system a general
glia activation was provoked by oxaliplatin administration. In particular,
microglia (Iba1 antigen) showed a round-shaped body, indicative of a
differentiation toward a macrophagic phenotype. Moreover, the astrocytes (GFAP antigen) showed an increased density and signs of activation, such as increase in the body size, thickening of the processes and
labelling intensity. These modications concerned the dorsal horn of the
spinal cord, as evidenced by DAB staining. Astrocytes and microglia
examined by confocal microscopy in different brain districts revealed an
increasing activation in amygdala, raphe nuclei, thalamic nuclei, somatosensorial area 1 and periacqueductal grey matter (PAG). Further understanding of the molecular mechanisms that underlie the effects of glia on
pain processing should lead to the development of new and more efcient approaches for the treatment of pain.
Paper No.: 2948

INTERACTIONS OF SDF-1 AND ADP IN PLATELET
ACTIVATION
Olof Gidlof, C Hogberg, B Olde, D Erlinge
Lund University, Department of Molecular Cardiology, Lund, Sweden
Introduction: Apart from playing a crucial role in maintaining hemostasis, platelets are also increasingly recognized as important mediators of
inammation. Platelets interact with endothelial cells and leukocytes
through an extensive repertoire of inammatory molecules. Several studies have shown that chemokines such as stromal cell-derived factor one
(SDF-1) are capable of inducing platelet aggregation. The mechanism by
which SDF-1 activates platelets is not yet fully understood and current
reports are partly contradictory. The aim of this study was to elucidate
the signalling pathways involved in SDF-1 mediated platelet activation.
Materials: Whole blood, platelet rich plasma and washed platelets were
301
obtained from healthy individuals. Results: Platelet aggregation induced
by 10 nM SDF-1b was reduced by 80% with the addition of 10 lM of
the P2Y12 receptor agonist AZD6140 (p < 0,01). In addition, cAMP levels in PGE1-treated platelets were decreased by 14% when adding 10
nM SDF-1b (p < 0,05). 5 lM of ADP caused a 12% upregulation of the
SDF-1 receptor CXCR4 on platelets (p < 0,05). Conclusion: We show
that SDF-1b mediated platelet activation is dependent on P2Y12 receptor
signalling. Treating platelets with the chemokine results in a decrease in
cAMP levels, conrming that activation requires Gai-coupled signalling.
We also demonstrate that stimulating platelets with ADP results in an
increased surface expression of the SDF-1 receptor, CXCR4. Taken
together, these data suggest possible signalling crosstalk between SDF-1
and ADP in platelet activation.
Paper No.: 1602

PHARMACOLOGICAL BASIS FOR THE MEDICINAL USE OF
PSYLLIUM HUSK IN CONSTIPATION AND DIARRHEA
Anwar-ul-Hassan Gilani(1), MH Mehmood(1,2)
(1) Aga Khan University, Department of Biological and Biomedical
Sciences, Karachi, Sind, Pakistan
(2) University of Karachi, Sind, Pakistan
The psyllium husk was studied to rationalize its medicinal use in gut
motility disorders using both the in-vivo and in-vitro assays. The aqueous
methanolic extract (Pop.Cr) caused laxative effect in mice at 100 and 300
mg/kg. The laxative effect was partially sensitive to atropine or
SB203186 (5-HT4 receptor antagonist). At higher doses (500 and 1000
mg/kg), it exhibited antisecretory and antidiarrheal activities. The antidiarrheal effect of Pop.Cr was increased when repeated in mice pretreated
with L-NAME (nitric oxide synthase inhibitor). When tested in guinea
pig-ileum, Pop.Cr caused a concentration-dependent stimulant effect at 110 mg/mL, which was partly inhibited in the presence of atropine or/
SB203186, suggesting partially cholinergic and 5-HT4 receptor-mediated
effect. In spontaneously contracting rabbit jejunum, Pop.Cr caused partially atropine-sensitive stimulatory effect followed by slight relaxation at
the highest tested concentration. The stimulatory effect was increased
when repeated in the presence of L-NAME or methylene blue and
Pop.Cr relaxed the K+ (80 mM)-induced contraction, indicating the
involvement of Ca++ channels and NO pathway. The relaxation was
potentiated with atropine pretreatment while inhibited in tissue pretreated
with L-NAME or methylene blue. This study indicates that psyllium
husk possesses a gut stimulant effect mediated through multiple pathways, which might be complementing the laxative effect attributed to its
ber contents. Moreover, the presence of gut relaxant components
explains its medicinal use in diarrhea and perhaps also meant by nature
to offset the excessive stimulant effect which could be harmful otherwise.
Paper No.: 1771

ANTIDEPRESSANT ACTIVITY OF ORCHIS MASCULA
Anwar-ul Hassan Gilani(1), S Khan(1), HS Siddiqi(1,2)
(1) Aga Khan University, Department of Biological and Biomedical
Sciences, Karachi, Pakistan
(2) University of Karachi, Karachi, Pakistan
Depression is most popular among the mental disorders and is one of the
leading causes of disability and morbidity around the world. Herbal medicines have been used by every human society to cure or treat variety of
health problems. Orchis mascula (O. mascula) has been used in folk
medicine for various aliments including neurological disorders. It is also
used as tonic for nervous disability and reported to be nutritious for
neurasthenia. In the present study O. mascula root methanolic extract

was evaluated for its antidepressant properties using forced-swim test
(FST), tail suspension test (TST), yohimbine potentiation test (YPT) and
locomotor test (LMT). Phenelzine, imipramine and uoxetine were used
as a reference drugs. Our results showed that O. mascula extract (10 100 mg/kg) caused dose-dependent reduction in the immobility time of
rodents using FST (EC50 = 47 5.1 mg/kg rats) and TST (EC50 = 31
3.7 mg/kg mice) indicating antidepressant property of extract. Data also
showed that the pretreatment of animals with extract exhibited marked
reduction in the immobility time which does not correlate with the spontaneous activity recorded by LMT e.g. at the dose of 30 mg/kg 51%
decrease (control counts: 7206 534 and test counts: 3508 421) was
found in the locomotor counts of mice, thus further conrming the present ndings. In conclusion, the O. mascula root extract possesses antidepressant activity. However, further studies are required to investigate the
active principle responsible for the antidepressant property using bioassay directed fractionation method.
Paper No.: 1772

PHARMACOLOGICAL USE OF BORAGO OFFICINALIS
Anwar-ul-Hassan Gilani
Aga Khan University, Department of Biological and Biomedical
Sciences, Karachi, Pakistan
Borago ofcinalis Linn. (family Boraginaceae), commonly known as
Borage, originally indigenous to Mediterranean region and now is found
all over the world. Different parts of the plants particularly the seed oil is
used for medicinal purpose. Borage is popular for its wide medicinal use,
such as, gastrointestinal (colic, cramps, diarrhea), airways (asthma, bronchitis), cardiovascular, (cardiotonic, antihypertensive and blood purier),
urinary (diuretic and kidney/bladder disorders), anti-dyslipidemic and
other metabolic and sexual disorders. Moreover, it is used as nerve tonic,
demulcent, while its oil is used as anti-arthritic, anti-inammatory and
anti-neurodermatitis. Borage contains multiple chemicals, such as coumarins, tannins, avonoids, polyphenolics including vanillic acid, p-coumaric acid phenolic acid, caffeic acid, rosmarinic acid, chlorogenic acid and
scopoletin, alkaloids including amabiline, lycopsamine and supinidine,
hydrocarbons mainly tetracosane and heptacosane, alcohols including
cis-3-hexenol and hexanol and predominant fatty acids including a-linolenic, stearidonic, c-linolenic, palmitic and linoleic acids other than niacin, thiamine, riboavin, ascorbic acid, salicylic acid. It is reported to
possess antioxidant, renal protective and cytotoxic. We observed that its
antispasmodic, bronchodilator and antihypertensive activities are mediated possibly due to calcium antagonist constituents. Borage oil, the main
source of c-linolenic possesses antihypertensive, anti-dyslipidemic and
immunomodulator activities by raising the level of 6-omega polyunsaturated fatty acids in the various tissues. Thus, Borage has a lot of therapeutic potential particularly in inammatory and metabolic disorders.
Borage is generally considered safe with minor side-effects except in
pregnancy; however, seed oil also contains pyrrolizidine alkaloids, thus
requiring further studies to establish the safety of Borage oil.
Paper No.: 3288

FROM PHYLOGENY TO PHARMACOLOGY;
CHEMOGENOMIC PHARMACOLOGICAL GPCR
CLUSTERING AND LIGAND-INFERENCE
David Gloriam(1), S Garland(2)
(1) University of Copenhagen, Department of Medicinal Chemistry,
Copenhagen, Denmark
(2) GlaxoSmithKline Pharmaceuticals PLC, Stevenage, UK
302
Conventional phylogenetic analysis utilises the whole receptor sequence.
This has limitations; however, from a ligand design perspective because
the majority of residues are not involved in ligand binding and thus evolutionary patterns dominate over ligand-binding properties. Clustering on
only the binding residues generate a pharmacological clustering that
resembles more closely experience from cross-screening of ligands. We
dened Family A ligand accessible residues (TM bundle inward-facing)
from all receptor with experimentally determined structures and compared with datasets extracted from literature[1]. Specic motifs of ligandbinding residues (interaction ngerprints) were identied and linked to
privileged structures. By comparing such motifs data could be inferred
and used in lead generation by cross-target ligand inference[2].
References: 1. Gloriam, D.E. et al.; Denition of the G protein-coupled
receptor...; PM:19537715 2. Gloriam DE, Garland SL.; Chemogenomic
analysis can rationalise and predict the binding prole of privileged
structures at GPCRs; In preparation
Paper No.: 1303

A RANDOMIZED CLINICAL TRIAL UTILIZING THE DRUG
BURDEN INDEX TO REDUCE EXPOSURE TO
ANTICHOLINERGIC AND SEDATIVE MEDICATIONS IN
OLDER PEOPLE
D Gnjidic(1), S Hilmer(1), D Le Couteur(1,2), D Abernethy(3)
(1) University of Sydney, and Royal North Shore Hospital, Royal North
Shore Hospital, Departments of Clinical Pharmacology and Aged Care,
Sydney, NSW,Australia
(2) Concord Hospital, Centre for Research and Education on Ageing,
Sydney, NSW, Australia
(3) Food and Drug Administration, Silver Spring, Maryland, USA
The DBI is a pharmacological model that measures individuals exposure
to anticholinergic and sedative drugs. Higher DBI has been associated
with functional impairment in observational studies of older people
(Hilmer SN et al, Arch Intern Med 2007; 167: 781-7; Cao YJ et al, Clin
Pharmacol Ther 2008; 83:422-9; Gnjidic D et al, Br J Clin Pharmacol
2009; 68:97-105). The aim of this study was to assess the impact of providing information about DBI to General Practitioners (GPs) on prescribing for older people. Participants were people aged 70 years, living in
self-care retirement villages in Sydney, Australia. The intervention
involved a letter and phone call to GPs, using DBI to prompt them to
consider cessation or dose reduction of anticholinergic and sedative medications. A total of 115 participants were enrolled. At baseline, 19 out of
57 participants in the intervention and 31 out of 58 participants in the
control group had a DBI >0 (p < 0.05). At follow up, DBI change was
observed in 16 participants. DBI decreased in 12 participants, six in
intervention (32%), and six in control group (19%). GPs identied the
following barriers to reducing anticholinergic and sedative drugs: uncomfortable altering prescriptions initiated by specialists; unable to inuence
patients attitudes; unaware of patients medications and strong clinical
indication. The intervention targeting GPs prescribing practices was less
effective than anticipated in reducing anticholinergic and sedative drugs,
and barriers were identied. Future studies should trial multidisciplinary
interventions, engaging patients, specialists, GPs and pharmacists.
Paper No.: 782

ANALGESICS
EVATUATION OF THE EFFECTS INDUCED BY NICORANDIL
AND ITS IMMEDIATE PRECURSOR IN THE MODELS OF
FORMALDEHYDE-INDUCED NOCICEPTIVE RESPONSE AND
CARRAGEENAN-INDUCED EDEMA IN MICE
Federal University of MInas Gerais, Department of Pharmacology &

NO-releasing compounds have been used as therapeutic agents
(MILLER, M.R. et al.; Br. J. Pharmacol.; 2007; v. 151: p. 305). Nicorandil [N-(2-nitroxyethyl) nicotinamide] has both the ability to release NO
and open ATP-dependent K+ channels. The lack of information about the
antinociceptive properties of nicorandil prompted us to synthesize it and
its immediate precursor [N-(2-hidroxyethyl) nicotinamide] and investigate their effects in models of nociceptive and inammatory pain as well
as edema. Nicorandil (50, 100 or 150 mg/kg) or its precursor (50, 100,
150, 250, 500 or 1000 mg/kg) were administered per os 1 h prior the
injection of formalin, or the induction of paw edema with carrageenan,
in female Swiss mice. The motor activity was investigated using rotarod. The results were analyzed by one-way Anova followed by Newman-Keuls test (p < 0.05). Nicorandil (50, 100 or 150 mg/kg) inhibited
both phases of the formalin model. Only the highest dose (1000 mg/kg)
of its precursor inhibited the second phase of this response. Neither nicorandil nor its precursor inhibited carrageenan-induced edema. The motor
activity of mice was not impaired by the compounds. Nicorandil, but not
its immediate precursor, presented antinociceptive activity that was not
associated to impairment of motor activity. None of them exhibited antiinammatory activity, suggesting that the antinociceptive activity may be
more related to a central action. These results demonstrate the role of nicotinamide derivatives or NO-releasing compounds as pain modulators.
The role of NO in the inammatory processes is still under debate (WEI,
XM, Int. Immunopharmacol.; 2003; v. 3: p. 1581).
Paper No.: 2996

AUTOCRINE EFFECTS OF EXTRACELLULAR CYCLIC AMP
IN SKELETAL MUSCLE
Rosely Godinho, E Aparecida-Santos, AL Andrade-Lopes, T Chiavegatti
University Federal of Sao Paulo, Department of Pharmacology, Sao
Paulo, Brazil
Cyclic AMP (cAMP)-mediated pathway regulates several skeletal muscle
processes such as muscle contraction and expression of synaptic proteins.
We have showed that AC activation mediated by GsPCR leads to extracellular accumulation and subsequent conversion of cAMP into adenosine. (Chiavegatti et al., Br J Pharmacol, 2008; 53:1331-1340). In the
present study, we evaluate possible existence of this pathway in vivo and
the autocrine action of extracellular cAMP on skeletal muscle. Firstly,
intra (cAMPi) and extracellular cAMP (cAMPe) were analyzed in rat
EDL muscles. 30-min treatment of muscle with forskolin or -adrenoceptor agonist isoproterenol increased by 4-13 fold the basal cAMPi and by
10-36 fold the cAMPe. The inhibitor of organic anion transporters probenecid (100 lM) attenuated by 60% the cAMPe rise, without interference in cAMP synthesis. In vivo, 2h-treatment of adult rats with 2adrenoceptor agonist fenoterol (3-10 mg/kg) increased by 84% plasma
cAMP content, an effect abolished by pre-treatment with probenecid.
Autocrine actions of cAMP were studied in L6 skeletal muscle cultures.
The membrane impermeable cAMP (1 or 10 lM) increased by 56% and
215% the cAMPi, which was inhibited by non-selective adenosine receptor CGS 14953. In summary, our results demonstrate the cAMP efux
through organic anion transporters in rat skeletal muscle, which might
contribute to the increment in plasma cAMP induced by fenoterol. More
importantly, the cAMPe is able to regulate cellular function in an autocrine fashion via its metabolite adenosine, activating receptors coupled to
Gas, which provides a positive feedback response to intracellular cAMP
signalling.
Adriana Godin, W Ferreira, F Oliveira, L Rocha, R Vieira,

E Nascimento Jr, J Seniuk, A de Fatima, M Coelho
303
Paper No.: 1177
P12 - ION CHANNELOPATHIES: NEW WINDOWS ON
COMPLEX DISEASE AND THERAPY
THE ANTIVASOCONSTRICTOR EFFECT OF POTASSIUM
CHANNEL OPENER P1075 IN THE HUMAN VASCULAR
GRAFTS
Ljiljana Gojkovic-Bukarica(1), A Novakovic(2), B Beleslin-Cokic(1),
M Peric(1), J Markovic-Lipkovski(1), S Cirovic(1), V Kanjuh(3)
(1) Belgrade University Faculty of Medicine, Department of Pharmacology, Belgrade, Republic of Serbia
(2) Belgrade University Faculty of Pharmacy, Belgrade, Republic of
Serbia
(3) Republic of Serbian Academies of Sciences and Arts, Belgrade,
Republic of Serbia
The human internal mammary artery (HIMA) and saphenous vein (HSV)
are used in coronary artery bypass grafting, but spasm of grafts may
occur. In order to nd agent that can prevent spasm, the aim of our study
was to evaluate the antivasoconstrictor effect of K-channel opener,
P1075 (N-cyano-N-(1,1-dimethylpropyl)-N-3-pyridylguanidine), on the
contractions of HIMA and HSV evoked by exogenously applied noradrenaline (NA) or by NA realized during electric eld stimulation (EFS,
20 Hz, adrenergic origin). P1075 induced a concentration-dependent
inhibition of both EFS-contractions, and contractions evoked by exogenous NA of the HSV (pEC50 values of 7.06 and 6.70, P > 0.05) and of
the HIMA (6.83 and 6.02, P < 0.05). Glibenclamide (1 microM), a selective blocker of ATP-sensitive K (KATP)-channels completely antagonized
the effect of P1075 on the EFS-contractions of HIMA and HSV and contractions evoked by exogenous NA on the HSV. However, 10 microM of
glibenclamide had to be used for antagonism of P1075 effect on exogenous NA contractions in HIMA. Immunomorphological study conrmed
presence of different subtypes of KATP channels (Kir6.1 and Kir6.2) in
the smooth muscle of HIMA and Kir6.2 in HSV. Thus, P1075 exhibits
potent inhibitory effect on NA-evoked contractions of the HIMA and
HSV. It seems that in the antivasoconstrictor effect of P1075 in the
HIMA and HSV are involved different subtypes of KATP channels.
Paper No.: 2308

THE EFFECTS OF LAVANDULA STOECHAS EXTRACT ON
CHOLESTEROL LEVELS AND CARDIAC FUNCTIONS ON
ISCHEMIA-REPERFUSION MODEL IN ISOLATED RAT
HEARTS
Sule Gok, E Olmez, A Sonmez, K Vural, E Daryverenli, M Soylar
Celal Bayar University, School of Medicine, Department of Pharmacology, Manisa,Turkey
Introduction: Lavandula stoechas (LS) has been commonly used as cholesterol reducing and vessel dilating in traditional medicine in Turkey. In
this study, we investigated the effects of LS extract on serum lipid prole
and myocardial ischemia-reperfusion injury of isolated rat hearts with
cholesterol-fed. Materials/Animals: Rats were fed with 2% cholesterolenriched or standard chow for 8 weeks. In treated groups, the extract of
LS (100 and 500 mg/kg/day, p.o.) or atorvastatin (20 mg/kg/day, p.o), a
HMG CoA reductase inhibitor, were also administered. At the end of 8
weeks, the hearts of anaesthetized rats were removed and perfused by
Langendorff system. After 30 min, the hearts were subjected to 30 min
global ischemia followed by 30 min reperfusion. Lipid proles of rat serums were also analyzed. Results: Total and LDL-cholesterol levels of
cholesterol-fed rats were signicantly higher than all other groups. Treatment with LS or atorvastatin signicantly reduced cholesterol. In hearts,
after ischemia, left ventricular developed pressure abolished and did not
recover by reperfusion in all groups. Left ventricular end diastolic pres-
sure signicantly elevated in ischemia. This increase was more in reperfusion in all groups, and signicanty greater in the higher dose of LS
and atorvastatin treated rats with cholesterol-fed. Perfusion pressure was
signicantly high in reperfusion compared to the preischemic period in
all groups. Ventricular tachycardia and ventricular brillation were not
prevented by LS and atorvastatin. Conclusion: These results suggest that
LS and atorvastatin reduces cholesterol levels in rats, but not provide
recovery on cardiac functions and arrhythmias in this model.
This Project was supported by TUBITAK (No:108S082)
Paper No.: 768

HEALTH AND DISEASE
ABCB1 C3435T GENETIC POLYMORPHISM ALTERS
CLINICAL RESPONSE TO LOSARTAN IN PATIENTS WITH
HYPERTENSION
MT Goktas(1), F Pepedil(2), G Sain-Guven(2), B Cakir(3), Melih
O Babaoglu(1), U Yasar(1), A Bozkurt(1)
(1) Hacettepe University, Faculty of Medicine, Department of Pharmacology, Ankara, Turkey
(2) Hacettepe University,Faculty of Medicine, Department of Internal
Medicine, Ankara, Turkey
(3) Hacettepe University, Faculty of Medicine, Department of Public
Health, Ankara, Turkey
Losartan is a selective angiotensin II receptor antagonist used in hypertension treatment. Losartan has been shown to be a substrate for the
drug-efux transporter ATP-binding cassette sub-family B member 1
(ABCB1, MDR1). ABCB1 is a genetically polymorphic transporter. The
ABCB1 3435T allele has been shown to be associated with altered transporter functions, leading to change in systemic bioavailability and clinical efcacy of its substrates. The aim of this study was to investigate the
effects of the ABCB1 C3435T genetic polymorphism on clinical efcacy
of losartan in hypertensive patients. Patients (n = 74) diagnosed to have
mild (Grade I) hypertension were included in the study. Genomic DNA
was extracted from peripheral blood lymphocytes. Genotyping was performed using polymerase chain reaction and endonuclease digestion
methods. Blood pressure measurements were recorded before and 6
weeks after losartan (100 mg/day) administration. Blood pressure
changes in patients with wild-type genotype as compared to those with
variant genotypes were analysed by using Students t test. The average
(mean SD) decrease in systolic blood pressure in patients carrying the
T allele (11.6 9.7 mmHg, n = 55) was signicantly higher when compared to the patients with homozygous wild-type (CC, 7.6 9.4 mmHg,
n = 19) genotype (P = 0.03). These results suggest for the rst time in literature that MDR1 C3435T genetic polymorphism may play an important role in clinical response to losartan in hypertension.
(The study was supported by Hacettepe University Research Centre
(0801101009) and Turkish Academy of Sciences, Young Scientist Award
MOB: TUBA-GEBIP/ 2007-06)
Paper No.: 843

THE INVOLVMENT OF ERK-DEPENDENT PROCESSES IN
ANTIDEPRESSANT DRUGS EFFECTS ON B-ARRESTINS
Moran Golan(1), G Schreiber(0), S Avissar(1)
(1) Ben Gurion University of the Negev, Department of Pharmacology,
Beer Sheva,Israel
(2) Ben Gurion University of the Negev, Department of Psychiatry, Beer
Sheva, Israel
(3) Barzilai Medical Center, Ashkelon, Israel
304
Preliminary data from our laboratory indicates that chronic exposure to
antidepressant drugs (ADs) results in a signicant increase in activated
b-arrestin1 protein levels while causing a major decrease in the protein
levels of both b-arrestin2 and functional ERK1/2. this study aims at elucidating ADs mechanism of action at the post-receptor level involving barrestin1&2 and functional ERK1/2. C6 glioma cells were treated chronically with various classes of ADs in the presence or absence of the
MEK1/2 inhibitor U0126 or the translation inhibitor cycloheximide. proteins function and levels were measured by confocal microscopy and
western blotting. mRNA levels were measured by RT-PCR. chronic
exposure to ADs resulted in a major increase of activated ERK1/2 in the
nuclear fraction of the cells. both U0126 and cycloheximide overturned
the previously reported elevation of b-arrestin1 protein levels following
ADs. ADs treatment caused an increase in b-arrestin1 mRNA levels, that
was reversed by U0126. Surprisingly, ADs increased b-arrestin2 mRNA
levels in the presence or absence of U0126. The present ndings support
our assumption that by reducing b-arrestin2 protein levels ADs enable
activated ERK1/2 translocation to the nucleus, thus increasing b-arrestin1
transcription and expression. the results also implicate that ADs may
induce a non-ERK1/2-dependent b-arrestin2 transcription pathway and
that the reduction in its protein levels is due to post-transcriptional/translational modication. the described ndings also indicate that b-arrestins
may serve as diagnostic markers for major depression.
Paper No.: 1488

EFFECT OF HYDROALCOHOLIC EXTRACT OF EMBLICA
OFFICINALIS (HAEEO) AGAINST KAINIC ACID INDUCED
SEIZURES, OXIDATIVE STRESS AND EXPRESSION OF HEAT
SHOCK PROTEINS IN RAT BRAIN
Mahaveer Golechha, J Bhatia, A Goyal
New Delhi, India
Introduction: The effect of standardized hydroalcoholic extract of Emblica ofcinalis (HAEEO), an Indian medicinal plant with potent antioxidant activity was studied against kainic acid (KA)-induced seizures,
effect on markers of oxidative stress and expression of heat shock protein
(HSP-72) in rat brain. Methods: Rats were administered KA (10 mg/kg
i.p.) and observed for behavioral changes, incidence and latency of convulsions over four hours. The rats were thereafter sacriced for estimation of oxidative stress parameters; malondialdehyde (MDA) and
glutathione (GSH) and the expression of HSP-72. The HAEEO was
administered for seven successive days at doses of 300, 500 and 700 mg/
kg i.p. Results: KA induced long-lasting seizures and associated symptoms. The brain level of MDA was signicantly raised after KA administration (361.29 10.79 nmol/g wet tissue) as compared to vehicle treated
group (184.86 3.94 nmol/g wet tissue) and signicantly decreased the
levels of GSH. Pretreatment with HAEEO (500 and 700 mg/kg, i.p) signicantly increased the latency of seizures (79.25 2.60 min and 99.85
2.64 min respectively) as compared to the vehicle treated KA group.
There was also increase in expression of HSP-72 in the KA group. HAEEO (500 and 700 mg/kg, i.p) signicantly prevented the increase in
MDA levels and ameliorated the fall in glutathione. HAEEO also
improved cognitive decit induced by kainic acid, evidenced by
increased latencies in passive avoidance task. Conclusion: The study
reports the potential antiepileptic effect of HAEEO along with amelioration of KA induced cognitive impairment.
Paper No.: 739

INVOLVIMENT OF 5-HT1A RECEPTORS IN THE EFFECTS OF
CANNABIDIOL INJECTED INTO THE BED NUCLEUS OF
THE STRIA TERMINALIS ON BEHAVIORAL AND
CARDIOVASCULAR RESPONSES INDUCED BY
CONTEXTUAL CONDITIONED FEAR
Felipe V Gomes, DG Reis, FHF Alves, FMA Correa, L Resstel,
FS Guimaraes
University of Sao Paulo, School of Medicine of Ribeirao Preto, Department of Pharmacology, Sao Paulo, Brazil
Systemic administration of cannabidiol (CBD), non-psychotomimetic
compound from Cannabis sativa, is able to attenuate cardiovascular and
behavioral (freezing) changes induced by re-exposure to a context that
had been previously paired with footshocks. Previous results from our
group suggest that the BNST may be involved in the anxiolytic effects
of the CBD. These effects could be mediated by facilitation of the endocannabinoid system or by the activation of 5-HT1A receptors. Materials:
Male Wistar rats (240-270g, n = 5-6) with cannulas implanted bilaterally
into the BNST were submitted to a 10 min conditioning session (6 footshocks, 1.5 mA, 3 s). The behavioral and cardiovascular responses to the
context were measured 24 h later in a 10 min test session. CBD (15, 30
or 60 nmol) or vehicle (V, 0.2lL) were administered 10 min before test.
The second experiment was similar to the rst one except that animals
received microinjections of the 5-HT1A receptor antagonist WAY100635
(WAY; 0.37 nmol) 5 min before CBD (30 nmol) treatment. Results are
expressed as meanSEM. Results: CBD (30 and 60 nmol) signicantly
decreased the % of freezing (V: 73.9 4.1, CBD 30: 33.1 3.4, CBD
60: 19.3 3.4) and attenuated the increased blood pressure and heart rate
induced by context. WAY100635 by itself did not change the cardiovascular and behavioral response to context, but blocked the effects of CBD
(WAY+V: 64.7 8.0, WAY+ CBD 30: 69.3 5.9) Conclusion: The
results suggest that BNST could be involved in the anxiolytic-like effects
of CBD observed after systemic administration by facilitating 5-HT1A
receptor-mediated neurotransmission.
Paper No.: 2966

TRANS-RESVERATROL ENHANCES ENOS EXPRESSION AND
DECREASES ITS INTERACTION WITH CAVEOLIN 1 IN
VASCULAR SMOOTH MUSCLE AND ENDOTHELIAL CELLS
JE Gomez, A Cunas, J Leiro, F Orallo, Manuel Campos-Toimil
University of Santiago de Compostela, Department of Pharmacology,
Santiago de Compostela, Spain
Caveolae are specialized membrane microdomains in which complexes
of signalling molecules are compartmentalized via their interactions with
caveolin. Endothelial nitric oxide synthase (eNOS) is a constitutive caveolar enzyme in vascular endothelial cells. We here report, for the rst
time, the effects of trans-resveratrol (t-RESV, a natural vasorelaxant polyphenol) on vascular caveolar integrity and on the protein interaction
between caveolin 1 (cav-1) and eNOS, using primary rat aortic smooth
muscle cells (RASMC), foetal rat aortic smooth muscle cells (A7r5), and
human umbilical vein endothelial cells (HUVEC). Caveolin-enriched
membrane domains were obtained using a discontinuous sucrose gradient
fractionation. The fractions were analysed by SDS-PAGE and western
blotting. cav-eNOS interactions and protein expression were study by
immunoprecipitation and reverse-transcription polymerase chain reaction
(rt-PCR), respectively. RASMC and A7r5 express three caveolin isoforms (cav-1, -2 and -3), while HUVEC only express cav-1 and cav-2.
eNOS and caveolin were found in the same light membrane fractions
(17-25% sucrose). t-RESV treatment did not affect the protein distribution
305
in any of the cell types tested, although it caused cav-1 down-regulation
and eNOS up-regulation in both A7r5 and HUVEC. eNOS interacts with
cav-1 in untreated HUVEC and A7r5. t-RESV treatment decrease this
interaction in a concentration dependent manner. In RASMC, t-RESV
induced eNOS expression at the mRNA level and the eNOS-cav-1 interaction depends on the concentration of this polyphenol. Our results suggest that t-RESV increases eNOS levels in the vascular system, and
contributes to free this enzyme from cav-1, and therefore eNOS becomes
more susceptible to activation.
Paper No.: 3175

EFFECT OF BIS-1-4-DIHYDROPYRIDINE IN KIDNEYS OF
DIABETIC RATS
Committee under number 24/09. Swiss female mice (10 weeks, 32g 5)
were subjected to surgery for implantation of membranes containing 1%
polyvinyl alcohol (PVA) or 1% PVA associated with LP (0.2% or 1%) in
the dorsal region. On days 2, 7 and 14 after surgery the animals were
sacriced and the implanted area was removed for macroscopic evaluation. Cylindrical fragments were collected for determination of nitrite
concentrations and preparation of histological slides. The macroscopic
evaluation showed that LP (0.2 and 1%) induced tissue neoformation
when compared to control groups. LP (0.2 and 1%) reduced the recruitment of polymorphonuclear cells to the tissue neoformated on day 2 and
stimulated the inltration of mononuclear cells on days 2 and 14 after
surgery (p < 0.05). Increased nitrite levels was detected in the tissue
neoformated of animals from LP groups (0.2 and 1%) on days 2, 7 and
14 (p < 0.05). LP 0.2% promoted a stimulus for collagen deposition in
the tissue neoformated on days 7 and 14 (p < 0.05). These data suggest
that the biomembrane with laticifers proteins of C. procera (LP) modulates the inammatory phase of healing, which seems to inuence the
subsequent phases of the healing process.
Raquel Gomez-Pliego(1), R Villalobos-Molina(2), M Ibarra-Barajas(2),

B Velasco-Bejarano(1), J Gomez-Zamudio(2)
(1) UNAM, FES-Cuautitlan, Mexico City, Mexico
(2) UNAM, FES-Iztacala, Mexico City, Mexico
Diabetes and hypertension are common diseases related with increased
risk of renal damage. Patients with both pathologies have higher risk to
develop chronic renal failure, than patients with diabetes alone. In this
regard, blood pressure control is important to prevent progression of
renal damage in diabetes. We explored the effects of bis-1-4-dihydropyridine (bis-1-4-DHP), a calcium channel blocker, on blood pressure and
kidney perfusion pressure in Wistar rats (control, diabetic, control and
diabetic + bis-DHP-treatment), at 2,4,6 and 10 weeks. bis-1-4-DHP (10
mg kg-1) started one week before diabetes, the compound was dissolved
in DMSO, and suspended in drinking water with carboximethyl cellulose. Diabetes was induced by STZ (55 mg kg-1, i.p.). Kidneys weight
of diabetic groups of rats signicantly increased vs control groups at 4, 6
and 10 weeks of treatment. The ratios kidney weight x 100/ body weight
were different between controls vs diabetics at all times (P < 0.05). Kidney perfusion pressure decreased by diabetes, while it partially recovered
by bis-1-4-DHP treatment in response to phenylephrine, this data indicate
that bis-1-4-DHP by antagonizing Ca2 + entrance, probably induce vasodilation at efferent and afferent arterioles, reducing glomerular injury and
thus causing nephroprotection. It is important to mention that we synthesized bis-1-4-DHP under the Green Chemistry protocol and is a non photosensitive molecule.
RG-P supported by postdoctoral fellowship from CONACYT ICyTDF.
Paper No.: 3401

DISEASES
BIOMEMBRANE WITH LATICIFERS PROTEINS FROM
CALOTROPIS PROCERA INDUCES TISSUE NEOFORMATION
IN MICE
Danilo Goncalves(1), I Figueiredo(1), I Calou(1), R Oliveira(2),
L Freitas(1), R Pinheiro(1), T Andrade(3), M Frade(3), GA Brito(1),
R Leitao(1), M Ramos(1), N Alencar(1)
(2) Federal University of Ceara, Department of Biochemistry and Molecular Biology, Fortaleza, Brazil
(3) University of Sao Paulo, Medical School, Ribeirao Preto, Brazil
The latex of Calotropis procera has been widely used in folk medicine
on dermatological disorders. In this study the effect of laticifer proteins
(LP) was evaluated on healing by rst intention. Animal handling and
experimental protocols were registered on the Institutional Ethics
Paper No.: 3402

DISEASES
COMPARATIVE STUDY OF THE EFFECTS OF LOVASTATIN
AND SINVASTATIN IN MODELS OF ACUTE INFLAMMATION
IN RODENTS AND HUMAN NEUTROPHILS
Danilo Goncalves, I Calou, A Araujo, I Figueiredo, T Olinda, LK Leal,
G Viana
Federal University of Ceara, Departement of Physiology and Pharmacology, Fortaleza, Brazil
The anti-inammatory effects of lovastatin (LOV) and sinvastatin (SINVA) were evaluated on animal models of peritonitis and paw edema
induced by carrageenan (CARR) and through the determination of percentage of inhibition of myeloperoxidase (MPO) activity on human neutrophils. Peritonitis was induced by a single administration of CARR 1%
after treatment of male Swiss mice (25-30g, n = 6) with vehicle (distilled
water, p.o.), dexametasone (1 mg/Kg, i.p.), LOV (2, 5, 10 mg/Kg, p.o.)
or SINVA (5 and 10 mg/Kg, p.o.). Both LOV (16,0 2,1; 16,1 2,0;
15,5 2,3 x 103/mm3, respectively) and SINVA (25,3 2,7 and 17,5
5,2 103/mm3) have signicantly (p < 0,05) reduced leukocite migration
when compared to vehicle (LOV: 58,7 10,5 and SINVA: 101,2 12,3)
and dexametasone (LOV: 14,3 3,5 and SINVA: 20,5 5,3). Paw
edema was induced by CARR application to the right paw (Wistar Rats,
150-200g, n = 6) and swelling was recorded by pletismography (mL).
Lovastatin reduced (p < 0,05) the paw edema, mL, at the doses of 2, 5
and 10 mg/Kg during the period of measures: second hour (1,33 0,12;
1,03 0,19; 1,19 0,21; respectively), third hour (2,30 0,17; 1,76
0,23; 1,93 0,21) and fourth hour (2,44 0,12; 1,85 0,21; 2,06
0,35) when compared to control group (1,83 0,21; 2,86 0,24 e 3,26
0,13). Sinvastatin has not reduced the paw edema. Both statins have
reduced MPO activity: LOV: 25, 50 and 100 micrograms/mL (70, 75
and 80% inhibitions, respectively) and SINVA at the same doses (24, 35
and 51%, respectively) (p < 0,05).
Paper No.: 1471

DISCOVERY
INFLUENCE OF POLYPHENOLS IN A COLON TUMORAL
CELL LINE: EFFECT ON BUTYRATE UPTAKE AND ON
PROLIFERATION, DIFFERENTIATION, VIABILITY AND
APOPTOSIS
Pedro Goncalves(1), JR Araujo(1), MJ Pinho(2), F Martel(1)
306
(1) University of Porto Faculty of Medicine, Department of Biochemistry, Porto, Portugal
(2) University of Porto Faculty of Medicine, Institute Pharmacology and
Therapeutics, Porto, Portugal
Butyrate (BT), one of the main end products of anaerobic bacterial fermentation of dietary ber within the human colon, is known to inhibit
colon carcinogenesis. Polyphenols have several properties, including
anti-carcinogenic activity. The aim of this study was to investigate the
inuence of some polyphenols upon the apical uptake of BT into human
colonic adenocarcinoma Caco-2 cells, and to evaluate their inuence
upon the effect of BT on proliferation, differentiation, viability and apoptosis. MCT1-mediated apical uptake of a low (10 lM) and of a high (20
mM) concentration of 14C-BT in Caco-2 cells is modulated by either
acute or chronic exposure to some polyphenols, and resveratrol acts as a
competitive inhibitor of 14C-BT uptake. BT (5 mM; 48 h) markedly
decreased cellular viability and proliferation while increasing cell differentiation and apoptosis. The polyphenolic compounds modulated some
of these parameters. However, in general, combination of polyphenols
with BT did not signicantly modify the changes induced by BT alone.
In conclusion, changes in uptake of BT induced by polyphenols do not
correlate with changes on cell viability, proliferation, differentiation and
apoptosis.
Supported by Fundacao para a Ciencia e a Tecnologia (FCT) and Programa Ciencia, Tecnologia e Inovacao do Quadro Comunitario de Apoio
(PTDC/SAU-FCF/67805/2006).
Paper No.: 3135

MOLECULAR DETERMINANTS OF WARFARIN
PHARMACOKINETICS AND RESPONSE
Inna Gong(1), R Tirona(1,2), U Schwarz(1,2), N Crown(2),
G Dresser(2), S LaRue(5), N Langlois(5), A Lazo-Langner(3),
P Wells(5), R Kim(1,2,4)
(1) University of Western Ontario, Department of Physiology and
Pharmacology, London, Ontario, Canada
(2) University of Western Ontario, Division of Clinical Pharmacology,
Department of Medicine, London, Ontario, Canada
(3) University of Western Ontario, Division of Haematology, Department
of Medicine, London, Ontario, Canada
(4) Lawson Health Research Institute, London, Ontario, Canada
(5) University of Ottawa, Division of Haematology, Department of Medicine, Ottawa, Ontario, Canada
Warfarin is a widely prescribed anticoagulant known for marked interindividual variation in drug responsiveness. Genetic polymorphisms in
genes affecting warfarin metabolism (CYP2C9) and activity (VKORC1)
together with patient clinical parameters explain 50% of dose variation.
Of the known relevant patient parameters such as age, weight and gender,
it was uncertain how these inuence pharmacokinetic (PK) and/or pharmacodynamic (PD) responses. Accordingly, we have characterized warfarin PK-PD responses in patients initiating therapy. On treatment days 3, 5
and 8, plasma was obtained to measure response (INR), R/S-warfarin levels (liquid chromatography-tandem mass spectrometry), and vitamin K
status (ELISA). The results demonstrate that S-warfarin clearance was
lower in CYP2C9 *2 and *3 variant carriers as expected. Notably, clearance of S-warfarin was different between females and males, suggesting
gender-dependent differences in the metabolic capacity for warfarin.
S-warfarin clearance and vitamin K status were negatively associated
with age indicating that both PK and PD differences are responsible for
the observed increase in warfarin sensitivity. In addition, warfarin dose
requirement related to genetic variation in the vitamin K metabolizing
enzyme CYP4F2 (V433M). Importantly, therapeutic S-warfarin plasma
levels segregated with VKORC1 haplotype. Furthermore, VKORC1 haplotype-dependent warfarin sensitivity signicantly related to increased
maximum inhibitory response (Imax). Lastly, western blot of our liver
bank microsomes demonstrated that VKORC1 haplotype relates to

altered VKOR protein levels. In summary, our data reveal important new
mechanistic insight regarding warfarin dose variability. Better understanding of warfarin PK-PD interaction will serve as a basis for creating
a more predictive algorithm for individualized warfarin therapy.
Paper No.: 797

DETERMINATION OF THE RATE OF RELEASE OF
IBUPROFEN AND DEXIBUPROFEN VIA TOPICAL ROUTE
Ana VM Gonzalez, G Ccalleja, LB Chavez, JJT Duran,
PB Papantonakis, ST Duran
Complutense University, Department of Pharmacy & Pharmaceutical
Technology, Madrid, Spain
The rate of release of ibuprofen and dexibuprofen is determined by
carrying out ow-through cell dissolution tests on their respective formulations and quantitatively analysing by spectrophotometry. The owthrough cell is chosen since it combines all the optimal characteristics
that mimic the skin conditions. This is achieved by setting the temperature to 32C, using buffers which are of pH 5.5 and 6.5 and a constant
ow rate of 5ml/min. Ibuprofen exists as a racaemic mixture, however
only the (S+) isomer possesses pharmacological activity and it has been
stated in literature that R-ibuprofen is converted to its stereoselective S
enantiomer inside the body via a metabolic reaction. Dexibuprofen is
believed to be advantageous because it is 160 times more effective in the
inhibition of prostaglandin synthesis, therefore it is possible to administer
lower doses containing less concentration of active ingredient, resulting
in a lower incidence of adverse effects. Ibuprofen and dexibuprofen are
poorly soluble in water (pka= 4.41) making it more difcult to produce
semi-solid formulations containing these active ingredients. B-cyclodextrin has proven to be an ideal candidate to aid the dissolution in water
without degrading ibuprofen and dexibuprofen. Cyclodextrins act by
modifying the external physical and chemical properties of the active
ingredient. Cyclodextrins are cyclic oligosaccharides that form an external hydrophilic surface and an internal zone that form a lipophilic cavity.
It has been demonstrated that are capable of encapsulating lipophilic
active ingredients within a hydrosoluble complexes.
Paper No. 2915

FOCUS GROUP: P15 - ENDOTHELIUM IN HEALTH AND
DISEASE
PHARMACODYNAMIC/PHARMACOKINETIC STUDY OF
TWO FORMULATION OF ACETYLSALICYLIC ACID IN
PATIENTS WITH CHRONIC CORONARY DISEASE
Jose Antonio Gonzalez-Correa(1), A Guerrero(1), E Rueda(2),
MM Arrebola(3), JA Lopez-Villodres(1), JP de la Cruz(1)
(1) University of Malaga, School of Medicine, Department of Pharmacology, Malaga, Spain
(2) University Hospital Virgen de la Victoria, Service of Cardiology.
Malaga, Spain
(3) University Hospital Carlos Haya, Service of Clinical Analysis,
Malaga, Spain
Aim: to establish the pharmacodynamic/pharmacokinetic of two formulation of acetylsalicylic acid in patients with chronic coronary disease.
Methods: clinical trial parallel, double-blind, randomized, comparative
pharmacodynamic/pharmacokinetic study or the long-term administration
of 150 mg of prolonged release ASA (ASA-PR) versus 150 mg of immediate release ASA (ASA-IR) on platelet function (Nordm; EudraCT:
2006-002357-74). Patients were divided into two groups: ASA-PR (n =
25) and ASA-IR (n = 25). Follow-up: baseline visit (clinical evaluation
and star of treatment for 21 days); visit 2 (characterisation of the kinetic
307
and dynamic prole); visit 3 (day 28, patients nal clinical follow-up
visit). We collected clinical, analytical and biochemical parameters
related to platelet function, endothelial and oxidative status. Statistical
analysis was performed uni and bivariate (chi square and t-student).
Results: Platelet aggregation is inhibited equally by both ASA formulations (1,78 0,50 vs. 2,56 0,61 ohms, p > 0,05). The serum thromboxane levels are inhibited equally by both forms of ASA (8,21 2,64 vs.
7,07 2,85 pg/mL, p > 0,05). The plasma prostacyclin levels was higher
with ASA-PR (1,42 0,24 vs. 0,84 0,12 pg/mL, p < 0,05). Both formulation increase nitric oxide levels, although they remained elevated for
longer in patients who received ASA-PR (17,19 1,07 vs. 12,01 3,61
lmol/L, p < 0,05). Pharmacokinetics parameters of ASA: Cmax 2,10
0,18 vs. 5,59 0,42 lg/mL, Tmax 4 vs. 1 h .ASA-PR maintaining elevated salicylic acid levels for longer respect to ASA-IR (after 9 hours of
drugs administration: 1,60 0,14 vs. 0,63 0,12 lg/mL, p < 0,05). Conclusions: ASA-PR provide sufcient levels of ASA to obtain adequate
anti-aggregation, as well as maintained SA levels that give higher prostacyclin and nitric oxide concentration.
Paper No.: 2993

THE OXIDATIVE STRESS PREDICTS A HIGHER CORONARY
RISK
Jose Antonio Gonzalez Correa(1), M Carnero(2), J Munoz-Marin(1),
F Reguillo(2), M Ramajo(3), J Navarro-Dorado(3), S Redondo(3),
JP De la Cruz(1), T Tejerina(3)
(1) Universidad de Malaga, School of Medicine, Department of
Pharmacology, Malaga, Spain
Spain
(3) Universitu Complutense of Madrid, School of Medicine, Department
Introduction: In the last 15 years, several reports have demonstrated the
important role of oxidative stress in the biomolecular pathways of atherogenesis. Most of these reports are based in animal or in vitro models. Up
to now, little is known about the implication of oxidative stress in atherogenesis and its possible inuence on the coronary risk in human beings.
Methods: During a two year period we measured the levels of multiple
oxidative stress markers (reduted glutation (GSH), mitochondrial superoxide dismutase (SOD-Mn), nitrites, peroxynitrites and malondialdehidic
acid (MDDA)) in a cohort of patients with coronary disease. We studied
if these markers were or not related to the Framinghan/ATP III coronary
risk scale punctuation. Results: 119 patients were included in the present
study. Hematite MDA and nitrite levels were independent predictors of
greater Framinghan/ATP III scale punctuations (B = 32.146 points /
mmol/mL (IC 95% 24.132- 40.16); B = 0.326 points / lmol/g (IC 95%
0.049- 0.604). On the other hand, GSH levels were inversely related to
Framinghn/ATP III punctuation (B= -0.175 points/ lmol/g (IC 95%=0.344- -0.005)). No signicant relationship was detected between SODMn (p = 0.559) or peroxynitrites (p = 0.721) and Framinghan/ATPIII
risk. Conclusions: MDA and nitrite levels predict greater Framinghn/
ATP III scale punctuations. On the contrary, GSH levels are related to
lower Framingham predicted coronary risk.
Paper No.: 2147

HEALTH AND DISEASE
EFFECT OF SULODEXIDE ON VASCULAR PERMEABILITY
IN THE KIDNEY OF DIABETIC RATS
Caroline Gonzalez-Serryn(1), D Fernandes(1), Y Mathison(2)
(1) Universidad Central de Venezuela, Escuela de Medicina Jose Mara
Vargas, Caracas, Venezuela
(2) Catedra de Farmacologa, Facultad de Farmacia, Universidad Central

de Venezuela, Caracas, Venezuela
Diabetic nephropathy continues to be the leading cause of end-stage
renal disease, despite therapies targeted towards glycemic and blood
pressure control. Drugs with endothelium protective effects have been
shown to decrease cardiovascular and renal complications in diabetics>.
In the present study we investigated the effect of sulodexide (SLX), a
glycosaminoglycans composed from heparin-like and dermatan fractions,
which previously have demonstrated renal protection in diabetic patients;
on vascular permeability in the kidney of diabetic rats by quantication
of extravasated albumin previously colored with Evans blue (EB). Diabetes was induced in male Sprague Dawley rats (200-250 g) by intraperitoneal administration of streptozotocin (50 mg/kg). Animals were divided
into four groups: control (injected with saline solution), control-treated
with sulodexide (SLX), diabetic (DM) and diabetic-treated with sulodexide (DM+SLX). SLX was administered 15 mg/kg/day s.c., 5 days/week.
Blood pressure, serum creatinine and urinary protein excretion were measured every 6 weeks. After 12 weeks, animals were anesthetized and the
Evans blue administered in the jugular vein; then were sacriced and the
kidney removed for histological analysis and quantication of EB extravasation. Diabetes increased signicantly EB extravasation in the diabetic
group (128% vs control) and the treatment with SLX partially prevented
it (40% vs control), showing a reducing effect on plasma protein extravasation. Additionally SLX prevented the increase in serum creatinine and
urinary albumin excretion at 6 and 12 weeks of the experimental stages.
These ndings suggest an endothelium-protective effect of sulodexide in
streptozotocin-induced diabetes, related with renal protection.
(Supported by LOCTI-UCV)
Paper No.: 1862

GENETIC VARIATION IN THE HTAS2R38 TASTE RECEPTOR
AND BRASSICA VEGETABLES INTAKE
Nela Gorovic(1), S Afzal(1), A Tjnneland(2), K Overvad(3), U Vogel(4),
C Albrechtsen(1), HE Poulsen(1)
Clinical Pharmacology Q7642, Copenhagen, Denmark
(2) Danish Cancer Society, Institute of Cancer Epidemiology,
Copenhagen, Denmark
(3) Aarhus University, Institute of Public Health, Department of
Epidemiology, Aarhus, Denmark
(4) National Food Institute, Department of Toxicology and Risk
Assessment, Technical University of Denmark, Sborg, Denmark
The human TAS2R38 receptor is believed to be partly responsible for
the ability to taste phenylthiocarbamide (PTC), a bitter compound very
similar to the bitter glucosinolates in Brassica vegetables. These vegetables and their active compounds have chemoprotective properties. This
study investigated the relationship between genetic variation in the
hTAS2R38 receptor and the actual consumption of Brassica vegetables
with the hypothesis that taster status was associated with intake of these
vegetables. Furthermore secondary intake information on alcohol and
chocolate, as well as measures of BMI and waist-circumference were
analysed for association with the hTAS2R38 receptor polymorphisms.
The subjects were selected from the Diet, Cancer and Health (DCH)
study, which is an ongoing prospective Danish cohort study. Two groups,
each consisting of 250 subjects were selected based on their Brassica
vegetables intake from the upper quartile ( 23 g/day) and the lower
quartile ( 7 g/day daily intake of Brassicas from a randomly selected
sub-cohort of DCH. DNA was extracted from blood and analyzed for
three functional SNP?s in the hTAS2R38 gene. The hTAS2R38 bitter
taste receptor haplotypes were not associated with the daily intake of
Brassica vegetables in our study, and no association between the haplotypes and any of the other variables tested was found. Our results do not
support previous ndings of an association between Brassica intake and
308
hTAS2R38 haplotypes, indicating that non-genetic factors may have
more inuence on dietary choice than genetics. The intake of Brassica
vegetables was independent of the hTAS2R38 taster status.
Paper No.: 1754

DISCOVERY
CHARACTERIZATION OF LEUT, A BACTERIAL MEMBER OF
THE NEUROTRANSMITTER:SODIUM SYMPORTER FAMILY,
IN AN INWARD-FACING CONFORMATION
Kamil Gotfryd(1), D Kruger(1), J Kniazeff(1), J Javitch(2), C Loland(1),
U Gether(1)
(1) University of Copenhagen, Molecular Neuropharmacology Group,
Copenhagen, Denmark
(2) Columbia University, Center for Molecular Recognition and Departments of Psychiatry and Pharmacology, New York, NY, USA
The leucine transporter (LeuT) from Aquifex aeolicus is a bacterial member of the neurotransmitter:sodium symporter family. Although several
crystal LeuT structures were solved, the inward-facing conformation has
so far only been hypothesized. We have previously described a dopamine
transporter (DAT) mutant (Y335A) which locks DAT in an inward-facing
conformation due to the disruption of ionic interactions constituting the
predicted intracellular gate. Here, we present a characterization of the
homologous mutation in LeuT (Y268A). Results from binding assays
performed on detergent-solubilized LeuT showed that the afnity for leucine in LeuT Y268A is markedly decreased, compared to WT. Moreover,
we found differences in Na+-dependency and accessibility for the leucine
binding sites between LeuT WT and Y268A. Furthermore, we were able
to specically label an inserted cysteine with tetramethylrhodamine
(TMR) maleimide in position 192 of LeuT (LeuT-192TMR), the domain
that has been suggested to be exposed to the intracellular environment in
the homologous serotonin transporter only in the inward-facing conformation. Quenching of uorescence from LeuT-192TMR by the hydrophilic quencher potassium iodide (KI) was markedly enhanced in the
presence of leucine, suggesting that leucine binding shifts position 192
to a more aqueous environment and thereby the LeuT conformation
towards the inward-facing state. Conversely, LeuT-192TMR-Y268A
showed a markedly less change in the KI quenching upon leucine binding, suggesting that the 192TMR is already exposed to the intracellular
side. Taken together, these results indicate that mutation of Y268 in LeuT
causes a shift in the conformational equilibrium towards an inward-facing state as compared to LeuT WT.
Paper No.: 2258

DISEASES
EFFECT OF RUPATADINE ON HISTAMINE-INDUCED RAT
PAW OEDEMA FORMATION AND THE CONCOMITANT
UPREGULATION OF H1R AND PAFR GENE EXPRESSION
Materials: Various concentrations of roupatadine fumarate were administered to Wistar rats either systemically (i.v., i.p.) or locally (s.c., into the
paw), 1 hour prior to intraplantar injection of histamine.Oedema formation was measured with the use of a plethysmometer, at 10-20 minute
intervals, with the contralateral paw serving as a control. Loratadine,
administered via the same routes, was used for comparison.H1R and
PAFR gene expression in paw tissue was evaluated with RT-PCR.
Results: Roupatadine effectively reduced histamine-induced rat paw
oedema in a concentration-dependent fashion, in a fashion not unlike to
that of loratadine. The histamine induced upregulation of H1R and
PAFR gene expression was similarly affected by both drugs. Conclusion:
Roupatadine exerts an inhibitory effect on histamine-induced rat paw
oedema, consistent with its anti-(H1) histamine action. Its effect on H1R
and PAFR gene expression is suggestive of a potential benecial effect
in the late phase of allergic inammation.
Paper No.: 1005

DISEASES
ADVERSE EFFECTS OF RITUXIMAB IN NON ONCOLOGICAL
PATIENTS
Aurore Gouraud(1), L Moachon(2), G Miremont(3), MJ Pastor(4),
C Villier(5), J Descotes(1), T Vial(1)
(1) Hospices Civils de Lyon, Centre de Pharmacovigilance, Lyon, France
(2) Centre de Pharmacovigilance, Cochin- Saint Vincent de Paul, Paris,
France
(3) Centre de Pharmacovigilance, Bordeaux, France
(4) Centre de Pharmacovigilance, Marseille, France
(5) Centre de Pharmacovigilance, Grenoble, France
Rituximab has been approved in various haematological malignancies and
in rheumatoid arthritis but rituximab is now used in a wide range of severe
autoimmune disorders. However, data on its safety in these unapproved
indications is scarce. We reviewed the adverse effects of rituximab in nononcological treated patients from an analysis of the French Pharmacovigilance database. Cases were retained only if patients were treated for non
oncological disorders and were analysed according to the MedDRA classication. Out of 737 extracted cases, 207 involved patients without cancer.
In 26 cases, patients were less than 18 years of age. In 55% of cases, rituximab was used to treat connective tissue diseases, mainly rheumatoid
arthritis and complicated systemic lupus erythematosus. Other indications
included autoimmune cytopenia, vasculitis, graft rejection and thrombotic
microangiopathy. A severity criterion was found in 70% of cases. Infusion
reactions were reported in 30.8% of cases, including 31 serious reactions
and one fatal case. Blood and lymphatic system disorders were found in
37 cases and they mainly consisted of cytopenia that occurred after a mean
of 82 days. Infections were noted in 15 patients and serum sickness disease was reported in 6 cases. Complete recovery was conrmed in 132
patients whereas death occurred in 10 patients with a possible causal relationship with rituximab treatment in 6. Rituximab is a promising therapeutic approach to treat resistant autoimmune diseases. Our preliminary data
suggest the safety prole of rituximab in these patients is very similar to
that observed in oncological patients.
Antonis Goulas(1), K Kalokasidis(1), D Molyva(1), E Varsamidou(1),

B Kokkas(1), K Varsamidis(2)
(1) Aristotle University of Thessaloniki, Medical School, 1st Department
of Pharmacology, Thessaloniki, Greece
(2) Alexandrian Technological Educational Institute of Thessaloniki,
Department of Physiotherapy, Thessaloniki, Greece
Introduction: Rupatadine, a dual histamine (H1R-selective), and platelet
activating factor (PAF) antagonist, was shown to be active in various
models of allergic/ inammatory response.In an effort to further explore
the effect of rupatadine in the acute and late phase inammatory
response, we examined its effect on histamine-induced oedema as well
as on H1R and PAF receptor (PAFR) gene expression, in the rat paw.
Paper No.: 637

EFFECT OF ATORVASTATIN ON PANCREATIC B-CELL
FUNCTION AND INSULIN RESISTANCE IN TYPE 2 DIABETES
MELLITUS PATIENTS
Aman Goyal(1), S Singh(1), N Tandon (2), N Gupta(2), YK Gupta(1)
(1) Department of Pharmacology, All India Institute of Medical Sciences,
New Delhi, India
309
(2) Department of Endocrinology and Metabolism, All India Institute of
Medical Sciences, New Delhi, India
Introduction: Statins are commonly used for the management of dyslipidemia in type 2 diabetes mellitus (T2DM) patients. On the one hand, statins have been reported to have adverse effect on glucose metabolism,
and on the other hand, may also improve the pancreatic b-cell function
mediated through its effects on b-cell apoptosis. This randomized, double-blind, placebo-controlled trial was conducted to evaluate the effect of
atorvastatin 10mg, using Homeostasis Model Assessment 2 on pancreatic b-cell function (HOMA-%b) and insulin resistance (HOMA2-IR) in
T2DM patients. Patients: 51 T2DM patients on oral anti-diabetic drugs,
not taking statins with baseline LDL-C between 100-160 mg/dl were
included. 43 patients (21 in placebo group and 22 in atorvastatin group)
completed the study and were taken up for nal analysis. Fasting blood
samples were obtained at baseline and 12 weeks to determine levels of
blood glucose, lipid prole, insulin, C-peptide and glycosylated haemoglobin (HbA1c). Results: Atorvastatin decreased total cholesterol by
37% (P < 0.001), LDL-C by 60% (P < 0.001) and triglycerides by 12%
(p = 0.09). There was statistically insignicant decrease in HOMA2-%b
(73.91 24.42 to 71.78 38.01, p = 0.72) and increase in HOMA2-IR
(1.18 0.68 to 1.35 0.76, p = 0.16), also the HbA1c levels (7.56
0.88 to 7.59 1.40%, p = 0.92) as compared to baseline in atorvastatin
group. Conclusion: Atorvastatin failed to produce any signicant
response on pancreatic b-cell function and insulin resistance in T2DM
patients. The baseline level of dyslipidemia in study cohort may not be
high enough to cause lipotoxicity on b-cells.
(Clinical Trials Registry-India number, CTRI/2008/091/000099).
Paper No.: 3269

COVARIATE ANALYSIS OF T-WAVE MORPHOLOGY AND
QTC PROLONGATION: NEW INSIGHTS INTO
DRUG-INDUCED ECG CHANGES
Claus Graff(1), E Toft(1), J Matz(2), JK Kanters(3), MP Andersen(1),
J Nielsen(4), JJ Struijk(1)
Paper No.: 2228

IMATINIB DETERMINATION IN HUMAN SERUM BY
HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY
Paula Granic, D Sertic, M Lovric, Z Lalic, N Bozina, B Labar, J Sertic
University Hospital Center Zagreb, Department of Laboratory Diagnosis,
Zagreb,Croatia
Introduction: Imatinib is a 2-phenylaminopyrimidine derivative that functions as a specic inhibitor of a number of tyrosine kinase enzymes. It is
used in chronic myelogenous leukemia (CML), gastrointestinal stromal
tumors (GISTs) and a number of other malignancies. Materials and
Methods: The aim of this study was to develop and validate sensitive
and specic high-perfomance liquid chromatography (HPLC) method for
the determination of imatinib in serum/plasma. Imatinib was extracted
from serum with methanol. Clozapine was used as an internal standard.
The sample was fractionated on a column MN EC Nucleosil 100-5-C-18
EC 250 x 4.6 mm with a mobile system consisting of ammonium acetate
buffer, methanol and acetonitrile (40:40:20). The ow rate was 0.75 ml
min-1. Quantitation was performed by measurement of UV detector at
the wavelength of 265 nm. Results: Calibration curve was linear in the
range of 40 - 5000 ng/ml, limit of detection (LOD) was 13.1 ng/ml and
limit of quantitation (LOQ) was 38.6 ng/m. Recovery ranged from
94.1% to 105.7%. Repeatability expressed as a coefcient of variation
was 4.90% and 1.95% for 75 and 1500 ng/ml, respectively, and the coefcient of variation for intermediate precision was <10.0%. Stability of
the analyte during several freeze/thaw cycles and long-term stability (during 21 days) was good. Conclusion: The method proved to be simple,
useful, enough sensitive and specic for routine monitoring of imatinib
concentrations in patients with chronic myelogenous leukemia.
Paper No.: 3153

SESSION - HEPATOLOGY
ROLES OF DNA DAMAGE AND INFLAMMATION IN THE
GENDER-SELECTIVE LIVER CARCINOGENICITY OF
4-AMINOBIPHENYL IN MICE
(1) Aalborg University, Aalborg, Denmark

(2) H. Lundbeck A/S, Copenhagen, Denmark
(3) University of Copenhagen, Copenhagen, Denmark
rhus University Hospitals, Aalborg, Denmark
(4) Aalborg Hospital, A
Denis Grant, K Sugamori, A Emami, S Wang, D Brenneman
QTc prolongation due to the administration of a drug is not a reliable surrogate of the drugs proarrhythmic potential. Consequently, the development of suitable indices for the characterization of drug-induced
repolarization changes might greatly improve risk assessment of new and
existing compounds. This study adds a T-wave morphology composite
score (MCS) to the QTc interval evaluation of drugs affecting cardiac
repolarization. Electrocardiographic recordings from 62 subjects on placebo and 400 mg moxioxacin were compared to recordings from 21
subjects receiving 160 and 320 mg d,l-sotalol. The antibiotic drug, moxioxacin has a favorable cardiovascular safety prole and is recommended as a positive control in thorough QT studies. In contrast, the
antiarrhythmic drug d,l-sotalol has a less favorable safety prole with a
reported incidence of TdP between 1.8% and 4.8%. This difference in
risk proles between moxioxacin and d,l-sotalol is indicated by T-wave
morphology changes, as assessed by DMCS. T-wave morphology
changes are larger for 320 mg d,l-sotalol than for 160 mg d,l-sotalol,
which are again larger than for moxioxacin and placebo. Covariate
analyses of DQTc and DMCS showed T-wave morphology changes as a
signicant effect of dl-sotalol. In contrast, there is no effect of moxioxacin on T-wave morphology (DMCS) at any given change in QTc. This
study offers new insights into the repolarization behavior of a drug with
low cardiac risk versus a high risk drug and suggests added benets of a
T-wave morphology composite score as a covariate to the assessment of
the QTc interval.
We recently observed a lower incidence of 4-aminobiphenyl (ABP)induced liver tumors in female than in male C57BL/6 mice. Male mice
lacking the arylamine N-acetyltransferases Nat1 and Nat2 (Nat1/2-/- mice)
were also partially protected. To determine whether these gender and
strain differences in ABP-induced tumor incidence relate to differences in
DNA damaging events and/or differences in tumor promotion due to
inammatory responses triggered by the acute liver toxicity of ABP, we
initiated studies to compare DNA damage, hepatotoxicity and inammation in male and female C57BL/6 and Nat1/2-/- mice following acute ABP
exposure. Liver DNA levels of neither covalent C8-deoxyguanosine-ABP
adducts nor of the oxidative DNA damage marker 8-oxoguanosine correlated with the strain or gender differences in ABP-induced tumor incidence. The acute in vivo hepatotoxicity of ABP was assessed by
measuring serum levels of the liver biomarker ALT and by histological
analysis. Results obtained so far suggest that, in contrast to recently
published studies using the liver carcinogen diethylnitrosamine, ABP at a
dose that produces liver tumors causes little or no acute liver damage in
either male or female mice. Levels of the inammatory mediators TNFa
and IL-6 also do not appear to be signicantly elevated after ABP exposure. Our results suggest either that the level of DNA damage does not
predict rates of tumor initiation, or that DNA lesion types other than those
measured by our assays are causative. The results also suggest that different tumor-promoting processes contribute to ABP-induced liver tumorigenesis than those of other liver carcinogens.
University of Toronto, Department of Pharmacology and Toxicology,

Toronto, Ontario, Canada
310
Paper No.: 1821
HEALTH AND DISEASE
ALTERATION OF THE EFFECTS OF GROWTH FACTORS BY
SIMULATED MICROGRAVITY
Daniela Grimm(1), C Ulbrich(2), J Pietsch(2), M Infanger(2),
G Aleshcheva(2), J Bauer(3)
(2) Charite Universitatsmedizin Berlin, Germany
(3) Max Planck Institute of Biochemistry, Martinsried, Germany
VEGF and bFGF interact with appropriate endothelial cell (EC) surface
receptors and initiate intracellular signal cascades. We investigated the
effects of bFGF and VEGF on signalling pathways of EA.hy926 cells
under 1g and under simulated microgravity (s-lg) using a random positioning machine. For this purpose, we added 10 ng/ml of either bFGF
or VEGF or of both together to EC cultures. In the presence of the
growth factors the expression of various genes changed after 4 hours.
The changes were rather similar, independently whether the cells were
cultured under 1g and s-lg similarly. After 24 h the growth factors
exerted effects on proteins regarding their accumulation within the cells
or their secretion into the supernatant. Some proteins, such as those
being involved in the VEGF signalling pathway, were enhanced similarly under both conditions. Other proteins, representing various interleukins or nuclear factors were secreted or accumulated at higher rates,
when bFGF or/and VEGF were present in the cultures under 1g, but at
lower rates under s-lg. During elongated incubation of cell cultures
externally added VEGF and bFGF were removed from the cell supernatant. But effects of their actions could be observed even after 7 days
of culturing. At this time, accumulation of a number of proteins in supernatants is enforced. This enforcement is only strong under normal
gravity but weak under s-lg. Therefore, we conclude that growth factors may turn on or shut off different intracellular signalling in ECs
depending on whether they are applied under microgravity or normal
gravity conditions.
Paper No.: 3202

AMBULATORY CONTROL OF ARTERIAL PRESSURE IN
PATIENTS WITH ISOLATED SYSTOLIC HYPERTENSION AND
ASSOCIATED ISCHEMIC HEART DISEASE
Paper No.: 2329

IMPACT OF CYP2C9, CYP2C19 AND VKORC1
POLYMORPHISMS ON ACENOCOUMAROL
TIME-TO-ACHIEVE STABILITY
Liliane Gschwind(1), V Rollason Gumprecht(1), M Rebsamen(2),
V Nepote(1), P Bonnabry(1), F Boehlen(3), C Combescure(4),
P Francis(5), P Chopard(5), P Dayer(1), J Desmeules(1)
(1) University Hospitals of Geneva (HUG), Division of Clinical Pharmacology and Toxicology, Geneva, Switzerland
(2) University Hospitals of Geneva (HUG), Division of Medical Genetic,
Geneva, Switzerland
(3) University Hospitals of Geneva (HUG), Division of Angiology and
Hemostasis,Geneva, Switzerland
(4) University Hospitals of Geneva (HUG), Division of Clinical Epidemiology, Geneva, Switzerland
(5) University Hospitals of Geneva (HUG), Quality of Care Service,
Geneva, Switzerland
Acenocoumarol is an oral anticoagulant commonly prescribed in Switzerland. The impact of CYP2C9, CYP2C19 and VKORC1 polymorphisms
on acenocoumarol time-to-achieve stability (dened as 3 INRs between 2
and 3 in a two week interval) was assessed in few studies but their
respective importance for the clinical practice is still difcult to determine. The objective of this study was to investigate the impact of these
polymorphisms on the acenocoumarol time-to-achieve stability. A prospective observational study was performed in the University Hospitals
of Geneva on patients beginning acenocoumarol treatment. CYP2C9,
CYP2C19 and VKORC1 genotypes were determined in 98 patients. Data
on INR, comedications, comorbidities, and doses of acenocoumarol were
collected during the rst 35 days of therapy. Carriers of mutations on
CYP2C9 or VKORC1 genes needed a statistically signicant lower dose
of acenocoumarol to reach a therapeutic INR (p < 0.05). A trend to a
lower probability to achieve stability in the rst 35 days of treatment in
carriers of CYP2C9*3 alleles was observed, compared with wild-type
subjects or CYP2C9*2 subjects. The same observations were made for
carriers of CYP2C19*2/*2 compared with carriers of wild-type CYP2C19
or CYP2C19 *1/*2. VKORC1 polymorphisms didnt seem to have an
inuence on acenocoumarol time-to-achieve stability. The knowledge of
CYP2C9, CYP2C19 and VKORC1 genotypes could be useful to identify
potential candidates requiring more frequent INR evaluations to minimize
problems in the initial phase of acenocoumarol treatment. We aim to further study the impact of these polymorphisms and others on the susceptibility to drug-drug interactions with acenocoumarol.
Jose Ernesto Groning(1), R Garcia(2), D Garcia-Barreto(2),

A Hernandez-Canero(2)
(1) ISCM Havana, Department of Pharmacology, Havana, Cuba
(2) Institute of Cardiology, Havana, Cuba
When ischemic heart disease is associated with arterial hypertension,
treatment becomes a complex task. A group of 126 non-controlled
patients with isolated systolic hypertension aged over 50 with associated
chronic ischemic heart disease was studied to assess the effect of antihypertensive treatment strategy based on the combination of b-blocker
(Atenolol), a diuretic, and an angiotensin-converting enzyme inhibitor
(ACEI) on the ambulatory control of arterial pressure. All the patients
were assessed at onset and 6, 24 and 52 weeks later. The percentage of
patients achieving blood pressure control was 52.38%; 54.76% and
71.42% at 6, 24 and 52 weeks respectively. At baseline, there was a
mean (SD) of 1.89 (1.54) angina episodes/wk. One year later, the frequency decreased to 0.14 (0.54) angina episodes/wk (P < 0.001). We
conclude that a strategy based on combination of some drugs with different action mechanisms, is very effective to treat the high blood pressure
in patients with isolated systolic hypertension and associated ischemic
heart disease.
Paper No.: 1052

DISEASE AND THERAPY
VOLUME-REGULATED CHLORIDE CHANNEL MEDIATES
FOAM CELL FORMATION IN ATHEROSCEROSIS
Yong-Yuan Guan
Sun Yat-Sen University Zhongshan School of Medicine, Department of
Pharmacology, Guangzhou, PR China
Introduce: The formation of foam cell during the development of atherosclerosis evokes the change of cell volume, which may result in the activation of volume-regulated Cl- channel (VRCC). However, we dont
really understand the role of VRCC in foam cell formation. In this study,
we investigated the alteration of VRCC in the formation of foam cell in
vivo and vitro. Methods: VRCC currents in cultured RAW264.7 macrophages and peritoneal macrophages isolated from ApoE-/- mice were
recorded by whole cell patch technique. The Cl- efux was measured by
6-Methoxy-N-ethylquinolinium iodide uorescent probe. In vitro, macrophages were incubated with 80 ug/ml ox-LDL for more than 24 hours,
311
and let foam formation. Results: With the form cell formation, the VRCC
currennt and Cl- efux were increased. There is a positive correlation
between intracellular cholesterol content and VRCC current / Cl- ux. In
peritoneal macrophages isolated from ApoE-/- mice that were fed a high
cholesterol diet for for 1, 7 and14 weeks, the VRCC current and Cl- ux
were enhanced with the time course. The values of atherosclerotic plaque
area was very related to the increases in VRCC current and Cl- ux.
Conclusion: present results suggest that the VRCC mediates foam cell
formation in atherosclerosis.
Paper No.: 1655

DATA MODELLING IN DRUG DEVELOPMENT. ..
A NEW DIMERIC LINEAR DIPEPTIDE MIMETIC OF NGF
ACTIVE ON ANIMAL MODELS OF PARKINSONS DISEASE
patients each, one group treated with Vimang (900 mg daily) and the placebogroup. The intervention was for 6 months. Serum concentration of
malonildialdehydo, peroxidation potential, extracellular superoxide
dismutase and catalase activities, reduced glutathione, fragmentation of
proteins and oxidation potential of proteins were determined in both
groups before treatment and 3 and 6 months after treatment. Vimang tablet supplementation increased superoxide dismutase activity (p = 0.01)
and decreased serum reduced glutationlevels (GSH) (P < 0.001). We
suggested that the antioxidant components of the extract could have been
utilized by the cells, sparing the intra- and extracellular antioxidant system and increasing serum peroxil scavenging capacity, thus preventing
neurodegeneration-associated increase in GSH oxidation and lipoperoxidation. Also, we conrmed the existence of an eurodegeneration-associated oxidative stress in human serum in all patients before treatment as
documented by an increase in serum lipoperoxides and GSH and a
decrease in serum antioxidant capacity.
Tatiana Gudasheva, P Povarnina, T Antipova, S Seredenin

Russian Academy of Medical Sciences, Zakusovs Institute of Pharmacology, Department of Chemistry, Moscow, Russian Federation
The potential of Nerve Growth Factor (NGF) as a therapeutic agent for
several neurodegenerative disorders including Parkinsons disease (PD)
has been indicated. Our goal was to synthesize linear dimeric dipeptide
with NGF-like activity that may be more perspective as pharmacological
agent in comparison with parent protein and NGF mimetics discribed.
The object of Modelling was NGF loop-4 < b>-turn formed by residues
93-96, -Asp-Glu-Lys-Gln-, which is most exposed outward. We included
in dipeptide composition the amino acid residues that belong to the central peptide fragment of the <b>turn. The preceding residue of aspartic
acid was imitated with its bioisoster, the residue of succinic acid. Further
two N-monosuccinyl-Glu-Lys-OH dipeptides mimicking the loop 4 were
linked together with an appropriate spacer. We synthesized several
dimeric dipeptides with different spacers. The peptide with hexametylendiamine spacer named GK-2 demonstrated good NGF agonist activity at
a nanomolar concentration. GK-2 increased survival of immortalized
mice hippocampal cells HT22 and stimulated tyrosine phosphorilation of
TrkA receptor. More important the peptide was shown to modulate motor
symptoms in rodent models of PD. We shown that oral (10 mg/kg) or
intraperitoneal administration (0.01; 0.5; 1.0 and 5.0 mg/kg) of GK-2
reduces galoperidol induced catalepsy in rats. GK-2 (1 mg/kg i.p.)
reverses apomorphine-induced rotations in unilateral 6-OHDA lesioned
rats and reduces MPTP-induced akinesia. Furture studies will explore
potential use of this compound in the neuroprotective therapy for PD.
Paper No.: 404

EVALUATION OF THE EFFECT OF AN EXTRACT OF
MANGIFERA INDICA L. IN PATIENTS WITH
SPINOCEREBELLAR ATAXIA TYPE 2
Mariela Guevara-Garca(1), L Velazquez(2), R Aguilera(2),
R Rodrguez(2), O Ramrez(2), D Almaguer(2), JC Rodrguez(2)
(1) National Center of Clinical Trial, Research Department, Havana,
Cuba
(2) Clinic of Rehabilitation and Researh of SCA2, Havana, Cuba
SCA-2 Ataxia is a genetic health problem for Cuba, due to the existence
of 168 affected families in all the national territory with 757 patients and
almost 8000 descendents in risk of suffering from this illness during the
next years. After a long lag period, therapeutic and other interventions
based on knowledge of redox biology are on the horizon for at least
some of the neurodegenerative diseases. In other hand, an extract of
Mangifera indica L. (Vimang) is used in Cuba as a nutritional antioxidant supplement. Twenty patients were clinically and genetically diagnosed as Type SCA-2 Ataxia were randomized in two groups, 10
Paper No.: 2858

EFFECTS OF DULOXETINE ON BLADDER AND EXTERNAL
URETHRAL SPHINCTER IN ANESTHETIZED FEMALE
GUINEA-PIGS WITH INTRAVESICAL ACETIC ACID
Veronique Guilloteau, S Palea, M Guerard, T Westfall, P Lluel
UPS Faculte des Sciences Pharmaceutiques, UROsphere, Department of
Pharmacology, Toulouse, France
Motoneurons innervating the external urethral sphincter (EUS) are
located in ventral horn of spinal cord in guinea-pigs, cats and humans. In
cats, acetic acid (AA) bladder infusion-induced bladder overactivity and
loss of bladder/EUS coordination were reversed by duloxetine. The aim
was to evaluate the effect of duloxetine on both bladder and EUS activities during intravesical AA in guinea-pigs. Catheters were implanted into
the bladder and jugular vein and wire electrodes into the EUS. Saline
was infused into the bladder (30min) followed by AA (30min). Duloxetine (3mg/kg) or vehicle (n = 6) were administered intravenously. Intercontraction interval (ICI) and maximal amplitudes of EUS activity
during the lling and voiding were analyzed over two consecutive 30min
periods. Intravesical AA induced a signicant decrease in ICI (197
27sec) compared to saline (649 61sec). Duloxetine (30-60min post
administration) partially reversed this effect (387 62sec). During intravesical saline, EUS activity increased during lling and stopped during
voiding. Following AA infusion, an increase in EUS activity during
voiding was observed (186% of saline values). Duloxetine reduced the
increased EUS activity during voiding (24% of saline values), while
vehicle had no effect (114% of saline values). In conclusion, under normal conditions, there is coordination between bladder and EUS activity
(activity during lling and inactivity during voiding). Intravesical AA
elicited bladder overactivity characterized by a decreased ICI, associated
with a loss of bladder/EUS coordination. Duloxetine reversed bladder
overactivity and restored coordination. Intravesical AA in guinea-pigs
appears to be a suitable model for evaluating the effects of pharmacological agents on bladder and EUS functions.
Paper No.: 1173

FOCUSED CONFERENCE GROUP: PW03 -TARGETING TRP
CHANNELS FOR PAIN RELIEF (AND MORE)
LASSBIO-881: AN N-ACYLHYDRAZONE TRPV1 ANTAGONIST
ORALLY EFFECTIVE AGAINST CAPSAICIN- AND PARTIAL
SCIATIC LIGATION-INDUCED HYPERNOCICEPTION
Marilia Guimaraes, J Tributino, M Santos, C Mesquita, C Lima, L Silva,
C Duarte, E Barreiro, C Fraga, N Castro, AL Miranda
Federal University of Rio de Janeiro, Institute of Clinical Biomedics, Rio
de Janeiro, Brazil
312
LASSBio-881 is an orally effective antinociceptive compound that binds
to CB1 receptors and is active mainly in the neurogenic component of
pain models. However, due to its chemical characteristics, we investigated whether LASSBio-881 had also effects on TRPV1 channels. CB1and TRPV1-expressing Xenopus oocytes were used to evaluate effects
on both receptors. In vivo effects were evaluated in capsaicin-induced
acute and inammatory changes in nociception as well as in a partial sciatic ligation model. LASSBio-881 inhibited capsaicin-elicited TRPV1
currents with an IC50 of 14 lM and inhibited proton-gated currents by
70% at 20 lM. Preliminary experiments indicate that this compound acts
as an inverse agonist at CB1 receptors as well. Locally applied LASSBio-881 decreased time spent in capsaicin-elicited nocifensive behaviour
by 30%, and given orally it reduced in about 50% measures of two models of inammatory thermal hypernociception. In addition, LASSBio881, at 300 lmol/kg/day p.o., decreased the paw withdrawal responses
to thermal stimuli of animals with sciatic neuropathy 7-11 days after
nerve ligation. At this dose, hyperthermia was not observed within 4
hours following administration. LASSBio-881 is a TRPV1 antagonist
that apparently competes with capsaicin. Accordingly, LASSBio-881
inhibited nociception in models of acute, inammatory and neuropathic
pain presumed to involve TRPV1 signalling. These in vivo actions were
not hindered by hyperthermia, a common side effect of other TRPV1
antagonists. We propose that the antinociceptive properties of LASSBio881 are due to TRPV1 antagonism and not CB1 agonism, although other
molecular interactions may contribute to the effects of this multi-target
drug candidate.
Paper No.: 2884

ENHANCEMENT OF CLONIDINE-INDUCED ANALGESIA BY
SULFISOXAZOLE IN MICE
Paper No.: 3295

ENZYMATIC INHIBITION, ANTIOXIDANT AND
ANTIBACTERIAL ACTIVITY OF MEDICINAL PLANT
EXTRACTS: POTENTIAL APPLICATION IN THE
MANAGEMENT OF TYPE 2 DIABETES
Vandana Gulati, I Harding, E Palombo
Swinburne University of Technology, Faculty of Life and Social Sciences, Melbourne, Victoria, Australia
Type 2 diabetes can be managed by reducing postprandial hyperglycemia
using oral medications which inhibit digestive enzymes involved in carbohydrate metabolism. Two such enzymes of signicance are a-amylase
and a-glucosidase. Plants are one of the richest sources of natural compounds which may provide the basis of such inhibitory medication. This
study investigated the inhibitory effects of thirty ethanolic plant extracts
on a-amylase and a-glucosidase. In addition, DPPH free radical scavenging effect and total phenolic and avonoid content were determined. The
percentage enzyme inhibition was calculated with respect to negative
control (ethanol). Santalum spicatum (120 mg/ml) and Acacia ligulata
(75 mg/ml) showed 100% inhibition of a-amylase. The same extracts
showed a-glucosidase inhibition in the range of 62.5 2.5% at a concentration of 1.56 lg/ml. The DPPH scavenging effect was found to be 86
2% at 100 lg/ml for the majority of the plant extracts tested. The total
phenolic and avonoid content was found to correlate with plants showing good a-amylase and a-glucosidase inhibition, and DPPH scavenging
effect. The same plant extracts also showed good antibacterial activity
against Gram positive and Gram negative bacteria. The extracts (or phytochemicals within extracts) may be developed into useful alternative
oral therapies for the management of Type 2 diabetes. The above extracts
will be screened for Angiotensin converting enzyme (ACE)-inhibitory
activity to determine whether they can be used to manage hypertension
related to type 2 diabetes.
Anil Gulati(1), G Matwyshyn(1), M Boxwalla(2), B Puppala(2),

S Andurkar(1)
(1) Midwestern University, Chicago College of Pharmacy, Downers
Grove, IL, USA
(2) Advocate Lutheran General Childrens Hospital, Park Ridge, IL,
USA
Clonidine, an a2-adrenergic agonist, produces signicant analgesia and
potentiates morphine analgesia. ETA receptor antagonists also potentiate
theantinociceptive response to morphine. An interaction between clonidine and ET inthe cardiovascular effects involving sympathetic nervous
system has been reported, but it is not known whether ETA receptor
antagonist will affect clonidine analgesia. This study examined the inuence of sulsoxazole (ETAreceptor antagonist) on clonidine analgesia.
Male Swiss Webster mice were used to determine antinociceptive
response of drugs by measuring tail ick latency. The effect of clonidine
(0.3, 1.0 and 3.0 mg/kg, i.p.) alone or in combination with sulsoxazole
(25, 75 and 225 mg/kg, p.o.) on analgesia and body temperature was
determined. Clonidine produced a dose-dependent analgesia and hypothermia. Sulsoxazole (25, 75 and 225 mg/kg) when administered with
clonidine (0.3 mg/kg) signicantly potentiated (31% increase in AUC)
the analgesic effect of clonidine. Yohimbine (a2-adrenergic receptor
antagonist) did not affect analgesic effect of clonidine plus sulsoxazole.
Idazoxan(I1-imidazoline and a2-adrenergic receptor antagonist) reduced
(47% decrease inAUC) the analgesic effect of clonidine plus sulsoxazole. Treatment with naloxone reduced (46% decrease in AUC) the analgesic effect of clonidine plus sulsoxazole. These results indicate that
sulsoxazole, an ETA receptor antagonist, potentiates the analgesic
effect of clonidine, which could be mediated through I1-imidazoline
receptors and opioid receptors. Combined administration of non-opioids
clonidine and sulsoxazole produces marked antinociception suggesting
that this therapeutic strategy might provide analternative in pain management.
Paper No.: 2866

HEALTH AND DISEASE
ENDOTHELIAL DYSFUNCTION OF UTERINE ARTERY IN
SURAMIN-INDUCED PREECLAMPSIA-LIKE SYNDROME IN
RATS
Sebile Guler(1), EG Cekic(1), G Soydan(1), D Ertoy Baydar(2),
M Tuncer(1)
(1) Hacettepe University, Faculty of Medicine, Department of
Pharmacology, Ankara, Turkey
(2) Hacettepe University Faculty of Medicine, Department of Pathology,
Ankara, Turkey
Since the pathogenesis of preeclampsia is not yet fully explained, animal
models are employed to nd a solution. Among these models, suramininduced preeclampsia-like syndrome is the most recent one. Endothelial
dysfunction of rat aorta has been shown in this model. The aim of the
study is to investigate whether suramin-induced preeclampsia-like syndrome induces endothelial dysfunction in uterine artery which is important to supply blood to placenta and fetus. Pregnant Wistar rats were used.
On the 10th and 11th day of pregnancy totally 200 mg/kg suramin or saline were given. Blood pressure and weight gain were measured till 20th
day of pregnancy in which rats were sacriced. Left kidneys were
removed for pathological examination. Urine protein levels were determined by sticks. Uterine arteries were isolated and placed on wire myograph to evaluate pharmacological responses to endothelin-1, prostaglandin
F2a, acetylcholine and sodium nitroprusside. Blood pressures signicantly increased in suramin-treated group. The number of the fetuses,
weight of the placentas and fetuses were decreased. Although urine sticks
did not show signicant differences for proteinuria, pathological examination
313
of the kidneys revealed severe tubular injury in suramin-treated group.
Proximal tubules in the renal cortex showed diffuse macrovacuolar degeneration. Endothelin-1 and prostaglandin F2a contractions of uterine artery
were increased and acetylcholine relaxations were decreased in the suramin-treated group, while sodium nitroprusside relaxations did not change.
These results indicate that suramin induced preeclampsia-like syndrome
and caused endothelial dysfunction in uterine artery.
Paper No.: 2752

ADMINISTRATIVE COMPLEXITIES FOR A EUROPEAN
OBSERVATIONAL STUDY
Sinem Ezgi Gulmez(1), S Lignot-Maleyran(1), S Micon(1), F Hamoud(1),
C De Vries(2), M Sturkenboom(3), P Blin(1), N Moore(1)
(1) University Victor Segalen, Department of Pharmacology, Bordeaux,
France
(2) University of Bath, Department of Pharmacology and Pharmacy.
Bath, UK
(3) Erasmus University Medical Centre, Department of Epidemiology &
Biostatistics, Rotterdam, The Netherlands
The development of pharmacoepidemiology in Europe, strongly supported by the EMEA, will need to rely much on multinational retrospective studies. Pharmacoepidemiology studies are non-interventional and
therefore they do not fall under the clinical trials directive (2001/20/EC).
Based on the 1995 Data Protection Directive (95/46/EC), accessing these
data should require only an authorisation by the relevant Data Protection
Committees and therefore one might expect similar procedures for study
approval across European countries. Experience from a simple retrospective multinational study across 7 countries shows that this is not the case.
In two countries, a single opinion from a single Data Protection Committee was enough. In three, there is a single multicentre Committee, but
some hospitals insist on approval by local Ethics Committees (EC). In
the rest, there is either no central Committee, or the advice of the central
Committee comes after that of local EC. The information requested by
the different Committees varies even within the same country. Some
Committees give their advice for free, others charge after having given
the advice, and some require payment even before considering the project. This degree of complexity and disharmony, and resulting cost, is
not required by the European Directive. It might be argued that a single
opinion should be enough to cover all of Europe. Regulators will need to
be aware that these time-consuming, expensive and useless complexities
must be factored in when estimating the time needed to do a study, and
the cost of the study.
Paper No.: 2753

SALOME: A PILOT STUDY TO TEST CAUSALITY
ASSESSMENT OF DRUG-EXPOSED ACUTE LIVER FAILURE
CASES USING DIFFERENT SCALES
Sinem Ezgi Gulmez(1), GP Pageaux(2), D Larrey(2), S Lignot-Maleyran(1), F Hamoud(1), S Micon(1), R Lassalle(1), J Jove(1), F Bissoli(3),
Y Horsmans(4), J Bernuau(5), B Stricker(6), D Thorburn(7), P Blin(1),
N Moore (1), on behalf of SALT-I team
(1) University Victor Segalen, Department of Pharmacology, Bordeaux,
France
(2) CHU Montpellier, Hopital Saint-Eloi, Department of Hepatogastroenterology,Montpellier, France
(3) Clinica San Gaudenzio, Department of Internal Medicine and Cardiology, Novara, Italy
(4) Universite Catholique de Louvain, Department of Gastroenterology,
Brussels, Belgium
(5) APHP, Hopital Beaujon, Federation dHepatogastroenterologie,
Clichy, France
(6) Erasmus University Medical Centre, Department of Epidemiology &
Biostatistics, Rotterdam, The netherlands
(7) Royal Free Hampstead NHS Trust, Liver Transplantation & Hepatobilliary Unit, London, UK
SALOME is a pilot study of the Study of Acute Liver Transplant, retrospectively evaluating cases of acute liver failure (ALF) patients exposed
to non-steroidal anti-inammatory drugs (NSAIDs). The objective is to
dene the level of drug name blinding for causality assessment, and the
causality scale to be used. Thirty randomly selected drug-exposed ALF
cases in France were submitted to 7 members of the Case Adjudication
Committee (CAC) for causality assessment. Individually they were asked
to dene what they considered as the index-date, then to determine causality of the drugs to which the patient was exposed 30 days prior to
index-date. Members used 3 different causality scales (WHO, RUCAM,
Venulet). Each case was sequentially assessed 3 times with increasing levels of detail: Assesment-1: Drug names blinded as A, B, C; doses therapeutic, overdose, not known; Assesment-2: Information from
assesment-1 + drug class; Assesment-3: Information from assesment-2 +
full drug data including the names except for NSAIDs (kept blinded as
NSAID). Subsequently, a consensus meeting of the CAC was held. All
members dened the index-date as the date of rst symptoms of liver disease. Overall, there were 429 assessments: WHO scale was completed for
97.4% of these; RUCAM for 32.6%, and Venulet for 96.3% (but only the
causality decision part). As the level of drug information increased, the
certainty of causality assessment increased. The CAC agreed to use
Assessment-3 and the WHO scale of causality, which may be the most
appropriate scale for such retrospective studies of drug-exposed cases.
Paper No.: 2667

SESSION - ONCOLOGY
HGF/C-MET OVEREXPRESSIONS, BUT NOT MET MUTATION,
CORRELATES WITH PROGRESSION OF NON-SMALL CELL
CARCINOMAS
Mukaddes Gumustekin(1), A Kargi(2), G Bulut(3), A Gozukizil(3),
C Ulukus(2), I Oztop(4), N Atabey(3)
(1) Dokuz Eylul University School of Medicine, Department of Pharmacology, Izmir,Turkey
(2) Dokuz Eylul University School of Medicine, Department of Pathology,
Izmir,Turkey
(3) Dokuz Eylul University School of Medicine Department of Medical
Biology andGenetics, Izmir, Turkey
(4) Dokuz Eylul University School of Medicine Department of Medical
Oncology, Izmir,Turkey
HGF/c-Met pathway is potential therapeutic target for cancer treatment.
Overexpression of HGF and c-Met have been shown in non-small-cell
lung cancer (NSCLC). However, their role in tumor progression is not
clearly dened. The aim of this study is to determine the role of HGF/cMet pathway and its association with invasion related markers and clinicopathologic parameters in NSCLC. Immunohistochemical analysis was
performed on 63 parafn-embedded NSCLC tumor sections which were
provided from Pathology archives of Dokuz Eylul University. The
expressions of invasion related markers such as MMP-2, MMP-9, TIMP1, TIMP-3 and RhoA were also examined. DNA sequencing for Met
mutations in both nonkinase and tyrosine kinase (TK) domain was performed. Overexpression ratio of c-Met was higher than those HGF.
Co-expression of HGF/c-Met was signicantly associated with lymph
node invasion and TIMP-3 and RhoA overexpressions. There were positive
correlation between TIMP-3 overexpression and advanced stage and negative correlation between RhoA overexpression and survival. A single
nucleotide polymorphism (SNP) in sema domain and two SNPs in TK
domain of c-Met were found. There was no statistically signicant correlation between the presence of c-Met alterations and clinicopathologic
parameters except shorter survival time in cases with two SNPs in TK
314
domain. These results suggest that HGF/c-Met might exert their effects
in tumor progression in association with RhoA and probably with TIMP3. The blockade of the HGF/c-Met pathway with RhoA and/or TIMP-3
inhibitors may be an effective therapeutic target for NSCLC treatment.
Keywords: Non-small cell lung cancer, HGF, c-Met, RhoA, TIMP-3,
invasion, c-Met mutations.
Paper No.: 3216

DOES HGF/C-MET SIGNALLING EXERT ITS EFFECTS ON
VASCULAR DYSFUNCTION BY SMALL GTPASES IN
DIABETIC RATS?
Essential Medicines, 16th list, March 2009 and National List of Essential
Medicines 2003, India, having different brands in the same strength, dosage form and quantity, were analysed for their costs in the Indian Market.
Results: Total no. of brands were 1209 for core drugs with possible cost
reduction from 1.37% to 88.85%, and 1039 for complementary drugs
from 44.57% to 96.63%. Possible cost reduction for 21 oral formulations
with 1395 brands was 12.68% to 96.63%, for 15 injections with 738
brands was 1.37% to 73.18% and for 5 topical formulations with 15
brands was 23.8% to 83.39%. Overall possible reduction in cost was
observed ranging from 1.37% to 96.63% (average 54.12%). Conclusion:
Antibacterial drugs are available in many brands with variable costs. Prescription of most economical brand of the required formulation can
reduce expenditure on majority of antibacterial drugs signicantly. Promotion of generic drugs and awareness about rational use of drugs may
have supraadditive effect in providing cost effective treatment.
M Gumustekin(1), A Arici(1), Feyza Arslan(1), M Karaman(2),

M Ates(3), Ei Guneli(2)
(1) Dokuz Eylul University Faculty of Medicine, Department of Pharmacology, Izmir, Turkey
(2) Dokuz Eylul University, Institute of Health Sciences, Department of
Laboratory Animal Science, Izmir, Turkey
(3) Dokuz Eylul University, Advanced Professional School of Health
Sciences, Izmir, Turkey
Paper No.: 474

TRANSCRIPTOME PROFILING REVEALS INVOLVEMENT OF
PKC E IN THE DEVELOPMENT OF INSULIN RESISTANCE IN
LIVER OF HFD+STZ FED RATS VIA RHO KINASE PATHWAY
The role of HGF/c-Met signalling in diabetes-induced vascular dysfunction has been reported. However, the downstream effector molecules take
place in this pathways havent been claried yet in diabetes. Since, the
RhoA and Rac1 are downstream effectors of HGF/c-Met pathway, we
planned to evaluate their expression levels and its correlation with HGF/
c-Met expressions and vascular reactivity studies in diabetic rat aortic
segments. Male Wistar rats were injected with streptozotocin (45 mg kg1, i.p.) to induce type-1 diabetes mellitus (SIDM). The effects of HGF/cMet signalling via their effector macromolecules small GTPases, RhoA
and Rac1 on vascular function were investigated in isolated aortic segments in organ bath chambers 8 weeks after diabetes induction. Local
SA and HGF, c-Met, p-Met, RhoA
HGF levels will be evaluated by ELY
and Rac-1 expressions will be evaluated by immunohistochemical analyses. Pretreatment with RhoA kinase inhibitor Y27632 (10-7 10-5M)
inhibited phenylephrine -induced contractions in a concentration-dependent manner in the control group. The relaxant response to cumulative
concentrations of acetylcholine was assessed after preconstriction with
phenylephrine (110lM). The response to acetylcholine was higher in
control rats than diabetic group. Pretreatment with Rac-1 inhibitor
NSC23766 (1lM) attenuated ACh-induced relaxation of control and diabetic rat thoracic aortic rings. The organ bath studies show that small
GTPases, RhoA and Rac1 may play a role in diabetes-induced vascular
dysfunction in SIDM rats. All the expression and functional analyses
must be completed for a denite result about relationship between HGF
and small-GTPases in diabetes-induced vascular dysfunction.
Jeena Gupta, K Tikoo
Paper No.: 2348

COST ANALYSIS OF ANTIBACTERIAL DRUGS IN A
DEVELOPING COUNTRY, INDIA
Paper No.: 1246

THE ROLE OF D1/D2 DOPAMINE RECEPTORS IN THE
DEVELOPMENT AND EXPRESSION OF NIGROSTRATAL DA
SENSITISATION IN RATS
Devi Dayal Gupta, M Gupta

Indira Gandhi Medical College, Department of Medical Education,
Shimla, India.
Introduction: In developing countries bacterial infections are very common due to poor socio-economic status. Antibacterial drugs are most
commonly prescribed group of drugs, consuming a major share of health
budget. Therefore the present study was proposed to explore the possibilities of reduction in expenditure on antibacterial drugs. Materials: Systemic and topical antibacterial formulations, from WHO Model List of
National Institute of Pharmaceutical Education and Research (NIPER),

Department of Pharmacology and Toxicology, Mohali, Punjab, India
Liver is the main organ of glucose disposal and it is the foremost to get
affected during the progression of Type 2 diabetes (T2D). However, the
detailed molecular mechanism underlying the development of insulin
resistance in liver of non genetic model of T2D has not been yet
explored. Hence, the present study was aimed gain insight into the mechanism of development of insulin resistance in the liver of non-genetic
model of T2D. We performed a global hepatic gene expression proling
study in a T2D model [high fat diet (HFD) fed Streptozotocin (STZ, 35
mg/kg, i.p.) treated rats] using microarray technology. The gene expression data was further validated by RT-PCR and protein expression.
Microarray analysis revealed that genes related protein/amino acid, carbohydrate/fat metabolism and cell adhesion were upregulated whereas
transcription, signal transduction and DNA repair related genes were
down regulated. Further, analyzing the expression proling data revealed
that PKC e plays an important role in the development of insulin resistance in liver of HFD+STZ fed rats by increasing Rho signalling pathway. This was further conrmed by increase in Rho kinase activity by
increase in Thr 853 phosphorylation of MYPT-1. To best of our knowledge this is the rst report that shows the involvement of PKC e in the
development of insulin resistance in liver of HFD+STZ fed rats.
Susanna Gyarmati(1), J Timar(1), A Horvath(1), A Fendt(1), S

Furst(1,2)
(1) Semmelweis University, Department of Pharmacology and Pharmacotherapy, Budapest, Hungary
(2) Hungarian Academy of Sciences, Neuropsychopharmacology
Research Group, Budapest,Hungary
Repeated administration of methylenedioxymethamphetamine (MDMA)
results in sensitisation of the mesolimbic dopamine (DA) transmission
315
measured by changes in the locomotor activity. The role of D1 receptors
in this DA sensitisation (DAS) was demonstrated (Ramos et al, Psychopharmacology 177, 2004). The aim of the present study was to prove the
development of nigrostriatal DAS as well, by following the changes in
the intensity of MDMA-induced stereotyped behaviour. Two weeks after
pretreatment with a single sc. 10 mg/kg MDMA a subsequent MDMA
challenge (10 mg/kg ip.) induced signicantly more marked stereotyped
behaviour than in control (saline-pretreated) rats. The role of D1 and D2
receptors in the development and the expression of DAS was analysed
by using selective D1 (SCH 23390) and D2 receptor (Sulpiride) antagonists. In order to check the development of DAS SCH 23390 (0.5 mg/kg
sc.) and Sulpiride (50 mg/kg sc.) were administered parallel with the
MDMA pretreatment. When the expression of DAS was measured SCH
23390 (0.05 mg/kg sc.) and Sulpiride (10 mg/kg sc.) were given 30 min
before MDMA challenge. SCH 23390 inhibited both the development
and expression of DAS. Sulpiride failed to affect the development, but
inhibited the expression. The results indicate that both D1 and D2 receptors are involved in the expression of nigrostriatal DA sensitisation,
while the development of it seems to be depending on the function of D1
receptors.
This study was supported by Hungarian grants OTKA K-60999 and
ETT-441/2006.
Paper No.: 3394

QUALITY-ANALYSIS OF AMINOGLYCOSIDE THERAPY IN
CHILDREN AT A BELGIAN UNIVERSITY HOSPITAL
Evelien Haegeman(1), P De Cock(1), N Goerlandt(1),
P Vanhaesebrouck(2), V Stove(3), A Verstraete(3), P Schelstraete(4),
H Robays(1)
(1) Ghent University Hospital, Pharmacy Department, Ghent, Belgium
(2) Ghent University Hospital, Department of Neonatology, Ghent,
Belgium
(3) Ghent University Hospital, Department of Clinical Chemistry, Ghent,
Belgium
(4) Ghent University Hospital, Department of Pediatric Pulmonology and
Infectious Diseases, Ghent, Belgium
Introduction: the compliance of aminoglycoside prescribing and therapeutic drug monitoring (TDM) in children with updated hospital guidelines was assessed by a retrospective analysis. Patients: a total of 93
aminoglycoside therapies in 80 patients (30 neonates, 50 children > 1
month) were evaluated. Results: the rst initiated dosing regimen was
incorrect in 5.4%. From the 88 correctly initiated dosing regimens,
only 10.2% led to a peak and trough level within therapeutic range,
while 5.7% led to a potentially toxic trough level, 20.5% to a subtherapeutic peak level and 63.6% could not be evaluated. 94 times there
was an indication for TDM, namely peak and trough monitoring of
the rst initiated dosing regimen (88.3%), remonitoring within a maximum of 7 days interval (32%) or remonitoring after adjustment of the
dosing regimen (8.5%). The majority of necessary trough levels were
taken at the right time (67.0%), whereas peak monitoring was often
performed too late (10.6%, ranging from 1 to 7 doses too late with a
median of 1.5 doses) or not at all (63.8%); although both are essential
for good interpretation of the plasma levels. TDM results showed that
adjustment of the dosing regimen was required in 29 cases, of which
no more than 10.3% was appropriately executed. Conclusion: dissemination and education of hospital guidelines should be encouraged.
Moreover, these results suggest that despite adequate dosing TDM
is still imperative to ensure efcacy and minimize toxicity of aminoglycoside treatments.
Paper No.: 1314

DISCOVERY
IL1B-MEDIATES TIME AND CONCENTRATION DEPENDENT
INDUCTION OF ABCC2 GENE EXPRESSION VIA P38 MAPK
AND AKT PATHWAY IN SK-HEP1 CELLS
Sierk Haenisch, S Lachelt, A Schmall, I Cascorbi
University Hospital Schleswig-Holstein, Institute of Experimental and
Clinical Pharmacology, Kiel, Germany
The efux transporter ABCC2 (MRP2) is luminally expressed at membranes of several tissues like liver, gut, kidney, and interestingly in epileptogenic brain tissue. It is involved in distribution and elimination of
drugs as well as in detoxication and response to oxidative stress. There
is evidence that in inammatory diseases, ABCC2 expression and activity could be altered thus contributing to changes in absorption, distribution and clearance of xenobiotics. To clarify the inuence of interleukin1b (IL1b) an important key player of inammation on gene expression
of ABCC2, we incubated SK-Hep1 cells with different concentrations of
IL1b (0.012 ng/ml, 1 ng/ml, 10 ng/ml) up to 48 hours. ABCC2 mRNA
and protein expression was determined by rtPCR and Western blotting.
The degree of phosphorylation was measured by human phospho MAPK
array. 12 hours after initiating IL1b treatment, a maximal increment of
ABCC2 mRNA expression was observed. The ratios compared to nonexposed cells were 2.59 0.24 at 0.012 ng/ml, 1.88 0.39 at 1 ng/ ml,
and 1.91 0.16 at 10 ng/ml, respectively. Maximal 1.7-fold ABCC2 protein expression was observed after 3 hours using 0.012 ng/ml. PhosphoMAPK array showed a more than 2-fold increase of phosphorylation for
Akt1, 2, P70S6 kinase and for p38 b and d after 3 hours at 0.012 ng/ml.
These results suggest an IL1b-mediated time and concentration dependent stimulation of ABCC2 mRNA and protein expression in the hepatic
cell line SK-Hep1. Furthermore the p38MAPK and Akt pathways seem
to be involved in induction of ABCC2 transcription and protein synthesis
on translational level.
Paper No.: 919
ANALGESICS
LACK OF CORRELATION BETWEEN PLASMA
ASYMMETRIC DIMETHYLARGININE (ADMA) LEVELS AND
PAIN-RELATED BEHAVIORS IN NEUROPATHIC RATS
E Hazoglu, Ahmet Ulugol
Trakya University Faculty of Medicine, Department of Pharmacology,
Edirne,Turkey
Recent research has demonstrated elevations of ADMA levels in many
cardiovascular and metabolic diseases, and ADMA has also been proposed as a modulator of nociception in opiate tolerance and addiction.
The present study was conducted to determine whether increased level of
ADMA is implicated in tactile allodynia in rats with partial tight ligation
of the sciatic nerve. Using high-performance liquid chromatography,
plasma levels of symmetric dimethylarginine (SDMA), L-homoarginine
and L-arginine to allodynia are also determined simultaneously. An electronic von Frey algometer was used to detect tactile allodynia, in which
paw withdrawal latency was taken as a behavioral index. Neuropathic
rats were divided into three groups of 2-, 4- and 8-week feeding duration
after the surgery. Beginning from the 1st measurement after the ligation,
paw withdrawal thresholds signicantly reduced compared with duration-matched control animals, indicating that mechanical allodynia developed in sciatic nerve-ligated rats. At the end of each period, animals
were anesthetized and blood samples were collected from carotid artery.
In both neuropathic and control groups, plasma levels of ADMA and the
other molecules remained unchanged throughout the experiment, and
there were no signicant differences between two groups in different
durations. These results suggest that endogenous ADMA, SDMA,
316
L-homoarginine and L-arginine levels do not change during the different
stages of neuropathic duration, and accordingly may not be implicated in
tactile allodynia in neuropathic rats.
Paper No.: 2678

POTENTIATION OF UTERUS-RELAXING EFFECTS OF
NIFEDIPINE WITH BETA-ADRENERGIC AGONISTS: STUDIES
ON RATS AND HUMAN MYOMETRIUM
Judith Hajagos-Toth(1), Z Kormanyos(2), G Falkay(2), R Gaspar(2)
(1) University of Szeged Faculty of Pharmacy, Department of Pharmacodynamics and Biopharmacy, Szeged, Hungary
(2) University of Szeged Faculty of General Medicine, Szent-Gyorgyi
Albert Clinical Center, Szeged, Hungary
Tocolysis is one of the greatest challenges in obstetrical practice. It is
known that the calcium channel antagonists abolish the intracellular calcium ion transients and myometrial contraction. However there is a
growing interest in experimental studies to use different tocolytic combination. Our aims were to investigate the modication of the effect of
nifedipine by progesterone and salmeterol in hormone-induced preterm
delivery in rats in vivo on different days of pregnancy (16, 18) and by
terbutaline and nifedipine on the contraction of isolated human myometrium. For human myometrial rings rhythmic contractions were evoked
with oxytocin in an isolated organ bath. Nifedipine treatment was effective to delay the hormone-induced preterm delivery in rats but its effect
could be tripled by addition of b-mimetics. The progesterone pre-treatment (0.5 mg/animal for 5-6 days) abolished the effect of nifedipine to
delay labour. In isolated organ bath both nifedipine and terbutaline inhibit the oxytocin-induced human myometrial concentration dose-dependently. A synergism was observed in the uterus-relaxing effect of
nifedipine and terbutaline, though the extent of potentiation depended on
the sequence of administration of the two compounds. When terbutaline
was added rst in a single dose, it worsened the effect of nifedipine, indicating the role of calcium channel activating effect of terbutaline. It is
concluded that the effect of nifedipine to delay labour is worsened by
progesterone and combination of nifedipine and b2-agonists should be
considered for clinical use. However, the administration of terbutaline
can not precede the administration of nifedipine.
Paper No.: 539
THE EFFECTS OF PHENCYCLIDINE NEW DERIVATIVES ON
ANXIETY BEHAVIORS IN RATS
Paper No.: 1785

EFFECTS OF HIGH-SALT DIET AND BIOACTIVE PEPTIDES
ON MRNA EXPRESSION OF COMMON MARKERS OF
RENIN-ANGIOTENSIN-ALDOSTERONE SYSTEM IN
GOTO-KAKIZAKI RAT AORTA
Anne Hakala(1), P Jakala(1), AM Turpeinen(2), R Korpela(1,2),
H Vapaatalo(1)
(1) University of Helsinki, Institute of Biomedicine, Department of
Pharmacology, Helsinki, Finland
(2) Valio Ltd, R&D, Helsinki, Finland
High-salt diet increases blood pressure whereas bioactive peptide-containing milk products have been shown to decrease it. These effects are
suggested to be controlled by renin-angiotensin-aldosterone system
(RAAS). Male Goto-Kakizaki (GK) rats were given high-salt diet (8%)
and divided into 3 groups which received bioactive peptide-containing
milk products A and B (Ile-Pro-Pro + Val-Pro-Pro 26.5 mg/ml and 21.2
mg/ml, respectively) and milk product without peptides (C) or water as
drinking uid for 8 weeks ad libitum. Control group received normal diet
(0.6% salt). The RNA expressions were determined from the aorta tissue
by qPCR. Angiotensin-converting enzyme 1 (ACE1) expression did not
differ between A-, B- and C groups. High-salt diet alone increased the
expression of ACE1 but lowered the expression of ACE2. A and B groups expressed ACE2 more than the water group. Ang-(17) receptor
Mas and arginase I expressions were similar in all groups. Cyclooxygenase 2 (COX-2) expression was lower in the A- and control groups than
in the water group. All milk products decreased COX-2 expression. As a
reference, same expression levels were analyzed from spontaneously
hypertensive rats (SHR) and Wistar-Kyoto (WKY) aortic samples. SHR
and WKY rats expressed ACE1 less than GK-rats. WKY rats expressed
ACE2 most. WKY and SHR rats had increased expression of COX-2
when compared to the GK-strain. High-salt diet affected RAAS markers
on mRNA level. It can be concluded that bioactive peptide-containing
milk products have mild positive effects on the markers of RAAS and
they may antagonize the effects of high-salt diet.
Paper No.: 1970

PSEUDO-IRREVERSIBLE ALLOSTERIC MODULATORS
MATHEMATICAL MODELS PREDICTING THEIR
BEHAVIOUR AND A PUTATIVE PRACTICAL EXAMPLE
David Hall
Ramin Hajikhani(1), J Solati(1), A Ahmadi(2), A-A Salari(1)

GlaxoSmithKline, Department of Respiratory Biology, Stevenage, UK
(1) Islamic Azad University-Karaj Branch, Department of Biology, Karaj,
Iran
(2) Islamic Azad University-Karaj Branch, Department of Chemistry,
Karaj, Iran
Anxiety is a common disorder that attacks many people in any society
and is often accompanied by physiological sensations such as tachycardia,
chest pain, shortness of breath, insensitivity, etc. The purpose of this study
was to evaluate the putative anxiolytic-like effects of Phencyclidine (1-(1phenylcyclohexyl) piperidine, CAS 956-90-1, PCP, I) and its methyl and
methoxy hydroxyl derivatives (II, III) using elevated plus maze test of
anxiety. Phencyclidine and its methyl and methoxy hydroxyl derivatives
(I, II, III) (hydrochloride, 1, 2, 5 mg/kg) were synthesized and administrated intraperitoneally (IP) on adult male wistar rats. The results demonstrated that, intraperitoneal (IP) administration of PCP analogues (I, II,
III) hydrochloride (1,2,5 mg/kg) increase the percentage of open arm time
(OAT%) and percentage of open arm entries (OAE%). This study
revealed that both derivatives of phencyclidine (II, III) were more effective than PCP (I) itself in modulation of anxiety behavior in rats.
It is well known that slow dissociation of competitive antagonists can

result in insurmountable antagonism in assay systems with relatively
short agonist contact times, a situation termed hemi-equilibrium (Paton
& Rang, 1966, Advances in Drug Research, Harper & Simmonds eds,
Academic Press, NY). Allosteric antagonists have been described at a
number of receptors, but as yet no one has considered how allosteric
modulators might behave under conditions of hemi-equilibrium. In this
study mathematical models based on the Operational Model were derived
to investigate the behaviour of irreversible allosteric antagonists. These
models should provide a good approximation to the behaviour of
pseudo-irreversible allosteric antagonists. The behaviours of these models
are quite distinct from those of (pseudo)-irreversible competitive antagonism. A pseudo-irreversible afnity-only modulator splits the receptors
into two populations with different afnities for the agonist. Hence, they
may atten agonist curves or may render them biphasic but do not affect
the maximal response. Efcacy-only modulators split the receptors into
populations with different transducer ratios for the agonist. Hence, they
tend to depress the maximal response although this effect is dependent
317
on the agonists efcacy. In human T cells, an allosteric antagonist of
CCR4 caused concentration-response curves to CCL22 in an actin polymerisation assay to become biphasic without affecting the maximal
response. Since the agonist contact time in this assay is only 15 sec, this
may imply that this molecule is an afnity-only modulator of CCR4.
Paper No.: 2939

DISEASES
INHIBITION OF INOS, COX-2 AND MPGES-1 EXPRESSION,
AND NO AND PGE2 PRODUCTION BY FLAVONOIDS IN
ACTIVATED MACROPHAGES
Mari Hamalainen(1), R Nieminen(1), MZ Asmawi(2), P Vuorela(3),
H Vapaatalo(4), E Moilanen(1)
(1) University of Tampere Medical School, Tampere University Hospital,
The Immunopharmacology Research Group, Tampere, Finland
(2) Universiti Sains Malaysia, School of Pharmaceutical Sciences,
Penang, Malaysia
bo Akademi University, Faculty of Mathematics and Natural
(3) A
Sciences, Department of Biochemistry and Pharmacy, Turku, Finland
(4) University of Helsinki, Institute of Biomedicine & Pharmacology,
Helsinki, Finland
Nitrix oxide (NO) and prostaglandin E2 (PGE2) have a central role in
inammation. Bacterial products and proinammatory cytokines upregulate the expression of inducible enzymes nitric oxide synthase (iNOS),
cyclooxygenase-2 (COX-2) and prostaglandin E synthase-1 (mPGES-1)
leading to increased production of NO and PGE2. In the present study,
we investigated the effects of 26 avonoids on iNOS expression and NO
production, and on PGE2 production and COX-2 and mPGES-1 expression in activated J774 macrophages. In the further studies, the mechanisms of action of the effective compounds were investigated. Eight of
the tested compounds: avone, daidzein, genistein, quercetin, isorhamnetin, kaempferol, naringenin and pelargonidin, inhibited NO and PGE2
production by more than 50% when compared to LPS (lipopolysaccharide)-treated control. All eight effective avonoids inhibited also iNOS
expression. Four avonoids (avone, isorhamnetin, daidzein and genistein) inhibited LPS-induced COX-2 mRNA expression, and mPGES-1
expression was down-regulated by kaempferol and isorhamnetin. The
activation of nuclear factor-jB (NF-jB), which is a signicant transcription factor for inammatory genes was inhibited by avone, daidzein,
genistein, quercetin, isorhamnetin, kaempferol, naringenin and pelargonidin. In addition, genistein, kaempferol, quercetin and daidzein inhibited
the activation of the signal transducer and activator of transcription-1
(STAT-1), another important transcription factor in inammation. The
present study characterises the effects and mechanisms of avonoids on
iNOS, COX-2 and mPGES-1 expression and on NO and PGE2 production in activated macrophages. The results partially explain the pharmacological efcacy of avonoids as anti-inammatory compounds, and
introduce kaempferol and isorhamnetin as compounds capable of downregulating the expression of mPGES-1 mRNA.
Paper No.: 1465

GINSENOSIDE RG-1 AND SALVIANOLIC ACID B MEDIATED
AUTOPHAGY AND APOPTOSIS IN NUTRIENT STARVATION
CARDIAC MYOCYTES
Xiao Han, J-X Liu, X-Z Li
Chinese Academy of Medial Sciences, Fundamental Medical Science
Center, Xiyuan Hospital, Beijing, PR China
Growing evidence reveals that autophagy contributes to cellular recovery
in an adverse environment to perform prevention role. Here we observed
autophagy and apoptosis in nutrient depletion cultured cardiac myocytes

and the effects of Ginsenoside Rg-1 and Salvianolic acid B on them.
Methods and Results: Cultured cardiac myocytes were incubated in glucose-free medium (GD) or GD in present of Ginsenoside Rg-1 or Salvianolic acid B for 3 hours with normal medium cultured myocytes as
control. In myocytes upon induction of GD, we demonstrated the
increased expression and punctated immunostaining of LC3 by using
Western blotting and uorescent staining. Autophagic vacuoles and
apoptotic body were detected by electron microscope. Dual labeling of
LC3 and Caspase-8 were further performed to observed the relationship
between autophagy and apoptosis in GD myocytes. Caspase-8 immunostaining was marked in cells which LC3 were observed as many distinct
dots in cytosolic. Ginsenoside Rg-1 and Salvianolic acid B inhibited the
expression of LC3 and Caspase-8. Myoctyes treated with Ginsenoside
Rg-1 and Salvianolic acid B were not detected the evidence of autophagy
and apoptosis. Conclusions: In our study, autophagy and apoptosis presented positive correlation. Autophagy, triggered by GD for 3 h, could
not be a survival mechanism and may lead to cell death, by which apoptosis was increased. The excessively stimulated autophagy and apoptosis
can be down regulated by Ginsenoside Rg-1 and Salvianolic acid B to
stabilize cardiac homeostasis.
Paper No.: 2189

HEALTH AND DISEASE
SEXUAL DIMORPHISM IN AORTIC ENDOTHELIAL
FUNCTION OF STREPTOZOTOCIN-INDUCED DIABETIC RATS
X Han(1), R Zhang(1), L Anderson(2), Roshanak Rahimian(1)
(1) University of the Pacic, Thomas J. Long School of Pharmacy,
Department of Physiology and Pharmacology, Stockton, CA, USA
(2) University of Pacic, Arthur A. Dugoni School of Dentistry,
Department of Anatomical Sciences, San Francisco, CA, USA
To date little is known of the interaction between diabetes and gender in
the vasculature. Thus, the objective of this study was to investigate
whether there is a gender difference in aortic endothelial cell function in
streptozotocin (STZ, 65 mg/kg, iv)-induced diabetic rats. The potential
roles of superoxide and cyclooxygenase (COX) metabolites in diabetesinduced vascular dysfunction were also studied. Endothelium-dependent
vasodilation (EDV) in response to acetylcholine (ACh; 10-8 to 10-5M)
was measured in aortic rings precontracted with phenylephrine (PE; 2`M)
before and after pretreatment with MnTMPyP (10mM), a superoxide
scavenger, or indomethacin (indo; 10`M), a COX inhibitor. Constrictor
response curves to PE (10-8 to 10-5M) were also generated before and
after pretreatment with indo. In addition, the level of endothelial nitric
oxide synthase (eNOS) mRNA expression was determined using real
time RT-PCR. STZ-induced diabetes impaired EDV, but its effect was
more pronounced in females. Similarly, eNOS mRNA expression was
lower in diabetic animals and the decrease was greater in females than in
males. Preincubation with MnTMPyP increased the sensitivity of aortic
rings to ACh-induced relaxation only in diabetic rats, and again the effect
was greater in females. In controls, indo signicantly reduced contraction
to PE in females, but in diabetic rats, males demonstrated the greater
reduction in PE response. These results suggest the predisposition of
female rat aorta to vascular injury in diabetes, possibly via altered NO
and superoxide production. Furthermore, COX metabolites also play a
role in the vascular reactivity in the diabetic aorta.
(Supported by NIH/NIDCR).
Paper No.: 966

HAISHENGSU EXTRACTED FROM TEGILLARCA GRANOSA
INHIBITS CELL GROWTH OF HUMAN HEPATIC CANCER IN
NUDE MICE
Yantao Han(1), X Chen(1), Z Han(2), P Li(2), J Tong(3), C Wang(1)
318
(1) Qingdao University Medical College, Functional Experiment Center,
Qingdao, PRChina
(2) Qingdao University, The Afliated Hospital of Medical College,
Qingdao, PR China
(3) The Afliated Hospital of Qingdao University, Qingdao, PR China
Haishengsu (HSS) is a seashell protein puried from Tegillarca granosa.
In the present study we investigated the anti-tumor effects of HSS on
human hepatic cancer BEL7402 cells in vivo and in vitro. HSS effectively inhibited the growth of BEL7402 cells as estimated by the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in a
dose-dependent manner. The maximum inhibition rate was 37.8% for
100 lg/ml of HSS for 72h. The inhibitory effects of HSS on carcinoma
cell growth were conrmed in mice bearing BEL7402 xenografts. HSS
delayed the growth of BEL7402 xenografts after three weeks of oral
administration. The negative and positive control group received PBS
and FT207. The inhibition rates of HSS of 62.5, 125, and 250 mg/kg
were 50%, 58%, and 60%, respectively. And the relative growth rates of
tumor (T/C %) were 32%-45% in tumor bearing nude mice. Administration of HSS induced an obvious apoptosis in tumor xenografts analyzed
after isolation from the mice by HE staining and transmission electron
microscopy, and no any apparent toxic symptoms in nude mice was
observed after oral intake of HSS. Our results showed that HSS could
suppress cell growth of human hepatic cancer in nude mice in dosedependent manner without apparent adverse effects and HSS may have a
therapeutic value in antitumor drug development.
Paper No.: 999

THERAPEUTIC TARGETS
INVOLVEMENT OF EGFR/AKT / CYCLIN D1 / CDK4
SIGNALLING PATHWAY IN INHIBITING UVA-INDUCED
APOPTOSIS OF HACAT CELLS BY POLYPEPTIDE FROM
CHLAMYS FARRERI
Zhiwu Han(1), L Zhu(1), J Tong(1), P Li(1), L Yan(2), C Wang(2)
Department of Pharmacy, Qingdao, PR China
(2) Qingdao University Medical College, Department of Pharmacology,
Qingdao, PR China
UVA is a potent inducer of reactive oxygen species (ROS), which can
induce cell apoptosis. Polypeptide from Chlamys farreri (PCF) is a natural marine antioxidant and our previous studies have reported that PCF
could effectively protect human keratinocyte cell line HaCaT cells from
apoptosis induced by UVA in vitro. The aim of present study is to investigate whether PCF protects HaCaT cells from apoptosis induced by 8
J.cm-2 UVA by EGFR/AKT/Cyclin D1/CDK4 pathways. We measured
the cell apoptosis by agarose gel electrophoresis. The expression of
EGFR and AKT were detected by RT-PCR. Phosphorylated AKT, activated CyclinD1, and CDK-4 were investigated by western blotting. The
results showed that AG1478 and PHZ1023, corresponding the inhibitor
of EGFR and AKT could signicantly prevente UVA-induced apoptosis
of HaCaT cells. PCF not only strongly reduced the AKT production, but
also diminished expression of phosphorylated AKT, CyclinD1 and
CDK4 in HaCaT cells radiated by UVA in a dose-dependent manner.
These results indicated that EGFR/AKT/Cyclin D1/CDK4 pathways participated the HaCaT cell apoptosis induced by UVA and the protective
effects of PCF may attribute to decreasing intracellular EGFR level and
blocking EGFR/CDK-4 apoptotic signalling pathway.
Supported by the Shandong Natural Science Foundation of China, No.
Z2007c09).
Keywords: Polypeptide from Chlamys farreri; EGFR; UVA; apoptosis;
HaCaT cells
Paper No.: 1975

DISEASES
RHO-KINASE INHIBITORS AUGMENT ANESTHETICSINDUCED RELAXATION IN RAT AIRWAY SMOOTH MUSCLE
Motohiko Hanazaki(1), A Kohjitani(2), S Mizobuchi(1), K Morita(1),
H Sakai(3), Y Chiba(3), M Misawa(3)
(1) Okayama University, Graduate School of Medicine, Dentistry &
Pharmaceutical Sciences, Department of Anesthesiology &
Resuscitology, Okayama, Japan
(2) Kagoshima University Graduate School of Medical and Dental
Sciences, Department of Dental Anesthesiology, Kagoshima, Japan
(3) Hoshi University School of Pharmacy, Department of Pharmacology,
Tokyo, Japan
Introduction: Airway smooth muscle (ASM) contraction is caused by
increasing both intracellular Ca2 + ([Ca2 + ]i) and the force at the same
([Ca2 + ]i) (increase in Ca2 + sensitivity). Small G-protein RhoA and
Rho-kinase (ROCK) play important roles in regulating Ca2 + sensitivity.
In this study, we investigated the effects of ROCK inhibitors on anesthetic-induced relaxation in rat ASM. Methods: Ring strips from intrapulmonary bronchus of male Wistar rats were placed in 400-micro-L organ
baths containing Krebs-Henseleit solution. After obtaining stable contraction with 30 microM acetylcholine, isometric forces were measured with
the following protocols : (1) Y-27632 (0.01-300 microM), fasudil (0.01100 microM), or H -1152 (0.01 - 100 microM) were cumulatively
applied. (2) propofol (1 microM-1 mM), with or without Y-27632, fasudil or H-1152 (0.03, 0.1 microM), was cumulatively applied. (3) isourane (0.5-4.0%), with or without Y-27632 (1 microM), was cumulatively
applied. Results: (1) All ROCK inhibitors, especially H-1152, produced
concentration-dependent relaxation. (2) 0.03 microM Y-27632 and fasudil did not affect the relaxation by propofol, while 0.1 microM both
agents signicantly shifted concentration-response curves to the left.
H-1152 (0.03 and 0.1 microM) signicantly shifted the concentrationresponse curve to the left. Combination of ROCK inhibitor and propofol
showed the great relaxation, which exceed the simple sum of relaxation
by both ROCK inhibitor and propofol. (3) Y-27632 signicantly shifted
the concentration-response curve for isourane to the left. Conclusions:
1) ROCK inhibitors augment anesthetics-induced relaxation of rat airway
smooth muscle. 2) Combined use of ROCK inhibit or and anesthetics
causes further relaxation.
Paper No.: 2964

N-ACETYLCYSTEINE AS A NOVEL PROPHYLACTIC TREATMENT FOR IFOSFAMIDE-INDUCED NEPHROTOXICITY IN
CHILDREN; TRANSLATIONAL PHARMACOKINETICS
Lauren Hanly(1,2,10), N Chen(1,2,10), B Baw(3), B Malkin(4),
M Culter(1,2), D Freeman(1,2), M Rieder(1,2,4,8,9),
G Koren(1,2,4,5,6,7,8,10)
(2) University of Western Ontario, Division of Clinical of Pharmacology,
London, Ontario, Canada
(3) McMaster University, Hamilton, Ontario, Canada
(4) London Health Science Centre, Department of Pediatrics, London,
Ontario, Canada
(5) Hospital for Sick Kids, Division of Clinical Pharmacology and
Toxicology, Toronto, Ontario, Canada
(6) University of Toronto, Department of Pharmacology, Toronto,
Ontario, Canada
(7) University of Toronto, Faculty of Medicine, Toronto, Ontario,
Canada
319
(8) University of Western Ontario, Facutly of Medicine, London,
Ontario, Canada
(9) Childrens Hospital of Western Ontario, CIHR-GSK Chair in Pediatric Clinical Pharmacology, London, Ontario, Canada
(10) University of Western Ontario, Ivey Chair in Molecular Toxicology,
London, Ontario, Canada
Ifosfamide (IFO) is a highly effective chemotherapeutic agent, treating
a wide variety of solid tumours . Its use is associated with 30% risk for
nephrotoxicity in children. Nephrotoxicity is believed to be due to oxidative stress; therefore use of an antioxidant for its attenuation is feasible. NAC is a synthetic thiol which is used clinically in children as an
antidote for acetaminophen overdose and has been demonstrated to be
effective in preventing IFO nephrotoxicity in a rodent model. Our
objective is to compare the systemic exposure of NAC in children treated for acetaminophen overdose, to the systemic exposure associated
with prevention of nephrotoxicity in rats. Blood samples were collected
from male Wistar Albino rats treated with the dose schedule of NAC
shown to prevent IFO-induced nephrotoxicity. In parallel, blood samples
were collected from children (n = 10) who had received NAC for acetaminophen overdose. For both the rat and pediatric patients NAC was
measured by High Performance Liquid Chromatography and systemic
exposure determined by calculating the area under the curve. The mean
AUC of NAC in rats who were given therapeutically effective levels of
NAC was 18.72 mMHr, similar to the AUC in children treated with
NAC for acetaminophen overdose (14.56 mMHr). The range of AUC
distribution was similar between the rats and pediatric patients. These
ndings support the use of NAC to prevent IFO-induced nephrotoxicity
in a clinical setting. This study is signicant in the advancement toward
effective prevention of life threatening IFO-induced nephrotoxicity in
children.
Paper No.: 1249

ROLE OF KERATINOCYTES GPR109A AND COX-2 IN
NICOTINIC ACID AND MONO-METHYL FUMARATE
INDUCED FLUSHING
Julien Hanson(1,2,3), A Gille(2), S Zwykiel(2), M Lukasova(2,3),
BE Clausen(4), K Ahmed(1,2), S Tunaru(1,2), A Wirth(1,2),
S Offermanns(1,2,5)
(1) Max-Planck-Institute for Heart and Lung Research, Department of
Pharmacology,Bad Nauheim, Germany
(2) University of Heidelberg, Institute of Pharmacology, Heidelberg,
Germany
(3) University of Lie`ge, Drug Research Center, Department of Medicinal
Chemistry, Lie`ge, Belgium
(4) Erasmus MC, University Medical Center Faculty, Department of
Immunology, Rotterdam, The Netherlands
(5) Goethe University Frankfurt, Medical Faculty, Frankfurt, Germany
The anti-dyslipidemic drug nicotinic acid and the anti-psoriatic drug
mono-Methyl fumarate induce cutaneous ushing through the activation
of the G-protein-coupled receptor GPR109A. Flushing is a troublesome
side effect of nicotinic acid, but may be a direct reection of the wanted
effects of mono-Methyl fumarate. Here we analysed the mechanisms
underlying GPR109A-mediated ushing and show that both Langerhans
cells and keratinocytes express GPR109A. Using cell ablation
approaches and transgenic cell type-specic expression of GPR109A in
Gpr109a-/- mice, we provide evidence that the early phase of ushing
depends on GPR109A expressed on Langerhans cells, whereas the late
phase is mediated by GPR109A expressed on keratinocytes. Interestingly, the rst phase of ushing is blocked by a selective cyclooxygenase-1 (COX-1) inhibitor, and the late phase is sensitive to a selective
COX-2 inhibitor. Both, mono-Methyl fumarate and nicotinic acid, induce
PGE2 formation in isolated keratinocytes through activation of GPR109A
and COX-2. Thus, early and late phases of the GPR109A-mediated cutaneous ushing reaction involve different epidermal cell types and prostanoid
forming enzymes. These data will help to guide new efcient approaches to
mitigate nicotinic acid-induced ushing and may help to exploit the potential anti-psoriatic effects of GPR109A agonists in the skin.
Paper No.: 1623

DISEASES
ELEVATION OF THE VENODILATATORY RESPONSIVENESS
TO NITRIC OXIDE IN NASAL MUCOSAE OF RATS WITH
ALLERGIC RHINITIS
Tatsuya Hara, J Enzaka, M Sakai-Oshita, H Sakai, Y Chiba, M Misawa
Tokyo, Japan
In patients with severe allergic rhinitis, the rhinostenosis is a criticalproblem for their quality of life. The principal cause of the rhinostenosis
isvenodilatation in nasal mucosa. Several chemical mediators includingleukotrienes (LTs), which induce rhinostenosis, have been reported.
Thevenodilatation by LTs is considered to be ascribed to nitric oxide
(NO)production. NO/PKG pathway is known as a mechanism that causes
potentvasodilatation. In addition to given specic antigen-induced nasal
symptoms,nasal hyperresponsiveness (NHR) to nonspecic stimuli is one
of thecharacteristics features of patients with allergic rhinitis. However,
the exactmechanism of NHR is still unclear. In the present study, the
mechanism ofaugmented venodilatation in nasal mucosa was investigated
in antigen-challengedrats. Leukotriene D4 (LTD4) induced a weak venodilatation in nasal mucosae ofthe control rats. However, the LTD4induced venodilatation was signicantlyaugmented in the antigen-challenged rats. The augmented LTD4-inducedvenodilatation was signicantly inhibited by pretreatment with L-NMMA, aninhibitor of NO
synthase (NOS). The LTD4-induced production of NOx in the nasalcavity was increased in the antigen challenged rats. The sodium nitroprusside
(aNO donor)-induced venodilatation in nasal mucosae of the antigen
challengedrats was also signicantly increased as compared to that of the
control group.In conclusion, not only an increased NO production but
also an enhanced NOresponsiveness might be involved in the development of NHR in allergic rhinitis.The NO/PKG pathway is estimated to
be involved in the augmented venodilatationin nasal mucosae of the
NHR rats.
Paper No.: 2109

METHODS FOR IMPROVING COMPLIANCE WITH MEDICINE INTAKE(MICMI): PILOT PROJECT TO EVALUATE THE
PERSONAL ELECTRONIC MEDICINE PROFILE[PEM]
Philipp Harbig(1), I Barat(1), P Lund Nielsen(2), EM Damsgaard(1)
rhus University Hospital, Department of Geriatrics, THG 2, Aarhus,
(1) A
Denmark
(2) Vejlby Apotek, Risskov, Denmark
Objective: Non-compliance is a major problem in medical treatment. The
aim of this project is to evaluate the national prescription database PEM
as an instrument to measure compliance. PEM is an internet based portal
which gives the general practitioners the opportunity to evaluate compliance of their patients. PEM calculates the length of time a single prescription lasts, based on the amount of tablets and the dose prescribed.
Delay in redemption of a prescription appears graphically in PEM as a
gap on the time axis. Methods: 16 patients consuming 66 drugs were
consecutively selected. Compliance was evaluated three times by pill
320
counts (golden standard) over a period of 12 months. Non-compliance
was said to be present when the doses consumed during the period were
less than 80%. Non-compliance according to PEM was dened to be
present when the sum of all gaps made up more than 20% of the total
length of the time axis for a year. Results: Non-compliance according to
pill counts was 13 drugs out of 66 (20%) and according to PEM 29 out
of 66 (44%). Conclusion: This preliminary study may indicate that PEM
is unreliable as an instrument to measure non-compliance. However, this
is a small number of persons and drugs. We plan to publish a larger
study including 945 patients.
Paper No.: 2447

ON PHARMACO-TOXICOLOGICAL APPROACHES TO
VESICAL DYSFUNCTION
Ulrich Harlin(1), E Neu(1,2,3), A Hofstetter(4), G Staehler(5),
S Magour(6), G Ernst(4), W Seidenbusch(3,1), MC Michailov(1,2,3),
D Huting(1), U Welscher(1,2,3), J Foltinova(7)
(1) Institute of Ecological Medicine c/o International Council for
Scientic Development (ICSD e.V.)/International Academy of Science,
Muenchen, Germany
(2) University of Erlangen-Nuernberg, Institute of Botany, Erlangen,
Germany
(3) University of Innsbruck, Institute of Experimental Physics, Innsbruck,
Austria
(4) University of Muenchen, Medical Faculty, Muenchen, Germany
(5) University of Heidelberg, Medical Faculty, Heidelberg, Germany
(6) Free University Berlin, Medical Faculty, Berlin, Germany
(7) Comenius University, Institute of Histology, Bratislava, Slovakia
Introduction: Therapeutic/pathophysiological motor reactions to drugs/
toxicants (b-sympathomimetics, herbal drugs, pyrethroids, Hg, etc.)
counteracting/leading to functional vesical disturbances (incontinence,
bladder hyperactivity), also supporting disposition to cystitis, pyelonephritis up to renal hypertension are discussed (Neu, Stiglmayr et al,
Chinese J.Pathophysiol. 2006;22/13:228; Neu, Harlin, Hofstetter et al,
Urology 2006;68/5A:78; Toxicol.&Appl.Pharm. 2004;197/3:297;
Fund.&Clin. Pharm. 1999;13/1:246s+364s; Eur.J.Pharmac.Sci. 1998;6/
1:219). Method. Spontaneous phasic (SPC: 1-5/min) contractions & to
electrical neurogenic stimulation (CES: 10/100Hz, 0.3ms, 3s), isolated
guinea-pig detrusor. Inuence of trichlorophenol (TCP: 10mM in acetone) & pentachlorophenol (PCP: 10mM in ethanol). Results: After
chlorophenols (1-100lM) are SPC&CES semi-reversibly decreased up
to 90%, e.g. for PCP 50lM 60-70%, for TCP 20-30%. Inhibitory
effects of acetone and ethanol (0.01-1%) on PCP&CES are about 520% resp. (for 100lM chlorophenols) (n = 128). Differences of effects
between chlorophenols and pyrethroids, also in rat-detrusor, are
observed (n = 75; p < 0.05 < 0.01). Discussion. These ndings are in
accordance with suggestions about participation of mechanosensitive
ion channels (Ca++-activated K+) in chlorophenol effects related to
transformation of spike- into burst-plateau electrical activity by ions
(after stretch, Ca++, Ba++), drugs (TEA, etc.), toxicants. Differences to
effects of pyrethroids, also in rat-detrusor are observed. Conclusion:
Detrusor motor reactions to chlorophenols (similar to pyrethroids, bsympathomimetics, herbal drugs, etc.) support proposal about application of SPC&CES for indicator of possible vesical dysfunctions by toxicants, also of pharmaco-therapeutic effects.
Dedicated to moral support (1989-2010; Hon. ICSD-members, *Nobel
Laureate: Helmuth Muller-Mohnssen medicine), Erwin Neher*, Walter
Stuhmer (biophysics), Dieter G. Weiss (biology)/Germany, Rita LevyMontalcini* (biol./pharmacol.: 100 years)/Italy, Bengt Samuelson* (medicine)/Sweden, Lord Alexander Todd* (chemistry)/UK
Paper No.: 2180

EXPRESSION OF A NOVEL TRUNCATED VARIANT OF NOX2
IN MACROPHAGES
Craig Harrison, GR Drummond, CG Sobey, S Selemidis
Monash University, Department of Pharmacology, Clayton, VIC, Australia
Evidence implicates the superoxide generating macrophage Nox2-containing NADPH oxidase enzyme complex in a variety of cardiovascular
diseases including atherosclerosis, and other diseases involving the innate
immune system, such as inuenza A virus- and bacteria-induced lung
disease. In the present study we aimed to identify novel variants of Nox2
using a variety of mouse tissues that we have previously shown to
express signicant amounts of the native 58 kDa form of Nox2, such as
heart, spleen, brain, lung, aorta, kidney and macrophagecells. Using tissues from Nox2-/- mice as controls we identied a novel, truncated, ~35
kDa variant of Nox2 expressed in high levels in mouse lung, primary
alveoli and peritoneal macrophages and in a mouse macrophage cell line
RAW264.7. This protein was not detectable in mouse circulating macrophage precursor cells, the Ly6Chi and Ly6Clo monocytes despite these
cells expressing high levels of the 58 kDa form of Nox2. Furthermore, in
aorta from hypercholesterolaemic apolipoprotein E-decient (ApoE-/-)
mice, we observedexpression of the ~35 kDa protein that correlated with
atherosclerosis severity, and it was absent in aorta from ApoE-/-/Nox2-/mice. RT-PCR experiments using specic Nox2 primers have identied a
truncated transcript calculated to yield a protein approximately 35 kDa in
size, which is currently awaiting sequencing. In conclusion, our studies
may have identied a novel ~35 kDa Nox2 splice variant which is
expressed predominantly by macrophages. We hypothesize that this protein is a novel regulator of superoxide production under physiological
and pathological settings.
Paper No.: 2432

HEALTH AND DISEASE
IS HYDROGEN SULFIDE AN ENDOTHELIUM-DERIVED
RELAXING FACTOR?
Joanne Hart(1), M Al-magableh(2)
(1) RMIT University, School of Medical Sciences, Bundoora West,
Australia
(2) Monash University, Victoria, Australia
Hydrogen sulde (H2S) is potentially an important endogenous vascular
signalling molecule. It is reported to be produced by the enzyme cystathionine-c-lyase (CSE) in vascular tissues and mice decient in CSE are
known to be hypertensive (Yang, Science, 322:587-590, 2008). This
study examines whether or not H2S is an endothelium-derived relaxing
factor (EDRF) in mouse aorta. Western blotting was used to determine
the expression of the 3 known H2S-producing enzymes, CSE, cystathionine-b-synthase (CBS) and 3-mercaptopyruvate sulphurtransferase (3MST) in mouse aorta. CSE and 3-MST, but not CBS were expressed in
mouse aorta, with and without endothelium. Mouse aorta was found to
produce H2S via CSE at a rate of 7 3lmol/g/min (n = 8). Immunohistochemistry showed that CSE was present in endothelial and vascular
smooth muscle cells, with more intense staining for CSE in the endothelial layer of the mouse aorta. Acetylcholine-mediated vasorelaxation in
mouse aorta was signicantly reduced by treatment with the CSE inhibitor PPG (20mM) (Maximum vasorelaxation (%): Control 68 3, PPGtreated 34 3, n = 5, P < 0.01, ANOVA) as was vasorelaxation mediated via the endothelium-dependent, but receptor independent calcium
ionophore A23187 (Maximum vasorelaxation (%): Control 41 2, PPGtreated 17 5, n = 5, P < 0.01, ANOVA). Thus, H2S-producing
321
enzymes are present and functional in mouse aorta. These data suggest a
role for H2S as an EDRF, via muscarinic receptor elicited and Ca2 + dependent activation of CSE in the endothelial cells. Further studies
should determine any role of 3-MST in endothelial generation of H2S
and the physiological relevance of these ndings.
Paper No.: 1494

DISEASES
RELATION OF HSPS EXPRESSION AND FORMATION OF
ATHEROSCLEROTIC LESIONS IN THE AORTA OF
APOE-DEFICIENT MICE
Naoya Hashikawa, N Hobara, C Yutani
Okayama University of Science, Department of Life Science, Okayama,
Japan
Atherosclerosis is a multifactorial disease characterized by inammation
and endothelial injury arising from infection, hemodynamic forces, oxidized LDL, dietary factors, toxins, and chemical insults, all of which can
activate the stress response and induce Heat Shock Proteins (HSPs).
HSPs are present in most cells, serving as molecular chaperones, and
they play a role in cell protection from damage in response to stress. In
this study, we investigated the effect of geranyl-geranyl-acetone (GGA),
which is HSPs inducer, on atherosclerotic lesion formation. We found
GGA increased HSPs expression in C57BL/6J aorta, especially Hsp20,
25, 70, and 90. Furthermore, HSPs expression in atherosclerosis was
explored with the use of apolipoprotein E decient (ApoE-/-) mice.
ApoE-/- mice were fed a high-cholesterol diet for 10 weeks after weaning. Mice on each diet were treated with GGA for 5 weeks in pre-atherogenic (5- to 10-week-old; Early-phase) or post-atherogenic (10- to 15week-old; Late-phase) process. After treatment, mRNA level of HSPs
was analyzed using RT-PCR. Atherosclerotic lesions in the aorta were
evaluated by oil red O staining. In Early-phase group, treatment with
GGA mildly prevented the formation of atherosclerotic lesions in ApoE-/mice aorta. By contrast, in Late-phase group, the atherosclerotic changes
were further exaggerated by GGA, with a signicant increase in the level
of HSPs mRNA transcription. These results suggested that the expression
of HSPs prevent atherosclerosis in pre-atherogenic but promote atherosclerosis in post-atherogenic, thus HSPs do a different working to the
level of atherosclerosis progress.
Paper No.: 1132

HEALTH AND DISEASE
CHANGE IN INTRACELLULAR CA2 + RESPONSE OF MIDDLE
CEREBRAL ARTERY OF STROKE-PRONE SPONTANEOUSLY
HYPERTENSIVE RATS
Terumasa Hashimoto, H Ohata, T Miyazaki, K Shibata, K Nobe,
K Honda
Showa University, Department Pharmacology, Tokyo, Japan
Calcium acts as a universal signal for various cellular functions including
vasoconstriction. In the present study, the change in intracellular calcium
([Ca2 + ]i) dynamics in middle cerebral artery (MCA) was assessed in relation to development of stroke in stroke-prone spontaneously hypertensive
rats (SHR-SP). Isolated MCA was loaded with 10 lM Fluo-4/AM and 10
lM Fura Red/AM. [Ca2 + ]i response was determined utilizing real-time
confocal laser microscope. Expression of connexin mRNA was determined by RT-PCR. At 3 months of age (pre-stroke), global [Ca2 + ]i oscillation and vasomotion were induced by application of 5hydroxytryptamine (5-HT). On the other hand, MCA lost the [Ca2 + ]i and
contractile responses at 5 months of age (post-stroke). Rhythmic changes
in [Ca2 + ]i preceded vasomotion; moreover, individual smooth muscle
cell rhythmic changes in [Ca2 + ]i was synchronized. Rhythmic change in
[Ca2 + ]i was abolished following treatment with nicardipine, a L-type Ca2

+
channel antagonist; furthermore, 100 lM carbenoxolone (CBX), a selective gap junction blocker, lowed [Ca2 + ]i rhythmic changes. Additionally,
connexin 45 (Cx 45) mRNA expression increased signicantly in MCA
isolated from SHR-SP at 3 months of age. These observations suggest that
synchronization of the rhythmic change in [Ca2 + ]i of individual smooth
muscle cells is necessary for global [Ca2 + ]i oscillation. Global [Ca2 + ]i
oscillation induced vasomotion. And that, synchronization of rhythmic
change in [Ca2 + ]i may be coordinated via gap junctions.
Paper No.: 3431

EFFECT OF COMBINED ORAL CONTRACEPTIVES ON THE
SERUM CONCENTRATION OF OLANZAPINE
Tore Haslemo(1), H Refsum(1), E Molden(1,2)
(1) Diakonhjemmet Hospital, Centre for Psychopharmacology, Oslo,
Norway
(2) University of Oslo, Department of Pharmaceutical Biosciences, Oslo,
Norway
Olanzapine is metabolised by CYP1A2, combined oral contraceptives
(COC) have been shown to inhibit the activity of this enzyme. The aim
of this study was to evaluate the effect of COC on the serum concentration of olanzapine in women aged 18-40 y. Serum concentrations of olanzapine measured at the routine therapeutic drug monitoring (TDM)
service at Diakonhjemmet Hospital were included in the study. For
women aged 18-40 y, treating physicians were invited to provide and
conrm information about smoking habits and whether the patients used
contraceptives (by brand/generic name) or not. Mean dose-adjusted
serum concentrations (CD ratio) of olanzapine in the COC group were
compared with females using progestogen and those not using contraceptives at all by Mann-Whitney tests. A total of 149 valid reports were
returned. Out of 20 patients using prescription contraceptives, 10 used
COC (the other 10 patients used progestogen without oestrogen). 8 out
of these 20 patients (40%) smoked cigarettes. In the COC group, 3 out
of 10 patients smoked cigarettes. Among the patients who did not use
prescribed contraceptives, 64 out of 129 patients (50%) smoked cigarettes. There were no signicant differences in mean CD ratios of olanzapine between the subgroups, i.e. 8.75 nM/mg (COC group), 9.89 nM/mg
(progesterone group) and 8.64 nM/mg (non-contraceptive group); P >
0.2. In conclusion, our ndings suggest that use of COC does not affect
the serum concentration of olanzapine.
Paper No.: 3313

DISEASES
COMPARISON OF POSITIVE REACTION OF TUBERCULIN
SKIN TEST AND QUANTIFERON AMONG HEALTHY ADULT
MALES IN JAPAN
Tomoko Hasunuma(1), M Kuwamata(1), M Takatsuki(1), N Kobayashi(1), A Aoki(1), Y Monuki(1), m Morikawa(1), T Fukushima(1), M Nishiwaki(1), H Iijima(1), Y Kumagai(1,2)
(1) Kitasato University, Research Center for Clinical Pharmacology,
Tokyo, Japan
(2) Kitasato University East Hospital, Clinical Trial Center, Tokyo, Japan
Purpose: It is essential to determine the risk of tuberculosis in the development of immunosuppressive biologic agents. Especially, in Japan, the
positive rate of the tuberculin skin test (TST) is rather high when compared to that in Europe and United States. Recently, a serological-based
test for tuberculosis infection, called heQuantiFERONhf (QFT) has
been available for clinical use. In this study, we examined both tests
322
among the healthy young males and examined the usefulness. Method:
123 males (mean age 32.5 years old) were examined. Present and past
medical history was taken as well as vital, blood, urine data and physical
condition, and both TST and QFT were done. Results: Strongly positive
reaction of TST was seen in 8 subjects out of 123 subjects. On the other
hand, QFT positive reaction was seen in 4 subjects, and doubtfully positive reaction was seen in 3 subjects. Positive rates of TST reaction were
as follows: in QFT positive (including doubtfully positive) subjects,
weakly positive was seen in 3 subjects (42.8%), moderately positive in 4
(57.1%), and negative was none; in QFT negative subjects (116 subjects), weakly positive in 44 (37.9%), moderately positive in 53 (45.7%),
strongly positive in 8 (7.9%), and negative in 11 (9.5%). Discussion:
From this study, there was no correlation between TST and QFT among
Japanese healthy male. QFT is a novel method to determine tuberculosis
infection using c-interferon production from lymphocytes reacting to the
tuberculosis specic proteins. From this study, checking tuberculosis
infection should be more careful in Japanese subjects.
Paper No. 1927

DO VASODILATING PROPERTIES OF AMIODARONE
CONTRIBUTE TO ITS ANTIARRTHYTHMIC ACTION IN
CLINICAL PRACTICE?
Apostolos Hatzitolios(1), N Katsiki(1), G Ntaios(1), C Savopoulos(1),
A Spiliopoulos(1), C Papadopoulos(1), V Mirtsou-Fidani(2)
(1) AHEPA University Hospital, 1st Propedeutic Department of Internal
Medicine, Thessaloniki, Greece
(2) Aristotle University of Thessaloniki, Department of Pharmacology,
Thessaloniki, Greece
Amiodarone, the major representative of class III antiarrhythmic agents,
is widely used in the treatment of ventricular and supraventricular arrhythmias. As a high percentage of cardiac arrhythmias are caused by
ischemic coronary heart disease, an agent that exerts both antianginal
and antiarrthythmic actions, such as amiodarone, presents an extremely
signicant advantage regarding the treatment of patients with chronic
atherosclerotic cardiopathy. In the present study, isolated rat aorta was
used to investigate the possible vasodilating effect of amiodarone and its
interaction with vasoconstrictor agents such as noradrenaline, potassium
chloride, serotonin and prostaglandin F2a in both calcium and calciumfree solutions. It was shown that the addition of amiodarone in both the
solutions promoted dose-dependent dilatation, mainly via the activation
of intracellular calcium binding mechanisms and that this effect was both
direct and indirect (antagonistic) against all vasoconstrictor agents. This
nding is in agreement with the results of other animal and human studies that have also conrmed the vasodilating properties of amiodarone.
In conclusion, amiodarone promotes coronary vasodilation by both direct
action on vascular smooth muscle and its sympatheticolytic properties
(partial a- and b- antagonist) and via increased synthesis of NO and cyclooxygenase-dependent endothelium factors along with inhibition of ablockers. In clinical practice, the vasodilating and cardioprotective
actions of amiodarone are especially important in patients with ischemic
cardiomyopathy complicated by sustained arrhythmias. Finally, as amiodarone and noradrenaline have been shown to exert antagonistic actions,
the application of amiodarone may be particularly indicated in conditions
characterized by sympathicotonia.
Paper No.: 3368

THE EFFECT OF CO-ADMINISTRATION OF CYP1A2 AND PGLYCOPROTEIN INDUCERS OR INHIBITORS ON SERUM
THEOPHYLLINE CONCENTRATION
Milka Hauta-aho(1,2), T Laurikainen(1), T Katiskalahti(3), T Tirkkonen(1,2), K Laine(1,2,4)
(1) University of Turku, Department of Pharmacology, Drug Development, and Therapeutics, Turku, Finland
(2) TYKSLAB, Healthcare District of Southwestern Finland, Department
of Clinical Pharmacology, Turku, Finland
(3) StatFinn Ltd., Turku, Finland
(4) Medbase Ltd., Turku, Finland
Theophylline is metabolized predominantly by CYP1A2 isoenzyme. Coadministration of drugs inducing or inhibiting CYP1A2 is known to alter
serum theophylline level. (Ohnishi A, Drugs&Aging 2003;20:71-84)
Instead, the role of P-glycoprotein (P-gp) in theophylline pharmacokinetics is unknown. We investigated the frequency of co-administration of
CYP1A2 and P-gp inducers or inhibitors with theophylline and the effect
of co-administration on serum theophylline level in in-patients of Turku
University Hospital. Medical records of 3,306 theophylline-treated
patients were utilized over the 8.5 years study period (July 1, 1996 to
December 31, 2004). Serum theophylline levels between the control (theophylline without interacting medication) and interaction groups were
analyzed by using ANCOVA model. Age, gender, ward, theophylline
dose and plasma levels of alkaline phosphatase and alanine aminotransferase were introduced as covariates in the model. Co-administration of
potentially interacting drugs was detected in 32% of all theophylline-treated patients. Most commonly used interacting drugs were P-gp inhibitors
verapamil, diltiazem, and quinine (8.5%, 6.6%, and 2.8% of all theophylline treatment periods, respectively) and CYP1A2 inducer carbamazepine
(2.1%). The mean serum theophylline levels were moderately, but signicantly higher (P = 0.04) during co-administration with P-gp inhibitors vs.
controls after oral, but not intravenous administration. No signicant differences were found with other interaction groups vs. control. Co-administration of theophylline and CYP1A2 and P-glycoprotein inducers or
inhibitors appears to have limited clinical signicance at population level.
However, as has been shown for CYP1A2, P-gp-mediated drug interactions may have clinically signicant effect on individual patients theophylline therapy, and thus, deserve further studies.
Paper No.: 1074

ENDOGENOUS HEPARIN LEVELS IN THE CONTROLLED
ASTHMATIC PATIENTS
Ivan Havlik(1), A Ahmed(1), M Mer(2), H Davids(1)
(1) University of the Witwatersrand, Department of Pharmacy & Pharmacology, Parktown, South Africa
(2) University of the Witwatersrand, Department of Pulmonology and
Critical Care, Parktown, South Africa
Heparin possesses anti-inammatory properties and heparin-like substances may show potential as a treatment option for asthmatic patients.
The authors thus hypothesized that asthmatic patients have decreased
levels of circulating endogenous heparin than the healthy individual. A
total of 53 adults, aged 18-78 yrs, with asthma controlled by combination
therapy were recruited from the Asthma Clinic at the Johannesburg General Hospital, South Africa. Medications prescribed included Budeam
(budesonide), Asthavent (salbutamol) and Beconase (beclometasone),
amongst others. Endogenous heparin levels in the asthmatic patient were
compared with 26 healthy controls, aged 18-57 yrs, recruited from the
general population. Heparin levels in the blood samples were tested
using the Chromogenix Coatest Heparin kit. Very low plasma heparin
concentrations were determined for all asthmatic patients (2.231 0.001
mIU/ml). Average plasma heparin levels in healthy volunteers (454.0
0.032 mIU/ml) were signicantly higher (p < 0.001) than that of the
asthmatic patients. These preliminary results indicate signicantly
decreased endogenous heparin levels in the asthmatic patient, thereby
potentially decreasing anti-inammatory effects. As it is not clear
whether the decreased levels of heparin is as a result of the medication
(in particular b2 agonists) being administered, or a manifestation of the
disease itself, the research is an ongoing endeavour to clarify this aspect.
323
Paper No.: 943
UNDERSTANDING ADRENOMEDULLIN BINDING TO ITS
RECEPTORS
Debbie Hay(1), T Qi(1), K Ly(1), D Rathbone(2), J Simms(3), D Poyner(2)
(1) University of Auckland, School of Biological Sciences, Auckland,
New Zealand
(2) Aston University, Birmingham, UK
(3) Monash University, Victoria, Australia
Adrenomedullin (AM), a 52 amino acid peptide, is produced by endothelial cells and vascular smooth muscle cells. AM protects the cardiovascular system from damage in cardiovascular disease and is involved in the
development of the blood and lymphatic vasculature. Therefore AM
receptors are potential drug targets. AM has two receptors. These are
dimeric complexes of a G protein-coupled receptor, the calcitonin receptor-like receptor (CLR) and receptor activity-modifying proteins
(RAMPs). CLR with RAMP2 forms the AM1 receptor and CLR with
RAMP3 forms the AM2 receptor. CLR also forms the calcitonin generelated peptide (CGRP) receptor, with RAMP1. As CLR is common to
CGRP and AM receptors but the RAMPs are different, the RAMP itself
must contribute to receptor pharmacology, either by directly forming part
of the binding pocket or by allosterically modulating the structure of
CLR. The distinctive interface formed between each RAMP/CLR combination yields unique binding pockets, which could be exploited for the
development of highly selective drugs, once dened. Through extensive
mutagenesis in RAMP3 and pharmacological characterisation of the
mutants, combined with molecular modelling, key regions and residues
within the AM2 receptor that are necessary for high afnity AM binding
have been identied. Helix two in RAMP3, along with the loop connecting helices two and three are particularly important; within these regions,
glutamic acid at position 74 and tryptophan at position 84 play key roles.
Paper No.: 2232

NEUROPROTECTIVE AND ANTITREMOR EFFECT OF BAICALEIN AGAINST 6-OHDA-INDUCED EXPERIMENTAL PARKINSONISM
Guorong He(1), X Mu(1), X Li(1), X Yu(2), K Wang(3), G Du(1)
(1) Institute of Materia Medica Chinese Academy of Medical Sciences &
Peking Union Medical College, Institute of Materia Medica, National
Center for PharmaceuticalScreening, Beijing, PR China
(2) Yantai University School of Pharmacy, Department of Pharmacology,
PR China
(3) National Engineering Research Center for the Development of New
Drugs; Beijing Collab Pharma Co. Ltd., PR China
The aim of this study was to explore the protective and antitremor effect
of baicalein on neurons against 6-OHDA-lesioned experimental parkinsonism. In in vitro experiments, we found that baicalein could signicantly ameliorate the 6-OHDA-induced SH-SY5Y cell apoptosis and
also promote neurite outgrowth in PC12 cell. In in vivo experiments, the
rats received unilateral lesions of the medial forebrain bundle made by
stereotaxic injection of 6-OHDA (4ug/ul saline, containing 0.04% ascorbic acid). Baicalein could signicantly attenuate muscle tremor of 6OHDA-lesioned rats. The frequency of tremors was decreased by 23%
and 40% at the doses of 200 and 400 mg/kg respectively and the amplitude of tremors was decreased by 7% and 29% respectively (P < 0.05 or
P < 0.01). Moreover, the number of tyrosine hydroxylase, dopamine
transport protein positive cells in substantial nigra of rats treated with baicalein is higher than that of model group. In addition, baicalein could
increase the levels of DA and 5-HT in striatum. This alteration affects
the lateral globus pallidus, which become hyperactive, ultimately leading

to an increase in the inhibitory control exerted over the subthalamic
nucleus. These results suggest that baicalein can be a promising candidate for prevention or treatment of Parkinsons disease, owing to its antiapoptotic, pro-differentiation, anti-inammatory action and attenuate the
frequency and amplitude of muscle tremors.
Acknowledgements: This study was supported by the 11th Five-Year
Program of MOST (No.2009ZX09302) and the Research Special Fund
for Public Welfare Industry of Health (No. 200802041)
Paper No.: 1639

HEALTH AND DISEASE
MATERNAL HIGH FAT DIET PROGRAMMED ENDOTHELIAL
DYSFUNCTION ON JUVENILE OFFSPRING BORN TO HIGH
FAT DIET ON NONHUMAN PRIMATE MODEL
Guo-Wei He(1,2), L Fan(1), P Sinnayah(3), KL Grove(3)
(1) Starr Academic Center, Providence Heart & Vascular Institute, Cardiovascular Research Laboratory, Portland, Department of Surgery, OR,
USA
(2) Nankai University Medical College, TEDA International Cardiovascular Hospital, Basic Medical Research Center, Tianjin, PR China
(3) ONPRC, OHSU, Division of Neuroscience, Beaverton, OR, USA
Objectives: We investigated the effects of exposure to a high fat diet
(HFD) during pregnancy and/or the early postnatal period on endothelial
function in juvenile (14 month-old) Japanese macaques Methods: Adult
female macaques were maintained on either control (CTR, 13% fat calories) or high fat/calorie diet (HFD, 35% fat calories) during pregnancies
and until weaning. The CTR offspring were then fed with CTR (CTR/
CTR) or HFD (CTR/HFD) while the HFD offspring, with HFD (HFD/
HFD) or CTR (HFD/CTR), and terminated at 14 months to collect
abdominal aortae and renal arteries for concentration-relaxation curves
for acetylcholine (ACh, -10 ~ -5.5 log M) against U46619. Gene expression levels of endothelial nitric oxide synthase (eNOS) and vascular
endothelial growth factor (VEGF) were determined by qRT-PCR.
Results: ACh-induced relaxation was remarkably depressed in vessels
from HFD/HFD (Rmax: 47.2 + /- 8.1%, n = 6, p < 0.05), partially
depressed in CTR/HFD (Rmax: 67.0 + /- 10.4%, n = 4, p < 0.05) but
reversed in HFD/CTR juvenile offspring (Rmax: 86.5 + /- 7.9%, n = 8),
compared to CTR/CTR (89.7 + /- 7.6%, n = 5). eNOS mRNA expression
level in HFD/HFD and CTR/HFD slightly decreased. VEGF expression
levels were highly expressed in other 3 groups compared to control (3.0
+ /- 0.6, 1.5 + /- 0.3, 1.3 + /- 0.2 vs 1.0 + /- 0.1). Conclusions: Maternal
and postnatal exposure to a HFD 1). impairs endothelium-dependent
vasodilatation, raising the risk of early onset of atherosclerosis and 2).
the endothelial dysfunction is associated with alteration of eNOS and
VEGF expression.
Supported by St. Vincent Medical Foundation, NIDDK 079194,
2009DFB30560, 2010CB529502, 09ZCZDSF04200. 079194.
Paper No.: 1640

THE INTERACTION OF HUMAN UROTENSIN II AND
VASODILATOR AGENTS IN HUMAN INTERNAL THORACIC
ARTERY: CLINICAL IMPLICATIONS
Guo-Wei He(1,2,3), X-Y Bai(1), X-C Liu(1), W-B Jing(1), Q Yang(2)
(1) Nankai University Medical College, TEDA International Cardiovascular Hospital,Basic Medical Research Center, Tianjin, PR China
(2) The Chinese University of Hong Kong, pPR China
(3) Starr Academic Center, Providence Heart & Vascular Institute, CardiovascularResearch Laboratory, Portland, Department of Surgery, OR, USA
324
Objectives: Graft spasm in the internal thoracic artery (ITA) may occur
after coronary artery bypass grafting (CABG). We investigated the effect
of human urotensin II (hU-II), a cyclic peptide present in human tissues,
and of vasodilators on hU-II-mediated response in human ITA. Methods:
Fresh ITA segments (n = 102) taken from 46 patients undergoing CABG
were studied in organ bath. The interaction between hU-II and various
calcium antagonists or glyceryl trinitrate (GTN) was investigated in two
ways: the relaxing effect of vasodilators on the hU-II-induced precontraction and the depressing effect of vasodilator agents on the contraction
caused by hU-II (n = 6 in each group). Results: hU-II caused contractile
response in all human ITA. In potassium chloride-contraction, full (nifedipine: 98.9 + /- 3.9%) or nearly full (diltiazem: 92.7 + /- 6.0%) relaxation
with 18.6-fold-higher potency to nifedipine than to diltiazem (EC50 8.01 + /- 0.20 vs. -6.74 + /- 0.22 logM, p = 0.002) and in hU-II-contraction, nearly full relaxation (nifedipine: 90.6 + /- 4.6%; diltiazem: 95.1 +
/- 2.1%) with 6.2-fold-higher potency to nifedipine than to diltiazem
(EC50 -7.25 + /- 0.25 vs. -6.46 + /- 0.18 logM, p = 0.014) were
observed. GTN caused nearly full relaxation (95.4 + /- 5.8%) but
pretreatment with GTN failed to alter, whereas diltiazem and nifedipine
pretreatment reduced subsequent contraction to hU-II. Conclusions:
hU-II is a potent vasoconstrictor and possible spasmogen in human ITA.
Calcium antagonists and GTN relax the contraction caused by hU-II with
different potencies. However, calcium antagonists are more effective in
preventing the contraction induced by hU-II than GTN.
Supported by Grants 2009DFB30560, 2010CB529502, 09ZCZD
SF04200.
Paper No.: 2004

SASANQUASAPONIN PRETREATMENT PROTECTS
CADIOCYTES FROM ACUTE ANOXIA DAMAGE VIA A ERK1/
2-HSP70 PATHWAY
M He(1), Qingxian Zhu(1), D Liu(1), L Tang(2), D Yin(1), Z Liao(1)
(1) Nanchang University School of Pharmaceutical Science, Department
of Pharmacology & Molecular Therapeutics, Nanchang, PR China
(2) Nanchang University, Jiangxi Provincial Key Lab of Molecular
Medicine at Second Afliated Hospital, Nanchang, PR China
Recent studies have demonstrated the cardioprotective abilities of Sasanquasaponin, an effective component was from the remainder of seeds of
Camellia sasanqua. We have observed ischemic preconditioning require
activation of heat shock protein(HSP)70, an endogenous cardioprotective
protein, to induced to cardioprotection against myocardial ischemia. In
the present study, we tested two hypotheses: (a) that cardioprotection
induced by sasanquasaponin preconditioning is associated with the
increase of HSP70. (b) that increased expression of HSP70 involves activation of ERK1/2 signalling pathway. Sasanquasaponin preconditioning
induced a rapid increase in HSP70 expression and a protective effect
against anoxia injury in cultured neonatal rat myocardial cells, which
was inhibited by using either ERK1/2 specic blocker PD184352 or
plasmid pSilencer-shHSP70. Both PD184352 and pSilencer-shHSP70
also abolished sasanquasaponin -induced cardioprotection, most likely
owing to an increase in MnSOD activity and an decrease in total antioxidant status activities, such as SOD, GSH-Px and catalase. As a molecular
chaperone, HSP70 have stabilized the structure of endogenous antioxidant enzymes and maintain their activities, thereby attenuated free radical
damage induced by anoxia injury. Our data suggest that sasanquasaponin
pretreatment can protect the cadiocytes against anoxia injury. The protective mechanism of sasanquasaponin involves an increased expression of
HSF70 by activating ERK1/2 signalling pathway.
Acknowledgement: Project supported by the National Science Foundation of China(No 30760075), the 973 Program(No 2009CB526405).
Paper No.: 2005

HEALTH AND DISEASE
SYNERGISTIC EFFECT OF IRON AND GLUCOSE ON
ENDOTHELIAL CELLS IS MEDIATED BY DDAH/ADMA/ROS
SIGNALLING PATHWAY
SYNERGISTIC EFFECT OF IRON AND GLUCOSE ON
ENDOTHELIAL CELLS IS MEDIATED BY DDAH/ADMA/ROS
SIGNALLING PATHWAY
Ming He(1), D Liu(1), D Yin(2), Z Liao(1), L Tang(1), Q Huang(1)
(2) Nanchang University, Jiangxi Provincial Key Lab of Molecular Medicine at Second Afliated Hospital, Nanchang, PR China
More and more clinical trial and epidemiological studies have revealed a
signicant relationship between iron metabolism and diabetes, futher to
aggravate endothelial dysfunction in patients with diabetes mellitus.
However, the molecular mechanisms underlying the synergistic damage
to endothelial function were poorly known. The growing evidence indicates that endogenous inhibitors of endothelial NO synthase (eNOS) such
as asymmetric dimethylarginine (ADMA), mainly degradated by the
dimethylarginine dimethylaminohydrolase (DDAH), are associated with
the eNOS uncoupled and endothelial dysfunction. We hypothesize: (a)
that iron and glucose results in synergistic damage to HUVECs, (b) that
dysfunctionally uncoupled eNOS is involved in producing reactive oxygen species (ROS) in the HUVECs, (c) that the burst of ROS is responsible for the irreversible mitochondrial permeability transition pore
(mPTP) opening, which causes mitochondrial swelling and cell apoptosis. The present study examined our hypothesis by using exogenous Larginine and CsA, a special mPTP inhibitor. The results showed that
exogenous L-arginine supplementation could compete with ADMA, prevent eNOS uncoupled-mediated ROS production, and endothelial function could be improved by preventing the opening of mPTP and then
inhibiting the burst of ROS caused by using CsA. We conclude that the
molecular mechanisms of cooperation is mainly that iorn and glucose
can mutually potentiate their effects on ROS production, impair DDAH2 activity, elevate ADMA levels, induce eNOS uncoupled-mediated ROS
production, and then lead to aggravate endothelial dysfunction.
Acknowledgement: Project supported by the National Science Foundation of China(No 30760075, 30860271), the 973 Program(No 2009CB
526405)
Paper No. 2009

FOCUS GROUP: P06 - THE HEART GONE WRONG;
STABILIZATION OF CARDIAC FUNCTION
THE ROLE OF VOLTAGE-DEPENDENT ANION CHANNEL IN
ISCHEMIA/REPERFUSION INJURY MEDIATED BY
CHLORIDE
Ming He(1), Z Liao(1), L Tang(2), D Yin(1), F Zou(1), S Yin(1)
Many evidences have demonstrated that the incidences of Cl- oscillation
play an important role in myocardial ischemia/reperfusion (I/R) injury.
However, the mechanism is not well-illustrated. Mitochondrial permeability transition pore (mPTP) opening is considered as a critical event in
the transition from reversible to irreversible reperfusion injury. Voltagedependent anion channel (VDAC), a major component of mPTP, may be
a potential channel for regulating Cl- homeostasis. In our study, the role
of VDAC1 in I/R injury mediated by Cl- was investigated by silencing
VDAC1 expression with a short hairpin RNA (shRNA)-expressing vector in cardiomyocytes. The dynamic change of cellular and mitochondrial
Cl- and Ca2 + concentration was detected by confocal microscopy.
325
Down-regulation of VDAC1 expression by shRNA decreased mitochondrial Cl- concentration signicantly, resulting in attenuation of calcium
overload and decrease in apoptosis in cardiomyocyt es subjected to I/R.
Moreover, we found that, with Cl- concentration decreased, mitochondrial Ca2 + concentration also reduced, and mitochondrial membrane
potential was well preserved. These results indicate that VDAC1 is an
key factor of I/R injury mediated by Cl-. During I/R process, cytoplasmic
Cl- ows into mitochondria through VDAC, subsequently inducing Ca2 +
release, which is termed chloride-induced calcium release (ClICR) ClICR
can induce mPTP opening, resulting in Ca2 + overload.
This study was supported by the Natural Scientic Foundation of China
(30760075, 30960449) and the Natural Scientic Foundation of Jiangxi
Province (2009GQY0133).
Paper No.: 2155

HEALTH AND DISEASE
PROTECTIVE EFFECT OF SODIUM NITROPRUSSIDE
AGAINST IN VITRO ISCHEMIA-REPERFUSION INJURY OF
RAT MESENTERIC ARTERY
X He, M Zhao, H-L Zhang, X-L Xu, L-Y Xie, L Sun, X-J Yu, Wei-Jin
Zang
Xian Jiaotong University, Department of Pharmacology, Xian, PR
China
Ischemia reperfusion (I/R) injury severely endangers human health and is
therefore a major focus of medical studies. Nitric oxide (NO), a principle
mediator of vasodilatation, plays an important role in the protection
against I/R injury. In the realm of vascular pharmacological research,
however, there are only sporadic functional studies investigating in vitro
vascular I/R. The present methodological study was designed to investigate the protective effect of NO donor sodium nitroprusside (SNP)
against in vitro I/R injury on rat mesenteric artery. Simulated I/R model
was established using an organ culture system. The ischemia was simulated using modied ischemia mimetic solution and anaerobic environment. The arterial rings of I/R group were pretreated with SNP (0.1, 0.5
and 1.0mM). The dose-dependent vasomotor responses were recorded by
the Multi Myograph System. The results showed that the in vitro I/R
model was coincident with in vivo I/R model. In the in vitro model, the
arterial tissue structure showed pathological abnormalities and the levels
of lactate dehydrogenase and creatine kinase dramatically increased. SNP
(1.0mM) could inhibit the vasoconstriction of mesenteric arteries associated with I/R. The maximum effect (Emax) values induced by KCl, 5hydroxytryptamine and U46619 (72%6%, 58%5% and 141%6%)
were lower than those of I/R group (91%8%, 238%8%, and
286%8%). This study demonstrates that NO possesses potent protective
effects against vascular I/R injury. The experimental model of in vitro
vascular I/R would offer great prospects for reliable investigation of
pharmacological attempts.
Paper No.: 3256

TRANSPORT: PERSPECTIVES FOR DISEASE AND DRUG DISCOVERY
CONFOUNDING FACTORS IN QUANTIFYING THE CONCENTRATION DEPENDENT P-GLYCOPROTEIN MEDIATED
TRANSPORT IN IN VITRO CELL PERMEATION EXPERIMENTS
Aki T Heikkinen(1,2), T Korjamo(3,4), V Lepikko(1,2), J Monkkonen(1,2)
(1) University of Eastern Finland, Department of Pharmacy, Kuopio, Finland
(2) Biocenter Kuopio, Finland
(3) Novamass Ltd, Oulu, Finland
(4) Current afliation: R&D, Orion Corporation, Orion Pharma, Espoo,
Finland
P-glycoprotein mediated efux is one of the barriers limiting the intestinal drug absorption. Predictions of the intestinal P-glycoprotein function
should account for the concentration dependency because high intestinal
drug concentrations may saturate P-glycoprotein. However, the substrate
binding site of P-glycoprotein lies within the cells and the drug concentration at the binding site cannot be measured directly. Therefore, in vitro
in vivo extrapolation of P-glycoprotein saturation is challenging. We
explored the effects of the aqueous boundary layers, extracellular pH and
cellular retention on the apparent saturation kinetics of P-glycoprotein
mediated transport of quinidine in in vitro permeation setting. The
changes in the experimental conditions caused one order of magnitude
and vefold variation to the apparent afnity to P-glycoprotein and to the
maximum effective P-glycoprotein mediated transport rate of quinidine,
respectively. Fitting the concentration data into a compartmental model
accounting for the aqueous boundary layers, cell membranes and cellular
retention suggested that the P-glycoprotein function per se was not
altered but the differences in the passive transfer of quinidine changed
the apparent transport kinetics. These results provide further insight into
the dynamics of the P-glycoprotein mediated transport and on the roles
of several confounding factors involved in in vitro experimental setting.
Paper No.: 1025

ALLOSTERIC MODULATION OF REPRODUCTIVE
GPCRS - A CASE FOR THE LUTEINIZING HORMONE
RECEPTOR
Laura Heitman
LACDR, University of Leiden, Department of Medicinal Chemistry, Leiden, TheNetherlands
Allosteric modulation of G protein-coupled receptors (GPCRs) has been
of great interest in the past decade. For GPCRs with proteins or peptides
as endogenous ligands, low molecular weight (LMW) allosteric modulators could provide additional benets, such as oral bioavailability. The
luteinizing hormone (LH) receptor is a peptide-activated GPCR and
plays an important role in fertility and certain cancers. We recently
reported on the rst radiolabeled LMW agonist for this receptor, [3H]Org
43553, which was now used to screen for new LMW ligands. This
screen yielded several compounds that were able to either allosterically
enhance (LUF5419) or inhibit (LUF5771) the binding of the allosteric
agonist, Org 43553, by decreasing or increasing its dissociation rate,
respectively. In a functional assay it was shown that LUF5419 increased
the efcacy of Org 43553, while the potency and efcacy of LH was
unaffected. On the other hand, the presence of LUF5771 resulted in
decreased potency of both Org 43553 and LH. Subsequent molecular
Modelling studies suggested that these novel compounds can be accommodated by an overlapping site in the receptor. This was conrmed by
the fact that the potency to increase radioligand dissociation of LUF5771
was decreased by the presence of LUF5419. Taken together, these results
demonstrate that LUF5419 and LUF5771 are allosteric modulators of the
human LH receptor that bind at the same allosteric site (different from
the Org 43553 site). This site may provide another opportunity for the
discovery of new orally bioavailable ligands for the human LH receptor.
Paper No.: 2656

FLUCONAZOLE-INDUCED INTOXICATION WITH
PHENYTOIN IN A PATIENT WITH ULTRA-HIGH ACTIVITY
OF CYP2C9
Anders Hellden(1), U Bergman(1), K Engstrom Hellgren(2), I Nilsson
Remahl(3), I Odar-Cederlof(1), M Ramsjo(1), L Bertilsson(1)
326
(1) Karolinska Institute, Karolinska University Hospital, Division of
Clinical Pharmacology, Department of Laboratory Medicine, Stockholm,
Sweden
(2) Karolinska Institute, Karolinska University Hospital, Department of
Rheumatology, Stockholm, Sweden
(3) Karolinska Institute, Karolinska University Hospital, Department of
Neurology, Stockholm, Sweden
Introduction: The cytochrome P450 enzyme CYP2C9 metabolizes several important drugs such as warfarin and oral antidiabetic drugs. The
enzyme is polymorphic and all known alleles, e.g. CYP2C9*2 and*3,
give decreased activity. Ultra-high activity of the enzyme has not been
reported before. Patient: We present a patient with Bechets disease, who
required treatment with high doses of phenytoin. When uconazole, a
potent inhibitor of CYP2C9, was added, the patient became intoxicated
by phenytoin. On suspicion of ultra-high activity of CYP2C9 a phenotyping test for CYP2C9 with losartan was performed. Results: The
patient was shown to have a higher activity of CYP2C9 than any of the
190 healthy Swedish Caucasians used as controls. Conclusions: Our nding of an ultrarapid metabolism of losartan and phenytoin may apply to
other CYP2C9 substrates, where inhibition of CYP2C9 might cause
severe adverse drug reactions.
Paper No.: 2485

DISCOVERY
MODULATING THE DOPAMINE TRANSPORTER
EXPRESSION THROUGH CHANGES IN THE PROTEOSTASIS
ENVIRONMENT
Freja Henriksen, I Ammendrup-Johnsen, U Gether
Copenhagen University, Panum Institute, Department of Neuroscience
and Pharmacology, Copenhagen, Denmark
The dopamine transporter (DAT) is a presynaptic plasma membrane protein that mediates reuptake of dopamine from the synaptic cleft. Disturbances in dopaminergic signalling are important for a number of
pathophysiological conditions including addiction, schizophrenia, attention decit hyperactive disorder, and Parkinsons disease. Nevertheless,
we only have sparse insight into the mechanisms controlling the surface
availability of DAT. We have investigated the role of the extreme C-terminus of DAT in folding and ER export, and the effect of modulating
the folding environment on the cellular DAT processing. We demonstrate
that the consequences of mutating the extreme C-terminal residues of
DAT are species dependent and furthermore depend markedly on the
expression system. These observations might be explained by differences
in the chaperone environment, reported to play a critical role in protein
folding and trafcking. We demonstrate that treatment of DAT transfected cells with MG-132, a modulator of the folding environment, increase
the surface targeting of wt DAT and C-terminal hDAT mutants. This provides proof of principle that the folding environment affects DAT expression. To further investigate the role of specic cochaperone interactions
in DAT processing, a shRNA screen was carried out on a selection of
Hsp90 and Hsp70 cochaperones. Knockdown of several cochaperones
markedly increased surface targeting of functional hDAT, and rescued an
ER retained DAT mutant to the cell surface. This indicates that cochaperones may have regulatory roles in hDAT processing and thereby supports
the hypothesis, that folding environment may constitute an unappreciated
level of hDAT surface expression regulation.
Paper No.: 3414

DISCOVERY
REGULATION OF MICROGLIAL IMMUNE FUCTION BY
STORE-OPERATED CALCIUM CHANNELS(SOCS)
Yon-sei Uniiversity, College of Medicine, Brain Korea 21 Project for

Medical Sciences, Department of Pharmacology, Seoul, South Korea
Microglia is the immune effector cell in central nervous system (CNS)
that participates in tissue repair, inammatory responses and neuronal
degeneration. Microglia can differentiate into active state which shows
not only amoeboid morphology but increased chemotaxis response,
phagocytosis capacity, and cytokine release. Most important signalling in
differentiation of immune cell is sustained cytosolic calcium increase
which was maintained by SOC. Recently, molecular identity of SOC was
revealed that Orais and Stims constitute SOC. In this study, we demonstrate that not TRPCs but Orais and Stim1 regulate differentiation,
phagocytosis activity, and cytokine secretion in microglia. We use primary microglia cells from neonatal mouse brain which show 99.5% purity with GFAP immune-staining. Primary microglia has Orai 1, 2, 3, and
Stim1, but not any TRPCs and Stim2. SOC blocker, SKF96365 inhibits
sustained [Ca2 + ]i elevation after a transient calcium evocation which
was mediated by PLC activation through P2Y receptor and SFK96365
inhibits UDP-induced phagocytosis activity of microglia. These data
indicates that SOC constituted by Orais and Stim1 controls phagocytic
function of microglia.
Paper No.: 2337

THE EFFECTS OF CANNABIDIOL AND THE CB1
ANTAGONIST AM251 ON THE DEPRESSOR RESPONSES TO
ACEA IN VIVO
Claire Hepburn(1), O Keown(1,2), S Watt(1), I Megson(2), S Leslie(3),
K Kane(4), C Wainwright(1)
(1) Robert Gordon University, School of Pharmacy and Life Sciences,
Schoolhill, Aberdeen, UK
(2) UHI Millennium Institute, Department of Diabetes & Cardiovascular
Science, Inverness, UK
(3) Raigmore Hospital Highland Heartbeat Centre, Inverness, UK
(4) Strathclyde Institute of Pharmacy & Biomedical Sciences, University
of Strathclyde, Glasgow, UK
Cannabidiol (CBD) is markedly anti-arrhythmic in an experimental
model of coronary occlusion. The aim of this study was to further characterise the cardiovascular pharmacology of CBD. Male Sprague Dawley rats were anaesthestised via i.p. injection of sodium pentobarbital
(60mg kg-1). Vessels were cannulated to permit drug administration,
MABP transduction and articial ventilation. To assess whether CBD
was acting via the CB1 receptor, rats pre-treated with CBD (50lg kg-1)
or saline by I.V bolus injection before blood pressure responses to
ACEA (3mg kg-1) were observed in the presence of either vehicle or
AM251 (1 and 3mg kg-1). To determine whether CBD was acting via
fatty acid amide hydrolase (FAAH) or GPR55, subsequent experiments
involved the replacement of (i) CBD with the FAAH inhibitor URB597
(1mg kg-1) or (ii) ACEA with the GPR55 agonist, O-1602 (30ng kg-1).
ACEA produced a consistent fall in (MABP) (~30-32%) which was signicantly diminished in the presence by AM251 (P < 0.001) while CBD
had no effect. ACEA antagonism by AM251 was lost in the presence of
CBD. Like CBD, URB597 (1mg kg-1), did not affect the ACEA
response, however antagonism of the ACEA-induced depressor response
by AM251 remained intact (p < 0.05). O-1602 produced a negligible
effect on blood pressure, however AM251 unmasked a signicant
depressor response to O-1602; CBD also tended to increase the O-1602
response. These results suggest that CBD is not acting as either as an
inhibitor of FAAH or as a GPR55 antagonist but may interact with the
CB1 receptor.
Dae Keon Heo, MG Lee, JY Kim

327
Paper No.: 2868
FACTORS ASSOCIATED WITH SHORT-TERM ADHERENCE
TO DIETARY AND LIFESTYLE GUIDELINES IN SECONDARY
PREVENTION OF ACUTE MYOCARDIAL INFARCTION IN
REAL LIFE PRACTICE IN FRANCE
S Hercberg(1), D Thomas(2), C Droz-Perroteau(3), C Dureau-Pournin(3), P Ducimetie`re(4), F Paillard(5), J Tricoire(6), M-A Bernard(3),
J Benichou(7), N Danchin(8), L Guize(8), P Blin(3), Nicholas Moore(3)
(1) INSERM U557-INRA-CNAM-CRNH, Bobigny, France
(2) Hopital Pitie-Salpetrie`re, Paris, France
(3) Universite de Bordeaux-INSERM CIC 0005, Bordeaux, France
(4) INSERM, Villejuif, France
(5) CHU de Pontchaillou, Rennes, France
(6) Cardiologist, Toulouse, France
(7) CHU de Rouen-INSERM U657, Rouen, France
(8) Hopital Europeen Georges Pompidou, Paris, France
Adherence to dietary and lifestyle guidelines (DLG) plays a major role
in secondary prevention after acute myocardial infarction (AMI). A
cohort study of patients included by cardiologists after recent AMI was
done to assess DLG adherence and to determine the factors associated
with DLG score. Adherence to DLG was evaluated by a 23-item composite score, estimated after 6 months of follow-up, taking into account
evolution since inclusion of diet (21 items), tobacco use (1 item), physical activity (1 item). Each item was scored -1 for worsening, 0 for no
change, +1 for improvement. Each patients DLG score was the mean of
the 23 items. Linear regression models were used to assess factors associated with DLG score. Inclusion variables tested were socio-demographic, clinical, dietary, lifestyle habits. From May 2006 to March
2008, 1575 subjects were included: 56% aged >60 years, 81% male,
9.3% smokers, 40% with a cardiovascular rehabilitation program (CRP).
This was the rst AMI for 87%. For 6.7%, LVEF was <40%. After 6
months, median DLG score was +0.35, i.e., an improvement in overall
DLG compliance. The variables most strongly associated with more
improvement of DLG score were: prescription of CRP at inclusion, education above primary school, high consumption of at-risk food before
inclusion, previous history of hypercholesterolemia, large waist circumference at inclusion. Age >60 years,smoking at inclusion, female gender
were associated with less improvement. The factor most strongly associated with 6-month DLG improvement was the prescription of a CRP:
this result underscores the interest of such interventions.
Paper No.: 3101

INFLUENCE OF NURSES ATTITUDES ON ADVERSE DRUG
REACTION REPORTING: A PILOT STUDY IN PORTUGAL
Maria T Herdeiro(2,3), S Pernas(1), O da Cruz e Silva(2),
A Figueiras(4)
(1) Universidade de Aveiro, Departamento de Biologia, Seccao Autonoma Ciencias da Saude, Aveiro, Portugal
(2) Universidade de Aveiro, Centro de Biologia Celular, Seccao Autonoma de Ciencias da Saude, Aveiro, Portugal
(3) Centro de Investigacao em Tecnologias da Saude (CITS), IPSN-CESPU, Gandra,Portugal
(4) Universidade de Santiago de Compostela, Departamento de Medicina
Preventiva e Saude Publica, Santiago de Compostela, Portugal
there is ahigh rate of underreporting by these professionals.Objective:

Test questionnaire reproducibility about attitudes and knowledgerelated
with report of adverse drug reactions (ADR) between nurses of the regioncorresponding to the Regional Health Administration Center of Portugal.Methods: This study focused on a sample of nurses in hospitals
and healthcenters in center Portugal. To each health professional was
given a structuredquestionnaire twice, with 2 weeks interval. Some of
the attitudes included inquestionnaire were based on the seven signs of
Inman, which were measured usinga visual analogue scale continuous.
The questionnaire was given to a sample of30 nurses: To test the reliability and reproducibility of the questionnaire wasdetermined by the intraclass correlation coefcient.Results: Financial incentives (ICC 0.77, CI
95% 0.57 - 0.88; p 0.0000);Complacency (ICC 0.70, Cl 95% 0.46 - 0.85
and p 0.0000); Insecurity (ICC 0.69,CI 95% 0.44 - 0.84 and p 0.0000);
Difdence (ICC 0.65, CI 95% 0.39 - 0.82 and p0.0000); Ignorance of
the notication system (ICC 0.65, CI 95% 0.38 - 0.82 andp 0.0000).Conclusion: This study demonstrates that there are attitudes with very goodintraclass correlation coefcient. We recommend based on these
resultscontinuity of studies including case-control studies.
Paper No.: 3102

PORTUGUESE PHARMACIST ATTITUDES TOWARDS THE
USE OF HERBAL COMPLEMENTARY AND ALTERNATIVE
MEDICINES IN ONCOLOGY
Maria T Herdeiro(1,2), E Mendes(1), A Figueiras(3), F Pimentel(4)
(1) Universidade de Aveiro, Seccao Autonoma de Ciencias da Saude,
Aveiro, Portugal
(2) Centro de Investigacao em Tecnologias da Saude (CITS), IPSN-CESPU, Gandra, Portugal
(3) Universidade de Santiago de Compostela, Departamento de Medicina
Preventiva e Saude Publica, Santiago de Compostela, Portugal
(4) Hospital Infante D. Pedro, Aveiro, Portugal
Introduction: The use of herbal complementary and alternative medicines
(CAM), is growing among Portuguese cancer patients, contributing to a
higher risk for unwanted interactions with conventional cancer therapies.
Although health professionals are becoming aware that patients use some
forms of CAM, in particular phytotherapy, few discuss these therapies
with them. Therefore, interactions may not be recognised as such, or
may only be reported if considered serious, leading to the under-reporting of herb-drug interactions. The aim of the study is to assess pharmacists perceptions and attitudes towards the use of herbal CAM by cancer
patients. Materials: Questionnaires included 3 questions related with 15
most common CAM therapies and 1 set of questions related with phytotherapy. To assess reproducibility, each questionnaire was used in a pilot
test, performed at 8 pharmacies in the surrounding area of the Hospital
Infante D. Pedro (Aveiro) on a sample of 15 pharmacists. The questionnaires were administered twice, with 3 weeks interval. The questionnaires reproducibility was assessed using Cohens kappa coefcient.
Results: Kappa was above 0,6 for the majority of the questions, revealing
a substantial degree of agreement. For some therapies, some physicians
gave the same answer in both phases of the pilot test, which conditioned
the determination of kappa. However this is only a theorical limitation
since a perfect agreement was observed. Conclusion: The level of reliability estimated suggests that pharmacists familiarity with CAM, and
attitude towards its use by cancer patients, can be adequately measured
by the developed questionnaires.
Introduction: It is essential monitoring the safety of medicines. Nurses

canplay a key role in this notication but, for reasons still unknown,
328
Paper No.: 2015
CONTINUOUS INFUSION OF A TINY DOSE OF
CORTICOSTERONE IMPROVES AGEING AND
HYPERTENSION RELATED HEART FUNCTION IMPAIRMENT
AND REDUCES CARDIAC FIBROSIS
JJ Rob Hermans, M Minnaard-Huiban, IPE Beugels, H van Essen,
JJ Debets, H AJ Struijker-Boudier, JFM Smits
Maastricht University, Cardiovascular Research Institute, Maastricht, The
Netherlands
Ageing and hypertension are major risk factors for pathological remodelling, brosis and function impairment of the heart. Glucocorticoids are
known anti-inammatory and anti-brotic agents, but when applied as a
high dose bolus, are suspected to deteriorate heart function. The effects
of continuous low dose glucocorticoid infusions on the brosis and function of the aged hypertensive heart to our knowledge are unknown.
Therefore, male lean spontaneously hypertensive heart failure prone
(SHHF) were treated by continuously infusing a minute dose (0.06 mg/
day) of corticosterone (hemisuccinate), either between 40 and 44 weeks
or 72 and 80 weeks of age. At treatment end (i.e. rats being 44 or 80
weeks) left ventricular (LV) heart function and brosis were assessed.
Glucocorticoid infusion reduced (P < 0.05) interstitial LV brosis by
50% in both age groups. Compared to 44 weeks old rats, 80 weeks old
SHHFs displayed elevated LV end diastolic pressures (P < 0.05) and
reduced basal LV contractilities (+dP/Pdt; P < 0.05) and dobutamine
stimulated LV relaxations (-dP/Pdt; P < 0.05). However, in glucocorticoid infused aged rats these parameters were signicantly improved (P <
0.05) to similar values as in younger rats. Plasma corticosterone and
daily urinary corticosterone excretion were not changed by the infusions,
indicating that the applied corticosterone dose is low. Remarkably, if the
daily dose would be compared to the reported daily rat adrenal corticosterone production it would just be a small fraction (1-3%) thereof. Thus
we conclude that continuous infusion of corticosterone at a tiny dose
improves the function and brosis of the aged hypertensive heart.
Paper No.: 3426

THE ROLE OF NITRIC OXIDE IN THE LOCAL ANTINOCICEPTIVE EFFECTS AND EXPRESSION OF CANNABINOID-2
RECEPTORS DURING NEUROPATHIC PAIN IN MICE
A Hervera(1), R Negrete(1), S Leanez(1), J Martn-Campos(2), Olga
Pol(1)
(1) Universitat Auto`noma de Barcelona, Grup de Neurofarmacologia
Molecular, Institut de Recerca de lHospital de la Sta Creu i Sant Pau &
Institut de Neurocie`ncies, Barcelona, Spain
(2) Grup de Bioquimica, Institut de Recerca de lHospital de la Sta Creu
i Sant Pau, Barcelona, Spain
The local administration of cannabinoid 2 receptor (CB2R) agonists
attenuates neuropathic pain but the precise mechanisms implicated in
these effects are not completely elucidated. Our aim is to evaluate if
nitric oxide synthesized by the neuronal nitric oxide synthase (NOS1)
could modulate CB2R antinociception through the peripheral nitric
oxide-cGMP-protein kinase G (PKG) pathway activation and affect their
gene expression during neuropathic pain. In wild type (WT) mice at 21
days after the chronic constriction of the sciatic nerve, we evaluated the
local antinociceptive effects of a CB2R agonist (JWH-015) and a NOS1
(N-[(4S)-4-amino-5-[(2-aminoethyl)amino]pentyl]-Na-nitroguanidine
tris(triuoroacetate) salt; NANT), L-guanylate cyclase (ODQ) or a PKG
(Rp-8-pCPT-cGMPs) inhibitor, alone or combined. The expression of
CB2R mRNA levels in the dorsal root ganglia and spinal cord of WT
and NOS1-KO mice at pre- and post-nerve injury was also assessed. The
subplantar administration of NANT, ODQ or Rp-8-pCPT-cGMPs dosedependently inhibited neuropathic pain and enhanced the local antinociceptive effects of JWH-015. Moreover, although the basal levels of
CB2R mRNA were similar between WT and NOS1-KO animals, nerve
injury only increased CB2R expression in the dorsal root ganglia of WT
but not in NOS1-KO mice. Our results suggest that the inactivation of
nitric oxide-cGMP-PKG peripheral pathway triggered by NOS1
improved the peripheral CB2R antinociceptive effects and that nitric
oxide synthesized by NOS1 is implicated in the peripheral up-regulation
of the CB2R gene transcription observed during neuropathic pain. This
work was supported by grants from FIS, Madrid (PS0900968) and Fundacio La Marato de TV3, Barcelona (070810), Spain.
Paper No.: 1245

N-METHYL-D-ASPARTATE CHANNEL BLOCKING
PROPERTIES OF DUAL REUPTAKE INHIBITORS
Peter Heusler, A Tourette, C Cathala, D Cussac
Pierre Fabre Research Institute, Department of Cellular and Molecular
Biology, Castres Cedex, France
Milnacipran and duloxetine are dual serotonin and noradrenaline reuptake inhibitors approved by the US FDA for the management of bromyalgia (FM), a pathology characterized by chronic pain symptoms. In
addition, potential N-methyl-D-aspartate (NMDA) type ionotropic glutamate receptor antagonist properties of these drugs may be of interest in
their MoA since ketamine, a non-competitive NMDA antagonist, is currently used as analgesic and has shown efcacy as an antidepressant drug
in man. In order to characterize the functional actions of these drugs and
other reference NMDA antagonists, NMDA receptors composed of the
recombinant human NR1 and NR2B subunits were expressed in Xenopus oocytes and whole-cell currents were recorded electrophysiologically
using the two-electrode voltage-clamp technique. All compounds concentration- and voltage-dependently inhibited NMDA receptor-mediated currents with a high potency for (+)MK-801 (IC50 = 9 nM) and
phencyclidine (57 nM), an intermediate potency for memantine (0.7 lM)
and ketamine (0.9 lM), and a lower potency for milnacipran (7 lM) and
duloxetine (32 lM). In assays on NMDA current recovery after blockade, milnacipran and duloxetine yielded rapid unblocking kinetics
similar to the well tolerated fast offset compound, memantine. In contrast, other antagonists such as ketamine and phencyclidine had clearly
slower unblocking kinetic properties, potentially associated with cognitive impairment. Thus, dual serotonin / noradrenaline reuptake inhibitors
with NMDA antagonist properties such as milnacipran may be of interest
for better management of chronic pain conditions such as FM.
Paper No.: 1324

CHANGE IN FEEDING BEHAVIOR BY SIRNAS FOR A NPY
RECEPTOR INTO PARAVENTRICULAR NUCLEI
Hiroshi Higuchi, T Shiiya, S-I Murase
Niigata University Graduate School of Medicine, Dentistry & Pharmaceutical Sciences, Division of Pharmacolgy, Niigata, Japan
NPY is the most potent peptidergic neurotransmitter for feeding regulation in the hypothalamus, among orexigenic peptides such as AgRP and
anorexigenic ones such as a-MSH. Although blockade of NPY Y1
receptor in the paraventricular nucleus (PVN) suppresses feeing, the
feeding behavior is increased in NPY Y1 receptor-knockout mice, probably due to compensatory mechanism. To investigate this mechanism, we
rst studied the effect of in vivo knockdown of Y1 receptor by a plas-
329
mid-based small interference RNA (siRNA). siRNA against Y1 receptors
was stereotaxically injected into the arcuate nucleus (Arc) and PVN.
Acute knockdown of Y1 receptor gene expression in PVN resulted in
decreased feeding clearly for 2 days, but not in Arc. Based on the anatomical structure of NPY neurons (which originate from Arc and terminate to PVN), knockdown in gene expression of Y1 receptor might
suppress the activity of anorexigenic neurons in PVN. There was no
compensatory increase in feeding on short-term knockdown. Thus siRNA plasmid-induced knockdown of NPY gene expression serves as a
powerful tool for regulation of endogenous feeding regulating genes in
the brain.
Paper No.: 1359

EFFECTS OF CALCITONIN GENE-RELATED PEPTIDE(CGRP)
ON THE UNDERPERFUSION WITH NOREPINEPHRINE
INJURIES IN DIABETIC RAT HEARTS
Makie Higuchi, T Akamine
Kyushu University of Nursing, Division of Pharmacology, Tamana,
Kumamoto, Japan
We have reported that norepinephrine (NE) during underperfusion aggravates myocardial stiffness and abnormal energy metabolism, particularly
in diabetic (DM) hearts (Higuchi M et al, J Mol Cell Cardiol 1991; 23:
137-148, J Cardiovasc Pharmacol 1995; 26: 899-907, Cardiovasc Res
1996; 35: 303-314). In this study we examined the effects of calcitonin
gene-related peptide (CGRP) on the underperfusion with NE 10-6M injuries in streptozotocin-induced DM and non-DM rat hearts. The isolated
paced hearts were normoperfused (75 cmH2O) for 30min, then underperfused (30 cmH2O: 1/3 of the control inow) with NE for 45min. CGRP
was infused from 15min before (2~3 nM) and during underperfusion
(9~10 nM). Increases in left ventricular (LV) stiffness were detected with
increases in LV end-diastolic tension and pressure. CGRP signicantly
increased the normoperfusion ow rate, but did not during underperfusion
in both heart groups. CGRP in DM hearts showed a slight but signicant
increase in peak (+)dP/dt during normo- and underperfusion, and tended
to exacerbate increases in LV stiffness during underperfusion. These
results suggest that CGRP does not alleviate the underperfusion injury,
accompanying with a slight positive inotropic effect in DM hearts.
Paper No.: 2193

SESSION - ONCOLOGY
ARACHIDONIC ACID PROMOTES GLUTAMATE-INDUCED
CELL DEATH ASSOCIATED WITH NECROSIS THROUGH
12-LIPOXYGENASE ACTIVATION IN GLIOMA CELLS
Yoshihiro Higuchi(1), M Hashii(2), H Tanii(2), Y Koriyama(2)
(1) Suzuka University of Medical Science, Faculty of Pharmaceutical
Sciences, Department of Molecular Pharmacology, Suzuka, Japan
(2) Kanazawa University Graduate School of Medicine, Kanazawa,
Japan
We have demonstrated that glutamate induced glutathione (GSH) depletion and thereby cell death in C6 rat glioma cells. This cell death seemed
to be apoptosis through accumulation of reactive oxygen species (ROS)
or hydroperoxides. We checked effect of arachidonic acid (AA) and 12lipoxygenase activity in the glutamate-induced cell death. A signicant
increase of 12-lipoxygenase activity was observed in the presence of AA
under the GSH depletion induced by glutamate. AA promoted the glutamate-induced cell death reducing caspase-3 activity and diminishing internucleosomal DNA fragmentation that is thought to represent
apoptosis. Furthermore, AA diminished intracellular NAD, ATP and
membrane potential revealing a dysfunction of mitochondrial membrane.
Protease inhibitors such as TPCK and DCI but no Ac-DEVD, caspase
inhibitor, suppressed the glutamate-induced cell death. These results suggest that AA promotes cell death by inducing to necrosis from caspase-3
independent apoptosis through lipid peroxidation initiated by ROS or
lipid hydroperoxides generated during the GSH depletion in C6 cells.
Paper No.: 2358

GENTAMICIN PHARMACOKINETICS AND DOSING IN
OLDER PEOPLE: IS FRAILTY A FACTOR?
Sarah Hilmer(1), K Tran(1), P Rubie(1), J Wright(1), S Matthews(2),
D Gnjidic(1), P Carroll(2)
(1) Royal North Shore Hospital and University of Sydney, Department
of Clinical Pharmacology and Aged Care, St Leonards, NSW, Australia
(2) University of Sydney, St Leonards, NSW, Australia
Frailty is associated with sarcopaenia and increased susceptibility to disease and may affect clinical pharmacology. We aimed to determine
whether frailty is associated with the pharmacokinetics of gentamicin
and its role in dosing guidelines. An observational study of gentamicin
pharmacokinetics was performed in 31 inpatients aged >65 years who
were administered gentamicin prophylaxis during urologic surgery in
three hospitals in Sydney, Australia. Frailty was assessed using the Edmonton Frail Scale. Creatinine clearance (CrCl) was calculated with the
Cockcroft Gault equation and estimated glomerular ltration rate (eGFR)
with the Modication of Diet in Renal Disease equation. Gentamicin levels were measured 30 minutes, 2 hours and 4-6 hours after administration. Gentamicin volume of distribution (Vd) and clearance were
calculated using Target Concentration Intervention software (www.tciworks.info). Bland Altman analyses were performed to compare CrCl
and eGFR with gentamicin clearance. Frail patients were older and
shorter than non-frail (frail 81 + /-6 years, 167.2 + /-4.6cm, n = 14; nonfrail 74 + /-7 years,172.1 + /-6.4cm, n = 17). Mean Vd was 14.8 + /1.4L in frail and 15.2 + /-2.2L in non-frail (ns). Gentamicin clearance
was signicantly lower in frail participants (frail 46.6 + /-10.7mL/min,
non-frail 58.2 + /-2.2mL/min, p = 0.01). In frail and non-frail participants, the best estimate of gentamicin clearance was by CrCl calculated
using ideal body weight (mean difference -0.16mL/min), with overestimates by CrCl calculated using actual body weight (10.62mL/min) and
by eGFR (16.49mL/min). In older people frailty is associated with a
trend towards reduced Vd and signicantly lower clearance of gentamicin. Gentamicin clearance is best estimated with CrCl calculated using
ideal body weight in frail and non-frail older people.
Paper No.: 2256

CAN DRUG REMOVALS INVOLVING CYCLOOXYGENASE
INHIBITORS BE AVOIDED? RESULTS FROM VOLUNTEER
STUDIES USING EX VIVO WHOLE BLOOD ANALYSES
Burkhard Hinz(1), K Brune(2)
(1) University of Rostock, Institute of Toxicology and Pharmacology,
Rostock,Germany
(2) University of Erlangen, Institute of Experimental and Clinical Pharmacology and Toxicology, Erlangen, Germany
Within the past 20 years many cyclooxygenase (COX) inhibitors were
removed for unwanted drug effects. As compared to COX inhibitors still
available, all these compounds share the same mode of action, but differ
considerably in their pharmacokinetics. Evidence is provided here that
the ex vivo human whole blood assay could serve as a valuable tool for
dening the lowest effective dose and adequate dosing intervals. Previous work indicates that an 80% COX-2 inhibition in this assay is necessary for pain relief, whereas only a greater than 95% COX-1 inhibition
confers clinically relevant inhibition of platelet aggregation. Using this
330
technique, we could show that oral administration of 50 mg lumiracoxib
to human volunteers inhibits COX-2 to a comparable degree as 100 or
200 mg suggesting doses lower than 100 mg as sufcient for pain therapy (Hinz et al., Ann Rheum Dis 2009;68:289-91). Oral administration
of 1 g dipyrone (INN, metamizol) elicits a greater than 80% inhibition of
COX-2, but also a transient, greater than 95% COX-1 inhibition, suggesting that higher doses may add little to the effect, but rather increase
unwanted drug effects (Hinz et al., FASEB J 2007;21:2343-51). Administration of 1 g acetaminophen, a drug long been claimed to work centrally only, revealed no COX-1 blockade relevant for platelet inhibition,
but an about 80% inhibition of intravascular COX-2 (Hinz et al., FASEB
J 2008:22:383-90), indicating that an evaluation of acetaminophens cardiovascular risk is warranted. Collectively, the ex vivo whole blood system may predict critical aspects of COX inhibition in man.
Paper No.: 1598

DISEASES
ROLE OF NITRIC OXIDE IN METHOTREXATE-INDUCED
ABNORMAL 5-HYDROXYTRYPTAMINE METABOLISM AND
DAMAGE IN THE RAT SMALL INTESTINE
Masahiko Hirafuji, Y Takano, M Hirano, A Kamiya, T Machida,
N Hamaue, K Iizuka
Health Sciences University of Hokkaido, Faculty of Pharmaceutical
Sciences, Department of Pharmacology, Hokkaido, Japan
Methotrexate (MTX) is a cytotoxic drug widely used for cancer chemotherapy of leukemia and other malignancies. However, chemotherapeutic
doses of MTX often cause severe side effects such as small intestinal
damage or moderate nausea and vomiting. 5-Hydroxytryptamine (5-HT)
synthesized by and released from enterochromafn (EC) cells in small
intestine is considered to play important roles in the mechanism of gastrointestinal damage by cytotoxic drugs. We have investigated the effect
of MTX administration on the 5-HT metabolism and small intestinal
damage in the rat. A single dose of MTX (50 mg/kg, i.p.) caused signicant increases in ileal 5-HT content and tryptophan hydroxylase1 (TPH1)
mRNA expression 96 hr after administration. Immunohistochemical
staining using anti-TPH antibody showed that the number of TPH-immunoreactive EC cells in the ileal surface epithelium and crypts were significantly increased by MTX administration. MTX also caused signicant
increases in mRNA and protein expression of inducible nitric oxide synthase (iNOS) with concomitant increase in nitric oxide (NO) production
96 hr after administration, but had no effect on mRNA or protein expression of endothelial NOS, neuronal NOS, cyclooxygenase-1 and cyclooxygenase-2. Daily administration of dexamethasone (1 mg/kg, s.c.) or NGnitro-L-arginine methyl ester (L-NAME; 20 mg/kg, s.c.), a NOS inhibitor, inhibited the increased ileal 5-HT content and EC cell hyperplasia
induced by MTX administration. L-NAME also inhibited MTX-induced
histological damage to the ileal mucosa. These results suggest that
iNOS-derived NO plays a key role in MTX-induced abnormal 5-HT
metabolism and small intestinal damage in the rat.
Paper No.: 3335

FISH OIL AFFECTS CLOCK GENE AND CLOCKCONTROLLED METABOLISM-RELATED GENE EXPRESSION
IN THE MOUSE LIVER
Akiko Hirao, H Nagahama, M Itokawa, M Shimoda, Y Tahara,
S Shibata
Waseda University School of Advanced Science and Engineering,
Laboratory of Physiology and Pharmacology, Tokyo, Japan
[Background] For a long time fat has been selected as a source of energy,
so there were no reasons for the selection of fat provided from animals
or plants. However, Fish oil has been closed up as The third oil
recently. The sh oil containing abundant unsaturated fatty acid shows
the decrease of cholesterol level and the density of the visceral fat. On
the other hand, circadian clock is reported to control the metabolism of
lipid and cholesterol, because mutation of clock gene causes obesity and
hypercholesteremia. Then, I asked whether sh oil improves the obesity
through affecting clock gene expression and clock-controlled metabolism
related gene expression. [Method and procedure] Mouse body weight
and the amount of food intake are measured under high sucrose containing diet. In order to examine which metabolic pathway is related to the
sh oil-induced anti-obesity effect, various clock genes and metabolism
related genes expression were observed by RT-PCR. In the following
experiments, EPA and DHA were prepared for the experiments, because
these chemicals were highly contained in the sh oil. [Results and Discussion] Mouse body weight gain in sh oil group decreased in comparison to soybean oil group, although the amount of food intake was same
between two groups. Glut2 gene, which is controlled under clock, and
Per2 gene expression were higher in sh oil group than in soybean oil
group, suggesting that sh oil may possess anti-obesity activity through
not only metabolism but clock systems.
Paper No.: 2859

ASSOCIATION OF GENETIC VARIANTS OF UGT1A1 GENE
WITH SERUM BILIRUBIN LEVEL IN THE JAPANESE
Akira Hirasawa(1), T Akahane(1), T Tsuruta(1), K Saito(2), T Zama(2),
Y Tanigawara(3), N Susumu(1), D Aoki(1)
(1) Keio University School of Medicine, Department of Gynaecology
and Obstetrics,Tokyo, Japan
(2) Keio University School of Medicine, Department of Otolaryngo,
Head/Neck Surgery, Tokyo, Japan
(3) Keio University School of Medicine, Department of Hospital Pharmacy, Tokyo, Japan
Irinotecan is metabolized to active form which is further conjugated and
detoxied by the UGT1A1 enzyme. The severe toxicities in patients who
receive irinotecan are related to its genetic variants, and the serum total
bilirubin (BIL) levels are useful for the prediction of adverse reactions to
irinotecan. However, the relationship between the specic UGT1A1
genotype and BIL levels has not yet been conrmed. Then, we studied
443 cases with no history of liver dysfunction to explore their relationship. Genomic DNAs were extracted from peripheral leukocytes after the
informed consent was obtained. The assays for genotyping the polymorphisms in the UGT1A1 (*6, *27, *28, *60) were based on either Invader
assay or direct sequencing. The frequencies of *28 and *60 variants were
much lower than those published results for whites, while the frequencies
of *6 and *27 variants were much higher. Furthermore, 89% (24/27)
subjects with hyperbilirubinemia had *6, *28 or *60 variants. This study
showed that several UGT1A1 genotypes were signicantly associated
with the increased BIL levels. These ndings will be useful for further
pharmacogenomical studies on adverse reactions to irinotecan.
Paper No.: 862

UBIQUITIN/PROTEASOME-DEPENDENT DOWN-REGULATION FOLLOWING CLATHRIN-MEDIATED INTERNALIZATION OF HISTAMINE H1-RECEPTORS IN CHINESE
HAMSTER OVARY CELLS
Shigeru Hishinuma(1), H Komazaki(1), H Fukui(2), M Shoji(1)
331
(1) Meiji Pharmaceutical University, Department of Pharmacodynamics,
Tokyo, Japan
(2) University of Tokushima, Graduate School of Health Biosciences,
Department of Molecular Pharmacology, Tokushima, Japan
We investigated the regulatory pathways responsible for agonist-induced
internalization and down-regulation of Gq protein-coupled histamine H1receptors in Chinese hamster ovary cells. Histamine-induced internalization and down-regulation of H1-receptors were detected as the loss of
[3H]mepyramine binding sites on intact cells accessible to hydrophilic
and hydrophobic H1-receptor antagonists, pirdonium and mepyramine,
respectively. Pretreatment of cells with 0.1 mM histamine for 30 min at
37C induced internalization as well as down-regulation of H1-receptors,
both of which were inhibited either in the presence of an inhibitor against
G protein-coupled receptor kinases (ZnCl2) or under hypertonic conditions where clathrin-dependent endocytosis is known to be inhibited, but
were not affected by inhibitors against caveolae/raft-dependent endocytosis (lipin and nystatin). Down-regulation of H1-receptors, but not their
internalization, was inhibited by protein kinase C inhibitors (chelerythrin
or GF109203X), a ubiquitin E1 inhibitor (UBEI-41) and proteasome
inhibitors (lactacystin and MG-132). Neither a Ca2 + /calmodulin-dependent protein kinase II inhibitor (KN-62) nor lysosomal protease inhibitors
(E-64, leupeptin, chloroquine and NH4Cl) affected the internalization
and down-regulation of H1-receptors. These results suggest that agonistinduced down-regulation of histamine H1-receptors is regulated by the
protein kinase C/ubiquitin/proteasome-dependent but the lysosome-independent degradation pathway, following G protein-coupled receptor
kinase/clathrin-dependent but caveolae/raft-independent internalization.
Paper No.: 1378

HEALTH AND DISEASE
VASCULAR ACTIONS OF THE CALCIMIMETIC,
CINACALCET
Wing Sze Vanessa Ho, Pratish Thakore
Paper No.: 1499

ANGIOTENSIN II TYPE 2 RECEPTOR ACTIVATION
MODULATES INSULIN RECEPTOR SIGNALLING PATHWAYS
IN ADULT SENSORY NEURONS
Narumi Hobara(1), N Hashikawa(1), Y Zamami(2), H Kawasaki(2),
C Yutani(1)
(1) Okayama University of Science, Department of Life Science,
Okayama, Japan
(2) Okayama University,Okayama, Japan
Diabetes and insulin resistance are associated with an increased risk of
hypertension. Previously, we reported chronic hyperinsulinemia enhances
adrenergic vasoconstiriction and decrease calcitonin gene-related peptide
(CGRP)-containing nerve-mediated vasodilation in fructose drinking rats
(FDR), which have insulin resistance. We also found that activation of
angiotensin II type 2 receptors (AT2R), which is well known to act as
mediate vasodilation, facilitates CGRP nerve regeneration. However, the
exact role of AT2 R with a focus on insulin resistance is not clear. In
present study, we examined AT2R functionally expressed in neuron with
insulin resistance. To investigate the association between hyperinsulinemia and nerve remodeling, we treated rats with 15% fructose solution
in drinking uid for 4 weeks, which have insulin resistance and assessed
effects of neurites outgrowth in relation to the expression of angiotensin
II receptors. We also examined whether AT2R stimulation modulates
insulin receptor signalling pathways. FDR dorsal root ganglia (DRG),
which is a prominent site of CGRP, was increased AT2R protein expression but not AT1R protein expression. Using FDR DRG neurons we have
demonstrated that effect of CGP42112 (AT2R agonist) compared with
control rats, in promoting extended CGRP-like immunoreactive nerve.
The neurites outgrowth effect of CGP42112 was much less in FDR DRG
neurons. LY294002, a phosphoinositide 3-kinase (PI3 kinase) inhibitor
decreased AT2R-mediated neurites outgrowth in control rats DRG. These
data demonstrate that chronic hyperinsulinemia decline AT2R function
and involve in PI3 kinase pathway activity related to nerve regeneration.
St Georges University of London, Division of Basic Medical Sciences,

London, UK
Calcium-sensing receptors (CaR) are G-protein-coupled receptors that
are activated by Ca2 + ions. Cinacalcet, which is a positive allosteric
modulator of CaR on parathyroid cells, suppresses parathyroid hormone
(PTH) secretion and is therefore used to treat hyperparathyroidism. However, cinacalcet and other calcimimetics can also modulate blood pressure independently of PTH levels, possibly due to CaR in the
vasculature (Smajilovic S & Tfelt-Hansen J, Hypertension 2008; 52:9941000). Here, we examined the vascular actions of cinacalcet in isolated
resistance arteries. Segments (2mm long) of the third generation of superior mesenteric artery from male Wistar rats (200-300g) were mounted in
a wire myograph for tension recording. Data are expressed as means.e.m. (n 4 rats) and analyzed by one-way analysis of variance or Students t-tests. P < 0.05 was considered statistically signicant. In vessels
precontracted with 10lM methoxamine, cinacalcet induced endothelium
(EC)-independent relaxation (+EC: pEC50 = 5.58 0.07; Rmax=97
5%; -EC: pEC50 = 5.59 0.06; Rmax=91 5%). The presence of calhex231 (negative allosteric modulator of CaR; 3lM) or iberiotoxin
(inhibitor of large conductance Ca2 + -activated K+ channels; 50nM), or
desensitization of perivascular sensory nerves by capsaicin (10lM for
1h) had no effect on relaxation to cinacalcet (-EC: control, pEC50 = 5.56
0.04; Rmax=92 3%; +calhex231, pEC50 = 5.50 0.04; Rmax=96
3%; +iberiotoxin: pEC50 = 5.49 0.1; Rmax=95 7%; +capsaicin,
pEC50 = 5.51 0.04; Rmax=94 4%). Reducing the extracellular [Ca2
+
] from 2 to 0.5mM also had no effect (data not shown). However, precontracting the vessels with 60mM KCl, instead of methoxamine, attenuated cinacalcet relaxations (KCl: pEC50 = 5.24 0.09; Rmax=103
5%; P < 0.05). To conclude, cinacalcet induced potent relaxation of rat
small mesenteric arteries independently of CaR, despite the reported
expression of CaR in endothelium and perivascular sensory neves.
Paper No.: 3386

SHORT TERM IMPACTS OF RATIONAL PHARMACOTHERAPY COURSE ON THE RATIONAL PRESCRIBING SKILS OF
FOURTH-YEAR MEDICAL STUDENTS
Nil Hocaoglu, H Guven, S Gidener, Y Tuncok, S Kalkan, M Guneli,
A Gelal
Dokuz Eylul University School of Medicine, Department of Pharmacology, Izmir, Turkey
In academic year 2008/9, University of Dokuz Eylul Medical School
decided to introduce a new course on rational pharmacotherapy (RPT).
Five day-RPT course was entegrated to the curriculum. The course was
organised in the beginning of the fourth-year. The aim of the present
study was to evaluate the short term impacts of RPT course on rational
prescribing skills of the students. The impact of the RPT course was
measured by pre/post-test design by an objective structured clinical
examination (OSCE) for 2008/9 (group 1) and 2009/10 (group 2) school
year. Problem solving steps (1- denition of the patients problem,
2- determination of treatment aims, 3- determination of non-pharmacological treatment, 4- selection of the personal drug and treatment options,
5- evaluation of drug eligibility for patient, 6- evaluation of prescription,
7-giving information for prescribed drugs a. instructions for drug use, b.
information about the adverse effects, c. next appointment; total 80
points) and communication skills (20 points) were assessed. One hundred
twenty nine (group 1) and 136 (group 2) students participated to the
332
courses. There was not any signicant difference in the total scores of
pre-tests among the groups (p > 0.05, 17.5 1.1 and 14.8 10.5 for
group 1 and group 2, respectively). In both groups, the mean scores of
the post-tests were signicantly higher than the pre-tests (p < 0.0001,
68.6 10.4 and 73.2 11.2, for group 1 and group 2, respectively). The
present study demonstrated that the fourth-year medical students markedly beneted from the RPT course in developing rational prescribing
skills.
Paper No. 879

FOCUS GROUP: P13 - MAXIMISING BENEFITS AND
PHARMACOKINETICS, TOLERABILITY, AND SAFETY OF
ALMOREXANT IN JAPANESE AND CAUCASIAN HEALTHY
MALE SUBJECTS
Matthias Hoch(1), P Hoever(1), F Alessi(2), J Marjason(3),
J Dingemanse(1)
(1) Actelion Pharmaceuticals Ltd, Department of Clinical Pharmacology,
Allschwil, Switzerland
(2) Actelion Pharmaceuticals Ltd, Department of Biometry, Imperia, Italy
(3) Q-Pharm Pty Limited, Herston, Australia
A single-center, open-label, parallel group study was conducted to compare the pharmacokinetics, tolerability, and safety of single doses of the
dual orexin receptor antagonist almorexant (200 mg) in Japanese and
Caucasian healthy male subjects (n = 10 in each group, body weight
matched [ 5%]). The plasma concentration-time prole of almorexant
was characterized by a rapid absorption and disposition. The pharmacokinetic parameters of Japanese versus Caucasian subjects were similar.
Peak plasma concentrations (C[max]) were reached at approximately 1.0
hour in both ethnic groups. C[max] was 13% lower (ratio of geometric
means [90% CI]: 0.87 [0.53, 1.43]), and the total exposure (AUC[0-])
was 15% smaller (0.85 [0.54, 1.35]) in the Japanese subjects. There was
no difference in the geometric mean ratio for t[] between the two ethnic
groups (1.03 [0.84, 1.25]). Almorexant was well tolerated. No clinically
signicant treatment-emergent abnormalities in clinical laboratory, vital
signs, or ECG parameters were observed. The only observed AEs were
somnolence, dizziness, and anemia. All events were of mild to moderate
intensity, without clear difference in the number of reported AEs between
the two ethnic groups. In conclusion, the pharmacokinetics, tolerability,
and safety proles of almorexant in Japanese and Caucasian healthy male
subjects are similar. No dose adjustments for clinical studies with almorexant in Japanese subjects appear to be needed.
Paper No.: 2362

THERAPEUTIC TARGETS
THE EFFECT OF CAPTOPRIL ON THE ACTIVITY OF SELENOENZYMES THIOREDOXIN REDUCTASE AND GLUTATHIONE PEROXIDASE, REDOX HOMEOSTASIS AND TRACE
ELEMENT LEVEL IN RAT LIVER
Anna Hodkova(1), D Kotyzova, V Eybl
3 days with CAP (50mg/kg b.w.). The experiment was nished at the
24th h after the last dose of CAP. The level of reduced glutathione
(GSH), lipid peroxidation (LP; MDA concentration), catalase (CAT) and
the activities of following enzymes: GPx-1, TrxR-1, glutathione reductase (GR) and superoxide dismutase (SOD) were estimated in liver homogenates. The content of trace elements (Cu, Fe, Zn) in the liver tissue
was determined by AAS. Results: The LP level and TrxR-1, GR, CAT
and SOD activities were decreased by CAP by 30,9% (p < 0,01), 22,0%
(p < 0,01), 11,6% (p < 0,01), 6,6% (p < 0,05) and 31,6% (p < 0,01)
respectively. GPx-1 activity was increased by CAP by 9,1% (p < 0,05).
The level of GSH was not changed. CAP didnt change the trace element
concentration. Conclusion: Captopril signicantly inuenced the redox
homeostasis in the acute experiment in rats. The acute inhibitory effect
of captopril on the activity of TrxR-1 represents an important result.
Supported by the Grant MSM of CZE 0021620819.
Paper No.: 2096

DISTINCT ROLES FOR THE NUCLEAR RECEPTORS NUR77
AND NOR-1 IN DOPAMINE-MEDIATED AND
PSYCHOSTIMULANT-INDUCED BEHAVIORS
Celine Hodler(1), B Paquet(1), J Baillargeon(1), P Seeman(2),
D Levesque(3), C Rouillard(1)
(1) Laval University, Department of Psychiatry and Neuroscience,
Quebec, Canada
(2) University of Toronto, Department of Pharmacology, Toronto,
Canada
(3) University of Montreal, Faculty of Pharmacy, Montreal, Canada
The nuclear receptors Nur77 and Nor-1 are both mainly expressed in target structures of dopamine systems. Using knock-out mice, we investigated the roles of these nuclear receptors in dopamine-mediated and
psychostimulant-induced behaviours. In untreated animals, Nur77 knockout mice showed an enhanced wheel-running activity compared to
Nur77 (+/+) animals whereas Nor-1 knock-out mice displayed a signicant lower basal activity compared to their wild-type littermates. When
exposed to multiple amphetamine administrations, Nur77 knock-out mice
displayed an enhanced response at every time point compared to Nur77
(+/+). On the contrary, Nor-1 knock-out mice had an equivalent or
slightly lower response to amphetamine compared to their WT littermates
and failed to demonstrate behavioural sensitization when challenged
again with amphetamine 5 days later. Interestingly, Nur77 knock-out
mice displayed an upregulated level of striatal high afnity D2 receptors
whereas Nor-1 knock-out animals are characterized by a reduced level of
D2-high receptor level. In addition, striatal enkephalin mRNA expression
is upregulated in Nur77 knock-out mice whereas it is down-regulated in
Nor-1 knock-out animals. All mouse strains developed a signicant
cocaine-induced place preference (CPP) which persisted for up to 21
days. However, CPP values in Nur77 knock-out mice returned to their
pre-conditioning level at day 21. These results demonstrate that Nur77
and Nor-1 have distinct roles in the regulation of DA-related behaviours
that is consistent with molecular differences observed in these mouse
strains.
Supported by the Canadian Institutes for Health Research (CIHR).
Charles University in Prague, Faculty of Medicine in Pilsen, Department

of Pharmacology and Toxicology, Pilsen, Czech Republic
Introduction: Captopril (CAP), ACE inhibitor, is a free radical scavenger,
antioxidant and Fe/Cu chelator. However, little attention has been paid to
the inuence of CAP on the activity of selenoenzymes, mainly thioredoxin reductase (TrxR) and glutathione peroxidase (GPx) - important
antioxidant defences. We investigated the effect of CAP on the activity
of antioxidative defences inclusive these selenoenzymes in an acute
experiment. Materials: This experiment was performed in male Wistar
rats (130140g b.w.), which were randomly divided into 2 groups of 8
animals each: I control, II CAP. Rats were treated po once daily for
Paper No.: 3159

THE PLATELET ACTIVATOR SUCCINATE - A POSSIBLY
DRUG CANDIDATE FOR THE FUTURE?
Carl Hogberg, O Gidlof, B Olde, D Erlinge
Lund University Hospital, Department of Cardiology Lund, Sweden
333
Our group has in a previous work, based on qRT-PCR on platelet membrane receptors, identied a few new unknown orphans GPCR. Amongst
these identied orphan GPCRs was the succinate receptor, GPR91. The
mediator to GPR91 is the dicarboxylic acid, succinate. Succinate is
known to potenciate the agonistic action of other more powerful ligands,
like ADP, and hence increases the platelet activation and aggregation.
Our main focus for this study was to investigate the capacity of succinate
to activate the human platelet, and if so, how does the intracellular signalling response, mediated through GPR91, behave functionally? Human
platelets was treated with succinate and used for; light transmission
aggregometry - for physiological experiment with succinate treated platelets alone with antagonists; ow cytometry - to study platelet activation
through PAC1 (marker for GPaIII/bII activation) and p-selectine (marker
for granular release of ADP and ATP etc.); cyclic adenosine mono-phosphate (cAMP) -assay, to measure succinate impact on the intracellular
cAMP production and calcium ux to measure the release of intracellular
calcium and further activation. The summarized results from this study
by these methods conclude that succinate truly has an ability of its own
to induce platelet aggregation. The results also show that the succinate
receptor, GPR91, most likely is a Gi-coupled receptor which attenuates
the cAMP production, leading to platelet activation which initiating
platelets aggregation.
Paper No.: 1000

A GERMAN LIST OF POTENTIALLY INAPPROPRIATE
MEDICATION IN THE ELDERLY
S Holt(1), S Schmiedl(1,2), Petra A Thurmann(1,2)
(1) University Witten/Herdecke, Department of Clinical Pharmacology,
Wuppertal, Germany
(2) Philipp Klee-Institute of Clinical Pharmacology, HELIOS Clinic,
Wuppertal, Germany
Several drugs are classied as potentially inappropriate medication (PIM)
for elderly patients due to their increased risk for causing adverse drug
reactions (Laroche ML et al. CP&T 2009;85:94-97). Since published
PIM lists from the US or France may not be suitable for Germany
because of differences in drug availability, prescription behaviour and
guidelines, we aimed to develop a German PIM list for elderly patients.
After an extensive literature search we created a preliminary German
PIM list. Thereafter 25 German speaking experts of 7 different specialties
rated these drugs with regard to e.g. comorbidities using a 5-point Likert
scale during a web-based, modied two round Delphi survey (RAND
Corporation 1969). For certain drugs, experts differentiated between high
and low doses and immediate vs. extended release preparations, respectively. 83 drugs were rated nally as PIM, e.g. indomethacin and uphenazin. In 46 drugs, experts rating failed to make a clear decision after
the second round. For several PIM-drugs alternatives were suggested. In
case the prescription of PIM cannot be avoided due to lack of alternatives, the nal German PIM-list includes recommendations regarding to
e.g. monitoring parameters or dose reduction. A considerable number of
frequently used drugs were considered to be potentially inappropriate for
elderly patients by a group of German speaking experts. The nal PIM
list will be validated evaluating the relationship between usage of PIM
and the occurrence of clinically relevant adverse effects in several
cohorts of elderly German patients.
Supported by BMBF Fo-Nr. 01ET0721
Paper No.: 1500

POTENTIALLY INAPPROPRIATE MEDICATION IN A GERMAN STROKE COHORT
S Holt(1), Jacek Szymanski(1,2), C Diederichs(3), PA Thurmann(1,2),
K Berger(3)
(1) University Witten/Herdecke, Department of Clinical Pharmacology,

Wuppertal, Germany
(2) Philipp Klee-Institute of Clinical Pharmacology, HELIOS Clinic
Wuppertal, Germany
(3) University of Munster, Institute of Epidemiology and Social
Medicine, Munster, Germany
Elderly patients receive the highest proportion of all drugs in most countries. Several of these drugs are classied as potentially inappropriate
medication (PIM) for elderly patients due to their increased risk for causing adverse drug reactions (ADR). Based on the newly developed German PIM list, which is adapted to the German market and prescribing
patterns (Holt S et al. BJCP 2009;68(Suppl.1):19), we aimed to analyze
PIM utilisation in elderly stroke survivors. Demographic data, medical
history, ADR and current medication from 756 patients of the Stroke
Database North-West-Germany (70 11 years, 40.5% females) were collected on average 3.7 years after the stroke during face-to-face interviews
or by postal questionnaires (Schneider K et al. Nervenheilkunde
2009;28:114-118). Medication use was evaluated with regard to polypharmacy and the prevalence of PIM. 728 of the 756 patients used at
least one drug, the average number of medication was 6 3 drugs (range
1-21). 65.1% of them used 5 or more drugs. In 137 patients (19%) at
least one PIM was found, 2% used two or more PIM. The most commonly observed PIM were acetyldigoxine, piracetam and amitriptyline
(11.8%, 8.5%, 6.5% of all PIM respectively). Polypharmacy occurs frequently in elderly stroke patients. The percentage of PIM reects the
19.8% average prevalence of PIM in a cross national survey of 8 European countries (Fialova D et al. JAMA 2005;293:1348-1358). Further
research with regard to the relationship between multimorbidity, clinical
functioning, polypharmacy, PIM and ADR is required.
Supported by BMBF grants 01ET0721 and 01ET0723
Paper No.: 1290

PHARMACOKINETIC DRUG INTERACTION OF ENOXACIN
AND TIZANIDINE VIA CYP1A2 INHIBITION
Masato Homma, K Momo, T Kobayashi, Y Kohda
University of Tsukuba, Department of Pharmaceutical Sciences, Tsukuba,
Japan
Enoxacin (ENX), a new quinolone antibiotic agent, possesses inhibitory
activity on CYP1A2, a principal enzyme for tizanidine (Tz) metabolism. Co-administration of ENX with Tz, therefore, potentially
enhances pharmacodynamics of Tz via pharmacokinetic drug interaction, though the details have been unclear. To assess the clinical impact
of this drug interaction, we investigated effects of ENX co-administration on Tz pharmacokinetics in healthy subjects. Open label cross-over
study was performed in seven males (non-smokers, 27.1 3.4 yrs).
Plasma Tz concentrations and reduction in blood pressure and heart
rate were examined after single dose of Tz (2 mg) with and without
pre-treatment of ENX (100 mg, 7 times for 3 days). Pharmacokinetic
parameters of Tz (area under the concentration - time curves: AUC,
maximum concentration: Cmax and clearance: CL) were compared
between control (Tz alone) and ENX phase. Urinary praxanthine/caffeine ratio as an index for CYP1A2 activity was also measured. AUC
and Cmax for Tz in ENX phase signicantly higher than control
(AUC: 27.4 9.5 vs. 5.1 2.8 2.8 nghr/mL, Cmax: 8.5 3.0 vs. 1.9
1.0 ng/mL, P < 0.01). CL for Tz and urinary praxanthine/caffeine
ratios in ENX phase were signicantly lower than control (CL: 1.3
0.6 vs. 8.3 5.4 L/hr/kg, P < 0.01, praxanthine/caffeine ratios: 7 5
vs. 50 43, p < 0.05). Reductions in blood pressure and heart rate
after Tz dose were also enhanced in ENX phase (systolic blood pressure: -32 7 vs. -10 10 mmHg, p < 0.05, heart rate: -24 9 vs. -9
8 beats/min, P < 0.01). It was conrmed that ENX co-administration
enhanced pharmacodynamics of Tz due to pharmacokinetic drug interaction via CYP1A2 inhibition.
334
Paper No.: 2327
FOCUSED CONFERENCE GROUP: P12 - ION CHANNELOPATHIES: NEW WINDOWS ON COMPLEX DISEASE AND THERAPY
SIMULATION OF THE EFFECT OF THE PHASPHORYLATION
ON THE GAP JUNCTION CHANNEL CLOSURE BY COMPUTATINAL CHEMISTRY
Paper No.: 3239

MODIFICATION OF PERIVASCULAR ADIPOSE TISSUE
(PVAT) BY DIABETES MELLITUS
Enrique Hong, O Echeverra
Nobuo Homma
Teikyo University of Science and Technology, Department of Occupational Therapy, Uenohara, Yamanashi, Japan
Six connexin43 (CX43) make a connexon (hemichannel), and two connexons couple and then make a gap junction channel. The intracellular
carboxyl terminal lesion (CT) of Cx43 follows to a ball and chain mechanism and is related to the closure of Cx43 channel. The phosphorylation
of carboxyl terminal lesion (CT S255-I382) contributes to the cell-cell
communication in various tissues. The CT is reported to be important for
channel closure even in expressed as an independent protein. A serine at
262 (S262) and a serine at 282 (S282) in CT seem to be involved in this
regulation. We applied the NMR data of rat CX43 to 3-dimensional (3D)
molecular Modelling and we caliculate and visualized eleven candidate
3D structure by computational chemistry method. We also imaginary
introduced phosphorylation into S262 and/or S282 and then calculated/
optimized the 3D structure of CT by computer simulation. The phosphorylation at S262 was related to the structural change of the proximal lesion
of CT but the phosphorylation at 282 less affected the 3D structure of CT.
Other functional lesions (helix1 A315-T362, Helix2 D340-A348, ZO1
binding domain D379-I382) were not affected in their 3D structure by the
imaginary phosphorylation. Those results indicate that the phosphorylation at S262 might be important for regulating the function of gap junction channel. It may be useful for molecular pharmacology to simulate
and understand the 3D behavior of a small-sized peptide in the cell.
Paper No.: 1101

EARLY INDICATIONS FOR CLINICAL EFFICACY OF
DIABETES DRUGS IN DEVELOPMENT IN PHASE 1 STUDIES:
HOW?
Center for Research and Advanced Studies of National Polytechnic Institute (CINVESTAV), Department of Pharmacology, Mexico DF, Mexico
PVAT modulates vascular smooth muscle contraction induced by several
agonists; this effect is endothelial dependent and due to release of a substance. Therefore, it was decided to observe if such mechanism was also
present in diabetic animals. Rats were treated with either 60 mg/kg of
streptozotocin for type 1 diabetes (T1D), or with 65 mg/kg of streptozotocin plus 230 mg/kg of nicotinamide for type 2 diabetes (T2D) and rats
were sacriced 10 or 20 weeks afterT1D or T2D, respectively. The aorta
was removed and aortic rings with and without PVAT were placed in isolated tissue baths. Rings of control rats with PVAT produced smaller
responses to noradrenaline, phenylephrine and 5-HT, than rings without
PVAT. Incubation of rings with captopril or losartan increases the modulatory effects of PVAT. Rings from rats with T2D showed a modulation
of the contractile response to norepinephrine, phenylephrine and 5-HT,
but such effect was of lesser magnitude than in controls. However, rings
from T1D rats showed an increase of the contractile responses to 5-HT,
phenylephrine and noradrenaline in rings with PVAT. Therefore, the renin
angiotensin system seems to be involved in these effects, since both captopril and losartan increases the modulatory effect of PVAT on the vasocontractil responses to various agonists.
Partially supported by CONACYT 14470 and by ICYTDF PICSD0824.
Paper No.: 1485

FOCUSED CONFERENCE GROUP: P16 - NATURAL PRODUCTS: PAST AND FUTURE?
GINKGOLIDE B SLOWS THE CYSTOGENESIS AND GROWTH
IN MDCK AND EMBRYONIC KIDNEY MODELS OF POLYCYSTIC KIDNEY DISEASE VIA PKA-DEPENDENT PATHWAY
GINKGOLIDE B SLOWS THE CYSTOGENESIS AND GROWTH
IN MDCK AND EMBRYONIC KIDNEY MODELS OF POLYCYSTIC KIDNEY DISEASE VIA PKA-DEPENDENT PATHWAY
Zhou Hong, G Jinsheng, Y Baoxue
Marcus Hompesch, L Morrow

Prol Institute for Clinical Research, Inc., Chula Vista, CA, USA
Based on the dramatically increasing number of patients with diabetes,
and hence the need to nd better treatments that can help to effectively
battle the diabetes pandemic, the arsenal of drug candidates is growing
rapidly. Picking up early indicators about a drug candidates potential
clinical efcacy is highly desirable in order to make the drug development process more effective. In looking at the early phase clinical
research toolbox, the pharmacodynamic (PD) characterization of diabetes
compounds is based on a number of different methods, with many of
them being applied in different variations. PD proling is done by means
of Oral Glucose Tolerance testing, Standardized Meal Challenges, Frequently Sampled Intravenous Glucose Tolerance testing, Glucose Clamps
and others. The most relevant downsides of using different and variably
applied methods to obtain information about a very limited number of
relevant early phase clinical PD outcomes are obvious: a) a potential bias
in selecting the method based on investigator awareness or preference; b)
outcome data that arent comparable between studies done on the same
or similar compounds. Data from different studies performed by, and
methods utilized by the authors will be discussed to identify methods that
are more likely than others to provide meaningful and comparable glucometabolic outcome data in early phase clinical studies. Conclusions will
be presented about the different methods regarding their potential utility.
Peking University School of Basic Medical Sciences, Department of

Pharmacology and Key Laboratory of Molecular Cardiovascular Sciences, Ministry of Education, Beijing, PR China
Ginkgolide B (Gink B), the major bioactive component of Ginkgo biloba
extracts, has been reported to play multiple pharmacological roles. The
mechanism it acts in is mainly concerned with the specic antagonism
against platelet-activating factor -stimulated acute pathological responses.
The goal of this study is to characterize the effect of Gink B on the progressive development of cysts induced by PKA-pathway activation in
MDCK and embryonic kidney models of polycystic kidney disease
(PKD). The results indicate that Gink B signicantly inhibited MDCK
cyst formation and expansion in dose-dependent manner (IC50 4.47X108 M). The data also show that Gink B suppressed cyst enlargement up to
74% in an embryonic kidney cyst model. Gink B promoted the MDCK
cell-derived tubule formation by inducing cell differentiation. Gink B
neither induced cytotoxicity and apoptosis, nor affected the cell proliferation rate in MDCK cells at testing concentrations. Furthermore, treating
MDCK cells with Gink B signicantly inhibited forskolin-stimulated the
activation of PKA-dependent pathway, including Ras, B-Raf/Raf-1,
MEK and MAPKs. This inhibitory effect of Gink B on PKA-pathway
may be its crucial mechanism of its preventing or slowing the cystogenesis and growth in MDCK and embryonic kidney model of PKD. These
ndings suggest Gink B could be a potential therapeutic agent used to
treat PKD through regulating PKA-dependent signalling pathways.
335
Paper No.: 2351
STRONG INHIBITION OF CYP2C8 WITHIN 1 HOUR AFTER
GEMFIBROZIL DOSING: ESTIMATION OF THE ONSET OF
CYP2C8 INACTIVATION USING REPAGLINIDE AS AN IN
VIVO PROBE
Johanna Honkalammi, M Niemi, PJ Neuvonen, JT Backman
ated Ca2 + entry via TRPC3 and TRPC6. The increases in [Ca2 + ]i via
TRPC3 and TRPC6 were abolished by deletion of CaM/IP3 receptor
binding (CIRB) domain at the C terminus of TRPC3 and TRPC6, indicating that CIRB domain is critical for ETAR-linked activation of TRPC3
and TRPC6. BiFC system based on the formation of a uorescent complex by two non-uorescent fragments of the yellow uorescent protein
visualized homomeric TRPC3 and heteromeric TRPC3/6 interaction in
the cytoplasm and homomeric TRPC6 interaction in the plasma membrane. These results indicate the multiplicity of activation mechanisms
for ETAR-operated TRPC3 and TRPC6.
Helsinki University and Helsinki University Central Hospital, Department of Clinical Pharmacology, Helsinki, Finland
The cytochrome P450 (CYP) 2C8 enzyme participates in the metabolism
of many drugs, including repaglinide, pioglitazone, rosiglitazone, loperamide, amiodarone, amodiaquine and paclitaxel. Gembrozil causes a
strong and long-lasting inhibition of CYP2C8 due to its metabolite gembrozil glucuronide, which is a mechanism-based inactivator of
CYP2C8. However, the time-course of the beginning of this effect is not
known. We studied the onset of the CYP2C8 inhibitory effect of gembrozil using repaglinide as a CYP2C8 probe substrate. In a randomized
5-phase crossover study, 10 healthy volunteers ingested 0.25 mg repaglinide alone (control phase) or with a single 600 mg oral dose of gembrozil taken 0, 1, 3 or 6 h before repaglinide. The plasma concentrations of
repaglinide, gembrozil and their metabolites were measured. Gembrozil taken 0, 1, 3 or 6 h before repaglinide increased the total AUC of repaglinide 5.1-, 6.6-, 6.8- and 5.5-fold (P < 0.001) and its Cmax 1.5-, 2.2-,
2.3-, and 2.2-fold, compared to control (P < 0.05). The concentrations of
the CYP2C8-dependent repaglinide metabolite M4 were not affected
when gembrozil and repaglinide were administered simultaneously, but
its Cmax and AUC(0-3) were reduced greatly, by more than 80-90%, with
longer intervals (P < 0.001). Overall, the changes in the pharmacokinetics of repaglinide were greatest when repaglinide was taken 1 or 3 h after
gembrozil. In conclusion, a strong inactivation of CYP2C8 is evident
already within 1 h after gembrozil administration. This has implications
in clinical practice and in drug interaction studies, when using gembrozil as a model inhibitor of CYP2C8.
Paper No.: 1179

MOLECULAR MECHANISM OF ETAR-OPERATED CA2 +
ENTRY VIA TRPC CHANNELS AND VISUALIZATION OF
HOMO- AND HETEROMERIC TRPC3/6 INTERACTION IN
LIVING CELLS USING BIFC
Paper No.: 2142

HEALTH AND DISEASE
ISCHEMIC SUPPRESSION OF CEREBRAL ENDOTHELIAL
ENOS: EFFECTS OF ESTROGEN
James Horne, S Duckles, D Krause
University of California School of Medicine University, Department of
Pharmacology, Irvine, CA, USA
Endothelial cells are major components of the neurovascular unit; their
role in maintaining brain and blood vessel function is essential. Recently,
we showed that 17 b-estradiol (E) protects cerebral endothelial cells from
ischemic damage during oxygen/glucose deprivation (OGD). Others have
shown that ischemia suppresses endothelial nitric oxide synthase (eNOS)
(J. Fish et al, ASBMB 2009; 285:810-826), impairing production of the
important endothelial vasodilator, NO. We have previously shown that E
increases eNOS in cerebral endothelium, augmenting endothelial vasodilator capacity. Therefore, we investigated the hypothesis that E treatment
affects levels of eNOS during OGD. Mouse brain microvascular endothelial cells (bEND3) were exposed to OGD for 6 hr in culture; cell viability, as assessed by lactate dehydrogenase leakage, decreased signicantly.
Pretreatment for 48 hr with 20 nM E increased cell viability after OGD.
eNOS protein during normoxia (measured by Western blot) was
increased by E treatment, but eNOS remained suppressed in E-treated
cells after 6 hour OGD, even though cell viability was improved. Thus,
in cerebrovascular endothelium, OGD causes a profound loss of eNOS
that does not appear to be prevented by treatment with E under these
conditions. Future experiments will be necessary to more completely
address the impact of OGD and E on eNOS and NO production and
explore the potential role of NO in the protective effects of E in cerebral
endothelial cells.
Supported by US NIH RO1 HL50775.
Takahiro Horinouchi, T Higa, H Aoyagi, H Asano, T Nishiya,

A Nishimoto, S Miwa
Hokkaido University Graduate School of Medicine, Department of
Cellular Pharmacology, Sapporo-City, Japan
Sustained Ca2 + entry via transient receptor potential canonical (TRPC)
channels (TRPC1-7), which is triggered by stimulation of G protein-coupled receptors including endothelin type A receptor (ETAR), is involved
in progressive reModelling processes of the cardiovascular systems.
However, it remains to be determined which TRPC isoforms are associated with ETAR-operated Ca2 + entry. The purpose of this study is to elucidate TRPC functioning as ETAR-operated Ca2 + channel and its
activation mechanism. In addition, we attempted to visualize interaction
of ETAR-operated TRPC in living cells by using bimolecular uorescence complementation (BiFC) analysis. Functional study with Ca2 +
measurement demonstrated that both TRPC3 and TRPC6 contribute to
ETAR-operated Ca2 + entry. The TRPC3-mediated increase in intracellular Ca2 + concentration ([Ca2 + ]i) induced by ETAR activation consisted
of transient and sustained phases, while Ca2 + inux via TRPC6 was
transient. Pharmacological approach with specic inhibitors revealed that
Gq/11 protein, phospholipase C, Src, phosphoinositide 3-kinase, myosin
light chain kinase, and calmodulin (CaM) are involved in ETAR-medi-
Paper No.: 1194

EFFECT OF CROCUS SATIVUS EXTRACT ON SEIZUREINDUCED EXTRACELLULAR CHANGES OF GLUTAMATE
AND ASPARTATE IN THE RAT HIPPOCAMPUS
Hossein Hosseinzadeh(1), H-R Sadeghnia(2), A Rahimi(1)
(1) Mashhad University of Medical Sciences, School of Pharmacy,
Department of Pharmacodynamics & Toxicology, Pharmaceutical
Research Center, Mashhad, IR Iran
(2) Mashhad University of Medical Sciences, Faculty of Medicine,
Mashhad, Iran
Crocus sativus stigma (saffron) has anticonvulsant effects in both pentylenetetrazole and maximal electroshock models in mice. In the present
study, we investigated the effect of a systemic administration of kainic
acid (KA) on the extracellular glutamate and aspartate levels in the
hippocampus of anesthetized rats. We also examined the effect of
Crocus sativus extract (CSE) and diazepam (DZP) pretreatment on the
336
extracellular levels of these neurotransmitters following KA administration. Under urethane anesthesia (1.2 g/kg), a concentric microdialysis
probe was stereotaxically inserted through implanted guide cannula in
the ventral hippocampus. The rats were then given either saline (10 ml/
kg, i.p., n = 5) or CSE (200 mg/kg or 400mg/kg, i.p., n = 5) and 40 min
later, saline-treated and CSE-treated animals were administrated with KA
(15mg/kg i.p.). A group of rats also received DZP (15mg/kg, i.p., n = 5)
20 min prior to KA administration. Analysis of glutamate and aspartate
in the dialysate was performed by reversed-phase high performance
liquid chromatography with precolumn derivatization with o-phtaldialdehyde (OPA) and uorescence detection. Basal excitatory amino acids
(EAA) levels were not affected by pre-treatment with CSE. Following
KA injection, there was a signicant increase (P < 0.001) in the extracellular EAA levels (about 5- and 3-fold, respectively) at 80 min after injection. Kainate-evoked release of EAA was signicantly reduced by DZP
and CSE (P < 0.001). The results of this study showed that acute systemic injection of Crocus sativus extract reduces the extracellular concentrations of glutamate and aspartate in the rat hippocampus during
seizures induced by KA.
Paper No.: 2173

DISEASES
INHIBITION OF OVALBUMIN-INDUCED ASTHMA-LIKE
REACTIONS IN BALB/C MICE BY THE
ANTI-INFLAMMATORY COMPOUND AMURENSIN H
Qi Hou(1), L Yang(1), S Yuan(1), Z Wu(2), M Wen(3), J Bai(1),
C Yao(1), Y Li(1), M Lin(1), G Cheng(1)
(1) Chinese Academy of Medical Sciences & Peking Union College,
Institute of Materia Medica, Department of Pharmacology, Beijing, PR
China
College, Institute of Cancer Research, Beijing, PR China
(3) Weifang Asthma Hospital, PR China
We investigated the inhibitory effects ofamurensin H (AH), a synthetic
dimer of resveratrol from Vitis amurensis, in ovalbumin (OVA)- induced
asthma mice. Methods: We conducted a 3-week asthmatic and allergic
OVA-induced animal model in BALB/c mice.Amuresin H was orally
administered at doses of 50, 75 and 100 mg/kg for 5 days, and inammatory and asthmatic responses in OVA-sensitized mice were examined.
Results: The experiments showed that AH could signicantly inhibit
TNFa production, and reduce WBC numbers of bronchoalveolar uid
and CD4 + /CD8 + ratio of splenic lymphocytes. AH could also reduce
the ear edema induced by 2,4-dinitro-1-uorobenzene (DNFB), compared to the control group. In addition, AH could reduce the activity of
NF-jB both in OVA-induced asthmatic mice and in NF-jB-luc transfected Raw264.7 cells, and signicantly downregulate ICAM-1 and MMP9
mRNA and protein expression levels. Furthermore, AH signicantly
inhibited TNFa-induced peripheral blood mononuclear cells PBMCs chemotaxis, migration and adhesion to human umbilical vein endothelial
cells HUVECs.Conclusions: These results suggest that inhibition of
NF-jB activity, pro-inammatory factors expression, PBMCs functions
and T lymphycytes activation may represent the underlying mechanisms
by which AH antagonizes inammation and asthma.
(1) Chang Gung Institute of Technology, Department of Nursing, TaoYuan, Taiwan

(2) Chang Gung Institute of Technology, Department of Pharmacology,
Tao-Yuan,Taiwan
Heme oxygenase-1 (HO-1) plays a crucial role in tissue pathological
changes such as brain injuries. Our previous studies have demonstrated
that bradykinin (BK) induces the expression of several inammatory proteins, including matrix metalloproteinase (MMP)-9 and cyclooxygenase
(COX)-2 via MAPKs and NF-jB in rat brain astrocytes (RBA-1). However, the molecular mechanisms underlying BK-induced HO-1 expression in RBA-1 cells remain poorly dened. Here we demonstrated that
BK induced HO-1 expression and enzymatic activity via a B2 BK receptor-activated reactive oxygen species (ROS)-dependent signalling pathway. ROS generation led to activation of extracellular signal-regulated
kinase 1/2 (ERK1/2) and c-Jun-N-terminal kinase (JNK) and then activated the downstream molecules NF-jB and c-Jun, respectively. The cFos, an AP-1 subunit, was up-regulated by activation of NF-jB and cJun, which bound to HO-1 promoter, and thereby turned on transcription
of HO-1 gene. The rat HO-1 promoter containing a putative AP-1 cisbinding site was identied as a crucial domain linking to BK action.
Taken together, these results suggested that in RBA-1 cells, activation of
ERK/NF-jB and JNK/c-Jun cascades by a ROS-dependent event
enhancing c-Fos/AP-1 activity is essential for HO-1 up-regulation and
activation induced by BK.
Paper No.: 1727

DISEASE AND THERAPY
MINOCYCLINE INHIBITS D-AMPHETAMINE-ELICITED
ACTION POTENTIAL BURSTS IN A CENTRAL SNAIL
NEURON
Hui-Yu Hsu, Y-H Chen
China Medical University, Graduate Institute of Acupuncture Science,
Taichung, Taiwan
Minocycline is a second-generation tetracycline that has been reported to
have powerful neuroprotective properties. In our previous studies, we
found that d-amphetamine (AMPH) elicited action potential bursts in an
identiable RP4 neuron of the African snail, Achatina fulica Ferussac.
This study sought to determine the effects of minocycline on the AMPHelicited action potential pattern changes in the central snail neuron using
the two-electrode voltage clamping method. Extracellular application of
AMPH at 0.3 mM elicited action potential bursts in the RP4 neuron. Minocycline (0.3 mM-0.9 mM) inhibited the action potential bursts elicited
by AMPH. Forskolin (0.05 mM), an adenylate cyclase activator, and dibutyryl cAMP, a membrane-permeable cAMP analog (1 mM), restored
the inhibitory effects of minocycline on AMPH-elicited action potential
bursts. Co-administration with forskolin (0.05 mM) plus tetraethylammonium chloride (TEA; 5 mM) or co-administration with TEA (5 mM) and
dibutyryl cAMP (1 mM) also elicited action potential bursts, while these
effects were inhibited by minocycline. In addition, minocycline prevented forskolin from eliciting action potential bursts at the high concentration of 0.1 mM. Notably, TEA (50 mM) and pentylenetetrazol (PTZ;
50 mM) elicited action potential bursts in the RP4 neuron, but these
effects were not affected by minocycline. These results suggest that the
cAMP-protein kinase A signalling pathway is involved in the inhibitory
effects of minocycline against AMPH-elicited action potential bursts.
Paper No.: 1042

ROS-DEPENDENT C-FOS/AP-1 INDUCTION UP-REGULATES
HEME OXYGENASE-1 EXPRESSION BY BRADYKININ IN
BRAIN ASTROCYTES
Paper No.: 1307

P53 IN AMYLOID-B PEPTIDE-INDUCED APOPTOSIS IN
CEREBRAL ENDOTHELIAL CELL
Hsi-Lung Hsieh(1), C-M Yang(2)
Ming-Jen Hsu, Y-F Chuang, P-T Chiu, C-Y Wang, J-R Sheu
337
Taipei Medical University School of Medicine, Department of Pharmacology, Taipei, Taiwan
Accumulation of amyloid-b peptide (A-b) in senile plaques has been
implicated in the pathogenesis of Alzheimers disease. Prevailing evidence
demonstrated that A-b induces neuronal cell death. Recent studies have
shown that Ab also induces non-neuronal cells death. These non-neuronal
cells include astrocytes, oligodendrocytes, and cerebral endothelial cells
(CECs). CECs and astrocytes constitute blood-brain barrier (BBB) which
prevents central nervous system (CNS) from deleterious insults. Therefore, A-b-induced CEC death may lead to BBB breakdown and cerebrovascular degeneration. Our previous study revealed that A-b may activate
p38MAPK-mediated signalling to cause p53 phosphorylation and Bax
expression leading to CEC death. However, the precise mechanism
involved in A-b-induced cell death remains unclear. We demonstrated in
the present study that compound C, an AMP-activated protein kinase
(AMPK) inhibitor, suppressed A-b-induced p38 MAPK-p53 signalling
cascade. Transfection of CECs with AMPK dominant-negative mutant
(DN) also inhibited A-b-induced p53 phosphorylation. Moreover, A-b
was shown to time-dependently cause AMPK phosphorylation in CECs.
In addition to phosphorylation, modication of p53 by acetylation has also
played a crucial role in p53 activation and subsequent events leading to
cell death. We demonstrated for the rst time that A-b induced p53 acetylation in a time-dependently manner in CECs. Taken together, these results
suggest that A-b may activate the AMPK-p38 MAPK-p53-Bax signalling
to cause CEC death. Furthermore, modication of p53 by phosphorylation
and acetylation may be attributed to A-b induced CEC death.
(1) Nanchang university, Faculty of Basic Medicine, Nanching, PR

China
(2) Nanchang University, Deparment of Pharmacy, Nanchang, PR China
In recent years, investigating for platelet aggregation inhibitor from snake
venom is a hotspot that was highly concerned by drug researchers. GPIb
and GPIIb/IIIa were the major drug targets in affecting platelet function.
Viper venom contains various proteins which were specic to these targets, such as GPIb analog and disintegrins. We had puried a novel C
type lectin protein (CLP, GPIb analog) by sequential Uno Q, Uno S ionexchange chromatography and RP-HPLC from ve-pace snake venom.
The novel protein was a 26.59 kD dimer that consists of two subunit
with Mw of 14.14 kD and 13.03 kD. The two chains share 45% identity
with each other, and high homologous to other CLPs when blast was
made in protein database. The protein was given a name as agacutegrin
because of its characteristics. Agacutegrin inhibited the aggregation of
human platelet induced by ADP and collagen with IC50 at 153.1 nmol/L
and 214.4 nmol/L, respectively. Besides, agacutegrin had an effective
inhibition to carcinoma cells proliferation in vitro with ID50 among
18~35 ug/mL. The possible mechanism was to inhibit cell adhere to
matrix protein, e.g. bronectin, laminin and collagen. Moreover, agacutegin had no thrombin like enzyme, phospholipase A2 and arginyl esterase
activities and hemorrhagic toxin. These results suggested that agacutegrin
was most likely a promising drug to treat arterial thrombus, atherosclerosis, even the metastasis of tumor cells.
Acknowledgement: This study was supported by the Natural Scientic
Foundation of China (30860347).
Paper No.: 2925

PRELIMINARY INVESTIGATION TO THE CLINICAL APPLICATION OF PHOSPHOLIPASE A2 INHIBITOR FROM SNAKE
SERUM
Paper No.: 2006

THE EXPRESSION OF 14-3-3 SUBTYPES IN UTERINE
LEIOMYOMA
Chunghong Huang, X Deng, K Chen
Ouping Huang(2), SF Fang(2), L Wang(3), D Yin(4), B Yi(1), L Tang(1),

M He(1)
Nanchang University, Faculty of Basic Medicine, Nanching, PR China

Phospholipases A2 (PLA2) are esterolytic enzymes which catalyze the
hydrolysis of phosphoglycerides at the sn-2-position of the glycerol
backbone releasing lysophospholipids and free fatty acids, which transformed into a series of inammatory mediators that take signicant part
in severe acute pancreatic (SAP), acute lung injury et al. PLA2 over-activation was the major cause to a series inammatory diseases. PLA2 is an
important target for clinical treatment of SAP and MODS. PLA2 family
was also known as the major toxic enzyme in viper venom. Bioinformatics study showed that venom PLA2 shares nearly 40% identity with
human sPLA2 in amino sequence, and higher homologous in conformation. An interesting nding was that natural PLA2 inhibitors (PLI) were
mostly lie in snake serum. In our preliminary study, the PLIs were chemically proteins, and were specic and effective to those PLA2 related diseases. Puried PLI from ve-pace snake serum was obtained by afnity
chromatography and gel ltration. The PLI inhibited PLA2 activities in
vitro and hemorrhagic toxicity in a dose-manner. SAP mice model was
replicated and used for determining snake PLIs protective effects against
SAP in vivo, with control groups of Chloroquine and/or glucocorticoid
treatment. The PLI group had a signicant effect to diminish SAP-caused
MODS and lethality than those non-specic drugs. Detailed investigations are ongoing and it will be an intriguing work for drug discovery.
Paper No.: 2926

SNAKE VENOM C TYPE LECTIN PROTEIN, AGACUTEGRIN,
INHIBITS PLATELET AGGREGATION AND CARCINOMA
CELL MIGRATION

(2) Jiangxi Maternal and Child Health Hospital, Nanchang, PR China
(4) Jiangxi Tumor Hospital, Nanchang, PR China
Although uterine leiomyomas are the most common gynaecological
benign tumour and greatly affect reproductive health and well being, the
pathophysiology and epidemiology of uterine leiomyomas are not well
known. 14-3-3 protein has emerged as critical regulators of diverse cellular responses. Previous studies found that 14-3-3 protein expression was
observed and associated with various tumors genesis and progression.
Here, we further to screen and identify the various subtypes of 14-3-3
protein that were expressed differently in patients with uterine leiomyoma versus normal myometrium. We have selected 20 consenting premenopausal patients scheduled to undergo hysterectomy for symptomatic
uterine leiomyomas. Paired samples of leiomyoma and adjacent myometrium were obtained and submitted to reverse transcription polymerase
chain reaction (RT-PCR) and western blot. We have observed 14-3-3
subtypes including b, c, e, zeta, eta and theta have no signicant change
in leiomyoma compared with normal myometrium. Interestingly, one of
the subtypes, which exhibited a marked down-regulation in leiomyoma
tissues, was identied 14-3-3-d. In conclusion, the 14-3-3-d reduction in
uterine leiomyoma suggests that 14-3-3-d may play a role in the origin
or growth of leiomyoma. This study was supported by the 973 Program(: 2009CB526405).
Chunhong Huang(1), S Tu(1), Z Liao(2), X Deng(1), K Chen(1)

338
Paper No.: 2159
HEALTH AND DISEASE
OREXINS DEPOLARIZE ROSTRAL VENTROLATERAL
MEDULLA NEURONS AND REGULATE CARDIOVASCULAR
FUNCTIONS IN THE RAT MAINLY VIA OREXIN 2 RECEPTOR
Shang-Cheng Huang(1), Y-W Dai(1), Y-H Li(1), C-T Chen(3), L-L
Hwang(1,2)
(1) Taipei Medical University, Graduate Institute of Medical Sciences,
Taipei,Taiwan
(2) Taipei Medical University, Department of Physiology, Taipei, Taiwan
(3) National Health Research Institutes, Division of Biotechnology andPharmaceutical Research, Taipei, Taiwan
Application of orexin A or B to the rostral ventrolateral medulla
(RVLM), where bulbospinal vasomotor neurons are located, elevated
arterial pressure and heart rate. To determine the site and mechanism of
action of orexins in the RVLM, whole-cell patch clamp recordings were
made from RVLM neurons in neonatal rat brainstem slices. Orexin A
and B concentration-dependently depolarized RVLM neurons. Orexin A
or B (100 nM) excited 42% of RVLM neurons. Tetrodotoxin failed to
block orexin-induced depolarizations. In the presence of SB-334867, an
orexin 1 receptor (OX1R) antagonist, orexin A excited 42% of RVLM
neurons with a smaller, but not signicantly different, amplitude of membrane depolarizations (4.9 0.8 vs. 7.2 1.1 mV). In the presence of
TCS OX2 29, an orexin 2 receptor (OX2R) antagonist, the percentage of
orexin A-excited neurons decreased to 25% and orexin A-induced depolarizations became signicantly smaller (1.7 0.5 mV). Co-application
of SB-334867 and TCS OX2 29 completely eliminated orexin A-induced
depolarizations in all neurons tested. An OX2R agonist, [Ala11,D-Leu15]orexin B, concentration-dependently depolarized RVLM neurons. In 60
RVLM neurons tested for their responsiveness to orexin A and B (100
nM), 27% was excited by either peptides, while 10% and 8% were
excited only by orexin A or B. Regarding neuron phenotypes, 88% of
adrenergic neurons and 43% of non-adrenergic neurons were depolarized
by orexins. The orexin-excited RVLM neurons also included rhythmically ring neurons. We conclude that orexins may directly excite RVLM
neurons, which include bulbospinal vasomotor neurons, mainly via
OX2R, with a smaller contribution from OX1R.
Paper No.: 1580

DISEASES
DIFFERENT MITOGEN-ACTIVATED PROTEIN
KINASE-DEPENTENT CYTOKINES AND INFLAMMATORY
MEDIATORS PRODUCTIONS INDUCED BY LPS IN
ALVEOLAR MACROPHAGE OF CHRONIC BRONCHITIS RATS
Yan Huang, J Li, X-M Meng, G-L Jiang
We found that PGE2 from AM of CB rats was increased and the synthesis was suppressed by PD98059, SB203580. We also found SB203580
and PD98059, the inhibitors of ERK and p38 MAPK, can signicantly
inhibited COX-2 mRNA and protein expression. Moreover, ERK and
p38 MAPK had synergistic effect on COX-2 protein expression. Inhibition of ERK MAPK signal transduction could inhibit TGF-b expression
in AM of CB rats. These results demonstrated that COX-2, PGE2 and
TGF-b productions in AM of CB rats were signicantly increased, which
might be regulated by the different MAPK signalling pathway.
Paper No.: 1123

ANTI-HEPATITIS B VIRUS ACTIVITY IN CULTURED CELL
LINE HEPG2.2.15 CELLS OF TOTAL PHENOLICS
EXTRACTED FROM OENANTHE JAVANICA
Zheng-Ming Huang(1), X-J Wang(0), X-B Yang(2), J-G Song(3)
(1) Hospital 302 of PLA, Department of Clinical Pharmacology, Beijing,
PR China
(2) General Hospital of PLA, Institute of Geriatrics, Beijing, PR China
(3) Wannan Medical College, Department of Pharmacology,Anhui Wuhu,
PR China
Introduction: In this study, human hepatoma HepG2.2.15 cells culture
system was used as in vitro model to evaluate the anti-HBV effects of
total phenolics extracted from Oenanthe javanica(OJTPA). Methods: The
content of HBeAg and HBsAg in medium and HBV-DNA, cccDNA (covalenced closed cricoid DNA) in cells were determined by enzyme
immunoassay kit and uorescent quantitation real-time PCR (RQPCR)after Hep G2.2.15 cells were treated with OJTPA for the 4th, 8th
and 4th day cessated treatment. Results: In the current study,OJTPA
could dose-and-time dependently inhibit the expression of HBeAg,HBsAg,HBV-DNA and cccDNA.The inhibition rates of OJTPA on
HBeAg,HBsAg,HBV-DNA and cccDNA in the HepG2.2.15 cells were
59.33%,90%,62.3%,62.7% on day 8,respectively. Conclution: These
results suggested that OJTPA therefore offered further investigation as a
potential therapeutic agent for HBV infection. Key words: total phenolics
acid of Oenanthe Javanica(OJTPA), HepG2.2.15 cells, HBeAg,HBsAg,
HBV-DNA,HBV cccDNA.
Paper No.: 3178

THE EFFECTS OF AZOLE-BASED HEME OXYGENASE
INHIBITORS ON BREAST CANCER GROWTH AND
METASTASIS IN VIVO
Maaike Hum(1), RA Dercho(1), S Nagy(1), JZ Vlahakis(2),
WA Szarek(2), K Nakatsu(1)
Anhui Medical University, School of Pharmacy, Hefei, PR China

Lipopolysaccharide (LPS), a potent stimulator of inammatory responses
in macrophages, activates several intracellular signalling pathways,
including mitogen-activated protein kinases (MAPK). Previous studies
have investigated the effect of LPS on MAPK activation in macrophage
of normal rats. In the current study, we investigated the effect of LPS
exposure in vivo on activation of MAPK in alveolar macrophage(AM) of
chronic bronchitis(CB) rats, researched the corresponding LPS-stimulated
COX-2, PGE2 and TGF-b production and their MAPK signal transduction pathways. CB was induced by endotracheal instillation of lipopolysaccharedes(LPS) followed by Bacillus Calmette Guein (BCG) injection
through caudal vein 1 week later. Special inhibitors of p38, ERK and
JNK MAPK signal transduction pathways were used to determined the
effect of MAPK activation on COX-2, PGE2, TGF-b production in AM
of CB rats via RT-PCR, western blotting, radioimmunoassay and ELISA.
(1) Queens University, Department of Pharmacology and Toxicology,

Kingston, Ontario, Canada
(2) Queens University, Department of Chemistry, Kingston, Ontario,
Canada
Heme oxygenases (HOs) are responsible for the breakdown of heme to
biliverdin, free iron, and carbon monoxide (CO). The two major isoforms, HO-1 (inducible) and HO-2 (constitutive) are involved in a variety of physiological functions, including apoptosis, inammation and
angiogenesis, which are important to the progression of some types of
cancer. Previous work using metalloporphyrin-based compounds has
identied HO inhibition as a potential target for the prevention of tumour
growth and progression. These compounds are limited by their lack of
selectivity for HO and have been shown to inhibit nitric oxide synthase
and soluble guanylyl cyclase. Novel azole-based HO inhibitors have
339
demonstrated increased in vitro selectivity for HO and were tested for
their effects on breast cancer growth and metastasis in vivo. GFP-labelled
AC2M2 cells were implanted into the mammary fat pad of female nude
mice which were treated on days 1, 3 and 5 of a 7 day cycle with either
an azole- or metalloporphyrin-based HO inhibitor, heme (HO inducer),
or saline (control). Primary tumours were removed on day 21 and metastases were allowed to grow for another 10 days. Results indicate that the
azole-based compound decreased primary tumour volume, as did the
metalloporphyrin, when compared to both saline and heme. Furthermore,
the incidences of lung metastasis were also decreased with the azolebased HO inhibitor. These ndings indicate that there is potential for the
use of azole-based HO inhibitors in the treatment of breast cancer growth
and prevention of metastasis.
(Funded by the Ontario Institute of Cancer Research 08NOV-142)
Paper No.: 2420

P19 - GENERAL SESSION - BIOLOGICS
EFFECTS OF (-)-EPIGALLOCATECHIN GALLATE ON RPE
CELL MIGRATION AND ADHESION
Chi-Feng Hung(1), C-M Chan(1), H-H Lin(1)
Fu Jen Catholic University, School of Medicine, Taipei, Taiwan
In diseases such as proliferative vitreoretinopathy (PVR), proliferative
diabetic retinopathy, and age-related macular degeneration, retinal pigment epithelial (RPE) cells can initiate proliferation and migration, and
secrete extracellular matrix proteins (ECM). (-)-Epigallocatechin gallate
(EGCG) has been shown to suppress the migration and adhesion of
many cell types, but its effects on RPE cell migration and adhesion were
not known. The proliferation and migration of RPE cells can be
enhanced by platelet-derived growth factor (PDGF), and bronectin is a
major ECM component of PVR tissue. Therefore, we investigated the
inhibitory effects of EGCG on PDGF-BB-induced migration and bronectin adhesion in RPE cells. The migration of RPE cells was detected
by an electric cell-substrate impedance sensing migration assay and a
Transwell migration assay. Cells were loaded with BCECF/AM and their
adhesion to bronectin was examined. The interactions of EGCG with
PDGF-BB were analyzed by a dot binding assay. Cytoskeletal reorganization was examined by immunouorescence microscopy. A PDGF-BBinduced signalling pathway was detected by Western blotting. In the
present study, we nd that EGCG can inhibit PDGF-BB-induced human
RPE cell migration and, in a dose-dependent manner, RPE cell adhesion
to bronectin. Our analysis demonstrates that EGCG does not directly
bind to PDGF-BB and the inhibition of EGCG against bronectininduced cytoskeletal reorganization is observed. Furthermore, EGCG is
shown to suppress PDGF-BB-induced PDGF-b receptor, downstream
PI3K/Akt and MARK phosphorylation. Our results provide the rst evidence that EGCG is an effective inhibitor of RPE cell migration and
adhesion to bronectin, and, therefore, may prevent epiretinal membrane
formation.
Paper No.: 1126

P16 - NATURAL PRODUCTS: PAST AND FUTURE?
MECHANISM OF INHIBITION OF TRANSCRIPTION FACTOR
SPECIFICITY PROTEIN 1 BY BETULINIC ACID
Jan-Jong Hung, W-C Chang
National Cheng-Kung University, Institute of Biosignal Transduction,
Tainan, Taiwan
Betulinic acid is a pentacyclic triterpene natural product identied as an
effective inhibitor of cell proliferation. However, the underlying mecha-
nism of these responses is not well understood. Since several tumorrelated genes, such as vascular endothelial growth factor and p21WAF1/
CIP1, are regulated by specicity protein 1 (Sp1), the primary goal of
this research is to investigate the mechanism in which betulinic acid
affects the levels of Sp1 in HeLa cancer cells. Using HeLa cervical cancer cells as a model, we found that betulinic acid dose-dependently
decreased the levels of Sp1. Besides, betulinic acid increased the ubiquitin levels of Sp1, and then the interaction between Sp1 and the proteasome subunit Rpt6 was enhanced. Previous studies in our laboratory
have shown that SUMOylation of Sp1 augments its degradation by
increasing the Sp1 proteolytic process. To further examine the possible
role of betulinic acid on the SUMOylated Sp1, the HA-SUMO-1-Sp1
was used to mimic the SUMOylated Sp1. We found that the decrease in
Sp1 protein within cells expressing SUMO-1-Sp1 was more signicant
than that in cells expressing wt-Sp1 under betulinic acid treatment. Taken
together, the results showed that betulinic acid decreases the levels of
Sp1 through both post-translational modication of Sp1, and a caspasesdependent cleavage of Sp1 during betulinic acid-induced apoptosis;
hence betulinic acid has the potential for use as a novel class of Sp1-targeting anticancer drugs.
Paper No.: 1601

M3-MUSCARINIC ACETYLCHOLINE RECEPTOR
STIMULATION OF GLUCOSE UPTAKE IN L6 SKELETAL
MUSCLE CELLS: EVIDENCE FOR A
CAMKKB-AMPK-DEPENDENT MECHANISM
Dana Hutchinson(1,2), J Merlin(1,2), R Csikasz(3), B Evans(1,2),
T Bengtsson(3), R Summers(1,2)
(1) Monash University, Department of Pharmacology, Parkville, Vic,
Australia
(2) Monash Institute of Pharmaceutical Sciences, Monash University,
Deppartment of Drug Discovery Biology, Parkville, Vic, Australia
(3) Stockholm University, The Wenner-Gren Institute, Department of
Physiology,Stockhom, Sweden
The role of muscarinic acetylcholine receptors (mAChRs) in regulating glucose uptake in L6 skeletal muscle cells was investigated.
Whole cell radioligand binding using [3H]-N-methyl scopolamine
chloride identied mAChRs in differentiated (Bmax 7.86 0.91
fmol/mg protein, pKD 9.66 0.16) but not undifferentiated L6 cells
and RT-PCR analysis identied the presence of M3 mRNA. M3
receptors are Gq-coupled, and cholinergic stimulation of L6 cells by
the mAChR agonists acetylcholine, oxotremorine-M and carbachol
increased Ca2 + in differentiated but not undifferentiated cells. This
was due to muscarinic and not nicotinic activation as responses were
antagonised by the muscarinic antagonist atropine but not the nicotinic antagonist tubocurarine. [3H]-2-Deoxyglucose uptake was
increased in differentiated L6 cells by insulin, and also by acetylcholine, oxotremorine-M and carbachol in a concentration-dependent
manner. This response via mAChR stimulation was inhibited by the
AMPK inhibitor Compound C but not the PI3K inhibitor wortmannin. Western blotting showed that both carbachol and the AMPK
activator AICAR increased phosphorylation of AMPK, with the effect
of carbachol sensitive to inhibition by compound C and the CAMKKb inhibitor STO609, but not by wortmannin. Using CHO-K1 cells
stably expressing each of the mAChR subtypes (M1-M5), it was
determined only the M1, M3 and M5 receptors (which signal through
Gq) are capable of phosphorylating AMPK, indicating a Gq-dependent mechanism. This study demonstrates that activation of mAChRs
in L6 skeletal muscle cells stimulates glucose uptake via a CaMKKb-AMPK-dependent mechanism, independent of the insulin-stimulated pathway.
340
Paper No.: 3111
LIGAND-DIRECTED SIGNALLING BY B-ADRENOCEPTOR
ANTAGONISTS AT THE HUMAN b1- AND
b3-ADRENOCEPTORS
Dana Hutchinson, M Sarwar, E Stanic, M Sato, B Evans, R Summers
Monash University, Department of Pharmacology and Monash Insituite
of Pharmaceutical Science, Parkville, Australia
The capacity of ligands to differentially block or stimulate signalling
pathways has been termed ligand directed signalling (LDS). Although it
is traditionally believed that an antagonist simply blocks receptor function, LDS suggests that antagonists themselves could have various effects
by recognising and binding to different conformations of the receptor
and hence activating different signalling pathways. We examined
whether several clinically used b-AR antagonists promote LDS in CHO
cells stably expressing the human b1-AR (160.8 13.5 fmol/mg protein)
or b3-AR (628.5 116.7 fmol/mg protein). We investigated the ability
of these ligands to inuence cAMP accumulation and ERK1/2 phosphorylation. All anatagonists examined behaved as classical competitive
antagonists with respect to cAMP accumulation in response to either isoprenaline (b1-AR) or L755507 (b3-AR). However we found that some
of the b-AR antagonists were able to activate MAPK signalling, particularly ERK1/2 phosphorylation. At the b1-AR, alprenolol, bucindolol,
carvedilol and propranolol concentration-dependently activated ERK1/2
as did isoprenaline, whereas nebivolol and bupranolol had no signicant
effect. Further investigation revealed that pertussis toxin inhibited ERK
responses to alprenolol, bucindolol, carvedilol or propranolol, but not
those to isoprenaline or noradrenaline, indicating different activation
pathways utilised by b-AR antagonists and agonists. At the b3-AR,
L755507, L748337, propranolol, carvedilol and SR59230A increased
ERK1/2 phosphorylation through a c-Src mediated mechanism that was
not affected by pertussis toxin, whereas bupranolol and nebivolol did not
activate ERK1/2. These results indicate that b-AR antagonists exhibit
LDS and can activate MAPK through pathways that are dependent upon
the ligand and receptor investigated.
Paper No.: 1840

SINGLE-LABEL TIME-RESOLVED FLUORESCENCE BASED
ESTROGEN RECEPTOR-LIGAND BINDING ASSAY
Roope Huttunen, Xxxx Shweta, E Martikkala, M Lahdenranta,
P Hanninen, H Harma
placed from the receptor and remained in the solution and its timeresolved uorescence was quenched using soluble quencher molecules.
The bound fraction of Eu-E2 was protected from quenching upon binding
to the receptor. The QRET method was compared with a commercial FP
assay. Our results show high sensitivity and S/B more than 2 times
higher than for the FP assay. We conclude that the QRET method provides an attractive new assay format for nuclear receptor ligand screening.
Paper No.: 2683

HEPATOTOXICITY ASSOCIATED WITH ANTITUBERCULOSIS
DRUGS: A COHORT OF INCIDENT PATIENTS RECEIVING
TREATMENT OR ON CHEMOPROPHYLAXIS
Luisa Ibanez(1), M Sabate(2), E Perez(1), X Vidal(1), Hepatox - TBC
Group (3)
(1) Autonomus University of Barcelona, Vall dHebron University
Hospital, FundacioInstitut Catala` de Farmacologia, Barcelona, Spain
(2) Fundacio Institut Catala` de Farmacologia, Barcelona, Spain
(3) Vall dHebron University Hospital. Surveillance Centre of
Tuberculosis (CAPDrassanes), Barcelona, Spain
Nowadays tuberculosis is a serious problem to public health due to an
increase in complications derived from its treatment, including hepatotoxicity. The aim of the study is to identify and characterize serum biomarkers which would be predictive of hepatotoxicity associated with
anti-TB agents. Patients who initiate treatment or prophylaxis with antiTB drugs are being included in a tertiary-care hospital and in a tuberculosis surveillance centre in Barcelona (Spain). Serum samples are being
collected from patients a) before initiation of anti-TB, b) on the 20th day
after the start of anti-TB and c) at the time when hepatotoxicity is diagnosed, or d) at the end of the anti-TB treatment. Prospective laboratory
data, anti-TB regimen and concomitant drug exposures are being
recorded. Mass spectrometric serum proling will be undertaken to identify potential biomarkers of hepatotoxicity at the end of the study. Out of
a total of 23 potential patients, 15 (6 female) have been included. Their
median age is 29 (20-44). Six patients are being treated with anti-TB
drugs (pulmonary TB 2, extrapulmonary 2, both 2) and 9 with chemoprophylaxis. Hepatotoxicity appeared in 2 patients after 1 month of treatment. A blood sample before the start of anti-TB treatment is available
in 11 patients, and a blood sample at the time of hepatotoxicity in 2
patients. One patient was lost to follow-up and 1 withdrew from the
treatment because of adverse effects. Preliminary results conrm the feasibility of the study and the importance of liver toxicity in patients treated with anti-TB drugs.
University of Turku, Laboratory of Biophysics, Turku, Finland

Homogeneous uorescence-based microplate assays are desirable in
high-throughput screening assays of new nuclear receptor regulators,
since they are safe, simple, cost-effective, miniaturizable and amenable
for automation. For example, uorescence polarization (FP) is a widely
used single-label homogeneous assay method for nuclear receptor ligand
screening. However, reagents, plastics and biological specimens decrease
signal-to-background (S/B) thus limiting dynamic range and sensitivity
of assays. Assays based on lanthanide chelate-conjugated probes provide
an advantage, since their detection by time-resolved uorescence results
in a high specic signal with low background. Standard lanthanide-based
homogeneous assays use time-resolved Forster resonance energy transfer
(TR-FRET) and require labeling of both ligand and receptor making the
assay format more complex. To overcome these limitations we have
developed a new single-label method for estrogen receptor (ER) ligand
binding. In this method based on quenching resonance energy transfer
(QRET), the ligand (E2) is labeled with a lanthanide (Eu) chelate. In the
QRET assay, unlabelled E2 competed with the labelled ligand Eu-E2
from binding to ER. By adding E2, the uorescent probe Eu-E2 was dis-
Paper No.: 1418

GPR120 MEDIATES HIGH-FAT DIET-INDUCED ADIPOCYTE
HYPERTROPHY AND OBESITY
Atsuhiko Ichimura, A Hirasawa, S Miyauchi, G Tsujimoto
Kyoto University Graduate School of Pharmaceutical Sciences,
Department of Genomic Drug Discovery Science, Kyoto, Japan
Free fatty acids (FFAs) are not only essential nutritional components but
they also function as signalling molecules. Recently, members of the
G-protein-coupled receptors have been shown to be activated by FFAs.
Among the FFA receptors, we previously showed that GPR120, which is
expressed in large intestine, functions as a receptor for unsaturated longchain FFAs such as a-linolenic acid (a-LA) (Hirasawa A et al, Nature
341
Medicine 2005, 11:90-94). The stimulation of GPR120 by a-LA promotes the secretion of glucagon-like peptide-1. Transcriptional analysis
and immunostaining using specic antibody against GPR120 showed
that GPR120 is expressed abundantly not only in large intestine but also
in several other tissues such as white adipose tissue (WAT). However,
the biological functions and physiological roles of GPR120 in these tissues have remained to be elucidated. In the present study, we have used
genetic approaches in mice to address the role of GPR120. We found that
GPR120-decient mice were obese and showed hypertrophic adipocyte,
fatty liver, glucose intolerance and reduced insulin sensitivity compared
with wild-type mice by high-fat diet (HFD) feeding. Microarray gene
expression analysis showed that repressed expression of genes relating
insulin signal such as insulin receptor in WAT and enhanced expression
of the gene relating lipid synthesis in liver of HFD-fed GPR120-defcient mice. Furthermore, insulin-dependent Akt phosphorylation was signicantly decreased in WAT and liver of HFD-fed GPR120-decient
mice. These results demonstrate that GPR120 play an important role in
lipid and glucose metabolism.
Paper No.: 1594

HEALTH AND DISEASE
EFFECT OF FLUVASTATIN ON CEREBRAL SALT WASTING
AFTER SUBARACHNOID HEMORRHAGE IN RATS
Takahiro Igarashi(1), M Yoneko(2), J Kojima(2), N Moro(1),
Y Kondo(1), Y Katayama(1)
(1) Nihon University School of Medicine, Division of Neurosurgery,
Department of Neurological Surgery, Tokyo, Japan
(2) National Center for Child Health and Development, Tokyo, Japan
Introduction: Cerebral salt wasting (CSW) frequently occurs concomitantly with subarachnoid hemorrhage (SAH). CSW induces excessive
natriuresis and osmotic diuresis, and reduces total blood volume. As a
result, the risk of symptomatic cerebral vasospasm and cerebral ischemia
may be elevated. Therefore, it is important to develop drugs inhibiting
CSW. Although it has been reported that statins inhibit cerebral vasospasm after SAH, the mechanisms of statins on cerebral vasospasm
remain unclear. The purpose of this study is to evaluate whether uvastatin prevents natriuresis and osmotic diuresis after SAH. Materials and
Methods: Male Wistar rats were used weighing 270 306 g. We used
SAH model by endovascular puncture (EP model) that exhibits natriuresis and osmotic diuresis, these phenomena was generally named by
CSW. Urine was cumulatively collected from SAH onset to 6 hours later
and sodium (Na) excretion was calculated. Fluvastatin at 30 mg/kg (n =
9) and 100 mg/kg (n = 12) orally were administered immediately after
SAH onset. Control animals (n = 9) were orally administered to water (5
mL/kg) after SAH. Results: Urine volume and Na excretion were
increased in control group. Fluvastatin signicantly prevented excess Na
excretion (P < 0.01) and urine volume (P < 0.01) and showed potent,
dose-dependent, anti-CSW effects. Also uvastatin reduced Na/K ratio in
urine. Conclusion: The present results demonstrate that uvastatin has
potency of inhibition of CSW. In conclusion, inhibitory effects of uvastatin on cerebral vasospasm are due to inhibit on CSW. Fluvastatin supports efcient uid management after SAH.
Paper No.: 435

ANTI-INFLAMMATORY ACTIVITY OF AQUEOUS FRUIT
PULP EXTRACT OF HUNTERIA UMBELLATA K. SCHUM IN
ACUTE AND CHRONIC INFLAMMATION
Ighodaro Igbe(1), P Ching(2), A Eromon(1)
(1) University of Benin, Department of Pharmacology and Toxicology,
Benin City,Nigeria
(2) Niger Delta University, Amassoma, Bayelsa State, Nigeria
Aim of study: Aqueous seed extract of the H. umbellata K. Schum (Apocynaceae) was investigated for hypoglycaemic activity in rats. Materials
and methods: Diabetes was induced by a single dose of streptozotocin
(50 mg/kg i.p). Daily doses of 400, 800 and 1000 mg/kg of extract were
orally administered to fasted normal and diabetic rats. Blood glucose levels were monitored after 0, 2, 4, 8, 12 h and on the 14th day post treatment. Liver glycogen levels were also estimated on the 14th day.
Results: In normal rats, only 400 mg/kg of the extract produced a signicant reduction in blood glucose at the 4th hour (P < 0.05) which was
22.15%. In diabetic rats, the extract (400, 800 mg/kg) caused signicant
reduction (P < 0.01), which were 51.87 and 43.47% respectively, with
maximum effect at the 8th hour. This reduction in blood glucose was
greater than that of Glibenclamide (31.03%). Only 400 mg/kg produce a
signicant reduction (P < 0.01) on the 14th day (43.60%). Liver glycogen levels were signicantly increased (P < 0.05) in diabetic rats by
extract (400 and 800 mg/kg and these were comparable to glibenclamide.
Acute toxicity data showed no mortality in mice up to 17.5 g/kg. Conclusion: The extract possesses marked hypoglycaemic effects in diabetic rats
possibly through increased glycogenesis, thus justifying its use in herbal
medicine for the treatment of diabetes.
Paper No.: 2251

EXPLORING GENDER DIFFERENCES IN THE RELAXANT
EFFECT OF QUERCETIN ON
PHENYLEPHRINE-CONTRACTED NORMAL AND DIABETIC
RAT AORTA
Aloysius Iguegbe(1), F Achike(2), R Mustafa(3)
(1) University Teknologi Mara, Faculty of Pharmacy, Kuala Lumpur,
Malaysi
(2) International Medical University, Kuala Lumpur, Malaysia
(3) University Malaya, Faculty of Medicine, Kuala Lumpur, Malaysia
Reactive oxygen species (ROS) are elevated in cardiovascular diseases
including diabetes for which gender is a risk factor. ROS incapacitates
endothelium derived nitric oxide leading to altered vascular responses for
which antioxidants, including quercetin (Q) have been advocated for its
management. We previously demonstrated a quercetin-enhanced vasodilatory effect on phenyleprine (PE)-contracted rat aorta which was eNOScGMP mediated predominantly in the euglycemic male compared to the
female WKY rat. We further investigated whether this gender difference
in Q action exists in PE- contracted diabetic aorta. Endothelium-intact
thoracic aortic rings isolated from age and sex-matched streptozotocininduced diabetic WKY rats were mounted in organ chambers for isometric tension recording. Q attenuated PE-evoked contractions signicantly
more in male and female diabetic aortae than the corresponding non-diabetic tissues. L-NAME or methylene blue inhibited Q relaxant effect in
diabetic aorta from both genders. Indomethacin enhanced contractions in
diabetic male but not the female and reduced Q action in diabetic aorta
from both genders. These results suggest that Q is more active in tissues
undergoing oxidative stress, such as the diabetic compared to the euglycaemic tissues. Whereas Q attenuated PE-contraction more in euglycaemic male than female tissues (perhaps because of greater ROS stress in
male compared to the oestrogen-mediated lesser stress in the female), in
the diabetic state, its effect is similar in both genders and is equally mediated by the eNOS-cGMP pathway. In addition, Q exerts its effect, in both
genders, partly through a vasodilator COX-product.
Paper No.: 1558

ANTI-OBESITY AND ANTI-DIABETIC EFFECTS OF ACACIA
POLYPHENOL
Nobutomo Ikarashi, T Toda, T Okaniwa, K Ito, W Ochiai, K Sugiyama
342
Hoshi University, Department of Clinical Pharmacokinetics, Tokyo,
Japan
Acacia polyphenol (AP) extracted from the bark of the black wattle tree
(Acacia mearnsima) is rich in unique catechin-like avan-3-ols, such as
robinetinidol and setinidol. The present study investigated the anti-obesity/anti-diabetic effects of AP using obese diabetic KKAy mice. KKAy
mice received either normal diet, high-fat diet or high-fat diet with additional AP for 7 weeks. After the end of administration, body weight,
plasma glucose and insulin were measured. Furthermore, mRNA and
protein expression of obesity/diabetic suppression-related genes were
measured in skeletal muscle, liver and white adipose tissue. As a result,
compared to the high-fat diet group, increases in body weight, plasma
glucose and insulin were signicantly suppressed for AP groups. Furthermore, compared to the high-fat diet group, mRNA expression of energy
expenditure-related genes (PPARa, PPARd, CPT1, ACO and UCP3) was
signicantly higher for AP groups in skeletal muscle. Protein expressions
of CPT1, ACO and UCP3 for AP groups were also signicantly higher
when compared to the high-fat diet group. Moreover, AP lowered the
expression of fat acid synthesis-related genes (SREBP-1c, ACC and
FAS) in the liver. AP also increased mRNA expression of adiponectin
and decreased expression of TNF-a in white adipose tissue. In conclusion, the anti-obesity actions of AP are considered attributable to
increased expression of energy expenditure-related genes in skeletal muscle, and decreased fatty acid synthesis and fat intake in the liver. These
results suggest that AP is expected to be a useful plant extract for alleviating metabolic syndrome.
Paper No.: 925

DISEASES
MICROSOMAL PROSTAGLANDIN E SYNTHASE-1 AND COX-2
ARE COORDINATELY CONTRIBUTE TO ISCHEMIC EXCITOTOXICITY THROUGH EP3 RECEPTORS
Yuri Ikeda-Matsuo(1), H Tanji(1), Y Hirayama(1), S Uematsu(2),
S Akira(2), Y Sasaki(1)
(1) Kitasato University School of Pharmaceutical Sciences, Laboratory
of Pharmacology, Tokyo, Japan
(2) Osaka University, Department of Host Defense, Research Institute for
Microbial Diseases, Osaka, Japan
We have demonstrated that microsomal prostaglandin E synthase-1
(mPGES-1) is a critical factor of stroke-reperfusion injury (PNAS, 2006;
103: p-11790-11795). In this study, we tried to elucidate the involvement
of COX-2, an upstream enzyme for PGE2 production, and EP receptors,
downstream effecters for PGE2, in the mPGES-1 neurotoxicity. In cultured rat hippocampal slice, in which the mRNAs of EP1-4 receptors
were constitutively expressed, the expressions of the mRNA and protein
of both mPGES-1 and COX-2 were induced after glutamate stimulation.
Excitotoxicity in CA1 neurons, as well as PGE2 production and PGES
activation, induced by glutamate was signicantly attenuated by either
MK-886 or NS-398, inhibitors of mPGES-1 and COX-2, respectively;
however, co-application of these inhibitors had neither an additive nor
synergistic effect. Among the EP1-4 antagonists and agonists, only the
EP3 antagonist ONO-AE3-240 attenuated and only the EP3 agonist
ONO-AE-248 augmented the glutamate-induced excitotoxicity. The protective effect of NS-398 and ONO-AE3-240 on the excitotoxicity
observed in wild-type (WT) slices was completely abolished in mPGES1 knockout (KO) slices, which showed less excitotoxicity than WT
slices. In the transient focal ischemia model, mPGES-1 and COX-2 were
co-localized in the infarct region of the cortex. Intraperitoneal injection
of NS-398 or ONO-AE3-240 reduced ischemic injuries in WT mice, but
not in mPGES-1 KO mice, which showed less dysfunction than WT
mice. These results suggest that mPGES-1 and COX-2 are co-induced by
excess glutamate in ischemic brain and act together to exacerbate stroke
injury through PGE2 production followed by activation of EP3 receptors.
Paper No.: 2803

POSSIBLE INVOLVEMENT OF B-ENDORPHIN IN
TOLERANCE TO FENTANYL-INDUCED
ANTI-HYPERALGESIA UNDER A PAIN-LIKE STATE
Satoshi Imai(1), M Narita(1), N Kuzumaki(1), LA Devi(2), Y Uezono(3),
T Suzuki(1)
(1) Hoshi University School Pharmacy & Pharmaceutical Science,
Department of Toxicology, Tokyo, Japan
(2) Mount Sinai School of Medicine, Department of Pharmacology &
System Therapy, New York, NY, USA
(3) National Cancer Center, Division of Cancer Pathophysiology, Tokyo,
Japan
Previously, we demonstrated that repeated treatment with fentanyl causes
a desensitization to its ability to anti-hyperalgesia associated with the
attenuation of monomeric l-opioid receptor (MOR) resensitization in
mice with neuropathic pain. On the assumption that b-endorphin might
be released within the spinal cord under a neuropathic pain, we examined
whether b-endorphin could be responsible for a desensitization of fentanyl under a pain-like state. In cultured cells, unlikely morphine, either
fentanyl or oxycodone induced a robust monomeric MOR internalization
and, in turn, its resensitization. In the presence of b-endorphin, the internalized monomeric MOR induced by fentanyl, but not oxycodone,
remained within the cytosol even after washing-out. This phenomenon
may explain the high degree of tolerance to fentanyl-induced anti-hyperalgesic effects. Unlike monomeric MORs, rapid internalized l-d heterodimers after co-treatment with b-endorphin and fentanyl came back to
the plasma membrane in cultured cells. Furthermore, in sciatic nerveligated mice following repeated injection of fentanyl, hyperalgesia was
completely abolished by injection of morphine. The present data indicate
that the resensitization of l-d heterodimers after co-treatment with bendorphin and fentanyl may be responsible for the anti-hyperalgesic
effects of morphine in nerve-ligated mice following repeated treatment
with fentanyl.
Paper No.: 1737

DISEASE AND THERAPY
THE REGULATION OF MELATONIN RELEASE BY BK
CHANNEL ACTIVITY IN PINEALOCYTES
Yuji Imaizumi, H Mizutani, H Yamamura, M Muramatsu, S Ohya
Nagoya City University Graduate School of Pharmaceutical Sciences,
Department of Molecular and Cellular Pharmacology, Nagoya, Japan
Pineal gland regulates circadian rhythm through synthesis and secretion
of melatonin. Although melatonin production is closely associated with
cytosolic Ca2 + signals, the Ca2 + dynamics coupled to Ca2 + -sensitive
channels have not been fully understood. The functional coupling
between cytoplasmic Ca2 + signal and large-conductance Ca2 + -activated
K+ (BK) channel activity during stimulation of nicotinic acetylcholine
receptors was examined in rat pinealocytes. Here we found that spontaneous and oscillatory changes in intracellular Ca2 + concentration generated in approximately 17% of pinealocytes. The spontaneous Ca2 +
oscillations were mostly abolished by extracellular Ca2 + removal or
application of nifedipine but not by blockers of intracellular Ca2 + releasing channels. Interestingly, the spontaneous Ca2 + oscillations were
enhanced by BK channel blockers, paxilline, penitrem A, or iberiotoxin.
In contrast, the enhancement of BK channel activity by 12,14-dichlorodehydroabietic acid (diCl-DHAA) or NS-1619 attenuated spontaneous
Ca2 + oscillations. Moreover, in about 23% of pinealocytes, Ca2 + oscillations were observed following the short application of nicotine, which
343
2
induced a transient Ca rise. The nicotine-evoked Ca2 + oscillations

were reduced or enhanced by the application of nifedipine or paxilline,
respectively. Outward currents upon depolarization were signicantly
increased in the presence of diCl-DHAA. The current activation was
reversed by the addition of paxilline. Expression analysis revealed that
rat pinealocytes predominantly expressed a and b3 subunits of BK channel. These results indicate that the spontaneous and evoked Ca2 + oscillations, which were well modied by BK channel activities, may be
involved in pineal functions and regulated by the parasympathetic innervation.
Paper No.: 2681

DISCOVERY
TOXICOKINETICS OF THE FOOD-TOXIN IQ IN HUMAN
PLACENTAL PERFUSION IS NOT AFFECTED BY ABCG2 OR
XENOBIOTIC METABOLISM
Elina Immonen(1,2), M Kummu(1,2), A Petsalo(3), T Pihlaja(1),
K Vahakangas(2), P Myllynen(1)
Human liver microsomes were incubated with losartan and AA, and the
reactions were terminated by adding ice-cold ethanol to the samples. AA
was detected by newly developed LC/MS/MS method using deuteriumlabeled AA as internal standard. This study was approved by the Ethics
committee of Karolinska Institute. Results and Conclusion: Linear calibration curves were obtained to determine AA up to 200 uM with r >
0.999. AA remaining after incubation was about 50% without any inhibitor, but about 80, 75, 65, and 60% with losartan, sulfaphenazole, quercetin, and ketoconazole, respectively. These results indicate that under
these conditions losartan is a more potent inhibitor of the metabolism of
AA compared to drugs with specic inhibitory effect on CYP2C9,
CYP2C8, and CYP3A4, respectively. The effect on the formation of
vasoactive agents derived from this pathway remains to be studied.
Paper No.: 863

PHARMACOKINETICS AND DOSAGE REGIMEN OF
CIPROFLOXACIN FOLLOWING SINGLE INTRAMUSCULAR
ADMINISTRATION IN SAHIWAL CATTLE
Zahid Iqbal(1), A Basit(2), I Javed(3), IU Jan(4), UA Khan(5)
(1) University of Oulu, Institute of Biomedicine, Department of Pharmacology & Toxicology, Oulu, Finland
(2) University of Eastern Finland, Department of Pharmacology and Toxicology, Kuopio, Finland
(3) Novamass Analytical Ltd., Medipolis Center, Oulu, Finland
Metabolizing enzymes and transporters affect toxicokinetics of foreign
compounds (e.g. drugs and carcinogens) in human placenta. The heterocyclic amine, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) is a foodborne carcinogen being metabolically activated by cytochrome P450
(CYP) enzymes, especially by CYP1A1/2. IQ is also a substrate for
ABCG2 transporter. Placental transfer and metabolism of 14C-IQ was
evaluated in 4-hour ex vivo human placental perfusions. Placentas were
perfused with 14C-IQ alone (0.5 lM, n = 6) or in combination with
GF120918 (inhibitor of ABCG2, 1 lM, n = 6) or Ko143 (specic inhibitor of ABCG2, 2 lM, n = 4) to study the role of ABCG2 inhibition in
transfer. Critical parameters (leak from fetal to maternal circulation, pH
values, blood gases, glucose consumption, the production of hCG hormone and transport of antipyrine) were analyzed during the perfusions.
14C-IQ on maternal and fetal sides was determined by liquid scintillation
counting. IQ and its metabolites in nal perfusates were determined also
by LC/TOF-MS. ABCG2 expression and EROD activity (CYP1A1/2)
were analyzed from perfused tissues. 14C-IQ was easily transferred
through the placenta from maternal to fetal side. Neither signicant
EROD activity nor IQ metabolites were found in placentas from nonsmoking mothers. Inhibition of ABCG2 by GF120918 (FM-ratio of IQ
0.95) or Ko143 (FM-ratio of IQ 0.94) did not affect 14C-IQ transfer
(FM-ratio of IQ in IQ only perfusions 0.97) implicating that placental
ABCG2 does not have a signicant role in protecting fetus from IQ.
Paper No.: 2177

EFFECT OF LOSARTAN ON METABOLISM OF
ARACHIDONIC ACID IN HUMAN LIVER MICROSOMES
Nobuo Inotsume(1), T Toda(1), K Sakamoto(1), S Takeuchi(1),
T Hayakawa(1), B Ask(2), E Eliasson(2), A Rane(2)
(1) Riphah International University, Islamic International Medical

College, Islamabad, Pakistan
(2) Bahauddin Zakaria University, Faculty of Veterinary Sciences,
Multan, Pakistan
(3) University of Agriculture, Department of Physiology and Pharmacology, Faisalabad, Pakistan
(4) Agriculture University, Department of Animal Health, Peshawar,
Pakistan
(5) Riphah International University, Riphah Academy of Research and
Education, Islamabad, Pakistan
The objective of this study was to determine the pharmacokinetics and
optimal dosage regimen of ciprooxacin in Sahiwal cattle. Ciprooxacin
was administered intramuscularly at 5 mg/kg body weight in each of
eight animals. Following drug administration, blood samples were collected at different time intervals and analyzed for ciprooxacin using
high performance liquid chromatograph (HPLC). Pharmacokinetic
parameters were calculated using two compartment open model. Peak
plasma concentration (Cmax) of ciprooxacin, 4.35 0.29 lg/mL was
achieved at 0.86 0.04 hours (Tmax). Values for half life of absorption
(t1/2 abs), distribution (t1/2 a) and elimination (t1/2 b) were 0.73 0.19,
0.40 0.04 and 3.25 0.46 hours, respectively. The value for apparent
volume of distribution (Vd) was 1.24 0.16 L/kg, area-under-the-curve
(AUC) 18.73 1.09 lg.hr/mL and total body clearance (ClB) was 0.31
0.02 L/hr/kg. Based on these results, it was concluded that calculated
dose should be higher than the dose recommended by the manufacturer
to treat susceptible bacteria in Sahiwal cattle.
Keywords: Pharmacokinetics, Dosage regimen, Ciprooxacin, HPLC,
Sahiwal cattle
Paper No.: 1161

TOXIC DOSE OF METHAMPHETAMINE INDUCES
ENDOPLASMIC RETICULUM STRESS RELATED GENE
CHOP/GADD153/DDIT3 IN DOPAMINERGIC NEURAL CELLS
Zahid Iqbal(1), A Basit(2), I Javed(3), IU Jan(4), UA Khan(5)
(1) Hokkaido Pharmaceutical University School of Pharmacy, Department of Pharmaceutics, Hokkaido, Japan
(2) Karolinska Institute, Stockholm, Sweden
Introduction: We have already found that losartan inhibits paclitaxel and
uvastatin metabolism using human liver microsomes with genetic variants of CYP2C8 and CYP2C9. We have studied the effect of losartan on
arachidonic acid (AA) metabolism in human liver microsomes. Methods:
(1) Riphah International University, Islamic International Medical

College, Islamabad, Pakistan
(2) Bahauddin Zakaria University, Faculty of Veterinary Sciences,
Multan, Pakistan
(3) University of Agriculture, Department of Physiology and Pharmacology, Faisalabad, Pakistan
344
(4) Agriculture University, Department of Animal Health, Peshawar,
Pakistan
(5) Riphah International University, Riphah Academy of Research and
Education, Islamabad, Pakistan
The objective of this study was to determine the pharmacokinetics and
optimal dosage regimen of ciprooxacin in Sahiwal cattle. Ciprooxacin
was administered intramuscularly at 5 mg/kg body weight in each of
eight animals. Following drug administration, blood samples were collected at different time intervals and analyzed for ciprooxacin using
high performance liquid chromatograph (HPLC). Pharmacokinetic
parameters were calculated using two compartment open model. Peak
plasma concentration (Cmax) of ciprooxacin, 4.35 0.29 lg/mL was
achieved at 0.86 0.04 hours (Tmax). Values for half life of absorption
(t1/2 abs), distribution (t1/2 a) and elimination (t1/2 b) were 0.73 0.19,
0.40 0.04 and 3.25 0.46 hours, respectively. The value for apparent
volume of distribution (Vd) was 1.24 0.16 L/kg, area-under-the-curve
(AUC) 18.73 1.09 lg.hr/mL and total body clearance (ClB) was 0.31
0.02 L/hr/kg. Based on these results, it was concluded that calculated
dose should be higher than the dose recommended by the manufacturer
to treat susceptible bacteria in Sahiwal cattle.
Keywords: Pharmacokinetics, Dosage regimen, Ciprooxacin, HPLC,
Sahiwal cattle
Paper No.: 3395

5-HT2A RECEPTOR MODELLING AND AGONIST DESIGN
Vignir Isberg, D Gloriam, T Balle, T Sander, FS Jrgensen
University of Copenhagen, Department of Medicinal Chemistry, Copenhagen, Denmark
Modelling of agonist bound G-protein coupled receptors (GPCRs)
remains a challenge as experimental structure data is limited. However,
recent developments in GPCR crystallography have resulted in novel
GPCR crystal structures and presented new opportunities in receptor
Modelling (Cherezov et al., Science 2007; 318: 1258-65; Scheerer et al.,
Nature 2008; 455: 497-502). We have modeled an agonist and G-protein
bound 5-HT2A receptor based on the crystal structures of the b2 adrenergic receptor and G-protein bound opsin. A homology model was build in
MODELLER and activated in silico using constrained molecular dynamics in Desmond. Based on the activated model, we have designed several
novel compounds that have now been synthesized and will be tested as
radioligands to be used for positron emission tomography (PET).
Paper No.: 1122

INVOLVEMENT OF A7 NICOTINIC ACETYLCHOLINE
RECEPTOR ON THE CONDITIONED PLACE AVERSION
INDUCED BY NALOXONE IN SINGLE-DOSE
MORPHINE-TREATED RATS
Shigeru Ishida(1,2), Y Ikuta(1), I Miyazaki(3), M Asanuma(3),
H Matsunaga(1), T Sendo(1), H Araki(4), H Kawasaki(2), Y Kitamura(5)
(1) Okayama University Graduate School, Department of Clinical
Pharmacy, Okayama, Japan
(2) Okayama University Graduate School, Department of Clinical
Pharmaceutical Sciences, Okayama, Japan
(3) Okayama University Graduate School, Department of Brain Science,
Okayama, Japan
(4) Ehime University Graduate School, Department of Clinical Pharmacology and Pharmacy, Ehime, Japan
(5) Okayama University Graduate School, Department of Pharmaceutical
Care and Health Sciences, Okayama, Japan
It is well known that opiate withdrawal symptoms can be precipitated by

an opioid receptor antagonist both in humans and rodents. Recently, there
has been renewed interest in the phenomenon of acute opiate dependence. While it is well documented in behavioral and neurochemical
studies of chronic opiate dependence, few attempts have been made to
examine a model of acute opiate dependence. We previously reported
that conditioned place aversion (CPA) could be observed when naloxone
was administrated 24 h after even a single-dose of morphine, and pretreatment nicotine attenuates this naloxone-precipitated CPA (Araki H et
al, Psychopharmacology 2004; 171: 398-404). Furthermore, we suggested the possible involvement of the central amygdaloid nucleus (CeA)
in the negative motivational component of this naloxone-precipitated
withdrawal from acute morphine dependence (Jin C et al, Psychopharmacology 2004; 175: 428-435). In the present study, we examined the
mechanism of nicotine-induced attenuation of CPA precipitated by naloxone in single-dose morphine-treated rats. In the behavioral study, nicotine
signicantly attenuated naloxone-precipitated CPA by microinjection into
the CeA. Tropisetron, an a7 nicotinic acetylcholine receptor (nAChR)
agonist, into the CeA, also signicantly attenuated CPA. In the immunohistochemical study, nicotine increased c-Fos expression in the CeA.
This increase was signicantly inhibited by methyllycaconitine, an a7
nAChR antagonist. These results suggest that the a7 nAChR subtype
plays a role in nicotine-induced attenuation of CPA, and CeA may be
one of the brain regions involved in the effect of nicotine on acutely opiate dependent subjects.
Paper No.: 2380

SEROTONERGIC GENE POLYMORPHISMS AND DRUG
PLASMA CONCENTRATION INFLUENCE THERAPEUTIC
RESPONSE TO PAROXETINE IN PATIENTS WITH PANIC
DISORDER
Shin Ishiguro(1), M Ueda(1), T Watanabe(1), Y Saeki(1), T Ayugase(1),
A Saito(2), K Akiyama(2), Y Inoue(3), S Morita(4), G Hirokane(4),
N Yamada(4), K Shimoda(1)
(1) Dokkyo Medical University School of Medicine, Department of
Psychiatry, Tochigi, Japan
(2) Dokkyo Medical University School of Medicine, Department of
Biological Psychiatry and Neuroscience, Tochigi, Japan
(3) Mitsubishi Tanabe Pharma Factory Ltd, Japan
(4) Shiga University of Medical Science, Department of Psychiatry,
Japan
Serotonin receptor gene polymorphisms such as -1019C/G promoter
polymorphism of serotonin 1A (5-HT1A) receptor gene and serotonin
2A (5-HT2A) receptor gene polymorphism have been found to be associated with panic disorder (PD), while 5-HTT gene-linked polymorphic
region (5-HTTLPR) is not associated with PD in meta-analysis of association studies. The objective of this study was to evaluate genetic and
pharmacokinetic factors for pharmacotherapeutic effect of paroxetine
(PAX) in patients with PD. Subjects were 65 drug nave patients who
fullled DSM-IV-TR criteria for a diagnosis of PD. Subjects were
received 10 mg/day of PAX for 4 weeks. Severity of PD was assessed
with the Panic and Agoraphobia Scale (PAS) at baseline and 2, 4 weeks
after the initiation of the treatment. Plasma concentration of PAX in 2
and 4 weeks after the initiation of pharmacotherapy was determined by
high performance liquid chromatography. -1019C/G 5-HT1A, -1438G/A
5-HT2A and 5-HTTLPR genotypes were determined by polymerase
chain reaction techniques. Multiple regression analysis revealed that the
plasma concentration of PAX, 5-HTTLPR genotype and -1019C/G 5HT1A gene polymorphism were signicant factors affecting clinical
response to PAX (reduction ratio of PAS score) in 2 weeks after the initiation of pharmacotherapy, while -1019C/G 5-HT1A gene polymorphism
was the only signicant factor affecting clinical response to PAX in 4
weeks after initiation of pharmacotherapy. The present study indicated
that plasma concentration of PAX, 5-HTTLPR genotype and -1019C/G
5-HT1A genotype may inuence the therapeutic response to PAX in PD.
345
Paper No.: 876
P19 - GENERAL SESSION - CELL BIOLOGY
DUAL REGULATION OF HEPATOCYTE APOPTOSIS BY
REACTIVE OXYGEN SPECIES: INCREASED BIMEL
EXPRESSION DOWNSTREAM OF ERK AND SUPPRESSIVE
BIMEL DEGRADATION BY PROTEASOME INHIBITION
nicantly greater than that of PC-fed rats. In PC-fed rats, AII-induced vasocontraction was augmented by L-NAME and endothelium-denudation.
In contrast, AII-induced contraction in CH rats was not affected by LNAME. These results suggest that chronic administration of PC in hypercholesterolemic rats increased an antioxidant GS, and improved the
endothelial function to produce the vasorelaxing factors such as nitric
oxide.
Yasuhiro Ishihara(1), M Sekine(1), K Takeuchi(2), F Ito(2),

N Shimamoto(1)
(1) Tokushima Bunri University, Faculty of Pharmaceutical Sciences at
Kagawa, Kagawa, Japan
(2) Setsunan University, Faculty of Pharmaceutical Sciences, Japan
Reactive oxygen species (ROS) are well known to induce apoptosis via
mitochondrial pathway. Because apoptosis induced by ROS is related to
various disorders such as neurodegenerative diseases, cardiac ischemiareperfusion injury and alcoholic liver injury, the elucidation of its execution mechanism is exceedingly important. Recently, stimuli of ROS and/
or accompanied with ROS generation activate ERK pathway, leading
apoptosis. However, this mechanism is rarely understood. Thus, in this
research, we attempted to explore the mechanism of apoptosis mediated
by ERK activation. Increased ROS levels and subsequent apoptosis in rat
primary hepatocytes were induced by the inhibition of catalase and glutathione peroxidase activities. ERK activation was necessary for the execution of hepatocyte apoptosis. A member of Bcl-2 family proteins, BimEL
was transcriptionally activated downstream of ERK pathway. Transfection of Bim siRNA inhibited BimEL expression and hepatocyte apoptosis. Active ERK is reported to phosphorylate BimEL, followed by its
degradation by protesome. However, in this apoptosis, proteasome activity was decreased by ROS resulting in the accumulation of BimEL
including phosphorylated and ubiquitinated forms inside hepatocytes.
Collectively, while ROS transcriptionally increase BimEL protein levels
via ERK pathway, ROS decrease proteasome activity and subsequently
the accumulation of BimEL inside hepatocyte, leading apoptosis. It is of
signicance in this research to demonstrate that dual regulation of ROS
in hepatocyte apoptosis, increased expression and decreased degradation
of BimEL.
Paper No.: 2223

EFFECTS OF POLYPHENOLIC COMPOUNDS ON
GLUTATHIONE LEVEL AND ENDOTHELIAL NO
PRODUCTION IN HYPERCHOLESTEROLEMIC RATS
Akira Ishihata, Y Igarashi, Y Shimizu, Y Kobayashi, A Yamada,
S Hishinuma, T Hosoda, Y Katano
Yamagata University School of Medicine, Department of Theoretical
Nursing and Pathophysiology, Yamagata, Japan
In hypercholesterolemia, oxidative stress can increase the platelet aggregability directly or through impaired endothelial function. We investigated whether chronic administration of polyphenolic compounds (PC)
reduced the plasma lipid concentration and oxidative stress in hypercholesterolemic rats, and whether PC improved endothelial function. Male
F344 rats were fed standard diet (ST), 4% cholesterol-rich-diet (CH), or
4% cholesterol-rich-diet plus red wine polyphenolic compounds (CH/
PC) for 3 months. Plasma lipids were higher both in CH and CH/PC
than ST, but were not different between CH and CH/PC. To evaluate
antioxidant levels in each rat, the reduced glutathione (GSH) content and
the superoxide dismutase (SOD) activity were measured. High cholesterol diet reduced the GSH level, but PC-fed rats increased it to the control level. PC did not increase the SOD activity in high cholesterol
conditions. In aortic vascular ring preparations, AII induced the vascular
smooth muscle contraction. In cholesterol-fed rats, the tension was sig-
Paper No.: 2238

HEALTH AND DISEASE
NO DILATES RAT RETINAL BLOOD VESSELS THROUGH
THE COX-1-DEPENDENT PROSTANOIDS-CAMP SIGNALLING
PATHWAY IN VIVO
Kunio Ishii, A Mori, K Sakamoto, T Nakahara
Kitasato University School of Pharmaceutical Sciences, Department of
Molecular Pharmacology, Tokyo, Japan
Previous studies demonstrated that nitric oxide (NO) stimulates the cyclooxygenase (COX)-dependent pathway in the ocular vasculature; however, importance of this mechanism in regulating retinal circulation
remains to be elucidated. Therefore, we investigated the role of COXdependent pathway in NO-induced vasodilation of rat retinal blood vessels in vivo. The retinal vascular responses were assessed by determining
changes in diameters of retinal blood vessels contained in the ocular fundus images that were captured with an original high-resolution digital
fundus camera for small animals. Both systemic blood pressure and heart
rate were continuously recorded. Localization of COX and soluble guanylyl cyclase (sGC) in the rat retina was also examined by means of
immunohistochemical methods. The NO donors (SNP and NOR3)
increased diameters of retinal blood vessels and decreased systemic
blood pressure in a dose-dependent manner. Treatment of rats with indomethacin or SC-560, an inhibitor of COX-1, markedly attenuated the
vasodilation of retinal blood vessels, but not the depressor response, to
the NO donors. However, both vascular responses to NO donors were
not affected by the COX-2 inhibitors NS-398 and nimesulide. The adenylyl cyclase inhibitor SQ 22536 reduced the retinal vascular responses
to the NO donors, whereas the sGC inhibitor ODQ failed to affect them.
The COX-1 immunoreactivity was found in blood vessels in the rat retina. On the other hand, the retinal blood vessels were faintly stained for
sGC, although the apparent immunoreactivities were observed on choroidal blood vessels. These results suggest that the COX-1-dependent prostanoid-cAMP signalling pathway plays an important role in the rat
retinal vascular responses to NO in vivo.
Paper No.: 1432

DYSFUNCTION OF PRESYNAPTIC REGULATION OF
NOREPINEPHRINE RELEASE FROM ADRENERGIC NERVES
OF CAUDAL ARTERY IN TYPE 2 DIABETIC
GOTO-KAKIZAKI RATS
Reiko Ishii-Nozawa(1), M Mita(2), M Shoji(2), K Takeuchi(1)
(1) Meiji Pharmaceutical University, Department of Clinical Pharmacology, Tokyo, Japan
(2) Meiji Pharmaceutical University, Department of Pharmacodynamics,
Tokyo, Japan
The spontaneously diabetic Goto-Kakizaki (GK) rat is a model of type 2
diabetes mellitus. In the present study, we examined endogenous norepinephrine (NE) release from caudal arteries of 12-week-old GK rats and
age-matched Wistar rats. NE was quantied by an HPLC-electrochemical
346
detection technique. Electrical eld stimulation (EFS: 1 Hz, 0.5 msec
duration, 50 V, for 3 min) evoked signicant NE release from caudal
arteries of both rats. The NE content of caudal arteries and plasma NE
concentration were signicantly lower in GK rats than Wistar rats,
though the amount of NE release was almost equal in both rats. We
examined the effects of an a2-adrenoceptor agonist clonidine and an A1adenosine receptor agonist 2-chloroadenosine on the release of endogenous NE evoked by EFS. These agonists signicantly reduced NE
release from Wistar rats; however, it did not affect NE release from GK
rats. Furthermore, we examined the effects of an uptake inhibitor desipramine (DES) on the release of endogenous NE evoked by EFS. DES signicantly facilitated NE release from both rats in a concentrationdependent manner; however, the amount of NE released in the presence
of DES was less in GK rats than Wistar rats. These results suggest that
the dysfunction of presynaptic receptors and uptake system on sympathetic nerves in GK rats may be related to the autonomic nervous system
dysfunction associated with diabetic complications.
Paper No.: 1289

CHARACTERIZATION OF RESISTANCE TO CYTOSINE
ARABINOSIDE (ARA-C) IN NALM-6 HUMAN B LEUKEMIA
CELLS
Masaaki Ishikawa, S-I Kanno
Tohoku Pharmaceutical University, Department of Clinical Pharmacotherapy, Sendai, Japan
Cytosine arabinoside (1-b-D-arabinofuranosylcytosine; Ara-C) is the
most important antimetabolite used for acute leukemia. We have established Ara-C (0.003-1 umol/l)-resistant NALM-6 leukemia cells,
attempted the characterization of their resistance, and examined whether
Ara-C resistance related to new markers, the activation of NF-jB, telomerase activity and Fas expression. The Ara-C-resistant cell lines were
developed by stepwise increases in the drug. NF-jB activation was
assayed by electrophoretic mobility shift assay. Telomerase activity was
measured by using a PCR-based telomeric repeat amplication protocol
(TRAP). Fas expression detected using ow cytometric analysis. Apoptotic cells were estimated based on changes in nuclear morphology. The
mRNA expression of human equilibrative nucleoside transporter-1
(hENT-1), which is an uptake transporter of Ara-C, was initially
decreased during the acquisition of resistance to Ara-C. There were no
differences in the cytotoxic effect of other anticancer drugs, but there
was similar resistance to nucleoside analogues via hENT-1 between the
parental and resistant cells. The activation of NF-jB was accompanied
by the acquisition of Ara-C resistance. Telomerase activity increased with
the acquisition of Ara-C resistance. Fas expression increased with the
acquisition of Ara-C resistance in the late stage. The induction of apoptosis and reduction of cell viability by cytotoxic anti-Fas antibody was
more susceptible in resistant cells than parental cells. Our results have
revealed that decreased hENT-1 expression and function is causatively
responsible for the acquisition of Ara-C resistance, and resistance to AraC up-regulates new markers: the activation of NF-jB, telomerase activity
and Fas expression.
Paper No.: 1583

APELIN IS MAINLY REGULATED BY HIF-1A IN
ASTROCYTES
Yuki Ishimaru, A Kasai, Y Yoshioka, A Yamamuro, S Maeda
Setsunan University Faculty of Pharmaceuticals, Department of Pharmacotherapuetics, Osaka, Japan
Apelin is an endogenous ligand for APJ, a newly deorphanized G proteincoupled receptor. Recently, we found that apelin expression was dramatically increased during retinal angiogenesis in oxygen-induced retinopathy
model, and that apelin was a crucial angiogenic factor for hypoxiainduced retinal angiogenesis. Expression of vascular endothelial growth
factor (VEGF), a potent angiogenic factor, was also increased during
hypoxia-induced retinal angiogenesis. Although both these factors were
upregulated by hypoxia, the difference in these expression patterns in retina was observed during hypoxia-induced retinal angiogenesis. Hypoxia
inducible factor (HIF) as a transcription factor plays an essential role in
regulating gene expression in response to hypoxia. It has been reported
that HIF-a subunit has three isoforms (HIF-1a, HIF-2a, and HIF-3a). In
this study, we investigated the involvement of HIF-1a and HIF-2a in the
regulation of the hypoxia-induced expression of apelin in astrocytes which
are secreting VEGF and guiding retinal angiogenesis. In hypoxic astrocytes, apelin expression was dramatically inhibited by a small interference
(si) RNA targeting HIF-1a but slightly by siRNA targeting HIF-2a. By
contrast, the inhibition of VEGF expression by siRNA targeting HIF-1a
was similar to that by siRNA HIF-2a in hypoxic astrocytes. The hypoxiainduced upregulation of both these factors were completely blocked by
siRNAs targeting both HIF-1a and HIF-2a, implying that the induction of
both these factors was not regulated by HIF-3a. These data suggest that
hypoxia-induced upregulation of apelin is mainly regulated by HIF-1a in
astrocytes although that of VEGF by HIF-1a and HIF-2a.
Paper No.: 3301

DISEASES
COMPARATIVE MECHANISMS UNDERLYING THE HEALING
EFFECTS OF CAPTOPRIL AND RANITIDINE ON
ASPIRIN-INDUCED GASTRIC LESIONS
Nafeeza Ismail(1), N Basri(2)
(1) University Technology MARA, Faculty of Medicine, Department of
Pharmacology, Shah Alam, Selangor, Malaysia
(2) University Kebangsaan, Selangor, Malaysia
Captopril, an angiotensin converting enzyme inhibitor, currently used as
an antihypertensive agent has been shown to have antioxidant properties.
The study aims to determine the effects of captopril on factors affecting
gastric mucosal integrity and the healing of aspirin-induced gastric
lesions. Twelve male Sprague-Dawley (200-250g) rats given aspirin (40
mg/100g body weight) were divided into two groups; Ranitidine and captopril. The dose of captopril used was 1 mg/100g body weight daily and
ranitidine was given 2.5 mg/100g body weight given twice daily. Ranitidine and captopril were given orally for 4 weeks after which, all rats were
killed and parameters measured. Gastric acidity and gastric mucosal content of malodialdehyde (MDA) was signicantly reduced (P < 0.05) in the
ranitidine group compared to the captopril group. Captopril, signicantly
increased the gastric mucosal content of prostaglandin (PGE2) compared
to ranitidine. The gastric mucosal content of GSH was comparable in both
groups. There was, however no difference between ranitidine and captopril on the gastric lesion index. Thus, captopril has comparable healing
effects to ranitidine on aspirin-induced gastric lesions. The mechanism by
which captopril heals gastric lesions is by increasing gastric PGE2
whereas ranitidine involves multiple mechanisms.
Paper No.: 3151

DISEASES
THERAPEUTIC TARGETING OF CCL17 VIA THE SYSTEMIC
ADMINISTRATION OF A MONOCLONAL ANTIBODY
AMELIORATES EXPERIMENTAL FUNGAL ASTHMA
Ugur Ismailoglu(1,2), A-P Moreira(1), M Ryan(3), A Das(3),
AL Coelho(1), CM Hogaboam(1)
347
(1) University of Michigan Medical School, Department of Pathology,
Ann Arbor, MI, USA
(2) Hacettepe University, Faculty of Pharmacy, Department of Pharmacology, Ankara, Turkey
(3) Centocor Inc., Philadelphia, PA, USA
Aspergillus fumigatus exacerbates asthma and other allergic lung diseases. The pulmonary immune response directed against Aspergillus is
skewed toward Th2 type immune cells, which are recruited into the lung
via chemokine receptor 4 (CCR4). CCL17 and CCL22 are natural
ligands for CCR4. Since we previously observed increased lung CCL17
and CCL22 levels during A. fumigatus-induced asthma in mice, we
hypothesized that systemic immuno-neutralization of CCL17 and/or
CCL22 might represent an attractive approach for the amelioration of this
disease. To induce chronic fungal asthma, mice that were previously sensitized with Aspergillus antigens were challenged with conidia (i.e. day
0). Anti-CCL17 or anti-CCL22 neutralizing mouse monoclonal antibodies were given (5 lg/dose/i.p.) separately or together every other day
from days 0 to 7, or days 7 to 14 after the conidia challenge. Similar
IgG2A treatment was used as isotype control. At day 7 and 14 time
points, airway resistance was evaluated, mice were sacriced, and lung
tissues were isolated for histological, molecular, and proteomic analysis.
Anti-CCL17 treatment signicantly decreased the contractility to methacholine, fungal retention, inammatory cell recruitment in the lung, and
mucus production compared with IgG2A treated group. However, antiCCL22 treatment had no such effects at either time point. Surprisingly,
at day 7 the combination of anti-CCL17 and anti-CCL22 exacerbated airway inammation and methacholine responsiveness, possibly due to
greater fungal retention in the lungs. Together, our results highlight the
therapeutic potential of targeting CCL17 alone in established fungal
asthma in mice. Thus, this chemokine could be an important target during clinical asthma.
Paper No.: 908

HEALTH AND DISEASE
ROLE OF AT1 RECEPTOR AND NAD(P)H OXIDASE IN THE
PRESSOR RESONSE AND HYPOTHALAMIC ANTIOXIDANT
ENZYMES ACTIVITY INDUCE BY FORCED
IMMOBILIZATION IN RATS
Anita Israel, J Silva, S DeJesus, I Medina, MR Garrido
Paper No.: 3326

PHARMACOTHERAPEUTICS SKILLS TRAINING DESIGN: A
CASE STUDY OF MEDICAL FACULTY - ANDALAS UNIVERSITY
Laila Isrona
Andalas University, Faculty of Medicine, Department of Pharmacology
and Therapeutics, Padang, Indonesia
Having done a curriculum analysis at Medical Faculty of Andalas University (MFAU) INDONESIA, apparently there are at least three weaknesses identied, that is: a lack of integration, a lack of evaluation and a
lack of organization. Regarding a lack of integration, we can see not only
in the acquisition of knowledge but also in the accomplishment of clinical skills. Skills training are concentrated at one block and weak of supporting of procedural knowledge. This paper has presented the design of
Pharmacotherapeutics skill training based on a thorough analysis of the
existing curriculum at MFAU context and academicals literatures. This
design is implemented in accordance to the theory of Spiral Curriculum.
The skills are applied at year 2 and continue until clerkship program.
The skills consist of taking a drug history, prescription writing, acquiring
inform consent to therapy, acquiring objective information which underpin safe and effective prescribing, selecting adverse drug reactions and
interactions, drug administration, drug allergy, monitoring drug therapy,
and managing new evidence.
Paper No.: 920

EFFECTS OF A2 TYPE BOTULINUM TOXIN ON
SPONTANEOUS MINIATURE AND EVOKED TRANSMITTER
RELEASE FROM THE RAT SPINAL EXCITATORY AND
INHIBITORY SYNAPSES
Yushi Ito(1), O Akaike(1), MC Shin(1), K Nonaka(1), T Harakawa(2),
A Ginnaga(2)
Universidad Central de Venezuela, Laboratory of Neuropeptides, Caracas,Venezuela
(1) Research Division for Life Sciences, Kumamoto Health Science

University, Kumamoto, Japan
(2) Institute Human Vaccine Production Department, The Chemo-SeroTherapeutic Research Institute, Kumamoto, Japan
The brain renin angiotensin system has an important role in stress

response. Abundant evidence suggest that a key mechanism by which
central angiotensin II (ANG) inuences blood pressure is via its ability
to produce reactive oxygen species (ROS) such as superoxide (02.-),
which is sequentially metabolized by the enzymes superoxide dismutase
(SOD), catalase (CAT) and glutathione peroxidise (GPx). The role of
ROS in the stress response is matter of research. We assessed the
involvement of AT1 receptor, NAD(P)H oxidase and 02.- in the pressor
response and in the hypothalamic antioxidant enzymes activity induced
by forced immobilization (IMO) in rats. Male Sprague-Dawley rats (220240g) were divided in the following groups 1.Control; 2. IMO (2 hr); 3.
Losartan (LST) (100 mg/kg, p.o, 14 days); 4.LST+IMO; 5.Apocinin
(APO) (5 mg/kg, i.p, 4 days); 6. APO+IMO; 7. Tempol: TEM (20 mg/
kg, i.p, 3 days) and 8. TEM+IMO. Mean arterial pressure (MAP) and
HR were recorded, before and after IMO, using tail-cuff plethysmograph.
Immediately, the rats were sacriced by decapitation and the hypothalamus dissected under stereomicroscopic control. CAT, SOD and GPx
activity were determined by spectrophotometric analysis. IMO signicantly increased PAM (35 2,4 mmHg), HR, and hypothalamic CAT,
SOD and GPx activity. LST, APO and TEM blunted the pressor response
and the increase of the three antioxidant enzymes activity induce by
IMO. Our results indicate a role AT1 receptor/NADPH oxidase/02.- pathway in the pressor and antioxidant enzyme activity response induced by
acute stress.
(Mision Ciencia, Proyecto ECCV No. 2007001585).
We observed effects of newly developed A2 type botulinum toxin

(A2NTX) on spontaneous miniature and evoked transmitter release from
inhibitory (glycinergic or GABAergic), or excitatory (glutamatergic)
nerve terminals in rat spinal cord, by use of synaptic bouton preparations, under voltage clamp condition. A2NTX (0.1-1 pM) initially augmented and then decreased amplitude and frequency of spontaneous
miniature release of glycine or GABA (mIPSCs) dose-dependently. At an
increased concentration (1-10 pM), A2NTX suppressed the amplitude of
glutamatergic mEPSCs. The rank order of inhibitory effects was glycinergic > GABAergic >> glutamatergic synapses. Focal electrical stimulation
of synaptic boutons elicited eIPSC or eEPSC with larger amplitude and
low failure rate (Rf). A2NTX (0.01-1 pM) initially enhanced amplitude
or decreased the failure rate of eIPSC or eEPSC, and then almost completely abolished generation of eIPSC or eEPSC. The rank order of the
inhibitory effects on the amplitude or Rf were glycinergic eIPSC ? GABAergic eIPSC > glutamatergic eEPSCs. Excess extracellular K+ or Ca2 +
(excess [K+]o or [Ca2 + ]o), and 4-AP restored glycinergic, GABAergic
or glutamatergic mIPSCs suppressed by A2NTX. We conclude that
A2NTX inhibits spontaneous miniature release at 0.1-10 pM and evoked
release at 0.01-1 pM in rat spinal cord, and the inhibition was much efcient in evoked rather than spontaneous miniature release. Excess [K+]o,
4-AP and excess [Ca2 + ]o, which raise the intracellular Ca concentration,
rescue the transmission suppressed by A2NTX poisoning, suggesting the
transmitter release machinery became less sensitive to intracellular Ca2 +
in A2NTX poisoned synaptic boutons.
348
Paper No.: 1937
ROLE OF NITRIC OXIDE/CYCLIC GMP AND CYCLIC AMP IN
THE BETA-ADRENOCEPTOR VASORELAXANT RESPONSE
EGCG still showed a signicant decrease in tube formation. The MMP-2

and MMP-9 activitis and MMP-2 proteins were inhibited by TF3 or
EGCG in a concentration-dependent mannar. Conclusion: The effect of
TF3 was similar to that of EGCG, indicating that the tube formation of
endothelial cells was suppressed by both MMPs in vitro. Our results suggest TF3 and EGCG have anti-tumor effect via antiangiogenesis in vitro.
Maria Dolores Ivorra, N Flacco, S Estrada, M Perez-Aso, P DOcon

University of Valencia, Department of Pharmacology, Burjassot, Spain
Cyclic AMP (cAMP) is the classical second messenger of b-adrenoceptors (b-ARs), but its role in vasorelaxation has not been clearly established and other mechanisms such as nitric oxide/cyclic GMP (NO/
cGMP) pathway have been proposed in several vessels. We examined
the role of these two pathways in the conductance and resistance vessels
of male Wistar rats (aorta and small mesenteric arteries, 2nd branch,
SMA). Isoprenaline-induced relaxations (1 nM-10 lM) upon 1 lM or 10
lM phenylephrine were performed in the presence/absence of adenylate
cyclase inhibitor 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ 22,536,
10 lM), guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 lM) or nitric oxide synthase inhibitor L-NAME
(100 lM). SQ 22,536 did not modify the vasorelaxant response in the
aorta, but signicantly shifted the isoprenaline concentration-response
curve in SMA rightwards (pD2 = 7.1 0.1 versus 5.7 0.9, n = 5).
ODQ and L-NAME inhibited the response in the aorta (Control:
Emax=102.6 4.1, pD2 = 7.5 0.1; ODQ: Emax=81.1 2.8, pD2 =
6.1 0.1; L-NAME: Emax=88.3 2.7, pD2 = 6.1 0.1, n = 4-5), but
not in SMA. In the aorta, isoprenaline (10 lM) increased cAMP and
cGMP levels (333 42% and 242 39% versus basal, respectively, n =
8-13). Propranolol (1 lM) inhibited these accumulations, CGP20712
(b1-AR antagonist, 1 lM) signicantly inhibited cAMP, but not cGMP,
whereas ICI118551 (b2-AR antagonist, 1 lM) inhibited cGMP, but not
cAMP isoprenaline-induced accumulation. We conclude that, in the
aorta, b1-ARs are preferentially coupled with adenylate cyclase and b2ARs are preferentially coupled with guanylate cyclase, being the NO/
cGMP the predominant pathway involved in the vasorelaxant response.
However, in SMA, cAMP plays a major role in b-AR mediated relaxation.
Paper No.: 1613

TEA POLYPHENOLS INHIBIT TUBE FORMATION IN
COCULTURED ENDOTHELIAL CELLS WITH FIBROBLASTS
Shinichi Iwai, A Tsuboi, C Kurahashi, A Yura, K Oguchi
Paper No.: 1966

ASSOCIATION BETWEEN AUTONOMIC NERVOUS SYSTEM
ACTIVITY IN SCHIZOPHRENIA AND ANTIPSYCHOTIC
MEDICATION: THE COMPARISON OF TYPICAL AND
ATYPICAL ANTIPSYCHOTIC DRUGS
Yohko Iwamoto(1,2), I Kisida(1,2), M Fujibayashi(3), S Tanaka(3),
C Kawanishi(1), C Ishii(2), N Ishii(2), Y Hirayasu(1), T Moritani(3)
(1) Department of Psychiatry, Yokohama City University School of
Medicine, Yokohama, Japan
(2) Fujisawa Hospital, Kanagawa, Japan
(3) Graduate School of Human and Environmental Studies, Kyoto
University, Kyoto, Japan
Objective: Reduced activity of autonomic nervous system (ANS) in
schizophrenic patients, which possibly increases prevalence of cardiovascular diseases, has been reported. In the present study, we investigated
the inuence of typical and atypical antipsychotic drugs on the ANS
activity in schizophrenic patients. Methods: One hundred and nineteen
Japanese patients with schizophrenia participated in this study. ANS
activity was assessed by means of heart-rate variability power spectral
analysis, which enables us to identify separate frequency components,
i.e., total power (TP: overall ANS), low-frequency (LF: sympatho-vagal)
power, and high-frequency (HF: vagal) power, during a resting condition.
Antipsychotic drug treatment was investigated in each participant, and
inuence of prescription patterns on ANS activity was analysed. This
study was approved by the ethical committee of Fujisawa Hospital, and
written informed consent was obtained by all participants. Results: The
atypical drug/total antipsychotic drug dose ratio was associated with
ANS activity (TP, p = 0.008; LF, p = 0.003), using multiple regression
analyses for ANS, sex, age, and antipsychotic drug dose calculated in
terms of chlorpromazine equivalents as independent variables. The ANS
activity of patients treated with atypical antipsychotic drugs alone was
higher compared to those treated with both atypical and typical antipsychotic drugs (LF, p = 0.047) although no signicant difference was found
in mean dose of antipsychotic drugs between those groups. Conclusion:
Our ndings indicate that inuence of atypical antipsychotic drugs on
ANS activity was less than that of typical antipsychotic drugs.
Showa University School of Medicine, Department of Pharmacology,

Tokyo, Japan
Introduction: Several cohort studies have reported that green tea extract
has potential as a natural anti-tumor medicine. Tea polyphenols are
thought to be responsible for most of the anti-tumor activity. Several tea
polyphenols, especially those with galloyl, have strong antioxidant and
antiangiogenic activities such as (-)-epigallocatechin-3-gallate (EGCG).
Theaavin-3, 3-digallate (TF3) also, a theaavin derivative in black tea,
has two galloyl groups. Matrix metalloproteinases (MMPs) are associated
with extracellular matrix degradation, cellular migration, and angiogenesis, and EGCG is an inhibitor of the activity and secretion of MMPs;
thus one of its major roles is the inhibition of angiogenesis. However,
there are few studies of angiogenesis in theaavin derivatives. We investigated the effects of TF3 and EGCG on angiogenesis in vitro. Materials:
Angiogenesis was assayed by cocultured human umbilical vein endothelial cells with broblasts. Cells were cultivated in various concentrations
of TF3 and EGCG. After 11 days, MMP-2 and MMP-9 activities and the
pro-MMP-2 protein in the medium were measured by gelatin zymography and immunoassay, respectively. Results: Tube formation was markedly inhibited by 100 loM TF3 or EGCG. Even at 10 microM, TF3 or
Paper No.: 1124

INHIBITORY EFFECTS OF OTC-DRUGS ON CYTOCHROME
P450 IN HUMAN LIVER MICROSOME
INHIBITORY EFFECTS OF OTC-DRUGS ON CYTOCHROME
P450 IN HUMAN LIVER MICROSOME
Mariko Iwase(1), Y Nishimura(1), N Kurata(2), Y Saito(1), N Uchida(1),
H Yasuhara(1)
(1) Showa University School of Medicine, Department of Pharmacology,
Tokyo, Japan
(2) Showa University, Faculty of Arts and Sciences at Fujiyoshida,
Tokyo, Japan
Over the counter (OTC) drugs play an important role in self-medication
which is the treatment of common health problems without medical
349
supervision. It has been believed that OTC drugs are relatively safe,
however, their information of the drug-drug interaction mediated by cytochrome P450(CYP) is not enough. This study was designed to evaluate
the inhibitory effects of OTC drugs on CYP1A2, CYP2C9, CYP2C19,
CYP2D6 and CYP3A enzyme activities by using human liver microsomes. Six rst generation H1 receptor antagonist (carbinoxamine(CBX),
clemastine(CLM), diphenhydramine(DPH), diphenylpyraline(DPP),
chlorpheniramine(CP) and triprolidine(TP)) and two secretolytic and
mucolytic agents(ambroxol(AX) and bromhexine(BH)) were estimated.
All of the drugs, particularly BH, CLM, AX and DPH showed competitive inhibition on CYP2D6 activity, their Ki values were 0.096, 0.379,
1.76 and 8.02 lM, respectively. BH and CLM also had inhibitory effect
on CYP2C19 activity(Ki = 0.88 and 1.64 lM respectively). In contrast,
weak inhibition of CYP1A2, CYP2C9 and CYP3A4 activities by these
drugs were observed. Preincubation analysis did not show any remarkable effects, suggesting that these eight drugs are unlikely mechanismbased inhibitor of these CYPs. Considering the hepatic and plasma concentration of these drugs, BH, CLM, AX and DPH may cause drug-drug
interaction with other CYP2D6 substrates. OTC drugs are available without Drs prescription and consumers use them self-responsibility. To
avoid the relevant adverse events caused during the medication with
them, further clinical studies are needed to provide accurate information
for their appropriate use.
Paper No.: 1136

INHIBITORY EFFECT OF PIPERLONGUMINE ON PLATELET
AGGREGATION VIA COLLAGEN RECEPTOR
Masaya Iwashita, Y Nasu, A Okuda, N Nakahata
Tohoku University, Graduate School of Pharmaceutical Sciences,
Department of Cellular Signal, Sendai, Japan
Piper longum L. has been used as a crude drug for the treatment of the
disorder of peripherally poor blood circulation in Asia. In the previous
study, we revealed that piperlongumine, a constituent of Piper longum
L., inhibited collagen-induced platelet aggregation. Therefore, in the
present study, we examined the inhibitory mechanism of piperlongumine on collagen-induced platelet aggregation. Fresh blood was
obtained from male rabbits or healthy volunteers. We prepared washed
platelets by a centrifuged method. To examine the mechanism of inhibitory activity of piperlongumine, we examined the effect of piperlongumine on collagen-induced generation of TXB2, a TXA2 stable
metabolite, because collagen-induced platelet aggregation was mediated
via TXA2 generation. Piperlongumine inhibited collagen-induced
TXB2 generation in a concentration-dependent manner. Next, because
TXA2 is generated from arachidonic acid liberated by platelet stimulation such as collagen, we examined the effect of piperlongumine on
collagen-induced arachidonic acid liberation by using [14C]arachidonic
acid-labeled platelets. As the result, piperlongumine inhibited collageninduced [14C]arachidonic acid liberation. Additionally, piperlongumine
also inhibited collagen-induced phosphatidylinositol hydrolysis. It is
suggested that collagen-induced platelet aggregation is mainly mediated
via glycoprotein VI (GPVI). Then, we examined the effect of piperlongumine on GPVI agonist convulxin-induced platelet aggregation,
phosphatidylinositol hydrolysis and protein phosphorylation. As the
result, piperlongumine inhibited the aggregation, phosphatidylinositol
hydrolysis and Syk phosphorylation. In conclusion, piperlongumine
inhibited collagen-induced platelet activation via GPVI with a novel
mechanism of action. Therefore, piperlongumine is a potential lead
compound for valuable antiplatelet drugs.
Paper No.: 1451

HEALTH AND DISEASE
INVOLVEMENT OF EARLY RELEASE OF HMGB1 IN
AGGRAVATION OF NEURONAL DAMAGE AFTER
TRANSIENT FOCAL ISCHEMIA IN DIABETIC RAT BRAIN
Naohiro Iwata, M Okazaki, S Kamiuchi, Y Hibino
Josai University Faculty of Pharmaceutical Sciences, Laboratory of
Immunobiochemistry, Sakado, Japan
Cerebral ischemic injury proceeds with acute neuronal death in the ischemic core and with delayed damage processes in penumbra. High-mobility group box 1 (HMGB1) is massively released extracellularly from
necrotic cells and evokes delayed inammatory processes via interaction
with the receptor for advanced glycation end products or toll-like receptors in the post-ischemic brain. Previously, we revealed that the diabetic
(DM) state aggravates cerebral ischemic injury following stroke in rats.
Thus, we examined the expression of HMGB1 or other inammatory
cytokines in DM rat brain after middle cerebral artery occlusion followed
by reperfusion (MCAO/Re) in the present study. Type1-diabetes was
induced by a single injection of streptozotocin in male Sprague Dawley
rats. In the sham-operated DM rat brain, the immunoreactivity of
HMGB1, which was located in the neuronal nuclei of the cortex, markedly increased compared with that in non-DM control rats. The DM state
aggravated MCAO/Re-induced neurological decits and cerebral injury
assessed by infarction volume. Enhancement of expression in neuronal
nuclei and translocation from the nucleus to the cytoplasm of HMGB1
induced by MCAO/Re was remarkably accelerated in the penumbral
region of DM rat cortex. Immunoblotting revealed that the ischemiainduced increase in release of HMGB1 into the cerebrospinal uid was
also enhanced in DM rats. Moreover, gene expression of interleukin-1b
and tumor necrosis factor-a were also enhanced in DM cortex compared
with in non-DM. These results suggest that early release of HMGB1
may be involved in the aggravation of neuronal damage by transient
cerebral ischemia in DM rats.
Paper No.: 3289

ENHANCEMENT OF ASPIRIN-INDUCED GASTRIC BLEEDING
BY PRETREATMENT OF CLOPIDOGREL, AN
ANTI-PLATELET DRUG, IN RATS: PROPHYLACTIC EFFECTS
OF ANTIULCER DRUGS
Chitose Izuhara, K Yamazaki, H Nishio, K Amagase, K Takeuchi
Kyoto Pharmaceutical University, Department of Pharmacology, Division
of Pathological Sciences, Kyoto, Japan
Background/Aim: We examined the effect of an anti-platelet drug clopidogrel, a P2Y12 receptor antagonist, on gastric bleeding induced by intraluminal perfusion with aspirin (ASA) in the rat stomach, and
investigated the prophylactic effect of antiulcer drugs on gastric bleeding
under such conditions. Methods: Male SD rats were used after 18 h fasting. Under urethane anesthesia, acid secretion was stimulated by i.v. infusion of histamine (8 mg/kg), and the stomach was superfused with saline
or 25 mM ASA for 60 min at a rate of 0.4 ml/min using an infusion
pump, and the perfusate was collected every 5 min. Gastric bleeding was
evaluated as hemoglobin concentration in the perfusate. Clopidogrel (30
mg/kg) was given p.o. 24 h before the perfusion. Omeprazole (30 mg/
kg), famotidine (10 mg/kg) or teprenone (300 mg/kg) was given i.d. 30
min before ASA perfusion. Results: Perfusion of the stomach with ASA
provoked gastric bleeding with an increase of luminal hemoglobin content, under stimulation of acid secretion. Pretreatment with clopidogrel,
despite provoking by itself neither bleeding nor damage, signicantly
increased the severity of gastric damage and bleeding caused by ASA.
Gastric bleeding caused by clopidogrel plus ASA was markedly reduced
350
by pretreatment with omeprazole and famotidine, while teprenone
showed a minimal effect. Conclusion: These results suggest that clopidogrel increases gastric bleeding induced by a low dose of ASA. Both famotidine and omeprazole are useful for preventing gastric bleeding
caused by ASA plus clopidogrel. This model may be useful for screening
of the protective drugs against gastric bleeding.
Paper No.: 1066

PARAQUAT ACTIVATES NRF2-ARE PATHWAY: IMPLICATION
OF DECREASE IN PROTEASOME ACTIVITY IN
DOPAMINE-MEDIATED CYTOTOXICITY
proposed as a possible source for the enhanced calcium levels, which

also implicates calcium-independent phospholipase A2b (iPLA2b), as the
enzyme has been shown to be involved in the regulation of SOC in several different cells. The aim of this work has been to analyze the role of
iPLA2b in the dystrophic mouse cell line EDL-MDX-2. PLA2 activity
has been assessed using either PED6 as substrate or by measuring 3Hlabeled arachidonic acid release. Furthermore, the inuence of PLA2
activity on calcium signalling has been investigated using Fura-2 and
45
Ca2 + . Pharmacological tools have been employed to suppress as well
as to induce iPLA2b specic activity. In accordance with previous ndings, it could be shown that modulating iPLA2 activity has a direct inuence on calcium entry in dystrophic muscle cells. Bromoenol lactone, a
selective iPLA2 inhibitor, markedly decreased thapsigargin induced calcium inux in EDL-MDX-2 myotubes. However, no conclusive evidence
could be obtained to prove that the iPLA2b isoform was in effect responsible for the SOC regulation.
Yasuhiko Izumi(1), S Matsushima(1), N Yamamoto(1), T Kume(1),

H Sawada(2), A Akaike(1)
(1) Kyoto University Graduate School of Pharmaceutical Sciences,
Department of Pharmacology, Kyoto, Japan
(2) Utano National Hospital, Center for Neurological Diseases, Department of Neurology and Clinical Research Center, Kyoto, Japan
Parkinson disease is one of the most common neurodegenerative disorders and characterized by a selective loss of dopaminergic neurons in the
substantia nigra pars compacta. The exact cause of the neuronal loss
remains unclear, although endogenous dopamine could serve as a vulnerability factor for dopaminergic neurons because dopamine itself exhibits
cytotoxicity. Since a decrease in proteasome activity was found in the
substantia nigra with sporadic Parkinson disease, the relationship
between ubiquitin-proteasome system and dopaminergic neuronal death
has been paid to attention, but has remained controversial. Therefore, we
investigated the role of proteasome activity in dopamine-mediated cell
death. Treatment of PC12 rat pheochromocytoma cells with the herbicide
paraquat, a potential risk factor for the development of Parkinson disease,
induced an increase in intracellular dopamine content, and depletion of
intracellular dopamine content suppressed paraquat-induced cytotoxicity.
The effect of paraquat on 26S proteasome activity was examined in
PC12 cells overexpressing a proteasome-sensitive uorescent protein,
ZsProSensor-1. Flow cytometry analysis showed that treatment with
paraquat decreased 26S proteasome activity. Proteasome inhibiton
induced the accumulation of NF-E2-related factor 2 (Nrf2) and antioxidant response element (ARE)-dependent transcriptional activity. Paraquat
also increased ARE activity in a Nrf2-dependent manner. Nrf2-ARE
pathway regulates induction of genes encoding phase II detoxifying
enzymes and antioxidant proteins, such as c-glutamylcysteine synthetase
and hemeoxygenase-1 (HO-1). Paraquat elevated intracellular glutathione
content and upregulated HO-1 protein expression. These results suggest
that reduction of proteasome activity by paraquat stimulates ARE-mediated gene expresssion.
Paper No.: 2284

DISEASE AND THERAPY
DOES IPLA2B PLAY A ROLE IN STORE-OPERATED
CALCIUM ENTRY IN EDL-MDX-2 MYOTUBES?
Karin Jacobson, O Petermann, E Roulet, U Ruegg
Paper No.: 1912

5-HT2C RECEPTOR SIGNAL PEPTIDE
Jan Anker Jahnsen, S Uhlen
University of Bergen, Department of Medicine/Centre for Pharmacy,
Bergen, Norway
Introduction: All G-protein coupled receptors (GPCRs) have seven transmembrane domains, and an extracellularly positioned N-terminal tail.
The GPCRs with short N-tails use the rst transmembrane domain as signal anchor for directing the receptor to the plasma membrane. GPCRs
with longer N-tails often have an additional N-terminal cleavable signal
peptide sequence. Whether a protein has a signal peptide can be predicted from its amino acid sequence. Among the GPCRs for monoaminergic transmittor substances, only the 5-HT2C serotonin receptor andthe
a2C-adrenoceptor are predicted to have signal peptides. The N-terminaltail of the 5-HT2C receptor contains a single nucleotide polymorphism
(SNP) site at amino acid position 23 (Cys23Ser). 87% of the Caucasian
population has a cysteine, while 13% has a serine. The Cys23Ser SNP is
potentially of great clinical importance, since the 5-HT2C serotonin
receptor is implied in psychiatric disorders. Materials: 11 5-HT2C receptor constructs and 14a2C-adrenoceptor constructs were created. The Nterminal tails were modied by truncation, addition of a cleavable signal
peptide, and addition of a FLAG epitope. An antibody could detect if
putative signal peptides were cleaved off. Results and Conclusion: The
results showed that the predicted signal peptide in the N-terminal tail of
the 5-HT2C receptor, including the Cys23Ser SNP site, indeed can act as
a cleavable signal peptide. On the other hand, the predicted signal peptide in the N-terminal tail of the a2C-adrenoceptor did not act as a functional cleavable signal peptide.
Paper No.: 1681

THE EFFECT OF KYP-2047, A PROLYL OLIGOPEPTIDASE
INHIBITOR, ON EXTRACELLULAR NEUROTENSIN- AND
SUBSTANCE P-LIKE-IMMUNOREACTIVITY IN THE RAT
BRAIN
University of Geneva, School of Pharmaceutical Sciences, Laboratory of

Pharmacology, Geneva, Switzerland
Aaro Jalkanen, K Savolainen, M Forsberg
Duchenne Muscular Dystrophy is a progressive muscle wasting disorder

resulting from dystrophin deciency. One characteristic of dystrophic
muscles is abnormally elevated levels of intracellular calcium, detrimental to the cells; however, the cause of the calcium misregulation remains
unresolved. Increased activity of store-operated channels (SOC) has been
University of Eastern Finland, School of Pharmacy, Kuopio, Finalnd

Prolyl oligopeptidase (POP) is a serine protease which cleaves prolinecontaining small peptides. Several peptides that are substrates of POP in
vitro are known to be involved in memory and learning. POP inhibitors
351
have also shown to improve cognitive functions in animals. This effect
may be mediated through the reduced breakdown of the promnesic neuropeptide substrates of POP, such as substance P (SP) and neurotensin
(NT) (Mannisto et al, Drug News Perspect 2007; 20: 293-305). Recent
ndings in peptidomics studies also support the role of POP in the
metabolism of neuropeptides in vivo (Nolte WM et al, Biochemistry
2009; 48: 11971-11981). However, it is not clear how this mainly cytoplasmic enzyme can regulate the extracellular levels and receptor-mediated effects of SP and NT. This study was designed to examine the
effects of a POP inhibitor on the brain extracellular neuropeptide levels
using in vivo microdialysis approach for the rst time in POP studies.
Young male Wistar rats were treated with a single dose of potent POP
inhibitor, KYP-2047 (50 lmol/kg i.p.), or vehicle. Microdialysates from
the striatum of conscious animals were collected for 240 minutes after
treatment. NT and SP like-immunoreactivities (LI) in microdialysates
were measured using a highly sensitive radioimmunoassay (LLOQ 1.2
fmol/100 ll sample). The results show that KYP-2047 had no effect on
the extracellular NTLI. Preliminary data indicates that also SPLI remains
unchanged. We suggest that POP does not signicantly regulate the
turnover of NT and SP in the brain extracellular compartment in vivo.
Paper No.: 971

THE EFFECT OF TRAMADOL ON CONCENTRATION OF
NORADRENALINE IN THE LOCUS COERULEUS OF RATS
DURING THE FORMALIN TEST
Akram Jamshidzadeh(1), MA Mobasher(1), J Sajedianfard(2)
(1) Shiraz University Faculty of Pharmacy and Pharmaceutical Sciences
Research Center, Shiraz, Fars, Iran
(2) Shiraz University Faculty of veterinary Medicine, Shiraz, Fars, Iran
Tramadol is a centrally-acting analgesic with opioid, noradrenergic and
serotonergic properties. The locus coeruleus (LC) is a pontine nucleus
clearly implicated in the regulation of pain, depression and anxiety. In
the present study, we evaluated the changes of noradrenalin (NA), in the
locus coeruleus before and after the injection of 20 mg/kg tramadol. The
rats were used for formalin test in four groups. Group I: control, group
II: formalin, group III: tramadol, and group IV: tramadol and formalin in.
The concentration of NA in microdialysis samples were measured by
HPLC-ECD every 15 minutes. The level of NA rises up to about four
folds in LC in the group II 45 minutes after the microdialysis, and after
the injection of tramadol in group IV it was a signicant reduction in NA
concentration. We observed that in group IV there was a signicant
reduction of pain scores ten minutes after the injection which was coincide with the peak of NA concentration in LC. We concluded that tramadol has an indirect effect on LC because the injection of tramadol by
itself could not change the level of NA in LC (group III) but in the conditions of pain, tramadol acts on l receptors in paraaquaductal grey matter (PAG), central amygdaloid nucleus and spinal cord, furthermore by
inhibiting the reuptake of NA and increase of its concentration in synaptic spaces and nerve terminals the drug could suppress the pain.
Paper No.: 1523

AMOXICILLIN PHARMACOKINETICS IN PATIENTS WITH
ASPIRATION PNEUMONIA AS A COMPLICATION OF OVERDOSAGE WITH PSYCHOTROPIC DRUGS: A PILOT STUDY
Pharmacokinetics of amoxicillin has been investigated in six severely

poisoned patients who had attempted suicide by ingestion of high doses
of various psychotropic drugs and had aspiration pneumonia as a complication. Amoxicillin has been administered every 6 hours, as a 30 min.
infusion of 2 grams as a loading dose and 1 gram of antibiotic afterwards. Blood samples were drawn just prior to dosing, at the end of infusion and at time points 1, 2, 3, 4 and 6 hours. Ultra pressure liquid
chromatography coupled to mass spectrometry was used to quantify
amoxicillin in plasma. Non-compartmental model was used to calculate
pharmacokinetic parameters at days 1 and 3 of treatment. Elimination
rate constant of amoxicillin signicantly decreased at the day 3 of treatment in comparison to the day 1 (0.40 0.12 h-1 vs 0.56 0.13 h-1,
respectively; P < 0.02). Both the apparent volume of distribution and
mean residence time were also signicantly higher at the 3rd day of treatment (22.5 7.12 l vs 14.89 7.54 l and 1.51 1.00 h vs 0.74 0.39 h,
respectively). Mean peak plasma concentrations of antibiotic were dosedependent during the treatment. Therefore, the pharmacokinetics of
amoxicillin that was used to treat aspiration pneumonia in patients with
overdose of psychotropic drugs was altered. This should be taken into
consideration in optimizing antibiotic therapy in that category of critically
ill subjects.
Paper No.: 2135

THE USE OF TRUNCATED AREA UNDER THE CURVE AS A
MEASURE OF RELATIVE EXTENT IN BIOEQUIVALENCE
STUDIES
Seong Bok Jang, YJ Lee, LA Lim, K Park
Yonsei University College of Medicine, Department of Pharmacology,
Seoul, South Korea
Objectives: The aim of this study was to investigate the translatability
of the truncated area under the curve (TAUC) as an approximate measure of the area under the curve (AUC) in bioequivalence studies.
Methods: Pharmacokinetic (PK) studies were conducted for 3 oral formulations, one hydroxychloroquine sulfateformulation (t1/2 = 28 days;
CV = 28%) with 48 subjects, and two leunomide formulations (t1/2 =
9 days; CV = 11%) with 25 subjects each. The 3 studies were a 2x2
cross-over design. The blood samples were collected up to 89 days for
the hydroxychloroquine sulfate study and 32 days for the leunomide
studies. The 3 data sets were then resampled 500 times each and
TAUCs were calculated at different truncated times. Bioequivalence test
results for TAUCs were obtained from resampled datas ets and compared with AUClast (AUC from 0 to last sampling time) from the original data sets. Results: At the truncation time of 72 hrs and thereafter,
the bioequivalence test results for the leunomide studies were found
100% identical to the original results with AUC last. Although the
accuracy decreased, for hydroxychloroquine sulfate which showed an
extraordinary long half-life, the test results at 72 hrs were still identical
to the original results up to 85%. Conclusions: This work illustrated
the feasibility of applying the proposed TAUC method to bioequivalence trials. It showed that sampling up to only 72 hrs might provide
results accurate enough for most of the drugs including those with a
half-life as long as 10 days.
Supported by Brain Korea 21 Project for Medical Science, Yonsei University
Dusan Jandric, V Dragojevic-Simic, B Ciric, D Jovanovic, V Kilibarda,

S Vucinic, S Dobric
Paper No.: 2121

TARGETING ANTIOXIDANTS TO SITES OF OXIDATIVE
STRESS USING DUAL ACTION DRUGS
Military Medical Academy, Center for Clinical Pharmacology, Belgrade,

Republic of Serbia
Nitya Jani(1), J Ziogas(1), C Schiesser(2), C Wright(1)
352
(1) University of Melbourne, Department of Pharmacology, Parkville,
Melbourne, VIC, Australia
(2) University of Melbourne School of Chemistry, Bio21 Molecular
Science and Biotechnology Institue, Parkville, Melbourne, VIC,
Australia
Compounds combining the AT1 receptor antagonist milfasartan with antioxidant pharmacophores (selenium, phenol or ebselen) may target antioxidant activity to sites of oxidative stress where AT1 receptors are
upregulated (Warnholtz et al, Circulation 1999; 99: 2027-33). This study
examined the relative antagonist and antioxidant properties of such compounds. The selenium and phenol-substituted milfasartan analogues, but
not the ebselen-milfasartan analogue, retained AT1 receptor antagonist
potency assessed in rat isolated right atria. Antioxidant properties were
examined in a mouse red blood cell haemolysis assay using AAPH as a
radical generator. Each of the antioxidant pharmacophores and the phenol-substituted milfasartan protected against radical-mediated lysis (P <
0.05 vs. vehicle). The other milfasartan analogues did not show antioxidant activity in the haemolysis assay. Antioxidant capacity was also
tested in mouse isolated paced left atria. In this case the free radical generator doxorubicin (30 lM), resulted in a 65% decrease in left atrial force
over 90 min. The reduction in force was prevented by pre-incubation
with ebselen and phenol, but not selenocystein (10 lL each) or the milfasartan analogues. However, pre-treating the atria with milfasartan (10
lM) revealed a protective effect of phenol-milfasartan (P < 0.05 vs. milfasartan). These data show that only the phenol-substituted milfasartan
retained AT1 receptor antagonist potency and antioxidant activity. However, binding to the AT1 receptor interferes with the antioxidant activity
of the phenol-milfasartan. Therefore, phenol-milfasartan needs to be
modied in a manner that exposes the phenol group when bound to the
AT1 receptor in order to generate a dual action drug.
Paper No.: 609

EFFECTS OF 3,5,7,3,4-PENTAMETHOXYFLAVONE ON
ISOLATED HUMAN CAVERNOSUM
Chaweewan Jansakul(1), K Techanaparukse(2), P Sirirugsa(3)
(1) Prince of Songkla University, Faculty of Science, Deaprtment of
Physiology, Hat-Yai, Thailand
(2) Yala Hospital, Department of Surgery, Yala, Thailand
(3) Prince of Songkla University, Faculty of Science, Department of
Biology, Hat-Yai, Thailand
3,5,7,3,4-pentamethoxyavone (PM) is a major avone isolated from
rhizomes of Kaempferia parviora (KP) that act as an aphrodisiac. The
effects and mechanisms of PM actions were examined in vitro using
human cavernosum (HC) obtained from patients that had undergone a
sex-change operation. PM caused a dose-dependent relaxation of the HC,
pre-contracted with phenylephrine (Phe). NG-nitro-L-arginine, ODQ or
TEA did not modify its relaxant activity whereas glybenclamide caused
a slight inhibition. PM did not modify the relaxant activity of glyceryl
trinitrate on HC pre-contracted with PE, but inhibited the relaxation
activity caused by acetylcholine. In normal Krebs solution, in the presence of nifedipine, nifedipine and SKF 96365, or in the Ca2 + -free Krebs
solution, PM caused a further inhibition of HC-contracted by Phe. In HC
with a thapsigargin Ca2 + -empty sarcoplasmic reticulum, whether in normal Krebs or in the Ca2 + -free Krebs solution, PM did not further suppress HC contraction induced by Phe. Y 27632 totally suppressed the
Phe induced HC contraction in the Ca2 + -free Krebs solution in both
normal and the Ca2 + -empty HC. These results indicate that PM caused
an opening of the ATP-sensitive K+ channel, and may also act as a Ca2 +
mobilizing inhibitor of the sarcoplasmic reticulum, but does not act as a
phosphodiesterase inhibitor, a dihydropyridine Ca2 + channel blocker, a
store-operated Ca2 + -channel blocker or a Rho-kinase inhibitor. These
studies indicate that PM is responsible for the therapeutic claims made
for the aphrodisiac activity of KP rhizomes.
Paper No.: 1190

HYPOTENSIVE CONSTITUENTS AND THEIR MECHANISMS
RESPONSIBLE FOR THE HYPOTENSIVE ACTIVITY OF
PHYLLANTHUS ACIDUS (L.) SKEELS
Chaweewan Jansakul(1), Y Leeya(1), M Mulvany(2), E Queiroz(3),
A Marston(4), K Hostettmann(3)
(1) Prince of Songkla University, Faculty of Science, Department of
Physiology, Hat-Yai, Thailand
(3) Laboratory of Pharmacognosy and Phytochemistry, School of
Pharmaceutical Sciences, University of Geneva, Geneva, Switzerland
(4) University of the Free State, Department of Chemistry, Bloemfontein,
South Africa
We previously reported that an n-butanol extract from leaves of Phyllanthus acidus (PA extract) caused a decrease in mean arterial blood pressure (MAP) and heart rate of anesthetized rats and caused an
endothelium-independent relaxation of thoracic aortic pre-constricted
with phenylephrine (Phe) by opening of ATP-sensitive K+ channels and
Ca2 + activated K+ channels, as well as stimulation of a soluble guanylate
cyclase in the vascular smooth muscle. The present study aimed to identify the active substances responsible for these effects of the PA extract.
Bioguided isolation of the pharmacologically active compounds in the
PA extract led to the identication of ve compounds identied by classical spectroscopic methods: adenosine, 4-hydroxybenzoic acid, caffeic
acid, hypogallic acid, and kaempferol. Each caused a decrease in MAP
and heart rate in anesthetized rats, and caused dilatation of the Phe-preconstricted aortic rings. NG-nitro-L-arginine or removal of the endothelium reduced the vasodilatory activity. ODQ and tetraethylammonium
attenuated the vasodilatory activity of adenosine whereas glybenclamide
and ODQ attenuated the effect of hypogallic acid. These results suggest
that the hypotensive and negative chronotropic activities of the PA
extract are the result of the direct action of these ve compounds on the
blood vessels. The hypotensive action is through stimulation of nitric
oxide release from the vascular endothelium, in part through stimulation
of soluble guanylate cyclase, and opening of ATP-sensitive K+ channels
and Ca2 + -sensitive K+ channels in the vascular smooth muscle.
Paper No.: 3344

ANALGESICS
EVIDENCE FOR CGRP UPTAKE IN RAT DURA MATER
ENCEPHALI: RELEVANCE TO MIGRAINE
Inger Jansen-Olesen(1), D Amrutkar(1), A Mataji(2), H Salmasi(2),
M Sheykhzade(2), K Messlinger(3), J Olesen(1), S Gupta(1)
(1) Glostrup Research Institute, Glostrup Hospital, Faculty of Health Sciences, University of Copenhagen, Department of Neurology, Glostrup,
Denmark
(2) University of Copenhagen, Faculty of Pharmaceutical Sciences,
Department of Pharmacology and Pharmacotherapy, Copenhagen, Denmark
(3) University of Erlangen-Nurnberg, Institute of Physiology and Pathophysiology, Germany
Background: Calcitonin gene-related peptide (CGRP), a potent vasodilator, is distributed in trigeminovascular sensory nerve bres and is
released upon activation. Uptake and re-release of peptide signalling
molecules is generally not believed to occur. We have previously found
functional evidence for uptake in a guinea pig basilar artery preparation.
CGRP release as well as uptake was investigated in dura mater, a tissue
highly relevant to migraine. Experimental approach: The hemi-sected
skull model was used to study CGRP release and uptake from rat dura
353
mater using an enzyme-linked immunoassay. We used four successive
capsaicin challenges to deplete CGRP followed by CGRP incubation to
allow uptake. Immunohistochemistry was performed to visualize depletion and uptake of CGRP from sensory nerves. Key results: Capsaicininduced CGRP release was attenuated by the TRPV1 antagonist capsazepine and by Ca2 + -free environment. Subsequent to depletion of CGRP,
skull halves were incubated with exogenous CGRP which caused an
increase in capsaicin-induced CGRP release as compared to the challenge just prior to incubation. The CGRP uptake was not inuenced by a
Ca2 + -free environment. Sumatriptan, olcegepant and CGRP(8-37) did
not affect the uptake of CGRP. However, a monoclonal CGRP-binding
antibody decreased CGRP uptake signicantly. Release of CGRP after
incubation was attenuated by Ca2 + -free environment and by capsazepine. Immunohistochemistry experiments also indicate CGRP uptake in
rat dura mater. Conclusion: We have shown evidence for CGRP uptake
in rat dura mater. The release of up-taken CGRP seems to be mediated
via the same mechanisms as CGRP release before uptake.
Paper No.: 2252

TYPE II DIABETES DRUG METFORMIN INDUCES
TRANSCRIPTIONAL COACTIVATOR PGC-1A IN LIVER
POSSIBLY THROUGH AMPK INDEPENDENT MECHANISM
Sanna-Mari Jarvenpaa, J Hakkola
University of Oulu, Department of Pharmacology and Toxicology, Oulu,
Finland
PGC-1a (PPARc- coactivator 1alpha) is a master regulator of energy
metabolism in many important tissues and highly regulated by external
stimuli. In liver PGC-1a and its target genes involved in gluconeogenesis: PEPCK (phosphoenolpyruvate carboxykinase) and G6Pase (glucose6-phosphatase) are upregulated by fasting via cAMP mediated mechanism. Metformin is a widely used oral anti-diabetic drug which exerts its
effect by activating AMPK (AMP-activated kinase). AMPK inhibits glucose production in liver by down-regulating gluconeogenic genes. However, the molecular mechanisms of this action are not completely
understood. Here we show that effect of metformin on hepatic PGC-1alphaexpression is different from other major tissues and probably AMPK
independent. PGC-1alphamRNA levels were induced 2 to 5 fold in
mouse and human primary hepatocytes by treatment with metformin,
dose and time-dependently. However, PEPCK and G6Pase were strongly
inhibited. In contrast, AMPK agonist AICAR inhibited both PGC-1a and
gluconeogenic genes suggesting that metformin acts also independent of
AMPK action. Induction of PGC-1a by cAMP signalling pathway was
diminished after introduction of metformin suggesting that the effect of
metformin also depends on cAMP status of the cell. Constitutive expression of PGC-1a is upregulated by metformin while cAMP stimulated
induction, activated in fasting and diabetes, is attenuated. However, the
effect of metformin on gluconeogenic marker genes is inhibitory regardless of the cAMP status. Thus we show a regulatory mechanism of
action which gives novel data about the complex function of metformin
in liver.
Paper No.: 2675

BI-TRANSGENIC MICE EXPRESSING HUMAN
HYDROXYSTEROID (17B) DEHYDROGENASE 1 (HSD17B1)
AND ESTROGEN RESPONSE ELEMENT (ERE) -REPORTER
GENE PROVIDE A NOVEL TOOL TO MONITOR ACTION OF
HSD17B1 INHIBITORS
(1) University of Turku, Institute of Biomedicine, Department of Physiology, Turku, Finland

(2) Hormos Medical Ltd. Subsidiary of QuatRx Pharmaceuticals, Turku,
Finland
(3) Hubrecht Institute, Utrecht, The Netherlands
(4) University of Turku, Turku Center for Disease Modeling, Turku,
Finland
Estrogens inuence the pathological processes of hormone-dependent
diseases in women, such as breast and endometrial cancers. Hydroxysteroid (17b) dehydrogenase 1 (HSD17B1) catalyzes the conversion of biologically low-active estrone to a highly potent estrogen, estradiol, in
various sex steroid target tissues. In the present study, transgenic mice
over-expressing human HSD17B1 (HSD17B1TG mice) were cross-bred
with reporter mice carrying three estrogen responsive elements (EREs)
combined with a luciferase reporter gene (ERELuc mice) for testing the
effect of two HSD17B1 inhibitors in vivo. The estrogen receptor activation of the intact bi-transgenic ERELuc-HSD17B1TG mice was studied
by measuring luciferase activity ex vivo in the adult bi-TG female mice.
The luciferase activity was signicantly increased in several tissues,
including liver, kidney and mammary gland, as compared with the ERELuc mice. Accordingly, treating the pre-pubertal mice with estrone (0,3
lg/kg) for ve days increased the uterus weight signicantly more in the
bi-TG mice as compared with the ERELuc mice. The efcacy of
HSD17B1 inhibitors was tested in the immature bi-TG female mice by
providing estrone (0,3 lg/kg) with or without HSD17B1 inhibitors (20
mg/kg) for ve days. In the model used, the HSD17B1 inhibitors prevented estrone induced uterus weight increase signicantly. Furthermore,
the luciferase activity measured ex vivo in the uterine tissues showed
comparable Results: As a conclusion, the HSD17B1TG mice crossed
with the ERELuc mice serve as a novel preclinical model for testing the
efcacy of HSD17B1 inhibitors in vivo.
Paper No.: 610

TO INVESTIGATE THE EFFECTS OF 5-HT AND ELECTRICAL
FIELD STIMULATION (EFS) IN THE BLADDER OF SUNCUS
MURINUS
Farideh A Javid(1), RJ Naylor(2)
(1) Division of Pharmacy and Pharmaceutical Sciences, School of
Applied Sciences, University of Hudderseld, Hudderseld, UK.
(2) Pharmacy, University of Bradford, Bradford, UK
The pattern of neurotransmission in the Suncus murinus bladder is very
similar to that of human (Mok et al., J. Aut. Nervous System, 2000; 80,
130-136). The aim of the present study was to investigate the effect of 5HT and EFS in the whole intact Suncus murinus bladder. The whole
intact bladder with its attached urethra and autonomic neurones were dissected and immersed in 80 ml organ baths containing Krebs solution
(37C, 95% O2, 5% CO2), and left to equilibrate for 60 min and washed
every 20 min. Applying a resting tension of 1.0 g, the contractions were
recorded isometrically. The effect of 5-HT and EFS (30 V, 0.5 ms width,
single pulse for 10 s) were studied in the absence and presence of methysergide (0.01 and 0.1 lM). 5-HT induced contractions which were significantly (p < 0.05, 0.001) attenuated by methysergide (0.01 and 0.1 lM).
However, EFS -induced contractions were signicantly (p < 0.05, 0.01)
potentiated in the presence of methysergide (0.01 lM). The data suggest
that the 5-HT1/2/7 receptors may be involved in mediating a contraction
response to exogenously added 5-HT. However, these receptors may act
differently upon the release of the endogenous 5-HT.
Paivi Jarvensivu(1), T Saloniemi(1), P Koskimies(2), P van der Saag(3),

M Poutanen(1,4)
354
Paper No.: 1110
ANTISPASMODIC EFFECT OF ACHILLEA FILLIPENDULA
EXTRACT ON THE GUINEA PIG ILEUM
Katayou Javidnia, R Miri, H Mirkhani, E Faghih Mirzaei, M Shari
Shiraz University of Medical Sciences, Medicinal & Natural Products
Chemistry Research Center, Shiraz, Iran
Achillea species has been used in traditional medicine as antispasmodic.
The aerial parts of the plant are used for medicinal purposes. The plant
materials soaked in a 90% aqueous-methanol solution in a largecontainer
for 3 days. combined ltrate was evaporated on a rotaryevaporator under
reduced pressure to a thick semi-solid paste. The spasmolytic activity of
Acillea llipendula was studied using isolated guinea pig ileum. Respective segments of 2 cm length were suspended in a 10 mL tissue bath
containing Tyrodes.K+ was used to depolarize the intestinal preparations.
High K+ (80 mM) was added to the tissue bath, which produced a sustained contraction. Samples were added in a cumulative fashion to obtain
concentration-dependent inhibitory responses. The relaxation of intestinal
preparations, pre-contracted with K+ (80 mM) was expressed as the percent of the control response mediated by K+. To conrm the calcium
antagonist activity of test substances. The extract caused a dose-dependent inhibition of spontaneous and K+ induced contractions of isolated
guinea pig ileum Incubating the intestinal preparations with Ac. (0.31.0 mg/mL) shifted the Ca+2 CRCs to the right .These effects were comparable to that produced by nifedipin. the extract caused inhibition of
spontaneous and high K+-induced contractions of guinea pig ileum, thus
showing that the antispasmodic action is mediated through a calcium
antagonist effect, which was conrmed when the Acillea llipendula
extract caused a concentration-dependent rightward shift in the Ca+2
CRCs, similar to that caused by nifedipin.
Paper No.: 748

ACUTE ORAL TOXICITY AND ACUTE DERMAL TOXICITY
OF THE AQUEOUS EXTRACT OF GMELINA ARBOREA ROXB.
(FAMILY VERBENACEAE) LEAVES
Maria Cesthrine Jane Javier
University of the Philippines, College of Medicine, Department of
Pharmacology and Toxicology, Manila, The Philippines
The use of herbal medicine has a long history and can be traced back to
the prehistoric period. Plants have been and still are primary sources of
drugs. Gmelina arborea Roxb. (Family Verbenaceae) is one of the herbal
medicines which have been used for ages especially in India and neighboring countries. All the plant parts of Gmelina arborea have been utilized in India for various illnesses. The leaves are used as demulcent,
bechic and for removing fetid discharges from ulcers or as wash for these
ulcers. Several studies involving the identication and isolation of the
active constituents of Gmelina arborea have been done. The chemical
constituents of Gmelina arborea include a variety of alkaloids, avonoids, iridoids and lignans. However, studies on the safety of Gmelina
arborea are limited. This study determines the toxicity of acute oral
administration and acute dermal administration of the aqueous extract of
Gmelina arborea leaves. The acute oral toxicity up-and-down procedure and acute dermal toxicity xed dose procedure from the OECD
guidelines for the testing of chemicals were used. The test animals survived at the 5000 mg/kg limit test dose showing an LD50 > 5000 mg/
kg. There are no toxidromes present. The test animals also survived the
5000 mg/kg dermal dose thus, making it safe for dermal administration.
Presence of skin irritation was not observed. Therefore, the aqueous
extract of Gmelina arborea leaves may be safe for acute administration
via the oral or dermal route; however, further studies are required to fully
establish safety.
Paper No.: 2117

DISCOVERY
INFLUENCE OF P-GLYCOPROTEIN POLYMORPHISM ON
NORTRIPTYLINE-INDUCED POSTURAL HYPOTENSION
Berit P Jensen(1), R Vyas(2), G Bonke(1), RR Roberts(1,3), D Jardine(4),
EJ Begg(1,2)
Zealand
(2) Christchurch Hospital, Department of Clinical Pharmacology,
Christchurch, New Zealand
(3) University of Otago, Department of Biochemistry, Dunedin, New
Zealand
(4) Christchurch Hospital, Department of General Medicine,
P-glycoprotein (P-gp) is a transporter protein encoded by the gene
ABCB1. In a previous study (Roberts R et al, Pharmacogenomics 2002,
2, 191-196), a single nucleotide polymorphism in ABCB1 (3435C>T)
was associated with symptomatic nortriptyline-induced postural hypotension in depressed patients. To further investigate this nding, a prospective study was undertaken. Following genetic screening of 67 healthy
volunteers, eight CGC homozygotes and nine TTT homozygotes of
ABCB1(1236-2677-3435) were identied. All had at least one functional
allele of CYP2D6, which is involved in nortriptyline metabolism. A single oral dose of 25 mg nortriptyline was administered. Blood pressure
and heart rate was monitored at 0, 2, 4, 6 and 8h post-dose while the
subject changed from supine to the upright position by active standing
and by using a tilt table. Multiple blood samples were taken over 72h to
determine the exposure of nortriptyline and its active metabolites. No difference was seen in pharmacokinetic variables between haplotype
groups. Both groups showed normal blood pressure and heart rate
response to standing. Following nortriptyline treatment, heart rate
increased on changing to upright position in both haplotype groups with
a maximum at 6h post-dose (p = 0.0001), but no difference was seen
between groups. Systolic blood pressure on standing following nortriptyline trended towards a greater decrease in CGC/CGC compared with
TTT/TTT, contrary to the hypothesis. The association between P-gp
polymorphism and nortriptyline-induced postural hypotension found in
the previous patient study could thus not be conrmed in this study in
healthy volunteers following a single oral dose of nortriptyline.
Paper No.: 2118

TOTAL AND FREE CLEARANCE OF R- AND S-WARFARIN IN
ELDERLY PEOPLE
Berit P Jensen(1), P Chin(2), RR Roberts(1,3), J Maddison(4,5),
EJ Begg(1,2)
Zealand
(2) Christchurch Hospital, Department of Clinical Pharmacology,
(3) University of Otago, Department of Biochemistry, Dunedin, New
Zealand
(4) Royal Adelaide Hospital, Adelaide, SA, Australia
(5) University of Adelaide, Discipline of Pharmacology, Adelaide, SA,
Australia
Metabolic drug clearance (CL) has been consistently shown to be
impaired in the older age group for ow-limited (high CL) drugs and also
for capacity-limited (low CL) drugs with low protein binding. However,
there have been conicting results for capacity-limited drugs with high
protein binding (Butler JM & Begg EJ, Clin Pharmacokinet 2008; 47:
355
297-321). A limitation of most of these studies is that CL has been based
on total drug concentrations, rather than free (unbound) concentrations.
As protein binding may decrease with age, impaired intrinsic CL may not
be revealed if only total CL is assessed. To test the hypothesis that free
CL of capacity-limited highly protein bound drugs is impaired in the
elderly, warfarin (~99% protein bound) was investigated as a model drug.
A steady-state blood sample was taken from 70 patients (age range 18-89
y) in routine treatment with warfarin. Concentrations of R- and S-warfarin were determined in plasma (total) and ultraltrate (free) by an LC-MS
assay developed for the study. Total and free CL were determined and
regressed against age. For R-warfarin a signicant decrease with age was
found for both total and free CL, with a greater effect on free CL. For Swarfarin a decrease in free CL was found with age but no change was
observed for total CL. The decrease in free CL of R- and S-warfarin was
found to be ~0.5% per year. This data supports the hypothesis that free
CL of highly protein bound drugs is impaired in elderly people.
Paper No.: 3331

CYP2C9*3 AND *13 ALLELES SIGNIFICANTLY AFFECTED
THE PHARMACOKINETICS OF MELOXICAM IN HEALTHY
SUBJECTS
Kyeong-Joo Jeon, C-I Choi, J-W Bae, M-J Kim, C-G Jang, S-Y Lee
Sungkyunkwan University, College of Pharmacy, Su-Won, South Korea
Meloxicam is a nonsteroidal anti-inammatory drugs (NSAIDs) that
exhibits anti-inammatory, analgesic and antipyretic effects via a selective inhibition of cyclooxygenase 2 (COX-2). Meloxicam is almost completely metabolized to four inactive metabolites, and CYP2C9 plays an
important role in the metabolism of meloxicam. It is reported that genetic
variants in CYP2C9 are associated with decreased enzymatic activity of
CYP2C9. So we investigated effects of CYP2C9 genetic polymorphisms
on the pharmacokinetics and pharmacodynamics of meloxicam in
healthy Korean subjects. Genotyping for CYP2C9 was performed by
using PCR-RFLP and direct sequencing methods, and 20 healthy Korean
volunteers were selected for this study. They were divided into two
groups according to CYP2C9 genotype, CYP2C9EM (CYP2C9*1/*1, n
= 10) and CYP2C9IM (CYP2C9*1/*3 and CYP2C9*1/*13, n = 10). A
single oral dose of 15 mg of meloxicam was administered to each subject
and plasma concentration of meloxicam was determined by using
HPLC-UV system. Pharmacodynamics of meloxicam was evaluated by
measuring serum thromboxane B2 (TXB2) concentration with enzyme
immunoassays (EIA). AUC0- of meloxicam in CYP2C9IM was 90%
higher than that in CYP2C9EM, respectively (P = 0.0001). Elimination
half-life (t1/2) of meloxicam in CYP2C9IM was 68% longer than that in
CYP2C9EM (P < 0.0001). Oral clearance of meloxicam in each group
was also signicantly different. The inhibition of TXB2 in CYP2C9IM
was greater than that in CYP2C9EM, but this different was not statistically signicant. CYP2C9 genetic polymorphisms can affect the pharmacokinetics and possibly, pharmacodynamics of meloxicam.
Paper No.: 3332

DISCOVERY
SIGNIFICANT PHARMACOKINETIC INTERACTION
BETWEEN PITAVASTATIN AND CLARITHROMYCIN IN
HEALTHY SUBJECTS
Pitavastatin is an inhibitor of HMG-CoA reductase, and is used for the
treatment of hypercholesterolemia. After intestinal absorption, pitavastatin
is rapidly taken up by the liver through the OATP1B1, one of hepatic

inux transporters. Clarithromycin is one of macrolide antibiotic agents
used for the treatment of respiratory and skin structure infections, and is
known as a potent OATP1B1 inhibitor. We investigated the effects of clarithromycin on the pharmacokinetics of pitavastatin in Korean subjects. 12
healthy volunteers with OATP1B1*1a/*1b genotype participated in this
open-labeled, two-phase parallel study. In control phase, they were
received a single oral dose of 2 mg pitavastatin. After wash-out period
(clarithromycin phase), 500-mg of clarithromycin was administered 12
hours before and 0, 12, 24 and 36 hours after the administration of a single oral dose of 2 mg pitavastatin. Plasma concentrations of pitavastatin
and its lactone were measured by using LC-MS/MS system. In clarithromycin, Cmax and AUC0- of pitavastatin (86.5 24.4 ng/mL and 213.0
50.3 nghr/mL, respectively) were signicantly higher than those in control phase (46.9 16.1 ng/mL and 165.7 57.7 nghr/mL, respectively).
Oral clearance of pitavastatin between control phase and clarithromycin
phase was also signicantly different (14.1 6.5 L/hr vs. 9.8 2.5 L/hr,
P < 0.01). Other parameters are not statistically signicant between two
phases. Pharmacokinetics of pitavastatin lactone was also not signicantly
different between two phases. In conclusion, clarithromycin signicantly
affects the pharmacokinetics of pitavastatin but not its lactone form.
Paper No.: 3333

THE ROLE OF CYP2C9 GENETIC VARIANTS IN THE
DISPOSITION OF GLIPIZIDE IN HEALTHY SUBJECTS
Glipizide is a second-generation sulfonylurea antidiabetic agent used for
the treatment of type II diabetes mellitus. Alike most of sulfonylurea
compounds, glipizide is also metabolized primarily by highly polymorphic cytochrome P450 2C9 (CYP2C9). CYP2C9*3 and CYP2C9*13
alleles have been reported to be associated with decreased CYP2C9
enzyme activity. So we investigated the effects of CYP2C9 genetic polymorphism on the pharmacokinetics and pharmacodynamics of glipizide.
Genotyping for CYP2C9 was performed using PCR-RFLP method, and
observed allelic frequencies of CYP2C9*3 and CYP2C9*13 were 5.3%
and 0.4%, respectively. Twenty-four healthy Korean volunteers were
selected for this study, and they were divided by two groups according
to CYP2C9 genotype, CYP2C9EM (CYP2C9*1/*1, n = 11) and
CYP2C9IM (CYP2C9*1/*3 and CYP2C9*1/*13, n = 13). A single oral
dose of 5 mg glipizide was administered to each subject and plasma concentration of glipizide was determined by using HPLC-UV system. Pharmacodynamics of glipizide was evaluated by the measurement of plasma
glucose and insulin concentration. AUC0- of glipizide in CYP2C9IM
(3937.3 502.4 nghr/mL) was signicantly higher than that in
CYP2C9EM (2625.3 782.3 nghr/mL). Oral clearance of glipizide in
CYP2C9IM was 37% lower than that in CYP2C9EM (P = 0.0001). But,
other parameters were not signicantly different between two groups.
Also, plasma glucose and insulin concentration between two groups were
not signicantly different. CYP2C9 genetic polymorphism affects the
pharmacokinetics of glipizide, but further studies are needed to evaluate
the effects on the pharmacodynamics of glipizide.
Paper No.: 3370

DISCOVERY
EVALUATION OF THE PHARMACOKINETIC PROFILE OF
EZETIMIBE IN HEALTHY KOREANS WITH DIFFERENT
SLCO1B1 GENOTYPE
356
Ezetimibe is the rst lipid-lowering agent that inhibits dietary and biliary
cholesterol absorption and is used for the treatment of hypercholesterolemia. Ezetimibe is known as a substrate of OATP1B1, one of the hepatic
uptake drug transporters encoded by SLCO1B1 gene. We investigated
the effects of SLCO1B1 genetic polymorphism, focused on
SLCO1B1*15 allele, on the pharmacokinetics of ezetimibe and its glucuronide metabolite. After genotyping for SLCO1B1 using PCR-RFLP
methods, 30 healthy Korean subjects were participated and grouped
according to number of SLCO1B1*15 alleles, group1 (SLCO1B1*1/*1, n
= 15), group2 (SLCO1B1*1/*15, n = 11) and group3 (SLCO1B1*15/
*15, n = 4). A single oral dose of 10 mg ezetimibe was administered to
each subject and plasma concentrations of ezetimibe and ezetimibe glucuronide were measured by LC-MS/MS analytical method. Maximum
plasma concentration (Cmax) of ezetimibe in group1, group2 and group3
was 4.71 2.91 ng/mL, 4.63 2.92 ng/mL and 3.61 2.63 ng/mL,
respectively. Area under the plasma concentration-time curve (AUC0-)
of ezetimibe in each genotype group was 88.1 36.6 nghr/mL, 90.7
31.6 nghr/mL and 55.6 30.3 nghr/mL, respectively. However, these
differences were not statistically signicant (P > 0.05). Other parameters
were also not signicantly different between three genotype groups. Similarly, pharmacokinetic differences of ezetimibe glucuronide between
three genotype groups didnt reached the level of statistical signicance.
In conclusion, SLCO1B1 genetic polymorphisms are not associated with
the pharmacokinetic changes of ezetimibe, although ezetimibe is known
as a substrate of OATP1B1.
Paper No.: 2748

ILEX LATIFOLIA PROTECTS AGAINST AMYLOID BETA
PROTEIN (25-35)-INDUCED NEUROTOXICITY IN CULTURED
NEURONS AND HAS ANTIDEMENTIA ACTIVITIES IN MICE
Ha Yeon Jeong, YH Seong
Chungbuk National University, College of Veterinary Medicine, Chungbuk, Korea
Ilex species (Aquifoliaceae) are distributed in china and have been used
to treat coronary heart diseases. Ilex latifolia (IL), called as Ku-Ding-Cha
has been used as a diuretic, remedy for sore throats, and for the treatment
of indigestion, various kinds of inammatory diseases and headaches.
The present study investigated protective effects of ethanol extract of IL
on b amyloid (Ab) (25-35)-induced neurotoxicity in cultured rat cortical
neurons and on memory impairments in mice. Exposure of cultured neurons to 10 uM Ab(25-35) for 36 h induced neuronal apoptotic death. At
10-100 ug/ml, IL inhibited 10 lM Ab (25-35)-induced neuronal cell
death, elevation of intracellular Ca2 + concentration ([Ca2 + ]i) and generation of reactive oxygen species (ROS) in primary cultured cortical neurons. Memory loss induced by intracerebroventricular injection of ICR
mice with 15 nmol Ab(25-35), which was assessed by passive avoidance
test and Morris water maze test was markedly prevented by chronic
administration of IL (25-100 mg/kg, P.O., 8days). Neuronal death measured histologically in the hippocampal and cortical regions of memory
impaired mice brain was also signicantly reduced by the treatment with
IL. From these results, we suggest that the antidementia effect of IL is
due to its neuroprotective effect against Ab-induced neurotoxidity and
that IL has a possible therapeutic role in preventing neurodegeneration in
Alzheimers disease.
Paper No.: 1242

THE ANGIOTENSIN II TYPE 1 RECEPTOR REGULATES
MICRORNA EXPRESSION BY GQ AND ERK1/2 DEPENDENT
MECHANISMS
Pia Jeppesen(1), GL Christensen(2,3), M Schneider(1), Y Nossent(1,2),
HB Jensen(1), DC Andersen(1), S Gammeltoft(3), JL Hansen(2,4),
SP Sheikh(1)
(1) University of Southern Denmark, Institute of Molecular Biology,
Department of Biochemistry, Pharmacology and Genetics, Odense
University Hospital, Odense, Denmark
(2) University of Copenhagen, Laboratory for Molecular Cardiology,
Danish National Research Foundation Centre for Cardiac Arrhythmia,
Department of BiomedicalSciences, Copenhagen, Denmark,
(3) Glostrup Hospital, Department of Clinical Biochemistry, Copenhagen, Denmark
(4) Copenhagen University Hospital, Copenhagen, Denmark
The Angiotensin II type 1 receptor (AT1R) is a key regulator of blood
pressure, body uid homeostasis, cardiac contractility and remodelling,
and is profoundly involved in development of cardiac disease. AT1R signalling has been studied in detail and involves both activation of heterotrimeric G proteins and signal cascades dependent on B-arrestins.
Angiotensin II (AngII) signalling has been suggested to involve regulation of microRNAs (miRNAs), which are small non-coding RNAs that
by sequence complementarity, negatively regulate the stability and translation of mRNA targets. In this study we confer a comprehensive analysis of AT1R mediated miRNA regulation and moreover focus on
separating the role of G-protein-dependent and -independent pathways.
Ang II regulation of miRNA expression was investigated by a global
miRNA array analysis in AT1R HEK293 cells, which showed upregulation of 7 miRNAs. These miRNAs were similarly found to be up-regulated by the AT1R in primary cultures of cardiac broblasts. The
expression of these Ang II induced miRNAs was sensitive to Gaq/11 and
MEK1 inhibition and was not induced by a biased AngII analogue,
[Sar1, Ile4, Ile8] Ang II (SII Ang II), which does not activate Gaq/11
protein signalling, but still activates G protein-independent signalling.
These ndings indicate that the AT1R directly regulates the expression of
several miRNAs and that this regulation is primarily mediated by Gaq/11
protein activation and is dependent on Erk1/2.
Paper No.: 2355
DISEASES
ARE THERE DIFFERENCES IN THE ACTIVITIES OF CYP3A,
CYP2D6 AND P-GLYCOPROTEIN BETWEEN
THERAPY-NAIVE HIV-INFECTED PATIENTS AND HEALTHY
VOLUNTEERS?
Alexander Jetter(1), G Fatkenheuer(2), D Frank(3), T Klaassen(3),
A Seeringer(4), O Doroshyenko(3), J Kirchheiner(4), W Hein(5),
E Schomig(3), U Fuhr(3), C Wyen(2)
(1) University Hospital Zurich, Division of Clinical Pharmacology and
Toxicology, Zurich, Switzerland
(2) University Hospital of Cologne, Department I of Internal Medicine,
Cologne, Germany
(3) University Hospital of Cologne, Department of Pharmacology &
Clinical Pharmacology, Cologne, Germany
(4) University of Ulm, Department of Pharmacology of Natural Products
and Clinical Pharmacology, Ulm, Germany
(5) University Hospital of Cologne, Department of Clinical Chemistry,
Cologne, Germany
Infection and inammation may lead to reduced activities of drugmetabolizing enzymes and drug transporters. Here, we investigated
357
whether these ndings can be corroborated in HIV-infected patients
using a cocktail phenotyping approach. A phenotyping cocktail consisting of 1.5 mg midazolam (intestinal and hepatic CYP3A), 30 mg
dextromethorphan (CYP2D6), and 0.5 mg digoxin (P-glycoprotein)
administered orally, and of 1.0 mg midazolam intravenously four hours
later (hepatic CYP3A activity), was administered to 30 therapy-nave
HIV-infected patients and to 12 healthy male controls. Plasma samples
were analysed using immunoassays for digoxin and LC-MS/MS for all
other analytes. Pharmacokinetics were calculated noncompartimentally.
A parallel-group average bioequivalence approach was chosen for comparisons between patients and volunteers. In patients, mean apparent
oral midazolam clearance (overall CYP3A activity) was 0.490-fold
lower (90% condence interval CI, 0.377-0.638) than in volunteers,
while midazolam clearance after intravenous dosing was not different
(point estimate 0.956, 90% CI, 0.807-1.131). In patients with at least
one active CYP2D6 allele, the molar ratio AUCdextromethorphan /
AUCdextrorphan was 1.289-fold higher than in volunteers (90% CI,
0.778-2.136) suggesting a lower CYP2D6 activity. In patients, digoxin
AUC and Cmax were 1.216 and 1.304 times the AUC observed in
volunteers, respectively (90% CIs, 0.969-1.526 and 1.034-1.644). In
conclusion, overall CYP3A activity was lower in therapy-nave HIVinfected patients than in healthy volunteers. The CYP2D6 and P-glycoprotein activities only tended to be lower, and differences were small.
Variabilities within the groups were higher than the discrepancies
between them, making it unlikely that dose adaptations based on infection status would be useful.
Paper No.: 1304

HEALTH AND DISEASE
THE EFFECT OF CONNEXIN 43 REMOLDING IN VASCULAR
RESTENOSIS
Liping Jiang(1), L Zhu(1), T Hong(2), J Feng(2)
(1) Nanchang University School of Medicine, Nanchang, Jaingxi, PR
China
(2) Nanchang University, First Afliated Hospital, Nanchang, Jaingxi,
PR China
To investigate the role of gap junction (GJ) in the pathogenesis of restenosis. Twenty-ve Sprague-Dawley rats were randomly assigned into
ve groups. The control group consumed regular chew and the other four
groups were exposed to an atherogenic diet for 8 weeks before balloon
distension injury in the common carotid arteries. According to the samples taken at different times after injury, rats were sacriced at 0d, 7d,
14d and 28d, respectively. All artery speciments were performed for morphometric analysis, including HE staining and immunohistochemistry,
RT-PCR and Western Blotting, respectively. Results showed that VSMCs
proliferation was markedly increased at 14 days after injury by using HE
staining. Disorders of VSMCs were observed in neointima with a growth
tendency toward luminal surface. At 28 days, lumen size was dramatically decreased. Immunohistochemistry of Cx43 showed a signicantly
strong immunostaining was found in the neointima zone, especially in 28
days group. Results showed that Cx43 mRNA and protein expression
were declined 7d after injury compared with 0d group and they were
markedly up-regulated with the progress of RS, especially in 28 days
group. Study shows for the rst time that arteries become re-narrowed
after balloon catheter injury with an atherogenic diet in rats. Both Cx43
mRNA and protein expression are subsequently increased during vascular restenosis. So the reModelling of Cx43 may be involved in the development of RS after PCI, which will provide a new therapeutic basis for
RS.
*Grant support: the National Natural Science Foundation of China, No
30760286 and the K.C. Wang Education Foundation, Hong Kong.
Paper No.: 1368

GENE EXPRESSION PROFILING OF CARDIOPROTECTIVE
EFFECTS OF QI-SHEN-YI-QI ON ACUTE MYOCARDIAL
INFARCTION
M Jiang(1), G Wang(1), P Cui(1), Z Zhao(1), Xiaoying Wang(2),
G Luo(1)
(1) Nankai University, College of Pharmacy, Tianjin, PR China
(2) Tianjin University of Traditional Chinese Medicine. Tianjin, PR
China
Introduction: To identify the gene-expression patterns that are involved
in treating acute myocardial infarction by Qi-Shen-Yi-Qi (QSYQ) drop
pills, the changes of global gene expression in a rat model of myocardial
infarction were evaluated by cDNA microarray which included 29,700
genes. Methods: Rats were divided randomly into 4 groups, sham-operated, myocardial infarction, QSYQ and nifedipine (n = 8). Both echocardiographic response and cardiac enzymes were evaluated at 7 days after
surgery. Genes involved in ischemia injury and responses to treatment in
ischemic hearts were identied by microarray analysis and veried by
quantitative Real-time RT-PCR. Results: 265 genes were identied that
showed differential expression in response to myocardial infarction by
the treatment of QSYQ. Gene Ontology, KEGG pathway analysis and
hierarchical cluster were applied in the gene expression exploring. Of
those genes exhibiting signicant differential expression, genes that were
involved in apoptosis, cell adhesion and metabolism were regulated
markedly. According to the pathway analysis, 31 pathways were regulated by QSYQ treatment on myocardial infarction which included Glycolysis/ luconeogenesis, Focal adhesion and PPAR signalling pathway.
Comparing with nifedipine, the most signicant expressed genes were
belonged to energy metabolism such as Ppp3r1 and Sdhc. Conclusion:
Microarray gene proling has revealed candidate genes by QSYQ treatment on myocardial infarction, some of them novel, which may account
for the therapeutical angiogenesis of QSYQ on coronary artery disease in
clinic that may lead to neovascularization and confer myocardial protective effects.
Paper No.: 1804

PROTEOMIC AND METABONOMIC-BASED
INVESTIGATIONS ON MECHANISMS OF AGING OF
SENESCENCE-ACCELERATED MOUSE AND ACTIONS OF
LIUWEI AND BAWEI DIHUANG DECOCTIONS
Ning Jiang(1), W-X Zhou(1), Y-X Zhang(1), X-Z Yan(2), Q Zhang(2)
(1) Beijing Institute of Pharmacology and Toxicology, Department of
Pharmacology on Traditional Chinese Medicine, Beijing, PR Chinah
(2) National Center of Biomedical Analysis, Beijing, PR Chinah
Introduction: Senescence-accelerated mouse (SAM) is a good model to
study mechanisms of aging. Liuwei Dihuang (LW) and Bawei Dihuang
(BW) decoctions are classical traditional Chinese medicinal prescriptions
for anti-aging. To better understand the mechanisms of aging of SAM
and actions of LW and BW, proteomic and metabonomic techniques
were employed in this study. Materials: SAM-prone/8 (SAMP8) were
orally administered with LW for consecutive 30 days. Two-dimensional
electrophoresis (2-DE) proteomic and 1H nuclear-magnetic-resonancespectroscopy (NMR) metabonomic prole data were collected on sera of
non-treated SAMP8 and SAMP8 treated with LW or BW. Results: The
results showed that the serum protein expressions of non-treated SAMP8
and SAMP8 treated with LW and BW were different. There were 21 and
13 differentially expressed proteins after LW and BW treatment, which
were relavant to the functions of transcriptional control, cytoskeleton, cell
signalling, etc.. Principle components analysis of 1HNMR spectra
358
showed clear separation among serum samples of non-treated SAMP8
and SAMP8 treated with LW and BW. Metabolites that LW and BW
affected were glucose, lactate, high density lipoprotein, etc. related to
glucose, lipids, lipoproteins and amino acids metabolism. Conclusion:
These ndings suggested abnormal serum protein expressions and
metabolism disequilibrium in SAMP8 caused by aging and the mechanisms of actions of the LW and BW may be related to the restoration of
the normal condition of specic proteins and metabolism equilibrium in
SAMP8.
(Supported by grants from the National Natural Science Foundation of
China 30701073, 90709012 and the National high Technology Research
and Development Program of China 2008AA02Z423).
Paper No.: 1146

HEALTH AND DISEASE
EVIDENCE FOR A NOVEL SIGNALLING MECHANISM
INVOLVING NAADP IN ENDOTHELIUM-DEPENDENT
HYPERPOLARIZATION OF RESISTANCE ARTERIES
Francesc Jimenez-Altayo, KA Dora, P Galindo, GC Churchill,
A Galione, CJ Garland
Recent studies suggest NAADP may serve as a novel and potent messenger to mobilize calcium from intracellular acidic stores in a range of cell
types. We investigated the possibility NAADP signalling might participate in the endothelium-dependent hyperpolarization that underlies vasodilatation in resistance arteries. Rat isolated mesenteric arteries were
mounted in either a wire or pressure myograph. Simultaneous changes in
smooth muscle membrane potential and tension were assessed with or
without a recently discovered NAADP receptor inhibitor, Ned-19 (10
lM) or vacuolar H+-ATPase inhibitor balomycin A1 (100 nM) present.
In some experiments, changes in smooth muscle and endothelial cell
[Ca2 + ]i were monitored in pressurized arteries using confocal microscopy. Concentration-dependent hyperpolarization to either acetylcholine
(0.001-10 lM) or cyclopiazonic acid (10 lM, to block SERCA) and
ongoing contraction (0.001-10 lM) to the a- and b-agonist norepinephrine or the a1-agonist phenylephrine were each reduced by Ned-19 or
balomycin A1. In contrast, neither hyperpolarization to the PAR-2
receptor agonist SLIGRL (0.001-10 lM) or the b-agonist isoprenaline
nor contraction to U46619 (thromboxane-mimetic; 0.001-1 lM) or high
KCl (45 mM) was altered. In pressurized arteries, elevated endothelial
cell [Ca2 + ]i to either acetylcholine or SLIGRL was unaffected by Ned19, but rises in smooth muscle cell [Ca2 + ]i stimulated by phenylephrine
were abolished. These preliminary data from mesenteric resistance
arteries, suggest that NAADP-induced calcium release from acidic
compartments may play a pivotal role in endothelium-dependent hyperpolarization and smooth muscle contraction to some agonists.
Paper No.: 3235

ANALYSIS OF SUSPECTED ADVERSE REACTIONS TO
ANTIDIABETIC DRUGS IN SPAIN,2006-2008
collected by the Pharmacovigilance Spanish System database FEDRA.

We have used the ATC classication for antidiabetic active ingredients.
The causality assessment has been covered by the algorithm of KarchLassagna modied. The statistical analysis was performed with SPSS 17.
Results: In Spain during the period 2006-2008, there have been collected
667 suspected adverse reactions to antidiabetic agents. The average
recorded age of patients is 65 years (64,72 14,38) and there is predominance in women (55,9%). The group A10B (Oral hypoglycaemic agents)
has been responsible for 81,7% of notications, whichever to metformin
in 30,9% of all cases, and increasing to 49,9% considering combinations.
Within the group A10A (Insulins and analogues), long-acting insulins
are the most frequent with 8,5% of cases. Disorders of metabolism and
nutrition are the most common adverse reactions (28,8%), followed by
gastrointestinal disorders (19,5%). According to the classication of
Karch-Lassagna modied, an adverse reaction to antidiabetic medication
is possible in 50,8% of cases, and probable in 49,2%. Conclusions: A
higher frequency of suspected adverse reactions to metformin may be
explained for the increased use in type 2 diabetes mellitus treatment and
its combinations with newly marketed drugs.
Paper No.: 3306

UTILIZATION STUDY OF ANTIDIABETIC DRUGS IN SPAIN,
2006-2008
Mara-Isabel Jimenez-Serrana, R Mateos-Campos
University of Salamanca Faculty of Pharmacy, Department of Preventive
Medicine and Public Health, Salamanca, Spain
Background: Currently, antidiabetic therapy experiences important
inclusions and withdrawals of active ingredients. The consequences of
these changes in prescribing proles require a detailed analysis. Objetive:
To analyze the use of antidiabetic drugs in Spain during 2006-2008.
Methods: Data of group A10 (ATC Classication System) were obtained
from the prescriptions redeemed by the Spanish National Health System.
Consumption analysis includes Dened Daily Dose per 1000 Inhabitants
(DDI), attributable use and estimated prevalence of diabetes. Economic
analysis includes attributable cost and cost treatment day (CTD). The statistical analysis was performed with SPSS 17. Results: Antidiabetic drug
use increased from 55,67 to 60,43 DDI (8,55%). Oral hypoglycaemic
agents utilization was twice that of insulins, and reached 45,68 DDI and
14,75 DDI respectively. The prevalence of diabetes mellitus was 6,0% in
2008. Insulin costs amounted to 360 million euros (61,4%), whereas oral
agents costs reached 225 million euros (38,6%). Within the group A10A,
the most attributable consumption was to biphasic insulins -over 33%-,
while its attributable cost was lower than the long-acting insulins (45,9%
in 2008). Within the group A10B, the attributable consumption of sulfonylureas decreased to 43% and represented 23,68% of the attributable
cost, opposite biguanides utilization increased to 40%- whose attributable cost was only 16,15%. The highest CTDs are for inhaled insulin
(9,04 in 2007) and exenatide (4,29 in 2008). Conclusions: In this period, antidiabetic prescription patterns may be dened by the inclusion of
new active ingredients with exacerbated costs, beside a higher use of low
cost biphasic insulins and biguanides.
Mara-Isabel Jimenez-Serrana, R Mateos-Campos

University of Salamanca Faculty of Pharmacy, Department of Preventive
Medicine and Public Health, Salamanca, Spain
Background: Adverse reactions to antidiabetic drugs have changed in
recent years. The system of Yellow Card allows the identication and
causality analysis of them. Objetive: To analyze suspected adverse reactions to antidiabetic drugs reported in Spain during 2006-2008. Methods:
The information was obtained from the spontaneous notication of
suspected adverse reactions to antidiabetic drugs made by Yellow Card,
Paper No.: 995

PHARMACOKINETICS OF L-CARNITINE,
ACETYL-L-CARNITINE AND PROPIONYL-L-CARNITINE
AFTER MULTIPLE-DOSE ORAL ADMINISTRATION OF
L-CARNITINE SOLUTION IN HEALTHY SUBJECTS
Fanbo Jing(1), H Qu(1), Z Sui(1), Y Cao(1), Z Han(1), L Zhu(1), C
Wang(2)
359
Department of Pharmacy, Qingdao, PR China
(2) Qingdao University Medical College, Department of Pharmacology,
Qingdao, PRChina
L-carnitine supplements have been used as adjuvant therapy in several
clinical disorders. To evaluate the pharmacokinetic characteristics of
L-carnitine, the plasma concentrations of L-carnitine, and its acyl esters,
acetyl-L-carnitine (ALC) and propionyl-L-carnitine (PLC) of 12 healthy
volunteers following multiple doses oral administration of L-carnitine
were detected by HPLC. The results showed that the maximum plasma
concentration (Cmax) and area under the curve AUC(0-) of L-carnitine
was114.21 27.95umolL-1h and 2650.01 258.48umolL-1h
respectively. The elimination half-life of L-carnitine and the time
required to reach the Cmax (Tmax) was 69.55 20.38 and3.29 0.84h
respectively. The Cmax and AUC(0-)of ALC and PLC was lower than
L-carnitine (P < 0.05). The half time of ALC and PLC was also shorter
than L-carnitine (P < 0.05) . The L-carnitine of multiple dose has a
higher Cmax than that of the single-dose admistration. The L-carnitine
was rapidly eliminated from plasma after the maximum concentration
within 72h. However, no signicant differences were observed in the
Cmax of the ALC and PLC between the single and multiple doses. These
data showed different characteristics following different admistration of
L-carnitine, which may be as reference in the designing of dosing regimens for L-carnitine or its analogues, such as ALC or PLC.
Q2008C04).
Key Words: L-carnitine, Acetyl-L-carnitine, Propionyl-L-cainitine,
Pharmacokinetics
Paper No.: 705

POTENTIALLY INAPPROPRIATE DRUG PRESCRIPTION IN
HOSPITALIZED GERIATRIC PATIENTS
Paper No.: 1413

COCKCROFT-GAULT AND MDRD IN ESTIMATING RENAL
FUNCTION IN OLDER HEART FAILURE
PATIENTS IMPLICATIONS FOR PRESCRIBING OF
PREDOMINANTLY RENALLY CLEARED MEDICATIONS
Sally Johns, J Maddison, S Shakib
Royal Adelaide Hospital, Department of Geriatric Medicine, Adelaide,
SA, Australia
Renal impairment is common in older patients and has implications for
prescribing of renally cleared medications. There is interest in using the
modied diet in renal disease (MDRD) equations in place of the Cockcroft-Gault (CG) equation to estimate glomerular ltration rate (GFR).
We aimed to compare estimations of renal function in older patients
using two methods and review implications for prescribing. Data were
reviewed from patients attending a tertiary hospital clinical pharmacology
outpatients department (age, weight, height (where available), serum creatinine and medications). The 30 most commonly prescribed medications
were reviewed for prescribing recommendations based on renal function.
CG and MDRD 175 were calculated and cases where there was a discrepancy of greater than 10% at the critical prescribing thresholds of 60,
50, 30, 25, 15 and 10ml/min were identied. 143 patients had a mean
age of 80.6 (SD 7.37) (58% females). Mean creatinine clearance calculated with CG was 44.6 (95% CI 40.7-48.6) and with MDRD 175 was
54.1 (95% CI 50.5-57.7). The mean difference between CG and MDRD
was 9.5mls/min (<0.0001, 95% CI 6.9-12.1). 51% of patients showed a
difference between the two methods which was around a potential critical
prescribing threshold. These patients were older and weighed less. CG
and MDRD give statistically signicant differences in estimated GFR.
These differences are clinically signicant as they occur around critical
prescribing thresholds. If MDRD is to be used for prescribing purposes,
older patients will be exposed to higher doses of renally cleared drugs
with the potential for increases in toxicity.
Marcela Jiron(1), P Jara(1), L Escobar(1), S Orellana(1), I Huala(1),

L Arriagada(2), MD Castillo(2), VH Carrasco(2)
(1) Universidad de Chile, Facultad de Ciencias Qumicas y
Farmaceuticas, Santiago, Chile
(2) Hospital Clnico de la Universidad de Chile, Seccion de Geriatra,
Santiago,Chile
Beers criteria have been widely used to identify potentially inappropriate prescription (PIP). In Chile, these criteria are not used routinely.
The purpose of this study was to assess PIP frequency in patients
hospitalized in the Geriatric Unit (GU) of the Clinical Hospital of the
Universidad de Chile. Clinical Pharmacists carried out a prospective
follow-up study in patients hospitalized in the GU between June 2006
and October 2009. Selection criteria were ages 65 years or older, hospitalization of at least 3 days, with more than 3 months of life expectancy. Inappropriate drug prescription was dened by 2003 Beers
Criteria. Outcomes of the study were PIP frequency; Beers estimated
severity of PIP and mean length of hospital stay. A total of 272
patients met selection criteria. The mean age of patients was 80.7
7.7 years and 177(65%) were women. During hospital stay, 2963
medications were prescribed; mean number of medication/patient was
10.9 4.5. Ninety four (34.7%) patients received at least 1 PIP. A
total of 110(3.7%) corresponded to PIP and high severity was found
in 89(80.9%) of cases. Use of inappropriate drugs was signicantly
associated to longer mean length of hospital stay and higher mean
number of medication than patients without PIP. This study showed a
longer mean length of hospital stay and higher number of medication
prescribed among patients with PIP. Other analyses must be performed to clarify this association and their consequences in geriatric
patients.
Paper No.: 1807

HEALTH AND DISEASE
REDUCED TWO-PORE DOMAIN POTASSIUM CHANNEL
(K2P) FUNCTION MAY CONTRIBUTE TO HYPERTENSION IN
SPONTANEOUSLY-HYPERTENSIVE RATS
Ian Johnson, E Porter, N Ashton, A Gurney, A Weston
University of Manchester, Faculty of Life Sciences, Manchester, UK
Polyunsaturated fatty acids (PUFAs) including arachidonic acid (AA)
activate the K2P potassium channels TREK and TRAAK and also
reduce blood pressure in hypertensive animals. Sharp microelectrode
electrophysiology and pressure myography were used to determine K2P
channels role in the development of hypertension in isolated mesenteric
arteries from 13-week old spontaneously-hypertensive rats (SHRs) and
age-matched Wistar-Kyoto (WKY) controls. Changes in myocyte membrane potential (Em) and vessel diameter were assessed after exposure to
AA in the presence or absence of the TREK/TRAAK inhibitor chlorpromazine. SHR resting Em was more depolarised than WKY (-49.2
0.5 vs -53.7 0.2mV, p = 0.0001, n = 4). In de-endothelialised SHR vessels, 30microM AA signicantly hyperpolarised resting Em (P < 0.001,
n = 4), a response unaffected by the KATP and BKCa inhibitors glibenclamide (10microM) and iberiotoxin (100nM), respectively. In isolated
WKY arteries, AA (1-100microM) dose-dependently relaxed U46619induced tone (log EC50 -5.4 0.1, n = 5), an action signicantly inhibited by 10microM chlorpromazine (log EC50 -4.9 0.1, p = 0.009). In
matched SHR vessels, AA-induced relaxations were shifted signicantly
compared to WKY controls (log EC50 -4.9 0.07, p = 0.0001, n = 4);
this reduced sensitivity was chlorpromazine-insensitive (log EC50 -4.7
360
0.1). Collectively, these data suggest that PUFA-sensitive K2P channels
are functional in WKY vessels and that loss of this function contributes
to the onset of hypertension in SHR animals.
Work funded by the British Heart Foundation.
Paper No.: 2386

FETAL DE NOVO CHOLESTEROL SYNTHESIS IS REQUIRED
FOR NEUROECTODERMAL SURVIVAL AND
CARDIOVASCULAR DIFFERENTIATION IN EARLY MOUSE
EMBRYOS
Heli Jokela(1), P Rantakari(1,2), T Lamminen(1,2), L Strauss(1,3),
R Ola(4), A-L Mutka(4), H Gylling(5), T Miettinen(6), P Pakarinen(1,2),
K Sainio(4), M Poutanen(1,2)
(1) University of Turku, Institute of Biomedicine, Turku, Finland
(2) University of Turku, Center for Disease Modeling, Turku, Finland
(3) University of Turku, Institute of Microbiology and Pathology, Turku,
Finland
(4) University of Helsinki, Institute of Biomedicine, Helsinki, Finland
(5) Kuopio University Hospital, Kuopio, Finland
(6) University of Helsinki, Institute of Internal Medicine, Helsinki, Finland
Cholesterol biosynthesis is essential for all living cells. During embryonic development, cholesterol is also needed for the nal modication of
sonic hedgehog into functional signalling molecule, which is one of the
main regulators of embryonic patterning and organogenesis. However,
little is known about the de novo biosynthesis of cholesterol during fetal
development. We analysed embryonic lethal mice decient for hydroxysteroid (17b) dehydrogenase 7 (HSD17B7), which catalyzes in vitro an
essential step in cholesterol biosynthesis, the conversion of zymosterone
to zymosterol. Our results provide in vivo evidence that the de novo cholesterol synthesis is required for neuroectodermal survival and cardiovascular differentiation. Interestingly, the total cholesterol concentration still
remains unaltered in the HSD17B7 knockout (KO) mouse embryos at
embryonic day (E) 10.5, while the pre-defect intermediates of cholesterol
biosynthesis (lanosterol, squalene) are accumulated and post-defect ones
(cholestenol, lathosterol, desmosterol) are markedly reduced. Morphological and histological analyses revealed that the endothelial cells failed to
form organized vascular structures in the HSD17B7 decient yolk sacs
and by E10.5 the embryos have cardiovascular defects including pericardial effusion and reduced number of cardiomyocytes. Furthermore, we
observed increasing apoptosis in the neural tube in the KO mice starting
at E9.5. Interestingly, at E8.5, sonic hedgehog down stream target
patched showed normal expression pattern, but at E9.5 the embryonic
development ceased and the embryos died by E11.5. In conclusion, the
data indicate a function for HSD17B7 in de novo cholesterol biosynthesis essential in developing mouse embryo during organogenesis.
Paper No.: 1143

HEALTH AND DISEASE
ROLES OF BINDING AFFINITY AND LIPOPHILICITY IN THE
KINETICS OF PROSTANOID RECEPTOR LIGANDS
Robert Jones(1), D Woodward(2), J Wang(2), R Clark(1)
(histamine H1) showing very slow onsets that correlate with their high
pA2 values (9.6 10.2). L-798106 (EP3), L-826266 (EP3 and TP), astemizole and terfenadine (H1) form a distinct cluster: moderate afnity
(pA2 7.5 8.2) and very slow onset are linked with high lipophilicity. The
more water-soluble EP3 antagonist (ASA)-3ap (see OConnell et al, Bioorg Med Chem Lett 2009; 19:778-782) had a relatively fast onset (pA2 =
7.9). The afnity trends may be explained under the limited diffusion
model of Rang and Colquhoun and colleagues. This model also accommodates partition of a lipophilic ligand into the cell membrane as a means
of retarding diffusion through the extracellular uid. However, the situation may be more complex, involving pKa/logP-dependent permeation of
an external tissue barrier and also lateral diffusion in the cell membrane
lipid followed by transfer to the transmembrane domains forming the
receptor binding site (plasmalemmal diffusion microkinetic model).
Paper No.: 2936

ANALGESICS
EFFECTS OF DICLOFENAC ON THE REPOLARIZATION IN
DOG VENTRICULAR MUSCLE
Norbert Jost(1), A Kristof(1), I Koncz(2), T Szell(2), P Biliczki(2),
J Papp(1,2), A Varro(0), L Virag(2)
(1) Hungarian Academy of Sciences, Division of Cardiovascular Pharmacology, Szeged,Hungary
(2) University of Szeged, Department of Pharmacology & Pharmacotherapy, Szeged,Hungary
The nonsteroidal anti-inammatory drug diclofenac are widely used for
chronic pain. There is, however, little information about the effects of this
drug on the ventricular repolarization. Therefore, the aim of our study
was to characterize the cellular electrophysiological effect of diclofenac in
dog right ventricular preparations by applying the standard microelectrode
and whole-cell patch clamp techniques at 37 C. At a stimulation cycle
length of 1000 ms diclofenac at 10 lM and 20 lM concentrations did not
lengthened signicantly the ventricular repolarization and at 20 lM
slightly decreased the maximal rate of depolarization (Vmax) in papillary
muscle indicating the sodium channel blocking property of the drug.
However, after impairment of the repolarization reserve by adding 30 lM
BaCl2, diclofenac at the concentration of 20 lM moderately and reverse
rate-dependently prolonged the action potential duration (APD90). The
effects of diclofenac on the rapid component of the delayed rectier (IKr),
the transient outward (Ito) and on the inward rectier (IK1) potassium currents were also investigated. The drug (50 lM) did not inuence the Ito
and IK1 currents. However, the IKr tail current was signicantly decreased
by 10 lM diclofenac (control: 68.9 8.3 pA, drug: 52.3 5.6 pA, test
potential: 20 mV, n = 7). The estimated EC50 value of IKr blockade was
approx 50 lM. We concluded that diclofenac at therapeutic concentration
did not inuence the ventricular repolarization in the heart. However, at
high dose and at impaired repolarization reserve, the proarrhythmic side
effects of the drug are cannot be excluded.
Paper No.: 3383

SYNTHESIS OF NEW DETERGENTS DERIVED FROM
QUATERNARY AMMONIUM SALTS AS A PART OF
DISINFECTION AND DECONTAMINATION MEANS
(1) University of Strathclyde, Strathclyde Institute of Pharmacy & Biomedical Sciences, Glasgow, UK
(2) Allergan Inc, Department of Biological Sciences, Irvine, CA, USA
Daniel Jun(1,2), J Marek(2,3), K Musilek(2), J Cabal(2), O Soukup(1,2),

M Hrabinova(2), M Pohanka(2), P Stodulka(2), K Kuca(1,2)
The factors involved in the slow onsets of prostanoid receptor ligands on

guinea-pig isolated tissues have been investigated. For EP1, EP3 and TP
agonists, the dominant factor appears to be receptor afnity (none is
highly lipophilic). For antagonists, afnity also has a strong inuence,
with GW-848687 (EP1), ICI-192605, BMS-180291 (TP) and doxepin
(1) University of Defence, Faculty of Military Health Sciences, Center of

Advanced Studies,Hradec Kralove, Czech Republic
(2) University of Defence, Faculty of Military Health Sciences,
Department of Toxicology, Hradec Kralove, Czech Republic
(3) Vakos XT a. s., Prague, Czech Republic
361
Biological and chemical warfare agents are still considered to be threat.
Production and storage of these agents is prohibited by the Biological
and Chemical Weapons Convention (BWC and CWC). Although the
probability of their military use is relatively low, their possible misuse by
terrorist groups or individuals remains high and many research groups
throughout the world are interested in development of novel and efcient
countermeasures against them. At our department, we are interested in
preparation of novel prophylactic and therapeutic antidotes against nerve
agents and in development of novel detergents which should be used as
active ingredients of the solutions for biological and chemical warfare
agent decontamination. For this purpose, we have prepared synthesized
series of new quaternary ammonium salt detergents structurally derived
from benzalkonium salts. Prepared compounds are belonging to the
group of cationic surface-acting agents. Instead of benzyl group in the
detergent molecule, we have used different substituted derivatives of pyridine. New compounds are nowadays tested for their disinfection and
decontamination efcacy.
This work was supported by the Ministry of Defense (Czech Republic) project OVUOFVZ200803 (Development of novel decontaminants and
disinfectants of skin based on micellar compounds).
Paper No.: 3374

DISCOVERY
CHARACTERIZATION OF TMEM16 FAMILY AND ITS
FUNCTIONAL IMPLICATIONS IN HUMAN PANCREATIC
ACINAR CELLS
Jinsei Jung(1), JH Nam(1), JS Yoon(2), MG Lee(1)
(1) Yonsei University, Department of Pharmacology and Research Center
for Human Natural Defense System, Brain Korea 21 Project for Medical
Sciences, College of Medicine, Seoul, South Korea
(2) Kwandong University, College of Medicine, Department of Pharmacology, Kangwon-do, South Korea
Calcium-activated chloride channels (CACCs) play a pivotal role in uid
and enzyme secretion in pancreatic acinar cells. This study aimed to
identify the expression patterns and physiologic roles of individual ANO
(TMEM16) family proteins in human pancreatic acinar cells. An RTPCR assay using ANO isoform-specic primers revealed that human
pancreas express high levels of ANO1 and ANO6 mRNA. To elucidate
physiological functions of these proteins, Cl- currents were measured in
HEK 293T cells transfected with ANO1 and ANO6 using the whole-cell
and inside-out patch clamp techniques. Both ANO1 and ANO6 showed
a CACC activity. However, they showed differences in several aspects,
such as, activation time and calcium sensitivity. These characteristics
were compared with currents obtained from human pancreatic acinar
cells. Isolated human pancreatic acinar cells had almost similar in CACC
properties of the ANO1 and ANO 6 and its localization is on the apical
regions specic. Our results rstly report that ANO1 and ANO6 encode
for CACC in the human pancreatic acinar cells, and they play specic
and complementary roles in pancreatic secretion.
Paper No.: 3164

ST. JOHNS WORT EXTRACT OR FLUOXETINE INFLUENCE
GENE EXPRESSION OF HYPOTHALAMIC AND
HIPPOCAMPAL TISSUES IN A MODEL OF STRESS INDUCED
DEPRESSION
Peggy Jungke(1,4), G Ostrow(2), J Li(2), C Kolb(3), O Kelber(3), D Weiser(3), K Nieber(4), V Butterweck(1)
(1) University of Florida, College of Pharmacy, Department of Pharmaceutics, Gainesville, FL, USA
(2) University of Florida, ICBR-Cancer Genetics Research Complex,

Gainesville, USA
(3) Steigerwald Arzneimittelwerk GmbH, Darmstadt, Germany
(4) University of Leipzig, Institute of Pharmacy, Leipzig, Germany
Introduction: Mild to moderate forms of depression are successfully treated with St. Johns wort (SJW). As chronic stress is a major risk factor
for psychiatric illnesses including depression, the effect of a SJW extract
with established clinical efcacy (STW 3-VI; 250 and 500 mg/kg; p.o.)
and uoxetine (10 mg/kg, p.o.) on gene expression was studied in a
chronic restraint stress (CRS) model in rats (1h for 21 days), for which
neuroethological and biochemical parameters have been documented
(Grundmann O et al, Neuropharmacology doi:10.1016/j.neuropharm.2009.12.014). Methods: Hypothalamic and hippocampal tissues
were analyzed using the Affymetrix gene chip Rat Genome 230 2.0
Array, Limma analysis and the PANTHER database. Results: Chronic
stress inuenced 256 genes in hippocampus compared to the nonstressed
control group, treatment in chronic stress with uoxetine 43, with STW
3-VI 250 mg/kg 140 and with STW 3-IV 500 mg/kg 223 genes compared to the untreated stress group. Several pathways were identied
which are functionally linked with the changes of neuroethological and
biochemical parameters. Conclusion: Gene expression proles provide
novel information about brain circuits involved in the onset of psychic
depression and in its therapy with St. Johns wort.
Paper No.: 2949

ERYTHROPOIETIN USE IN CANCER-RELATED ANEMIA IN
CROATIA
Danica Juricic, S Badzek, L Bielen, V Erdeljic, K Makar-Ausperger,
M Radacic-Aumiler, R Likic, I Francetic
University Hospital Center, Deparment of Clinical Pharmacology,
Zagreb, Croatia
Anemia is a common problem among cancer patients. Fatigue resulting
from anemia has a detrimental effect on the patients quality of life, and
anemia itself has a negative effect on both radio and chemotherapy.
Treatment options for the correction of cancer-related anemia include red
blood cell (RBC) transfusions and the use of erythropoiesis-stimulating
agents (ESAs). While RBC transfusions lead to a quick raise in the
patients hemoglobin level, there is a problem of iron overload and the
RBC transfusions availability. ESAs are an efcient way of reducing the
need for RBC transfusions. However, clinical trials in the past few years
have raised many questions about the serious adverse effects of ESAs
including the possibility of adverse cancer outcome. Availabe international guidelines limit the use of ESAs to symptomatic patients with
non-myeloid malignancies when the anemia is due to chemotherapy,
while RBC transfusion is the preferred method od anemia correction in
other cases. In Croatia, our National Institute for health insurance does
not cover the costs of erythropoietin use for the treatment of cancerrelated anemia. However, ESAs can be used if recommended by an
oncologist and approved by the Hospital treatment committee. In 2008.,
erythropoietin was the 6th highest expenditure-drug in Croatian hospitals.
The controversies, costs and the wish to provide adequate patient care
has led to the development of the guidelines for eythropoietin use in cancer-related anemia in Croatia which are reviewed in this article.
Paper No.: 2364

DYSKINESIA AND PLASMA DOPA LEVELS IN
HEMI-PARKINSONIAN RATS AFTER CHRONIC ORAL
ADMINISTRATION OF MUCUNA PRURIENS SEED EXTRACT
OR L-DOPA METHYL ESTER
Monica Jrgensen(1,2), JB Gramsbergen(1), LP Christensen(2)
362
(1) University of Southern Denmark, Institute of Molecular Medicine,
Neurobiology Research, Odense, Denmark
(2) University of Southern Denmark, Institute of Chemical Engineering,
Biotechnology and Environmental Technology, Odense, Denmark
In traditional Ayurvedic Indian Medicine, seeds of Mucuna pruriens are
used to treat Parkinsons Disease (PD). Mucuna seeds are rich in L-DOPA
and it has been suggested that Mucuna preparations induce less adverse
effects in PD patients than synthetic DOPA preparations. We recently studied the development of L-DOPA-induced abnormal involuntary movements
(AIMs, dyskinesias) in the 6-hydroxydopamine rat model of PD after
chronic treatment (12.5 mg DOPA/kg, ip, daily during 3 weeks) with Mucuna seed extract. We observed fewer AIMs in Mucuna treated rats as compared to L-DOPA methyl ester (LDME) treated animals, though peak
plasma DOPA levels were similar. Here we studied plasma DOPA levels
during chronic oral administration of LDME or Mucuna seed extract (1-2
mg DOPA/ml in drinking water) and monitored effects on forelimb akinesia,
rotational behavior and AIMs. Peak plasma DOPA levels were increased up
to approximately 400 fold (relative to baseline) with LDME and up to about
90 fold with Mucuna seed extract, suggesting a different drinking pattern for
Mucuna seed extract. Forelimb akinesia was abolished using 1.5-2 mg/ml
DOPA and six out of eleven rats developed AIMs of variable severity (1 mg/
ml - 2 mg/ml) using LDME. Whether oral chronic administration of Mucuna
seed extract induces AIMs remains to be investigated.
Paper No.: 2797
DISCOVERY
IDENTIFICATION OF KINASES REGULATING MONOAMINE
TRANSPORTER FUNCTION BY KINOME-WIDE SIRNA
SCREENING
Trine Nygaard Jrgensen, I Ammendrup-Johnsen, U Gether
Catechol-O-methyltransferase (COMT) is a centrally and peripherally

expressed enzyme that metabolizes compounds containing catecholstructures. In the PFC, COMT is thought to have a special role in the
regulation of dopamine levels due to low levels of DAT. In addition,
NET has been claimed to compensate low levels of DAT in the clearance of dopamine in the PFC. We have studied the roles of the monoamine transporters and metabolizers using COMT knock-out mice and
selective DAT, NET and a non-selective MAO inhibitor as tools. The
samples produced by PFC microdialysis in conscious mice were analyzed of dopamine and DOPAC by a highly sensitive HPLC-system.
Lack of COMT increased the levels of dopamine in the PFC during
NET [genotype effect F1,25 = 5.998, P < 0.05] and MAO [genotype
effect F1,20 = 11.084, P < 0.01] inhibition. These effects were not sex
dependent. During DAT inhibition no sex or genotype dependent
changes in dopamine levels were seen. Genotype, but not sex, had a
signicant effect on DOPAC levels in the PFC where COMT knockout mice showed higher levels than their wild-type littermates after
NET [genotype effect F1,25 = 90.050, P < 0.001], MAO [genotype
effect F1,20 = 63.898, P < 0.001] and DAT [genotype effect F1,22 =
56,945, P < 0.001] inhibition. We conclude that the contribution of
both NET and subsequently MAO on dopamine levels in the PFC is
about 50%, whereas uptake2 followed by COMT eliminates another
50%. The contribution of DAT is minor if any.
Paper No.: 2025

HEALTH AND DISEASE
ARTERIAL VASODILATION MEDIATED BY PROTEINASEACTIVATED RECEPTOR-2 IN OBESE DIABETIC MICE
Satomi Kagota(1), JJ McGuire(2)
University of Copenhagen, Panum Institute, Department of Neuroscience

and Pharmacology, Copenhagen, Denmark
The transporters for dopamine (DAT), serotonin (SERT) and norepinephrine (NET), collectively termed the monamine transporters, are known to
be regulated by protein kinases such as protein kinase C, phosphatidylinositol 3-kinase, and calcium/calmodulin-dependent protein kinase II.
However, little is known about the entire ensemble of kinases and kinase
pathways controlling monoamine transporter function. To address this
question, we carried out a small interfering RNA (siRNA) screen of the
entire human kinome and assessed the effect of knockdown on the
uptake properties of human DAT, NET or SERT stably expressed in
human embryonic kidney 293 cells. We used a human siRNA kinome
library from Ambion consisting of three siRNAs per gene, targeting 710
genes. As a negative control, siRNA not targeting any annotated genes
in the human genome was applied. An assay testing for cell viability was
performed in parallel. To eliminate possible off-target effects and to focus
on kinases with the most signicant effect on the function of DAT, NET
and SERT, we focused on genes for which at least two out of three siRNAs showed a minimum of 30% reduction or increase in uptake for one
of the three transporters. Based on this criterion, a plethora of 99 kinases
that directly or indirectly regulate the activity of the monoamine transporters were identied. Ongoing studies in cell lines as well as in primary neuronal cultures are needed to validate these preliminary ndings.
Paper No.: 958
INHIBITION OF DOPAMINE AND NORADRENALINE TRANSPORTERS (DAT & NET) AND MONOAMINE OXIDASE (MAO) IN
THE PREFRONTAL CORTEX OF COMT KNOCK-OUT MICE
Mikko Kaenmaki, C Amberg, PT Mannisto
(1) Mukogawa Womens University School of Pharmaceutical Science,

Department of Pharmacology, Nishinomiya, Japan
(2) Memorial University, Cardiovascular Research Group, Division of
BioMedicalSciences, St. Johns, Canada
Activation of proteinase-activated receptor-2 (PAR-2) can cause vascular inammation injury and endothelium-dependent vasodilation. We
have reported that PAR-2-induced relaxations persisted in resistance
arteries from hypertensive rodent models despite endothelial dysfunction to other agonists. In type 1 non-obese diabetic mice aortas an
increased sensitivity to PAR-2 relaxation was attributed to upregulated
COX-2, however, in type 2 non-obese diabetic Goto-Kakizaki rat mesenteric arteries it was only NO. In this study we investigated whether
vasodilation by PAR-2 activating peptide (2-furoyl-LIGRLO-amide;
2y) was reduced and tested the effects of COX inhibitors on vasodilator mechanisms in small caliber arteries of obese diabetic mice
(B6.BKS(D)-Leprdb/J; dbdb) compared to non-diabetic mice. Under
wire myograph conditions, second order mesenteric arteries from mice
(males, 12 weeks of age) were precontracted submaximally by
U446619 and then exposed to vasodilators in the presence and absence
of inhibitors. Acetylcholine (ACh)- and nitroprusside-induced relaxations were decreased in dbdb arteries compared to C57BL/6J arteries.
In contrast 2y-induced relaxations were not different between strains.
L-NAME inhibited ACh- and 2y-induced relaxations in C57BL/6J,
but did not inhibit these relaxations in dbdb. Neither selective inhibitors of COX-1 (FR122044, SC560) nor COX-2 (NS398, SC58125)
affected these relaxations in both strains. Protein expression of endothelial NOS was greater, and soluble guanylyl cyclase was less in
dbdb mesenteric arteries. Our results suggest that in resistance arteries
of dbdb mice, PAR2-mediated relaxation was sustained even though
NO release and NO responsiveness were reduced. The mechanism
underlying PAR-2 resistance to diabetic vascular dysfunction was not
due to coupling to COX pathway.
University of Helsinki Faculty of Pharmacy, Division of Pharmacology

and Toxicology, Helsinki, Finland
363
Paper No.: 2935
DISEASES
WHOLE BLOOD CHALLENGE TESTS AS TRANSLATIONAL
TOOLS FOR EARLY ASSESSMENT OF ANTIINFLAMMATORY/ IMMUNOSUPPRESSIVE EFFECTS OF
DRUGS
Andrea J Kales, J Burggraaf, C Kluft, M Moerland
CHDR, Department of Cardiovascular Disease, Leiden, The Netherlands
Development of anti-inammatory/immunosuppressive compounds is
challenging as effect assessment is difcult and the concept of maximally
tolerated dose (MTD) for these drugs often inappropriate. This could be
remedied if tools were available allowing effect assessment across different populations. The ex-vivo LPS test may be such a tool, stimulating
monocytes via TLR-4. It would be an advantage if challenge tests that target other inammatory routes would be available. We therefore explored
the superantigen staphylococcal enterotoxin B (SEB) test which provides
information on T-cell-driven inammation. We used both tests to characterise the anti- inammatory effects of Mitogen activated protein
kinase (MAPK) inhibition in vitro. The tests were performed in whole
blood samples from healthy volunteers and rheumatoid arthritis patients
using TNF-a as read-out. For both challenge agents dose-response curves
were constructed and the inhibition was studied using MAPK inhibitor
VX-745. Preliminary results show that 75 ng/ml SEB induces a TNFrelease of ~90% of the maximal response. VX-745 dose-dependently
inhibited TNF-a release in healthy volunteers more potently after LPS
induction (EC50 = 0.12lM) compared to SEB induction (EC50 =
5.9lM). Inhibition of LPS-induced TNF-a release by VX-745 was comparable between healthy volunteers (EC50 = 0.12 lM) and RA patients
(EC50 = 0.10lM). These data suggest that the combination of different
in-vitro challenge tests can be used to obtain mechanistic insight into different inammatory pathways that may be inhibited with different
potency. The tests also allow comparison of drug potency between populations. Further development of this approach may be a useful translational tool early in drug development, making the MTD concept obsolete.
Paper No.: 2946

EXPRESSION OF CYTOCHROME P450 METABOLIZING
ENZYMES DEPENDS ON GESTATIONAL AGE IN
CRITICALLY ILL PRETERM INFANTS
Betty Kalikstad(1), H Goransson(2), T Kristoffersen(3), A Isaksson(2)
(1) University of Oslo Faculty of Medicine & Oslo University Hospital Rikshospitalet, Division of Paediatrics, Oslo, Norway
(2) Uppsala University Academic Hospital, Department of Medical
Sciences, Uppsala, Sweden
(3) Oslo University Hospital - Rikshospitalet, Centre for Molecular
Biology and Neuroscience and Institute of Medical Microbiology, Oslo,
Norway
Introduction: The neonatal period is a critical time for premature born
infants and they often require drug therapy. Variability in drug response
may be due to differences in expression of the metabolizing CYP450
genes. Comprehensive studies of CYP450 gene expression in clinical relevant settings are lacking. We aimed to compare the gene expression levels of all CYP450 genes simultaneously in relation to gestational age
(GA) and gender. Materials/Patients: Peripheral blood at subsequent time
points was withdrawn from premature born infants between GA 25.2
37.0, in a level III Neonatal Intensive Care Unit at Oslo University Hospital, Rikshospitalet. We used the RiboPure-Blood-, GLOBINclearHuman protocol, Nugene Ovation RNA Amplication system, and Gene
Chip Human Genome U133 Plus 2.0 arrays from Affymetrix to measure
global gene expression patterns. Ethical approval was granted by the

Regional Committees for Medical and Health Research Ethics in Norway. Results: CYP450 gene expression was signicantly altered at about
6 weeks postnatally between infants born at early (n = 18) versus late (n
= 23) GA (25.2-28.6 vs. 29.0-33.6 weeks). Thirteen CYP450 genes,
including those metabolizing commonly used drugs, such as CYP1A2 (p
= 0.0112) and CYP3A4 (p = 0.0424), differed in expression levels and
are therefore interesting candidates for explaining variable drug response.
No signicant differences in CYP450 gene expressions among preterm
born males and females were found.
Paper No.: 2947

MODULATION OF GENE EXPRESSION IN CRITICALLY ILL
PRETERM INFANTS FOLLOWING STRESS
Betty Kalikstad(1), H Goransson(2), T Kristoffersen(3), A Isaksson(2)
(1) University of Oslo Faculty of Medicine & Oslo University
Hospital - Rikshospitalet, Division of Paediatrics, Oslo, Norway
(2) Uppsala University Academic Hospital, Department of Medical
Sciences, Uppsala, Sweden
(3) Oslo University Hospital - Rikshospitalet, Centre for Molecular
Biology and Neuroscience and Institute of Medical Microbiology, Oslo,
Norway
Introduction: Preterm infants are exposed to various forms of stress by
advanced intensive care treatment. For this reason, gene expression studies, might elicit processeses involved in drug metabolism. We aimed to
examine the inuence of stress at the molecular level represented by
gene expressions. Materials/Patients: Critically ill premature infants in a
level III Neonatal Intensive Care Unit at Oslo University Hospital, Rikshospitalet, exposed to stress dened by surgery, catheter angiography,
and high intensive care treatment, were studied at subsequence time
points. For peripheral blood preparation, RiboPure-Blood, GLOBINclear-Human protocol, and Nugene Ovation RNA Amplication system was used. Gene expressions were assessed using Gene Chip Human
Genome U133 Plus 2.0 arrays from Affymetrix to identify genes differentially expressed. Ethical approval was granted by the Regional Committees for Medical and Health Research Ethics in Norway. Results: A
total of 107 peripheral blood samples were assessed. 13/20 infants
exposed to high level of stress had divergent gene expression levels for
200 genes. Further investigation showed a correlation with a group of
genes related to stress activated protein kinase signaling pathway. We
also found signicant differences for CYP450 genes investigated, including CYP1A2 (p = 0.0000), CYP2D6 (p = 0.0003), and CYP3A4 (p =
0.0000 and p = 0,0178), which are involved in drug metabolism. Conclusion: These results highlight the signicance of critical illness, pathophysiological mechanisms following stress, and their effect on gene
expression levels. Thus, molecular biomarkers reecting such ngerprints
are of importance to improve our understanding of the impact of stress
on gene expressions, including CYP450 genes, in preterm infants.
Paper No.: 1736

INTERECEPTIVE EFFECTS OF ATYPICAL ANXIOLYTICS
WITH NEUROPROTECTIVE PROPERTIES
Tatiana S Kalinina, AV Volkova, TA Voronina, SB Seredenin
SI Zakusov Institute of Pharmacology RAMS, Department of
Psychopharmacology, Moscow, Russian Federation
Studies with newer types of anxiotytics as intereceptive cues direct
toward characterizing stimulus effects of new drug and determining a
possible commonality of effect with classical anxiolytics. Intereceptive
effects of anxiolytics mexidol (2-ethyl-6-methyl-3-oxypyridine succinate)
364
and afobazol (5-ethoxy-2-[2-(morpholino)-ethylthio] benzimidazole dihydrochloride) were compared to those of diazepam in drug discrimination
and state dependent learning paradigms. To study discriminative stimulus
effects, animals were trained to discriminate diazepam (0.5 mg/kg, i.p.)
vs. diazepam (5.0 mg/kg, i.p.), mexidol (50-100 mg/kg, i.p.) or afobazol
(20 mg/kg, i.p.) vs. saline (i.p.) in a liquid-maintained two-lever discrimination procedure. State dependent effects of mexidol (150 mg/kg, i.p.),
afobazol (20 mg/kg, i.p.) and diazepam (5.0 mg/kg, i.p.) were investigated in one-trail step through passive avoidance procedure. In contrast
with diazepam-trained rats, afobazol-trained animals did not show any
stimulus control, 10% of mexidol-trained rats demonstrated high stimulus
control. Afobazol (20 mg/kg) occasioned saline lever responding in diazepam-trained rats and did not render on generalization of diazepam stimulus properties. Afobazol does not produced the state dependence, while
diazepam and mexidol evoked asymmetric state dependent learning.
Mexidol is necessary to include in group of moderately discriminated
substances while afobazol - in group of non-discriminated agents. The
data suggest the absence of overlap in receptor mechanisms of afobazol
and diazepam in vivo and low probability of drug dependence development after long course of afobazol treatment.
Paper No.: 1700

ADENOSINE MEDIATED - CARDIOVASCULAR TOXICITY IN
AMITRIPTYLINE POISONING RATS
S Kalkan, Nil Hocaoglu, K Oransay, M Buyukdeligoz, Y Tuncok
Dokuz Eylul University School of Medicine, Department of Pharmacology, Izmir,Turkey
This study was designed to clarify the contribution of endogenous adenosine in the amitriptyline-induced cardiovascular toxicity mechanisms.
Rats (n = 24) were randomized into three groups. Control group received
5% dextrose i.p 1 hour ago before the amitriptyline infusion (0.94 mg/
kg/min/60 minutes). Other rats pretreated 1 hour prior to experimental
protocol with EHNA (inhibitor of adenosine deaminase, 10 mg/kg i.p)
and NBTI (inhibitor of facilitated adenosine transport, 1mg/kg i.p). After
EHNA/NBTI administrations, Group 2 received 5% dextrose, while
Group 3 received amitriptyline infusion. Mean arterial pressure (MAP),
heart rate (HR), QT and QRS durations were recorded. Plasma adenosine
concentrations were measured (HPLC). In the control group, plasma
adenosine concentrations signicantly increased after amitriptyline infusion (p < 0.05). In EHNA/NBTI administered groups, plasma adenosine
concentrations signicantly increased (P < 0.001). In the control group,
amitriptyline infusion caused a signicant decrease in the MAP, HR and
prolongation in QT and QRS durations after 10th min. (P < 0.001). In
EHNA/NBTI administered group, a signicant decrease was found in the
MAP (after 30th min.) and HR (after 40th min.) and prolongation in the
QRS at 60th min.(p < 0.05). Amitriptyline infusion following EHNA/
NBTI administrations, caused a signicant decrease in the MAP, HR and
prolongation in the QT and QRS durations after 10th min. (P < 0.001).
In EHNA/NBTI administered groups, amitriptyline-induced MAP (P <
0.01, after 20th min) and HR reductions (p < 0.05, after 40th min) and
QRS prolongations (p < 0.05, after 10th min) were more signicant than
dextrose-induced reductions and prolongations. These results indicated
that amitriptyline enhanced plasma levels of adenosine and potantialized
cardiovascular effects of endogenous adenosine.
Paper No.: 1701

DOES AMITRIPTYLINE, A TRICYCLIC ANTIDEPRESSANT,
BIND TO ADENOSINE A1 OR A2A RECEPTORS?
S Kalkan(1), H Gurdal(2), Nil Hocaoglu(1), M Buyukdeligoz(1)
(1) Dokuz Eylul University School of Medicine, Department of
Pharmacology, Izmir,Turkey
(2) Ankara University School of Medicine, Department of Pharmacology

and Clinical Pharmacology, Ankara, Turkey
In previous studies, we have shown that adenosine receptors play a role
in the pathophysiology of amitriptyline-induced cardiovascular toxicity.
Also, selective adenosine A1 and A2a receptor antagonists (DPCPX and
CSC) were found to be effective in improving hypotension, QRS and
QT prolongation and survival time in in-vivo and in-vitro models of amitriptyline toxicity. The aim of this study was to investigate the afnity of
amitriptyline to adenosine A1 or A2a receptors by radioligand binding
assay. The membranes which contain adenosine A1 or A2a receptors were
labeled with specic radioactive ligand (DPCPX or CSC, respectively)
and displacement binding of the specic ligand were determined in the
presence of different concentrations of amitriptyline or selective adenosine receptor antagonists in the binding buffer for 60 min at 27C or 90
min at 25C. Reactions were terminated by rapid ltration using Whatman GFB lters. Filters were washed with ice-cold binding buffer. The
lter-bound radioactivity was determined in a b-counter (1450 Microbeta
WALLAC Trilux). Assays were conducted in duplicate. The binding of
specic A2a radioligand was inhibited by amitriptyline (IC50: 51.42
15.87 uM). The Ki value calculated for amitriptyline was 4.8 0.11.
Also, the binding of spesic A1 radioligand was inhibited by only high
concentrations of amitriptyline (10-4 and 10-3 M). In conclusion, the current data suggest that the binding of amitriptyline to adenosine A2a receptors may play a role in amitriptyline-induced cardiovascular toxicity.
Paper No.: 3377

DRUGS USED FOR THE TREATMENT OF TYPE 2 DIABETES
MELLITUS IN FINLAND DURING THE YEARS 2004-2009
Annikka Kalliokoski, LK Saastamoinen, JE Martikainen
The Social Insurance Institution, Helsinki, Finland
Pharmacological treatment of type 2 diabetes mellitus (T2DM) includes
antidiabetics, statins, and antihypertensives. We studied the pattern of
drug use in patients with T2DM in Finland during 20042009. Data was
retrieved from the nationwide prescription registry at the Finnish social
insurance institution (Kela) covering purchases of medicines used in outpatient care. Patients 40 years or older at the time they were granted a special reimbursement status (entitling for a higher rate of refund) due to DM
were considered to have T2DM. The number of patients with T2DM
increased steadily from 127,389 in 2004 to 173,077 in 2009. Of these
patients, about 90% used antidiabetics. Metformin use (either alone or in
combination) increased from 64% to 77% of patients, whereas the use of
insulin remained stable (about 35% of patients). Use of thiazolidinediones
increased from 2004 to 2007 (from 3% to 13%), after which it has slightly
decreased. In 2009, 5% of patients used dipeptidylpeptidase-4 inhibitors.
The proportion of T2DM patients with special reimbursement status for
hypertension has remained stable (about 56%) whereas that for chronic
coronary disease has slightly decreased (from 23% to 20%). Of the
patients who purchased antidiabetics in 2009, 65% also purchased ACE
inhibitors/angiotensin 2 receptor antagonists (54% in 2004) and 60% statins (41% in 2004). Based on the register data, it is not possible to evaluate, whether the treatment of an individual patient is in accordance with
the care guidelines. However, increasing use of metformin, ACE inhibitors/angiotensin 2 receptor antagonists and statins is reassuring.
Paper No.: 3292

EVIDENCE FOR FUNCTIONAL HETEROMERIZATION
BETWEEN MELATONIN MT1 AND MT2 RECEPTORS AND
SEROTONIN 5-HT2C RECEPTORS
Maud Kamal(1), J-L Guillaume(1), A Daulat(1), P Chen(1),
C Mannoury La Cour(2), MJ Millan(2), L Vincent(2), O Nosjean(2),
JA Boutin(2), M Spedding(2), P Delagrange(2), R Jockers(1)
365
(1) Universite Paris Descartes, Institut Cochin, INSERM U1016, CNRS
8104, Paris, France
(2) IDR Servier, Croissy/Seine, France
Introduction: A combination of MT1 and MT2 melatonin receptor activation and serotonin 5-HT2C receptor (5-HT2C) blockade is associated
with robust antidepressant properties, as exemplied by the innovative
agent, agomelatine. The objective of this work was to verify the existence of heteromeric complexes between human MT1 and MT2, and
non-edited human 5 HT2C(INI) receptors. Methods & Results: Using
bioluminescence resonance energy transfer (BRET) assays in HEK-293T
cells, 5-HT2C preferentially formed heterodimers with MT2 and, with
slightly lower propensity, with MT1 receptors. As monitored by BRET,
-arrestin1 constitutive recruitment to 5-HT2C receptors increased in the
presence of MT1 and MT2 receptors. As shown by [3H]-inositol-phosphate formation, 5-HT-induced stimulation of Gq/Phospholipase C
(PLC) via 5-HT2C receptors was potentiated in the presence of MT2
and, less markedly, MT1 receptors. MT2 receptors expressed alone did
not couple to PLC. However, suggesting heterodimer transactivation,
melatonin enhanced PLC activation via Gaq in cells co-expressing MT2
and 5-HT2C(INI) receptors. Conclusions: These data provide physical
and functional evidence for the formation of MT2/5-HT2CINI and MT1/
5-HT2CINI heterodimers in HEK-293T cells, with consequent alterations
in coupling to -arrestin and PLC. Formation of such heterodimers in
vivo might have important implications for the pathogenesis and treatment of depression.
Paper No.: 1991

PREDICTION OF NON-COMPLIANCE WITH ANTIBIOTIC
THERAPY IN AMBULATORY PATIENTS
Kristina Kandrotaite(1), V Neverauskas(1), M Jievaltas(2), V Saferis(3),
V Briedis(4), R Maciulaitis(5)
(1) Kaunas University of Medicine, Faculty of Pharmacy, Kaunas,
Lithuania
(2) Kaunas University of Medicine, Department of Urology, Kaunas,
Lithuania
(3) Kaunas University of Medicine, Department of Physics, Mathematics
andBiophysics, Kaunas, Lithuania
(4) Kaunas University of Medicine, Department of Social Pharmacy and
Pharmacy Technology, Kaunas, Lithuania
(5) Kaunas University of Medicine, Department of Basic and Clinical
Pharmacology, Kaunas, Lithuania
Objective. Non-compliance is one of causes of non-rational use of medications and prediction of this behaviour is in the interest of rationalisation of therapy. Until now there has been no research made on prediction
of non-compliance in Lithuania. The purpose of this research was to
identify possible risk factors that may cause non-compliance. Methods.
Questionnaire was developed according to the WHO recommendations
for minimal information about medications to the patient. All of participated patients (N = 36) had to take some antibiotics for treatment or for
prophylaxis at home. For most of the patients it was a short-time therapy
(from 5 to 7 days). By the end of the treatment those patients were questioned at home about usage of antibiotics; during the visit, there were
counted taken/untaken pills of prescribed antibiotics. Data were analyzed
statistically by descriptive and comparative statistic analysis by parameter
of Pearsons chi square (statistically signicant result was when chi
square >4, p < 0.05). Results. There were 31% of patients who failed to
comply with prescribed antibiotic therapy. Several statistically signicant
risk factors of non-compliance were identied representing both educational and social constrains, most important ones lack of knowledge
about using medications and maximal dose and better knowledge about
contraindications; having less than 10 different medications at home;
negligence of self-medication with prescription NSAIDs; frequent usage
of herbal medications et al. Conclusions. This exploratory study revealed
new hypothesis about non-compliance with antibiotic therapy. For verication and conrmation of these results further investigations are needed.
Paper No.: 731

PHAPMACOTHERAPY OF PATIENTS WITH ISCHEMIC
HEART DISEASE AND LEFT VENTRICLE DYSFUNCTION
TARGETING TO RESTORE INOTROPIC ACTION AND
BIONERGETIC RESERVE OF MYOCARDIUM
Ketevan T Kapanadze, GV Sukoyan, MA Rogava
N. Kipshidze National Centre of Therapy, Department of Cardiomypathy,
Tblisi, Georgia
Introduction: The therapeutic concept of rational treatment of CHF with
cardiotropic drug Adenocin, suggested in this work, leads to the increasing of cardiac output with adequate improvement in bioenergetic reserve,
to activation extra- and intracellular NAD-signalinig mechanism, to
restoring homeostasis of redox-states and Ca-transport through cellular
membranes. The goal of investigation was to study efcacy of Adenocin
treatment on the cardiac hemodynamics, bioenergetic and endothelial
function disturbances parameters in patients with ischemic heart disease
(IHD) and left ventricle (LV) dysfunction. Materials/Patients: 156
patients, had CHF with left ventricle ejection fraction 0.35 and received
optimal standard therapy, were included in the study. All patients were
randomized by double-blind method to Adenocin (n = 78) main or placebo (standard therapy, n = 78) - control group, for 2 weeks. Symptoms
of CHF, ECG, Echo-ECG, entothelial dysfunction, redox-state, NADHoxidase activity of platelets were clinically compared in control and main
group. Results: Treatment with cardioprotective, inotropic drug, Adenocin signicantly decreased NYHA functional class, increased the distance
walked in 6-min tests, decreased LV end-systolic and end-diastolic
dimensions and improved LV ejection fraction (increase from 28 to 44%,
and didnt change in control) and redox-potential of platelets, decreased
the content of endothelin-1 and activity of NADH-oxidase in the platelets
and plasma. The drug was well tolerated. Conclusion: Received data
allow us to recommend Adenocin in the treatment of the patients with
IHD and LV and endothelial dysfunction.
Paper No.: 1866

REGULATION OF GPR55 BY THE G PROTEIN COUPLED
RECEPTOR ASSOCIATED SORTING PROTEINS
Julia Kargl(1), N Balenga(1), L Martini(2), J Whistler(2),
M Waldhoer(1)
(1) Medical University of Graz, Institute of Experimental and Clinical
(2) University of California, Ernest Gallo Clinic and Research Center,
USA
GPR55 is a member of the G-protein coupled receptor (GPCR) subfamily A and is activated by different cannabinoid ligands as well as by the
lipid ligand lysophosphatidylinositol (LPI). Generally, the activity of
GPCRs is coordinated by receptor signaling, receptor desensitization and
receptor resensitization. The latter two mechanisms are typically associated with the sorting of the GPCRs between degradation or recycling
pathways and are highly regulated. Several sorting proteins have recently
been identied, for example the G-protein coupled receptor associated
sorting protein-1 (GASP-1). GASP-1 was originally found to target d
opioid receptors to lysosomes and hence a degradative pathway. Here
we show that GPR55 can internalize after ligand activation and is
366
subsequently targeted to intracellular vesicles of the lysosomal compartments. This result and the close similarity of GPR55 to the cannabinoid
receptor CB1 - which is targeted to lysosomes via the GASP-1 protein suggested that GASP-1 may be involved in targeting GPR55 to lysosomes. In fact, GPR55 binds GASP-1 and GASP-2 (the closest homologue to GASP-1) in vitro. This work provides rst evidence that GPR55
is targeted to lysosomes after prolonged agonist stimulation and this
mechanism is regulated by members of the G-protein coupled receptor
associated sorting proteins.
Paper No.: 558
INHIBITION BY LITHIUM OF THE NITRERGIC
RELAXATION OF RAT ANOCOCCYGEUS MUSCLE
Alireza Karimollah(1), M Ghasemi(2), MH Ghahremani(3),
AR Dehpour(2)
(1) Yazd Medical University, Department of Pharmacology, Yazd, Iran
(2) University of Tehran, School of Medicine, Medical Sciences,, Tehran,
Iran
(3) University of Tehran, Faculty of Pharmacy, Medical Sciences,
Tehran, Iran
We evaluated the effect of lithium on the nitric oxide (NO)-mediated
nonadrenergic noncholinergic (NANC) relaxation of rat anococcygeus
muscle. The isolated precontracted (phenylephrine,7.5lM) rat anococcygeus muscle were relaxed via electrical eld stimulation (5 Hz) in the
absence or presence of lithium (0.5, 1, and 5 mM) or in tissues excised
from lithium (600 mg/L indrinking water for 30 days)-treated animals.
Effects of L-NAME (0.03 and 100 lM) or guanylyl cyclase inhibitor
ODQ (1 lM) and NO precursor L-arginine (1 mM) on relaxations were
investigated. Effect of either in vitro (1 and 5 mM) or ex vivo lithium
treatment on relaxation to the NO donor sodium nitroprusside (0.11000lM) was also investigated. The NANC relaxation was signicantly
reduced by in vitro and ex vivo lithium treatment (P < 0.001). L-NAME
(100 lM and 1 mM) and ODQ (1 and 10 lM) signicantly inhibited
NANC relaxations in either control orlithium-treated strips. Combination
of lithium (0.5 mM) with L-NAME (0.03 lM) signicantly reduced the
NANC relaxation. Although 1 mM L-arginine had no effect on relaxations, it prevented their inhibition by both in vitro (1 and 5mM) and ex
vivo lithium of relaxations. SNP induced relaxation in precontracted muscles was not altered by lithium treatment. RT-PCR revealed a signicant
increase in the neuronal NOS expression in the anococcygeus muscle of
lithium-treated animals. Our experiments suggested that both ex vivo and
in vitro lithium administration attenuated the NO-mediated neurogenic
relaxation osolated rat anococcygeus muscle.
Paper No.: 1501

TWO DISTINCT PATHWAYS, PKA AND TRKA, MEDIATE
ACTIVATION OF CREB DURING A2-AR INDUCED NEURONAL
DIFFERENTIATION
the present study we have investigated the role of CREB in a2-AR

induced differentiation (Karkoulias et al., J. Neurochem. 2007; 103: 882895). We employed a luciferase assay and Western blotting analysis and
we have found sustained activation of CREB by epinephrine during the
differentiation process in a subtype-specic manner that closely resembles the differentiation properties of these subtypes. Expression of a
dominant negative mutant of CREB abrogated the a2-AR induced differentiation as indicated by the effect on the expression of Cyclin D1 and
markers of neuronal differentiation, whereas overexpression of CREB in
PC12a2A clone triggered differentiation comparable to that of PC12a2B/
a2C, by prolonging CREB activation. Furthermore, employing pharmacological inhibition of Protein Kinase A (PKA) and TrkA receptor tyrosine
kinase, we have dissected the pathway involved in the a2-AR induced
differentiation: CREB activation primarily occurs via a pathway involving PKA activation, whereas the sustained activation of CREB is mediated by the delayed transactivation of TrkA.
Paper No.: 2273
GEMFIBROZIL MARKEDLY INCREASES THE PLASMA
CONCENTRATIONS OF MONTELUKAST: A PREVIOUSLY
UNRECOGNIZED ROLE FOR CYP2C8 IN THE METABOLISM
OF MONTELUKAST
Tiina Karonen, J Laitila, M Niemi, P Neuvonen, J Backman
University of Helsinki and Helsinki University Central Hospital,
Department of Clinical Pharmacology, Helsinki, Finland
According to previous information, montelukast is metabolised by cytochrome P450 (CYP) 3A4 and CYP2C9. Montelukast is a selective competitive inhibitor of CYP2C8 in vitro, yet having no signicant inhibitory
effect on CYP2C8 in vivo. Our aim was to study the signicance of
CYP2C8 in the pharmacokinetics of montelukast using gembrozil as a
model CYP2C8 inhibitor. 10 healthy subjects took in a randomized
cross-over study gembrozil 600 mg or placebo twice daily for 3 days,
and on day 3, 10 mg montelukast. The plasma concentrations of montelukast and its metabolites were measured. In addition, the effects of
gembrozil and gembrozil glucuronide on montelukast (0.04 micromol/
l) metabolism were investigated in vitro. Gembrozil increased the mean
total AUC, Cmax and T1/2 of montelukast 4.5-fold, 1.5-fold, and 3.0fold, respectively (P < 0.001). Moreover, gembrozil impaired the formation of M6, the 36-hydroxylated primary metabolite of montelukast:
its Tmax was prolonged 3-fold (P = 0.005) and its AUC(0-7) was
reduced by 40% (P = 0.027). Further, gembrozil greatly reduced the
concentration of the secondary metabolite, M4b, which is formed from
M6: its AUC(0-24) and Cmax were reduced by >90% (P < 0.001). On
the other hand, gembrozil increased the AUC(0-24) of the 21-hydroxylated metabolite diastereomers M5a and M5b by 9.3-fold (P < 0.001) and
4.8-fold (P < 0.001), respectively. In human liver microsomes, gembrozil glucuronide (1-64 micromol/l), but not gembrozil, concentrationdependently inhibited the formation of M6 (up to 50%), but not of M5b.
In conclusion, gembrozil markedly increases the plasma concentrations
of montelukast, indicating that CYP2C8 has a major role in the elimination of montelukast.
G Karkoulias(1), P Papathanasopoulos(2), Christodoulos Flordellis(1)

(1) University of Patras, School of Medicine, Department of Pharmacology, Patras, Greece
(2) University of Patras, School of Medicine, Department of Neurology,
Patras, Greece
Recent studies have demonstrated subtype-specic neuronal differentiation of PC12 cells transfected with a2-adrenergic receptors (Taraviras et
al., J. Cell Biol. 2002; 81: 363-374). Many aspects of neuronal development, such as precursor proliferation, neuronal survival and differentiation are regulated by the transcription factor cAMP-response elementbinding protein (CREB) (Lonze et al., Neuron 2002; 35: 605-6234). In
Paper No.: 505

APELIN IS A CRUCIAL FACTOR FOR HYPOXIA-INDUCED
RETINAL ANGIOGENESIS
Atsushi Kasai(1), Y Ishimaru(1), T Kinjo(1), Y Yoshioka(1),
A Yamamuro(1), N Shintani(2), A Baba(2), S Maeda(1)
(1) Setsunan University Faculty of Pharmaceutical Sciences, Department
of Pharmacotherapeutics, Osaka, Japan
(2) Graduate School of Pharmaceutical Sciences, Osaka University
367
Progression of ischemic retinal diseases, such as diabetic retinopathy, is
closely associated with pathological retinal angiogenesis, which is mainly
induced by vascular endothelial growth factor (VEGF) and erythropoietin
(Epo). Antiangiogenic therapies with anti-VEGF drugs are effective in
treating retinal neovascularization. However, these therapies show the
transient efcacies and may cause the general side effects. Therefore,
new therapeutic target molecules are needed to resolve these issues. The
involvement of the apelin/APJ system, a newly deorphanized G proteincoupled receptor system, has been recently demonstrated in physiological
retinal vascularization during the early postnatal period in mice (Kasai et
al., ATVB 2008; 28:1717-1722). We investigated the role of endogenous
apelin in pathological retinal angiogenesis using a mousemodel of oxygen-induced retinopathy (OIR). In the OIR model, vaso-obliteration of
the central retinal vessel during the hyperoxic phase was accompanied
by subsequent upregulation of VEGF and Epo during the hypoxic phase.
Expression of retinal apelin was dramatically increased during the hypoxic phase. APJ in the retina of the OIR model was highly expressed in
capillary endothelial cells, most of which were proliferative. Retina of
wild-type mice showed a signicant increase in capillary density accompanied by abnormal vessel growth in the hypoxic phase, but there was
no increase in capillary density and abnormal vessels in retinas of apelinKO mice despite upregulation of VEGF and Epo mRNA. These results
suggest that apelin is a prerequisite factor for hypoxia-induced retinal
angiogenesis.
science in Serbia, cooperating closely together and with the whole

worlds doctors.The Section of Clinical Pharmacology of the Serbian
Medical Association coordinates educational meetings for all doctors eg.
training in Good Clinical Practice organized twice a year, with teachers
who are internationally recognized specialists in clinical pharmacology
and other specialties. The First Simposium Days of Hospital Clinical
Pharmacology was held 26 and 27 nov 2009 in SMA, participating over
100 doctors from Serbia, USA, France, Grrece, Bulgaria. It will be traditionally in Belgrade every year in November. Doctors from Serbia connected to ECRIN project The major goal of the Section is to improve
position and spread the activities of clinical pharmacologists as health
professionals in tertiary, secondary as well as in primary health care. We
work as clinicians, scientists and educators.
Paper No.: 1067

POTENTIAL DRUG-DRUG INTERACTIONS OF LOPINAVIR/
RITONAVIR IN ANTIRETROVIRAL PRESCRIPTIONS WITH
PRESCRIBED DAILY DOSES NOT ACCORDING TO THE
RECOMMENDED ANTIRETROVIRAL DOSES IN
SOUTH AFRICA
Norah Lucky Katende-Kyenda(1), M Lubbe(2), J Serfontein(2),
I Truter(3)
Paper No.: 3213

FOCUSED CONFERENCE GROUP: PW34 - STATE-OF-THE-ART
OF BASIC AND CLINICAL PHARMACOLOGY IN SERBIA
THE NEW ASSOCIATION IN CLINICAL PHARMACOLOGY:
SECTION OF CLINICAL PHARMACOLOGY SERBIAN
MEDICAL ASSOTIATION
Dragana A Kastratovic(1), MM Mikov(2), VV Stanimirovic(3),
AJ Sabo(2), SM Jankovic(4), N Japundzic-Zigon(5), DR Milovanovic(4),
R Lilic(6), MH Vukovic(7), VM Jugovic(8)
(1) ical Centre of Serbia, Belgrade, Serbia
(2) Medical Faculty Novi Sad, Serbia
(3) y for Drugs and Medical Devices of Serbia, Belgrade, Serbia
(4) Clinical Centre of Kragujevac, Serbia
(5) Medical Faculty Belgrade, Serbia
(6) Clinical Centre of Nis, Serbia
(7) Health Centre Valjevo, Serbia
(8) General Hospital, Sabac, Serbia
Clinical pharmacology has been a recognized medical specialization in
Serbia since 1983. Over 60 specialists in clinical pharmacology are being
engaged in hospitals of Serbia, where they work as clinicians. The rst
generation of specialists in clinical pharmacology passed their board
examination in the year 1986. Clinical pharmacologists work at departments of clinical pharmacology of the school of medicines in Belgrade,
Novi Sad, Kragujevac and Nis, contributing to the implementation of the
modern concept of treatment of hospitalized patients. The initiative to
submit the application to the Serbian Medical Association for the establishing the Section of Clinical Pharmacology has been launched at the
request of the specialists in clinical pharmacology, working in the different medical institutions. In October 9th, 2008. leading people engaged in
clinical pharmacology gathered together and reached the conclusion that
the establishment of the Section of Clinical Pharmacology of the Serbian
Medical Association was necessary. The Chairmanship of the Serbian
Medical Association Acad Prof R. Colovic, on February 19th, 2009,
reached the uninomous decision of all Serbian medical specialists to
establish the Section of Clinical Pharmacology of the Serbian Medical
Association. In this way, the specialists, who had completed the medical
specialty training in Clinical Pharmacology in Serbia, earned for the rst
time their own section within the Serbian Medical Association - the oldest professional association of doctors of medicine and dentistry in Serbia. The Serbian Medical Association was founded in Belgrade in 1872
and it is the largest professional association of doctors in Serbia, who,
through 82 sections, work on development of the medical profession and
(1) Walter Sisulu University, Department of Pharmacology, Mthatha,

South Africa
(2) North West University, Potchesfstroom, South Africa
(3) Nelson Mandela Metropolitan University, Port Elizabeth, South
Africa
Clinically signicant drug-drug interactions (DDIs) do occur when lopinavir/ritonavir (LPV/RTV) is coadministered with other antiretrovirals
(ARVs) that are substrates for or inducers or inhibitors of cytochrome
P450 isoenzymes. Potential DDIs between LPV/RTV and other ARVs in
general practitioners (GPs) and specialists (SPs) prescriptions with inappropriate prescribed daily doses (PDDs) were determined for 2005-2007.
A non-experimental, retrospective quantitative, drug-utilisation study was
performed on 49995, 81096 and 88988 ARV prescriptions claimed
through a pharmacy benet management company in a private healthcare
sector in South Africa. Potential DDIs between ARVs were identied and
classied according to a clinical signicance rating as described by Tatro.
Of 2638 GPs and 472 SPs ARV prescriptions claimed, 505 (19.14%)
were GPs and 143 (30.30%) SPs prescriptions with DDIs and PDDs not
according to the recommended ARV dosing. Potential DDIs were identied between LPV/RTV and other ARVs with PDDs not according to the
recommended ARV dosing accounting for 88.92% (n = 449) for GPs and
98.60% (n = 141) for SPs. The highest percentage of potential DDIs were
between LPV/RTV at PDDs 1066.4 mg/264 mg and efavirenz (EFV) at
PDD 600 mg with 61.47% (n = 276) for GPs and 38.30% (n = 54) for
SPs prescriptions, prescribed to patients 19-45 years. The standard dose
for LPV/RTV is 400 mg/100 mg twice daily or LPV/RTV 800 mg/200
mg once daily. The dosage for LPV/RTV prescribed by GPs and SPs to
HIV patients was high as compared to the recommended LPV/RTV dosing. It is therefore recommended that dosage adjustment be done.
Paper No.: 3286

TIME COURSE OF CHANGES IN TRPV1 CHANNEL
FUNCTION IN RAT URINARY BLADDER FOLLOWING
STREPTOZOTOCIN INDUCED DIABETES
Teeraporn Katisart, M Parsons, C Benham
University of Hertfordshire, Department of Life Scieces, Hateld, UK
368
Peripheral neuropathy, one of the consequences of diabetes, is reproduced in the streptozotocin (STZ) model of diabetes in rats. We now
report the time course of onset of this dysfunction. Male Wistar rats were
sacriced between 24 hours and 8 weeks after a single intra-peritoneal
administration of 65mg/kg STZ or citrate buffer. The bladders were
removed and tissue strips were equilibrated for 30 minutes before adding
capsaicin. Capsaicin was added at concentrations of 10-10 M to 10-6 M
to the organ bath in a cumulative manner. As early as 36 hours after
induction of diabetes by STZ, the contractile responses to capsaicin were
signicantly reduced (9.1 3.3 g/g wet weight of tissue) in comparison
to those of the controls (36.54 4.8 g/g) and this reduction persisted
until the eight week time point. In contrast, responses to the TRPA1 agonist allyl isothiocyanate were not affected at early timepoints but were
reduced eight weeks after STZ treatment. To explore the mechanism by
which STZ affects TRPV1 channels, bladder strips from non-STZ treated
rats were exposed to elevated glucose concentrations. Contractile
responses to the TRPV1 agonist capsaicin were not affected by exposure
(30 minutes or 2 hours) to elevated (44.4 mM) glucose. These results
suggest that there are distinct early effects of STZ treatment on TRPV1
channel function at a time when other afferent nerve terminal channels
(TRPA1) are functioning normally. This suggests that early onset of dysfunction in TRPV1 signalling may not merely be the consequence of
nerve damage.
Paper No.: 2370

DISEASES
INFLUENCE OF STW 11, A FIXED COMBINATION
MEDICINAL PRODUCT, ON CYTOKINE PRODUCTION OF
HUMAN MACROPHAGES
Careen Katryniok(1), G Bonaterra(0), D Weiser(1), O Kelber(1),
R Kinscherf(3)
(1) Steigerwald Arzneimittelwerk GmbH, Scientic Department,
Darmstadt, Germany
(2) University of Heidelberg, Section Macroscopic Anatomy, Heidelberg,
Germany
(3) University of Marburg, Department of Anatomy and Cell Biology,
Marburg, Germany
Extracts of Echinacea (purple coneower) are used in the prevention and
therapy of infectious diseases. The medicinal product STW 11 (Inuex)
contains the extract from purple coneower, and in addition extracts of
monkshood, venom of honey bee and bushmaster snake in homeopathic
dilutions. Previous studies showed a stimulation of the cellular and
humoral immune response. In this study we determined the inuence of
STW 11 on the production of cytokines in human macrophages. Differentiated (20 nM PMA, 24h) human U937 macrophages were used to
study anti-inammatory effects (stimulation with 10lg/ml LPS, 2h) and
activating effects (unstimulated cells). Cells were preincubated with
STW 11 (30min.) in different concentrations and mRNA level of cytokines (e.g. TNF-a IL-1b, IL-6 and IL-8) were analysed by quantitative
RT-PCR. Inhibition of IL-1b; expression by 30-50% was detectable in
LPS-activated macrophages after incubation with 1-100 ll/ml STW 11.
In unstimulated U937 macrophages, the mRNA expression levels of
TNF-a, IL-1b, IL-6, IL-8, CD40 and COX-2 were induced by STW 11
in a concentration-dependent manner, but much less than induction by
LPS. In summary, these data demonstrate anti-inammatory effects of
STW 11 in activated human macrophages combined with immunmodulating aspects being essential for antigen presentation in non-activated
human macrophages.
Paper No.: 1070

FOCUSED CONFERENCE GROUP: P19 - GENERAL SESSION AIRWAY
IN VIVO [11C]PK11195 PET IMAGING AS A BIOMARKER TO
ASSESS LIPOPOLYSACCHARIDE-INDUCED LUNG INFLAMMATION IN RAT
Sidath Katugampola, A Zhu, K Zasadny, M Callahan, C Proctor-Brown,
M Skaddan, K Kuszpit, T Bocan
(1) Pzer Global Research & Development, Department of Biomarkers,
Sandwich, UK
Chronic obstructive pulmonary disease (COPD) is a major public health
problem. As COPD progresses, polymorphonuclear neutrophils, macrophages and B cells increase. Therefore, detecting inammatory processes
of lung non-invasively is our primary motivation for using positron emission tomography (PET) imaging. Objective: To access the usefulness of
[11C]PK11195 PET imaging in evaluating acute lung inammation
induced by aerosol Lipopolysaccharide (LPS) exposure. Experiments and
Results: Four groups of jugular-cannulated (JVC) male Sprague-Dawley
rats were studied: treatment by aerosol exposure of phosphate buffered
saline (PBS), treatment by aerosol exposure (1mg/ml) of LPS, no treatment, and rats without JVC. All animal study procedures conducted were
approved by an institutional review committee (IACUC). At 8 hours post
LPS treatment, dynamic PET imaging was conducted for 90 min after iv
administration of [11C]PK11195 with serial blood sampling. At completion of PET, lung was collected for histology. Lung sections were stained
with hematoxylin and eosin (H&E stain) for general morphology assessment. PET imaging of lung was quantied using Logan plot analysis
yielding distribution volume (DV) of tracer. H&E stain results show
increases in neutrophils of LPS treated lungs, while [11C]PK11195 PET
imaging did not show any signicant change in [11C]PK11195 DV
across groups. JVC surgical procedure did not produce a detectable signal associated with possible inammation. Conclusions: Macrophage and
neutrophil densities at 8 hours induced by 1 mg/ml LPS exposure,
though physiologically relevant to levels associated with COPD in
patients, is not sufcient to be detectable by [11C]PK11195 PET imaging.
Paper No.: 1071

MEASURING AMINO ACID RELEASE FROM RAT BRAIN
AND SPINAL CORD TISSUES
S Katugampola, Emma Winter, R Fish, N Andrews, H Bye
Pzer Global Research & Development, Department of Biomarkers,
Sandwich, UK
The release of endogenous amino acids from rat cerebral cortex slices,
following stimulation with veratrine, has been previously investigated in
vitro (Leach M.J. et al., Epilepsia, 1986, 27, 490-497). The aim of this
study was to determine the effects of veratrine, veratridine in the absence
or presence of lamotrigine, pottasium and retigabine in rat cerebral cortex, hippocampus and spinal cord slices. Tissue slices (0.4 mm) was
obtained using a tissue chopper. Slices were incubated in Tyrodes in the
absence or presence of compound, samples spun and resulting supernatant analysed using HPLC. In cortex, veratridine and pottasium produced
a signicant (p < 0.05) increase in glutamate, GABA and glycine release.
The effect of veratridine was antagonised by lamotrigine and by tetradatoxin. In contrast, veratrine caused a signicant (p < 0.05) increase in
GABA release only. In hippocampus, veratridine produced a signicant
(p < 0.05) increase in GABA, glutamate and glycine, while retigabine
had no effect on any of the amino acids measured. Veratridine and pottasium produced a signicant (p < 0.05) increase in GABA and glycine
release in spinal cord.and the effect of veratridine was antagonised by
lamotrigine. In conclusion, a rat tissue (cortex, spinal cord and hippo-
369
campus) slice preparation has been successfully used to determine native
mechanistic pharmacology for a range of targets. This assay may provide
an opportunity to increae condence of novel CNS targets and their
impact on neurotransmitter release, which may provide an opportunity
for biomarker identication.
Paper No.: 1579

ANALGESICS
INHIBITORY EFFECTS OF DOPAMINE ON SPINAL REFLEX
POTENTIALS OF RAT
K Kawamoto, Ken-ichi Otsuguro, S Ito
Hokkaido University, Graduate School of Veterinary Medicine, Laboratory of Pharmacology, Sapporo, Japan
The synaptic transmission in the spinal cord is modulated by monoamines such as noradrenaline, serotonin (5-HT) and dopamine (DA), which
are released from terminals of descending bers. However, the effect of
DA in the spinal cord is not understood. In the present study, the effect
of exogenous and endogenous DA on synaptic transmission in the spinal
cord was examined. In the isolated spinal cord from neonatal rats, monosynaptic reex potential (MSR) and nociceptive reex potential (slow
ventral root potential; sVRP) were recorded from the lumbar ventral root
in response to electrical stimulation of the corresponding dorsal root. DA
(0.01-3 uM) inhibited sVRP but not MSR. RT-PCR analysis showed that
all DA receptor subtypes were expressed in the spinal cord. The inhibition of sVRP by DA was markedly reversed by D1-like-receptor (D1like-R) antagonists, SCH23390 and LE300, but not D2-like-receptor
antagonists, haloperidol and raclopride. A D1-like-R agonist, SKF83959
mimicked the inhibitory effect of DA. Methamphetamine (MAP), an
endogenous amine releaser, showed inhibitory effects on both sVRP and
MSR, which were reduced by pretreatment of reserpine. The MAPevoked inhibition of sVRP was reversed by D1-like-R antagonists, while
the inhibition of MSR was reversed by a 5-HT2A-R antagonist, ketanserin. Our data indicate that both endogenous and exogenous DA selectively inhibits sVRP via D1-like R. Endogenous DA is suggested to
inhibit nociception in the rat spinal cord.
Paper No.: 1757

NERVE GROWTH FACTOR (NGF) PREVENTS TUMOR
GROWTH
Hiromu Kawasaki(1), M Goda(1), S Takatori(1), Y Zamami(1), N Hobara(2), Y Kitamura(3)
(1) Okayama University Graduate School of Medicine, Dentistry & Pharmaceutical Sciences, Department of Clinical Pharmaceutical Sciences,
Okayama, Japan
(2) Okayama University of Science, Department of Life Science, Okayama, Japan
(3) Okayama University Graduate School of Medicine, Dentistry and
Pharmaceutical Sciences, Department of Pharmaceutical Care and Health
Sciences, Okayama, Japan
The angiogenesis is essential for growing tumor. Previous studies
showed that NGF facilitates innervation of perivascular nerves in neovessels and maturation of neovessels. Since perivascular nerves play an
important role in regulation of vascular tone and local blood ow, neuronal regulation of tumor blood ow may suppress tumor growth. Therefore, the aim of this study was to investigate whether NGF prevents
tumor growth in vivo. DU145 prostate carcinoma cells or HT1080 brosarcoma cells were subcutaneously implanted into nude mice, and NGF
or saline was subcutaneously infused using an osmotic minipump for 2
weeks from 21 days after implantation. The density of immunocytochemically stained SMA in tumor was quantied by computer-assisted
image processing. The proliferation effect of NGF against tumor cells
was examined by in vitro prolifelation assay.Tumor volumes of DU145
and HT1080 in saline group gradually increased within 56 days. Both
DU145 and HT1080 tumor volumes in NGF group at 25-56 days after
implantation were signicantly smaller than those in saline group. Significant suppression of each tumor growth was continued even after withdrawal of NGF. Vascular smooth muscle density of DU145 and HT1080
tumor in NGF group was signicant higher than that of saline groups,
while microvessel density of both tumors in NGF group was not different
from saline groups. NGF increased proliferation of HT1080 cells, but it
reduced DU145 cell proliferation in vitro. These results suggest that
NGF prevents tumor growth via the indirect effect, probably innervation
or maturation of tumor neovasculature.
Paper No.: 3284

DETECTING STIMULANT AND SEDATIVE PROPERTIES OF
DRUGS USING HOME CAGE TRACKING IN RATS
John Kelly, F Dunne, A OHalloran, K Lane, J Simpson, S OBrien
NUI Galway, Department of Pharmacology and Therapeutics, Galway,
Ireland
The introduction of automated locomotor activity methodologies has
been extremely useful in removing the subjectivity and bias from measuring this parameter in rodents. However, many of these behavioural
studies are still conducted in novel environments, rather than in ones that
the animals are familiar with, such as their home cage. The purpose of
the present series of experiments was to develop an automated home
cage tracking (HCT) prole using EthoVision software and assessing
the acute effects of stimulant (amphetamine and methamphetamine, 0-5
mg/kg, sc) and sedative (diazepam, 0-20 mg/kg, sc and chlordiazepoxide,
0-50 mg/kg sc) drugs in this apparatus. HCT was recorded for 11-60
minutes following administration (n = 4 per group) to young adult male
Sprague-Dawley rats. For amphetamine and methamphetamine, a dosedependent increase in home cage activity, followed by a reduction at
higher doses. Methamphetamine was more potent, whereas amphetamine
produced greater maximal responses. Both diazepam and chlordiazepoxide, dose-dependently reduced locomotor activity, with diazepam exhibiting a greater potency and stronger sedative effects than chlordiazepoxide.
Such dose response relationships were absent when using the open eld
test, a commonly evaluated novel method for measuring locomotor activity. Further modications using darkened bedding enabled the tracking
of the stimulant effects of amphetamine to be improved upon. In conclusion, these studies have successfully developed a sensitive HCT methodology that has been validated using stimulant and sedative properties in
the same test conditions, with relatively small numbers of animals
required to produce statistically signicant results.
Paper No.: 2427

DISEASES
PACAP DEFICIENCY ENHANCES OXAZOLONE-INDUCED
ALLERGIC CONTACT DERMATITIS IN MICE
A Kemeny(1), R Cseharovszky(1), D Reglodi(2), N Shintani(3),
H Hashimoto(3), A Baba(3), Erika Pinter(1), J Szolcsanyi(1),
Z Helyes(1)
(1) Department of Pharmacology and Pharmacotherapy, University of
Pecs, Pecs, Hungary
(2) Department of Anatomy, University of Pecs, Pecs, Hungary
(3) Laboratory of Molecular Neuropharmacology, Osaka University,
Osaka, Japan
370
Objectives: Pituitary adenylate cyclase-activating polypeptide-38 (PACAP-38) and its receptors (PAC1 and VPAC) are widely distributed in
capsaicin-sensitive primary afferents as well as several immune cells.
The aim of the present study was to investigate oxazolone-induced
delayed-type hypersensitivity reaction in the skin of PACAP knockout
(KO) and wild-type mice. Methods: Sensitization was induced by 2%
oxazolone on the shaved abdomen on two consecutive days. Hypersensitivity was elicited by oxazolone smeared on both sides of the ear 6
days later and the thickness was measured with a micrometer. Histological examination, cytokine prole (IL-2, IL-4, IL-5, Monocyte Chemoattractant Protein-1, IFN-c, TNF-a) and myeloperoxidase activity
correlating with the number of neutrophils and macrophages were
determined from the ear samples 24 and 48h later. Results: Oxazolone
induced a 110-130% ear oedema after 24-48h in wildtype mice, which
was signicantly greater, 140-150%, in PACAP knockouts. Histological
analysis showed more intensive oedema and inammatory cell accumulation in the KO group, but there was no difference in the myeloperoxidase activity of ear homogenates. MCP-1 elevation was signicantly
higher in the KO samples, while TNF-a, IL-4 and IFN-c concentrations
were similar in the two groups after 24h. Conclusion: These results
suggest that PACAP exerts anti-inammatory, especially oedema-inhibiting effects in allergic contact dermatitis. Histological and cytokine
data revealed its protective role in the accumulation of mononuclear
cells.
Paper No.: 3149

CHARACTERIZATION OF A SPHINGOSINE 1-PHOSPHATE
RECEPTOR ANTAGONIST PRODRUG
Perry Kennedy(1), R Zhu(2), J Tomsig(1), T Huang(2), T Mathews(2),
T Macdonald(2), K Lynch(1)
(1) University of Virginia, Department of Pharmacology, Charlottesville,
VA, USA
(2) University of Virginia, Charlottesville, Department of Chemistry,
VA, USA
Sphingosine 1-phosphate (S1P) is a pleiotropic signalling phospholipid
that acts as an agonist on ve G protein-coupled receptors (S1P1
S1P5). Through receptor signalling, S1P stimulates cell survival, proliferation and migration. Physiological effects of S1P receptor signalling
extend to immune cell trafcking, vascular barrier integrity, angiogenesis
and heart rate. We manipulated the chemical structure of FTY720 (ngolimod), an S1P receptor agonist prodrug, to create an S1P receptor antagonist prodrug, VPC 03090. Using GTP[c-35S] binding and calcium
mobilization assays, we found that the active agent, VPC 03090-phosphate, while having no activity at S1P2, is a competitive antagonist at
S1P1, 3, and 5, but an agonist at S1P4. Whole-cell radioligand binding
studies documented that VPC 03090-P has high afnity for the S1P
receptors, with a rank order potency of S1P5 > S1P4 > S1P1 > S1P3
(afnity constant 2 59 nM). Quantication of compound levels in
mouse plasma by liquid chromatography mass spectrometry revealed
in vivo phosphorylation of VPC 03090 (orally available) to a long-lived
VPC 03090-P (active species). We hypothesize that VPC 03090 will
inhibit agonist-driven responses at the S1P receptors in vivo. Given the
paucity of S1P1, 3, 5 antagonists with drug-like properties, we anticipate
this research tool will aid in revealing not only the contributions of S1P
to normal physiological tone, but also to aberrant pathological conditions. This research was supported by NIH grants R01 GM067958, T32
GM007055 and T32 GM008715.
Paper No.: 2361

THERAPEUTIC TARGETS
DRUGS WITH KNOWN PHARMACOLOGICAL PROFILES,
SUCH AS MONENSIN, DISULFIRAM AND SALINOMYCIN,
SHOW CANCER-SELECTIVE INHIBITION OF PROSTATE
CANCER CELL GROWTH BY INCREASING OXIDATIVE
STRESS
Kirsi Ketola(1), P Vainio(1), P Halonen(1,2), P Kohonen(1,2),
V Fey(1,2), RC Grafstrom(2,3), M Perala(1,2), O Kallioniemi(2,4),
K Iljin(1,2)
(1) University of Turku, Centre for Biotechnology, Turku, Finland
(2) VTT Technical Research Centre, Department of Medical Biotechnology, Turku, Finland
(3) Karolinska Institute, Institute of Environmental Medicine, Stockholm,
Sweden
(4) University of Helsinki, Institute for Molecular Medicine (FIMM),
Helsinki,Finland
To identify novel therapeutic opportunities for patients with prostate cancer, we carried out a high-throughput cell-based screening (HTS) of
4,910 most currently marketed drugs and drug-like molecules to systematically explore for their efcacy in four prostate cancer (VCaP, LNCaP,
DU 145, and PC-3) and two non-malignant prostate epithelial cell lines
(RWPE-1 and EP156T). The EC50 values were determined for each cell
type. Gene microarray studies, measurements of oxidative stress induction and cancer stem cell activity were used to explore the mechanism of
selected compounds. Monensin and disulram (DSF) were identied as
nanomolar inhibitors of VCaP and LNCaP growth. In addition, two compounds structurally similar to monensin, salinomycin and nigericin, were
included in studies and found to inhibit prostate cancer cell growth. Interestingly, all these compounds induced the expression of metal binding
and oxidative stress responsive genes. DSF, monensin and salinomycin
increased the level of oxidative stress and decreased the aldehyde dehydrogenase activity, suggesting deactivation of pathways linked to stem
cell processes. Our results indicate that several drugs with known pharmacological and toxicological prole showed unsuspected cancer-selective growth inhibitory potential in human prostate cancer cells. Analysis
of the molecular mechanisms of action for these drugs indicated
increased sensitization to oxidative stress as a common denominator.
Such mechanistic understanding could further help to design pre-clinical
and clinical studies.
Paper No. 3319

CYP2D6 GENETIC POLYMORPHISM HAS AN IMPACT ON
THE PHARMACOKINETICS OF CARVEDILOL IN KOREAN
SUBJECTS
Seul-Ki Keum, J-W Bae, C-G Jang, S-Y Lee
Carvedilol is a non-selective a- and b-adrenergic receptor blocking agent,
which is indicated for the treatment of hypertension, chronic heart failure, and left ventricular dysfunction following myocardial infarction.
CYP2D6 is major enzyme that mediates the metabolism of carvedilol via
4- and 5-hydroxylation, and a lesser extent, carvedilol is also metabolized by CYP2C9, 3A4, 2C19, 1A2, and 2E1. We investigated the effects
of CYP2D6 genetic polymorphism on the pharmacokinetics of carvedilol. After genotyping for CYP2D6 with volunteers, 34 healthy Korean
volunteers were selected and they were divided into four groups according to CYP2D6 genotype, group1 (CYP2D6*1/*1, n = 8), group2
(CYP2D6*1/*5 and CYP2D6*1/*10, n = 15) and group3 (CYP2D6*5/
*10 and CYP2D6*10/*10, n = 11). A single oral dose of 25 mg of carvedilol was administered to each subject and plasma concentration of
371
carvedilol was determined by using HPLC system with uorescence
detection. AUC0- of carvedilol in group3 was 270.2 110.4 ng/ mLhr,
that is 96% and 60% higher than that in group1 and group2 (P < 0.01).
Cmax of carvedilol in group3 was also 69% and 26% higher than that in
group1 and group2, but these differences were not statistically signicant. In conclusion, CYP2D6 genetic polymorphism is found to affect
the pharmacokinetics of carvedilol.
Paper No. 3320

GLICLAZIDE PHARMACOKINETICS IN RELATION TO
CYP2C19 GENOTYPE IN HEALTHY KOREANS
Seul-Ki Keum, C-I Choi, J-W Bae, C-G Jang, S-Y Lee
Gliclazide is a second-generation sulfonylurea hypoglycemic agent used
for the treatment of type II diabetes mellitus (non-insulin-dependent diabetes mellitus, NIDDM). Most of sulfonylurea compounds, including
gliclazide, are known to be metabolized mainly by CYP2C9, but recent
studies have presented that CYP2C19 is also involved in the hepatic
metabolism of gliclazide. We investigated the effects of CYP2C19
genetic polymorphism on the pharmacokinetics of gliclazide. After genotyping for CYP2C19 using PCR-RFLP method, 27 healthy Korean volunteers were selected. They were divided by three groups according to
CYP2C19 genotype, group1 (CYP2C19*1/*1, n = 9), group2
(CYP2C19*1/*2 and CYP2C19*1/*3, n = 11) and group3 (CYP2C19*2/
*2, CYP2C19*2/*3 and CYP2C19*3/*3, n = 7). A single oral dose of 80
mg gliclazide was administered to each subject and plasma concentration
of gliclazide was measured by using HPLC-UV system. AUC0- of gliclazide in group2 and group3 (95.4 31.9 lg/mLhr and 124.6 19.1 lg/
mLhr, respectively) was signicantly higher than that in group1 (58.0
15.3 lg/ mLhr). Oral clearance of gliclazide in group2 and group3 (0.9
0.2 L/hr and 0.7 0.1 L/hr, respectively) was signicantly lower than
that in group1 (1.5 0.4 L/hr). Elimination half-life of gliclazide was
also signicantly different between three genotype groups (11.4 1.9 hr,
15.1 3.7 hr and 18.8 2.5 hr, respectively). In conclusion, CYP2C19
genetic polymorphism clearly affects the pharmacokinetics of gliclazide.
Paper No.: 3321

DISCOVERY
INFLUENCE OF MRP2 C-24T POLYMORPHISM ON THE
DISPOSITION OF ORALLY ADMINISTERED EZETIMIBE IN
HEALTHY SUBJECTS
Seul-Ki Keum, C-I Choi, J-W Bae, M-J Kim, C-G Jang, S-Y Lee
Ezetimibe has a lipid-lowering property by modulating the intestinal
uptake of dietary and biliary cholesterol. It has been reported that ezetimibe is a substrate of MRP2 (ABCC2), a member of ATP-binding cassette
(ABC) drug efux transporters. MRP2 C-24T genetic polymorphism is
known to be associated with the decrease in the plasma concentration of
MRP2 substrates. In this study, we investigated the effects of MRP2 C24T genetic variant on the pharmacokinetics of ezetimibe and its major
metabolite, ezetimibe glucuronide. After genotyping for MRP2 using
PCR-RFLP method, 23 healthy Korean volunteers were selected. They
were grouped according to MRP2 C-24T genotype, MRP2 -24CC (CC, n
= 12), MRP2 -24CT (CT, n = 5) and MRP2 -24TT (TT, n = 6). A single
oral dose of 10 mg ezetimibe was administered to each subject, and
plasma concentrations of ezetimibe and ezetimibe glucuronide were
determined by using LC-MS/MS system. AUC0- of ezetimibe in each
MRP2 genotype group (CC, CT and TT group) was 68.9 15.3 nghr/
mL, 75.4 6.5 nghr/mL and 105.4 18.8 nghr/mL, respectively, and
these differences were statistically signicant (P = 0.0004). Oral clearance (CL/F) of ezetimibe in CC, CT and TT genotype group was also
signicantly different (151 29 L/hr, 134 12 L/hr and 97 16 L/hr,
respectively, P = 0.0008). Pharmacokinetic parameters of ezetimibe glucuronide were not signicantly different among three genotype groups.
MRP2 C-24T polymorphism is found to affect the plasma concentration
of ezetimibe.
Paper No.: 3369

PHARMACOKINTIC AND PHARMACODYNAMIC
ALTERATIONS OF OMEPRAZOLE IN RELATION TO
CYP2C19 GENOTYPE
Seul-Ki Keum, C-I Choi, J-W Bae, M-J Kim, C-G Jang, S-Y Lee
Omeprazole, one of proton pump inhibitors (PPIs), is used for the treatment of peptic ulcer, gastroesophageal reux disease (GERD), and is also
used in the therapy for the eradication of Helicobacter pylori with antimicrobials. The metabolism of omeprazole is mediated by several cytochrome P450 enzymes, primarily mediated by CYP2C19 enzyme and a
lesser extent, CYP3A4. CYP2C19 is known as one of highly polymorphic drug metabolizing enzymes, so we investigated the effect of
CYP2C19 genetic polymorphism on the pharmacokinetics and pharmacodymanics of omeprazole in healthy Koreans. After genotyping for
CYP2C19 with volunteers, 39 healthy Korean volunteers were selected
and were divided into three groups according to CYP2C19 genotype,
group1 (CYP2C19*1/*1, n = 16), group2 (CYP2C19*1/*2 and
CYP2C19*1/*3, n = 13), and group3 (CYP2C19*2/*2, CYP2C19*2/*3,
and CYP2C19*3/*3, n = 10). A single oral dose of 40 mg omeprazole
was administered to each subject and plasma concentration of omeprazole was measured by using HPLC-UV system. Pharmacodynamics of
omeprazole was evaluated by the measurement of intragastric pH. Cmax
and AUC0-of omeprazole in group3 was signicantly higher than that
in group1 and group2 (P < 0.0001, respectively), In addition, elimination
half-life and oral clearance of omeprazole were signicantly different
between three genotype groups (P < 0.0001, respectively). Pharmacodynamics of omeprazole was also affected by CYP2C19 genotype. The differences of mean pH, median pH and the fraction time pH >4 in each
genotype group were all statistically signicant (P < 0.0001, respectively). In conclusion, CYP2C19 genetic polymorphism affects pharmacodynamics as well as pharmacokinetics of omeprazole.
Paper No.: 910

HEALTH AND DISEASE
CESSATION OF THE METABOLIC DISTURBANCES
PROGRESSING AND ENDOTHELIAL DYSFUNCTION IN
PATIENTS WITH ISCHEMIC HEART DISEASE
Tamara Kezeli, N Gongadze, M Chipashvili, M Mirziashvili,
K Bakuridze
Tbilisi State Medical University, Department of Pharmacology, Tbilisi,
Georgia
Introduction. Formation of metabolic vicious circle plays a pivotal role
in the progression of ischemic heart disease (IHD) despite an optimal
therapy used. Therefore, we hypothesize that eliminated of this vicious
circle and improvement in endothelial function by cardioprotective drug
nadcin with antiischemic and antianginal properties, may lead to additional clinical benet in patients with stable IHD and refractory form of
372
angina pectoris. Materials/Patients. The population consisted of 87
patients with IHD and angina pectoris III-IV functional class by CCS,
hypertension, that required therapy with anti-hypertensive medication,
and 14 healthy controls. The content of pyridine nucleotides was measured by enzymatic methods, the content of ATP with luciferin-luciferase
test and endothelin-1 (ET-1) - using the R&D Systems of Human ET-1
Immunoassay (Great Britain). Results. The content of ATP in plasma and
erythrocytes, ratio of NAD/NADH, as the important mediators of energy
supply system, were signicantly improved after treatment with nadcin
and associated with the decreasing in the level of vasoconstriction
enzyme, ET-1, up to normal level. The direct correlation was found
between functional class of angina pectoris and NAD/NADH (r = 0,87, p <
0,001), the increasing of the content of ET-1 and decreasing the level of
redox potential NAD/NADH ([ET-1]= 5,1 [NAD]/[NADH] +6,1, r = -0,71,
p < 0,01) have been observed. Conclusion. Improved metabolic state of the
blood is coupled with the recovery of the endothelial function in patients
with IHD and leads to a benecial positive therapeutic action on the symptoms of IHD and angina pectoris under the treatment by nadcin.
Paper No.: 583

PHARMACOLOGICAL BASIS FOR THE MEDICINAL USE OF
ORIGANUM VULGARE LINN. IN UROLITHIASIS
A Khan, Anwar-ul-Hassan Gilani
Aga Khan University Medical College, Department of Biological &
Biomedical Sciences, Karachi, Pakistan
Urolithiasis is one of the oldest known diseases with constant rise in its
incidents and recurrence. Dawn the ages, medicinal plants have been
used as a source of relief and cure from various diseases including kidney stones. Therefore we investigated the crude extract of Origanum
Vulgare (Ov.Cr) for possible antiurolithic effect, to rationalize its traditional medicinal use, by using the in vitro experiments and in vivo rat
models. In the in-vitro experiments, Ov.Cr exhibited a dose-dependent
(0.25-4 mg/ml) inhibitory effect on the slope of nucleation and aggregation and also caused a decrease in number of calcium oxalate (CaOx)
monohydrate crystals in the CaOx metastable solutions. It also showed
dose-dependant antioxidant effect against DPPH free radical and lipid
peroxidation induce in rat kidney tissue homogenate. Ov.Cr relaxed high
K+ induced contraction in rabbit urinary bladder strips, showing calcium
channel blocking effect. In animal model of urolithiasis, developed in
male Wistar rats by giving 0.75% ethylene glycol in drinking water for 3
weeks along with NH4Cl for the rst 5 days, Ov.Cr treatment (10-30
mg/kg) prevented as well as reversed the toxic changes caused by stone
inducing treatment like loss of weights, high urinary volume due to renal
impairment, increased crystalluria, oxaluria, raised serum urea and creatinine levels and crystal deposition in kidneys in both preventive and curative groups compare to their respective controls. The data clearly indicate
the antiurolithic activity in Ov.Cr, possibly mediated through inhibition
of CaOx crystallization, antioxidant and antispasmodic activities which
rationalize its medicinal use in urolithiaisis.
(0.5, 1.0, 2.0 mg/kg) alone or in combination with alprazolam (0.25 mg/
kg), whilst the control group received only the vehicle for 21 days, followed by being forced to undergo swimming endurance tests, with measurements taken of various biochemical parameters including glutathione
(GSH) and thiobarbituric acid reactive substance (TBARS) in rats brain.
Results showed that propranolol and its combination with alprazolam
exhibited antianxiety effects by increasing the percentage preference,
number of entries and time spent in open arm in the EPM. Pretreatment
with propranolol (1 & 2 mg/kg) but not (0.5 mg/kg) signicantly
reversed the FST-induced increase in TBARS, dose dependently. When
combined, a signicant decrease in TBARS was observed even at propranolol doses 0.5 & 1 mg/kg. Alprazolam (0.25 mg/kg) combination
with propranolol (2 mg/kg) signicantly reversed FST-induced reduction
in GSH levels in rats. Thus, the combined treatment of alprazolam &
propranolol exhibited an enhanced effect on oxidative stress and showed
protective effect on brain by reducing TBARS level and increasing glutathione level. Taken together, these results suggest that propranolol and its
combination with alprazolam is able to improve the antianxiety effects
and reduced the oxidative stress in forced swimming rats.
Paper No.: 724

EVALUATION OF ANTIHYPERLIPIDEMIC &
CARDIOPROTECTIVE EFFECT OF METHANOLIC EXTRACT
OF LAGENARIA SICERARIA
Amitesh Khare, VV Khadke, AW Patil
Shri Bhausaheb Hire Government Medical College, Department of
Pharmacology, Greater Noida, Uttar Pradesh, India
Hyperlipidemia is a highly predictive risk factor for atherosclerosis, coronary artery disease, and cerebral vascular diseases. Lagenaria siceraria
commonly known as bottle gourd, which is ofcial in Ayurvedic Pharmacopoeia of India, and having composition of variety of essential phytoconstituents, so that the fruits are traditionally used for their
cardioprotective, cardiotonic, general tonic, diuretic, aphrodisiac, antidote
to certain poisons and scorpion strings, alternative purgative, and cooling
effects. In the present study methanolic extract of L. siceraria was used
in 18 healthy rabbits in which 12 were drug induced hyperlipidemic rats
using Triton-X-100 and 6 served as controls. Serological investigations
were performed. The study exhibited that elevated levels of blood cholesterol, triglycerides, LDL, were signicantly reduced and decreased HDL
was signicantly increased by administration of methanolic extract. Creactive proteins were also seen signicantly reduced in the test group
than in the group controls. In the present study direct relation was
observed in reduction of levels of triglycerides,LDL, Blood cholesterol,
C-reactive proteins owing to the study formulation given. Thus the overall effects can be considered as a good marker of the the antihyperlipidemic effects and reduction in associated morbidity with use of
Lagenaria siceraria. Clinical trial in humans is advocated to validate the
effect. (Wang J et al Curr Ther Res 1997;58:12., Vogel G et al SpringerVerly: Berloin; 1997. p. 604-8).
Paper No.: 3203

EFFECT OF PROPRANOLOL ALONE AND IN COMBINATION
WITH ALPRAZOLAM ON ELEVATED PLUS MAZE AND
FORCED SWIMMING INDUCED-STRESS IN RATS
Paper No.: 1401

STW 5, AN HERBAL PREPARATION, IS EFFECTIVE IN
EXPERIMENTAL INFLAMMATORY BOWEL DISEASE
Razia Khanam, F Ahmed, D Vohora, K Pillai
Mohammed T Khayya(1), H Abdel-Aziz(2), W Wadie(1), DM Abdallah(1), O Kelber(3), B Vinson(3), D Weiser(3)
Hamdard University, Department of Pharmacology, New Delhi, India

The present study aimed to examine the effects of propranolol alone and
in combination with alprazolam on elevated plus maze test and oxidative
stress in forced swimming rats. Rats were orally treated with propranolol
(1) Cairo University Faculty of Pharmacy, Department of Pharmacology,

Cairo, Egypt
(2) Heliopolis University, Faculty of Pharmacy, Department of Pharmacology, Cairo, Egypt
373
(3) Steigerwald Arzneimittelwerk GmbH, Scientic Department,
Darmstadt, Germany
Introduction: A multi-component herbal medicinal preparation, STW 5
(Iberogast), has established clinical efcacy in functional dyspepsia
and irritable bowel syndrome, conditions often triggered by gastrointestinal inammatory processes. The present study was performed on
an experimental model of inammatory bowel disease (IBD) to investigate its potential usefulness to treat colonic inammatory conditions.
Materials: Male rats were injected intra-colonically with trinitro benzene sulfonic acid (TNBS) in 50% ethanol under light anaesthesia,
leading to development of lesions within 4 days. STW 5 was given
orally either prophylactically before TNBS induction or curatively after
establishing IBD. Results: The drug dose dependently reduced the area
of lesions and colonic mass index. It prevented changes in myeloperoxidase and reduced glutathione levels in the colon and protected
against changes in ICAM-1, TNFa, IL-1b, IL-10, LTB4, and PGE2 in
blood and colonic tissue. The effect was comparable to that of sulfasalazine, used as reference drug. STW 5 was also effective in treating
established IBD, showing a positive correlation between therapeutic
efcacy and the effect on the measured mediators. The therapeutic
value of STW 5 was conrmed by colonic histopathology. Conclusion:
The ndings provide experimental evidence to support the potential
usefulness of STW 5 in inammatory conditions of the lower gastrointestinal tract.
Paper No.: 2254

FOCUSED CONFERENCE GROUP: P04 PHARMACOEPIDEMIOLOGY, CURRENT CONTROVERSIES
AND OPPORTUNITIES
EMERGENCE OF ARTEMISININ RESISTANT MALARIA IN
WESTERN CAMBODIA
Phisit Khemawoot(1), L Chanthap(1), Y Se(1), D Bethell(1),
D Saunders(1), S Sriwichai(1), P Teja-isavadharm(1), S Tyner(1),
D Walsh(1), H Noedl(2), D Socheat(3), M Fukuda(4)
(1) Armed Forces Research Institute of Medical Sciences, Bangkok,
Thailand
(2) Medical University of Vienna, Vienna, Austria
(3) National Center for Parasitology, Entomology and Malaria Control,
Phnom Penh,Cambodia
(4) Armed Forces Health Surveillance Center, Global Emerging Infections Surveillance Program, Maryland, USA
Artemisinin-based combination therapies (ACTs) are recommended as
rst-line treatment for falciparum malaria worldwide. ACTs were introduced as standard regimens along Thai-Cambodian border in the late
1990s, with subsequent resistance suspected in this region since the
mid-2000s. We compared clinical and laboratory data from two prospective drug efcacy trials using the same antimalarial regimen and
conducted at the same site in western Cambodia to determine the progression of artemisinin resistance. Sixty patients with uncomplicated
falciparum malaria were enrolled in 2006 and thirty eight patients were
enrolled in 2009; all received artesunate monotherapy 4 mg/kg/day for
7 days and were followed up for at least 28 days. Parasite clearance
times were longer and parasite reduction ratios reduced in 2009 compared to 2006. Signicantly more patients remained persistently parasitemic after 72 hours of therapy in 2009 than 2006 (56.8% versus
31.7% respectively, p = 0.015). The median 50% inhibitory concentration of dihydroartemisinin, against Plasmodium falciparum was signicantly increased from 1.29 ng/ml in 2006 to 2.86 ng/ml in 2009 (P <
0.001). However, we did not nd any signicant differences in mean
plasma dihydroartemisinin level or in clinical outcomes; PCR-adjusted
adequate clinical and parasitological responses occurred in more than
90% of patients in both studies at Day 28. Delayed parasite clearance
after artemisinin treatment in western Cambodia is a worrisome sign
that warrants continued monitoring.
Paper No.: 1808

HEALTH AND DISEASE
CHRONIC INTAKE OF RWPS IMPROVES EDHF-MEDIATED
RELAXATION IN THE MESENTERIC ARTERY OF OLD RATS:
ROLE OF ANGIOTENSIN II AND AT1 RECEPTORS
N Idriss Khodja, T Chataigneau, C Auger, Valerie B Schini-Kerth
University of Strasbourg, Faculty of Pharmacy, UMR CNRS 7213,
Illkirch, France
Aging is associated with blunted nitric oxide (NO)- and endotheliumderived hyperpolarizing factor (EDHF)-mediated endothelium-dependent
relaxations. This study determined whether red wine polyphenols
(RWPs), which stimulate the endothelial formation of NO and EDHF,
improve the endothelial function in old rats. Old male Wistar rats (46
weeks) received RWPs (100 mg/kg/day) in the drinking water. Old and
young (16 weeks) control rats received the solvent (ethanol, 3% v/v).
Reactivity of mesenteric artery rings was determined in organ chambers.
The expression level of angiotensin II and AT1 receptors was assessed by
immunohistochemistry. Acetylcholine-induced endothelium-dependent
relaxations were blunted in mesenteric artery rings of old rats compared
to young rats and this effect affected predominantly the EDHF component in comparison to the NO component. Blunted EDHF-mediated
responses were associated with an increased expression of angiotensin II
and AT1 receptors in the mesenteric artery of old rats. Intake of RWPs
for either 14, 21 or 28 days restored EDHF-mediated relaxations and
normalized the expression level of angiotensin II and AT1 receptors in
old rats. Moreover, the protective effect of a 14-day RWPs intake period
persisted after a 14-day washout period in old rats. Thus, chronic intake
of RWPs restores the blunted EDHF-mediated relaxation in the mesenteric artery of old rats and this effect is associated with the normalization
of the expression of angiotensin II and AT1 receptors. Since angiotensin
II has been shown to impair EDHF-mediated responses in the mesenteric
artery, an increased local renin-angiotensin system may contribute to the
altered endothelial function in aging.
Paper No.: 1285

NEPHROPROTECTIVE ACTIVITY OF THE LOW-ESTERIFIED
PECTINS
Maxim Khotimchenko(1), A Kropotov(1), Y Khotimchenko(2)
(1) Vladivostok State Medical University, Department of Pharmacology,
Vladivostok, Russian Federation
(2) Institute of Marine Biology, Laboratory of Pharmacology,
Vladivostok, Russian Federation
Pectins are the ionic plant polysaccharides exerting numerous pharmacological effects due to their pronounced binding activity regarding various
exogenous and endogenous substances. Presumably, pectin can reduce
uremic toxemia induced by renal function. Binding activity of pectins is
strictly dependent on their degree of esterication. In our studies the
effects of pectin with the degree of esterication about 1% on the renal
function were estimated. Under in vitro conditions low esteried pectin
exerts high binding activity regarding such uremic toxin as urea and signicantly lower activity regarding creatinine. In laboratory animals low
esteried pectin was shown to be effective for healing as well as for prevention of experimental renal failure. Administration of the pectin to the
rats with acute renal failure contributed to normalized volume of diuresis
and signicant reduction of blood levels of urea and creatinine. Administration of the low esteried pectin before ingestion of a nephrotoxic agent
prevented disturbances of renal function manifesting in less reduction of
diuresis and lower blood levels of urea and creatinine. In clinical observations supplementation of low esteried pectin into the complex therapy
of patients with chronic renal failure contributed to faster reduction of
374
clinical manifestations of uremia and decreased blood level of creatinine
and urea. Also supplementation of the low esteried pectin makes possible to prolong periods between hemodialysis procedures in patients with
severe renal failure. Therefore, it can be concluded that low esteried
pectin exerts nephroprotective activity in experiment presumably through
the binding of uremic toxins in intestine.
Paper No.: 1564

BASIC FIBROBLAST GROWTH FACTOR UPREGULATES
HYPOXIA INDUCIBLE FACTOR 1A AND GLUCOSE
TRANSPORTER 1 IN ADIPOSE CELLS
Sertraline 5mg/kg i.p. One hour before the experiment drugs were injected
intraperitoneally. Rats were examined at elevated plus maze at rst and
forced swiming test was used afterwards. The results were evaluated statistically by using Mann-Whitney U tests. Results: The immobility periods
were signicantly reduced by all doses of simvastatin. There was no signicant difference in simvastatin 10, 30 and 50 mg/kg doses. There was no signicant difference in spending time in open branches of elevated plus maze
in all simvastatin groups. Single dose of amitriptyline (10mg/kg) showed
signicant antidepressant and anxiolytic activities, but sertraline (5mg/kg)
did not. When amitriptyline and simvastatin were used together, its antidepressant and anxiolytic activities did not increase. Any antidepressant and
anxiolytic effects were not observed when sertraline and simvastatine were
used together. Discussion: In this study, it can be considered that simvastanin have a signicant but dose-independent antidepressant activity, while it
does not have an anxiolytic effect in rats. Amitriptyline did not change in
the antidepressant effects of simvastatine.
Yoshitaka Kihira(1), S Tomita(1), Y Ikeda(1), K Ishizawa(2),

K Tsuchiya(2), T Tamaki(1)
(1) The University of Tokushima Graduate School Institute of Health
Biosciences, Department of Pharmacology, Tokushima, Japan
(2) The University of Tokushima Graduate School Institute of Health
Biosciences, Department of Medical Pharmacology,, Tokushima, Japan
A previous study has shown that serum basic broblast growth factor
(bFGF) levels were reduced in obesity, indicating that bFGF is related to
the deterioration of the metabolic syndromes. However, the roles of bFGF
on adipose tissue are poorly understood. It has also been reported that
hypoxia occurs in adipose tissue in mice and patients and modulates the
progression of metabolic syndrome. To characterize how bFGF functions
in adipose tissue, we studied bFGF effects on the function of adipocytes
under hypoxic conditions by using cultured 3T3-L1 adipocytes. 10 day-differentiated 3T3-L1 adipocytes were treated with 20 ng/ml bFGF for 4 hours
in hypoxic chambers, and then cell lysates were prepared for Western blot
analysis. The result showed that the protein levels of glucose transporter 1
(GLUT1), a modulator of blood glucose concentration, were signicantly
increased in the bFGF-treated 3T3-L1 adipocytes to 1.74-fold compared
with the controls. Real time PCR analyses showed that this increase was
caused at the transcriptional level. Immunouorescent studies showed that
GLUT1 was localized in cell membranes of bFGF-treated cells, whereas no
uorescence in the membrane of control cells was observed. Furthermore,
bFGF was found to enhance protein levels of hypoxia inducible factor-1a
(HIF-1a), which is a transcriptional factor induced in hypoxic conditions
and known to be transcriptional regulator of GLUT1 gene. These results
suggest that bFGF induces GLUT1 in adipocytes possibly via HIF-1a transcriptional factor. The reduced levels of bFGF in obesity probably contribute to the impaired glucose tolerance in metabolic syndromes.
Paper No.: 2756

INVESTIGATION OF ANTIDEPRESSANT AND ANXIOLYTIC
EFFECTS OF SIMVASTATINE IN RATS
Fatma Sultan Kilic(1), Y Ozatyk(1), B Kaygisiz(1), C Baydemyr(2),
K Erol(1)
(1) Eskipehir Osmangazi University Medical School, Department of
Pharmacology, Eskipehir, Turkey
(2) Eskipehir Osmangazi University Medical School, Department of Biostatistics, Eskipehir, Turkey
Introduction: There are clinical studies about statins being antioxidant and
the use of depression treatment besides hypercholesterolemia. The aim of
this study is to evaluate antidepressant and anxiolytic effects of simvastatine, in rats. Material and Method: Forced swiming test was used for testing
antidepressant effect and elevated plus maze for anxiolytic effect. It was
used male rats weighing 200-250g. Groups (n:8) were: Control (Saline),
10, 30, 50mg/kg Simvastatine; Amitriptyline (10mg/kg); Sertraline 5mg/
kg; and Simvastatine (30 mg/kg) combined with Amitriptyline (10mg/kg),
Paper No.: 2779

THE ATIDEPRESSANT AND ANXIOLYTIC ACTIVITIES OF
GABAPENTINE
Fatma Sultan Kilic(1), S Ismailoglu(1), C Baydemir(2), K Erol(1)
(1) Eskipehir Osmangazi University School of Medicine, Department of
(2) Eskipehir Osmangazi University School of Medicine, Department of
Biostatistics, Eskipehir, Turkey
Introduction: Gabapentine is an analogue of caminobutyric acid and third
generation of antiepileptic drugs. Recently there are some clinical reports
about it could be used for treatment of depression and anxiety disorders. In
the present study, it was evaluated the anxiolytic and and antidepressant
activities of gabapentine. Material and Methods: Forced swiming test and
elevated plus maze were used for testing antidepressant effect and anxiolytic effect respectively. Female rats weighing 200-250g were used. Groups
(n:8) were: Control (Saline), Gabapentine (5, 10, 20, 40mg/kg); Amitriptyline (10mg/kg); Sertraline (5mg/kg); Benzodiazepine (5mg/kg), and
Gabapentine (40 mg/kg) combined with Amitriptyline, Sertraline, Benzodiazepine, i.p. One hour before the experiment drugs were injected intraperitoneally. Rats were examined at elevated plus maze at rst and forced
swiming test was used afterwards. The results are evaluated statistically by
using Mann-Whitney U test. Results: The immobility periods were signicantly reduced by high dose of gabapentine. All doses of gabapentine have
dose-independent anxiolytic activity. Single dose of amitriptyline and benzodiazepine showed signicant antidepressant and anxiolytic activities, but
sertraline did not. When amitriptyline and benzodiazepine were used with
gabapentine, its antidepressant and anxiolytic activities did not change.
Any antidepressant and anxiolytic effects were not observed when sertraline and gabapentin were used together. Conclusions: In this study, it can be
considered that gabapentine have a signicant but dose-independent anxiolytic activity, while it has antidepressant activity at high doses of gabapentine in rats. Amitriptyline and Benzodiazepine did not change the
antidepressant and anxiolitic effects of gabapentine.
Paper No.: 1298

THE PROTECTIVE EFFECTS BY THE EXTRACT FROM
PLATYCODON GRANDIFLORUM AGAINST MYOCARDIAL
ISCHEMIA IN RATS
Eun-Joo Kim(1), M-H Lee(1), K-S Kim(1), B-Y Byun(2)
(1) Korea Institute of Toxicology (KIT), Department of Pharmacology,
Daejeon, South Korea
(2) Daegu Haany University, Department of Oriental Medicine, Daegu,
South Korea
375
The purpose of the present study was to determine the cardioprotective
effects of the extract from Platycodon grandiorum. 8~10 week old S.D
male rats were anesthetized by intraperitoneal injection of Pentobarbital
Sodium(75mg/kg), intubated and mechanically ventilated while the body
temperature was maintained at 37.5 0.5C. Left thoracotomy was performed in order to accomplish the sequence of coronary artery occlusion
(45min), which was followed by 90min reperfusion. The test article was
orally administered 30 min before the coronary artery occlusion at doses
of 300 and 500mg/kg.
To measure the infarct size, at the end of reperfusion period, the heart
was excised after 2% Evans blue perfusion. The area at risk was identied using Evans blue staining, and the infarct area was identied by 1%
2,3,5-triphenylthetrazolium Chloride(TTC) staining. The infarcted areas
was identied as the TTC negative zone and expressed as a percentage
of the area at risk. To determine the lipid peroxidation level in the
plasma, the malondialdehyde (MDA) level was measured using the
MDA KIT (Northwest life science, USA). Oral administration of the test
article (0, 300, 500mg/kg) signicantly decreased the infarct size/ area at
risk (%): they were 53.4 1.98, 48.43 1.40, 36.36 2.70, respectively.
MDA content was 5.1 0.05, 3.9 0.05, 2.8 0.01lm/ml, respectively,
demonstrating a dose dependency. These results suggest that the extracts
from Platycodon grandorum have a great potential for the therapy of
myocardial ischemia diseases.
Paper No.: 1053

HEALTH AND DISEASE
3,4-DIHYDROXYFLAVONOL DECREASES VASCULAR
CONTRACTION THROUGH INHIBITION OF THE RHOA/
RHO-KINASE PATHWAY IN ENDOTHELIUM-DENUDED RAT
AORTA
InKyeom Kim
Kyungpook National University School of Medicine, Department of
Pharmacolgy, Daegu, South Korea
3,4-Dihydroxyavonol (DiOHF) is an effective vasodilator with
antioxidant activity. We hypothesized that DiOHF decreases vascular
contraction through inhibition of the RhoA/Rho-kinase pathway in endothelium-denuded rat aorta. Rat aortic rings were denuded of endothelium,
mounted in organ baths and treated with either DiOHF (10 or 30 uM) or
vehicle (DMSO) after vascular contraction induced by NaF, KCl, phenylephrine or U46619 reached plateaus. The phosphorylation levels of the
myosin light chain (MLC20), myosin phosphatase target subunit 1
(MYPT1) and protein kinase C (PKC)-potentiated inhibitory protein for
heterotrimeric myosin light chain phosphatase of 17-kDa (CPI17) were
determined by western blotting. We also measured the amount of GTPRhoA. DiOHF decreased vascular contraction induced by NaF, U46619,
phenylephrine or KCl. Blockers of K+ channel such as 4-aminopyridine,
charybdotoxin, apamin, or glibenclamide did not affect the vascular
relaxation induced by DiOHF. DiOHF decreased the activation of RhoA
and subsequent phosphorylation of MYPT1Thr855, CPI17Thr38, and
MLC20. In conclusion, DiOHF decreases vascular contraction through
inhibition of RhoA/Rho kinase pathway in endothelium-denuded rat
aorta. These results suggest that DiOHF is a good candidate for therapy
of cardiovascular diseases.
Korea Science and Engineering Foundation (R01-2008-000-12198-0),
Ministry of Education, Science and Technology, Republic of Korea, the
Brain Korea 21 Project in 2010, and the Australian Research Council.
Paper No.: 1054

HEALTH AND DISEASE
CALCIUM SENSITIZATION INDUCED BY SODIUM
FLUORIDE IN PERMEABILIZED RAT MESENTERIC
ARTERIES
InKyeom Kim
We hypothesized that NaF induces calcium sensitization in Ca2 + -controlled solution in permeabilized rat mesenteric arteries. Rat mesenteric
arteries were permeabilized with b-escin and subjected to tension measurement. NaF potentiated the concentration-response curves to Ca2 +
(decreased EC50 and increased Emax) in a dose-dependent manner.
Cumulative addition of NaF (4.0, 8.0 and 16 mM) also increased vascular tension in Ca2 + -controlled solution at pCa 7.0 or pCa 6.5, but not at
pCa 8.0. NaF-induced vasocontraction and GTPcS-induced vasocontraction were neither additive nor inhibited by GDPbS. NaF-induced vasocontraction at pCa 7.0 was inhibited by pretreatment with Rho kinase
inhibitors H1152 or Y27632 but not with a Ca2 + -ATPase inhibitor cyclopiazonic acid, a MLCK inhibitor ML-7 or a PKC inhibitor Ro318220. NaF induces calcium sensitization in a Ca2 + -dependent manner
in b-escin-permeabilized rat mesenteric arteries. These results suggest
that NaF is a selective activator of Rho kinase signalling pathway during
vascular contraction.
Paper No.: 1055

EFFECTS OF PHYTOALEXIN GLYCEOLLIN ISOLATED
FROM SOYBEAN, ON VASCULAR CONTRACTION IN RAT
AORTAS
InKyeom Kim
Glyceollins are the phytoalexin compounds produced in soy under
stressed conditions and synthesized from daidzein via pterocarpan. Present study was undertaken to investigate the molecular mechanisms by
which glyceollin I inhibits vascular contraction in rat aortas. Rat aortic
rings were mounted in organ baths and treated with either glyceollins or
vehicle (dimethyl sulfoxide) when vascular contraction reached plateaus.
The phosphorylation level of the myosin light chain (MLC20), myosin
phosphatase target subunit 1 (MYPT1) and protein kinase C (PKC)potentiated inhibitory protein for heterotrimeric myosin light chain phosphatase of 17-kDa (CPI17) were determined by western blotting. We also
measured the RhoA activity and Rho GTPase-activating protein (RhoGAP). Glyceollin I reduced vascular contraction whether endothelium is
present or denuded. Glyceollin I reduced vascular contraction induced by
NaF, KCl, U46619, or phenylephrine (PE). Blockers of K+ channels such
as 4-aminopyridine (4-Ap), charybdotoxin (CTX), tetraethylammonium
chloride (TEA), or glibenclamide (GB) did not affect the vasorelaxation
induced by glyceollin I. Glyceollin I reduced not only the activation of
RhoA but also subsequent phosphorylation of MYPT1Thr855, CPI17Thr38
and MLC20. However, glyceollin I had no effect on RhoGAP activation.
Glyceollin I reduced vascular contraction through inhibition of RhoA/
Rho-kinase signalling pathway. These results suggest that DiOHF is a
good candidate for therapy of cardiovascular diseases.
376
Paper No.: 1811
ARONIA JUICE, A POLYPHENOL-RICH BERRY JUICE,
INDUCES REDOX-SENSITIVE ACTIVATION OF
ENDOTHELIAL NITRIC OXIDE SYNTHASE AND
RELAXATION IN PORCINE CORONARY ARTERIES
orally). These animals exhibited a corresponding improvement in neurological function and a rduction of neuronal death, as determined histologically from the cortex and hippocampal regions. These results suggest
that anti-excitotoxic properties of IL may be responsible for its neuroprotective effects against focal cerebral ischemia and provide the pharmacological basis of IL as a promising agent for the treatment of
neurodegeneration in stroke.
J-H Kim, C Auger, T Chataigneau, Valerie B Schini-Kerth

University of Strasbourg, Faculty of Pharmacy, UMR CNRS 7213,
Illkirch Graffenstaden, France
Polyphenol-rich sources such as red wine strongly increase the formation
of endothelial nitric oxide (NO), a potent vasoprotective factor, via the
redox-sensitive activation of the Src/PI3-kinase/Akt pathway leading to
activation of endothelial NO synthase (eNOS) by phosphorylation at
Ser1177. The present study examined the potency of Aronia juice, a
non-alcoholic rich source of polyphenols, to induce redox-sensitive activation of eNOS and relaxation. Vascular reactivity was assessed using
porcine coronary artery rings in the presence of indomethacin and charybdotoxin plus apamin to prevent the formation of vasoactive prostanoids and inhibit endothelium-derived hyperpolarizing factor-mediated
responses, respectively. The phosphorylation level of Src Tyr418, Akt
Ser473 and eNOS Ser1177 was assessed in cultured coronary artery
endothelial cells by Western blot analysis. Aronia juice caused endothelium-dependent relaxations in coronary artery rings and induced phosphorylation of Src, Akt and eNOS in cultured endothelial cells. Both of
these responses were inhibited by membrane permeant analogues of
superoxide dismutase (SOD) and catalase, MnTMPyP and PEG-catalase,
respectively, but not by native SOD and catalase. Inhibitors of Src (PP2)
and PI3-kinase (wortmannin) also inhibited both responses. Aronia juice
induced the formation of reactive oxygen species as assessed by dihydroethidine staining, and of NO by electron paramagnetic resonance in
endothelial cells. Thus, the present ndings indicate that Aronia juice
is a potent inducer of endothelium-dependent relaxations of coronary
arteries via the redox-sensitive Src/PI3-kinase/Akt pathway-induced
phosphorylation of eNOS leading subsequently to the endothelial NO
formation.
Paper No.: 2743

HEALTH AND DISEASE
PROTECTIVE EFFECT OF ILEX LATIFOLIA ON
GLUTAMATE-INDUCED NEUROTOXICITY IN VITRO AND
FOCAL CEREBRAL ISCHEMIA IN VIVO
Joo Youn Kim, YH Seong
Ilex latifolia (IL, Aquifoliaceae), called as Ku-Ding-Cha has been consumed in southern china as a tea-like beverage and known to possess
many cardiovascular benets, such as antihypertension, increasing the
blood ow of coronary artery, protecting cardiac function from ischemic
injury. IL contains large amount of caffeoylquinic acid (CQA) which
have various pharmacological properties including antioxidant, anti bacterial and antiallergic effects. We performed in vitro and in vivo analyses
on the neuroprotective effect of an ethanolic extract of IL IL (10-100 ug/
ml) inhibited 500 uM glutamate-induced elevation of intracellular Ca2 +
concentration, generation of reactive oxygen species and neuronal cell
death in cultured rat cortical neurons. Since 3,5-diCQA, 3,5-diCQA
methyl ester and 3,4-diCQA isolated from IL also inhibited 500 uM glutamate-induced neuronal cell death, some of the neuroprotective effects
of IL might be attributable to these compounds. In rats, prevented cerebral ischemic injury induced by 2-h middle cerebral artery occlusion, followed by 24-h reperfusion. Ischemic infarct and edema volumes were
signicantly reduced in rats that received IL IL (100 and 200 mg/kg,
Paper No.: 2442

KNOWLEDGE, ATTITUDE AND PERCEPTION (KAP) STUDY
ON CLINICAL TRIAL OF PHARMACEUTICALS AND
INFORMED CONSENT IN KOREA
Kyung-Soo Kim(1), SH Lee, BI Choe, CM Kim
Catholic Medical Center, Clinical Research Coordinating Center, Soel,
South Korea
This study was performed in order to see KAP of Clinical Trial of Pharmaceuticals and Informed Consent in Korea. The number of subjects
was 310(137 patients and their family members, 109 administrative staffs
in the hospitals, and 64 personnels involved with clinical trials). Most of
them were well acquainted with clinical trial, and knew that it is the processes to identify the safety and efcacy. With regard to the cognition on
the informed consent, it was well understood about its basic concept and
procedures. But there were some differences of KAP of clinical trial and
informed consent depending on their position, disease state and experience state of participating in clinical trial, etc. Regarding the participation
in clinical trials, the persons with diseases showed more experience to
participate in it, by 5.8 times, rather than those without diseases, and the
persons with medication had 4.8 times more experience in participate in
clinical trials than those who do not take the drugs. In addition, the persons who have participated in clinical trials showed more willingness to
participate in clinical trials than those who have not participated in it. In
conclusion, the blind negative perception about clinical trials has been
changed to be relatively more favorable now compared to the past in
Korea. It is necessary to make efforts to give the right information about
clinical trials to the public.
Paper No.: 845

ASSOCIATION STUDY OF SINGLE NUCLEOTIDE POLYMORPHISMS IN THE COMT GENE AND KOREAN AUTISM SPECTRUM DISORDERS
Soon Ae Kim(1), HJ Yoo(2), IH Cho(3), M Park(4)
(1) Eulji University, Department of Pharmacology, Daejeon, South Korea
(2) Seoul National University Bundang Hospital, Department of Psychiatry, Seongam, South Korea
(3) Gachon Medical School, Department of Psychiatry, Incheon, South
Korea
(4) Eulji University, Department of Preventive Medicine, Daejeon, South
Korea
Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder with strong genetic components. The present study comprises the
detection of seven single nucleotide polymorphisms (SNPs) in COMT
followed by a family based association analysis of the SNPs in 151 Korean ASD trios by using the transmission disequilibrium test (TDT) and
haplotype analysis. We found preferential transmission of the G allele at
the rs6269 (P = 0.011) of COMT in ASD though haplotype analysis did
not revealed a signicant association (P = 0.023). These results suggest a
potential association between COMT and ASD in the Korean population.
377
Paper No.: 846
GENOME-WIDE ASSOCIATION SCAN OF KOREAN AUTISM
SPECTRUM DISORDERS WITH LANGUAGE DELAY
Soon Ae Kim, SY Yang
Eulji University, Department of Pharmacology, Daejeon, South Korea
Objective: Autism spectrum disorders (ASDs) are complex neurodevelopmental disease characterized by severe abnormality in language, social
interaction, and behavior. The genetic architecture of ASDs is complex
and the objective of this study is to explore possible association related
SNP marker of autism spectrum disorder in Korean population by SNPbased genome scan. Methods: Subjects consist of 42 male patients with
autism (Age 96.0-32.8 months, range 49~157 months), all of whom has
signicant language delay, i.e., lack of meaningful single words until 48
months, and their biological parents. Subjects with ASD were diagnosed
by 2 board-certied child psychiatrists using the Korean version of
ADOS and ADI-R. A SNP-based genome scan was done using the Affymetrix 5.0 SNP array. We evaluated call rate and minor allele frequency for quality control. And SNPs with minor allele frequency <0.1
and call rate <0.95 were discarded. We evaluated with total 326903
SNPs which are sufcient for quality control criteria. Transmission disequilibrium test was done using the Haploview program. Results: We
found total 16818 number ASD association signicant SNP markers
which p-values in TDT analysis are less than 0.05 in this study. However, after FDR, p-value of these all SNP is more than 0.05.
Paper No.: 865

HEALTH AND DISEASE
FUNCTIONAL ANALYSIS OF SINGLE NUCLEOTIDE
POLYMORPHSIMS IN NOS2A
Soon Ae Kim, SY Yang
(1) Sungkyunkwan University School of Medicine, Samsung Medical

Center, Departmentof Laboratory Medicine and Genetics, Seoul, Korea
(2) Samsung Biomedical Research Institute, Samsung Medical Center,
Seoul, South Korea
(3) Korea Advanced Institute of Science and Technology, Department of
Bio and Brain Engineering, Daejeon, South Korea
(4) Samsung Medical Center, Clinical Trial Center, Division of ClinicalPharmacology, Seoul, South Korea
(5) Sungkyunkwan University School of Medicine, Samsung Medical
Center, Department of Medicine, Seoul, South Korea
(6) Department of Neurology, Samsung Medical Center, Sungkyunkwan
University School of Medicine, Seoul, South Korea
(7) Department of Pharmacology, College of Medicine, Dankook University, Chonan, South Korea
Atorvastatin is a potent 3-hydroxy-3-ethylglutarylcoenzyme A reductase
inhibitor for the treatment of hyperlipidemia. However, there is wide variation in interindividual response to statin therapy, and it has been hypothesized that genetic differences may contribute to this variation. We
investigated the pharmacokinetic and pharmacodynamic characteristics of
atorvastatin administration in Korean subjects in relation to multiple
genetic markers. 51 healthy Korean volunteers were given single oral dose
(80 mg) of atorvastatin. Blood samples were collected up to 48 hrs (0, 0.25,
0.5,0.75,1,1.5,2,3,4,6,8,10,12,24,36,48 hrs) to measure plasma drug levels
and lipid proles (TC, TG, HDL, LDL). Subjects were genotyped for 1936
variants in 225 genes involved in drug metabolism, excretion, and transport, using DMET plus array (Affymetrix, CA, USA). Pharmacokinetic
(PK) parameters were determined by WINNONLIN (Pharsignt Co., CA,
USA). PK properties were as follows; mean AUC 171.5 ng eqhr/ml
(range, 17.8 - 508.8), Cmax 36.91 ng eqhr/ml (6.5 - 106.3), Tmax 1.53 hrs
(0.5 - 12.0), and T1/2 7.01 hr (2.6 - 10.9), showing signicant interindividual variability. Focusing the pharmacokinetic effect on the polymorphism of
ABCB1 and SLCO1B1 transporters, subjects with ABCB1 c.2677G>A
had larger AUC (P = 0.009). In subjects with SLCO1B1 N130D and
V174A haplotype, Cmax were higher (P = 0.018). Changes of the cholesterol levels were affected by CBR1, COMT, ADH1A, NAT2 and ABCC2
genetic polymorphisms. GSTA3, EPHX1, CHST13 and SLC28A1 genetic
polymorphisms were associated to TG change (P < 0.01). The result suggested some potential association between the generic polymorphisms
genes and the pharmacokinetics and lipid lowering effect of atorvastatin.
Eulji University, Department of Pharmacology, Daejeon, South Korea

Nitric oxide (NO) is a messenger molecule with diverse functions
throughout the body. NO is generated from L-arginine by the enzyme
nitric oxide synthase (NOS). Three isoforms of NOS have been described:
neuronal NOS (NOS1; nNOS), inducible NOS (NOS2A; iNOS), and
endothelial NOS (NOS3; eNOS). NOS2A produces much more NO than
NOS1 and NOS3 under the regulation of several cytokines such as interferon c, tummor necrosis factor, and interleukin 1 b related to activating
macrophage. The NOS2A gene is transcriptionally regulated through various signals and so the Polymorphisms within 5 region in NOS2A gene
may inuence gene expression. SNPs, SNP haplotypes, and microsatelites
in 5 region of NOS2A have been associated with human disease in many
reports. And functional effects have also been dened in vitro for some
SNP alleles and microsatellites. We have reported that SNPs in the promoter region of NOS2A was associated with Korean autism trios. We
constructed vector with the promoter region of NOS2A which includes
two single nucleotide polymorphisms (rs2779249, rs2779248) for luciferase assay and we studied in 293T cell line.
Paper No.: 2689

COMPREHENSIVE GENOTYPING OF DRUG METABOLIZING
ENZYMES AND TRANSPORTERS FOR THE ASSESSMENT OF
ATORVASTATIN
Suk-Ran Kim(1), H-H Won(2,3), J-W Kim(1), J-W Ko(4), W Huh(4,5),
O-Y Bang(6), S-C Lee(5), H-G Kim(7), S-Y Lee(1,4)
Paper No.: 1114

AN ACYCLIC RETINOID, NIK-333, ACCELERATES LIVER
REGENERATION AND LOWERS SERUM TRANSAMINASE
ACTIVITIES IN 70% PARTIALLY HEPATECTOMIZED RATS
IN VIVO
Mitsutoshi Kimura(1), N Ishibashi(2), S Yanagida(2), M Ogihara(1)
(1) Josai University Faculty of Pharmaceutical Sciences, Department of
Clinical Pharmacology, Sakado City, Japan
(2) Tokyo New Drug Research Laboratories, Pharmaceutical Division,
Kowa Co. Ltd., Tokyo, Japan
Effects of an acyclic retinoid (NIK-333), a synthetic analogue of retinoids, on restoration of liver mass and the recovery of liver function
were compared with those of natural retinoids in 70% partially hepatectomized rats in vivo. The NIK-333 (0.4 mg/kg/day, p.o.)- and all-trans-retinoic acid (ATRA: 4 mg/kg/day, p.o.)-treated rats, respectively, showed
an approx. 1.3- and 1.3-fold increase in the ratio of liver weight (LW) to
body weight (BW) over the solvent-administered control rats on day 2
and 3 after 70% partial hepatectomy. Accordingly 5-bromo-2hf-deoxyuridine (BrdU)-labeling index in the regenerating liver was signicantly
higher in NIK-333- or ATRA-treated rats compared with the control rats
on day 0.5 and 1. Retinol (40 mg/kg/day, p.o.) did not signicantly
increase both the ratio of LW/BW and BrdU labeling index compared
with control rats. The liver-related serum transaminase activities such as
alanine aminotransferase and aspartate aminotransferase were elevated
rapidly on day 1 and decreased to near pre-operative level on day 5 after
378
70% partial hepatectomy in the solvent-administered control rats. Both
NIK-333 (0.4 mg/kg/day, p.o.) and ATRA (4 mg/kg/day, p.o.) signicantly lowered the serum transaminases on day 2 and 3 after 70% partial
hepatectomy compared with the control rats. The transaminase-lowering
effects of NIK-333 were more effective than those of ATRA. Retinol (40
mg/kg/day, p.o.) did not signicantly decrease the serum transaminases
compared with the control rats. These results demonstrate that among the
three retinoids NIK-333 gave the most potent effects in promoting regeneration of liver mass and function with recovery from the liver injury.
Paper No.: 2161

INDIVIDUAL DIFFERENCES OF CYP2C19 MRNA
EXPRESSION IN THE SMALL INTESTINE OF JAPANESE
SUBJECTS
Yuichi Kinoshita, M Hayashi, N Matsuomoto, Y Tkeba, T Kumai,
S Kobayashi
St. Marianna University, Kawasaki City, Miyamae, Japan
CYP2C19 contributes to the metabolism of proton pump inhibitors as a
drug metabolizing enzyme in the liver. CYP2C19 in the liver shows individual differences. Some CYPs are also expressed in the small intestine,
and it may play some role in rst pass metabolism of a large number of
drugs in the small intestine. Particularly, CYP3A4 in small intestine is
forming a intestinal absorption barrier against xenobiotics. However, the
data of CYP2C19 mRNAexpression pattern in the small intestine of Japanese population is still unclear. In this study, we investigated CYP2C19
mRNA expression in the small ntestine of Japanese. Sample of small
intestine was obtained from 18 patients underwent pancreatoduodenectomy. The study protocol has been approved by the ethics committee of St.
Marianna University School of Medicine, and written informed consent
was obtained from each patient. CYP2C19 mRNA was measured by
quantitative real-time PCR methods. CYP2C19 mRNA was expressed in
small intestine from every patient. There was individual difference of
CYP2C19 mRNA in the small intestine of Japanese subjects. Genetic
polymorphism of CYP2C19 in these subjects were (*1/*1 n = 4, *1/*2 n
= 8, *1/*3 n = 4, *2/*3 n = 2). However, CYP2C19 mRNA expression
pattern was not correlated to these genetic polymorphisms.
Paper No.: 1392

DISCOVERY
INTERACTIONS OF ATAZANAVIR (ATV) WITH SOLUTE
CARRIER (SLC) TRANSPORTERS IN CACO-2 CELLS, AN
IN VITRO MODEL OF HUMAN INTESTINAL EPITHELIUM
O Kis(1), J Zastre(2), M Ramaswamy(1), Kevin Robillard(1),
R Bendayan(1)
(1) University of Toronto, Leslie Dan Faculty of Pharmacy, Department
ofPharmaceutical Sciences, Toronto, Ontario, Canada
(2) University of Georgia, College of Pharmacy, Department of
Pharmaceutical andBiomedical Sciences, Atlanta, GA, USA
Atazanavir, an HIV protease inhibitor (PI) used in rst-line highly active
antiretroviral therapy (HAART) regimens, has been implicated in several
drug-drug interactions at the level of the intestinal mucosa. At present,
limited information is available on the mechanism of intestinal absorption of ATV and other PIs. In this study, we examined the potential role
of SLC transporters, such as organic anion transporting polypeptides
(OATPs), in the intestinal uptake of ATV and antiretroviral drug-drug
interactions. Transporter mRNA and protein expression in Caco-2 cells
was evaluated by RT-PCR and immunoblotting, respectively. Cellular
accumulation of [3H]atazanavir in Caco-2 cells was measured in the
absence (control) or presence of standard inhibitors of SLC transporters
or antiretroviral drugs. The effect of extracellular pH (5.5, 7.4, 8.5) and

intracellular acidication with NH4Cl preincubation on ATV uptake was
also evaluated. Caco-2 cells demonstrated robust OATP2B1 expression
and functional activity. ATV uptake by Caco-2 cells was pH dependent
(uptake at pH 5.5 > pH 7.4 = pH 8.5) and susceptible to inhibition by
established OATP family inhibitors, estrone-3-sulfate, rifamycin SV,
MK571, and pravastatin. Although, HIV nucleoside analogs, tenofovir
DF, abacavir, lamivudine, and emtricitabine did not inhibit ATV uptake
by Caco-2 cells at clinical concentrations, PIs such as ritonavir, amprenavir, and tipranavir demonstrated potent concentration-dependent inhibition of ATV uptake by Caco-2 cells at concentrations below 10lM.
These data provide further understanding of PIs intestinal absorption and
potential mechanisms of antiretroviral drug-drug interactions at this site.
Funding support: Canadian Foundation for AIDS Research and Ontario
HIV Treatment Network studentship.
Paper No.: 1176
DYSFUNCTION OF AUTONOMIC NERVOUS SYSTEM
ACTIVITY IN SCHIZOPHRENIA AND THE INFLUENCE OF
NEUROLEPTICS
Ikuko Kishida(1), M Fujibayashi(3), S Tanaka(3), C Kawanishi(2),
Y Iwamoto(1), T Furuno(2), C Ishii(1), N Ishii(1), Y Hirayasu(2),
T Moritani(3)
(1) Fujisawa Hospital, Department of Psychiatry, Fujisawa, Japan
(2) Yokohama City University School of Medicine, Department of
Psychiatry, Yokohama, Japan
(3) Kyoto University, Graduate School of Human and Environmental
Studies, Kyoto, Japan
Objective: Schizophrenic patients have been reported to have an increased
prevalence of cardiovascular diseases, and antipsychotic medication is
one of the causes of these diseases. On the other hand, we reported
reduced autonomic nervous system (ANS) activity in schizophrenia,
which was considered a possible risk factor for cardiovascular diseases.
In the present study, we sought to replicate the reported positive association between low ANS activity and schizophrenia in a larger number of
patients, and we investigated the inuence of neuroleptics on ANS activity. Methods: Subjects were 189 Japanese patients with schizophrenia and
32 healthy controls. All subjects received an explanation of our study and
written informed consent was obtained. ANS activity was assessed by
means of heart-rate variability power spectral analysis, which enables us
to identify separate frequency components, i.e., total power (TP: overall
ANS), low-frequency (LF: sympatho-vagal) power, and high-frequency
(HF: vagal) power, during a resting condition. Statistical analyses were
performed using t-tests to determine the presence of differences in the
ANS activity. Results: We found signicantly lower TP (p = 0.028) in
schizophrenic patients than controls. Multiple regression analyses for
ANS, sex, age, and neuroleptics dose calculated in terms of chlorpromazine equivalents as independent variables revealed signicant correlations
between ANS activity and neuroleptics dose (TP, p = 0.001; LF, p =
0.014; HF, p = 0.002). We observed a trend toward associations of low
ANS activities in patients treated with olanzapine, although no signicant
difference was found. Conclusion: Our ndings suggest that neuroleptics
dose inuence on reduced ANS activity in schizophrenic patients.
Paper No.: 1085
HEALTH AND DISEASE
EXPRESSION OF SOLUBLE VEGF RECEPTOR-1 IN HUMAN
MONOCYTE-DERIVED MATURE DENDRITIC CELLS
Masatoshi Kishuku(1), Y Nishioka(2), K Teraoka(1), K Kawazoe(1), H
Hochi(3), S Sone(2), K Minakuchi(1)
(1) Tokushima University Hospital, Department of Pharmacy,
Tokushima, Japan
379
(2) Tokushima University Hospital, Department of Respiratory Medicine
and Rheumatology, Tokushima, Japan
(3) Kagawa University Hospital, Kagawa, Japan
The soluble form of vascular endothelial growth factor receptor-1
(sVEGFR-1) is produced from endothelial cells by alternative splicing of
VEGFR-1 mRNA, and can inhibit angiogenesis by blocking the biological effects of VEGF. we examined the expression, bioactivity and inhibition effect to tumor growth of sVEGFR-1 in human monocyte-derived
mature dendritic cells (mDCs). As compared to monocytes and immature
DCs (imDCs), mDCs generated by TNF-a or soluble CD40L (sCD40L)
with IFN-c, but not LPS or other stimuli, preferentially produce
sVEGFR-1. The production of sVEGFR-1 showed a distinct contrast to
those of VEGF in each DC matured with various stimuli. The supernatant of DCs matured with TNF-a or sCD40L with IFN-c showed inhibition of the tube formation of HUVECs, which was neutralized by antiVEGFR-1 Ab, indicating that sVEGFR-1 secreted from mDCs was biologically active. Interestingly, the supernatant of mDCs generated with
LPS increased HUVEC capillary-like formation in vitro. The ratio of
sVEGFR-1 to VEGF clearly reected the net angiogenic property of
mDCs. Administration of mDCs induced by TNF-a into the subcutaneous tumor of PC-14 cells implanted in SCID mice demonstrated the inhibition of tumor growth via reduction of the number of CD31-positive
vessels, indicating their in vivo anti-angiogenic potential. These results
suggest that sVEGFR-1 produced by mDCs contribute to their anti-angiogenic property, and the ratio of sVEGFR-1 to VEGF might be a useful
tool for evaluating their ability to regulate angiogenesis mediated by
VEGF.
Paper No.: 2995

EFFECTS OF CARIPRAZINE ON D2 AND D3 DOPAMINE
RECEPTOR SIGNALLING MECHANISMS IN VITRO
B Kiss(1), S Peterson(2), Zsolt Nemethy(1), K Fazekas(1), B Masri(2),
N Adham(3), M Caron(2)
(1) Gedeon Richter, PLc, Department of Pharmacological Research,
Budapest, Hugary
(2) Duke University Medical Center, Department of Cell Biology and
Medicine, Duke Durham, NC, USA
(3) Forest Research Institute, Jersey City, NJ, USA
Dopamine D2 and D3 receptors are major targets of currently used antipsychotics. These receptors mediate their effects through both a Gi/odependent pathway and a G protein-independent pathway via b-arrestin
and Akt/GSK-3. Cariprazine was evaluated for its effects on both G proteindependent and -independent pathways. G protein-dependent signalling: cariprazine displayed D2 and D3 silent antagonist activity in
[35S]GTPcS binding assays. In hD2L-expressing cells with or without
coexpression of Gqo5, cariprazine displayed partial agonist activity for
stimulating inositol phosphate (IP) production (Emax ~20%) and forskolin-stimulated cAMP inhibition (Emax ~60%), respectively. Similarly, in
hD3-expressing cells measuring forskolin-stimulated cAMP inhibition,
cariprazine behaved as a partial agonist (Emax ~70%). Consistent with
its partial agonist property, cariprazine antagonized agonist-stimulated
effects in the above systems. G protein-independent signalling: in the D2/
b-arrestin assay, cariprazine displayed weak agonist activity (Emax ~10%)
but behaved as a potent antagonist. In all the above systems, the intrinsic
efcacies of cariprazine and aripiprazole were comparable. However, cariprazine displayed approximately 5-fold greater potency and 3- to 7-fold
higher selectivity for the D3 versus D2 receptor as compared to aripiprazole. This prole may be of central importance in the clinical action of cariprazine.
Paper No.: 568

INFLUENCE OF THE SUPPRESSION OF CELL
PROLIFERATION AND NEUROGENESIS IN THE ABILITY OF
ANTIDEPRESSANTS IN AN ACTH-INDUCED ANIMAL MODEL
OF TREATMENT-RESISTANT
Yoshihisa Kitamura, M Doi, H Hayashi, I Miyazaki, M Asanuma,
H Kawasaki
Okayama University, Department of Pharmaceutical Care and Health
Sciences, Okayama, Japan
We previously reported that the antidepressant-like effects of tricyclic antidepressants in the forced swim test are blocked by chronic treatment
with adrenocorticotropic hormone (ACTH) in rats. ACTH-treated rats
serve as a valuable animal model for tricyclic antidepressant-resistant
depressive conditions. However, effect of ACTH on neurogenesis and
glial cells in the brain is obscure. The present study was undertaken to
investigate changes in neurogenesis and glial cells, particularly astrocytes
in the hippocampus of ACTH-treated rats. Chronic treatment of ACTH
decreases the number of BrdU- and Ki-67-labeled cells in the dentate
gyrus. This result suggests that chronic ACTH treatment causes hippocampal morphological changes, including suppression of neurogenesis.
Furthermore, the present study also demonstrated that chronic ACTH
treatment decreased in the expression of GFAP, the major intermediate
lament protein in astroglia. This nding suggested that the changes of
astroglia are likely to have an important functional signicance for the
etiology of depression and treatment-resistant depression. We design further experiments to clarify the effect of antidepressants on BrdU- and Ki67- staining and GFAP-positive activated astrocytes in the chronic treatment of ACTH in the hippocampus.
Paper No.: 914

TETRODOTOXIN INDUCES MOUSE COLONIC
CONTRACTION IN VITRO THROUGH BLOCKADE OF
ENTERIC INHIBITORY INNERVATION
Takio Kitazawa, Y Okuno
Rakuno Gakuen University School of Veterinary Medicine, Department
of Pharmacology, Ebetsu, Hokkaido, Japan
Tetrodotoxin (TTX) is a useful pharmacological tool for distinguishing
neural and muscle responses of drugs in visceral smooth muscle organs.
Although TTX generally does not affect muscle tonus, we observed that
TTX caused contraction of the mouse colon. The aim of the present
study was to characterize the TTX-induced contraction in mouse gastrointestinal strips. Longitudinal muscle strips from the stomach, small
intestine and caecum showed almost no mechanical responses to TTX.
However, TTX caused contraction of colonic strips in a concentrationdependent manner (1 nM-1lM). Other Na+ channel blockers (QX-222,
lidocaine) also caused contraction of colonic strips. The ranking order of
contraction is consistent with the Na+ channel blocking activity. Pretreatment with TTX, L-NAME or ODQ signicantly decreased the TTXinduced contraction. Both L-NAME and ODQ caused contraction of
colonic strips but not gastric and small intestinal strips. Region-dependency of L-NAME and ODQ-induced contraction correlated with that of
TTX-induced contraction. Sodium nitroprusside caused strong relaxation
of colonic strips compared with muscle strips from other regions. DMPP
(a ganglionic stimulant) caused an atropine-sensitive transient contraction
in gastric and small intestinal strips but relaxed colonic strips even in the
presence of physostigmine, suggesting that neural inhibitory regulation is
dominant in the colon. In conclusion, TTX induces contraction of mouse
colonic strips through blockade of strong and tonic inhibition by nitrergic
nerves.
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Paper No.: 2725
DISCOVERY
TRANSPORT OF CYCLIC NUCLEOTIDES IN HUMAN
COLONIC EPITHELIUM
Karen Kleberg(1), GM Jensen(1), JG Meyer(1), S Knuhtsen(2),
MB Hansen(2), N Bindslev(1)
(1) University of Copenhagen, Panum Institute, Department of
Biomedical Sciences, Copenhagen, Denmark
(2) University of Copenhagen, Bispebjerg Hospital, Department of
Surgery K, Copenhagen, Denmark
The colonic epithelium is important for the excretion of harmful xenobiotics and metabolites. A specialized system exists where basolateral
OATPs and luminal ABC transporters interplay in a both promiscuous
and specic excretion of xenobiotics. Dysfunctions of this system might
be related to development of disease, e.g. cancer. This study represents a
functional characterization of the serosal inward transport of cyclic nucleotides in human colonic epithelium; likely mediated by OATPs. Macroscopically normal appearing colonic epithelial biopsies were obtained
during endoscopy and mounted in micro-Ussing chambers (Osbak PS et
al, BMC Gastroenterol 2007;7:37), perfused with an oxygenated bicarbonate-Ringer solution. The biopsies were treated with amiloride, theophylline and indomethacin in order to prevent the endogenous
production and degradation of cAMP. The individual transports of basolaterally added nucleotides; cAMP, cGMP, and the more lipophilic dibutyryl(db)-cAMP, and dibutyryl(db)-cGMP were recorded as induced
chloride secretion causing an increase in short circuit current (SCC), N =
6. cAMP, cGMP and db-cGMP produced SCC increments of similar size
(10 2, 23 15, and 8 1 lA-cm-2, respectively). cGMP signals were
signicantly larger than db-cGMP signals but not cAMP-signals. DbcAMP produced signicantly larger signals (66 2 lA-cm-2) than the
other three. Halftimes of the generated signals were 66 2, 129 5, 292
25 and 100 6 s for cAMP, cGMP, db-cAMP, and db-cGMP, and were
mutually signicantly different. Values of signal size and halftimes represent meanSEM. In conclusion, the pattern of induced SCC by the 4 nucleotides indicates that the inux of the more lipophilic nucleotides, dbcAMP and db-cGMP is also transporter mediated.
Paper No.: 1952

DISEASE AND THERAPY
ADMINISTRATION OF CLARITHROMYCIN AND FUROSEMID
ALTERS CARDIAC ION CHANNEL GENE EXPRESSION AND
INDUCES B-ADRENERGICALLY EVOKED TORSADES DE
POINTES IN RAT
Jan Klimas, J Kmecova, K Mackovicova, P Vavrinec, P Krenek,
J Kyselovic, P Ochodnicky
rERG, KvLQT, CACNA1c and SCN5a genes in left ventricles. Results.

The duration of QTc was increased as follows: CON, FUR, CLA, CLA+FUR (89 1 ms, 95 2 ms, 107 3 ms, 118 4 ms; p < 0.05). The
occurrence of polymorphic ventricular arrhythmias and TdP has been
registered during b-adrenergic stimulation in 1/10 of rats in CLA+FUR
group. We observed decreased expression of KvLQT and CACNA1c in
CLA (by 37% and 56%, resp., p < 0.05) and FUR (by 28% and 49%,
resp., p < 0.05) groups but paradoxically not in CLA+FUR. Moreover,
the expression of rERG and SCN5a remained unchanged in all of the
groups. Conclusion. The co-administration of claritromycin and furosemide strongly increases the duration of QT and can induce TdP in the
rat. We suppose that this might be linked to alterations in ion channel
genes expression.
Supported by grants VEGA 1/0377/09, 1/0707/09 and 1/0357/09.
Paper No.: 1072

PK/PD MODEL FOR PEGYLATED, LONG-ACTING HUMAN
GROWTH HORMONE IN ADULTS WITH GROWTH
HORMONE DEFICIENCY
Thomas Klitgaard(1), J Madsen(2), BS Hansen(1), MH Rasmussen(3)
(1) Novo Nordisk A/S, Department of Clinical Pharmacology,
Copenhagen, Denmark
(2) Novo Nordisk A/S, Department of Biostatistics, Copenhagen,
Denmark
(3) Novo Nordisk A/S, Department of Medical and Science,
Development Projects, Copenhagen, Denmark
Introduction: Recombinant human Growth Hormone (rhGH) is normally
administered as daily subcutaneous (s.c.) injections. NNC126-0083 is a
pegylated rhGH developed to prolong the exposure of the product in the
blood circulation, allowing for a once weekly treatment. The objective of
this analysis was to develop a population PK/PD model for NNC 1260083 and its effects on insulin-like growth factor-I (IGF-I) in adults with
growth hormone deciency (AGHD) and to use the model in the design
of a long-term trial. Materials/Patients: The model was developed using
the software NONMEM and was based on data from 30 AGHD patients
(age 26-63 years; weight 54-103 kg). Subjects were dosed in four cohorts
of 7-8, once weekly for three weeks, with s.c. injections of NNC1260083 (n = 5-6) or placebo (n = 2). Doses escalated between cohorts (levels: 10, 20, 40, and 80 ug protein/kg). Individual PK/PD proles were
sampled for 168 and 240 hours after the rst and the third dose, respectively. Results: In the PK model, drug is absorbed into a central compartment by two serial rst-order processes. Elimination takes place by a
rst-order and a saturable pathway. For IGF-I, an indirect response model
was used. Model validation showed good predictive performance. Simulation showed approach to target IGF-I levels during the titration period
in a planned long-term trial. Conclusion: A population PK/PD model for
NNC 126-0083 in AGHD patients was developed and used to guide the
design of a planned long-term trial.
Comenius University Faculty of Pharmacy, Department of Pharmacology

Toxicology, Bratislava, Slovak Republic
Introduction. The drug-induced QT prolongation may trigger life-threatening arrhythmias, such as Torsades de Pointes (TdP), predominantly
under b-adrenergic stimulation. We studied the inuence of two stimuli
prolonging QT - claritromycin (blocker of IKr current) and furosemide
(inducing hypokalemia). We hypothetized that altered expression of
genes encoding K+ channels (rERG and KvLQT), L-typ Ca2 + channel
(CACNA1c) and/or Na+ channel (SCN5a) might be involved. Methods.
Wistar rats were treated for 7 days with claritromycin (CLA, 200mg/kg/
d, p.o.), furosemide (FUR, 200mg/kg/d, p.o.) or both (CLA+FUR). Controls (CON) received vehicle. ECG was monitored under basal conditions and under cumulative b-adrenergic stimulation (isoproterenol i.v.,
doses of 5-60 ng/min). Using RT-PCR, we examined the expressions of
Paper No.: 2734

PHARMACOLOGICAL RESTING STATE-FMRI STUDY USING
THC
Linda E. Klumpers(1), RP Soeter(2,3,4), N Khalili-Mahani(2,3,4), SARB
Rombouts(2,3,4), MA van Buchem(2,3), JMA van Gerven(1,5)
(1) Centre for Human Drug Research, Leiden, The Netherlands
(2) Leiden University Medical Center, Department of Radiology, Leiden,
The Netherlands
381
(3) Leiden Institute for Brain and Cognition, Leiden, The Netherlands
(4) Leiden University, Institute of Psychology, Leiden, The Netherlands
(5) Leiden University Medical Center, Department of Neurology, Leiden,
The Netherlands
Resting state (RS)-FMRI is a novel neuroimaging technique that allows
repeated task-independent assessments of functional connectivity. We
studied the suitability of RS-FMRI for pharmacological research, by
investigating the effects of the cannabinoid receptor type 1 (CB1)-receptor agonist ?9-tetrahydrocannabinol (THC) on functional brain connectivity. Nine male volunteers inhaled 2, 6 and 6 mg THC or placebo with
90-minute intervals in a randomised, double blind, cross-over trial. Eight
RS-FMRI scans of 8 minutes were obtained per occasion. Subjects rated
subjective feeling high on a visual analogue scale after each scan, as a
pharmacodynamic effect measure. Drug-induced effects on functional
connectivity were examined using double regression (Beckmann OHBM
2009) with FSL software (FMRIB Analysis Group, Oxford)). Eight maps
of voxelwise connectivity throughout the entire brain were provided per
RS-FMRI series with eight predened resting-state networks of interest.
These maps were used in a mixed effects model group analysis to determine brain regions with a statistically signicant drug-by-time interaction, and regions associated with the subjective effect scores (p < 0.05,
cluster corrected). THC administration decreased connectivity in different
brain regions, including cerebellum, cuneus and different cortical regions.
The subjective effect scores correlated with effects in the brainstem, cerebellum, medial frontal gyrus and parietal lobe. This study shows that RSFMRI is able to demonstrate THC-related decreases in functional brain
connectivity, mainly in brain regions with high densities of CB1-receptors. Some changes were related to (subjective) pharmacodynamic THC
effect. We conclude that RS-FMRI is a promising technique to study
pharmacologically induced changes in brain activity.
Paper No.: 1746

NEUROPROTECTIVE EFFECT OF MILDRONATE,
A MITOCHONDRIA-TARGETED DRUG, IN PARKINSONS
DISEASE MODEL IN RATS
Vija Klusa(1), J Pupure(1), S Isajevs(1), D Svirina(1), U Beitnere(1),
B Jansone(1), J Rumaks(1), S Svirskis(1), R Vilskersts(2), I Kalvinsh(2)
(1) University of Latvia, Department of Pharmacology, Riga, Latvia
Recently the novel strategy to protect pathological events in the nigrostriatal system of Parkinsons disease (PD) patients by mitochondriatargeted drugs has been put forward (DiMauro and Mancuso, Biosci Rep
2007; 27:125-37). The aim of this study was to evaluate the effects of
mildronate [3-(2,2,2-trimethylhydrazinium) propionate dihydrate], a representative of aza-butyrobetaine class, in PD model, since previously we
have revealed its mitochondria-regulating effects (Pupure et al, Cell Biochem Func 2008; 26:620-31). PD was modeled by unilateral intrastriatal
injection of 6-OHDA (20 mkg) in rats pretreated by mildronate (50 mg/
kg i.p., two weeks). Brain slices (10 microns) of the striatum (ST) and
s.nigra (SN) were examined immunohistochemically to assess the expression of tyrosine hydroxylase (TH), iNOS, caspase-3, glial brillary acidic
protein (GFAP). Mildronate completely protected cells against 6-OHDAinduced death in the lesioned ST by increasing the number of TH-positive cells to control level; decreasing iNOS overexpression in the SN,
and caspase-3 and GFAP overexpression in the ST. The obtained data
demonstrate mildronates neuroprotective/neuroregenerative effects by
reducing inammatory, degenerative and reactive astrogliosis processes,
probably through its mitochondria-targeted action. The clinical usefulness
of mildronate in the treatment of neurodegenerative diseases is suggested.
Acknowledgements:
LOREAL/Latvian
National
Commission
UNESCO/Latvian Academy of Sciences fellowship grant For Women
in Science-2008; ESF Nr.2009/0217/1DP/1.1.1.2.0/09/APIA/VIAA/031.
Paper No.: 1075

INHIBITORS OF CA2 + -ATPASE THAPSIGARGIN AND
TRILOBOLIDE POSSESS IMMUNOSTIMULATORY
PROPERTIES
Eva Kmonckova(1), J Harmatha(2), K Vokac(2), Z Zdek(1)
(1) Institute of Experimental Medicine, Acad. Sci., v.v.i., Department
of Pharmacology, Prague, Czech Republic
(2) Institute of Organic Chemistry and Biochemistry, Acad. Sci, v.v.i.,
Prague,Czech Republic
Thapsigargin (TG) and trilobolide (TB) are sesquiterpene lactones isolated from the plants Thapsia garganica and Laser trilobum, respectively. The both of them are recognized inhibitors of mammalian
sarco-endoplasmic reticulum Ca2 + -ATPase (SERCA). TG has been
found to exhibit prospective immunotherapeutic properties. It kills slowly
proliferating and non-proliferating cells, and inhibits replication of
viruses. The aim of our work was to investigate possible immunostimulatory potential of these compounds. Their effects were analyzed in cultures of rat and mouse resident peritoneal cells (PEC), and human
peripheral blood mononuclear cells (PBMC). Supernatant levels of interferon-c (IFN-c) were determined by ELISA after 2-24 h of culture. Production of nitric oxide (NO) by animal PEC was assayed using the
Griess reagent after 24 h of culture. The as low concentrations of TG
and TB as 40 nM and 1 lM were highly effective to activate secretion
of IFN-c in rat PEC and PBMC, respectively. The rat PEC also produced
high amounts of NO upon stimulation with TG and TB. The effects were
only marginally expressed in mouse PEC. The immunostimulatory activity is mediated by transcription factor NF-jB and depends on the activation of MAP kinases p38 and ERK1/2. Activation of IFN-c remained
unchanged in the presence of calcium chelating agents BAPTA/AM and
TMB-8. The immunostimulatory mode of action is thus unlikely to result
from the intracellular calcium enhancing effects of TG and TB.
Acknowledgements. The work was supported by the grant GACR 305/
07/0061.
Paper No.: 2978

DISCOVERY
TISSUE EXPRESSION AND FUNCTION OF ORGANIC ANION
TRANSPORTING POLYPEPTIDE 2B1 SPLICE VARIANTS
Michael Knauer, R Kim, R Tirona
University of Western Ontario, Department of Physiology and
The human Organic Anion Transporting Polypeptide 2B1 (OATP2B1) is
a membrane transporter that facilitates the cellular uptake of a number of
endogenous compounds and drugs. OATP2B1 is expressed in several tissues including the small intestine, liver, kidney and skeletal muscle, and
is considered to play a role in the pharmacokinetics of substrate drugs.
Recently, it has been shown that differential promoter usage in tissues
results in the expression of several OATP2B1 splice variants which utilize 5 distinct rst exons but share common subsequent exons. These
splice variations are expected to encode either a full length or truncated
protein missing 22 amino acids from the N-terminus. Little is known
regarding the relative expression of OATP2B1 splice variants in key tissues responsible for drug absorption and elimination, as well as the transport function of the truncated variant. Using variant-specic polymerase
chain reaction, both the predicted full length and shortened forms of
OATP2B1 were detected in liver, kidney and small intestine, albeit in differing proportions. With heterologous expression in cultured cells, we
compared the transport kinetics of the two forms of OATP2B1. Importantly, we demonstrate that the truncated variant was capable of transporting the known OATP2B1 substrates, estrone sulfate and rosuvastatin.
382
These ndings indicate that differential regulation of OATP2B1 splice
variant expression in tissues could contribute to variation in drug
response.
Paper No.: 2873

THE GABAB DIMERS OF HETERODIMERS: A WAY TO
INCREASE THE FUNCTIONAL COMPLEXITY?
Julie Kniazeff(1), L Comps-Agrar(0), D Maurel(0), E Trinquet(2),
J-P Pin(1)
(1) University of Montpellier, CNRS, ISERM, Institute of Functional
Genomics, Montpellier, France
(2) Cisbio Bioassays, Marcoule, France
The metabotropic receptor for c-minobutyrate (GABA), the GABAB
receptor, belongs to class C G-protein coupled receptors (GPCR), which
also includes the glutamate, calcium sensing or sweet and umami taste
receptors. Like most class C GPCRs, the GABAB receptor is dimeric,
but has the originality to be an obligatory heterodimer: it is constituted
of the GABAB1 subunit that bears the agonists binding site, and the GABAB2 subunit responsible for G-protein activation. In a recent study, we
showed that, unlike metabotropic glutamate receptors, the GABAB
receptor was actually expressed at the cell surface as dimers of heterodimers, with GABAB1 as the central element of the oligo-heterodimers
formation. We could also demonstrate the existence of these assemblies
in native tissues. However, the question of GPCRs functional units as
dimers or higher ordered oligomers remains a subject of intense debate.
Here, we propose to assess what would be the functional rational of the
GABAB dimer of heterodimers. To that aim, we used two approaches:
the dissociation of the dimers of heterodimeric GABAB receptors or the
use of one non-functional subunit per tetramer (unable to activate G-proteins, or unable to bind agonists). We noticed that, upon disturbance of
the tetrameric assembly, a higher G-protein coupling efcacy was
detected. Our data indicate that either only one heterodimeric GABAB
receptor was functional within a dimer of GABAB heterodimers or the
coupling efcacy of both heterodimers is somehow decreased in the
assembly.
Paper No.: 2916

EFFECT OF TEMPERATURE AND GLUCOSE
CONCENTRATION ON ANOXIC DOPAMINE EFFLUX IN THE
MOUSE CAUDATE
Steve Knight, N Chauhan, A Young, C Gibson, C Davidson
University of Leicester, School of Psychology, Leicester, UK
Hyperglycaemia and hypothermia have both been shown to be neuroprotective. Here we examined the effect of varying glucose concentration
and temperature on hypoxia evoked dopamine (DA) release in the mouse
caudate. Adult male mice (C57Bl/6 ~30 g) were killed by cervical dislocation and their brains rapidly removed. Brain slices (400 lm) were kept
in oxygenated articial cerebrospinal uid (aCSF) at room temperature
(22C). In a proportion of slices, the aCSF contained either 4mM glucose
or 10mM. After at least 45 min equilibration, slices were transferred to a
brain slice chamber and perfused with oxygenated aCSF at a range of
temperatures (30 36 C). After a further 45 min equilibration, they
were then perfused with ischaemic aCSF comprising hypoxia (N2/
CO2) and variable glucose (0 or 2 mM) for 15 minutes. Ischaemiainduced DA efux was measured using fast cyclic voltammetry at carbon
bre electrodes. We measured a) time from start of hypoxia to start of
DA release; b) time from start of DA release to peak DA release; c) peak

DA release and d) initial rate of DA release (dDA/dt). Caudate DA content was measured by HPLC. Data were analyzed by regression analysis.
We found a signicant correlation between varying temperature and the
time to onset of DA release (r = -0.723; p < 0.0001), but ischaemic aCSF
glucose concentration had no effect (t(19) = 0.06; p > 0.05). This data
suggests that during ischemia, temperature affects caudate dopamine
release but varying glucose has no effect.
Paper No.: 3408

ACUTE ALLOPREGNANOLONE ADMINISTRATION MAY
EXACERBATE, RATHER THAN ATTENUATE, STRIATAL
DAMAGE DURING ISCHAEMIA IN VITRO
Steve Knight(1), C Davidson(2), A Young(1), C Gibson(1)
(1) University of Leicester, School of Psychology, Leicester, UK
(2) St Georges University of London, UK
Few effective clinical treatments for cerebral stroke, a signicant cause
of morbidity and mortality, exist. Sex steroid hormones such as progesterone have been shown in vivo to produce signicant neuroprotection,
but its mechanism(s) remain unknown. Allopregnanolone (AlloP), an
active progesterone metabolite, may explain some of progesterones
actions. Using an in vitro model, we evaluated AlloPs action at the
GABAA receptor during ischaemia hypothesised as its main neuroprotective mechanism. Dopamine efux during ischaemia is neurotoxic.
Using adult male mouse (C57Bl/6~30 g) caudate brain slices, we
recorded ischaemia-induced DA efux using fast cyclic voltammetry at
carbon bre microelectrodes. We measured a) time from start of hypoxia
to start of DA release (Ton); b) time from start of DA release to peak DA
release (Tpeak); c) peak DA release (DAmax) and d) initial rate of DA
release (dDA/dt). Data were analysed with either paired t-test or one-way
ANOVA. As AlloP is proposed to be a GABAA agonist we initially evaluated the effect of the GABAA agonist Muscimol (25 lm) in our model
and found it to produce a neuroprotective prole, signicantly affecting
three release parameters - TPeak, DAmax and dDA/dt (p < 0.05, 11 d.f.).
In contrast, AlloP (100 nM), signicantly increased DA efux (p < 0.05,
F = 4.009) versus control. This result was neither predicted from reported
neuroprotective effects of AlloP in vivo, or the effect of a GABAA agonist in our model of ischaemia, and may suggest that AlloP actually
exacerbates striatal damage during the acute phase of ischaemia.
Paper No.: 1568

PHYSIOLOGICAL SIGNIFICANCE OF EXTRACELLULAR ATP
IN HACAT KERATINOCYTES
Daisaku Kobayashi(1), S Ohkubo(2), N Nakahata(1)
(1) Tohoku University Graduate School of Pharmaceutical Sciences,
Department of Cellular Signalling, Sendai, Japan
(2) National Institute of Health Sciences, Division of Pharmacology,
Japan
Skin, the biggest organ in the body, has a role in keeping our body from
drying, heats, invasion of various bacteria, or other physical stimuli.
Recently, it has been shown that physical stimuli result in ATP release in
various kinds of cells including keratinocytes. Although ATP is known
as an energy donor in the cells, it also exerts as an extracellular signalling molecule for cell-to-cell communication. Released ATP binds to
P2X or P2Y receptor on cell membranes. We have shown that shear
stress induces ATP release and ATP/UTP causes interleukin-6 (IL-6) pro-
383
duction via mRNA expression through P2Y2/P2Y4 receptors in human
HaCaT keratinocytes. In the present study, we examined the mechanism
of UTP-induced IL-6 production in HaCaT cells. UTP caused phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) in a concentration- and time-dependent manner. PD98059, a MEK inhibitor, and
BAPTA-AM, an intracellular Ca2 + chelator, reduced ERK1/2 phosphorylation and IL-6 synthesis. AG1478, an epidermal growth factor (EGF)
receptor kinase inhibitor, partially decreased UTP-induced ERK1/2 phosphorylation and IL-6 mRNA expression. These results suggests that
UTP-induced IL-6 production is in part mediated via phosphorylation of
ERK1/2 through Gq/11/IP3/[Ca2 + ]i and transactivation of EGF receptor.
On the other hand, FK506 and cyclosporine A, calcineurin inhibitors,
partially inhibited UTP-induced IL-6 production. In addition, the combined application of FK506 or cyclosporine A and PD98059 synergistically inhibited the UTP-induced IL-6 production. These results suggest
that ERK1/2 and calcineurin are cooperatively involved in UTP-induced
IL-6 production.
(Kobayashi, D., Ohkubo, S., Nakahata, N., Eur J Pharmacol 2007; 573:
249-252)
Paper No.: 3191
CYP2D6 MRNA EXPRESSION AND GENETIC
POLYMORPHISM IN THE SMALL INTESTINE OF JAPANESE
Shinichi Kobayashi, S Takenoshita-Nakaya, Y Kinoshita, N Matsumoto,
Y Takeba, T Kumai
St. Marianna University School of Medicine, Department of
Pharmacology, Kawasaki Kanagawa, Japan
Cytochrome P450 (CYP) mediates biotransformation of therapeutic
drugs in the liver. CYP2D6 contributes to the metabolism of dextrometorphan, antidepressant and antipsychotic drugs. Recently, it was reported
that CYPs are also expresse in the small intestine, and it may play a role
of rst-pass metabolism of a large number of drugs through the small
intestine. However, the data of CYP2D6 mRNA expression pattern in
the small intestine in Japanese population is still unclear. In this study,
we investigated CYP2D6 mRNA expression and genetic polymorphism
in the small intestine of Japanese. The tissue of small intestine was
obtained from patients underwent pancreatoduodenectomy by surgical
operation. The study protocol has been approved by the ethics committee
of St. Marianna University School of Medicine, and written informed
consent was obtained from each patient. CYP2D6 mRNA was measured
using quantitative real-time PCR methods. CYP2D6 mRNA was
expressed in the small intestine in each patient. There was individual difference of CYP2D6 mRNA in the small intestine of Japanese patient.
Genetic polymorphism of CYP2D6 in these subjects is (*1/*1n=7, *1/
*10 n = 10, *10/*10 n = 12, *5/*10 n = 1). CYP2D6 mRNA expression
of *10 carriers were signicantly higher than that of *1/*1. Such information is important for further research and clinical practice.
Paper No. 2158

CYP2D6 MRNA EXPRESSION AND GENETIC
POLYMORPHISM IN THE SMALL INTESTINE OF JAPANESE
Shinichi Kobayasi, S Takenosihta-Nakaya, Y Kinoshita, N Matsumoto,
Y Takeba, T Kumai
St. Marianna University School of Medicine, Department of
Pharmacology, Kawasaki City, Kanagawa, Japan
Cytochrome P450 (CYP) mediates biotransformation of therapeutic
drugs in the liver. CYP2D6 contributes to the metabolism of dextrometorphan, antidepressant and antipsychotic drugs. Recently, it was reported
that CYPs are also expresse in the small intestine, and it may play a role
of rst-pass metabolism of a large number of drugs through the small

intestine. However, the data of CYP2D6 mRNA expression pattern in
the small intestine in Japanese population is still unclear. In this study,
we investigated CYP2D6 mRNA expression and genetic polymorphism
in the small intestine of Japanese. The tissue of small intestine was
obtained from patients underwent pancreatoduodenectomy by surgical
operation. The study protocol has been approved by the ethics committee
of St. Marianna University School of Medicine, and written informed
consent was obtained from each patient. CYP2D6 mRNA was measured
using quantitative real-time PCR methods. CYP2D6 mRNA was
expressed in the small intestine in each patient. There was individual difference of CYP2D6 mRNA in the small intestine of Japanese patient.
Genetic polymorphism of CYP2D6 in these subjects is (*1/*1 n = 7, *1/
*10 n = 10, *10/*10 n = 12, *5/*10 n = 1). CYP2D6 mRNA expression
of *10 carriers were signicantly higher than that of *1/*1. Such information is important for further research and clinical practice.
Paper No.: 2094

ENHANCED BRET TECHNOLOGY FOR THE MONITORING
OF AGONIST-INDUCED AND AGONIST-INDEPENDENT
INTERACTIONS BETWEEN GPCRS AND B-ARRESTINS
Martina Kocan(1,2), HB See(2), NG Sampaio(2), BJ Feldman(3),
KA Eidne(2), KDG Peger(2)
(1) Monash Institute of Pharmaceutical Sciences and Department of
Pharmacology, MonashUniversity, Melbourne, VIC, Australia
(2) Western Australian Institute for Medical Research (WAIMR) and
Centre for Medical Research, University of Western Australia,
Nedlands,WA, Australia
(3) Stanford University, Department of Pediatrics, Pediatric
Endocrinology, San Francisco, CA, USA
The bioluminescence resonance energy transfer (BRET) technique has
becomeextremely valuable for the real-time monitoring of protein-protein
interactionsin live cells. This method is highly amenable to the detection
of Gprotein-coupled receptor (GPCR)-protein interactions, especially involvingscaffolding, regulatory and signalling proteins such as b-arrestins.
The BRETmethod utilises heterologous co-expression of fusion proteins
linking oneprotein of interest (GPCR) to a bioluminescent donor enzyme,
a variant ofRenilla luciferase, and a second protein of interest (b-arrestin)
to anacceptor uorophore. If in close proximity, energy resulting from
the rapidoxidation of coelenterazine substrate by the donor will transfer
to theacceptor, which in turn uoresces. With the aim of monitoring transient, weakor hardly detectable agonist-induced and constitutive GPCRprotein interactions,we utilised a number of improvements in the BRET
technology. Using novel formsof luciferase (Rluc2, Rluc8) with
advanced uorophores (Venus, GFP10) we wereable to detect interactions not detectable using less sensitive BRETcombinations in the same
conguration. For example, we were able to showreceptor/b-arrestin
interactions in an agonist-independent manner usingRluc2- or Rluc8tagged mutant receptors, in contrast to when using Rluc. Insummary, the
enhanced BRET methodology has not only enabled live cell drugscreening as we have recently published, it now provides a new level of
sensitivity for monitoring protein-protein complexes previously beyond
detectionlimits, including constitutive GPCR/b-arrestin interactions.
Paper No.: 2095

LIGAND-DIRECTED SIGNALLING AT THE RELAXIN FAMILY
PEPTIDE RECEPTOR 3 (RXFP3)
Martina Kocan(1), ET van der Westhuizen(1), MA Hossain(2), JD
Wade(2), RJ Summers(1)
384
(1) Monash Institute of Pharmaceutical Sciences and Department of
Pharmacology, Monash University, Melbourne, VIC, Australia
(2) University of Melbourne, Howard Florey Institute, Melbourne, VIC,
Australia
Relaxin Family Peptide Receptor 3 (RXFP3) is a G protein-coupled
receptor expressed in brain areas important for processing sensory
information and in feeding suggesting that it may be a target for antianxiety and anti-obesity agents. RXFP3 and its cognate agonist relaxin3 have important central roles, particularly in the modulation of
behavioural responses to stress and appetite regulation. Selective agonists and antagonists are potentially important tools to examine the physiological roles of the RXFP3 system and as novel therapeutic agents.
We recently discovered that multiple ligands interact with RXFP3 to
stabilise receptor conformations coupled to different signalling pathways. On treatment with relaxin-3, RXFP3 inhibits forskolin-stimulated
cAMP accumulation (Liu C et al, J Biol Chem 2003; 278: 5075450764) and stimulates ERK1/2 phosphorylation (van der Westhuizen
ET et al, Mol Pharmacol 2007; 71: 1618-1629). In addition, we showed
activation of the activator protein (AP)-1 and nuclear factor of jB cells
(NF-jB) reporter genes following stimulation of RXFP3 with relaxin-3
but surprisingly AP-1 was also stimulated by relaxin. The Gai/o inhibitor pertussis toxin blocked all reporter gene activation except for
relaxin-3-stimulated activation of AP-1. The novel peptide antagonist
R3(BD23-27)R/I5 blocked relaxin-3-stimulated AP-1 reporter activation
but had no inhibitory effect on relaxin-stimulated AP-1 activation or
relaxin-3-stimulated NF-jB activation. Additionally, R3(BD23-27)R/I5
inhibited relaxin-3-stimulated ERK1/2 phosphorylation and activated
the SRE reporter gene. These ndings indicate ligand-directed signalling at RXFP3 that is of crucial importance for the potential discovery
and development of novel anti-anxiety and anti-obesity agents acting
on this receptor.
Paper No.: 2377

HEALTH AND DISEASE
ESTROGEN-INDUCED RELAXATION OF RAT TAIL ARTERY
WAS ATTENUATED IN RATS WITH PULMONARY
HYPERTENSION
Ivan Kocic, R Szczepazka, I Pawlowska
Medical University of Gdansk, Department of Pharmacology, Gdansk,
Poland
Background: The mechanisms involved in estrogen effects on arterial
smooth muscle contractility are very complex and not fully claried.
Therefore, the aim of this paper was to examine the mechanisms of estrogen-induced relaxation of rat tail artery and how pulmonary hypertension
affects this action. Material and methods: We used male rats and performed experiments on isolated tail arteries in control group and group
with pulmonary hypertension (PAH) induced by monocrotaline (60 mg/
kg B.W. i.p.). Results: The pD2 value (-log EC50) of phenylephrine was
signicantly decreased in the presence of 20 mM of 17-b-estradiol (5.4
0.13 vs. 4.9 0,12, P < 0.05, N = 6).Estrogen-induced relaxation of
phenylephrine-precontracted tail artery has two components: endothelium-dependent (ED) and endothelium-independent (EI). Moreover,
estrogen effect was independent on ATP-sensitive K+ channels, vasoactive prostanoids and nitric oxide. PAH augmented maximal effect of
phenylephrine on the tail artery contractility, but did not affect estrogeninduced ED-relaxation. However, EI component of relaxation induced by
estrogen was completely abolished in tail arteries obtained from animals
with pulmonary hypertension. Conclusion: Pulmonary hypertension
affects the sensitivity of rat tail artery to phenylephrine and estrogen
leading to impairment of endothelium-independent mechanisms of relaxation.
Paper No.: 2376

ANTIPLATELET ACTION OF STATINS IN DIABETIC
RABBITS: INTERACTION WITH LEPTIN
Paper No.: 1346

FOCUSED CONFERENCE GROUP: PW18 - SYNAPTIC EVENTS
IN BRAIN FUNCTION
EFFECTS OF ACUTE AND CHRONIC TREATMENT WITH
ATTENTION-DEFICIT/HYPERACTIVITY DISORDER DRUGS
ON MONOAMINERGIC TRANSMISSION IN MICE
Ivan Kocic, B Racek-Krol, I Rusiecka, I Pawlowska, R Wasilewski,

M Bitel
Ken Koda(1), Y Ago(1), Y Cong(1), K Takuma(1), T Matsuda(1,2)
Medical University of Gdansk, Department of Pharmacology, Gdansk,

Poland
Background: The aim of the present study was to nd out whether
statins have direct antiplatelet action and to compare that in control
and diabetic rabbits.Methods: We measured platelet aggregation in the
blood samples obtained from the marginal ear rabbit veins. Aggregation
was induced by 10 mM of ADP. We used an aggregometer type
M-490 according to the turbidimetry method of Born. After obtaining
control results, the tests were performed in the presence of 4 different
statins (cerivastatin, mevastatin, lovastatin and pravastatin) at concentrations range from 0.01 to 1 mM. The tests were repeated in diabetic
rabbits (alloxon, 120mg/kg. B.W. i.v.) and in the presence of leptin
(125 ng/ml). Statistical evaluation was done by unpaired Mann-Whitney test. Results: All examined statins inhibited platelet aggregation
induced by ADP by 17% (pravastatin at 1mM) to 25% (mevastatin at
1 mM). Diabetes increased platelet aggregation rate from 48 5% to
72 8%, however statins (especially mevastatin and lovastatin at 1
mM) inhibited that aggregation by about 60%. Addition of leptin
(125ng/ml) to the blood samples obtained from healthy rabbits
increased aggregation rate to about 64%, but in the presence of statins
it was decreased to about 26%.Conclusion: Alloxon-induced diabetes
increases platelet aggregation rate in rabbits. Statins have direct antiplatelet action which is enhanced in diabetic rabbits probably due to
interaction with leptin.
(1) Osaka University Graduate School of Pharmaceutical Sciences, Laboratory of Medicinal Pharmacology, Suita, Osaka, Japan
(2) Osaka University Graduate School of Pharmaceutical Sciences, Center The Osaka-Hamamatsu Joint Research Center for Child Mental
Development, Department of Experimental Disease Model, Suita, Osaka,
Japan
Atomoxetine and methylphenidate are widely used in the treatment of
attention-decit/hyperactivity disorder (ADHD). However, it is not
known about the long-term effects of exposure to the drug on extracellular concentrations of monoamine neurotransmitters in the brain. The
present study investigated, using an in vivo microdialysis technique, the
effects of acute and chronic treatment with these ADHD drugs on the
extracellular levels of noradrenaline (NA), dopamine (DA) and serotonin
(5-HT) in the prefrontal cortex (PFC) and striatum (STR) of adolescent
mice. Acute treatment with atomoxetine caused robust increases in extracellular NA and DA, but not 5-HT, levels in the PFC. Unlike the PFC,
these monoamine levels in the STR were not affected. Acute treatment
with methylphenidate also increased NA and DA levels in the PFC, but
not in the STR, and it did not affect 5-HT levels in both brain regions.
Atomoxetine-induced increases in NA, but not DA, levels in the PFC
were reduced in mice pretreated chronically with atomoxetine. On the
other hand, pretreatment with methylphenidate did not affect the acute
effect of methylphenidate on extracellular monoamine levels in the PFC.
Immunohistochemical studies showed that acute treatment with these
drugs increased the c-Fos expression in the PFC, and this effect was not
385
affected by chronic exposure to each drug. These observations suggest
that ADHD drugs selectively affect catecholaminergic systems in the
PFC of mice, and atomoxetine, but not methylphenidate, induces neurochemical desensitization of noradrenalinergic neurons in the PFC.
Paper No.: 2496

STRATEGIES TO INDUCE CARDIAC ELECTROPHYSIOLOGICAL PROPERTIES IN C2C12 SKELETAL MYOBLASTS
X Koenig(1), E Zebedin-Brandl(1), M Mille(1), M Schnuerch(2),
M Koley(2), MD Mihovilovic(2), R Cervenka(1), P Lukacs(1), H Todt(1),
Karlheinz Hilber(1)
(1) Medical University of Vienna, Center for Physiology and Pharmacology, Vienna, Austria
(2) Vienna University of Technology, Institute of Applied Synthetic
Chemistry, Vienna, Austria
Skeletal myoblast transplantation into the heart is a new therapy after
myocardial infarction. Its success, however, is at present impeded by the
very limited capacity of the transplanted myoblasts to differentiate into
functional cardiomyocytes in the heart. A strategy to overcome this problem would be the induction of cardiomyogenic function in myoblasts
prior to transplantation. Here, we tried two different in vitro strategies to
achieve this goal. Mouse C2C12 skeletal myoblasts were incubated in
differentiation media preconditioned by primary cardiomyocyte cultures
(cardiac preconditioning), or treated with the cardiomyogenic small molecule Cardiogenol C for 2-14 days. Thereafter, their Na current properties were measured using the whole cell patch clamp technique, and
compared with those of untreated control cells. Simulation of a cardiac
cell environment by cardiac preconditioning altered the Na current
activation and inactivation properties of C2C12 cells from skeletal muscle to more cardiac-like ones. Tetrodotoxin and RT-PCR experiments
suggest that an upregulation of the expression of the cardiac sodium
channel isoform Nav1.5 versus the skeletal muscle isoform Nav1.4 is
responsible for the observed changes in sodium current function. Similar
effects on the Na currents of C2C12 cells, which can be explained by an
upregulation of cardiac Nav1.5 channels, were observed in cells treated
with the cardiomyogenic small molecule Cardiogenol C. We conclude
that cardiac preconditioning and treatment with Cardiogenol C affect the
electrophysiological properties of skeletal myoblasts. Such strategies may
be useful to generate myoblasts with more cardiac-like electrophysiological properties.
Supported by Austrian Science Fund P19352-B11.
Paper No.: 2794

DISRUPTED ION CHANNEL FUNCTION IN DYSTROPHIC
CARDIOMYOCYTES
trophic neonatal mice were studied by using the whole cell patch clamp
technique. Besides the most common mouse model for human DMD, the
dystrophin-decient mdx mouse, we also used mice additionally carrying
a mutation in the utrophin gene. The mdx-utr double mutant mouse
exhibits a more severe disease phenotype than the mdx mouse, and may
represent a more suitable animal model for human DMD. We found that
dystrophic cardiomyocytes show a 30% reduction in sodium current density compared to wild type cardiomyocytes. In addition, extra utrophindeciency altered sodium channel activation and inactivation properties,
which was not observed in only dystrophindecient (mdx) cardiomyocytes. Finally, calcium channel inactivation was impaired in both dystrophic mdx and mdx-utr cardiomyocytes. In conclusion, we found
signicant impairments in ion channel function in dystrophic cardiomyocytes. These may perturb electrical impulse propagation in the dystrophic
heart, and thus contribute to cardiac complications associated with the
muscular dystrophies.
Supported by the Austrian Science Fund (P19352-B11 and P21006B11).
Paper No.: 515

PHARMACOKINETIC AND PHARMACODYNAMIC
MODELLING OF DENOPAMINE IN PATIENTS WITH
CONGESTIVE HEART FAILURE
Yuichi Koike(1), N Yonehara(2)
(1) Ohu University, School of Pharmaceutical Sciences, Department of
Drug Metabolism and Clinical Pharmacokinetics, Koriyama, Japan
(2) Ohu University, Department of Drug Metabolism and Cllinical
Pharmacokinetics, Koriyama, Japan
Acute and chronic effects of denopamine were studied in six patients
with congestive heart failure (CHF) using pharmacokinetic-pharmacodynamic model. Six patients with NYHA grade II and III received a single
oral dose of 20 mg and 10 mg in every eight hour for twelve weeks.
Nine plasma samples were collected over 24 h and were assayed by
HPLC method. Swan-Ganz catheter was placed into the pulmonary
artery for measurement of hemodynamic parameters in each patient. The
pharmacokinetic (PK)-pharmacodynamic (PD) model consisted of twocompartment open model and an effect compartment linked to central
compartment. A sigmoid Emax model (Hill equation) described the relationship between the drug concentration in the effect compartment and
the drug effect. The cardiac index (CI) and heart rate (HR) were signicantly increased after single oral administration. The pulmonary vascular
resistance (PVR) was decreased signicantly. The relationship between
denopamine concentration and CI demonstrated counter clockwise hysteresis. The Emax of CI was 6.75}1.14 L/min/m2 (mean}SD) and EC50
of it was 12.4}6.2 ng/ml. The chronic effects of denopamine on the hemodynamic parameters did not change signicantly in comparison with
acute effects. In conclusion, the effect compartment model was found to
be useful in describing denopamine kinetics and pharmacodynamic
effects.
Xaver Koenig, M Mille, S Kimbacher, R Cervenka, P Lukacs, H Todt,

RE Bittner, K Hilber
Medical University of Vienna, Center for Physiology and Pharmacology,
Vienna, Austria
The muscular dystrophies, e.g. Duchenne Muscular Dystrophy (DMD),
comprise a heterogeneous group of inherited diseases that are characterized by progressive muscle weakness and degeneration. In many dystrophy types, the cardiac muscle is also affected- cardiomyopathy and/or
cardiac arrhythmias regularly represent life threatening complications.
The current understanding of the pathomechanisms underlying these cardiac problems is still very limited. Here we tested the hypothesis that
dysfunctional ion channels may be critically involved in dystrophy- associated cardiac disease. The functional properties of voltage-gated sodium
and calcium channels in cardiomyocytes derived from normal and dys-
Paper No.: 1343

MTA IS A SELECTIVE A2B-ADRENOCEPTOR ANTAGONIST
Katja Koivula, S Rondinelli, J Nasman
bo Akademi University, Department of Biosciences, Biochemistry and
A
Pharmacy, Turku, Finland
Muscarinic toxins (MTs) are three-nger folded peptides isolated from
mamba snake venoms. It is generally believed that MTs are highly
386
specic for muscarinic acetylcholine receptors (mAChRs) showing
unusually high selectivity among these receptor subtypes. For example
MT7 is a potent and selective antagonist of the M1 mAChR subtype (Kd
value <1 nM) with no detectable binding to other mAChR subtypes. To
further test the specicity of MTs we chose to study the interaction of
MTa with different receptors. In [Ca2 + ]i mobilization assays different
mAChRs and a-adrenergic receptors (a-AR) were incubated with MTa.
The only affected receptor type was the a2B-AR, for which there was a
right-shift of the concentration-response curve and a strong suppression
of the maximum response. This suggested a non-competitive mode of
binding. To further explore the binding of MTa to the a2B-adrenoceptor,
we performed radioligand binding experiments which revealed a noncompetitive and reversible binding mode of MTa to a2B-AR with an
IC50 of 3.2 nM. With the help of different a2-AR chimeras it was found
that MTa binds to the second extracellular loop of a2B-AR. The results
indicate that MTa is a selective and high afnity antagonist of a2B-AR
and thus the rst peptide ligand found to act on a2-ARs. Other a-ARs or
mAChRs do not appear to be targets for MTa. These ndings should
open up new views in terms of selective adrenoceptor drug design and
help to elucidate a2-adrenoceptor physiology.
Paper No.: 3310

RACEMIC KETOPROFEN INDUCES MORE INJECTION PAIN
THAN DEXKETOPROFEN
Introduction. It is reported that GB-115, a dipeptide analogue of endogenous cholecystokinin, at low doses potentiated the morphine-induced
analgesia in thermal models of nociception in rodents. The aim of the
present work was to examine GB-115 antinociceptive properties per se at
high doses. Methods. The analgesic activity of GB-115 was measured in
mice in chemical and thermal assays after oral administration. Results: It
was shown that GB-115 (0.1 - 20.0 mg/kg) dose-dependently attenuated
abdominal constrictions induced by intraperitoneal acetic acid. Its relative
potency was compared with diclofenac (20.0 mg/kg, p.o.) effect, yielded
to morphine and was prevented by pretreatment with nor-Binaltorphimine (5.0 mg/kg, i.p.), a selective antagonist of j-opioid receptors
(KOP), and with naltrexon iodide (10.0 mg/kg, s.c.), a non-selective
antagonist of peripheral opioid receptors. In contrast to indomethacin
(25.0 mg/kg, p.o.), GB-115 (10.0 mg/kg) appeared to exert an analgesia
in the rst but not in the second chronic phase of inammation induced
by formalin. In tests measured pain responses to heat stimuli GB-115
was not active in the hot plate (supraspinal analgesia) but it reduced
pain perception in the tail ick (spinal analgesia) and this effect was
antagonized by naltrexon iodide completely. Moreover, the KOP agonistic effects of GB-115 were conrmed in vitro receptor binding and functional assays. Conclusion. The data obtained indicate that GB-115
possesses signicant analgesic activity in animal tests through interaction
with peripheral j-opioid receptors. In conclusion, the present results provide a new way for further developing of novel short peptide analgesic
compounds.
Hannu Kokki, T Saarelainen, M Kokki

Kuopio University Hospital, University of Eastern Finland, Department
of Anaesthesiology and Intensive Care, Kuopio, Finland
Non-steroidal anti-inammatory analgesic drugs are essential part of
multimodal pain management. The rst doses should be administered
intravenously (iv) to ensure a rapid onset of action and to guarantee a full
bioavailability. Dexketoprofen is claimed to cause less frequently and
less severe injection pain than racemic ketoprofen. However, there is neither data how often and how severe injection pain iv racemic ketoprofen
causes nor data whether dexketoprofen causes less injection pain than
racemic drug. We conducted an open label clinical comparison of ketoprofen and dexketoprofen iv-injection pain in 511 patients, undergoing
elective surgery in Kuopio University Hospital. There were 70 children,
379 adults and 62 elderly patients. The patients were given either racemic ketoprofen 1 mg/kg (n = 271) or dexketoprofen 0.5 mg/kg (n = 240)
injected over 2-5 minutes (n = 138), over 5-10 minutes diluted in 10-20
ml of saline (n = 147) or as an intravenous infusion over 30-60 min
diluted in 100 ml of saline (n = 226). Injection pain was signicantly
more common and more severe with racemic ketoprofen (36% reporting
moderate/severe pain) than that with dexketoprofen (14%). Elderly
patients had more and children less injection pain than adults. However,
in all age-groups and with all administration modes the frequency injection pain was 3-times higher (p < 0.00001) and the severity was more
common with racemic ketoprofen than that with dexketoprofen. We conclude that injection pain is common and rather severe with racemic ketoprofen and thus, dexketoprofen-injection, that causes 3-times less
injection pain, is more feasible option for intravenous administration in
the management of acute pain.
Paper No.: 1662

EXPERIMENTAL STUDY OF THE ANTINOCICEPTIVE
EFFECTS OF GB-115, A DIPEPTIDE ANALOGUE OF
CHOLECYSTOKININ
Paper No.: 824

IN VITRO EVALUATION OF LINEZOLID AND NOVEL
OXAZOLIDINONE ACTIONS ON CENTRAL NERVOUS
SYSTEM NEURONES
Samuel Kombian, O Phillips
Kuwait University, Department of Applied Therapeutics, Kuwait City,
Kuwait
The use of linezolid, an oxazolidinone to treat Gram-positive bacteria
infections that are resistant to standard antibiotics is accompanied by
peripheral, central and optic neuropathies. The cellular mechanism(s)
underlying these side effects is/are not known. This study examined the
acute effects of linezolid and selected potent triazolyl- oxazolidinones on
neuronal responses to determine their mechanism of action. Using in vitro slices of the rat nucleus accumbens (NAc) and hippocampus, we
examined the effects of linezolid, PH027, PH036, PH084 and PH108 on
synaptic transmission and postsynaptic responses recorded in voltage or
current clamp mode. Bath application of PH084 and PH027 produced
concentration-dependent suppression of evoked non-NMDA receptormediated excitatory postsynaptic currents (EPSCs). PH084 was the most
potent in suppressing EPSC; 10 uM produce a depression of 36.9
4.0% (n = 9). PH027 depressed the EPSC by 19.4 4.0% (n = 5).
PH036, PH108 and Lzd did not produce a signicant suppression of the
evoked EPSC. Similar depression was also observed on fast EPSCs
recorded in the hippocampus. PH084 (10 uM) suppressed NMDA synaptic current by 43.7 6.8% (n = 5) while PH027 (10 uM) also suppressed
it by 15.1 7.7% (n = 3). Furthermore, PH084 (10 uM) suppressed
evoked IPSC by 25.4 7.7% (n = 6) while PH027 (10 uM) suppressed
it by 22.4 3.6% (n = 4). The synaptic depressant effects were blocked
by selective GABAB, a-adrenoceptor and D1-like dopamine receptor
antagonists but not by an adenosine A1 receptor antagonist (28.0
4.1%; n = 6). Thus, PH036 and PH108 and related analogs may be less
neurotoxic.
This work was supported by KURA grant # PT02/06.
Larisa Kolik, V Zhukov, T Gudasheva, S Seredenin

SI Zakusov Institute of Pharmacology RAMS, Laboratory of Pharmacological Regulation of Addictions, Moscow, Russian Federation
387
Paper No.: 1308
FOCUSED CONFERENCE GROUP: PW11 - NEW
OPPORTUNITIES IN THE PHARMACOLOGY OF
NEURO-IMMUNE INTERACTIONS
EXPRESSION CHANGE IN NICOTINIC RECEPTOR
ACCORDING TO CELLULAR MATURITY OF ANTIGEN
PRESENTING CELL LINE
Yukiko Kondo(1), E Tachikawa(2), S Ohtake(1), K Kudo(1),
K Mizuma(1), T Kashimoto(1), Y Irie(1), E Taira(1)
(1) Iwate Medical School, Department of Pharmacology, Morioka, Japan
(2) Tokyo University of Pharmacy and Life Science, Tokyo, Japan
There are a lot of reports about the interaction of the immune system and
the neuro-endocrine system and it is well known that the central nervous
system adjusts innate immune reactions. It is also reported that the parasympathetic nervous system controls the systemic inammatory
responses through the acetylcholine receptor a7 subunit. As for the antigen-presenting cells (APCs), consist of the dendritic cells (DCs), the
macrophages, and B lymphocytes, a7 subunit of the nicotinic receptor
appears in DCs and the activation of DCs with nicotine reinforces the
proliferation potency and cytokine secretion of T lymphocytes. However,
the role of the nicotine receptor on the DCs is not clear yet. In the present study, we had analyzed expression and the function of the nicotinic
receptor in a p53-decient APC cell line (JawsII) derived from a mouse
bone marrow. Next, we analyzed the inuence that nicotine and some
inammatory peptide on the expression status of the cell surface antigen
similar to the inuence of LPS or TNF-a. In addition, we had analyzed
the inuence of them on the proinammatory cytokines production from
the cells by the LPS stimulation. We found expression of CD80, CD86,
and MHC-I increasing and the nicotine receptor decreasing by LPS or
TNF-a in the JawsII cells. In addition, nicotinic stimulation had no effect
on the decrease of nicotinic receptor on JawsII cells by LPS.
Paper No.: 1589

BIOEQUIVALENCE STUDY OF TWO ORAL PREPARATIONS,
THYROSIT (100 UG LEVOTHYROXINE SODIUM) VS
ELTROXIN (100 LG ANHYDROUS THYROXINE SODIUM) IN
PATIENTS WHO HAVE HYPOTHYROIDISM WITH
EUTHYROID IN STEADY STATE
Supornchai Kongpattanakul, S Vannasaeng, K Sathirakul,
S Chatsiricharoenkul
Mahidol University Faculty of Medicine, Siriraj Hospital, Department of
Pharmacology, Bangkok, Thailand
The aim of this study was to determine the bioequivalence of 100 lg levothyroxine sodium tablet formulations in 20 patients who diagnosed
hypothyroidism with euthyroid in steady state. The study was carried
with an open-label, two-treatment, two-period, two-sequence, randomized crossover design without washout period. Tablets strength of 100 lg
levothyroxine sodium either the test or reference formulation was given
daily along 57 days of each period. At steady state (Day 57 on period 1
and Day 114 on period 2), plasma samples were obtained over 24-h
interval and the concentrations of T4 were determined by a Microparticle
Enzyme Immunoassay technique. Pharmacokinetic parameters were evaluated from plasma concentration-time proles of T4 using the noncompartmental analysis without the adjustment of baseline levels. The 90%
condence interval of geometric mean ratio of primary target parameters
(Cmax,ss and AUCss,24) between the test and reference formulations were
entirely within the acceptance bioequivalence limits of 80.00% to
125.00% which were 103.32%(97.99% - 108.93%) for Cmax,ss ratios and
102.32% (96.97% - 107.96%) for AUCss,24 ratios together with the
power more than 80%. In addition, non-parametric Friedmans test for
Tmax,ss were also demonstrate no signicant difference between the both
formulations (p > 0.05). All subjects were well tolerated. No serious
adverse effects were observed. It can be concluded that the test product
is bioequivalent to the reference product in term of rate and amounts of
drug absorption and it could be interchangeable in the majority of
patients receiving thyroxine replacement therapy.
Paper No.: 1625

THE EFFECTS OF NATURALLY OCCURRING SINGLE
NUCLEOTIDE POLYMORPHISMS (SNPS) AT THE
GLUCAGON-LIKE PEPTIDE 1 RECEPTOR (GLP-1R)
Cassandra Koole, J Simms, D Wootten, A Christopoulos, PM Sexton
Monash University, Drug Discovery Biology Laboratory, Department of
Pharmacology and Monash Institute of Pharmaceutical Sciences, Parkville, VIC, Australia
Type II diabetes mellitus is one of the largest health risks in the developed world and despite several drug therapies being available, is often
poorly controlled in patients. The glucagon-like peptide 1 receptor
(GLP-1R) is one of the most promising targets in the development of
treatments for the management of this disease, exhibiting many desirable
characteristics when stimulated, including augmenting insulin release and
b-cell mass. However, this receptor possesses multiple single nucleotide
polymorphisms (SNPs) leading to individual amino acid changes in the
receptor coding sequence. The distribution of these polymorphisms
across different populations, and the effects on receptor function, remain
largely unstudied. We have determined the functional proles of 11
GLP-1R SNPs in response to the orthosteric agonists, GLP-1, exendin
and oxyntomodulin. In addition, we have determined the effects of the
reported GLP-1R allosteric modulator Compound 2 (Knudsen et al.,
PNAS, 2007; 104:937-942) on the functional proles of each SNP. We
have demonstrated that several SNPs result in agonist dependent changes
in potency and efcacy in signalling pathways. These data represent a
detailed examination of the pharmacological effects of reported GLP-1R
SNPs, and the roles they may play in inuencing receptor function in the
presence of endogenous GLP-1 agonists, GLP-1 analogues, and the allosteric modulator Compound 2.
Paper No.: 2306

PHARMACOKINETIC STUDY OF IMMEDIATE AND
PROLONGED GENERIC MOLSIDOMINE FORMULATIONS
Jiri Kopecky(1), D Kholova(1), A Polaskova(1), J Zoulova(1),
I Zubata(1), L Vyslouzil(1), K Macek(2), J Chladek(1), J Kvetina(1)
(1) Institute of Experimental Biopharmaceutics, Joint Research Centre of
PRO.MED.CS Praha a.s. and Academy of Sciences of the Czech Republic, Hradec Kralove, Czech Republic
(2) University Hospital, Hradec Kralove, Czech Republic
Molsidomine is a nitrovasodilator used in angina pectoris. The aim of
the study was to investigate the pharmacokinetics of a new oral 8 mg
prolonged release (PR) molsidomine formulation: (i) in comparison with
4 mg immediate release (IR) formulation, (ii) after single-dose and
repeated (5 days) administration, and (iii) under fasting and fed conditions. The results come from our three bioequivalence studies conducted
in healthy male and female volunteers (18-51 years old, BMI 19.0-24.9
kg/m2). They are presented as means.d. PR formulation (8 mg orally)
demonstrated the same extent but slower rate of bioavailability in comparison with IR formulation (4 mg orally). Cmax was 42 15 ng/ml and
35 13 ng/ml and Tmax was 0.55 0.30 h and 1.58 0.83 h in IR formulation and PR formulation, respectively. The increase of the parame-
388
ters HVD (half-value duration) from 2.5 0.7 h in IR formulation to 5.7
1.6 h (2.3-fold) in PR formulation and T1/2 from 1.8 0.5 h to 2.9
0.5 h (1.6-fold) are manifestations of medium retardation. Neither pharmacokinetic cumulation nor decreasing of absorption and/or increasing
of elimination of molsidomine was produced by repeated administration
of PR formulation. Standard high-fat, high-calorie meal did not inuence
the extent but decreased the rate of molsidomine bioavailability in PR
formulation (Tmax increased from 1.6 0.8 h to 3.7 1.9 h). Retardation (parameter HVD) was not inuenced signicantly by food.
Paper No.: 1587

INHIBITION OF HEAT SHOCK PROTEIN 90 ATTENUATES
ADENYLATE CYCLASE SENSITIZATION AFTER CHRONIC
MORPHINE TREATMENT
Takaaki Koshimizu, H Tsuchiya, S Fujimura, Y Fujiwara, A Fujimura
Jichi Medical University, Department of Pharmacology, Shimotsuke,
Tochigi, Japan
Cellular adaptations to chronic opioid treatment result in enhanced
responsiveness of adenylate cyclase and an increase in forskolin- or agonist-stimulated cAMP production. It is, however, not known whether
chaperone molecules such as heat-shock proteins contribute to this adenylate cyclase sensitization. Here, we report that treatment of cells with
geldanamycin, an inhibitor of heat shock protein 90 (Hsp90), led to
effective attenuation of morphine-induced adenylate cyclase sensitization.
In SK-N-SH human neuroblastoma cells, morphine signicantly
increased RNA transcript and protein levels of type I adenylate cyclase,
leading to sensitization. Whole-genome tiling array analysis revealed that
cAMP response element-binding protein, an important mediator for cellular adaptation to morphine, associated with the proximal promoter of
Hsp90AB1 not only in SK-N-SH cells but also in rat PC12 and human
embryonic kidney cells. Hsp90AB1 transcript and protein levels
increased signicantly during morphine treatment, and co-application of
geldanamycin (0.1-10 nM) effectively suppressed the increase in forskolin-activated adenylate cyclase activation by 56%. Type I adenylate
cyclase, but not Hsp90AB1, underwent signicant degradation during
geldanamycin treatment. These results indicate that Hsp90 is a new pharmacological target for the suppression of adenylate cyclase sensitization
induced by chronic morphine treatment.
Paper No.: 1409

POSSIBLE REGULATION OF GABAB RECEPTORS
EXPRESSION BY PHOSPHATASES
Akira Koutani, H Niihara, N Kuramoto, K Ogita
Setsunan University, Department of Pharmacology, Osaka, Japan
GABAB receptors mediate slow and prolonged synaptic inhibition in the
brain, and are members of the G protein-coupled receptor (GPCR) superfamily. To date 2 GABAB receptor subunits have been identied as GABABR1 (R1) and GABABR2 (R2), and unlike other members of the
GPCR superfamily, functional GABAB receptors are heterodimers
formed between these 2 proteins. Among the 2 subunits, R2 is highly
phosphorylated at serine 892 by protein kinase A. Similar to the other
proteins, phosphorylated and dephosphorylated states of R2 are crucially
regulated and are probably important for stabilities, localizations, and
functions of GABAB receptors. While R2 and phosphorylated R2 (p892)
were strongly detected in cerebral cortex, hippocampus, and cerebellum,
relative phosphorylation of p892 to total R2 was constant among the
brain regions. In cultured cortical neurons, the phosphorylation was
increased by treatment with forskolin or okadaic acid, but not with cyclo-
sporine A, sodium uoride nor orthovanadate. In acute hippocampal

slices, both R2 and p892 were increased by the incubation with okadaic
acid (OA) at a concentration of 1000 nM in the Neurobasal medium for
15 min, while only total expression of R2 was increased by OA at lower
concentration of 100 nM. These data suggest that GABAB receptors are
functionally and on their protein expression regulated by 2 phosphatases
including PP1a and PP2a.
Paper No.: 1400

SURGICAL SITE INFECTIONS INCIDENCE AS A QUALITY
INDICATOR FOR PROPER ANTIBIOTIC PROPHYLACTIC
USE IN TERTIARY HEALTH CARE HOSPITAL INPATIENTS
Aleksandra Kovacevic(1), V Dragojevic-Simic(1), V Suljagic(2),
M Jevtic(3), J Vukovic(1), D Jandric(1)
(1) Military Medical Academy, Centre for Clinical Pharmacology,
Belgrade, Republic of Serbia
(2) Military Medical Academy, Department for Infection Control,
(3) Military Medical Academy, Clinics for Vascular Surgery, Belgrade,
Republic of Serbia
The objectives of the study were to analyze antimicrobial consumption
in surgical clinics of tertiary health care hospital after new prophylaxis
policy and restricted antibiotics list were approved, at the beginning of
2008, as well as to correlate it with incidence of surgical site infections
(SSIs). Antibiotic consumption was monitored in the period 2006
2008, expressed by WHO method of Dened Daily Doses per 1000 bed
days (DDD/1000 BD); incidence of SSIs was expressed as a percentage
of total surgical procedures per observed year. Total antimicrobial consumption in year 2008 decreased (807, 69; 784, 44; 668, 46 DDD/1000
BD, in 2006, 2007 and 2008, respectively). Signicant differences in
consumption of cephalosporins were observed. Consumption in 2006,
2007 and 2008 was as following: rst-generation cephalosporins
80,87; 69,69; 73,53; second-generation cephalosporins 52,18; 31,93;
76,96; third-generation cephalosporins 195,58; 234,43; 137,88; fourthgeneration cephalosporins 6,15; 1,50; 1,25, respectively. Signicant
decrease of ceftriaxone consumption occurred in 2008 comparing with
2007 and 2006 (74,3; 207,1; 125,3) and increase of cefazoline (35,12; 0,
00) and cefuroxime (60,91; 28, 46) consumption was observed in 2008
comparing 2007. Incidence of SSIs signicantly decreased (CI 95%) for
3 observed years, and was 6, 3% in 2006, 5% in 2007 and 3, 6% in
2008. Although it is known causes of SSIs are multi-factorial, monitoring
of them would serve as a quality indicator of proper antibiotic surgery
prophylactic use in inpatients and enable evaluation of adherence to
guidelines and recommendations for rational antimicrobial use.
Paper No.: 1704

COMPLETE OVERHAUL OF THE AUTHORIZATION AND
SINGLE-CENTRAL ETHICAL COMMITTEE APPROVAL
PROCEDURE OF CLINICAL TRIALS IN HUNGARY
Peter Kovacs(1), A Vas(2)
(1) University of Debrecen, Medical and Health Science Center, Department of Clinical Pharmacology, Debrecen, Hungary
(2) Gedeon Richter Plc., Budapest, Hungary
To ensure trial subject safety and GCP compliance since 2002 Hungary
required a national single ethical opinion and authority approval for all I
to IV phase trials involving medicines. This procedure was updated and
streamlined in 2009 to decrease red tape and shorten the approval times,
while enhancing public safety, professional and ethical conduct of clini-
389
cal trials. The new legislation developed further the previous dual parallel
system, consisting of a national regulatory authority (NIP) review concerning medical professional aspects and an independent ethical review
performed by a central ethics committee. The ethical opinion of the 30
member Ethics Committee for Clinical Pharmacology (ECCP) of the
Hungarian Medical Research Council (with a newly elevated sphere of
authority) is binding, the NIP can authorize only with the ECCP recommendation. Both the NIP and the ECCP rulings on a trial are based on
separate expert reviews. A dully constituted subcommittee of the ECCP
acts as a standing committee for emergencies and simple requests. Yearly
about 400 clinical trials and amendments are processed this way, total
authorization time maximized in 60 days. Requests can be continuously
submitted to the NIP, the full ECCP meets regularly every 21 day, the
subcommittee as required. The NIP checks the chemical pharmaceutical
documentation, manufacturing and analytical certicates and the scientic merit. This dual procedure, while using the national single ethics
opinion ensures a standardized and highly competent framework for the
authorization of clinical trials.
Paper No.: 2760

COGNITIVE ENHANCERS: DIFFERENT TARGETS COMMON OBJECTIVE
Nitric oxide sensitive guanylyl cyclase (NOsGC) is a heterodimeric

enzyme formed by an a- and a b1 subunit. There is conicting evidence concerning the role of the amino-terminus of the a1 subunit in
heterodimerization. Wagner et al. (J Biol Chem 2005; 280:1768717693) show that amino acids 61-128 of the a1 subunit are mandatory
for quantitative heterodimerization. In a study primarily intended to
analyze heme binding and activation by nitric oxide (NO) we have
previously shown that the deletion 259 of the a1-subunit leads to an
enzyme complex with b1 that is very similar to the full length a1/b1
complex and can be stimulated by NO (Koglin and Behrends 2003;
278:12590-12597). In the same study we show that deletion of 364
amino acids of the a1-subunit leads to a basally active enzyme complex. In the current study we puried the b1-subunit after co-expressing the different a1 deletion mutants using an analogous strategy to
the study of Wagner et al. 2005. We found preserved quantitative
dimerization with b1 despite truncation of 259 or 364 amino acids of
the a1-subunit. In addition we used uorescence resonance energy
transfer (FRET) based on uorescence lifetime imaging using fusion
proteins of the b1 subunit with EYFP and the a1-subunit and its deletion mutants with ECFP. Analysis of the respective combinations in
HEK-293 cells showed that FRET efciency was signicantly higher
for a1/b1, a1DN259/b1 and a1DN364/b1 than the negative control.
We conclude that the amino-terminus of the a1 subunit is dispensable
for quantitative dimerization with b1.
Georgy Kovalev, J Firstova

Russian Academy of Medical Sciences, Zakusov Institute of
Pharmacology, Laboratory of Radioisotopic Researches, Moscow,
Russian Federation
Acetylcholine and glutamate brain systems and neurotrophines are
involved in memory and learning. Cognitive enhancing drugs affect these
components of exploratory behavior in animals. This work aimed to
study the effects of cognitive enhancers (nootropics) Piracetam, Fenotropil, Panthocalcine, Semax, Nooglutil on brain NMDA- and nicotinic receptor binding, and BDNF content in mice. Radioligand assay
provided using [3H-G]-MK-801 and [3H-G](-)nicotine in hippocampal
and cortical membranes. BDNF content measured with sandwich format
of ELISA method. Behavioral pattern evaluated in plus-maze test (Salimov et al., 1995). No drug demonstrated direct effects on NMDARs
binding in vitro. Piracetam and Fenotropil (but not other nootropics) displaced the [3H](-)nicotine from specic binding sites in micromolar concentrations in vitro. After sub-chronic administration in equipotential
antiamnestic doses (once daily for 7 days, IP) the clear effects of all the
drugs observed only in mice sub-population with low exploratory activity
(EL). In this sub-population the drugs ameliorated the maze exploration,
increased the number of NMDARs and decreased the number of nAChRs in comparison with mice demonstrating high exploratory activity. Piracetam and Fenotropil modulated the nAChRs density acting on both
high-afnity (a4b2) and low-afnity (a7) binding sites, while Semax only
on the latter. Piracetam and Semax increased hippocampal and cortical
levels of BDNF in EL mice, while Fenotropil normalized BDNF content
selectively in hippocampus. Thus, nAChRs can be considered as primary
targets for Piracetam and Fenotropil. Positive modulatory effect of most
the nootropics on cognitive decit is accompanied by lowering of
nAChRs, growth of NMDARs, and increase of BDNF content.
Paper No.: 1231

HEALTH AND DISEASE
DIMERIZATION OF NITRIC OXIDE SENSITIVE GUANYLYL
CYCLASE
Jan Robert Kraehling, M Busker, T Haase, M Seeanner, S Behrends
Paper No.: 940

EFFECTS OF POTASSIUM CHANNEL OPENERS ON
PARAMETERS OF URETERAL FUNCTION AN IN VIVO
ANIMAL STUDY
Carsten Krahtz(1), AJ Becker(2), ME Mayer(2), MA Kuczyk(1),
CG Stief(2)
(1) Hannover Medical School, Division of Surgery, Department of
Urology & Urological Oncology, Hannover, Germany
(2) Ludwig-Maximilians-University, Faculty of Medicine, Department of
Urology, Munich, Germany
Introduction: The use of compounds (nitric oxide donors, a-blocking
agents) assumed to facilitate the passage of ureteral calculi by inhibiting ureteral smooth muscle contraction is being discussed for the treatment of urolithiasis, including colic pain. In vitro, the potassium
channel openers (PCOs) rimakalim, S0121 and levcromakalim have
also been shown to exert relaxation of isolated human ureteral smooth
muscle (J. Urol. 153, 4, Suppl., 1995). The aim of the present study
was to characterize the effects of the PCOs on parameters of ureteral
function. Materials/Patients: In the anesthetized rabbit the effects were
investigated of rimakalim, S0121 and levcromakalim (10 lg/kg - 60
lg/kg BW) on the parameters of ureteral peristalsis (frequency, intraureteral pressure, pressure amplitude) and on blood pressure. The parasympatholytic drugs copolamine butylbromide was used as reference
compound. Results: Levcromakalim and S0121 induced only a minor
reversion of the phasic contractile activity of ureteral smooth muscle.
In contrast, the effects of the drugs on the cardiovascular system were
more pronounced. Rimakalim (12 lg/kg KW) induced complete inhibition of the amplitude and frequency of ureteral peristalsis. This effect
was accompanied by a pronounced drop in blood pressure. Scopolamine butylbromide (0.67 mg/kg BW) had no impact on ureteral peristalsis or blood pressure. Conclusion: It seems likely that
levcromakalim or S0121 cannot facilitate the passage of an intraluminal ureteral stone and inhibit local spasms of ureteral smooth muscule.
The use of rimakalim in the treatment of urolithiasis is limited by the
vascular side effects of the drug.
Technical University of Braunschweig, Institute of Pharmacology,

Toxicology and Clinical Pharmacy, Braunschweig, Germany
390
Paper No.: 1768
ACTIVATION OF CAMP-RESPONSE-BINDING-ELEMENT
PROTEIN (CREB) IN THE NUCLEUS ACCUMBENS REDUCES
RELAPSE TO METHAMPHETAMINE-SEEKING BEHAVIOUR
IN RATS
ACTIVATION OF CAMP-RESPONSE-BINDING-ELEMENT
PROTEIN (CREB) IN THE NUCLEUS ACCUMBENS REDUCES
RELAPSE TO METHAMPHETAMINE-SEEKING BEHAVIOUR
IN RATS
Niree Kraushaar, L Hunt, J Cornish
Macquarie University, Department of Psychology, North Ryde, NSW,
Australia
Methamphetamine abuse is a large problem within society however little
is known about the underlying neurobiology that contributes to relapse
to methamphetamine use. Recent studies have suggested a role for the
cAMP-response-binding-element protein (CREB) in drug reward processes which can be activated by the release of cAMP-dependent-kinase
PKA. This study aimed to elucidate the role of CREB in relapse to drug
use by using a reinstatement model of drug-seeking behaviour. In particular, we examined the effect of activating CREB in a key brain reward
area, the nucleus accumbens (NAc), in mediating methamphetamineseeking behaviour. Male Sprague Dawley rats (374 6g, n = 12) were
surgically implanted with a jugular vein catheter and bilateral cannulae
into the NAc while under isoourane anaesthesia. One week following
surgery, rats were trained to self-administer intravenous methamphetamine during daily 2 hour sessions. Methamphetamine was self-administered at a rate of 0.1mg/kg/infusion, on a xed ratio schedule, for 14
days. Following behavioural extinction rats underwent 3 reinstatement
test days where they were treated with an intracranial infusion of the
PKA activator Sp-cAMPs (10 nmol/0.5ll/side, 20 nmol/0.5ll/side) or
aCSF (0.5ll/side) 30 minutes prior to a methamphetamine priming injection (1mg/kg, i.p.). Treatment with the PKA inhibitor Sp-cAMPs produced a dose-dependent decrease in methamphetamine-induced drug
seeking behaviour when compared to infusions of aCSF. These data suggests that PKA mediated activation of CREB in the NAc is involved in
the relapse to methamphetamine use in rats. This research is supported
by the Australian Research Council DP0986021.
Paper No.: 1351

STUDY OF TIME DEPENDENCE OF AFOBAZOL AND MEXIDOL PROTECTIVE ACTIVITY IN ITRACEREBRAL POSTTRAUMATIC HEMATOMA MODEL
Valentina Krayneva, A Fedorenko, S Seredenin
Zakusov Institute of Pharmacology RAMS, Department of Psychopharmacology, Moscow, Russian Federation
The time dependence of afobazol (5-ethoxy-2-[2-(morpholino)-ethylthio]
benzimidazole dihydrochloride) (MT1 (Ki = 1,6*105M), MT3 (Ki =
9,7*107M), sigma-1 (Ki = 5,9*106M), MAO-A (Ki = 3,6*106M))
effects were studied in comparison to an antioxidant mexidol (2-ethyl-6methyl-3-oxypyridine succinate) under an itracerebral posttraumatic
hematoma (IPH) model evoked by destruction of the brain tissue in the
area of internal capsule. In rats with IPH within the rst day death-rate
was 17%, and by 14 day increased up to 50%. In sham-operated rats the
survival was 100%. Afobazol (1.0 mg/kg, intraperitoneally 6 hours after
operation, and then twice a day within 14 days) increased survival in rats
with IPH up to 100%. Mexidol (50 mg/kg, intraperitoneally, 6 hours after
operation, and then daily within 14 days) was less effective in comparison to afobazol, death-rate by 14 day was 20%. The protective effects of
afobazol (1.0 mg/kg) and mexidol (50 mg/kg) administered 12 hours
after IPH procedure decreased. The protective effects was absent in IPH
rats treated with afobazol or mexidol 24 hours after IPH surgery. Thus, it
was established that afobazol demonstrated pronounced neuroprotective
effect in intracerebral posttraumatic hematoma model when administered
6 hours after the surgery.
Paper No.: 2398

CARDIOTROPHIN-1 IS UPREGULATED, BUT LEFT VENTRICULAR FUNCTION IS NOT AFFECTED AFTER ACUTE DAUNORUBICIN TREATMENT IN THE SPONTANEOUSLY
HYPERTENSIVE RATS
Peter Krenek, K Turcekova, H Cernecka, L Mesarosova, M Snopkova,
P Ochodnicky, P Musil, J Kyselovic, J Klimas
(1) Comenius University Faculty of Pharmacy, Department of Pharmacology and Toxicology, Bratislava, Slovak Republic
Introduction. Anthracyclines are potent cytostatic drugs with a pronounced cardiotoxic potential. Cardiotrophin-1 (CT-1) is a cytokine with
hypertrophic and cardioprotective properties and may be involved in
heart failure. We examined the effect of daunorubicin on cardiac function
and expression of CT-1 in normotensive Wistar (W) and spontaneously
hypertensive rats (SHR). Male 18-weeks old W or SHR rats received six
doses of daunorubicin (D, 3 mg/kg, i.p., every 48 hours). Materials. Control rats received vehicle. Left ventricular function was measured using
catheterization under tribromoethanol anesthesia, under basal conditions
and after b adrenergic stimulation with dobutamine. Gene expression of
CT-1 was measured using RT-PCR. Results. Daunorubicin caused a similar reduction in body (W 21%, SHR 18%; P < 0.05 vs Control) and
heart weight (Wistar, SHR 17%, P < 0.05 vs Control) both in the Wistar and SHR. Left ventricular dysfunction was present in daunorubicintreated normotensive Wistar rats (LVP, W: 155 3, WD: 126 8 mmHg,
P < 0.05; dP/dtmax, W: 7085 365 WD: 3940 435 mmHg/s; P <
0.05)), but not in the SHR (LVP, SHR: 151 6, SHR-D: 157 6 mmHg,
P=NS; dP/dtmax, SHR: 6396 823 SHR-D: 6386 64 mmHg/s; P <
0.05). CT-1 mRNA levels in the left ventricle were similar in untreated
Wistar and SHR and were increased more than twofold by daunorubicin
treatment in both strains (W: 1.00 + -0.21, WD: 2.25 0.33; SHR: 0.94
0.10, SHR-D: 2.23 0.54; both P < 0.05 vs control). Conclusion.
SHR appear to be protected against anthracycline-induced cardiac dysfunction in spite of similar regulation of CT-1 by the cardiotoxic insult.
(Grant support: 1/0377/09, 1/0707/09, 1/0357/09, APVV RPEU-0023-06)
Paper No.: 2685

ANALGESICS
LOCAL ANAESTHETIC INFUSED AT THE INCISION SITE
FOR POST-OPERATIVE PAIN MANAGEMENT FOLLOWING
ABDOMINAL SURGERY
Sumithra Krishnan(1,2), R Morris(1), P Hewett(2), A Karatassas(2),
J Tonkin(3), J Field(4)
(1) The Queen Elizabeth Hospital, Clinical Pharmacology, Adelaide, SA,
Australia
(2) The University of Adelaide, Department of Surgery, Adelaide, SA,
Australia
(3) The Queen Elizabeth Hospital, Department of Anaesthesia, Adelaide,
SA, Australia
(4) The University of Adelaide, Faculty of Health Sciences, Adelaide,
SA, Australia
Current pharmacological approaches used for pain management consist
mainly of opioids, which cause serious adverse effects (Klopfenstein et
al 2000). This may be reduced with appropriately delivered local
391
anaesthesia. The aim of this study was to test whether a continuous 96h
infusion of levobupivacaine, using a Painbuster can eliminate the need
for opioid analgesia following abdominal surgery. Patients (mean sd
age= 64 5 years, n = 81) scheduled for either laparoscopic or open
abdominal surgery and consented in this randomised double-blinded placebo-controlled trial, were allocated into either a treatment group (0.5%
levobupivacaine infusion) or control group (0.9% saline infusion) in the
Painbuster. At the end of surgery the Painbuster catheter was inserted into
the incision site. Patients received opioids via a patient-controlled-analgesia (PCA) system for break-through pain. Pain scores and total opioid
PCA consumption were recorded as an index of efcacy of the treatment
delivered in the Painbuster. Blood samples were assayed to measure
potential levobupivacaine toxicity. For those patients undergoing open
surgery, the active treatment reduces pain scores, whereas in patients
undergoing laparoscopic surgery there was a tendency for the active treatment to increase pain scores in comparison to the control group. There
was a trend toward lower PCA consumption in patients receiving levobupivacaine for open operations, whereas there was little difference
between the two treatments for patients undergoing laparoscopic surgery.
The continuous infusion of levobupivacaine reduces pain scores and opioid consumption in patients undergoing open abdominal surgery.
Paper No.: 1816

HEALTH AND DISEASE
EXPRESSION AND FUNCTIONAL STUDIES OF SMALL - AND
INTERMEDIATE CONDUCTANCE CALCIUM-ACTIVATED
POTASSIUM CHANNELS IN HUMAN SMALL PULMONARY
ARTERIES AND BRONCHIOLES
Christel Kroigaard(1), T Dalsgaard(1), B Elmedal Laursen(1),
H Pilegaard(2), U Simonsen(1)
(2) Aarhus University Hospital Skejby, Aarhus, Denmark
Small (SKCa) and intermediate (IKCa) conductance calcium-activated
potassium channels are involved in endothelium-dependent vasodilation
in systemic arteries. This study investigated whether IKCa and SKCa
channels are located to the human bronchial epithelium and pulmonary
vascular endothelium and whether activation of these channels leads to
broncho- and vasodilation. Expression of IKCa and SKCa3 was examined
by quantitative PCR and immunoblotting, and location by immunohistochemistry. Functional studies were conducted in human small pulmonary
arteries and bronchioles mounted in microvascular myographs for isometric tension recordings. SKCa3 protein and mRNA levels were similar
in pulmonary arteries and bronchioles, while IKCa protein was highest
expressed in pulmonary arteries. IKCa and SKCa3 immunoreaction was
found both in the endothelium and epithelium. In pulmonary arteries
contracted to U46619 (9,11-dideoxy-9a,11a-epoxymethanoprostaglandin
F2a) and bronchioles contracted to histamine, NS309, an opener of IKCa
and SKCa channels, induced concentration-dependent relaxations, which
were reduced by blocking both IKCa (charybdotoxin) and SKCa channels
(apamin). Relaxations evoked by a nitric oxide (NO) donor, GEA 3175,
in small pulmonary arteries and by GEA 3175 and the b2-adrenoceptor
agonist, salbutamol, in bronchioles were more pronounced than NS309evoked relaxations. These ndings provide evidence for expression and a
functional role of both IKCa and SKCa3 in the endothelium of human pulmonary arteries and epithelium of bronchioles. The location and modulation of IKCa and SKCa channels suggest that they are potential targets for
treatment of pulmonary disease.
Paper No.: 1817

HEALTH AND DISEASE
SKCA AND BKCA EXPRESSION IS UPREGULATED IN
PULMONARY ARTERIES FROM CHRONIC HYPOXIC RATS
Christel Kroigaard(1), T Dalsgaard(1), S-P Olesen(2), U Simonsen(1)

(2) University of Copenhagen, Denmark
Pulmonary arterial hypertension (PAH) is a life-threatening disease characterized by abnormal constriction of pulmonary arteries, proliferative
vasculopathies, and nally heart failure. The present treatments of PAH
are insufcient, and therefore there is interest in new pharmacological
approaches. We hypothesized that an increase in calcium-activated potassium (KCa) channel expression could be observed as a compensatory
mechanism to counteract PAH. For this purpose we investigated the
expression of small (SKCa), intermediate (IKCa) and large (BKCa) conductance KCa channels in lungs of normotensive/normoxic rats (n = 6)
and chronic hypobaric hypoxic rats (n = 7) that develop PAH and right
ventricular hypertrophy. Bronchioles and intrapulmonary arteries were
isolated and the expression of KCa channels investigated by QPCR,
immunohistochemistry and Western blotting. IKCa and SKCa3 immunoreaction was found in the arterial endothelium and bronchial epithelium.
At mRNA level, KCa channels were expressed in the following order in
rat bronchioles and arteries: BKCab1 > >SKCa1 > IKCa=BKCaa>SKCa3 >
>SKCa2. In arteries, BKCab1 mRNA and BKCaa mRNA and protein
expression was upregulated by hypoxia. IKCa mRNA expression was
higher in arteries than bronchioles and unaltered in tissue from chronic
hypoxic rats. mRNA for SKCa1, SKCa2 and SKCa3 was unaltered by
hypoxia, whereas SKCa3 protein was upregulated in arteries exposed to
hypoxia. In conclusion, in pulmonary arteries from chronic hypoxic rats
both SKCa3, BKCab1, and BKCaa was upregulated. Based on these
results and previous ndings that KCa openers dilate pulmonary arteries,
drugs that open KCa channels may be benecial for the treatment of
PAH.
Paper No.: 1793

EFFECTS OF ANTIOXIDANT XANTHONES FROM GENTIANA
DINARICA ON ACANTOSCELIDES OBTECTUS AGING IN
VIVO
Dijana Krstic-Milosevic(1), I Spasojevic(2), A Nikolic-Kokic(1),
M Spasic(1), D Blagojevic(1)
(1) University of Belgrade, Institute for Biological Research Sinisa
Stankovic, Department of Psyhiology, Belgrade, Republic of Serbia
(2) University of Belgrade, Institute for Multidisciplinary Research,
Free radical scavenging activities of xanthones: norswertianin-1-Oprimeveroside and norswertianin-8-O-primeveroside isolated from the
roots of G. dinarica towards stable free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) were proved in the comparison to ascorbic acid.
Xanthones have similar antioxidative capacity against OH generation
in metal involving Fenton reaction, when compared to synthetic analog
of vitamin E - Trolox. Neither Trolox nor xanthones showed any signicant capacity to scavenge OH radical generated in Haber-Weisslike metal-free generating system indicating their antioxidative action
preferentially via sequestration of transition metals preventing OH
generation. According to its antioxidant properties, xanthones were
offered as daily supplement (15 lM water solution) to an animal insect
model - bean weevil Acantoscelides obtectus. Bean weevil adults have
nutritional reserves for the whole lifespan and they only soak water.
Treatment did not change endogenous antioxidant level in weevils, but
inuence the rate of aging. In females, norswertianin-8-O-primeveroside
in early age slows down the rate of aging, but accelerates in the late.
In males, norswertianin-1-O-primeveroside showed light, but continuous
effect on slowing down the rate of aging resulting that supplemented
individuals lived longer. The results showed that potential health benets of exogenous nutritional antioxidants are combination of its individual antioxidant properties, composition, amount, metabolic fate, and the
inuence on antioxidant and other physiological processes, not antioxidant activity per se.
392
Paper No.: 2886
DISCOVERY
APPLICATION OF FLUORESCENCE SPECTROSCOPY TO
STUDY CONFORMATIONAL DYNAMICS OF LEUT, A
BACTERIAL HOMOLOGUE OF MAMMALIAN
NEUROTRANSMITTER TRANSPORTERS
Dinna B Kruger(1), J Kniazeff(2), L Shi(3), K Gotfryd(1),
CC Billesblle(1), CJ Loland(1), JA Javitch(4), H Weinstein(5),
U Gether(1)
(1) University of Copenhagen, Department of Neuroscience and Pharmacology, Copenhagen, Denmark
(2) University of Montepellier, Department of Molecular Pharmacology,
Montpellier, France
(3) Weill Medical College of Cornell University, Institute for Computational Biomedicine, New York, USA
(4) Columbia University College of Physicians and Surgeons, Center for
Molecular Recognition, New York, USA
(5) Weill Medical College of Cornell University, Department of
Physiology and Biophysics, New York, USA
The bacterial amino acid transporter LeuT belongs to the class of Neurotransmitter: Sodium Symporters (NSS) and displays homology to mammalian NSS such as transporters for dopamine, serotonin and
norepinephrine. They play key roles in controlling synaptic transmission
and are targeted by psychoactive drugs, e.g. antidepressants and psychostimulants (cocaine and amphetamine). High resolution crystals of LeuT,
revealed a central substrate-binding site (S1). However, the existence of
an extracellular second substrate binding site (S2) has been suggested.
The LeuT has also been crystallized with the antidepressant clomipramine bound to the S2 site. Here we apply uorescence spectroscopy
approaches to study the conformational dynamics of LeuT and the possible implications of the existence of a second substrate binding site. Single cysteine mutants were introduced in domains predicted from
computational simulations to undergo major conformational rearrangements during the transport process. The resulting mutants were puried
and the introduced cysteines were site-selectively labeled with tetramethylrhodamine maleimide. In uorescence quenching experiments, we were
able in response to substrate (leucine) binding, to detect conformational
changes corresponding to position E192C at the cytoplasmic site of
transmembrane segment 5. The change was consistent with increased
accessibility to E192C and thereby transition of the transporter, towards
inward facing conformation. Interestingly, substitution of sodium for lithium preserved the leucine induced conformational change, suggesting
that lithium can substitute for sodium in certain translocation steps. To
investigate the importance of S1 and S2 for substrate induced conformational changes in LeuT we currently analyze mutants that specically disrupt binding to S1 and S2, respectively.
Paper No.: 2307

DISCOVERY
HISTAMINE TRANSPORT INTO NEONATAL RAT ASTROCYTES: AN EXPERIMENTAL AND COMPUTATIONAL STUDY
Mojca Krzan(1), K Perdan-Pirkmajer(1), J Mavri(2)
(1) University of Ljubljana, Faculty of Medicine, Department of Pharmacology and Experimental Toxicology, Ljubljana, Slovenia
(2) National Institute of Chemistry, Ljubljana, Slovenia
Astrocytes participate in the clearance of neurotransmitters by their
uptake and subsequent enzymatic degradation. Histamine as a polar and/
or protonated molecule must use a carrier to be transported across the
cell membrane, although a specic histamine transporter has not been
elucidated, yet. In this work we combined the kinetic studies of hista-
mine uptake into neonatal rat cultured type 1 astrocytes with quantum
chemical calculations of histamine pKa values in conjunction with
Langevin dipoles solvation model as the rst step toward microscopic
simulation of transport. Our results indicate that astrocytes transport histamine by at least two carrier mediated processes, a concentration gradient dependent passive and a sodium-dependent and ATP-driven active
transport. We also demonstrated that histamine protonation states
strongly depend on the polarity of the environment. In conclusion we
suggest that histamine, a monoprotonated ion at physiological pH uses at
least two different mechanisms for its uptake into astrocytes -an electrodiffusion and Na (+) -dependent and ouabain sensitive active process.
We emphasize relevance of knowledge of histamines protonation states
at the rate limiting step of its transport for microscopic simulation that
will be possible when structure of histamine transporter is known.
Paper No.: 2372

PREDICTING STEADY STATE ORAL METHADONE DOSAGE
WITH CYP3A4 ACTIVITY? PRELIMINARY RESULTS
Kamilia Ksouda(1), F Vorspan(1), S Mouly(2), V Bloch(1), A Hajj(2),
K Peoch(2), J-L Laplanche(2), P Houze(3), Xxxx Lepine(1)
(1) Hopital Fernand Widal AP-HP et Neurospychopharmacologie des
Addictions INSERM U705, CNRS UMR 7157, Universite Paris-CiteDiderot et Paris-Cite-Descartes, Paris, France
(2) Hopital Lariboisie`re AP-HP et Neurospychopharmacologie des
Addictions, INSERMU705, CNRS UMR 7157, Universite Paris-CiteDiderot et Paris-Cite-Descartes, Paris, France
(3) Hopital Saint-Louis, AP-HP, Paris, France
Introduction: Oral methadone dosage at the steady state of heroin maintenance treatment is highly variable. CYP 3A4 is involved in methadone
disposition. This enzyme can be induced and inhibited by chronic intake
of medications as well as street drugs. CYP 3A4 activity has been shown
to inuence methadone plasma level, but clinicians are in search of
guidelines to predict the oral dosage of methadone that should be given
to each patient. Methods: We are conducting a study collecting clinical
and genetic variables in methadone maintained patients at the steady state
(stable oral dosage for at least 3 months, no other current drug dependence). Among those variables, a CYP3A4 functional test using 2mg
oral midazolam challenge was performed with 30 minutes and 4 hours
4OHmidazolam/midazolam ratio calculation. Results: Here we present
the preliminary results on the rst 20 patients. The sample was composed
of 15 men. The mean age was 41 7 years. The methadone oral dosage
ranged from 35 to 320 mg/day in this sample. Half of the patients were
HCV positive and 4 HIV positive (and treated). Methadone oral dosage
was statistically higher in case of HIV positive status (mean 167 135
vs. 48 27, ANOVA F(1df)=12,2 p=.003). This was explained by a statistically higher CYP 3A4 function repeated measure ANOVA
(F(2df)=4.7, p=.024). But the CYP3A4 function was not different
according to methadone oral dosage. Conclusion: CYP 3A4 function
may be predictive of methadone oral dosage in HIV treated patients, but
other independent variables have to be identied.
Paper No.: 987

DPP IV DEFICIENCY PRESERVES CARDIAC FUNCTION OF
ENDOTOXEMIC RATS VIA GLP SIGNALLING
Hui Chun Ku, MJ Su
National Taiwan University College of Medicine, Institute of Pharmacology, Taipei, Taiwan
Introduction: Dipeptidyl peptidase IV (DPP IV) is an enzyme responsible
for the degradation of GLPs, which is known to enhance glucose-depen-
393
dent insulin release, and contribute to lowering of glucose level. This
study aimed to characterize the role of GLP in regulation of cardiac function during endotoxemia. Materials: DchcHsd-DPP IV- rats were used as
DPP IV decient animals. Pressure volume loop in rat ventricles were
measured before and after LPS (10 mg/kg, i.v.) treatment. In some studies, exendin-4, a GLP analogue was subcutaneously injected 30 minutes
before LPS administration. Results: LPS treatment for 4 h resulted in a
reduction in rate of contraction development and relaxation of left ventricle in wild group of control, 52.7% and 63.6%, respectively. DPP IV
decient rats were more resistant to LPS. The rate of contraction development remained at 72.5%, and the rate of relaxation remained
unchanged. The LPS induced suppression of cardiac function in wild rats
was associated with a signicant reduction in cAMP level and phosphorylation of phospholamban. The cAMP level and phospholamban phosphorylation of LPS treated DPP IV mutant rats was higher than that in
wild rats. The deterioration of cardiac function and the reduction of
cAMP level and phospholamban phosphorylation in wild rats were signicantly reversed by pretreatment with exendin-4. Conclusion: Our
results suggest that higher GLP in DPP IV mutant rat may contribute to
the resistance to LPS, and exogenous GLP analogue may be useful in
the preservation of cardiac function of endotoxemic animal.
Paper No.: 2413

DISCOVERY
ACETYLCHOLINESTERASE REACTIVATORS - IN VITRO
PREDICTION OF PASSIVE DRUG TRANSPORT THROUGH
THE BLOOD-BRAIN BARRIER
Kamil Kuca(1), J Zdarova-Karasova(2), P Stodulka(1), K Musilek(2),
M Pohanka(1)
(1) University of Defence, Faculty of Military Health Science, Center of
Advanced Studies, Hradec Kralove, Czech Republic
(2) University of Defence, Faculty of Military Health Science, Department of Toxicology, Hradec Kralove, Czech Republic
21 structurally different drugs were used for development of in vitro
HPLC method for prediction of blood-brain barrier (BBB) penetration.
Then, the developed in vitro method was used for the prediction of BBB
penetration of selected acetylcholinesterase reactivators drugs used as
antidotes against nerve agent and pesticide poisonings. Owing to the fact
that these compounds have in their structures quaternary nitrogens, their
BBB penetration is expected to be limited. Our in vitro method conrms
this fact. Moreover, it is able to distinguish monoquaternary and bisquaternary compounds and it is able to show also some structural requirements needed for better BBB penetration. Obtained results are in very
good agreement with literature in vivo data. In summary, this method is a
very good approach for prediction of BBB penetration using passive
transport.
Acknowledgement: This work was supported by the project of Ministry
of Health of the Czech Republic - No.NS9748.
Paper No.: 2414

NOVEL CANDIDATES FOR REPLACEMENT OF STANDARD
ACETYLCHOLINESTERASE REACTIVATORS ANTIDOTES
IN CASE OF NERVE AGENT AND PESTICIDE POISONINGS
Kamil Kuca(1), K Musilek(2), D Jun(1), J Kassa(2), M Pohanka(1),
L Novotny(1), J Cabal(2), J Zdarova-Karasova(2)
(1) University of Defence, Faculty of Military Health Science, Center of
Advanced Studies, Hradec Kralove, Czech Republic
(2) University of Defence, Faculty of Military Health Science,
Department of Toxicology, Hradec Kralove, Czech Republic
Currently, only ve acetylcholinesterase (AChE) reactivators (pralidoxime, obidoxime, trimedoxime, MMB-4 and HI-6) are clinically used
as antidotes against nerve agents and pesticides around the world. However, their reactivation potency is limited. Due to this, many laboratories
around the world are aimed at development of novel candidates able to
replace standard therapy. Within last decade, hundreds of novel reactivators were synthesized and tested for their reactivation potency. Among
them, several candidates were selected and further investigated. In this
contribution, three promising candidates (oximes K027, oxime K203 and
DMS salt of the oxime HI-6) will be thoroughly discussed. These compounds are currently investigated in many countries throughout the world
for their extraordinary efcacy. Summary of results obtained with these
compounds will be presented.
Acknowledgement: This work was supported by the project of Ministry
of Health of the Czech Republic - No.NS9748.
Paper No.: 1559

DIFFERENCES IN THE PHARMACOKINETICS OF CYP3A
SUBSTRATES IN TSOD MICE AND STREPTOZOTOCININDUCED DIABETIC MICE
T Kudo, T Toda, T Ushiki, K Ohi, N Ikarashi, W Ochiai,
Kiyoshi Sugiyama
Hoshi University, Department of Clinical Pharmacokinetics, Tokyo,
Japan
Clinically, the pharmacokinetics of drugs may differ between type I diabetes (T1DM) and type 2 diabetes (T2DM). In this study, we evaluated
whether differences in drug pharmacokinetics between T1DM and
T2DM were due to altered CYP expression in the small intestine. We
used 7-month-old TSOD (Tsumura, Suzuki, Obese, Diabetes) mice with
advanced disease as a T2DM model and streptozotocin (STZ)-induced
T1DM model mice. As triazolam was administrered orally to TSOD
mice and STZ mice, CL/F in TSOD mice did not differ compared with
the control (TSNO) mice. In STZ mice, CL/F was greater than that in
the normal mice. The Cyp3a expression and activity were increased in
both the liver of TSOD mice and STZ mice compared with the control
mice. Small intestinal Cyp3a expression in TSOD mice was signicantly
lower than that in TSNO mice. No signicant difference existed in small
intestinal Cyp3a expression between STZ mice and the normal mice. We
evaluated whether the decrease in small intestinal Cyp3a expression in
TSOD mice was due to insulin. In small intestinal microsomal fractions
of insulin-treated mice, Cyp3a expression was signicantly lower than in
control mice. However, with insulin treatment, Cyp3a expression in
hepatic microsomal fractions did not change. These ndings suggest that
the differences in changes in small intestinal Cyp3a expression between
T1DM and T2DM are due to differences in plasma insulin concentrations. This could be a factor in the difference in the drug pharmacokinetics between patients with T2DM and those with T1DM.
Paper No.: 3276

HEALTH AND DISEASE
SEX DIFFERENCE IN EXPERIMENTAL HYPOXIC
PULMONARY HYPERTENSION IN RATS
Olga Kudryavtseva(1), N Medvedeva(2), V Koshelev(3)
rhus, Department of Pharmacology, A
rhus, Denmark
(1) University of A
(2) M.V. Lomonosov Moscow State University, Faculty of Biology,
Department of Human and Animal Physiology, Moscow, Russian Federation
(3) M.V.Lomonosov Moscow State University, Medical Faculty, Department of Physiology and General Pathology, Moscow, Russian Federation
394
The aim of current research was to determine whether sex hormones
play any role in development of hypoxia-induced pulmonary hypertension in rats. Male and female Wistar rats were used. Animals designated for exposure to chronic hypoxia were housed in a hypobaric
chamber at simulated altitude of 5000 m, 10 h a day, 2 wk. Agematched normoxic control animals were housed at ambient air. Right
ventricular systolic pressure (RVSP), right ventricular (RV) hypertrophy,
mean arterial pressure (MAP) and heart rate (HR) were measured.
Baroreex sensitivity was assessed using phenylephrine (PE) and
sodium nitroprusside (SNP). Two weeks after hypoxia exposure both
male and female rats developed RV hypertrophy and increase in RVSP.
The rst pathological change was more pronounced in female rats, the
second in males. Only male rats developed systemic hypertension in
response to chronic hypoxia exposure, but female rats exhibited marked
increase in HR. Pulmonary hypertension was associated with differential changes in baroreex regulation in males and females. Male rats
exhibited marked reduction in BP response. In clear contrast vascular
reactivity was unaffected in female rats, but they demonstrated potentiating of HR response to both agents, which resulted in changes in
baroreex sensitivity. Thus, there are gender-related differences in
hypoxia-induced pulmonary hypertension development. Male rats demonstrate the complex of pathological changes associated with chronic
hypoxia exposure: increase of RVSP, RV hypertrophy and systemic
hypertension, associated with decreased vascular reactivity. In female
rats its rather heart than vasculature that is affected by the pathological
inuence of chronic hypoxia.
Paper No.: 412

SUPPRESSION OF NF-JB SIGNALLING PATHWAY BY
EPIGALLOCATECHIN GALLATE CAN PREVENT DIABETES
ASSOCIATED COGNITIVE DEFICITS
Anurag Kuhad, M Bishnoi, V Tiwari, K Chopra
Panjab University, Institute of Pharmaceutical Sciences, Chandigarh,
India
Objective: The etiology of diabetes associated cognitive decline is multifactorial and involves insulin receptor down regulation, neuronal apoptosis and glutamatergic neurotransmission. The study was designed to
evaluate the impact of epigallocatechin gallate on cognitive function and
neuroinammatory cascade in streptozotocin-induced diabetes. Research
Design & Method: Streptozotocin-induced diabetic rats were treated with
epigallocatechin gallate for 10 weeks. Morris water maze was used for
behavioral assessment of memory. Cytoplasmic and nuclear fractions of
cerebral cortex and hippocampus were prepared for the quantication of
acetylcholinestrease activity, oxidative-nitrosative stress, tumor necrosis
factor-a (TNF-a), interleukin-1b (IL-1b), NFjB and caspase-3. Results:
After 10 weeks of steptozotocin injection, the rats produced signicant
increase in transfer latency which was coupled with enhanced acetylcholinestrease activity, increased oxidative-nitrosative stress, TNF-a, IL-1b,
caspase-3 activity and active p65 subunit of NFjb in different regions of
diabetic rat brain. Interestingly, co-administration of epigallocatechin gallate signicantly and dose-dependently prevented behavioral, biochemical and molecular changes associated with diabetes. Moreover, diabetic
rats treated with insulin-epigallocatechin gallate combination produced
more pronounced effect on molecular parameters as compared to their
per se groups. Conclusions: Collectively, the data reveal that activation
of NFjB signalling pathway is associated with diabetes induced cognitive impairment and point towards the therapeutic potential of epigallocatechin gallate in diabetic encephalopathy.
Paper No.: 830

PREPARATION, CHARACTERIZATION & EVALUATION OF
SOLVATOMORPHS OF ATORVASTATIN
Astha Kuhad, R Chadha
Panjab University, University Institute of Pharmaceutical Sciences,
Chandigarh, India
Preparation, identication and characterization of different forms of atorvastatin were carried out by crystallization from different solvents. Five
different forms of the drug were obtained. Appearance of a desolvation
endotherm in the DSC accompanied by mass loss in TGA for forms I, II,
IV and V showed these forms to be acetonitrile solvate hydrate (form I),
trihydrate (forms II and IV) and dimethylformamide solvate (form V),
respectively. However, the desolvation peak was absent in form III
(obtained from methanol) indicating the absence of any solvent of crystallization. This form was found to be partially crystalline by its XRPD
pattern. Solution calorimetry was further used to differentiate between
the forms as they differ in lattice energy, resulting in different enthalpies
of solution. The dissolution and solubility proles were correlated with
the enthalpy of solution and subsequently with crystallinity of all the
forms; the least endothermic form (form III) had the highest dissolution
rate. Further, these solvatomorphs were evaluated against triton-induced
hyperlipidemia in rats.
Paper No.: 1442

TYPE AND PREVALENCE OF SERIOUS ADVERSE EVENTS IN
PHASE I TRIALS PERFORMED IN JAPAN
Yuji Kumagai(1,2), T Momma(2), I Fukazawa(2), H Iijima(2)
(1) Kitasato University East Hospital, Clinical Trial Center, Sagamihara,
Kanagawa, Japan
(2) Japan Association of Contract Institutes for Clinical Pharmacology
(JACIC), Japan
Introduction: The major interest of investigators in phase I trials is safety
of subjects, since originally healthy subjects are generally included in the
trials. In carefully designed phase I trials, most of observed adverse
events will be minor in intensity, but several serious adverse events
(SAE) may also be found. The Japan Association of Contract Institutes
for Clinical Pharmacology (JACIC) was organized by Japanese clinical
pharmacology units, in which most of Phase I trials in Japan have been
performed, and JACIC is collecting data of SAEs in phase I trials in
Japan. Methods: JACIC surveyed SAEs found in trials performed from
1993 to 2009 by sending questionnaires to member units. Results: More
than 100,000 subjects were included in phase I trials in the units. Thirty
SAEs (0.027%) were assessed as side effects, which consist of excessive
pharmacological effects, anaphylaxis, abnormal liver function tests, infectious diseases and so on. Most of the SAEs were found in the cases of
extravascular administration and recovered rapidly. Discussion and conclusion: The low prevalence compared with reports from other countries
may be due to the lower doses used in the Japanese studies.
Paper No.: 488

HOW TO VIEW JUPITER
Cyrus Kumana, HF Tse, CP Lau
The University of Hong Kong, Department of Medicine, Hong Kong,
PR China
395
The JUPITER study (Ridker PM, et al 2008; NEJM 359:2195-207) that
was terminated prematurely after a median follow-up of 1.9 years, should
be viewed in the context of absolute event rates in other major clinical
trials with statins. As previously described (Kumana CR et al 1999,
JAMA 282:1899-901), crude relative risk (RR) and number needed to
treat (NNT)/year values with their 95% condence intervals (CIs) for
key events, were therefore calculated. JUPITER enrolled 17,802 subjects
with C-reactive protein levels 3 2mg/L and median-age 66 years; oral
rosuvastatin 20 mg/day was the active treatment. For its primary endpoint [a composite of the rst occurance of nonfatal myocardial infarction (MI), nonfatal stroke, hospitalization for unstable angina,
revascularisation procedure, or cardiovascular death] the RR was 57 (4670)% and the (NNT)/year 155 (144 to 242). Regarding fatal or non-fatal
MI, the RR was 46 (30-70)% and the NNT/year 457 (298 to 985). This
event was closest to the primary endpoint of the 4S trial, in which simvastatin treated patients with coronary heart disease and hypercholesterolemia attained corresponding values of 69 (61-79)% and 63 (49 to 89). In
JUPITER however, the RR and NNT/year values for physician reported
diabetes were 125 (104-150)% and -313 (-173 to -1607); negative NNTs
indicate harm. The high NNT/year (low absolute benet) of statin therapy for this indication is a concern, as about seven-fold more persons
than in 4S were exposed to potential side/adverse-effects and inconvenience to prevent one experiencing a fatal/non-fatal MI, whilst 1-2 more
subjects also became diabetic.
Paper No.: 450

EFFECT OF MASTITIS ON DISPOSITION KINETICS OF
LEVOFLOXACIN FOLLOWING SINGLE DOSE I.V.
ADMINISTRATION IN GOATS
Birendra Kuamr Roy(1), M Ram(1), V Singh(1), R Prtasad(1), KK
Singh(2)
(1) College of Veterinary Science & A.H., Department of Pharmacology
& Toxicology, Ranchi, India
(2) College of Veterinary Science & A.H., Department of Pathology,
Ranchi, India
The present study was conducted after single dose (10mg/kg) i.v. administration of levooxacin in six healthy and mastitic Black Bengal goats.
Mastitis was induced in six goats after 15 days wash out periods by
coagulase sensitive Staphylococcus aureus by the modied method of
Sar et al., 2006. Blood and milk samples were collected at 0, 2.5, 5, 10,
15, 20, 30, 45 minutes and 1,2,3,4,6,8,12,24,24, 36, 48 and 60 hours following drug administration. The concentrations of levooxacin were
determined by reverse phase HPLC. The mean maximum milk concentration (Cmax) was higher in mastitic goats as compared to healthy
goats.The mean t b in plasma (5.08 0.44hour) and in milk (7.28
0.23hour) of mastitic goats were longer than in healthy goats (4.04
0.59 hour and 4.16 0.30 hour).The ClB of levooxacin in milk was signicantly reduced in mastitic goats (1.67 0.21 ml/kg/min) than in
healthy goats (2.04 0.16 lg/ml/min). The AUC milk/AUC Plasma ratio
(3.36) in mastitic goats indicated extensive penetration of levooxacin
from plasma to milk. The AUC/MIC ratio in plasma and milk of mastitic
goats were 296.22 53.49 and 1014.90 152.63 respectively. Based on
the plasma kinetic parameters levooxacin may be administered i.v. @
5.12mg/kg at 25 hour interval for treatment of mastitis in goats.
Paper No.: 1016

NEUROPROTECTIVE EFFECT OF SEROFENDIC ACID ON IN
VITRO AND IN VIVO NEURONAL INJURY
Toshiaki Kume, Y Izumi, A Akaike
We have previously reported that a neuroprotective substance, serofendic

acid, was isolated from fetal calf serum. In the present study, we investigated the effect of serofendic acid against glutamate neurotoxicity using
in vitro assay and the effect of serofendic acid on ischemic injury
induced by a transient occlusion of the middle cerebral artery (MCA)
using in vivo assay. First, we examined in vitro effect of serofendic acid.
Exposure to glutamate induced neuronal death and nuclear fragmentation. Glutamate exposure also induced a transient increase in caspase-3
activity. Serofendic acid markedly prevented glutamate-induced neuronal
death and nuclear fragmentation. To elucidate the protective mechanisms
of serofendic acid, we examined the effect on the glutamate-induced
increase in intracellular Ca2 + concentration. Serofendic acid did not
affect the Ca2 + inux induced by glutamate. We investigated the effect
of serofendic acid on the loss of mitochondrial membrane potential
induced by glutamate. Serofendic acid prevented the loss of mitochondrial membrane potential following glutamate exposure. Moreover, serofendic acid suppressed the activation of caspase-3 induced by glutamate.
These in vitro results suggested that serofendic acid prevented glutamateinduced cytotoxicity by the prevention of loss of mitochondrial membrane potential and the suppression of the process of caspase-3 activation. Next, we examined the effect of serofendic acid on in vivo ischemic
injury induced by MCA occlusion. Serofendic acid, administered intracerebroventricularly, reduced total infarct volume. Moreover, serofendic
acid improved neurological decit scores. These results suggest that intracerebroventricular administration of serofendic acid prevents the neurodegeneration induced by a transient focal cerebral ischemia and
reperfusion.
Paper No.: 3144

DISCOVERY
ABC TRANSPORTERS AND HEAVY METALS IN BEWO
HUMAN CHORIOCARCINOMA CELLS
Maria Kummu(1,2,3), A Rautio(2), K Vahakangas(3), P Myllynen(1)
(1) University of Oulu, Institute of Biomedicine, Department Pharmacology and Toxicology, Oulu, Finland
(2) University of Oulu, Centre for Arctic Medicine, Oulu, Finland
(3) University of Eastern Finland, Department of Pharmacology and Toxicology, Kuopio, Finland
Heavy metals such as cadmium, lead and methylmercury may cause
reproductive toxicity. ABCG2 which is an efux transporter located in
the maternal facing membranes of the human placenta protects fetus
from xenobiotics by transferring compounds from the placenta to the
maternal circulation. The aim of this study was to clarify whether
heavy metals (CdCl2, PbCl2, MeHgCl) affect the expression and function of ABCG2 transporter in human placental BeWo choriocarcinoma
cells. The expression of ABCG2 was determined by immunoblotting
and functional activity of using uorescent substrates mitoxantrone and
pheophorbide A. Cytotoxicity was assessed by MTT assay. All compounds showed cytotoxicity MeHgCl and CdCl2 being the most cytotoxic. According to preliminary results CdCl2 and possibly PbCl2
seemed to upregulate ABCG2 protein expression while MeHg had no
effect. CdCl2 inhibited mitoxantrone efux suggesting inhibition ABC
transporter function. At the initial experiments CdCl2 did not inhibit
efux of specic ABCG2 substrate pheoborbide A suggesting that
CdCl2 probably affects ABC transporter other than ABCG2. PbCl2
had no effect on mitoxantrone efux. In conclusion, it seems that
CdCl2 affects both expression of ABCG2 and function of ABC transporter other than ABCG2. PbCl2 may affect the expression of
ABCG2. Further studies are needed to conrm these results and characterized whether heavy metals affect transplacental kinetics of foreign
compounds.
Kyoto University, Department of Pharmacology, Kyoto, Japan

396
Paper No.: 2340
HEALTH AND DISEASE
INVOLVEMENT OF LARGE-CONDUCTANCE CA2 + ACTIVATED K+ CHANNELS IN ACETYLCHOLINE-EVOKED
VASODILATION OF HUMAN PENILE SMALL ARTERIES
Attila Kun(1), I Kiraly(2), A Varro(1,3), U Simonsen(4), J Pataricza(1),
JG Papp(1,3), L Pajor(2)
(1) University of Szeged, Department of Pharmacology and
Pharmacotherapy, Szeged, Hungary
(2) University of Szeged, Department of Urology, Szeged, Hungary
(3) Hungarian Academy of Sciences, Division of Cardiovascular
Pharmacology, Szeged, Hungary
(4) University of Aarhus, Department of Pharmacology, Aarhus,
Denmark
We have previously found that activation of large-conductance Ca2 + activated K+ (BKCa) channels play an important role in relaxing corporal
smooth muscle function during erection. However we have scarce information about the activation of BKCa channels in endothelium-dependent
vascular relaxation. The purpose of the present study was to investigate
the involvement of BKCa channels in the nitric oxide (NO)- and endothelium derived hyperpolarizing factor (EDHF)-mediated vasodilation in
human penile small arteries. Erectile tissue was obtained in connection
with transsexual operations. Human penile small arteries were mounted
in microvascular myographs for isometric tension recording. In phenylephrine-contracted preparations, acetylcholine (ACh, 1 nM - 10 lM)
induced concentration-dependent relaxations (92 2%), which were
decreased but not abolished in the presence of NO synthase and cyclooxygenase enzyme inhibitors (49 6%). IbTX (0.1 lM), a selective
inhibitor of BKCa channels, inhibited ACh relaxation in the absence and
abolished ACh relaxation in the presence of NO synthase and cyclo-oxygenase enzyme inhibitors. NS11021 (0.1 - 100 lM), an opener of BKCa
channels, induced dose-dependent vascular relaxation with a maximum
of 93 7%. The present ndings demonstrate the contribution of both
NO and EDHF to ACh-evoked vasorelaxation in human penile small
arteries and suggest that BKCa channels are involved in both NO and
EDHF-mediated relaxation of human penile arteries.
Paper No.: 1749

FOCUSED CONFERENCE GROUP: P19 - GENERAL SESSION BIOLOGICS
INVOLVEMENT OF 5-AMP-ACTIVATED PROTEIN KINASE IN
PROLIFERATION AND SURVIVAL OF NEURAL STEM/
PROGENITOR CELLS
Nobuyuki Kuramoto, S Sano, N Tanaka, M Yoneyama, R Nagashima,
K Ogita
Neural stem/progenitor cells (NPCs), which are dened by their ability
to self-renew and differentiate into the three major cell types, neurons,
astrocytes, and oligodendrocytes, play a key role in the development and
maturation of the central nervous system. 5-AMP-activated protein
kinase (AMPK) acts as an efcient sensor for cellular energy state. To
elucidate roles of AMPK in proliferation of NPCs, we evaluated the
effect of an activator and inhibitor for AMPK on proliferative activity in
NPCs derived from the neocortex of embryonic mice. Neurospheres were
prepared from the neocortex of 15-days-old embryonic mice by primary
culturing in DMEM/F12 medium with EGF and bFGF for 9 days in vitro
(DIV). After replating, the cells were further cultured for 6 DIV under
the same condition in the absence or presence of metformin (AMPK activator) or compound C (AMPK inhibitor). Immunoblot analysis revealed
that the level of phospho-AMPK (p-AMPK), but not AMPK, was progressively increased in a culture time-dependent manner. Treatment with
metformin produced enhancement of the proliferative activity and sur-
vival of neurospheres during culture, as well as an increase in the level

of p-AMPK. In contrast, treatments with compound C produced a
decrease in the proliferative activity and survival of neurospheres during
culture. In addition, compound C had the ability to facilitate cell damage.
These results suggest that AMPK is involved in the proliferation and survival in NPCs of embryonic mouse neocortex.
Paper No.: 1281

THE EFFECT OF ESTROGEN SUPPLEMENTATION ON CELL
PROLIFERATION AND DISTRIBUTION OF PROSTATIC
INTERSTITIAL CELLS IN THE RAT PROSTATE
Snezana Kusljic(1,2), B Exintaris(2)
(1) University of Melbourne, Department of Nursing, Carlton, VIC,
Australia
(2) Monash Institute of Pharmaceutical Sciences, Parkville, VIC,
Australia
Benign prostatic hyperplasia (BPH) is associated with proliferation of
prostate tissue and an increase in smooth muscle tone. However, the way
in which the hormonal environment affects cell proliferation and prostatic interstitial cells (PIC) responsible for the maintenance of the smooth
muscle tone is not clear. The present study investigated the effect of
estrogen supplementation on cell proliferation, androgen/estrogen ratio
and expression and/or distribution of PIC. Male Sprague-Dawley rats
were anaesthetized with isourane/oxygen breathing mixture and subcutaneously implanted with silastic capsules. These capsules were either
empty or lled with a 10 or 20 mg of crystalline estrogen. Estrogen
exerted a potent effect on ventral prostate weight which was manifested
as a signicant decrease between controls and the E10 and E20 treated
rats. Active cell proliferation detected as Ki67-positive nuclei was
observed in the stromal and epithelial cells of the ventral prostatic lobes
from estrogen-treated rats and controls. Estrogen supplementation caused
a signicant and dose-dependent increase in prostatic estradiol and 5a-dihydrotestosterone (DHT) concentration but the ratios of either DHT:E2
or E2:DHT were not signicantly affected. PIC were observed in the
region between the bromuscular stroma and the glandular epithelium in
all three experimental groups. E20 treated rats showed a higher expression of PIC than controls and E10 treated rats. The present study provides
novel information regarding the synergistic role of estrogens and androgens in the prostate: estrogen may prevent prostatic hyperplasia via
mechanisms other than affecting cell proliferation or DHT/estrogen ratio.
Paper No.: 3146

IDENTIFICATION AND ELECTROPHYSIOLOGICAL
CHARACTERIZATION OF NEW NR3-CONTAINING NMDA
RECEPTOR MODULATORS
Trine Kvist(1), JR Greenwood(2), KB Hansen(1,3), RP Clausen(1),
SF Traynelis(3), H Brauner-Osborne(1)
Department of Medicinal Chemistry, Copenhagen, Denmark
(2) Schrodinger Inc., New York, NY, USA
(3) Emory University School of Medicine, Department of Pharmacology,
Atlanta, GA,USA
NMDA receptors are ligand-gated ion channels that mediate a slow component of excitatory synaptic transmission in the central nervous system.
Seven different NMDA receptor subunits (NR1, NR2A-D, NR3A-B)
have been cloned that form tetrameric complexes comprised of glycinebinding NR1 and NR3 subunits and glutamate-binding NR2 subunits.
The NMDA receptors are critically involved in physiological and patho-
397
logical processes in the central nervous system, including neuronal development, synaptic plasticity, and some forms of neurodegeneration.
Recombinant NMDA receptors composed of NR1 and NR3 subunits differ from conventional NMDA receptors in that they only require glycine
for activation. The activation mechanism and physiological function of
these receptors are largely unknown. Subtype-selective ligands could be
useful pharmacological tools for investigating the physiological function
and therapeutic prospects of NR3-containing NMDA receptors. Here, we
describe the development of a pharmacological assay for NR3-containing
NMDA receptors in the search for subtype selective modulators. The
pharmacology of the NR3 subunit was isolated by recombinant expression of NR1/NR3 receptors in Xenopus oocytes containing mutated nonfunctional NR1 subunits (NR1-mut). Based on a virtual screen, 103 compounds were selected and investigated by two-electrode voltage-clamp
recordings on the NR1-mut/NR3 receptor. Of the 103 compounds, 12
compounds were identied that either inhibits or positively modulates
the function of the NR3 subunits.
Paper No.: 1964

HEALTH AND DISEASE
VASOCONSTRICTION IN OMENTAL ARTERIES FROM
OBESE HUMAN SUBJECTS
Hye Chung Kwak(1), M Larvin(1,2), P Leeder(2), J Ahmed(2),
S OSullivan(1)
(1) University of Nottingham School of Graduate Entry Medicine,
Department of Surgery, Derby, UK
(2) Royal Derby Hospital, East Midlands Regional Bariatric Surgical
Services, Derby,UK
In vitro studies of human arteries are difcult, because healthy vessels
are rarely available and are inherently variable (Vanhoutte PM, J Hypertension 1999; 17: 1047-1058). Thus few laboratory studies of human
vessels have been published. The aim of this present pilot study was to
examine the effects of various vasoconstrictors on isolated human omental arteries yielded from severely obese patients, to establish a model for
future research into the effect of obesity on vessel function. The study
was approved by Derbyshire Research Ethics Committee. Human omental arteries were dissected from specimens removed atraumatically from
consenting bariatric patients. Vessel rings were mounted using 40lm
wires onto myographs (Danish Myo Technology, Aarhaus). Contractions
in response to vasoconstrictors were recorded using Labchart (AD Instruments Ltd, Oxford). Cumulative dose response curves were obtained to
endothelin (Rmax 9.1 mN +/) 1.1, LogEC50 of -10.1 + /- 0.2, n = 10)
and U4 (Rmax 6.2 mN +/) 0.7, LogEC50 -7.3 + /- 0.2). Vessels were
found to be unresponsive to methoxamine, whilst angiotensin and vasopressin did not lead to sustained or large contractions. These results are
distinct to those of previous studies. Omental vessels in cirrhosis
(Vaughan RB & Angus PW, Gut 2004; 53: 1544-1545) and pregnancy
(Ashworth JR et. al., Am J Obstetrics and gynaecology 1996; 175: 13071312) contracted to noradrenaline and methoxamine, and vasopressin
respectively. Further work will establish what vasorelaxant agents are
effective in these arteries, and focus on whether the effects of vasoactive
agents on omental arteries varies according to the severity of obesity.
Paper No.: 1825

DISEASES
THE ROLE OF ADHESIVE MOLECULES IN EXPERIMENTAL
SABACACHNOID HEMORRHAGE
Aij-Lie Kwan, C-Z Chang, C-L Lin, S-L Howng
Kaohsiung University Medical Hospital, Department of Neurosurgery,
Kaohsiung, Taiwan
ICAM-1, VCAM-1, and E-selectin are important inammatory mediators, they were elevated in the serum of SAH patients . Previous study
found that 6-mercaptopurine (6-mp)was effective in preventing and treatment arterial spasm in experimenntal SAH animal. This study examined
whether levels of adhesion molecules were altered after treatment with 6mp. Animals were injected with autologous blood to induce SAH, intraperitoneal treatment with 6-mp (2 mg/kg) was initiated 1 hr before (prevention) or later (treatment). The compound were given at 24 and 48 hr
post-SAH. ICAM-1, VCAM-1, and E-selectin levels were measure at 72
hr after SAH. Basilar arteries were sliced, their cross-sectional areas were
measured. Corugation of theelastic intima, damage of endothelial layer,
and the smooth muscle necrosis were prominently observed in the SAH
group and vehicle-treated SAH groups, but not in the 6-mp Vtreated
SAH group or in normal controls. No signicant differences were found
in the levels of VCAM-1 among all groups. E-selectin level were
increased in all animals subjected to SAHand SAH plus vehicle groups
compared with control group, but not in the 6-mp group. The levels of
ICAM-1 in the SAHand SAH plus vehicle groups were signicantly elevated (p < 0.001), prevention and treatment with 6-mp reduced ICAM-1
to control levels.ICAM-1 and E-selectin may play an important role in
mediating SAH-induced vasospasm, the reduction of both adhesive molecules after SAH may partly contribute to the antispastic effect of 6-mp.
Paper No.: 2778

ANALGESICS
INVESTIGATION OF ANTINOCICEPTIVE ACTIVITY OF
MIRTAZEPINE AND ITS MECHANISM
Fatma Sultan Kylyc, AE Doan, K Erol
Eskipehir Osmangazi University School of Medicine, Department of
Introduction: It is known that TAD group antidepressants have analgesic
effects. In this study, it was aimed whether the tetrasiclic antidepressant
mirtazapine has antinociceptive activity. Material and Method: In the rst
step, Swiss albino female mice weighing 25-35 g were used. (n:8) Groups
were established Control: Saline, (Mirtazapine 5mg/kg, 10mg/kg, 20mg/
kg), (Mirtazapine 10mg/kg and its combinations L-NAME 100mg/kg; Larginine 100mg/kg; Naloxone 1mg/kg; and Cyproheptadine 50 lg/kg).
One hour after the drugs were given intraperitoneally, Hot Plate, Tail Clip,
and Tail Flick tests were used for evaluating central, and 0,6% acetic acid
writting test for peripheral antinociceptive effects. In the second step;
brain hypocampus of Sprague Dawley type male rats weighing 250 20
g were isolated, and with the Chooper 0,6lm slices were obtained. In perfusion system, they were incubated for one hour at the presence of Krebs
solution. Perfusates were used for nNOS measurements. Brain slices were
homogenized and were used for protein measurements. In vitro groups
were established as Control (Saline), (Mirtazapine 3x10-3M, 4x10-3M,
5x10-3M), (Mirtazapine 4x10-3M and its combinations L-NAME, Larginine, Naloxone, Cyproheptadine 4x10-3M). Results: Mirtazapine did
not show central spinal analgesic activity, but had signicant peripheral
and biphasic analgesic effects at suprasipinal level. In addition, there was
not any signicant difference between different groups in nNOS levels at
brain slices. Conclusion: It is considered that nitrergic pathway does not
have an effect at central supraspinal analgesic activity of mirtazapine,
while opioidergic and serotonergic pathways have a signicant role.
Paper No.: 3430

FAST AORTA REMODELLING FOLLOWING THE ADMINISTRATION AND WITHDRAWAL OF CALCIUM ANTAGONISTS
IN SPONTANEOUSLY HYPERTENSIVE RATS
Jan Kyselovic, V Vaja, P Krenek, J Klimas, P Ochodnicky
398
Comenius University Bratislava, Faculty of Pharmacy, Department of
Pharmacology and Toxicology, Bratislava, Slovak Republic
Background: Long-term calcium antagonists therapy is associated with
morphologic and functional benecial changes in peripheral vasculature.
AIM: We analyzed the vasculoprotective effect of short-term administration of three calcium antagonists in spontaneously hypertensive rats
(SHR). Methods: Morphometrical (wall thickness, cross-sectional area,
media-to-lumen ratio, internal diameter) and functional (KCl- or noradrenaline-induced contraction, endothelium-dependent acetylcholineinduced relaxation) characteristics of isolated aorta were measured in 14week-old control WKY rats, SHR and SHR rats treated either with
verapamil (4 mg/kg), diltiazem (5 mg/kg) or nifedipine (1 mg/kg) twice
daily by subcutaneous injection for 7 days (n = 14 per vehicle treated
rats, n = 6 per CCB treated group). In the nifedipine arm, the animals
were additionally sacriced 24, 72 and 120 hours after the last dose of
nifedipine. Results: Systolic blood pressure was measured by tail cuff
and thoracic aorta was collected for histomorphometric and functional
analysis including acetylcholine-induced endothelium-dependent relaxation. Elevated systolic blood pressure in SHRs was signicantly reduced
by 7-day administration of nifedipine (158 4 mmHg) and diltiazem
(168 8 mmHg), but not of verapamil (196 4 mmHg). In all three
treated groups we observed a regression of vascular hypertrophy. All
drugs signicantly attenuated abnormal aortic wall thickness, cross-sectional area and media-to-lumen ratio, but only nifedipine improved
impaired endothelium-dependent relaxationShort-term administration of
antagonists improved relaxation function of the aorta. After discontinuation of nifedipine treatment we observed a rebound phenomenon. Three
days after withdrawal of nifedipine vascular parameters returned to pretreatment values. Therefore, vasculoprotection by calcium anatgonists is
not restricted to a prolonged blood pressure modulation, but occurs rapidly.
Paper No.: 2965

FOCUSED CONFERENCE GROUP: P18 - NUCLEAR RECEPTOR TARGETS FOR TREATMENT OF DISEASES
DOWNREGULATION OF PPARA EXPRESSION BY GLUCOSE
IN PANCREATIC B-CELLS IS MEDIATED BY CHREBP
Lars la Cour Poulsen(1), M Brgesen(1), SF Schmidt(1), F Frigerio(2),
P Maechler(2), S Mandrup(1)
(1) University of Southern Denmark, Department of Biochemistry and
Molecular Biology, Odense, Denmark
(2) University of Geneva, Department of Cell Physiology and Metabolism, Geneva, Switzerland
The reduction in insulin secretory capacity and b-cell mass observed in
type2 diabetes mellitus (T2DM) is thought to be caused at least in part
byglucolipotoxicity secondary to hyperglycemia and hyperlipidemia. Yet,
themolecular mechanism underlying the pathogenesis of glucolipotoxicity inpancreatic b-cells is not fully understood. The peroxisome proliferatoractivated receptor (PPAR)-a is a key activator of fatty acid
oxidation.PPARa has been shown to protect against lipid induced b-cell
dysfunction.Accordingly, it has been shown by our laboratory that
PPARa potentiatesglucose stimulated insulin secretion (GSIS) in b-cells.
PPARa expressionis downregulated by glucose in b-cells which may be
detrimental to b-cellfunction in the long term. In this work we show that
glucose repression ofPPARa in pancreatic b-cells is mediated by the carbohydrate responseelement binding protein (ChREBP), a glucose stimulated key activator oipogenesis. First, we nd that ectopic expression of
a constitutive activeform of ChREBP represses PPARa expression at low
glucose conditions inINS-1E cells and in pancreatic islets. Second, siRNA mediated knockdown ofChREBP relieves glucose repression in INS1E cells. Further, chromatinimmunoprecipitation combined with deep
sequencing (ChIP-seq) experiments reveala ChREBP binding site located
33 kb upstream the PPARa promoter in rats. Forthat reason, we suggest
that ChREBP mediates repression through binding to thissite. The present work is an early demonstration that ChREBP mediates glucoserepression of gene expression.
Paper No.: 2980

IDENTIFICATION OF DRUG-INDUCED ANAPHYLAXIS USING
THE FRENCH HOSPITAL ADMINISTRATIVE DATABASE
L Lacoin(1), C Lecomte(1), A Kostrzewa(2), V Gilleron(2), N Moore(1),
G Miremont-Salame(1), Francoise Haramburu(1)
(1) Regional Pharmacovigilance Centre, Bordeaux, France
(2) PMSI, CHU de Bordeaux, France
The objective was to assess the interest of the French hospital administrative database (PMSI) for identifying drug-related anaphylactic and anaphylactoid reactions leading to hospitalization or occurring at hospital.
Among the 68572 hospital stays registered in the PMSI of our University
Hospitals from October 1 to December 31 2008, we selected all hospital
stays including an ICD10 code of anaphylactic shock (adverse drug reaction (ADR) or not otherwise specied (NOS), angioneurotic edema, urticaria or allergy (ADR or NOS). The proportion of cases identied for
each code or combination of codes were calculated. For the ADRs identied, clinical features, suspected drugs and management were described.
We found 20 cases of anaphylactic reactions among the 334 selected hospital stays. Five of them were drug-induced anaphylactic shocks, but
were not coded with specic ADRs codes. Seven adverse effects were
identied by general codes of allergy, due to ADR or NOS. The positive
predictive value of each selected code was low, mainly due to the coding
of past medical history of allergy. Five of the six cases reported to the
pharmacovigilance centre were identied in the PMSI. Among the 20
cases, three patients had a previous history of anaphylaxis with the suspected drug. Only one of the ve cases of anaphylactic shock and one of
the nine adverse effects leading to hospitalization were reported the pharmacovigilance centre. The PMSI cannot yet accurately identify druginduced anaphylactic or anaphylactoid reactions, but could be easily
improved with some guidelines for coding.
Paper No. 3122

FOCUS GROUP: P10 - DRUGS FOR HALF THE WORLD:
PAEDIATRIC CLINICAL PHARMACOLOGY
GENOTYPE-SPECIFIC PHARMACODYNAMICS IN BH4
RESPONSIVE PHENYLKETONURIA
Florian B Lagler(1), C Zsifkovits(1), SW Gersting(2), R Fingerhut(3),
H Glossmann(1), C Muntau(2)
(1) Innsbruck Medical University, Department of Medical Genetics,
Molecular and Clinical Pharmacology, Innsbruck, Austria
(2) Ludwig-Maximilians-University,Department of Molecular Pediatrics,
Dr. von Hauner Childrens Hospital, Munich, Germany
(3) University Childrens Hospital, Newborn Screening Laboratory,
Zurich, Switzerland
(4) University of Innsbruck, Institue of Pharmacy and Center for Molecular Biosciences (CMBI), Department of Pharmaceutical Chemistry,
Innsbruck, Austria
Sapropterin (Kuvan), the synthetic form of BH4, was recently
approved as the rst pharmacological treatment for phenylketonuria
(PKU). The in vivo evaluation of the response to sapropterin is essential
for both the detection of responsive individuals and optimal therapy
adjustment. Current protocols for BH4 loading tests are diverse and have
several limitations. Consequently, the understanding of sapropterins
pharmacodynamics is limited. We established a comprehensive test set to
study pharmacodynamic features of sapropterin in living animals and
used it in different PAH decient mice. The pharmacodynamic characteristics varied substantially between our two murine models of human
BH4 responsive PKU. We showed that individualized treatment leads to
good treatment efcacy. Our ndings may provide the basis to establish
improved protocols not only for the safe detection but also for an optimized, genetically driven and individually tailored pharmacological treatment of BH4-responsive PKU.
399
Paper No.: 1141
ROLE OF METABOTROPIC GLUTAMATE RECEPTOR 5 IN
ETHANOL REGULATION OF NMDA RECEPTOR FUNCTION
AND IN ETHANOL-INDUCED CHANGE IN BLOOD PRESSURE
IN RATS
Chih-Chia Lai, W-K Hsieh
Tzu Chi University, Department of Pharmacology, Hualien, Taiwan
Our recent study showed that prolonged ethanol (alcohol) exposure may
activate PKC and Src tyrosine kinase leading to increases in the phosphorylated levels ofNMDA receptors in the lateral horn of spinal cord
where sympathetic preganglionic neurons are located (Hsieh, WK et al,
Br. J. Pharmacol. 2009; 158:806-818). Emerging evidence indicated that
metabotropic glutamate receptors subtype 5 (mGluR5) may play a crucial
role in regulation of several neurobiological effects of ethanol. Because
PKC and Src tyrosine kinase are downstream signals of mGluR5, the
present study were carried out to examine the role of mGluR5 in ethanol
regulation of NMDA receptor phosphorylation and inethanol-induced
cardiovascular effects. Ethanol-induced increases in the total protein
kinase C activity and in the levels of phosphoserine 896 on NR1 subunit
and phosphotyrosine 1336 on NR2B subunit in the lateral horn of spinal
cord were inhibited by post-treatment with MPEP (an mGluR5 antagonist) in vitro and in vivo. Intraperitoneal ethanol (3.2g/kg) caused a
decrease in blood pressure in rats anesthetized with urethane. MPEP
applied by intracerebroventricular injection 5 min after ethanol administration, signicantly increased ethanol-induced depressor effects at 60
and 90 min post-injection of ethanol. The results suggest that activation
of mGluR5 during prolonged ethanol exposure may contribute to ethanol
regulation of NMDA receptor function and of cardiovascular function.
Paper No.: 2477

CYP2D6 GENOTYPE: EFFECT ON SERUM LEVELS,
RESPONSE AND SIDE EFFECTS OF VENLAFAXINE
A Lalla(1), Lazara Montane Jaime(1)
The University of the West Indies, Department of Pharmacology, St
Augustine,Trinidad & Tobago
Introduction: Venlafaxine, a CYP2D6 substrate, is one of the most commonly prescribed antidepressant drugs in Trinidad and Tobago. In this
paper we investigated the inuence of CYP2D6 genotype on venlafaxine
steady-state concentration, subjects response and side effects. This has
not been investigated in Indian or African populations which constitute
the two major ethnic groups of the islands. Methods: Fifty-nine patients
included in the study received venlafaxine orally daily doses between 75
and 225 mg. Steady-state trough levels were taken on day 14, 28, 42 and
56 for therapeutic drug monitoring. Plasma concentrations of venlafaxine
enantiomers (VEN), O-desmethylvenlafaxine (ODV), N-desmethylvenlafaxine (NDV) and NO-didesmethylvenlafaxine (NODDV) were measured using GC-MS. Additionally, the CYP2D6 genotype and
dextromethorphan metabolic ratio (MR) was determined. Results: Of the
patients included, three were genotyped as ultrarapid metabolizer (gUM),
36 as extensive metabolizers (gEM) and 20 as intermediate metabolizers
(gIM). High inter-individual variability in serum levels of VEN was
detected and signicantly associated with the CYP2D6 genotype. Despite
signicant different VEN and NDV concentrations, the ODV and the
serum concentration of VEN and ODV were not signicantly different
between the CYP2D6 genotype groups. The ODV/VEN ratios as a measure of O-demethylation were determined and were signicantly correlated with the CYP2D6 genotype as well as the metabolic ratio.
Conclusions: Clinical outcome measurements revealed that patients with
gIM showed a better and faster therapeutic response starting at Day 14
on the CGI and HAMD-17 score than gEMs. Differences in side effects
were not signicant between the different genotype groups.
Paper No.: 2103

ROLE OF CORTISTATIN IN MODULATING THE GROWTH OF
COLON CANCER CELLS
Francis FY Lam, ESK Ng, CH Cho
The Chinese University of Hong Kong, School of Biomedical Sciences,
Shatin, Hong Kong, PR China
Cortistatin is a neuropeptide structurally related to somatostatin. The
inuence of cortistatin on cancer development is uncertain, but since it
has agonist activities on all somatostatin receptor types (SSTR1-5), and
somatostatin analogues are known to be useful in treatments of some
cancers, it is suspected that cortistatin might have useful anti-cancer
properties. In this study, we have investigated the actions of cortistatin
and somatostatin on growth of HT29 and SW1116 colon adenocarcinoma cells. Expressions of mRNA and proteins of the peptides and their
receptors were detected by RT-PCR and Western blot, respectively. Their
actions on cell proliferation were determined by the 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction method for
24 and 48 hours. In both HT29 and SW1116 cells, RT-PCR studies demonstrated positive expressions for cortistatin, somatostatin, SSTR1,
SSTR2, SSTR3, and SSTR5, but not SSTR4. Western blot also conrmed this pattern of protein expressions in the two types of cells, except
that proteins of somatostatin and SSTR3 were not found, suggesting that
cortistatin and the SSTR1,2,5 could play a role in cancer growth. Indeed
in the MTT studies cortistatin but not somatostatin produced a more consistent and signicant inhibition on cell proliferation in both colon cancer
cell lines. These ndings suggest that cortistatin but not somatostatin, is
the endogenous activator of SSTRs in modulating the growth of colon
cancer cells.
Paper No.: 2195

THE EFFECT OF IMATINIB MESYLATE ON SPONTANEOUS
ACTIVITY IN THE GUINEA-PIG PROSTATE
Michelle Lam(1), A Dey, B Exintaris
Action, Melbourne, VIC, Australia
Benign prostatic hyperplasia is a common disease amongst aging men
that can result in lower urinary tract symptoms brought on by an
enhanced smooth muscle tone. This may be attributed to spontaneous
contractions generated by cells identied in the human and guinea-pig
prostate that are similar to the interstitial cells of Cajal (ICCs) termed
prostatic interstitial cells (PICS). This study aimed to investigate the role
of PICs using imatinib mesylate, a Kit tyrosine kinase receptor inhibitor,
in spontaneous electrical and contractile activity of the guinea-pig prostate. Standard tension recording and microelectrode techniques were used
on mature guinea-pig prostate (750 850g) to study imatinib mesylate at
1lM, 5lM, 10lM and 50lM for 30 minutes. Spontaneous slow wave
activity of the prostate was not signicantly affected by imatinib mesylate; however, while statistically insignicant, imatinib mesylate at 10lM
(n = 8) reduced the control slow wave parameters of amplitude, duration
and frequency to 90.2%, 87.5% and 90.7%, respectively. Spontaneous
contractile activity also showed the greatest effects at 10lM (n = 4),
where the amplitude, duration and frequency of control contractions were
reduced to 71.4% (P < 0.05), 47.9% (P < 0.05) and 47% (P < 0.05),
400
respectively. Imatinib mesylate also gradually slowed control contractions to 89.4% at 1lM (n = 7), 59.4% at 5lM (n = 4), 34.8% at 10lM
(n = 4) and increased to 97% at 50lM (n = 4). While imatinib mesylate
signicantly reduces spontaneous contractions at 10lM, no signicant
effect is seen in spontaneous electrical activity, suggesting that imatinib
mesylate affects Ca2 + mobilisation in the guinea-pig prostate.
Paper No.: 1941

CYTOCHROME P450 2A5 EXPRESSION IS DEPENDENT ON
REDOX-SENSITIVE TRANSCRIPTION FACTOR NRF2 IN
LIVER
Virpi Lamsa(1), A-L Levonen(2), H Hurttila(2), S Yla-Herttuala(2),
M Yamamoto(3), J Hakkola(1)
(1) University of Oulu, Department of Pharmacology and Toxicology,
Oulu, Finland
(2) Kuopio University, Department of Biotechnology and Molecular
Medicine, Kuopio, Finland
(3) Tohoku University Graduate School of Medicine, Department of
Medical Biochemistry, Tohoku, Japan
Nuclear factor erythroid-derived 2 -like 2 (Nrf2) activates transcription of
numerous cytoprotective antioxidant response element (ARE) target
genes, including several Phase II detoxication genes. Potential Phase I
target genes are poorly characterized. Murine Cytochrome P450 2a5
(Cyp2a5) is upregulated by various hepatotoxic compounds and pathophysiological hepatic conditions. Recently, we discovered a functional
ARE in the distal Cyp2a5-5 -2,4 kb promoter region, which is activated
in hepatocytes by cadmium. We hypothesize that the various CYP2A5
inducing phenomena might be connected via common regulatory mechanism involving sensitive activation of Nrf2 in altered cellular redox status. In our studies, we have extensively characterized regulation of
Cyp2a5 by Nrf2 and compared it to a well characterized target gene
heme oxygenase-1 (Hmox1). Nuclear Nrf2 accumulation was detected in
mouse primary hepatocytes treated with PbCl2, methyl-Hg or phenethyl
isothiocyanate. Both CYP2A5 and HMOX1 were induced by each compound, although more rapid and transient induction was detected for
HMOX1. In Nrf2(-/-) primary hepatocytes, Nrf2 was found to be crucial
for CYP2A5 induction but not for HMOX1. Both CYP2A5 and HMOX1
were upregulated by Nrf2 overexpression and downregulated by Keap1
or Bach1 overexpression, although CYP2A5 response was consistently
more potent. Nrf2 decient animals showed signicantly attenuated
hepatic CYP2A5 basal expression, while HMOX1 expression was unaffected. Our results imply a key role for Nrf2 both in constitutive and
inducible activation of CYP2A5. Therefore, CYP2A5 upregulation could
be used as a sensitive indicator for hepatic activation of Nrf2/ARE pathway to predict hepatotoxicity in early drug development studies.
Paper No.: 2283

VIRAL CHEMOKINE RECEPTOR US28 ACTIVATES
B-CATENIN SIGNALLING PATHWAY
and in vivo (Maussang D, PNAS 2006; 103: 13068-13073). US28

induces a pro-angiogenic and transformed phenotype, binds a spectrum
of chemokines and constitutively activates Gaq/11 signaling pathways.
Injection of US28-expressing NIH-3T3 cells into nude mice promotes
tumorigenesis and angiogenesis via Cyclooxygenase-2 (Maussang D,
Cancer Res 2009; 69: 2861-2869). By means of microarray studies with
the 3T3 cells stably expressing US28 the oncogenic Wnt (or b-catenin)
signaling pathway is found to be differentially expressed. US28 is shown
to dose-dependently increase Tcf-Lef transcriptional activation. Mechanistically the canonical b-catenin pathway does not seem to be involved.
We are currently investigating the underlying mechanism of the US28
induced b-catenin signaling pathway.
Paper No.: 3429

LACK OF BAMBI (BMP AND ACTIVIN MEMBRANE-BOUND
INHIBITOR) IN MUTANT MICE INCREASES THE ANALGESIC
EFFECT OF MORPHINE, WITHOUT FAVOURING
TOLERANCE DEVELOPMENT OR WORSENING
WITHDRAWAL SEVERITY
Aquilino Lantero(1), D Merino(1), ML Laorden(2), A Daz(3),
MA Hurle(1)
(1) University of Cantabria Medical School, IFIMAV, Department of
Physiology and Pharmacology, Santander, Spain
(2) University of Murcia Medical School, Murcia, Spain
(3) University of Cantabria Medical School, CIBERSAN, IBBTEC,
Santander, Spain
BAMBI is a transmembrane pseudoreceptor that modulates negatively
cellular signals mediated by cytokines of the transforming growth factor
(TGF-b) family. Absence of BAMBI results in increased levels of TGFb signalling and, in our previous experience, BAMBI-KO mice unravels
the role of TGF-b as negative modulators of nociception, under physiological (acute pain) and pathological (neuropathic pain) conditions, by
transcriptional regulation of genes encoding endogenous opioid peptides.
Herein, we analyzed whether the absence of BAMBI has also consequences on the postsynaptic responses elicited by exogenously administered opioid drugs. Opioid receptor coupling with G-proteins, as
determined in spinal cord membranes by measuring [35S]GTPcS- binding stimulation with either l- (DAMGO), d- (DESLET) or j- (U69593)
agonists, was similar in BAMBI-KO and WT mice. Thermal nociceptive
responses (tail-ick test) were lower in KO than in WT mice and tail
withdrawal latencies following acute morphine (0.1 to 10 mg/kg) were
higher, in absolute terms, in the KO group. Similarly, KO mice treated
chronically with 10 mg/kg/day morphine displayed longer tail withdrawal latencies than WT all throughout the 8 days of treatment. There
was no signicant effect of genotype in the time course of tolerance
development or in the intensity of the withdrawal syndrome precipitated
with naltrexone on the 8th day. Our results suggest that the increased levels of TGF-b signalling, characteristic of BAMBI-KO mice, result in a
lower basal sensitivity to acute painful stimulus that increases the analgesic effect of morphine, but without favouring tolerance development or
worsening withdrawal severity.
Supported by RD06/001/1016 and SAF2007-65451.
Ellen Langemeijer(1,2), E Slinger(1), D Maussang(1), M Siderius(1),

R Leurs(1), M Smit(1)
(1) VU University Amsterdam, Department of Medicinal Chemistry,
Amsterdam, The Netherlands
(2) Leiden/Amsterdam Center for Drug Research, Amsterdam, The Netherlands
Human cytomegalovirus (HCMV), a widely spread herpes virus, is suggested to play a role in tumor progression. We have shown that US28, a
HCMV-encoded chemokine receptor, induces tumorigenesis both in vitro
Paper No. 3382

INFLUENCE OF GENDER ON CLOPIDOGREL DISPOSITION
IN HEALTHY VOLUNTEERS
O Laosa, Alberto M Borobia, B Mosquera, SH Lei, JL Lopez-Duran, B
Duque-Bascunana, P Guerra-Lopez, AJ Carcas-Sansuan, J Fras-Iniesta
401
La Paz University Hospital, School of Medicine, Autonoma University
of Madrid, Clinical Pharmacology Service, Madrid, Spain
Objetive: To evaluate the effect of gender on the variability and disposition of clopidogrel. Methods: Ninety-one healthy young (18-32 years)
volunteers (58.2% males) were randomized in an open, single-dose,
two-sequence, crossover trial with. 75 mg tablets were administered as
a single dose after at least 8 hours fast with a wash out of 7 days.
Serial blood samples were collected up to 32 h. and plasma analysed
by HPLC. Non-compartmental pharmacokinetic analysis was performed; AUC and Cmax were log-transformed for statistical analysis.
Statistical analysis: The inuence of gender on clopidogrel disposition
was analyzed by a linear mixed model for repeated measures including,
formulation, period, sequence and sex as xed effects and subject
nested sequence*sex as random effect. Pharmacokinetic parameters
were dose/weight adjusted. AUC(0-) and Cmax Test/References (T/R)
were calculated to analyse gender variability differences. Results: Both
formulations were found bioequivalent according to international standards. Statistical signicant gender effects were observed for AUC(0-)
(p = 0.013), but not for Cmax (p = 0.624). AUC(0-innity) (pg*h/ml)/
(mg/kg) was higher in males than in females: 1176.15 (95%CI 953.37,
1750.99) vs 772.78 (95%CI 607.89, 992.27). No statistical signicant
differences were observed between T/R AUC(0-) (p = 0.126) and Cmax
(p = 0.145) when male and female ratios were compared. Conclusion:
Gender-related differences in pharmacokinetic parameters (dose/weight
adjusted) indicate an about 52% higher clopidogrel disposition in males
than in females.
Paper No.: 1960

USE OF SEROTONINERGIC MEDICATIONS AND CARDIAC
VALVULOPATHY: A NESTED CASE-CONTROL STUDY IN
THE HEALTH IMPROVEMENT NETWORK (THIN) DATABA
Francesco Lapi(1), F Nicotra(2), A Rubino(3), L Scotti(2), M Thomson(3), A Vannacci(1), A Zambon(2), G Corrao(2), A Mugelli(1)
(1) University of Florence, Department of Pharmacology, Florence, Itlaly
(2) University of Milan-Bicocca, Department of Biostatistics, Milan,
Italy
(3) EPIC, London, UK
Background: The 5-HT2B serotonin receptors mediate the valvular tissue differentiation into broblast-like cells. The association between
Cardiac Valvulopathy (CV) and cabergoline or pergolide has been demostrated. Objectives: To quantify the risk of CV associated with use of
Serotoninergic Medications (SM) enable to stimulate 5-HT2B receptors.
They comprise selective serotonin reuptake inhibitors; tricyclic antidepressants; monoamine oxidase inhibitors; and other antidepressants
(e.g., venlafaxine, mirtazapine). Material/Patients: Cohort patients aged
18-89, with an incident prescription of SM between 1th January 1990
and 30th June 2008 and at least 1 year of registration before the prescription, were selected in THIN database. Cases were dened by medical coding for CV during follow-up and matched 1:10 to controls by
sex, age (3 years), and month and year of cohort entry. A conditional
logistic regression was used to estimate Odds Ratios (OR) and 95%
Condence Intervals (CI). Exposure status was assessed by cumulative
drug use for < 1; 1-5; 6-24 and > 24 months. Results: Among 862,600
SM users, incidence rate of CV was 4.5 per 10,000 person years at
risk. Cases (n = 2,200) and controls (21,930) aged 61.3 (+14.2) and
61.1 (+14.2) years old, respectively; females (62%) outnumbered males
among cases and controls. Compared with subjects who took SM for
less than one month, odds ratio was 0.97 (95%CI: 0.85-1.13), 0.99
(0.86-1.16) and 0.99 (0.85-1.16) among subjects who were treated with
SM for 1-6 months, 6-24 months and >24 months, respectively. Conclusion: These ndings do not reveal any relationship between use of
SM and the risk of CV.
Paper No.: 2491

DISEASES
EFFECTS OF BIO-LIBERATED GOLD IONS ON HUMAN
MONONUCLEAR CELLS. AN IN VITRO STUDY OF
DISSOLUCYTOTIC GOLD LIBERATION FROM METALLIC
GOLD IMPLANTS
Agnete Larsen(1), T Christensen(2), C Hst(2), M Stoltenberg(1),
G Danscher(1), AM Larsen(2)
(1) University of Aarhus, Institute of Anatomy, Department of
Neurobiology, Aarhus,Denmark
(2) University of Aarhus, Institute of Microbiology and Immunology,
Aarhus, Denmark
The anti-inammatory properties of gold salts have been used in medicine for over 75 years. The pharmaco-dynamic mechanisms include prevention of NF-  B activation in synovial cells reducing the
transcription of COX-2 and other pro-inammatory genes. Current use of
gold compounds are limited as up to 15-45% of patients have been
known to discontinue treatment due to side effects involving skin, liver,
lung, blood and kidney. Dissolucytosis is an macrophage-induced extracellular process of bio-liberation of gold ions known taking place both in
vitro (Larsen A et al Histochem Cell Biol 2007 128 1-6) and in vivo
(Larsen A et al Histochem Cell Biol 2008 130 681-92). Due to dissolucytosis, metallic gold compounds can serve as permanent gold ion
source of highly-localized gold liberation directed directly towards and
inamed tissue. The present study investigates whether bio-liberation of
gold affects the interaction between macrophages and T-lymphocytes
which are central for the function of the immune system. Human mononuclear cells exposed to inactive varizella zoster vira (vzv) were cultivated with or without access to a gold surface for six days.
Autometallography showed gold uptake in monocytes and to a minor
degree in lymphocytes. A lymphocyte transformation test (LTT) showed
a statistically signicantly smaller T-cell response following gold treatment. This study is the rst to show that bio-liberation of gold ions exhibit immunosuppression on human cells by interfering with the
macrophage- T-lymphocyte interaction and further studies into the clinical implications of this discovery seems warranted.
Paper No.: 1592

DISEASES
ADENOSINE A2B RECEPTOR MEDIATED INHIBITION OF
ANTI-IGE INDUCED HUMAN MAST CELL ACTIVATION
Hang Yung Alaster Lau, KH Yip, H Wise
Hong Kong,Shatin, PR China
The endogenous nucleoside adenosine is recognised as an anti-inammatory and immunosuppressive agent and has been demonstrated to suppress IgE-receptor mediated activation of human lung mast cells. In the
current study, we employed primary human cultured mast cells
(HCMCs), which are phenotypically similar to human lung mast cells, to
investigate the adenosine receptor subtype and the mechanisms underlying this inhibitory action of adenosine. IgE-receptor mediated HCMC
activation was induced by anti-IgE and the levels of histamine and IL-8
release were assayed for functional responses. For evaluating the cellular
mechanisms, activities of MAPKs and NF-jB signalling pathways were
determined by western blot while adenylate cyclase activities and intracellular calcium concentration [Ca2 + ]i were measured by cAMP accumulation and Fura 2-AM uorescence respectively. Adenosine exhibited
dose-dependent inhibition of anti-IgE induced release of histamine and
IL-8 at concentrations above 0.1 lM. This inhibitory action of adenosine
402
was reversed by non-selective (CGS15943) and specic A2B (PSB1115,
MRS1506) but not specic A2A (SCH442416) adenosine receptor antagonists. Following incubation of HCMC with adenosine, an elevation of
cAMP level was observed while the anti-IgE induced [Ca2 + ]i increase
as well as activation of p-ERK, p-JNK and NF-jB pathways proteins
were reduced. In conclusion, the current study suggests that adenosine
inhibits human mast cells activation through the G -coupled A2B receptor. The subsequent cAMP accumulation coupled with the suppression of
MAPKs and NF-jB activation as well as [Ca2 + ]i increase contribute to
the reduced degranulation and cytokine synthesis.
Paper No.: 3400

MECHANISTIC MODELLING OF ANTIGEN-SINK EFFECT
FOR THERAPEUTIC MONOCLONAL ANTIBODIES: A CASE
STUDY
YY Lau, L Roskos, M Liang, H Lu, M Zusmanovich, Bing Wang
MedImmune LLC, Global PK-PD & Bioanalysis, Hayward, CA, USA
Monoclonal antibodies (mAb) against cell membrane-associated antigens
are usually subject to the target mediated clearance, or the antigen-sink
effect. In this investigation, a mechanistic population model was developed to describe the PK of CAM-3001, a fully-human human monoclonal IgG against GM-CSFRa, in rheumatoid arthritis (RA) patients.
Serum CAM-3001 concentration data from a single-dose Phase 1 study
in RA patients were modeled using NONMEM VI. In this model other
than the rst-order elimination pathway by the reticuloendothelial system, CAM-3001 binds to GM-CSFRa and the antibody-receptor complex is subsequently internalized and degraded. To avoid model overparameterization, the internalization rate of GM-CSFR/CAM-3001 complex was xed to a value determined from in vitro quantitative confocal
uorescent imaging studies. The quasi-steady-state assumption assumes
that the elimination rate of the antibody-receptor complex is similar or
faster than the dissociation of the complex. The pharmacokinetic proles
of CAM-3001 in RA patients were adequately described by the mechanistic model. The estimated clearance of the linear elimination pathway
is 3.79 mL/kg/d, typical for the clearance of endogenous IgG in humans
by the reticuloendothelial system. The estimated central volume of distribution is 39.8 mL/kg, close to the serum volume. The baseline receptor
level and steady-state constant were 0.0780 and 1.03 nM, respectively.
The model also provided predictions for GM-CSFRa receptor occupancy
following CAM-3001 treatment. In conclusion, the mechanistic modelling of the antigen-sink effect provides insight into the target receptor
occupancy and supports rational clinical trial design for therapeutic
monoclonal antibodies.
Paper No.: 1139

INVOLVEMENT OF THE FIRST TRANSMEMBRANE
SEGMENT OF THE HUMAN 2A-ADRENOCEPTOR IN THE
SUBTYPE SELECTIVITY OF BULKY ANTAGONISTS
Jonne M Laurila(1), H Xhaard(2), G Wissel(3), JO Ruuskanen(1),
MS Johnson(3), M Scheinin(1)
(1) Department of Pharmacology, Drug Development and Therapeutics,
University ofTurku, Finland
(2) University of Helsinki, Faculty of Pharmacy, Centre for Drug
Research,University of Helsinki, Faculty of Pharmacy, Helsinki, Finland
bo Akademi University, Department of Biochemistry and Phar(3) A

macy, Turku, Finland
The human a2A-adrenoceptor binds several large antagonist ligands
(ARC239, chlorpromazine, prazosin, spiperone, spiroxatrine) with 10100 fold lower afnity than the a2B- and a2C-adrenoceptors. Previous
mutagenesis studies of the binding pocket (TM2-7 and XL2) have not
been able to explain the subtype selectivity of these bulky a2-adrenoceptor antagonists. We have now elucidated the possible involvement of the
extracellular amino terminus and TM1 in subtype selectivity with the chimaeric receptor approach. Eight receptor chimaeras were made: six
where TM1 and the N-terminus were swapped between the three a2adrenoceptor subtypes and two where only TM1 was swapped between
a2A- and a2C-adrenoceptors. The receptor chimaeras and all wild-type
human a2-adrenoceptors were expressed in CHO cells and tested for
ligand binding using [3H]RX821002 as radioligand and nine competitor
ligands. The afnities of three antagonists (spiperone, spiroxatrine and
chlorpromazine) were statistically signicantly improved by TM1 substitutions of the a2A-adrenoceptor. The improvements in afnity for these
ligands were up to two-, eight- and ve-fold. Reciprocal effects were not
seen for a2B/C-adrenoceptor based chimaeras. We conclude that TM1 is
involved in dening the specic pharmacological prole of the human
a2A-adrenoceptor. The exact mechanism of its involvement is not known,
but molecular modelling suggests indirect effects rather than specic side
chain interactions.
Paper No.: 1203

PROBING THE BINDING AND FUNCTION OF ORTHOSTERIC,
ALLOSTERIC AND BITOPIC AGONISTS AT THE M4
MUSCARINIC ACETYLCHOLINE RECEPTOR
Katie Leach, AE Davey, PM Sexton, A Christopoulos
Monash Institute of Pharmaceutical Science, Drug Discovery Biology
Laboratory, Melbourne, VIC, Australia
The increasing discovery of selective small molecule agonists for G protein-coupled receptors (GPCRs) raises questions with regards to whether
such ligands utilize all or part of an allosteric site(s) as part of their
mechanism. It is also unknown whether putative allosteric agonists promote similar activating GPCR conformations to those promoted by classic orthosteric agonists. The current investigation used mutagenesis to
show that, in contrast to the prototypical orthosteric agonists, ACh and
pilocarpine, and the bitopic (orthosteric/allosteric hybrid) agonist, McNA-343, the binding of the novel allosteric agonist/modulator,
LY2033298, does not involve orthosteric site amino acid residues, such
as the highly conserved Asp112(3.32) in transmembrane domain 3
(TM3) of the M4 muscarinic acetylcholine receptor. This suggests that
LY2033298 exerts its agonism purely via interaction with an allosteric
site. Although the afnity of LY2033298 was unaffected by substitution
of Asp112(3.32) for Glu or Asn, the ability of LY2033298 to allosterically potentiate the binding of ACh was greatly reduced at this mutant
receptor. Substitution of Trp108(3.28) and Leu109(3.29) with Ala
reduced the binding and/or cooperativity of LY2033298, suggesting that
these residues may also form part of the allosteric binding site. Substitution of Asp106(3.26) for Ala, Leu109(3.29) for Ala or Asp112(3.32) for
Glu reduced the ability of orthosteric, allosteric and bitopic agonists to
activate the receptor, indicating an important role for these residues in
the global activation mechanism. Nonetheless, at each of these mutants,
LY2033298 restored function when co-administered with ACh, suggesting that allosteric and orthosteric sites can cooperate to overcome large
GPCR activation barriers.
403
Paper No.: 2184
PHARMACOKINETIC COMPARISONS OF SUSTAINED- AND
IMMEDIATE-RELEASE ORAL FORMULATIONS OF
CILOSTAZOL IN HEALTHY KOREANS
Donghwan Lee(1,2), LA Lim(1,2), SB Jang(1,2), YJ Lee(1,2),
H Son(1,2), S Cho(1,2), J Chung(1,2), K Park(1,2)
(1) Yonsei University College of Medicine, Department of Pharmacology, Seoul, Korea
(2) Yonsei University College of Medicine, Brain Korea 21 Project for
Medical Science, Seoul, Korea
This study investigated the safety and pharmacokinetic proles of a
newly developed sustained-release (SR) formulation of cilostazol in
healthy subjects. A randomized, single-dose, open-label, 2-period crossover study was conducted in 26 healthy Koreans. They were randomly
assigned to two groups to receive either a 200 mg SR tablet once or a
100 mg immediate-release (IR) tablet twice 12 hours apart in period 1,
and the alternate formulation in period 2 with a 7-day washout period.
Blood samples were collected up to 72 hours and AUClast(AUC
between 0 and 72 hour), AUC AUC0-, Cmax, tmax, and t were
obtained for plasma concentrations of cilostazol and its active metabolites (OPC-13015 and OPC-13213). The geometric mean ratios(GMR)
(90% CI) of SR to IR formulation were 92% (83.7 ~ 101.3%) for AUClast, 91% (82.5 ~ 99.7%) for AUCinf, and 109% (96.2 ~ 123.2%) for
Cmax for cilostazol, 90% (82.0 ~ 98.1%) for AUClast, 91% (83.4 ~
100.1%) for AUC AUC0-, and 100% (88.7 ~ 112.1%) for Cmax for
OPC-13015, and 89% (81.3 ~ 97.0%) for AUClast, 93% (83.8 ~
102.4%) for AUC AUC0-, 100% (89.2 ~ 112.8%) for Cmax for OPC13213. For SR versus IR formulation of Cilostazol, t was 12.71 versus
10.22 hours, and tmax was 6.12 versus 3.9 hours. The results for the
metabolites were similar to cilostazol. Adverse events were also similar
in nature and frequency between the 2 formulations. No serious adverse
events were reported. This study shows that the new SR cilostazol tablet
shows comparable pharmacokinetic and safety proles.
Paper No.: 2750

NEUROPROTECTIVE EFFECT OF RICE BRAN OIL ON
AMYLOID B PROTEIN (25-35)-INDUCED NEUROTIXICITY:
IN VITRO AND IN VIVO
Chronic administration of RBO (2 and 3 ml/kg, 7 days, P.O) inhibited

Ab (25-35)-induced memory impairment. From these results, we suggest
that the antidement ia effect of RBO is due to its neuroprotective effect
against Ab (25-35)-induced neurotoxicity and that RBO may have a benecial effect in treatment of neurodegeneration in AD.
Paper No.: 2171

CYP2C9*3 AND *13 ALLELES SIGNIFICANTLY AFFECTED
THE PHARMACOKINETICS OF CELECOXIB IN HEALTHY
KOREAN SUBJECTS
Seok-Yong Lee, C-I Choi, J-W Bae, M-J Kim, C-G Jang
Sungkyunkwan University, College of Pharmacy, Laboratory of Pharmacology, Su-Won, South Korea
Celecoxib, a selective cyclooxygenase 2 (COX-2) inhibitor, is in a class
of nonsteroidal anti-inammatory drugs (NSAIDs) and is used for the
treatment of rheumatoid arthritis and osteoarthritis. Celecoxib is hepatically eliminated mainly by hydroxylation, and it is primarily mediated by
CYP2C9, a polymorphic drug metabolizing enzyme. So we investigated
effects of CYP2C9 genetic polymorphisms on the pharmacokinetics of
celecoxib in healthy Korean subjects. After genotyping for CYP2C9 with
volunteers, 26 healthy Korean subjects were selected for this study. They
were grouped according to CYP2C9 genotype, CYP2C9EM (n = 12,
CYP2C9*1/*1), CYP2C9IM (n = 12, CYP2C9*1/*3 or CYP2C9*1/*13),
and CYP2C9PM (n = 2, CYP2C9*3/*3). A single oral dose of 200 mg
of celecoxib was administered to each subject, and plasma concentration
of celecoxib and celecoxib carboxylic acid (CCA) was determined by
using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) system. AUCinf of celecoxib in CYP2C9IM group
(6302 1417 ngohr/mL) was signicantly higher than that in
CYP2C9EM group (3960 1340 ngohr/mL, P = 0.0004), and oral clearance (CL/F) of celecoxib in CYP2C9IM group was signicantly lower
than that in CYP2C9EM group (P = 0.0013). Other parameters of celecoxib were not signicantly different between two groups. Notably, Cmax,
t and AUC of celecoxib in CYP2C9PM group were dramatically
increased (159%, 273% and 691% of CYP2C9EM group), and CL/F
was decreased compared to those in other two groups. The AUC ratio of
celecoxib over CCA was higher in CYP2C9IM (1.6-fold) and
CYP2C9PM groups (6.9-fold) compare with the CYP2C9EM group. In
conclusion, CYP2C9 genetic polymorphisms can affect the pharmacokinetics of celecoxib in Koreans.
Hong-Kyu Lee, YH Seong

Alzheimers disease (AD) is the progressive neurodegenerative disorder
and the leading cause of senile dementia (selkoe et al., Physiol Rev.
2001: 81(2):741-66). Deposition of b-amyloid protein (Ab), a major constituent of the senile plaque, is thought to be an important cause in developing AD. Rice bran, the major byproduct of the rice milling industry, is
the source of a high quality vegetable oil, rice bran oil (RBO). RBO has
attracted much medicinal attention due to its strong hypocholesterolemic
properties known to be attributable to its balanced fatty acid composition
and high levels of antioxidant phytochemicals such as oryzanols, tocopherols and tocotrienols. We demonstrated that RBO extracted from
Korean rice plant (Cheongwon, Chungbuk, Korea) has protective effect
on Ab (25-35)-induced neurotoxicity in cultured neurons and memory
impairment in mice in the present study. Neurons cultured from 16-days
old rat fetus brain and exposed to 10 lM Ab (25-35) for 36 h induced
apoptotic neuronal death. RBO (0.01-10 ul/ml) inhibited 10 uM Ab (2535)-induced neuronal cell death, increase of intracellular Ca2 + concentration and reactive oxygen species generation in cultured neurons. Memory
impairment was established by intracerebroventricular microinjection of
Ab (25-35) (15 nmol) and measured by a passive avoidance test in mice.
Paper No.: 2712

GENETIC POLYMORPHISMS OF CYP2C9 AND THEIR
INFLUENCES ON WARFARIN SENSITIVITY IN MECHANICAL
HEART VALVE REPLACEMENT PATIENTS
Su-Jun Lee(1), YJ Jang(1), E-Y Cha(1), H-S Kim(1), M-S Hwang(1),
SS Lee(1), J-G Shin(1,2)
(1) Inje University College of Medicine, Department of Pharmacology
andPharmacogenomics Research Center, Busan, Korea
(2) Inje University College of Medicine, Department of Clinical Pharmacology, Busan, Korea
CYP2C9 single nucleotide polymorphisms (SNPs) are important in safe
and effective oral anticoagulation with warfarin use.The objectives of this
study were to determine the distribution of CYP2C9 variants in Koreans
and investigate their association with warfarin dose requirements in
patients who received mechanical heart valve replacements (MHVRs).
All 9 exons, intronexon junction, and promoter region of CYP2C9 were
amplied and the amplied fragments were directly sequenced in 50
healthy normal Koreans. Additional direct DNA sequencing of the
404
CYP2C9 gene was conducted in 36 of the 267 MHVR patients who
required low maintenance warfarin doses without carrying CYP2C9*3 and
VKORC1 1173T mutations. The effects of CYP2C9 genetics on warfarin
maintenance dose was assessed in 267 MHVR patients. Thirty-nine SNPs
including seven previously unidentied SNPs were identied in 50 Koreans by direct DNA sequencing. One of the CYP2C9 haplotypes exhibited
an association with warfarin low dose requirement. This haplotype consisting of -1565C>T, -1188T>C, IVS3 + 197G>A, IVS3-334C>T, IVS365G>C, IVS4-115A>G, and IVS5-73A>G was found in 15% of 36
MHVR patients who required low warfarin doses, while 4% of 50 normal
healthy subjects exhibited this haplotype. One of the SNPs comprising this
haplotype, -1565C>T, apparently changed a protein binding pattern as
observed in electrophoretic mobility shift assay. The haplotype including 1565C>T, -1188T>C, IVS3 + 197G>A, IVS3-334C>T, IVS3-65G>C,
IVS4-115A>G, and IVS5-73A>G seems to be associated with lower warfarin dose requirement and this haplotype could be considered in the
development of warfarin dose prediction model for Asian populations.
Paper No.: 2713

IDENTIFICATION OF CYP19A1 SINGLE NUCLEOTIDE
POLYMORPHISMS IN KOREANS AND COMPARATIVE
ANALYSIS OF FUNCTIONALLY IMPORTANT CYP19A1
VARIANTS WITH DIFFERENT ETHNIC GROUPS
Su-Jun Lee(1), W-Y Kim(1), I-S Jung(1), J-Y Choi(1), SS Lee(1),
J-G Shin(1,2)
(1) Inje University College of Medicine, Department of Pharmacology
andPharmacogenomics Research Center, Busan, Korea
(2) Inje University College of Medicine, Department of Clinical
Pharmacology, Busan,Korea
Aromatase, encoded by the CYP19A1 gene, is a key enzyme in the biosynthesis of estrogen. In an effort to screen for CYP19A1 single nucleotide polymorphisms (SNPs) in Koreans, the CYP19A1 gene was directly
sequenced in 50 normal subjects. A total of 19 variations were identied:
four in exons, ten in introns, six in 5-untranslated region (UTR), and
one in 3-UTR. The distribution of CYP19A1 (TTTA)n polymorphisms,
was such that the most frequent allele was (TTTA)7 (66%), followed by
(TTTA)11 (30%), (TTTA)12 (3%), and (TTTA)13 (1%). The order of the
frequency distribution of CYP19A1 variations, other than the (TTTA)n
variant, was: IVS6-106T>G and IVS7-79A>G (57%), 1531C>T (56%),
IVS5-16T>G and IVS6 + 36A>T (54%), -196A>C and -77G>A (49%),
IVS2-59A>G and 240A>G (48%), -278C>T (31%), 27TCTI>D (29%), 144C>T and -588G>A (19%), 790C>T (16%), and other minor alleles
with less than 5% frequency. Nineteen variations were used to characterize linkage disequilibrium (LD) structures at the CYP19A1 locus, which
resulted in three LD blocks. Nine tagging SNPs in the CYP19A1 were
determined. Ethnic different frequencies of functionally important
CYP19A1 SNPs were also collected and compared. Identication of
CYP19A1 SNPs with LD blocks and the tagging SNPs creates an important resource for genotype/phenotype association studies for estrogenrelated phenotypes.
Paper No.: 1436

PROGESTERONE RECEPTOR ACTIVATION OF
EXTRA-NUCLEAR SIGNALLING PATHWAYS IN
REGULATING P53 EXPRESSION IN VASCULAR
ENDOTHELIAL CELLS
Wen-Sen Lee(1), S-P Hsu(2)
(2) National Taiwan University, Graduate Institute of Physiology, Taipei,

Taiwan
Previously, we showed that progesterone exerted an anti-angiogenic
activity and inhibited the proliferation of human umbilical vein endothelial cells (HUVEC) through a p53-dependent pathway (Hsu et al., Cellular and Molecular Life Sciences. 2008, 65:3839-3850). Now we
investigate further the molecular mechanism underlying the hormone
activity. In cultured HUVEC, progesterone increased the formation of
progesterone receptor (PR)-Src complex and Src activity. These effects
were abolished by pretreatment with RU486, suggesting the involvement
of PR in these progesterone-induced activities. Luciferase reporting assay
demonstrated that the progesterone-increased p53 transactivity was prevented by PP2 (a Src inhibitor), Src Kinase Inhibitor I (SKI-I), Ras inhibitory peptide, or a farnesyltransferase inhibitor (FTI). Pretreatment with
PP2 or SKI-I prevented the progesterone-induced increase of p53 protein
level and membrane translocation of Kras. Transfection of HUVEC with
dominant-negative Erk2 prevented the progesterone-induced decrease of
thymidine incorporation and increases of the p53, p21 and p27 protein
levels. Moreover, progesterone-induced phosphorylation of Raf-1 and
Erk1/2 was abolished by pretreatment with Ras inhibitory peptide (Ras
InP) or sulindac sulde. Semiquantitative RT-PCR analysis revealed that
the administration of Src antisense oligonucleotide prevented the progesterone-increased in p53 mRNA level. Our data suggest that the Src/Kras/
Raf-1/Erk signalling pathway contributes to the progesterone-induced
up-regulation of p53 in HUVEC. These ndings highlight PR activation
of extra-nuclear signalling pathways in regulating p53 and cell cycle progression in HUVEC.
Paper No.: 2116

ANALGESICS
DOES PHENY-N-TERT-BUTYLNITRONE (ROS SCAVENGER)
HAS THE ANTINOCICEPTIVE EFFECT THROUGH SPINAL
MECHANISM IN FORMALIN RAT?
Won Hyung Lee(1), YK Ko(2)
(1) Chungnam National University School of Medicine, Department of
Anesthesiology and Pain Medicine, Daejon, PR China
(2) Chungnam University Hospital, Daejon, PR China
Introduction: This study is to assess the effects of ROS scavenger, pheny-N-tert-butylnitrone (PBN) and 5, 5-dimethylpyrroline-N-oxide
(DMPO), by systemic or intrathecal administration in rats subjected to
the formalin test. Methods: 5% formalin 50ul was injected in the left
hind paw of sprague-Dawley rats (N = 100, 250-300g) after injection of
various dose of intraperitioneal PBN (saline, 20mg/kg, 50mg/kg, 100mg/
kg), intraperitoneal DMPO (saline, 30mg/kg, 100mg/kg), or intrathecal
PBN (saline, 0.3mg, 1mg). Number of inches was measured for 60 min
after formalin injection.To verify the oxidative stress, nitration of protein
- nitrotyrosine - in lumbar spinal cord was evaluated by the immunohistochemistry. Results: Formalin injected into the rat hind paw induced a
biphasic nociceptive behavior in PBN and DMPO group. Systemic PBN
and DMPO IP group, nor PBN IT group diminished the nociceptive
behaviors dose-dependently during the phase. The immunohistochemical
staining of nitrated proteins was nearly undetectable in normal (naive)
rats. The immunohistochemical evaluation in saline pre-treated rats
revealed the increase of nirtated proteins in gray matter in the lumbar
spinal cord, and signicantly diminished upon systemic and intrathecal
PBN pre-treatment. So there are a discrepancy in the effect on pain
behavior and the result of inmmunohistochemistry of intrathecal PBN.
Conclusion: This study suggest that systemic nor intrathecal ROS scavenger posses the antinociceptive property in rats subjected to the formalin
test and ROS are involved in the development and maintenance of pain.
However the antinociceptive spinal mechanism of ROS scavengers is
still needed further study in formalin rat.
(1) Taipei Medical University, Graduate Institute of Medical Sciences,

Taipei, Taiwan
405
Paper No.: 2690
A PHASE I CLINCICAL STUDY OF CKD-732 IN
COMBINATION WITH CAPECITABINE AND OXALIPLATIN
IN PATIENTS WITH METASTATIC COLORECTAL CANCER
Yoon Jung Lee(1,2), S Shin(1), HC Chung(1), K Park(1,2)
(1) Yonsei University College of Medicine, Department of
Pharmacology, Seoul, Korea
(2) Brain Korea 21 Project for Medical Science, Yonsei University,
Seoul, Korea
CKD-732 is a recently developed synthetic analog of fumagillin with antiangiogeneic activity. This study was conducted to investigate tolerability and pharmacokinetics of CKD-732 in combination with capecitabine
and oxaliplatinin Korean patients with advanced or metastatic colorectal
cancer after failure to standard therapy. A total of 9 patients were
enrolled. For each 3-week cycle, the patients recevied a 1-hour i.v infusion of CKD-732 twice a week, a 2-hour i.v infusion of oxaliplatin and
an oral capecitabine b.i.d. for 2 weeks. The dose escalation scheme was
2mg/m2 (n = 3), 5mg/m2 (n = 4) and 10mg/m2(n = 2) of CKD-732 incombination with oxaliplatin (130mg/m2) and capecitabine (2000mg/
m2). Bloodsamples were collected at 1, 2, 4, 6, 10, 12, 13, 15hr (day 1)
and 0, 1, 2, 3,4, 10hr (day 11) after the start of CKD-732 infusion. For
CKD-732 on day 1, area under the concentration-time curve and maximum concentration per unit dose were 11.92lghr/L/mg and 6.94lg/L/
mg, respectively, and half-life was 1.83hr, which were not signicantly
different from day 11. Overall, dose proportionality was apparent in
CKD-732 pharmacokinetics and no evidence of pharmacokinetic interactions was found among the 3 drugs. 2 patients who received the dose of
10mg/m2 experienced a dose limiting toxicity, yielding the maximum
tolerated dose of 5mg/m2 in CKD-732 for the given combination treatment in Korean patients. Further studies incluidng efcacy assessment
are needed to validate the use of this new agent.
Paper No.: 1830

HEALTH AND DISEASE
METHYL PALMITATE IS THE RETINA-DERIVED RELAXING
FACTOR
Yuan-Chieh Lee(1,3,4), H-H Chang(7,8), C-H Liu(7,8), M-F Chen(2,8),
P-Y Chen(2,5,8), H-S Kuo(2,5), TJ-F Lee(2,5,6,7,8,9)
(1) Buddhist Tzu Chi General Hospital, Departments of Ophthalmology,
Hualien, Taiwan
(2) Buddhist Tzu Chi General Hospitalm, Departments of Research,
Hualien, Taiwan
(3) Tzu Chi University, Department of Medicine, Hualien, Taiwan
(4) Tzu Chi University, Graduate Institute of Medical Sciences, Hualien,
Taiwan
(5) Tzu Chi University, Institutes of Pharmacology and Toxicology, College of Life Sciences, Hualien, Taiwan
(6) Tzu Chi University, College of Life Sciences, Department of Neuroscience, Hualien, Taiwan
(7) Tzu Chi University, College of Life Sciences, Department of of Life
Sciences, Hualien, Taiwan
(8) Tzu Chi University, College of Life Sciences, Center for Vascular
Medicine, Hualien, Taiwan
(9) Southern Illinois University School of Medicine, Department of Pharmacology, Springeld, IL, USA
The retina has been shown to spontaneously release a vasodilating substance (Delaey and Van de Voorde, Circ Res. 1998). The identity of this
retina-derived relaxing factor (RRF) is not known. We determined if palmitic acid methyl ester (PAME) or methyl palmitate is the RRF. Using a
superfusion bioassay cascade technique with rat isolated retina as donor
tissue and rat aortic ring as detector tissue, we demonstrated that the ret-
ina upon superfusion with Krebsj solution spontaneously released RRF

(indicated by aortic ring relaxation) and PAME (measured by GC/MS).
The RRF and PAME release was calcium-dependent, since the release
was almost abolished when the retinas were superfused with calcium-free
Krebs solution. Furthermore, aortic dilations induced by RRF and
PAME were not affected after heating their solutions at 70 C J for 1 hr,
suggesting that both are heat stable. Exogenous PAME concentrationdependently induced aortic dilation with EC50 of 0.82 0.75 pM. The
dilation induced by RRF and PAME was inhibited in parallel by 4-aminopyridine (4-AP, 2 mM) and tetraethyl ammonium (TEA, 10 mM), but
was not affected by TEA at 1 mM or 3 mM, glibenclamide (3 ugM), or
iberiotoxin (100 nM). Following hexane extractions, RRF- and PAMEcontaining Krebs solutions caused no or reduced aortic dilations, which
were abolished by 4-AP. It is concluded that RRF and PAME act similarly on the voltage-dependent K+ (Kv) channels on aortic smooth muscle cells, causing aortic dilation. The close similarities in biochemical
and pharmacological properties of PAME and RRF suggest that PAME
is the RRF.
Paper No.: 1417

ACTIVATION OF GROUP II METABOTROPIC GLUTAMATE
RECEPTORS INHIBITS NEURONAL EXCITABILITY AND
EPILEPSY IN THE ENTORHINAL CORTEX VIA
DOWN-REGULATION OF PROTEIN KINASE A PATHWAY
Saobo Lei, C Yang, Z Xiao, J Pribula
University of North Dakota, Department of Pharmacology, Physiology
and Therapeutics. Grand Forks, ND, USA
The entorhinal cortex (EC) is an essential structure involved in temporal
lobe epilepsy. Here we examined the roles of group II metabotropic glutamate receptors (mGluRs) in the EC and explored the underlying cellular and molecular mechanisms. Application of LY354740, a highly
selective and potent agonist of group II mGluRs, generated hyperpolarization and decreased the excitability of the neurons in the EC.
LY354740-induced hyperpolarization required the functions of Gi proteins, adenylyl cyclase (AC) and protein kinase A (PKA). LY354740mediated depression on neuronal excitability was mediated by activation
of a background K+ channels. Application of LY354740 dramatically
depressed the epileptic activity induced by bath application of picrotoxin
in the EC slices. Our results demonstrate that activation of group II
mGluRs may represent a novel approach for the treatment of epilepsy.
Paper No.: 536

THE EFFECT OF PHELA, A TRADITIONAL MEDICINE
EXTRACT, ON INTERLEUKIN-2 IN A RAT MODEL
Makhotso R Lekhooa(1), A Walubo(1), J Du plessis(1), G Matsabisa(2)
(1) University of the Free State, Department of Pharmacology,
Bloemfontein, South Africa
(2) Medical Research Council, Indigenous Knowledge Systems Lead
Programme, CapeTown, South Africa
Phela is a traditional medicine extract that is being investigated for
immune boosting properties in patients infected with the human immune
deciency virus (HIV). Therefore, the aim of this study was to determine
the effect of Phela on interleukin 2 (IL-2), a major stimulant of cell mediated immunity, in a rat model. Sprague Dawley rats were used and
approval from the animal ethics committee was obtained. Three groups
of 14 rats each were treated daily with either normal-saline (control),
Phela-only (test-group), Phela+cyclosporine-A (Phela-CycA: negative
406
control), and a fourth group was inoculated once with inuenza vaccine
(positive control). Thereafter, 7 animals from each group were sacriced
after 7 and 14 days of treatment. Serum IL-2 was measured by ELISA.
On days 7 and 14, IL-2 concentrations (pg/ml: median [range]) in the
control, (232 [167-460] and 274 [215-328]) and Phela-only treated group
(233 [174-355] and 224 [217-295]) were similar. However, although not
statistically signicant, IL-2 levels (pg/ml: median[range]) were higher in
the Phela+CycA treated group (325 [268-433] and 280 [206-518]) compared to the Phela-only-treated group. The response to inuenza stimuli
was moderate at 7 days (294 [245-373]) and this returned to normal by
14 days (214 [183-309]. These results show that Phela did not stimulate
IL-2 of the normal immune system but stimulated it when the immune
system was supressed by cyclosporine-A. This implies that Phela may
stimulate IL-2 in patients with a comprimised immune system such as in
HIV positive patients. In conclusion, Phela is an immune stimulant.
Paper No.: 1208

A COMPREHENSIVE METHOD FOR IDENTIFICATION OF
PHELA, A TRADITIONAL HERBAL MEDICINE, USING
DIFFERENT CHROMATOGRAPHIC TECHNIQUES
Makhotso R Lekhooa(1), A Walubo(1), JB Du Plessis(1),
MG Matsabisa(2)
(1) University of the Free State, Department of Pharmacology,
Bloemfontein, South Africa
Programme, CapeTown, South Africa
Phela is a herbal mixture of four African traditional medicinal plants that
has been used for decades in wasting conditions and, after successful
observation studies in humans, is now being developed by the Medical
Research Council as an immune booster for patients with compromised
immune system. Therefore, a chromatographic ngerprint of Phela was
needed for quality control purposes. Here, a comprehensive method for
ngerprinting of Phela is described. It involved extraction of the product
by either acidic extraction or a simple salting-out method, followed by
Thin Layer Chromatography (TLC) and/or preparative Column Chromatography (CC) that were supported by High Performance Liquid Chromatography with UV-detector (HPLC-UV), HPLC with uorescencedetector (HPLC-FL), HPLC with Photo Diode Array detector (HPLCPDA) and Gas-Chromatography-Mass Selective Detector (GC-MSD)
spectrometry. An internal standard was used where appropriate. Peak
area and retention times were used for validation of the methods and ngerprints, respectively. The ngerprints were successfully used to differentiate Phela from another herbal product made from Hypericum
perforatum (St. Johns Wort) thereby illustrating their high potential for
use in differentiating Phela from other herbal medicines. By generating
ngerprints of phela using different chromatographic techniques based
on a standardized extraction process, this approach provides a variety of
methods from which laboratories can select and use according to availability of expertise and/or instruments and cost-benet analysis. Furthermore, it will enable wide application in quality control of the product
using validated methods, thereby promoting effective monitoring of the
nished product in all countries that will use it.
Paper No.: 1209

THE USE OF METABOLITE PEAKOKINETICS TO MONITOR
PHELA, A HERBAL MEDICINE, IN PLASMA AFTER ORAL
ADMINISTRATION IN A RAT MODEL
Makhotso R Lekhooa(1), A Walubo(1), JB Du Plessis(1),
MG Matsabisa(2)
(1) University of the Free State, Department of Pharmacology, Bloemfontein, South Africa
Programme, Cape Town, South Africa
Phela is a herbal traditional medicine that is under development for use
as an immune booster in immune compromised individuals. Therefore,
before major in vivo investigations could be done, there was a need to
establish a plasma marker for concentration monitoring of Phela. Chromatographic separation was achieved on a reverse phase column with
70% acetonitrile at 0.5 ml/min.. Emission and excitation wavelengths
were 210 and 290 nm, respectively. Sprague Dawley rats were used and
approval from the animal ethics committee was obtained. Three groups
of 5 rats each were administered with Phela (15.4 mg/kg) and 1 rat from
each group was sacriced at 1, 2, 4, 6 and 8 hours. On the HPLC analysis, a new peak in samples from treated animals was observed at 9.2
minutes, which implied that it was a metabolite of Phela. Plasma concentration was expressed as peak area per unit plasma volume (PK-area/L)
and this was used to derive the relevant pharmacokinetic parameters. The
metabolites half-life was 3.47 0.35 hours and reached maximum concentration at 4.67 1.15 hours. It was estimated that the concentration at
steady state (Css) would be 47.52 5.94 PK-area/L with no drug accumulation (Acc index =.009 0.004) on chronic dosing. The use of peak
area per unit volume to derive pharmacokinetics of unknown compounds
(peakokinetics) as well as to conrm ingestion of Phela were demonstrated with a hope that they may appeal to those experiencing similar
problems with monitoring of herbal products of which little is known.
Paper No.: 1561

TREATMENT OF CHILDRENS ASTHMA WITH A LIPID
EXTRACT OF THE NEW ZEALAND GREEN LIPPED MUSSEL
(PERNA CANALICULUS) (LYPRINOL) - A DOUBLE BLIND,
RANDOMISED CONTROLLED TRIAL IN CHILDREN WITH
MODERATE TO SEVERE CHRONIC OBSTRUCTIVE ASTHMA
James Lello(1,4,5), A Liang(2), E Robinson(3), D Leutenegger(1),
A Wheat(1)
(1) Asthma New Zealand The Lung Association (Inc.), Department of
Clinical Trials, Auckland, New Zealand
(2) Paediatric Allergy Specialist, St Heliers, Auckland, New Zealand
(3) University of Auckland, Medical and Health Science Campus
(Tamaki), Department of Community Health, Auckland, New Zealand
(4) University of Auckland, Medical and Health Science Campus
(Tamaki), Department of General Practice and Primary Healthcare,
Auckland, New Zealand
(5) Marsden Medical Ltd., Auckland, New Zealand
Shellsh are rich in the omega-3 fatty acid eicosapentaenoic acid (EPA).
EPA may potentially act to reduce airway inammation and bronchoconstriction.The efcacy and safety of an oral standardised lipid extract of
New Zealand green lipped mussel (Perna canaliculus) marketed as Lyprinol was assessed as maintenance therapy for children with asthma. A
total of 71 children aged 6 to 13 years were enrolled in a 16-week, single
centre, double-masked, placebo-controlled, parallel-group trial and randomly assigned to receive either Lyprinol or placebo (2 capsules twice
daily). Patients were maintained on inhaled corticosteroid (ICS) and asneeded b-agonist therapy throughout the study. If stable at monthly study
visits ICS dose was reduced by 25%. Safety monitoring included endtidal breath nitric oxide measurement. Primary outcome measures were
inhaled corticosteroid dose reduction with secondary outcome measures
of asthma exacerbation rate, asthma symptom score, Juniper quality of
life score and use of short acting b agonist. Lyprinol improved the percentage of children reporting little or no trouble with their asthma at three
months of treatment (97% vs. 76% p < 0.06). Both groups were able to
tolerate a dose reduction of ICS with no signicant difference between
palcebo and Lyprinol group. There were fewer mild and moderate asthma
exacerbations overall in the Lyprinol group. Lyprinol was well tolerated.
We conclude that Lyprinol is a safe nutritional supplement taken by chil-
407
dren with moderate asthma and that larger prospective controlled studies
should explore its potential use as an addition to conventional treatment.
Paper No.: 1828

HEALTH AND DISEASE
DUAL ECE/NEP INHIBITION AND ETA/B RECEPTOR
ANTAGONISTS: TWO WAYS OF INHIBITING ENDOGENOUS
ET-1 EFFECTS IN RESISTANCE ARTERIES
Pieter Lemkens, J Nelissen, JGPJ Bost, BJA Janssen, PMA Schiffers,
JGR De Mey
Maastricht University, Department of Pharmacology and Toxicology,
Endothelin-1 (ET-1), a 21 amino acid vasoconstrictor peptide, can be
formed by endothelin converting enzyme (ECE) and by neutral endopeptidases (NEP). The DOCA-salt hypertension model is reported to display
enhanced ET-1 function. Hypothesis: We investigated in isolated rat mesenteric resistance arteries (MA) whether (1) synthesis of endogenous ET1 can be inhibited by the dual ECE/NEP inhibitors phosphoramidon
(PRD) and SOL-1 (an analogue of SLV-309) and whether (2) the mixed
ETA/B-receptor antagonist bosentan (BOS) and PRD affect vasomotor
responses to a candidate stimulus of ET-1-release (BJP 156, 1239, 2009).
Methods: Vascular synthesis of ET-1 was induced by thrombin (TRB, 5
IEU for 3 hours) in isolated MA from 16 week old WKY rats and monitored by RIA. Arterial contractile responses to K+ were recorded by wire
myography. Results: Incubation with TRB increased ET-1 content in MA
by 3-fold. This increase was signicantly reduced in the presence of
PRD (10 uM) or SOL-1 (10 uM). BOS (10 uM) as well as PRD (10
uM) reduced K+-induced contractions in isolated 3th order MA of
DOCA-salt hypertensive rats, no effect of BOS was measured in arteries
of control rats. After mechanical removal of the endothelium, BOS had
no effect on K+-induced contractions in arteries of DOCA-salt hypertensive rats. Conclusions: (1) PRD and SOL-1 are effective in inhibiting
ET-1 synthesis and (2) in resistance arteries of DOCA-salt hypertensive
rats, endothelium-derived ET-1 enhances arterial smooth muscle tone.
Paper No.: 1883

THE THIOPURINE METHYLTRANSFERASE TPMT *1/*3A
GENOTYPE IS ASSOCIATED WITH A BETTER TREATMENT
OUTCOME FOR CHILDREN WITH ACUTE LYMPHOBLASTIC
LEUKAEMIA
Lynne Lennard(1), C Cartwright(1), R Wade(2), S Richards(2),
C Mitchell(2), S Kinsey(3), T Eden(4), J Lilleyman(5), A Vora(1)
(1) University of Shefeld, Department of Clinical Pharmacology,
Shefeld, UK
(2) University of Oxford, UK
(3) University of Leeds, UK
(4) University of Manchester, UK
(5) University of London, UK
The UK ALL97 trial for children with acute lymphoblastic leukaemia
compared the efcacy and toxicity of thioguanine versus mercaptopurine
during maintenance chemotherapy. Both thiopurines form cytotoxic thioguanine nucleotide (TGN) metabolites. Thiopurine methyltransferase
(TPMT) methylates the thiopurine ring and reduces TGN formation. The
ALL97 trial included a pharmacogenetic study which investigated the
impact of TPMT on treatment outcome. TPMT was genotyped in blood
samples obtained from children (aged 1 18 years) entered onto ALL97
between January 1997 and June 2002. Samples were screened for the
TPMT*3 family of low-activity variant alleles. The log-rank statistic was
used to compare groups for relapse-free (RFS) and event-free survival
(EFS). Samples were received from 1565 children (81% of children in
the trial), median age 4 years. TPMT genotype data was available for
1002 children. Five year RFS (83%) and EFS (81%) did not differ by
randomised thiopurine. Both RFS and EFS differed signicantly by
TPMT genotype. At 5 years, RFS for TPMT wild-type, homozygous
TPMT*1/*1, children (n = 913) was 82.6%, for TPMT*1/*3A (n = 77)
89.5% and for TPMT*1/*3C (n = 11) 40.0%. Five year EFS was 79.7%,
88.3% and 36.4% respectively. The difference in outcome between the
TPMT*1/*3A and TPMT*1/*3C children (RFS p = 0.01, EFS p = 0.004)
remained after stratifying for ethnic origin. The improved RFS and EFS
for TPMT*1/*3A children compared with TPMT*1/*1 children was of
weak statistical signicance (p = 0.09 and p = 0.06 respectively). Within
the ALL97 trials the TPMT*1/*3A genotype was associated with a better
treatment outcome.
This work was supported by the Leukaemia Research Fund.
Paper No.: 984

HEALTH AND DISEASE
IMPAIRMENT OF BOTH NITRIC OXIDE AND
ENDOTHELIUM-DERIVED HYPERPOLARIZING FACTOR
IN STREPTOZOTOCIN-INDUCED DIABETIC SMALL
MESENTERIC ARTERY
Chen Huei Leo(1), J Ziogas(2), JL Favaloro(1), OL Woodman(1)
(1) RMIT University School of Medical Sciences, Health Innovation
Research Institute, Bundoora, Victoria, Australia
(2) University of Melbourne, Victoria, Australia
Nitric oxide (NO) and endothelium-derived hyperpolarizing factor
(EDHF) contribute to endothelium-dependent relaxation in small vessels
such as mesenteric artery. Diabetes is known to impair endotheliumdependent relaxation, however, it is not established whether it affects
either or both NO and EDHF. The aim of this study is to evaluate the
mechanism of endothelium-dependent relaxation of mesenteric arteries
from type 1 diabetic rats. Wire myography was employed to examine
ACh-induced relaxation of mesenteric arteries. Superoxide levels, measured by L-012 and lucigenin-enhanced chemiluminescence, were significantly increased in diabetic mesenteric arteries compared to normal
arteries. Diabetes signicantly reduced the sensitivity to ACh (pEC50,
diabetic, 6.77 0.14 vs normal, 7.74 0.09, n = 10-11, p < 0.0001) in
mesenteric arteries. When the contribution of NO to relaxation was abolished by L-NNA (100 lM) and ODQ (10 lM), the sensitivity to ACh
was signicantly decreased in the diabetic arteries compared to normal
arteries (pEC50 diabetic, 6.68 0.12 vs pEC50 normal, 7.08 0.11, n =
11-12, p < 0.05), indicating an impaired contribution of EDHF. Conversely, when the contribution of EDHF was inhibited with the combination of TRAM-34 (1 lM), apamin (1 lM) and iberiotoxin (100 nM), the
maximum relaxation (Rmax) to ACh was signicantly decreased in diabetic arteries (Rmax diabetic 17 5 vs Rmax normal, 81 5, n = 8-9, p
< 0.0001), suggesting that the contribution of NO was also impaired by
diabetes. Western blot analysis also demonstrated eNOS uncoupling in
diabetic mesenteric arteries. Taken together, this study demonstrated that
endothelium-dependent relaxation was impaired at 10 weeks of diabetes
due to a reduced contribution of both NO and EDHF.
Paper No.: 985

HEALTH AND DISEASE
7 DAY TREATMENT WITH 3,4-DIHYDROXYFLAVONOL
PREVENTS ENDOTHELIAL DYSFUNCTION IN MESENTERIC
ARTERIES FROM STREPTOZOTOCIN-INDUCED DIABETIC
RATS
Chen Huei Leo(1), J Ziogas(2), JL Favaloro(1), OL Woodman(1)
(1) RMIT University School of Medical Sciences, Health Innovation
Research Institute, Bundoora, Victoria, Australia
(2) University of Melbourne, Victoria, Australia
408
3, 4-dihydroxyavonol (DiOHF) prevents diabetes-induced endothelial
dysfunction in aorta, however, it is unclear whether it is vasoprotective
in the microvasculature. The aim of this study was to evaluate the effect
of treating diabetic rats with DiOHF (7 days, 1mg/kg per day, s.c) on
mesenteric artery function. Superoxide levels, determined by lucigeninenhanced chemiluminescence, were signicantly increased in diabetic
mesenteric arteries but treatment with DiOHF reversed that effect. AChinduced relaxation of mesenteric arteries was assessed using wire myography. Diabetes signicantly reduced the sensitivity to ACh and treatment with DiOHF prevented endothelial dysfunction (pEC50 diabetic,
6.86 0.12 vs diabetic+DiOHF, 7.49 0.13, n = 11, p < 0.05) in mesenteric arteries. When the contribution of nitric oxide (NO) to relaxation
was eliminated by L-NNA (100 lM) and ODQ (10 lM), the sensitivity
to ACh was signicantly decreased in the diabetic arteries (pEC50 diabetic, 6.63 0.15 vs normal, 7.14 0.12, n = 12, p < 0.05), and treatment with DiOHF had no effect (pEC50 diabetic, 6.85 0.12). Thus,
DiOHF did not affect the contribution of endothelium-derived hyperpolarizing factor (EDHF) to relaxation. When the contribution of EDHF
was inhibited with the potassium channel blockers, TRAM-34 (1 lM),
apamin (1 lM) and iberiotoxin (100 nM), the maximum relaxation
(Rmax) to ACh was signicantly decreased in diabetic arteries (Rmax
diabetic 31 9 vs normal, 68 10, n = 8-9, p < 0.05), suggesting that
diabetes impaired NO activity but DiOHF treatment prevented that effect
(Rmax diabetic+DiOHF 69 6, n = 11). Treatment with DiOHF in normal rats had no effect on superoxide levels or endothelial function. The
antioxidant DiOHF prevents endothelial dysfunction by preserving NO
activity.
Paper No.: 1893

ANALGESICS
ANTINOCICEPTIVE EFFECTS OF BIPHALIN AND ITS
ANALOG AM 94
Sheila Leone, L Brunetti, G Orlando, A Mollica, L Recinella,
C Ferrante, A Chiavaroli, C Di Nisio, P Di Michele, R Shohreh,
M Vacca
Biphalin is an opioid linear octapeptide [(Tyr-D-Ala-Gly-Phe-NH-)2] displaying high potency both in vivo and in vitro. When administered intracerebroventricularly (ICV), biphalin was shown to be 3-fold more potent
than morphine and almost 7-fold more potent that etorphine in eliciting
antinociception. AM 94 [(Tyr-D-Ala-Gly-Phe)2-Piperazine] is a selective
agonist of mu-and d-opioid receptors. We assayed the analgesic activity
of biphalin and AM94 (1 nmol) in rats, after ICV administration in male
adult Wistar rats (200250 g). Responsiveness to nociceptive stimulation
was measured by means of a conventional hot plate apparatus, set at the
temperature of 54.0 0.4C. Under these conditions, the baseline latencies obtained in pre-experimental tests, with group of 6-8 rats, ranged
from 5.1 0.7 to 6.2 0.8. A cut-off time of 30s was adopted. Antinociceptive responses were expressed as % maximum possible effect
(%MPE) and calculated according to the following formula: (TL-BL)/
(30-BL) x 100, where TL=test latency, BL=baseline latency, 30 = cut-off
time in seconds. Data were expressed as the mean %MPE SEM for
each measurement time. Differences between groups were analyzed
using Anova followed by Student-Newman-Keuls test. P < 0.05 was
considered statistically signicant. Results indicate that biphalin had the
maximal antinociceptive effect (60% MPE) at 30 min, while AM94 had
a higher maximal antinociceptive effect (95% MPE) at 90 min, and sustained analgesia up to 180 min. Our data suggest that AM94 could effect
a powerful analgesia after ICV administration.
Paper No.: 1504

POSSIBLE ROLE OF ENDOCANNABINOIDS IN THE
CARDIOPROTECTIVE EFFECTS OF A POLYUNSATURATED
FATTY ACID-RICH DIET IN RATS
Istvan Lepran, N Morvay
University of Szeged Faculty of Medicine, Department of Pharmacology
and Pharmacotherapy, Szeged, Hungary
Polyunsaturated fatty acid-rich diets inuence the substrate availability
for the synthesis of endogenous cannabinoids, eg. arachidonoyl-ethanolamide. The aim of present investigations was to study the possible contribution of endocannabinoids to the effect of linoleic acid-rich diet during
ischemia-reperfusion arrhythmias in rats. Male, Sprague-Dawley rats
were fed a diet supplemented with 10% sunower seed oil (rich in linoleic acid, SSO) or pork fat (rich in saturated fats, SF) for 4 weeks. At the
end of the feeding period in pentobarbitone anaesthesia and articial ventilation myocardial ischemia was produced by ligation of the left main
coronary artery for 6 min, followed by reperfusion. SSO diet signicantly decreased the incidence of ventricular brillation (VF; 24% vs.
76%) and ventricular tachycardia (VT; 43% vs. 92%) during reperfusion
after 6 min myocardial ischemia. Pretreatment with AM251 (cannabinoid
CB1 antagonist; 3 mg/kg ip.) or with AM630 (CB2 antagonist; 3 mg/kg
ip.) increased the incidence of VT (100% and 79%, respectively) in SSO
fed animals. On the other hand, inhibition the transport of endogenous
cannabinoids (VDM11, 5 mg/kg ip.), thereby prolonging their effect at
the receptor, offered signicant protection against the development of VF
(36% vs. 76%) in SF fed animals, and improved the survival rate (79%
vs. 40%). These ndings may suggest that endogenous cannabinoids
have a cardioprotective effect during myocardial ischemia-reperfusion,
and their increased synthesis may contribute to the development of the
cardioprotective effect of polyunsaturated fatty acid rich diet.
Supported by ETT 494/2006, ETT 233/2009 and TAMOP-4.2.2-08/12008-0013.
Paper No.: 1536

LATIN AMERICA A SUCCESSFUL EMERGING MARKET FOR
CLINICAL RESEARCH
Federico Lerner
PRA International, Department of Clinical Operations and Project
Management, Buenos Aires, Argentina
As clinical trials become more globalized, pharma and biotech companies are redrawing their maps and rethinking their strategies. By doing
so they are forced to explore, to them, unknown regions. During this presentation Dr Lerner will educate and inform the audience about the benets and challanges of conducting clinical trials in Latin America. This
region is becoming an important part of global clinical development with
trial growth increasing steadily each year. The reasons for this growth
are explained by the resources available in Latin America: experienced
investigators with large patient populations, nave patient populations,
well equipped study institutions, relatively streamlined regulatory processes, strong clinical research infrastructure (laboratories, drug depot
services, and logistics), internationally recognized treatment standards
and quality data proven by various audits and inspections. Attendees will
receive practical advice on critical points to take into account when conducting trials in Latin America and in depth knowledge on how Latin
America as a region can be a successful region for your next clinical
trial.
409
Paper No.: 2053
CARDIOVASCULAR DYSFUNCTION AND THE ROLE OF
COX-2 IN TWO RAT MODELS OF DIABETES
Joanne YT Leung, SL Lim, CCY Pang
University of British Columbia, Department of Anaesthesiology, Pharmacology & Therapeutics, Vancouver, BC, Canada
Chronic diabetes is associated with low-level inammation, as evident
by the up-regulation of pro-inammatory cytokines and enhanced expression of the inducible isoform of cyclooxygenase (COX-2). This study
investigated if selective inhibition of COX-2 by nimesulide restores cardiovascular responses to adrenaline in two rat models of diabetes,
induced through an injection of streptozotocin (STZ, 60 mg/kg i.v.)
alone, or a combination of high fructose (FRU) feeding (60% of caloric
intake) and subsequent injection of STZ. Wistar rats (5 weeks old) were
fed a normal or high FRU diet starting day 0 until the end of the study.
After 2 weeks, half of the rats in each diet regimen were given STZ. Following 6 weeks of feeding, the effects of adrenaline on cardiovascular
responses were determined in the four groups of anesthetized rats (control, FRU, STZ, FRU-STZ) before and after acute i.v. injection of nimesulide (3 mg/kg). Both the STZ and FRU-STZ groups exhibited
hyperglycemia and signicantly (P < 0.05) reduced left ventricular contractility, mean arterial pressure (MAP) and mean circulatory lling pressure (MCFP, index of body venous tone) responses to adrenaline,
relative to the two control groups. Nimesulide did not affect responses in
the control rats, but increased the MAP and MCFP responses in both
groups of diabetic rats. Therefore, selective inhibition of COX-2 partially
restored vascular contractile responses to adrenaline in rats with STZand FRU-STZ-induced diabetes.
(Support: CIHR and Heart & Stroke Fdn of BC & Yukon)
Paper No.: 2316

HEALTH AND DISEASE
INHIBITION OF CYCLOOXYGENASE ACTIVITY IMPROVES
ENDOTHELIUM-DEPENDENT RELAXATION IN MESENTERIC ARTERIES OF OVARIECTOMIZED RATS FOLLOWING
CHRONIC INHIBITION OF NITRIC OXIDE SYNTHASE
Susan WS Leung, MLY Chan, GSK Man, RYK Man
The University of Hong Kong, Department of Pharmacology and Pharmacy, Hong Kong, PR China
Gender differences exist in the incidence and manifestation of vascular
diseases, of which endothelial dysfunction is the underlying cause. Endothelial dysfunction is associated with a reducd bioavaliability of nitric
oxide. In the present study, the effect of estrogen on endothelial function
were examined in rats follwoing chronic inhibition of nitric oxide synthases. Female Sprague Dawley rats were ovariectomized at 12 weeks old,
and they were supplemented with 17b-estradiol (25 lg/kg, intramuscularly) or its vehicle (olive oil). At 18 weeks old, they were administered
daily with L-NAME (a nitric oxide synthase inhibitor, 60 mg/kg, by
gavage) or its vehicle (drinking water) for 6 weeks. 17b-estradiol supplementation increased the plasma level of high density lipoprotein without
affecting the low density lipoprotein level in ovariectomized rats. Chronic
L-NAME treatment did not cause a signcant increase in blood pressure
in all these rats [unlike in male rats of same ages; unpublished data]. In
mesenteric artery contracted with phenylephrine, chronic L-NAME treatment impaired endothelium-dependent relaxation to A23187 (a calcium
ionophore) in ovariectomized rats with or without 17b-estradiol supplement. This impairment was reversed in the presence of indomethacin (a
cyclooxygenase inhibitor), which did not affect A23187-induced relaxation in mesenteric arteries of rats without L-NAME treatment. Therefore,
the present nding suggests that ovariectomy enhances the impairment of
endothelium-dependent relaxation in L-NAME-treated rats through activation of a cyclooxygenase-dependent signaling pathway, and that 17bestradiol may not be responsible for this detrimental effect of ovariectomy.
[This study was supported by a General Research Fund from the
Research Grant Committee, HKSAR]
Paper No.: 3165

ASSESSING POTENTIAL FOR DRUG ABUSE IN HUMANS:
DEFINING CLINICAL IMPORTANT DIFFERENCES IN
SUBJECTIVE PHARMACODYNAMIC MEASURES
N Levy-Cooperman(1), K Schoedel(1), N Chen(1), D Feltner(2),
CB Billing Jr.(2), E Sellers(1), Denise Milovan(1)
(1) Kendle Early Stage, Department of Clinical Pharmacology, Toronto,
Ontario, Canada
(2) Pzer Global Research & Development, Groton, CT, USA
Drug abuse potential is evaluated in humans using randomized, doubleblind crossover studies in which single doses of a test drug are compared
to placebo and active control(s) with established abuse liability. A standard for clinically important differences (CID) in abuse potential studies
has not yet been established. CID can be identied using epidemiological
data; however, data from negative controls (non-abused drugs) and placebo may also be useful. Visual Analogue Scales (VAS) data, including
Drug Liking (Disliking=0, neutral=50, Liking=100), from abuse potential
studies in recreational drug users (N = 1844) were combined. Descriptive and dose response data for placebo, two negative controls and seven
active controls of varying classes (opioids, dissociatives, stimulants,
depressants, cannabinoids) were investigated in a few commonly used
VAS. The mean maximum (Emax) placebo response on Drug Liking
VAS was 51.8 and the upper-condence limit was 57.8 (Range=52.761.3). Mean Emax for negative controls was 63.2, with a difference from
placebo of 5.8 points (range=5.5-6.25). In contrast, mean Emax for
active controls was 76.5, with an average difference from placebo of
24.5 points (range=6.3-37.7, low range associated with low doses). The
-pooled-SD and pooled-SEM were 7.6 and 3.3 for negative controls
and 9.1 and 5.1 for active controls (distribution-based approach). Other
VAS (e.g., high, good effects) data were also examined. Non-abused
drugs showed a distinct range of responses compared to high doses of
drugs with established abuse liability, suggesting that negative control
data may be a useful clinical anchor in establishing CIDs for investigating abuse potential of novel drugs.
Paper No.: 737

MAXIMISING BENEFITS AND MINIMIZING SIDE EFFECTS
OF ACETYLSALICYLIC ACID BY USING ANTIOXIDANT
DRUG THIOTRIAZOLIN
Anton Levykh, V Mamchur
Dnipropetrovsk State Medical Academy, Department of Pharmacology &
Clinical Pharmacology, Dnipropetrovsk, Ukraine
The aim of this work was to research the inuence of combination of
anti-aggregative drug acetylsalicylic acid (ASA) and domestic antioxidant drug thiotriazolin on indicators of platelets aggregative ability and
gastric ulceration. Research was carried out on Wistar rats, which were
divided on 3 groups: I intact rats; II ASA (10 mg/kg); III ASA (10
mg/kg)+thiotriazolin (2,5 mg/kg). Drugs were introduced intragastrically
in mentioned doses once per day during 7 days. Platelet aggregometry
test was carried out by G.V.Born turbidimetric method and using the
solutions of collagen 2 mg/ml, arachidonic acid (AA) 1 mmole/l and
adenosine diphosphate (ADP) 20 and 5 lmole/l as inductors. Urine 11dehydro-thromboxane B2 levels were determined using ELISA kit.
410
Ulcerogenic action of investigated drugs was expressed as gastric ulcerative index. ASA administration in rats leads to platelets aggregative ability reduction. So, degree and rate of aggregation induced by collagen,
AA and ADP was decreased signicantly. Co-administration of thiotriazolin led to intensication of anti-aggregative effects of ASA. So, degree
and rate of collagen-induced aggregation in ASA+thiotriazoline group
(III) in comparison with ASA group (II) were decreased by 20,1% and
52,5% (p < 0,001), ADP (5 lmole/l)-induced by 28,6% and 29,5% (p
< 0,05), ADP (20 lmole/l)-induced by 38,2% and 11,1% (p < 0,01)
accordingly. Urine 11-dehydro-TxB2 level in group III was 24,5% (p <
0,05) down from group II. Co-administration of thiotriazolin also led to
2,35-fold decreasing of gastric ulcerative index. Thus, thiotriazolin possesses a considerable anti-aggregative and gastroprotective action. Its coadministration in ASA-treated rats intensies anti-aggregative and
decreases ulcerogenic properties of this drug.
Paper No.: 1632

DISEASES
DYSFUNCTION OF CHOLINERGIC ANTI-INFLAMMATORY
PATHWAYS MEDIATES ORGAN DAMAGE IN
HYPERTENSION
DJ Li, Fu-ming Shen
Second Military Medical University, Department of Pharmacology,
Shanghai, PR China
Chronic morphine treatment caused adaptive changes in the glutamatergic system appears a critical element involved in morphine tolerance and
dependence. There is now compelling evidence showed that AQP4
knockout attenuated morphine tolerance and dependence and inhibited
the glutamate transporter 1 (GLT1) expression in cerebral compared with
wide-type mice. In the present study, we found that chronic morphine
treatment mainly affected the expression of AQP4 and GLT-1, but not
glutamate/aspartate transporter (GLAST) and excitatory amino acid carrier 1 (EAAC1). AQP4 and GLT1 expression in prefrontal cortex, striatum and cerebellum was signicantly decreased in wide-type mice
during chronic morphine treatment. However, the GLT1 expression was
unchanged in AQP4 knockout mice after chronic morphine treatment.
Furthermore, the reduction of glutamate uptake was at least in part correlated to the expression of glutamate transporter GLT1. These ndings
reveal that AQP4 knockout inhibiting the down-regulation of GLT1
expression and the reduction of glutamate uptake might be a signicant
event in the altered excitatory neurotransmission during chronic morphine treatment, and it is one of possible mechanisms of AQP4 knockout
modulate morphine pharmacological actions.
Paper No.: 3399

DISEASES
THE EXPRESSION OF FIVE CYTOKINES (IFN-A, IFN-C, IL-18,
IL-10, IL-12) IN PAM212 CELL LINES AND L929 CELL LINES
INFLUENCED BY RHU IFN-A 2B CREAM
Background: Inammatory responses are associated with the genesis and

progression of end-organ damage (EOD) in hypertension. A role for the
a7 nicotinic acetylcholine receptor (a 7nAChR) in inammation has
recently been identied. We tested the hypothesis that a7nAChR dysfunction contributes to hypertensive EOD. Methods and Results: In both
spontaneously hypertensive rats (SHR) and in rats with hypertension
induced by aortic coarctation, atropine-induced tachycardia was blunted
compared with normotensive controls. Both models of hypertension were
associated with decits in expression of the vesicular-acetylcholine-transporter (VAChT) and the a7nAChR in cardiovascular tissues. In hypertension induced by aortic coarctation, decits in aortic VAChT and
a7nAChR were present both above and below the coarctation, indicating
that they were independent of the level of arterial pressure itself. Hypertension in 40 week old SHR was associated with cardiac and aortic
hypertrophy. Morphological abnormalities consistent with EOD, along
with elevated tissue levels of pro-inammatory cytokines (tumor necrosis
factor-a interleukin-1b and interleukin-6), were observed in the heart,
kidney and aorta. Chronic treatment of SHR with the a7nAChR agonist
PNU-282987 reduced EOD and tissue levels of pro-inammatory cytokines. Two-kidney-one-clip hypertension in a7nAChR-null mice was
associated with greater serum levels of pro-inammatory cytokines and
more severe damage in the heart, aorta and kidney than in wild-type
mice. Conclusions: A decit in cholinergic anti-inammatory pathways
appears to contribute to the pathogenesis of EOD in models of hypertension of varying etiology. These pathways may provide a new target for
preventing cardiovascular disease resulting from hypertension.
Key Words: acetylcholine, a7 nicotinic acetylcholine receptor, inammation, hypertension, end-organ damage
Jin Li(1), Q Zhu(2), J Liu(2), J Hu(2)
Paper No.: 2139

AQUAPORIN 4 DEFICIENCY MODULATES GLUTAMATE
TRANSPORTERS EXPRESSION AND GLUTAMATE UPTAKE
IN CHRONIC MORPHINE-TREATED MICE
Paper No.: 924

EFFECTS OF LENALIDOMIDE ON OB/OB MICE CARDIAC
FUNCTION:THE ROLE OF TNF-A INHIBITORS
(1) General Hospital of Guangzhou, Military Command of PLA,

Pharmaceutical Department, Guangzhou, PR China
(2) The Second Military Medical Univerisity, Guangzhou, PR China
Objective: The skin immune system (SIS) has been brought by Bos in
1986, which is supplied and developed by dermal micrangium unit
(DMU) theory put forward by Sontheimer and dermal immune system
(DIS) concept proposed by Nicholoff. To approach the effects of the SIS,
we research the expression of ve cytokines (IFN-a, IFN-c, IL-18, IL10, IL-12) in PAM212 cell lines which is an epidermal cell and L929 cell
lines which is a broblast after administration of rHu IFN-a 2b cream,
which is a model drug and an antiviral drug used in clinical medication.
Method and Results: MTT colorimetry detection shows that rHu IFN-a
2b cream, clobetasol propionate and alltrans tretinoin have inhibition or
induction effort at vary degrees on PAM212 cell lines and L929 cell lines
proliferation. ELISA double antibody sandwich method results and RealTime PCR demi-quantitates results show, the protein and mRNA level of
the ve cytokines was signicantly difference in rHu IFN-a 2b treated
PAM212 cell lines and L929 cell lines and cell pretreated with clobetasol
propionate and alltrans tretinoin. Discussion: The ve cytokines
expressed by the PAM212 cell lines and L929 cell lines with or without
clobetasol propionate and alltrans tretinoin both can be inuenced by
rHu IFN-a 2b cream signicantly. As the part of skin, the immunoloregulation of PAM212 cell lines and L929 cell lines can be activated by
immunological drug and maybe act as the important roles in the SIS.
Linlin Li(1), R Jun(2), Y Hua(2)

Jin Li, H-T Yan, N Wu, R-B Su, J-Q Zheng
Beijing Institute of Pharmacology and Toxicology, Department of New
Drug Evaluation, Beijing, PR China
(1) Xinjiang Medical University, Department of Pharmacology, Urumqi,

PR China
(2) University of Wyoming, USA
411
Levels of the TNF-a are often elevated in obesity and may contribute to
obesity-induced cardiovascular complications. However, the role of
TNF-a in obesity-associated cardiac abnormalities has not been clearly
dened.Lenalidomide is a TNF-a inhibitor. This study was designed to
determine the inuence of Lenalidomide on ob/ob cardiac contractile
response of it. Mechanical Ca2 + properties were evaluated using an IonOptix system in cardiomyocytes from adult ob/ob mice. Cardiomyocyte
contractile was examined including peak shortening, duration and maximal velocity of shortening/relengthening (TPS/TR90, dp/dt) and
Expression of the TNF-a,ikb3,perk/erk,fas,fasl,caspase8, caspase12,bcl2,p38,bax,bip,gpr41 were evaluated by western blot analysis.
The level of blood glucose in 0min and 30min were decreased signicantly after ad lenalidomide ig for 3 days. PS and dl/dt were depressed
in ob/ob mice, but PS and dl/dt were increased after ad lenalidomide.
TNF-a,ikb3, perk/erk,fas, bcl2 were increased in ob/ob mice, but TNFa,fas,bcl2 decreased signicantly after ad lenalidomide. Caspase12 were
decreased in ob/ob mice, but it increased signicantly after ad lenalidomide ig for 3 days. These data indicate presence of inamation in
ob mice possibly associated with TNF-a receptor signalling pathway.
Paper No.: 1557

THE TEST DOSE - HOW TO BE A MORE VALUABLE AND
RELIABLE PREDICTOR?
Nianzhi Li(1), J Aronson(2)
(1) University of Oxford, Department of Pharmacology, Oxford, UK
(2) University of Oxford, Department of Clinical Pharmacology, Oxford,
UK
The test dose is a small amount of drug given prior to the administration of the rst therapeutic dose in order to prevent potentially harmful
consequences like anaphylactic reactions. However, the lack of evidence
about an optimal selection of drug and the suggested size of dose is
leading to an increasing concern that the test dose is unable to act like
a reliable predictive marker in clinical practice. The author systematically reviewed the major aspects of the application of test dose, including detecting misplaced needles in epidural analgesia, avoiding allergic
reactions in iron deciency therapies and amphotericin treatments, identifying hypersensitive individuals to quinidine and octreotide, diagnosing the side effects of drugs like gelofusine, and estimating overdose
when using deferoxamine. Generally it is a practical and dependable
tool, but at the same time, a test dose might also increase the risk for
anaphylactic reactions or even result in sudden death, hence it is important to consider the risk/benet ratio before the administration of a test
dose. More importantly, to achieve its maximal benets, drugs and pretreatment regimens should be available for treating in conjunction with
the test dose, thereby minimizing the potential adverse events and
reducing the risks.
Chemotherapy for malignant tumors fails because of multidrug resistance

(MDR). The overexpression of glutathione S-transferase (GST) with
MDR is considered one of the major obstacles to successful cancer chemotherapy. We performed high-throughput screening to search for inhibitors of GST from natural products. Two compounds isolated from Radix
Angelicae sinensis had an inhibitory effect on GST, with 50% inhibitory
concentration (IC50) values of 16.80 and 7.30 lM. Kinetic analyses
showed that the 2 compounds acted as reversible noncompetitive inhibitors of GST. They also increased apoptosis of B-MD-C1 (ADR+/+) cells
induced by adriamycin with low cytotoxic activity. Radix Angelicae sinensis may be a potential source of GST inhibition for pharmaceutical
use.
Keywords: cancer, multidrug resistance, glutathione S-transferase, 11-angeloylsenkyunolide F, Tokinolide B, Radix Angelicae sinensis
Paper No.: 1423

PROTECTIVE EFFECTS OF GANODERMA LUCIDUM
POLYSACCHARIDES ON INTESTINE DAMAGE IN VIVO AND
IN VITRO AND MECHANISMS APPROACH
Wei Dong Li, LH Chen, J Ma, Y Qin, JQ Zhang, J Yang, ZB Lin
Peking University Health Science Center, Department of Pharmacology,
Beijing, PRChina
Aim: To investigate the protective effect of Ganoderma lucidum polysaccharides Gl-PSand to identify potential mechanisms. Methods: Mice
were divided into normal, MTX control, Gl-PS 50, 100 and 200mg/kg
groups. Gl-PS was administrated for 10 days. On the 3 days before the
end of experiment MTX50mg/kg, ipwas administrated for two days.
The mice were sacriced at day 11. The jejunum were xed for light
(LM) and transmission electron microscope (TEM) assay. MDA and
SOD were determined by Biochemical method. Serum IgA was examined by ELISA. Non-transformed IEC-6 cells were used for in vitro
study. Cell proliferation was assessed by MTT assays. Epithelial cell
migration was assessed using a wounding model. Effects on epithelial
cell differentiation were observed by H&E staining. The ODC and cMyc mRNA expression were determined by RT-PCR. Results: LM and
TEM in the MTX group revealed desquamation of surface epithelium,
while the epithelium was slightly damaged in the Gl-PS treatment group.
MTX caused decrease in SOD and increase in MDA. These were
reversed by Gl-PS. Gl-PS could dose dependently increase serum IgA
under MTX stress and signicant stimulation of IEC proliferation, migration and differentiation. Gl-PS increased the expression of ODC and cMyc mRNA in IEC-6 cells. Conclusion: Gl-PS decreased the MTXinduced damage to the small intestine and induced improvement of intestinal wound healing was possibly due partly to the induction of ODC
and c-Myc gene. It clue to Gl-PS may potentially be useful for the treatment of intestinal disorders.
Paper No.: 3280

TWO GLUTATHIONE S-TRANSFERASE INHIBITORS FROM
RADIX ANGELICAE SINENSIS
Paper No.: 1784

EFFECT ON THE EXPRESSION OF CYP450 ENZYME IN RAT
LIVER INDUCED BY TOTAL PANAX NOTOGINSENOSIDE
AND THE POTENTIAL DRUG-DRUG INTERACTION STUDY
Shaojing Li(1), F Huang(2), XH Lu(3), AL Liu(4), GH Du(5)
Xiao-yu Li, G-L Liu, J-C Zhou, G Qian, B Sun, Y-K Guo, J-W Gao
(1) Insititute of Chinese Materia Medica, China Academy of Chinese

Medical Sciences, Beijing, PRChina
(2) Medical College of Shantou University, Shantou, PR China
(3) New Drug Research and Development Center of North China Pharmaceutical Group Corporation, Hebei, PR China
(4) Insitute of Materia Medica, Chinese Academy of Medical Sciences
and Peking Union Medical College, Beijing, PR China
Shanghai Jiao Tong University First Peoples Hospital, Department of

Clinical Pharmacology, Shanghai, PR China
Objective: To investigate the CYP450 enzyme protein expression in rat
liver induced by total panax notoginsenoside injection (TPNSi) and evaluate drug-drug interaction in rats when nimodipine and warfarine
co-administration with TPNSi. Methods: TPNSi was administered by
412
intravenous at 20 mg/kg to rats for ve weeks. CYP2E1, CYP3A4,
CYP1A2, CYP2C9 protein expressed in rat liver were investigated by
western blot. HPLC method DAS software was used for the pharmacokinetic study of nimodipine and warfarin in rats. Results: Expression of
CYP2E1 increased in rat liver induced by TPNSi compared with normal
rats (p < 0.05). There were potential inhibition for CYP3A4, CYP1A2
and CYP2C9 expression induced by TPNSi. Pharmacokinetic parameters
were displayed as followed: nimodipine: t1/2b was 0.556 and 0.452h,
AUC(0-t) was 1.379 and 1.555 mg/L*h, V1 was 1.307 and 0.902 L/kg,
CL was 3.082 and 2.809 L/h/kg, MRT(0-t) was 0.563 and 0.524h. Warfarin: t1/2b was 21.77 and 27.72h, Cmax was 66.73 and 56.22 lg/mL, Tmax
was of 1.8 and 2.7h, AUC(0-t) was of 1321.59 and 1401.41 mg/L*h, V1
was of 0.395 and 0.475 L/kg, CL was of 0.013 and 0.013 L/h/kg,
MRT(0-t) was of 17.01 and 19.92h in normal or TPNSi rats, respectively.
Conclusion: Inhibition on CYP3A4, CYP1A2 and CYP2C9 enzyme
activities maybe induced by TPNSi and the potential drug-drug interaction maybe occurred in clinic when nimodipine or warfarine was coadministration with TPNSi.
Keywords: total panax notoginsenoside, nimodipine, warfarine pharmacokinetic, drug-drug interaction.
Paper No.: 820

SESSION - ONCOLOGY
XJ-6-A INHIBITS TUMOR INVASION AND METASTASIS AND
ITS POTENTIAL MOLECULAR MECHANISMS
Xuejun Li, Y Cao
Peking University School of Basic Medical Sciences, Department of
XJ-6-A is a newly designed and synthesized compound. We investigated
its effects on tumor growth and metastasis and tried to elucidate its
potential mechanism. XJ-6-A (20 and 40 mgkg-1d-1 for 20 d, ig.) was
found to decreased the primary tumor weight and lung metastases in
mice bearing Lewis lung carcinoma and mitigate the damage of lung
alveolar. The inhibitory rate of pulmonary metastasis produced by XJ-6A at the dose of 20mgkg-1d-1 was approximately 50%. Moreover, the
expression of AQP1, osteopontin (OPN) and MMP-2 protein were obviously decreased observed by immunoblotting. In vitro, MTT assay
showed that low concentrations (below 10-5 M) of XJ-6-A did not suppress cell viability, whereas scrap-wound healing assay displayed inhibitory effect on tumor cell motility. XJ-6-A dramatically inhibited invasion
and migration of PC-3M carcinoma cells at the concentrations of 0.1, 1
and 10lM by Transwell assay. XJ-6-A inhibited the osmotic water permeability in HEK 293 cells transfected with pEGFP/AQP1. Interestingly,
AQP1 and MMP-2 protein of PC-3M carcinoma cells was decreased by
indirect immunouorescence staining and ow cytometric analysis.
These results indicate that the inhibitory effects of XJ-6-A on tumor
growth and metastasis AQP1 osmotic water permeability and distribution
might be involved in AQP1, OPN and MMP-2 protein function and
expression. This nding is very promising for the drug design to focus
on tumor metastasis as a novel agent in clinical tumor therapy.
Paper No.: 1452

EFFECT OF BICYCLOL ON HIGH FAT DIET (HFD)-INDUCED
FATTY LIVER IN RATS
Yan Li, H Yu, H Chen
scriptional factors regulating the hepatic lipid metabolism. Previous

studies showed that bicyclol protected against tetracycline-induced fatty
liver partially through the modulation on PPARb and its target genes
involved in fatty acid oxidation. SREBP-1c, the transcriptional factor
mainly modulating the synthesis of fatty acid, has been identied as an
attractive target for the therapeutic intervention of fatty liver. In the present study, we investigated the effect of bicyclol on high fat diet (HFD)induced fatty liver and its possible relationship with SREBP-1c pathway.
Bicyclol (150, 300mg/kg) was given by gavage daily for 4 weeks to
Male SD rats fed with HFD. SREBP-1c and its target genes were determined by RT-PCR and western blotting analysis. It was found that bicyclol signicantly protected against HFD-induced fatty liver by reducing
the accumulation of hepatic lipids biochemically and pathologically. The
increased protein expression of SREBP-1c and nuclear SREBP-1c
(n-SREBP-1c) were inhibited by bicyclol. The higher expression of its
lipogenic target genes including acetyl-CoA carboxylase (ACC), fatty
acid synthase (FAS) and StearoylCCoA desaturase (SCD) were also
inhibited after bicyclol treatment. In addition, bicyclol also inhibited the
protein expression of ACC and FAS. Bicyclol protected against HFDinduced fatty liver mainly through the inhibition of SREBP-1c pathway
and thus inhibiting the synthesis of fatty acid.
Paper No.: 3224

PHARMACOKINETIC STUDY OF RECOMBINANT
HUMANIZED ANTI-VEGF MONOCLONAL ANTIBODY
(BEVACIZUMAB) IN CHINESE SUBJECTS WITH ADVANCED
MALIGNANCIES
Yang Li(1), X Hu(1), G Cheng(1), YL Nie(2), YB Zhao(1), JY Wu(1), KX
Li(1), C Qi(3)
(1) Beijing Hospital Ministry of Health, Department of Pharmacy,
Beijing, PR China
(2) Chinese Academy of Medical Sciences, Beijing, PR China
(3) Roche Pharma Development Center, Beijing, PR China
Objective: To characterize the PK prole of bevacizumab following IV
administration in Chinese patients, and comparison of the PK prole of
bevacizumab to that of Caucasian patients enrolled in the phase I study.
Methods: This is a open-label, three-period (screening, treatment and follow up), study. All subjects were assigned to one of the three cohorts (n
= 12) according their enrollment sequence to receive bevacizumab at
dose levels of 5 mg/kg, or 10 mg/kg, or 15mg/kg. At each dose level,
dosing will begin with a single IV infusion of bevacizumab on Study
Day 1. Following the initial administration, subjects will be monitored
for safety for 20 days (screening period). Subjects who tolerate the initial
exposure will advance to treatment period to receive the same dose of bevacizumab IV on Study Day 22, 36, 50 and 64 (5 mg/kg, 10 mg/kg) and On
Study Day 22, 43, 64 and 85(15 mg/kg). The serum bevacizumab concentration was determined with a validated ELISA assay. Results: The pharmacokinetic parameters of bevacizumab, q2w/q3w, were similar to those after a
single dose. Conclusion: The bevacizumab PK parameters were comparable
between Chinese, obtained from this study, and Caucasian, which were
obtained from a historical Phase I study.
Paper No.: 1780
PROTEOMIC STUDY OF HEPATOTOXICITY INDUCED BY
RETRORSINE, A REPRESENTATIVE PYRROLIZIDINE
ALKALOID
Yanhong Li(1), WCS Tai(2), N Li(1), WLW Hsiao(2), G Lin(1)
Chinese Academy of Medical Sciences, Institute of Materia Medica,

Department of New Drug Development, Beijing, PR China
Peroxisome proliferators-activated receptor b (PPARb) and Sterol regulatory element binding proteins-1c (SREBP-1c) are two important tran-
(1) Chinese University of Hong Kong Faculty of Medicine, School of

Biomedical Sciences, Department of Pharmacology, Hong Kong, PR
China
413
(2) Hong Kong Baptist University, School of Chinese Medicine,
Kowloon Tong, Hong Kong, PR China
Pyrrolizidine alkaloids (PA)-induced hepatotoxicity has been well-established. However, the detailed mechanism of such toxicity is not fully
understood. The aim of this study was to investigate the progression of
hepatotoxicity induced by retrorsine, a representative PA, in rats and the
potential biomarkers of the toxicity using a proteomic approach. Hepatotoxicity in rats induced by retrorsine (140 mg/kg) was evaluated by
determining plasma alanine aminotransferase (ALT) activity and hepatic
glutathione (GSH) system, including GSH level, oxidized glutathione
(GSSG)/GSH ratio and glutathione reductase (GR) activity. Protein proles of livers obtained from treated and control rats were analyzed by
two-dimensional electrophoresis (2D-DIGE) followed by MALDI-TOFMS analysis. Retrorsine-induced liver damage was revealed by signicant elevation of ALT level and alteration of GSH system. Comparing
with control group, levels of 28 proteins signicantly changed in the retrorsine treated rats, among them 15 were up-regulated while 13 were
down-regulated. Most of these proteins are involving in the protein and
lipid metabolism. The modication of the proteins related to apoptosis
and inammation (such as heat shock protein b-8) might be associated
with the alteration of hepatic GSH system. The changes in hepatic levels
of a 2l globulin and spectrim a 2, which involve in blood coagulation
and brinolysis, might be related to retrosine-induced hemorrhagic hepatomegaly. The results suggested that proteomic study provide a powerful
technology for the investigation of the mechanism of PA intoxication
and also development of biomarkers of PA-induced hepatotoxicity.
[Supported by Hong Kong Jockey Club Charities Trust Fund (JCICM15-07)]
Paper No.: 1609

HEALTH AND DISEASE
INHIBITION OF IKB KINASE A/B (IKKA/B) IMPROVED
INSULIN RESISTANCE OF ENDOTHELIAL CELLS INDUCED
BY HIGH GLUCOSE IN VITRO
ZX Li(1), FH Chen(1), LL Liu(1), RX Zhan(1), Y Gao(1), SR Peng(1),
CX Chen(1), Qi-Ren Huang(1,2)
(1) Nanchang University, Department of Pharmacology & Molecular
Therapeutics, Nanchang, PR China
(2) Nanchang University, Key State Laboratory of Food Science & Technology, Nanchang, PR China
Insulin resistance (IR) of endothelial cells is a critical trigger of cardiocerebrovascular events in diabetes. Ikb kinase a/b (IKKa/b), a kinase
complex of NF-jB pathway, is implicated in the processes of inamation, cell apoptosis etc. However, it is well unknown whether IKKa/b is
involved in the regulation of endothelial IR induced by hyperglycemia
and its mechanisms. The purpose of this study is to ascertain whether
high glucose (HG) affects the IKKa/b expression level, and to investigate
the relation between IKKa/b and endothelial IR in vitro. HUVEC were
exposed to HG (35.6 mmol/L), vitamine E (Vit E, 100 umol/L) +HG,
myxothiazole (MTZ, 100umol/L) +HG, Bay11-8702 (BAY, 20 umol/L)
+HG, and mannitol (MNT, 35.6 mmol/L) for 48 h, respectively. The levels of nitric oxide (NO), endothelin-1(ET-1), and IKKa/b expression were
detected. The results revealed that HG signicantly reduced the level of
NO, whereas increased the levels of ET-1 and IKKa/b expression
(P<0.01, vs 5.5mmol/l of glucose). However, the individual treatment
with Vit E (an antioxidant), MTZ (an inhibitor of ROS generation) and
BAY (an inhibitor of IKKa/b) normalized the decrease in the level of
NO and the increase in the levels of ET-1 and IKKa/b expression induced
by HG (P<0.01, vs HG). The ndings suggested that the inhibition of
IKKa/b could improve endothelial IR induced HG via a ROS-dependent
pathway.
Keywords: Insulin resistance; Endothelial cells; Ikb kinase; Nitric oxide;
Reactive oxygen species
This work was supported by grants from the Natural Scientic Foundation of China (No.30660058 and No.30860111).
Paper No.: 2335

DISEASES
TOLL-LIKE RECEPTOR 4 MEDIATES THE
VASOCONSTRICTOR RESPONSE INITIATED BY ADIPOSE
TISSUE IN MICE ARTERIES
Chao Fan Liang(1), A Xu(1,2), PM Vanhoutte(1)
(1) The University of Hong Kong,Department of Pharmacology and
Pharmacy, Hong Kong,PR china
(2) The University of Hong Kong, LKS Faculty of Medicine, Department of Medicine,Hong Kong, PR China
Adipose tissue modulates vascular tone by secreting a number of proand anti-inammatory cytokines including tumour necrosis factor-a
(TNFa) and adiponectin, respectively. Toll-like receptor 4 (TLR4) is a
major target for lipopolysaccharide and saturated fatty acids, both of
which are potent inducers of inammation. The present study investigated whether or not TLR4 mediates vascular dysfunction induced by
obese adipose tissue. Aorta and carotid arteries from 12-week-old wild
type mice on standard diet were isolated. White peri-aortic and epididymal adipose tissues from TLR4 mutant mice or wild type littermates fed
a high fat diet (HFD) were dissected for ex vivo culture. Isometric tension
was measured in artery rings in the absence or presence of conditioned
medium collected from the fat explant culture. Several pro- and
anti-inammatory cytokines in adipose tissues were assayed by RT-quantitative PCR. In the presence of N(G)-nitro-L-arginine methyl ester
(nitric-oxide synthase inhibitor), epididymal fat from wild type but not
TLR4 mutant mice potentiated acetylcholine-mediated endotheliumdependent contraction in carotid arteries. Indomethacin (cyclooxygenase
inhibitor) and S18886 (TP receptor antagonist) inhibited the contraction.
In addition, inactivation of TLR4 restored the impaired anti-contractrile
effect of perivascular fat in aorta caused by HFD feeding. These changes
were associated with increased adiponectin expression, but decreased
expression of TNFa and interleukin-6 in the adipose tissues of TLR4
mutant mice. Epididymal fat from obese mice facilitates endotheliumdependent contractions to acetylcholine through activation of TLR4,
which in turn triggers the release of pro-inammation cytokines. Perivascular fat from TLR4 mutant mice curtails the contraction possibly by
increasing adiponectin.
Paper No.: 1591

TRAMADOL REVERSES RESERPINE-INDUCED
HYPOTHERMIA VIA l OPIOID RECEPTOR IN MICE
Jian-Hui Liang, H-L Sun, M Zhang, K Wang
Peking University, National Institute on Drug Dependence, Department
of Neuropharmacology, Beijing, PR China
Tramadol is a centrally acting analgesic with opioid agonist properties
and effects on monoamine transmission. Here we try to establish that
there might be an interaction between opioid and monaminergic components of tramadol, so as to provide an expanded view to better understand the mechanisms of action of tramdol. The effects of tramadol on
reserpine-induced hypothermia were investigated in mice. Tramadol
dose-dependently reversed reserpine-induced hypothermia, and morphine
potentiated the reversal effect of tramadol, while nonselective opioid
receptor antagonists, naloxone and diprenorphine (M5050) counteracted
414
the reversal by tramadol of reserpine-induced hypothermia. Additionally,
in contrast to the selective d opioid receptor antagonist naltrindole (NTI)
and the selective j opioid receptor antagonist nor-binaltorphimine (norBNI), b-Funaltrexamine hydrochloride (b-FNA), a selective l opioid
receptor antagonist, antagonized the reversal effect of tramadol. In conclusion, these results suggest that tramadol reverses reserpine-induced
hypothermia in mice via l opioid receptor, implicating that there might
be an opioid/monoaminergic interaction between the dual mechanisms of
action of tramadol.
Paper No.: 2792

HEALTH AND DISEASE
PROTECTIVE ROLE OF THE ADRENOMEDULLIN
RECEPTOR IN VASCULAR HYPERTROPHY
Lihuan Liang(1), C Tam(1), G Pozgai(1), R Siow(1), K Husmann(2),
W Born(2), J Fischer(2), R Poston(1), A Shah(1), S Brain(1)
(1) Kings College London, Cardiovascular Division, London, UK
(2) University of Zurich, Zurich, Switzerland
Adrenomedullin (AM) levels are raised in hypertension. The receptor
consists of calcitonin receptor-like receptor and receptor-activity-modifying-proteins 2 (RAMP2). We investigated the role of RAMP2 in angiotensin II (AngII) induced hypertension model using smooth muscle
cell-targeted RAMP2-overexpressing (RAMP2TG) mice and respective
wildtypes (WT). AngII (0.9 mg/kg/day) or vehicle (0.01% 10 mM acetic
acid in saline) was infused for 13 days via osmotic mini-pumps. The
mean arterial pressure was similar between WT and TG mice after 13
days infusion of AngII, but signicantly greater than that of vehicle
mice. However, a signicant increase (P<0.001) in aortic wall width and
wall area was observed in AngII-treated WT mice, but not in RAMP2TG
mice. VCAM-1 and MCP-1 expression on the aortic wall was examined
by immunohistochemistry. Both VCAM-1 and MCP-1 expression
induced by AngII were signicantly reduced in RAMP2TG mice compared to WT mice (P<0.001). Proliferation studies were performed using
WT and RAMP2TG vascular smooth muscle cells (VSMCs) in vitro. AngII (10-7M) signicantly increased (P<0.001) the proliferation of WT but
not RAMP2TG cells. To conclude, RAMP2TG mice are protected
against AngII-induced vascular hypertrophy and inammation. The ndings reveal the VSMC AM1 receptor as a novel target for protective
treatments aimed at reversing vascular hypertrophy and inammation.
Study was supported by BHF and a Capacity Building Award in Integrative Mammalian Biology funded by the BBSRC, BPS, HEFCE, KTN,
MRC and SFC.
Paper No.: 1309

ISOLATION REARING ALTERS PROTEIN EXPRESSION OF
SYNAPTIC MARKERS, MYELIN MARKERS AND GABAB1
RECEPTOR IN THE RAT BRAIN
Ann Li Lim, DA Taylor, DT Malone
Monash Institute of Pharmaceutical Sciences, Monash University,
Parkville, VIC, Australia
The isolation rearing paradigm which involves raising weaned rat pups
in individual cages for at least 8 weeks produces a range of behavioural
and neuropathological alterations compared with group-reared rats. Some

of these changes mimic ndings manifested in schizophrenia. Thus, isolation rearing provides a neurodevelopmental non-drug induced animal
model relevant to some features of this psychiatric disorder. Prepulse
inhibition and locomotor activity of both group and isolation-reared rats
were assessed following a regimen of vehicle injections. Upon completion of the behavioural experiments, the relative expression of synaptic,
myelin and GABA related proteins was determined using semi-quantitative uorescence immunohistochemistry. Isolation-reared rats displayed
disruption of prepulse inhibition and hyperlocomotion compared to
group-reared rats. The disruption of prepulse inhibition was lost following repeated testing and multiple injections whereas the hyperlocomotion
observed in isolation-reared rats persisted. Decreases in the relative protein expression of synaptophysin, synapsin I, myelin basic protein and
GABAB1 receptor, along with an increase in 2, 3-cyclic nucleotide
3-phosphodiesterase were observed in isolation-reared rats compared
with those in group-reared rats. Among the brain regions studied the protein expression changes were specic to the hippocampal subelds and
substantia nigra. Alterations in the expression of these neuroproteins suggest disruptions in synaptic function, myelination and GABAergic neurotransmission in isolation-reared rats that also had behavioural
abnormalities. Since these dysfunctions are also implicated in schizophrenia, the present ndings provide additional evidence to support the use
of isolation rearing as an animal model to aid further research of this
debilitating illness.
Paper No.: 2682

PEGYLATED INTERFERON-A2A IN HEALTHY MALE
VOLUNTEERS
Lay Ahyoung Lim(1,2), H Son(1,2), S Jang(1,2), Y Lee(1,2), JY
Chung(1,2), K Park(1,2)
(1) Yonsei University College of Medicine, Department of
Pharmacology, Seoul, Korea
(2) Brain Korea 21 Project for Medical Science, Seoul, Korea
DA3021 is a pegylated interferon (IFN)-a2a agent recently developed.
By pegylation, it is expected to have increased half-life while immunogenicity is reduced. A randomized, open-label, 2-period cross-over study
was conducted in 34 healthy Koreans, for a single subcutaneous administration of DA3021 or PEGASYS to evaluate pharmacokinetics and pharmacodynamic (serum 2,5-oligoadenylate synthetase; OAS) activities.
Pharmacokinetic and pharmacodynamic samples were collected up to
336 hours after drug administration. The data were analyzed by a
non-compartment method using WinNonlin. All subjects received acetaminophen after drug administration to prevent u-like symptoms. For
pharmacokinetics, the geometric mean ratios(GMR) (90% CI) of
DA3021 to PEGASYS were 70.72% (45.16~110.75) for maximum concentration, and 71.08% (40.86~ 123.67) for area under the concentrationtime curve. Half-lives were 157.05 hours for DA3021 and 77.38 hours
for PEGASYS and time to reach the maximum concentration were 69.41
hours and 83.37 hours respectively. For pharmacodynamics, the serum
OAS concentration increased over time after the dose and remained elevated until the last observation time. The GMR (90% CI) of DA3021 to
PEGASYS was 90.08% (91.68~104.82) for maximum effect, and
101.53% (94.52~109.06) for area under the effect-time curve. Most common adverse events were u-like symptoms, with no serious adverse
event reported. This study shows that DA3021 is comparable to PEGASYS in pharmacodynamics and safety proles while pharmacokinetic
prole was slightly different. A possible explanation for this discrepancy
may include high inter-subject variability and structural difference of
pegylation.
415
Paper No.: 1205
THE LIGAND BINDING PROPERTIES OF HUMAN
MEMBRANE ESTROGEN RECEPTORS
oxidative injury, including elevated lipid peroxidation in the infused SN

and the depleted DA level in the ipsilateral striatum was attenuated in
the pioglitazone-treated rats in a time-dependent manner. Pioglitazone
may inhibita-synuclein aggregation and neuroinammation and can be
considered as a neuroprotective agent against iron-induced apoptosis via
both mitochondrial and ER pathways.
Amanda HY Lin, GPH Leung, SWS Leung, RYK Man

The University of Hong Kong Faculty of Medicine, Department of Pharmacology and Pharmacy, Hong Kong, PR China
Estrogen (17b-estradiol) exerts rapid non-genomic effects in various cell
types. Surface binding sites for estrogen has been demonstrated using
confocal microscopy. However, the identity of the putative membrane
estrogen receptor (mER) and how well they bind with estrogen remained
elusive. The present study aims to determine the binding afnities of
estrogen to the mER candidates recently identied and to investigate the
importance for mERs to be transported to the membrane for estrogen
binding. Moreover, the relative binding afnities of the mERs with various estrogenic chemicals and phytoestrogens were evaluated. Human
estrogen receptor-a66 (ER66), estrogen receptor-a46 (ER46), estrogen
receptor-a36 (ER36) and G protein-coupled receptor 30 (GPR30) were
cloned and expressed using cell-free expression systems in the presence
of nanolipoprotein molecules as the membrane substitute. Expressed
receptor proteins were used in radioactive binding assay. ER66 and
ER46 have similar binding afnities towards estrogen (KdG79 pM),
whereas ER36 and GPR30 displayed no specic binding. ER66 and
ER46 expressed in prokaryotic system have lower binding afnities than
the receptors expressed in eukaryotic system. Removal of nanolipoprotein molecules also reduced the binding afnities of ER66 and ER46.
Our results suggested that post-translational modication and membrane
trafcking of mERs are important for proper conformation for binding.
Moreover, relative binding afnities to ER66 and ER46 are similar for
the compounds tested, except for the estrogen antagonist, ICI 182 780.
Paper No.: 2187

DISEASES
NEUROPROTECTIVE EFFECT OF PIOGLITAZONE ON
IRON-INDUCED NEURODEGENERATION: A-SYNUCLEIN
AGGREGATION
Anya Maan-Yuh Lin, HJ Yu, SF Fang, PL Chao
National Yang-Ming University, Graduate Institute of Pharmacology,
Taipei,Taiwan
The anti-neuroinammatory effects of pioglitazone, involving a-synuclein aggregation were studied in the nigrostriatal dopaminergic system of
rat brain. Ferrous citrate (iron) was locally infused in the substantia nigra
(SN) of chloral hydrate-anesthetized rats. In addition to the elevated
heme oxygenase-1 (HO-1), a-synuclein was time-dependently aggregated
in the iron-infused SN. Both DNA fragmentation and TUNEL-positive
cells were observed in the infused SN. Reduction in procaspases 3 and
12 levels and elevation in activated capase-3 levels in the infused SN
were noted. Pioglitazone, a peroxisome proliferative activated receptor
(PPAR)-c agonist, was orally administered 4 days prior to an intranigral
infusion of iron. Daily administration of pioglitazone enhanced ironinduced elevation in nuclear PPAR-clevels and attenuated iron-induced
elevation in a-synuclein aggregation and HO-1 levels. Iron-induced elevation in interleukin (IL)-1a and IL-6 mRNA levels as well as cyclo-oxygenase II and nitric oxide synthase protein levels were diminished after
pioglitazone treatment. ED-1 immunoreactivity, an indicator of activated
microglia was reduced in the pioglitazone-treated rats. Both iron-induced
activation of the mitochondrial pathway and activation of the ER pathway were attenuated in the pioglitazone-treated rats. Systemic pioglitazone inhibited iron-induced apoptosis in the infused SN. Iron-induced
Paper No.: 1641

DISEASES
THROMBIN-INDUCED CONNECTIVE TISSUE GROWTH
FACTOR EXPRESSION REQUIRES C-SRC/JAK2/STAT3
SIGNALLING PATHWAY IN HUMAN LUNG FIBROBLASTS
Chien-Huang Lin(1), H-C Pai(1), B-C Chen(2)
(1) Taipei Medical University College of Medicine, Graduate Institute of
Medical Sciences, Taipei, Taiwan
(2) Taipei Medical University College of Medicine, School
of Respiratory Therapy, Taipei, Taiwan
Introduction: Thrombin is a multifunctional serine protease and an
important brotic mediator that induces connective tissue growth factor
(CTGF) expression in human lung broblasts. In this study, we have
investigated the role of JAK2 and STAT3 in thrombin-induced CTGF
expression in human lung broblasts. Materials: We use human lung
broblast cell line (WI-38) in this study. Results: Thrombin induced an
increase in CTGF expression and CTGF-luciferase activity in WI-38
lung broblasts. AG490 (JAK inhibitor) and dominant negative of JAK2
(JAK2DN) inhibited thrombin-induced CTGF expression and CTGFluciferase activity in a concentration-dependent manner. Thrombin also
caused a time-dependent increase in JAK2 phosphorylation at Tyr1007/
1008. Transfection of cells with STAT3DN inhibited CTGF expression
and CTGF-luciferase activity. Thrombin induced STAT3 phosphorylation
at Tyr705 was inhibited by AG490 and JAK2DN in a time-dependent
manner. Thrombin induced STAT3 binding to CTGF promoter by DNAbinding afnity pull down assay. We also found that thrombin-induced
CTGF expression was inhibited by STAT3-1 ODN and STAT3-2 ODN.
Furthermore, thrombin-induced CTGF expression and CTGF-luciferase
activity were inhibited by c-SrcDN. Thrombin induced STAT3 phosphorylation and JAK2 phosphorylation also inhibited by c-SrcDN. Conclusion: The result of this study suggests that thrombin might activate c-Src/
JAK2/STAT pathway to induce CTGF expression in human lung broblasts.
Paper No.: 1317

HEALTH AND DISEASE
INTERMITTENT HYPOXIA ALTERS THE EXPRESSION OF
NMDA RECEPTOR SUBUNITS AND ANTIOXIDANT ENZYMES
IN THE RAT ROSTRAL VENTROLATERAL MEDULLA
Hsun-Hsun Lin, C-C Lai
Tzu Chi University, Department of Physiology, Hualien, Taiwan
Exposure of animals to chronic intermittent hypoxia (IH) in a manner
similar to that seen in obstructive sleep apnea patients resulted in
enhanced hypertension development. The IH induced-systemic hypertension is associated with increased sympathetic outow. IH also increases
reactive oxygen species (ROS) generation and ROS-mediated signalling
mechanisms contribute to hypertension. There are several studies that
observed an increase of expression of NMDA receptors in rat central or
peripheral neurons after IH. It interests us to know whether NMDA
receptor or ROS contributes the cardiovascular effects of IH in conscious
rats. Male WKY/SHR rats (10-14 weeks) were used in the study. The IH
416
was designed by applied 30s pure nitrogen followed by 45s air to rats
during the light phase (6 h) for 7 consecutive days. Radiotelemetry system was use to record the blood pressure during IH treatment. Western
blot analysis was used to detect the expression of NMDA receptor
subunits or antioxidant enzymes in the vasomotor centre, rostral
ventrolateral medulla (RVLM). Our preliminary results showed that the
expression of NR1 and NR2B subunits were elevated signicantly, yet
the SOD2 and CAT decreased signicantly after 7 days IH treatment in
SHR. In WKY rats, the NR2A subunits increased signicantly after 7
days challenge and the expression of SOD1, SOD2 decreased signicantly after 7 days IH treatment. We speculated that the changes of
NMDA receptor subunits and antioxidant enzymes in both strains of rat
may contribute the cardiovascular regulation of the animals after IH treatment.
Paper No.: 2133

DISEASES
MOLECULAR MECHANISMS FOR ER STRESS MEDIATED
ANTI-INFLAMMATORY ACTIONS IN MACROPHAGES
Wan-Wan Lin, HR Ho
Natioanl Taiwan University College of Medicine, Department of
Pharmacology, Taipei, Taiwan
In this study we used tunicamycin and brefeldin A as ER stressors, and
murine RAW264.7 macrophages and BV-2 microglia as cell models to
clarify the role of endoplasmic reticulum (ER) stress in lipopolysaccharide (LPS)- and interferons (IFNs)-mediated inammatory responses.
Results revealed the ability of ER stressors to attenuate LPS-elicited
iNOS gene expression. ER stressors do not affect early upstream signals
evoked by LPS. However, ER stressors can block NF-jB binding to specic DNA elements in the iNOS promoter in late phase signaling evoked
by LPS, attenuate RelB nuclear translocation and co-activator p300
expression. Furthermore, ER stressors could block LPS- and IFNs-mediated STAT1 phosphorylation; the latter has synergistic effect on LPSinduced iNOS expression. When treating cells with BAPTA/AM,
calmodulin inhibitor or Ca2+ free medium, the inhibitory effect of ER
stressors on STAT1 phosphorylation was abrogated. Studies suggest activation of tyrosine phosphatase is involved in the inhibitory effect of ER
stressors on IFNs signaling. In addition, ER stressors could induce MKP1 expression, which is a newly identied protein phosphatase targeting
on STAT1. Cells pretreated with calcineurin inhibitor could block the
effect of TM on STAT1 phosphorylation. Taken together, these results
suggest that attenuation of p300 expression, RelB nuclear translocation
and induction of Ca2+/CaM/calcineurin-dependent MKP-1 contribute to
the anti-inammatory actions of ER stress in LPS- and IFNs-activated
macrophages.
Paper No.: 2921

FOCUSED CONFERENCE GROUP: P11 - G PROTEIN-COUPLED 7TM RECEPTORS: FROM MOLECULAR TO
OCTOPAMINE RECEPTORS FROM THE BARNACLE
BALANUS IMPROVISUS EXHIBIT CAMP-MEDIATED SIGNALLING BY THE ANTIFOULING SUBSTANCE MEDETOMIDINE
Ulrika Lind(1), M Rosenblad(1), L Frank(2), S Falkbring(1),
L Brive(1,3), J Laurila(4), K Pohjanoksa(4), L Gunnarsson(1),
M Scheinin(4), L Martensson-Lindblad(2,5), A Blomberg(1)
(1) University of Gothenburg, Department of Cell and Molecular
Biology, Gothenburg, Sweden
(2) University of Gothenburg, Department of Zoology, Gothenburg,
Sweden
(3) University of Gothenburg, Department of Biomedicine, Gothenburg,

Sweden
(4) University of Turku, Department of Pharmacology, Drug
Development and Therapeutics, Turku, Finland
(5) I-Tech AB, Gothenburg, Sweden
Biofouling of ship hulls has many harmful consequences. The novel
antifouling agent medetomidine, previously known as an a2-adrenoceptor agonist, inhibits the settling of barnacles, the most problematic of
all biofouling species. We show that medetomidine induces a hyperactivity in the barnacle cyprid larvae that probably disrupts the settling
process. The hyperactivity is assumed to be mediated by G-protein coupled octopamine receptors. To identify the cellular targets for medetomidine, we cloned ve octopamine receptors from the barnacle Balanus
improvisus. We show by phylogenetic analyses that one receptor (BiOcta) belongs to the a-adrenoceptor like and the other four (BiOctb-R1,
BiOctb-R2, BiOctb-R3 and BiOctb-R4) to the b-adrenoceptor like
octopamine receptor subfamily. The phylogenetic analyses also indicated that B. improvisus might have a different repertoire of b-adrenoceptor like octopamine receptors than insects. Functional
characterization of the cloned receptors in CHO cells showed that they
were activated by both octopamine and medetomidine resulting in
cAMP formation. Our b-like octopamine receptors were also activated
by tyramine, but with less potency than octopamine. A hypothesis for
receptor discrimination between tyramine and octopamine was generated from a homology 3D model of the ligand-binding pocket. mRNA
expression analysis revealed that BiOctb-R3 and BiOctb-R4 were more
highly expressed in the cyprid stage as compared to adults. The characterization of B. improvisus octopamine receptors is important for a better functional understanding of these receptors in crustaceans, as well
as of biotechnological signicance.
Paper No.: 1011

EXPRESSION OF ADEA GENE IS ASSOCIATED WITH
MULTIDRUG-RESISTANCE IN ACINETOBACTER BAUMANNII
CLINICAL ISOLATES
Baodong Ling, G Liu, X Zhu, L Lin, Y Zeng, Y Xie
North Sichuan Medical College, Institute of Materia Medica and Department of Pharmacology, Nanchong, PR China
Acinetobacter baumannii are non-fermentative, Gram-negative bacilli
that cause a multitude of severe infections worldwide. Infections sustained by multidrug-resistant and even pandrug-resistant Acinetobacter
baumannii have become a growing public-health problem (Peleg AY et
al, 2008; 21: 538-82). In an attempt to assess the multidrug-resistance
in Acinetobacter baumannii isolated from our major teaching hospital
in the northern area of Sichuan Province, China, we have initiated to
investigate the distribution of drug efux system genes responsible for
antimicrobial resistance (Lin L et al, 2009; 33:27-32). In this study, we
report the resistance phenotype of Acinetobacter baumannii as well as
the expression of efux pump gene adeA in multidrug-resistant strains.
Of 86 non-repetitive clinical isolates of Acinetobacter baumannii,
63.95% were resistant to a variety of structurally unrelated antimicrobials although all isolates were susceptible to polymyxin B. Resistance to
carbapenems occurred in 27.91% of the isolates and 5 strains were
resistant to all the antimicrobial agents except polymyxin B. The presence of adeA gene was assessed by PCR and the mRNA expression of
adeA gene were quantied by real time uorescent quantitative
RT-PCR. 84.88% of the isolates detected was positive in adeA gene
and the levels of mRNA expression of adeA gene in multidrug-resistant
strains were higher compared with the susceptible ones. Our data reveal
high levels of mRNA expression of adeA gene plays a major role in
the development of multidrug-resistance in Acinetobacter baumannii
clinical isolates.
417
Paper No.: 2997
DISEASES
INHALED GLUCOCORTICOIDS DECREASE RAPIDLY
BRONCHIAL NO FLUX IN CHILDHOOD ASTHMA
Laura Linkosalo(1,2), L Lehtimaki(0), K Holm(4), M Kaila(2),
J Laitinen(4), E Moilanen(1)
(1) University of Tampere and Tampere University Hospital,
Immunopharmacology Research Group, Tampere, Finland
(2) University of Tampere and Tampere University Hospital, Paediatric
Research Centre, Tampere, Finland
(3) Tampere University Hospital, Department of Respiratory Medicine,
Tampere, Finland
(4) Tampereen Laakarikeskus Oy, Koskiklinikka, Tampere, Finland
Exhaled nitric oxide (NO) is a marker of lung inammation, and is
increased in patients with asthma. Exhaled NO has usually been assessed
at a single exhalation ow rate of 50 ml/s, but when NO is measured at
multiple exhalation ows, alveolar and bronchial NO output, and inammation, can be assessed separately. Twenty-one atopic children (15 boys,
age 6-12 years) with newly diagnosed steroid-nave asthma participated
in the study. Exhaled NO measurements and spirometry were carried out
before and 7, 28 and 56 days after starting treatment with inhaled uticasone (125lgx2). Exhaled NO was measured at three exhalation ow
rates (100, 200 and 300 ml/s), and bronchial NO ux and alveolar NO
concentration were calculated according to the linear method (Tsoukias
and George J Appl Physiol 1998;85:653; Lehtimaki et al AJRCCM
2001;163:1557), with correction for axial diffusion (Condorelli et al J
Appl Physiol 2007;102:417). Bronchial NO ux was increased in
patients with atopic asthma, and it reduced signicantly (by more than
50%, P<0.01) already in seven days of treatment. No changes in alveolar
NO concentration were detected. The 56 days follow-up was too short
to show any major changes in lung function even though symptoms
improved signicantly. The pre-treatment bronchial NO ux correlated
negatively to VC% (rho=-0.555, p=0.049) and to FVC% (rho=-0.584,
p=0.014). The results show that inhaled corticosteroids decrease rapidly
bronchial NO output in atopic asthma, and support its role as a useful biomarker of glucocorticoid effect in childhood asthma.
Paper No.: 1977

DOES DEFINITION OF MEDICATION ERRORS HAVE ANY
IMPACT AT PREVALENCE? A SYSTEMATIC REVIEW OF
DEFINITIONS
Marianne Lisby(1), LP Nielsen(1,2), B Brock(1,2), J Mainz(3)
(1) Aarhus University Hospital, Aarhus Sygehus, Department of Clinical
Pharmacology, Aarhus, Denmark
(3) University of Southern Denmark, Institute of Public Health, Odense,
Denmark
Objective: Multiplicity in terminology has been suggested as a possible
explanation for the variation in the prevalence of medication errors. So
far, no studies have challenged this assertion. The objective of this
review was, therefore, to describe the extent and characteristics of
medication error denitions in hospital settings and to reveal possible
tendencies with regard to the prevalence of medication errors. Methods:
Studies were searched for in PubMed, PsychINFO, Embase and
CINAHL employing primary search terms such as medication errors
and adverse drug events. Peer-reviewed articles containing these terms
as primary end-points were included. Study country, year, aim, design,
data-collection methods, sample-size, interventions and main results were
extracted. Results: Forty-ve of 203 relevant studies provided a generic
denition of medication errors including 26 different forms of wordings.

The studies conducted in 9 countries represented a variety of clinical settings and the approach was mainly descriptive. Of utmost importance is
the prevalence of medication errors, which ranged from 2-75 % with no
associations found between individual denitions and prevalence.Conclusion: Inconsistency in dening medication errors has been conrmed. It
appears, that denitions and detection of medication errors, rather than
being reproducible and reliable methods, are subjected to the individual
researchers preferences. Thus, application of a clear-cut denition, standardized terminology and reliable methods will greatly improve the quality and consistency in medication error ndings. Efforts to achieve a
common accepted denition, that denes the scope and content, are
therefore needed in future studies.
Paper No.: 1978

HOW SHOULD MEDICATION ERRORS BE DEFINED: A
SYSTEMATIC APPROACH TO TERMINOLOGY, CONCEPTS
AND DEFINITIONS APPLIED IN A CLINICAL SETTING
Marianne Lisby(1), J Mainz(2), B Brock(1,3), LP Nielsen(1,3)
Pharmacology, Aarhus, Denmark
Denmark
Introduction: Current literature reects inconsistent use of denitions of
medication errors revealing prevalence from 2-75 %; thus clarication of
the concept is urgently needed. The objective of this study was to
develop a denition of medication errors that separates minor errors from
serious medication errors and to apply it at data from clinical
settings.Methods:Based on existing taxonomy and through a modied
Delphi-process consensus of denition and error types were reached
among an interdisciplinary group of experts appointed by thirteen
Healthcare Organisations in Denmark and by the project group. The
expert panel prioritised ve denitions of medication errors and, subsequently, scored the relevance of 76 error types. The project group settled
non-consensus cases based on explicit criteria. Results: The panel
consisted of 12 physicians, 7 pharmacists and 6 nurses. Consensus was
reached for the denition, An error in the stages of the medication process - ordering, dispensing, administering and monitoring the effect - that
causes harm or implies a risk of harming the patient. Moreover, consensus for 60 of 76 error types was achieved. Applied to historical data the
denition reduced the number of medication errors from 34% to 7%.
Conclusion: Experts deemed a denition using harm or risk of harm as
the crucial cut-of point as most appropriate in Danish hospital settings.
Interestingly, a substantial reduction of medication errors was demonstrated, when applied to previous data. Since the denition is in accordance with international taxonomy, it is assumed to be applicable to
modern healthcare settings abroad.
Paper No.: 1979

THE EFFECT OF SYSTEMATIC MEDICATION REVIEW IN
PATIENTS ADMITTED TO AN ACUTE MEDICAL
DEPARTMENT
Marianne Lisby(1), A Thomsen(2), LP Nielsen(1,3), NM Lyhne(1),
C Breum-Leer(1), U Fredberg(4), H Jrgensen(2), B Brock(1,3)
Pharmacology
(2) Aarhus University Hospital, The Pharmacy Department, Aarhus,
Denmark
418
(4) Central Region Denmark, Regionshospital Silkeborg, Department of
Internal Medicine, Silkeborg, Denmark
Introduction: Elderly patients are exposed to medication errors and
adverse drug events due to morbidity, poly-pharmacy and inappropriate
interactions. Here we investigate the effect of additional medication
review measured as in-hospital length of stay (LOS) in elderly patients
admitted to an acute ward of medicine. Methods: The study was conducted as a randomised controlled study of 100 patients aged 70 years
or older. Intervention arm: a clinical pharmacist conducted systematic
medication reviews after a medical physician had prescribed the
patients medication. Information was collected from medical charts,
interview with patients and database registrations of drug purchase.
Subsequently, results were conferred with a clinical pharmacologist.
Control arm: Usual routine in the ward. Primary end-point was in-hospital LOS. Moreover, re-admissions, mortality, contacts to primary
healthcare and quality of life were measured at a 3-month follow-up.
A subgroup analysis addressed outcome in patients in whom recommendations were followed as compared to the to the remaining intervention group. Results: In the intervention arm the mean in-hospital
LOS was 239,9 hrs (95%CI: 190,2-289,6) and in the control arm:
238,6 hrs (95%CI: 137,6-339,6). No differences were observed for any
of the secondary endpoints. The subgroup analysis indicated that
patients receiving the intervention were admitted for almost twice as
long time. Conclusion: Overall, systematic medication review and
medication counselling did not show effect on in-hospital LOS in
elderly patients, when admitted to an acute ward of internal medicine.
Suggested interventions were more prone to be followed in patients
with complicated illness.
Paper No.: 1719

DISCOVERY OF NOVEL INFLUENZA NEURAMINIDASE
INHIBITORS FROM NATURAL PRODUCTS
Ailin Liu(1), S Lee(2), Y Wang(2), G Du(1)
(1) Chinese Academy of Medical Sciences, Institute of Materia Medica,
National Center for Pharmaceutical Screening, Beijing, PR China
(2) University of Macau, Institute of Chinese Medical Sciences, Macau,
PR China
Inuenza is the acute respiratory disease which was caused by the
inuenza A and B virus. It was reported that more than 500 million
persons were infected by inuenza virus annually. Although anti-viral
drugs play important role in inuenza treatment and prevention, especially for pandemic inuenza, the supply of effective drugs for inuenza treatment is limited. Therefore, new drug research and
development for inuenza treatment becomes very important and
urgent. Among the drug targets of inuenza virus, neuraminidase (NA)
is the most attractive drug target for new drugs for inuenza treatment.
In order to discover new anti-inuenza drugs, NA activity assay was
established, and high throughput screening (HTS) for NA inhibitors
from natural products was performed. In the end, several active
extracts from plants were discovered, and two of them, including Elsholtzia rugulosa Hemsl. and Caesalpinia sappan L. were chose for the
isolation of active constituents. Finally, 11 active compounds were
obtained. In order to discover more active compounds, based on the
pharmacophore of the active compounds, virtual screening was performed, and 5 active compounds with novel scaffolds were discovered.
In order to further improve their activity, we performed structure modication by drug design, chemical synthesis and bioassay evaluation,
their SAR was also studied. Several compounds with highly increased
activity were obtained, one CoMFA model can be used to new drug
design. These studies provide important theory and information
for further development of new drugs for inuenza treatment and
prevention.
Paper No.: 2719

HEALTH AND DISEASE
PLASMA FIBRINOGEN MEDIATES THE BINDING OF
STREPTOKINASE TO ACTIVATED HUMAN PLATELETS
Chao-Zong Liu(1), P-S Jiang(2), L-Y Chang(1)
(1) Tzu Chi University, Department of Pharmacology, Hualien City,
Taiwan
(2) Tzu Chi University, Institute of Pharmacology and Toxicology,
Hualien City, Taiwan
Clinical studies revealed that hemorrhage was slightly common with tissue-type plasminogen activator (t-PA) than with streptokinase (SK) in
treating arterial thrombosis, implying that SK may have some specicity
in lysing platelet-rich thrombi. This study aims to see if SK exhibits a
high afnity toward activated human platelets, the major component of
arterial thrombi. SK, produced by recombinant technology, was tagged
with a uorescent dye (FITC) and tested for its ability to bind resting
and ADP-activated human platelets with ow cytometry. In platelet-rich
plasma, FITC-SK bound to ADP-activated human platelets, but not to
resting platelets, in a dose-dependent manner. It failed to bind ADP-activated washed human platelets. These results indicate that plasma factor(s) is needed for the binding of SK to activated human platelets.
Blocking either platelet glycoprotein IIb/IIIa or brinogen gamma chain
C-terminus, which contains the glycoprotein IIb/IIIa recognition
sequence, diminished markedly the binding of FITC-SK to activated
human platelets. FITC-SKs platelet binding activity was also abolished
by aminocaproic acid, a lysine analogy that blocks the binding of plasminogen or plasmin to brin. From these results, we proposed that SK
formed a complex with plasminogen in the plasma and this complex then
was recruited to the surface of activated human platelets by interacting
with the brinogen that was immobilized onto the glycoprotein IIb/IIIa.
Our ndings may help understand the action mechanism of SK in lysing
arterial thrombi.
Paper No.: 708

IN VIVO AND IN VITRO MECHANISTIC STUDIES OF THE
WOUND-HEALING EFFECTS OF RADIX REHMANNIAE
TOWARDS DIABETIC FOOT ULCER USING
BIOASSAY-GUIDED FRACTIONATION
Cheuk-Lun Liu(1,2), L Cheng(1), HF Kwok(1), KM Lau(1), CM Koon(1),
CP Lau(1), KP Fung(1,2), BS Lau(1)
(1) The Chinese University of Hong Kong, Institute of Chinese
Medicine, Hong Kong, PR China
Sciences, Jong Kong, PR China
Radix Rehmanniae (RR) is one of the herb commonly used in traditional
Chinese medicine for diabetic wound healing. Our previous studies demonstrated a reduction of wound area by RR aqueous crude extract in diabetic foot ulcer rat model. In wound-healing, granulation formation is
crucial, including angiogenesis, accumulation of macrophage and broblast proliferation. Hence, the present study was to evaluate the pharmacological mechanisms of RR. Using bioassay-guided fractionation, the
angiogenesis, anti-inammatory and broblast proliferative effects of RR
aqueous extract were investigated in in vivo transgenic zebrash embryos
through quantifying sprouts formation, in vitro mouse macrophage cell
line RAW264.7 through measuring nitrc oxide production, and human
broblast Hs27 cell line via examining broblast proliferation, respectively. With sequential partition and column chromatography of the
freeze-dried aqueous crude extract of RR, a sub-fraction of the dichloromethane fraction of RR exhibited the most sprouts formation in the
sub-intestinal vessel region of zebrash embryos revealing its potency
in angiogenesis effect. Besides, up to 50% inhibition of nitric oxide
419
production was found in the dichloromethane fraction treated macrophage cells revealing that the fraction exhibited the most signicant antiinammatory activity (p < 0.05). Furthermore, the ethyl acetate fraction
(after sequential extraction with dichloromethane) showed the most
potent human broblast proliferative effect (p < 0.05). In conclusion, our
studies illustrated that RR seemed to contain relatively non-polar active
component(s) for its angiogenesis, anti-inammatory and broblast proliferative activities. Further work is required to characterize the active
principle(s) responsible for these activities.
Paper No.: 2003

OXIDATIVE STRESS-INDUCED STIMULATION OF
AMP-ACTIVATED PROTEIN KINASE INVOLVES IN SODIUM
FERULATE-INDUCED CARDIOPROTECTION
generation of nitric oxide (NO), the activity of nitric oxide synthase

(NOs), the phosphorylation of extracellular signal-regulated kinase1/2
(ERK1/2), and the expression of matrix metalloproteinases-9 (MMP-9)
and G protein-coupled receptor kinase 2 (GRK2) in cortical microvascular homogenates were evaluated. The ultrastructural morphology of cortical microvascular endothelial cells (CMEC) were observed. Results:
Transient global cerebral ischemia (20 min), followed by 24 h of reperfusion, signicantly promoted the generation of nitric oxide (NO) and the
activity of nitric oxide synthase (NOs) and led to serious edema with
mitochondrial injuries in CMEC. Crocin (20 mg/kg, 21days), pretreatment with WNK (20-10 mg/kg, 21days) markedly inhibited nitrative
injury and modulated the ultrastructure of CMEC. Furthermore, WNK
inhibited GRK2 translocation from cytosol to membrane (at 20 mg/kg)
and reduced ERK1/2 phosphorylation and MMP-9 expression in cortical
microvessels. Conclusion: We propose that WNK and its active compounds (Crocin) are effective to suppress reperfusion-induced vascular
injury to cerebral microvessels after global cerebral ischemia with the target on GRK2 pathways.
Dan Liu(2), D Yin(1), L Tang(1), B Zhong(1), D Sun(1), M He(1)

Medicine at Second Afliated Hospital, Nanchang, PR China
Sodium Ferulate (SF), one of effective antioxidant components derived
from Chinese medicinal herbs, has been show to protect against cardiomyocyte injury, possibly through the association with several signalling
pathways. The release of reactive oxygen species (ROS) is implicated in
cardiovascular diseases such as anoxia/reoxygenation(A/R) injury and
there is evidence that the production of ROS is linked to the stimulation
of AMP-activated protein kinase (AMPK). We hypothesized that AMPK
is involved in SF-induced cardioprotection and that this activation is
mediated by reactive oxygen species (ROS). To address this hypothesis,
we constructed AMPKa2-shRNA, transfected it into rat cardiomyocytes,
and a ROS-scavenger was used to assess the involvement of ROS. In the
A/R group, A/R injury resulted in a 1.4 fold increase in phosphorylation
levels of AMPK. SF preconditioning almost doubled the increase in
AMPK phosphorylation. AMPKa2-shRNA reduced the SF-medicated
increase in phosphorylation of AMPK and its target eNOS and abolished
cardioprotection. The ROS-scavenger NAC(500 lmol/L) blunted the SFmediated increase in AMPK and eNOS phosphorylation and prevented
cardioprotection. Overall, these results suggest that SF-induced AMPK
activation protects the cardiomyocyte against A/R injury and relies on
upstream production of ROS.
Acknowledgement: Project supported by the National Science Foundation of China(No. 30860271), the 973 Program(No 2009CB526405)
and the National Science Foundation of Jiangxi Province(No
2007GQY1110).
Paper No.: 930

EFFECTS AND MECHANISM OF WNK ON REPERFUSIONINDUCED VASCULAR INJURY TO CEREBRAL
MICROVESSELS AFTER GLOBAL CEREBRAL ISCHEMIA
Jianxun Liu, Y Zheng, X Li, L Xu
China Academy of Chinese Medical Sciences, Xiyaun Hospital,
Department of Pharmacology, Beijing, PR China
Introduction: Wei Nao Kang (WNK) is an active compounds extracted
from Ginkgo, Ginseng and suffron. The aim of present research is to
explore the effects of the WNK on ischemia/reperfusion (I/R) induced
vascular injury to cerebral microvessels after global cerebral ischemia.
Methods: Male mice were randomly divided into 5 groups: the sham
group, the ischemia/ reperfusion (I/R) model group, I/R+crocin (20 mg/
kg), and I/R+ WNK (10, 20 mg/kg). Bilateral common carotid artery
occlusion (20 min) in mice, followed by 24 h reperfusion, was built. The
Paper No.: 2156

ROLE OF ACETYLCHOLINE IN PROTECTING H9C2 CELLS
AGAINST OXIDATIVE STRESS INDUCED BY
ANGIOTENSIN II
J-J Liu, D-L Li, L Sun, M Zhao, X-J Yu, Wei-Jin Zang
China
Increasing studies and evidence suggested that excessive generation of reactive oxygen species (ROS) induced by rennin-angiotensin-aldosterone system (RAS) played a crucial role in different kinds of cardiovascular diseases
(CVD). Recent study showed vagus nerve stimulation modulated the cardiac redox status and adrenergic drive, thereby suppressed free radical generation in the failing heart. However, whether improving cardiac vagal
activity is benet for CVD and the exact underlying mechanism is still
unclear. H9c2 cells were treated with 10-5M acetylcholine (ACh) for 1 hour
before the addition of 10-6M angiotensin II (Ang II) for 6 hours, then measured the level of ROS, SOD, activity of caspase-3, ratio of Bax/Bcl2 and
index of apoptosis by TUNEL. The results showed Ang II signicantly
increased the expression and activity of NADPH oxidase, otherwise, compared with control group, production of ROS induced by Ang II increased
about 7-fold (vs control group, P<0.01). ACh reduced these value to 2-fold
(vs control group, P<0.01), but this effect could be inhibited by atropine.
Moreover, index of apoptosis were about 4-fold in Ang II group than control
group (7.8%1.6% vs 1.8%0.6%, P<0.01) and 3-fold higher than Ang
II+ACh group (2.4%0.9%, P<0.01). The activity and expression of caspase-3 and ratio of Bax/Bcl2 also appeared similar trend. Furthermore, we
observed ACh could reduce level of p38 MAPK phosphorylation induced
by Ang II. Our nding indicated that ACh could inhibit oxidative stress
induced by Ang II and decrease index of apoptosis through p38 MAPK signal pathway.
Paper No.: 998

DISRUPTION OF BBB IN RAT MCAO AND A PROTECTIVE
EFFECT OF ANTI-HMGB1 ANTIBODY
Keyue Liu, J Zhang, HK Takahashi, Y Tomono, H Wake, T Maruo,
A Otsuka, S Mori, M Nishibori
Okayama University Graduate School of Medicine, Department of
Pharmacology, Okayama City, Japan
The high mobility group box1(HMGB1), originally identied as an
architectural nuclear protein, exhibits an inammatory cytokine-like
420
activity in the extracellular space. In the previous study we showed that
treatment with anti-HMGB1 monoclonal antibody (mAb; 200 microgram/iv, twice) remarkably ameliorated brain infarction induced by 2hour MCAO in rats, even when the mAb was administered after the start
of reperfusion. In the present study, we focused on the protective effects
of anti-HMGB1 mAb on the disruption of blood-brain barrier (BBB) and
brain edema by using electron-microscopic observation and magnetic resonance imaging (MRI). Treatment with anti-HMGB1 mAb signicantly
reduced the water contents in ipsilateral side of the cerebral cortex, striatum, hypothalamus and hippocampus. Electron-microscopic observation
revealed that anti-HMGB1 mAb strongly inhibited the endfeet swelling
of brain capillaries and prevented detachment of the endfeet from basement membrane. Also, mAb reduced the edematous area in the T2weighted image of MRI at 3, 6, and 12-hour after reperfusion. Laserscanning confocal microscopic observation revealed that Evans blue/
albumin conjugate extravasated from capillary vessels into the ischemic
region was dramatically increased 3 hour after reperfusion and that the
treatment with mAb signicantly inhibited the extravasation of Evans
blue/albumin. Extravasated Evans blue/albumin conjugate appeared to
enter into neurons in ischemic region as well as perivascular structures.
These ndings indicate that anti-HMGB1 mAb efciently inhibits the
development of brain edema through the protection of BBB structure
and reduction of permeability to albumin in the early reperfusion phase
following focal ischemia of brain tissue.
Paper No.: 3212

ANTIOXIDANT ACTIVITY OF OLIGOSACCHARIDE ESTER
EXTRACTED FROM POLYGALA TENUIFOLIA ROOTS IN
SENESCENCE-ACCELERATED MICE
Ping Liu, Y Hu
Chinese PLA General Hospital, Department of Clinical Pharmacology,
Pharmacy Care Center, Beijing, PR China
The constituents of the ethanol extract from the root of Polygala tenuifolia Willd (Polygalaceae) were investigated for antioxidant activity in
senescence-accelerated mice. Consequently, two relevant samples were
obtained: a fraction separated by macroporous resin (YZ-OE), and a
major pure crystal of 3,6?-disinapoylsucrose (DISS). Based on HPLCESI-MS analysis, the most constituents in the YZ-OE fraction from the
extracted of P. tenuifolia were oligosaccharide esters. The antioxidant
activities of these two samples were evaluated using the accelerated
senescence-prone, short-lived mice (SAMP) in vivo. The activities of
superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) were
increased signicantly in SAMP mice fed with oligosaccharide esters
(YZ-OE 50mg/kg) and its constituents (DISS 50mg/kg). However, the
content of malondialdehyde (MDA) was increased in the blood and liver
of SAMP mice. But when given YZ-OE, it could be decreased, by 44.3
and 47.5%, respectively, when compared with SAMP model. Results
from the analyses indicated that the oligosaccharide esters (YZ-OE) from
roots of P. tenuifolia had a high antioxidant activity in vivo.
Paper No.: 1297

ESTABLISHMENT OF IN VITRO BINDING ASSAY OF HMGB1
AND S100A12 TO RAGE: EFFECTS OF HEPARIN AND DOMAIN
PEPTIDES ON THE BINDING
Rui Liu(1), S Mori(2), H Wake(1), J Zhang(1), K Liu(1), Y Izushi(1),
M Okamura(1), HK Takahashi(1), M Nishibori(1)
(1) Okayama University Graduate School of Medicine, Dentistry & Pharmaceutical Sciences, Department of Pharmacology, Okayama, Japan
(2) Shujitsu University School of Pharmacy, Japan
Interaction of the receptor for advanced glycation end products (RAGE)

with its ligands has been implicated in the pathogenesis of various
inammatory disorders. In this study, we establish in vitro binding assay
in which recombinant human high mobility group box 1 (rhHMGB1) or
S100A12 (rhS100A12) immobilized on the microplate binds to recombinant soluble RAGE (rsRAGE). The rsRAGE binding to both rhHMGB1
and rhS100A12 was saturable and dependent on the immobilized
ligands. The binding of rsRAGE to rhS100A12 was dependent on Ca2+
and Zn2+ whereas that to rhHMGB1 was not. Scatchard plot analysis
showed that rsRAGE has higher afnity for rhHMGB1 than for
rhS100A12. Structurally, HMGB1 is composed of two DNA-binding
domains (A-box and B-box), and a negative charged C-terminus. In the
present study, both recombinant A- box and AB-box domain were found
to possess binding-inhibitory effects on rsRAGE and rhHMGB1 binding.
Furthermore, rsRAGE was demonstrated to bind to heparin, and
rhS100A12 was also found to bind to heparin in the presence of Ca2+.
We examined the effects of heparin preparations with different molecular
size, unfractionated native heparin (UFH), low molecular heparin
(LMWH) 5000 Da and LMWH 3000 Da, on the binding of rsRAGE to
rhHMGB1 and rhS100A12. All three preparations concentration-dependently inhibited the binding of rsRAGE to rhHMGB1 preferably compared to rhS100A12. These results suggested that anti-inammatory
effects of heparin can be partly explained by blocking the interaction of
HMGB1 or S100A12 to RAGE. On the other hand, heparin would be a
promising effective remedy against RAGE-related inammatory disorders.
Paper No.: 1603

QUERCETIN PROTECTS CORTICAL NEUROVASCULAR UNIT
AGAINST AMYLOID B25-35 PEPTIDE-INDUCED AMNESIA IN
MICE
Rui Liu, GF Qiang, Y Jian, X Lan, HG Yang, GH Du
National Centre for Pharmaceutical Screening, Institute of Materia
Medica,Chinese Academy of Medical Sciences & Peking Union Medical
College, Beijing, PRChina
Alzheimers disease is the commonest age-related neurodegenerative
disorder characterized by a progressive loss of cognitive functions.
One feature is the accumulation of amyloid b proteins both in the
cerebral cortex and in the walls of blood vessels. A growing number
of studies have paid attention to neurovascular coupling, which provides an essential clue for better understanding of pathologic properties
of the brain and contributes to nd new strategy for drug treatment.
This study deals with the anti-amnesic effects and underlying mechanism of quercetin against Ab25-35-induced toxicity in mice. The
learning and memory capabilities, cortical neurovascular unit morphology, microvascular function, ultrastructure of cerebral cortices and cholinergic transmission were detected after oral administration of
quercetin continuously for 8 days. Our results demonstrated that quecertin administration conferred robust neurovascular unit protection in
Ab25-35-induced amnesic mice. In these effects, the learning and
memory capabilities were improved; major components of neurovascular unit were maintained, involving the preservation of microvascular
integrity and the attenuation of serious neuronal loss; regional cerebral
blood ow was gradually recovered; blood-brain barrier was well protected; tight junction expressions were up-regulated, keeping in step
with blood-brain barrier changes; the cholinergic neuronal transmission
was also improved, concerning an increase of acetylcholine level and
the modication of BNDF, TrkB and phosphor-CREB levels in cerebral cortex. In conclusion, quercetin induced reversal from the impairment of learning and memory performance induced by Ab25-35 might
be related to the preservation of neurovascular unit.
(Project is supported by National Natural Science Foundation of China,
NO. 30472015, 30772749.)
421
Paper No.: 1967
DISEASES
ANISODAMINE AUGMENTS ITS CHOLINERGIC
ANTI-INFLAMMATORY ROLE THROUGH POTENTIALIZING
STIMULATION EFFECT OF IL10 ON A7NACHR IN SEPSIS
X Liu, Q Li, A-J Liu, Ding-Feng Su
Second Military Medical University School of Pharmacy, Department of
Pharmacology, Shanghai, PR China
Anisodamine, a muscarinic acetylcholine receptor antagonist, has been
used clinically in China for treatment of various shocks, but the mechanism has not been well dened. Our previous studies demonstrate that
anisodamine plays its cholinergic anti-inammatory role through activation of a7 nicotinic acetylcholine receptor (a7nAChR). IL10 is a very
important anti-inammatory factor. Whether IL10 is involved in antishock role of anisodamine, and the relationship of anisodamine,
a7nAChR and IL-10 remains to be investigated. In this study, we found
that anisodamine could dose-dependently increase 24h survival rate of
C57BL/6 mice treated by LPS, and antishock role of anisodamine was
obviously attenuated in IL10-/- mice. However, anisodamine signicantly
decrease serum TNF-a and IL-1b level, but did not affect IL-10 level is
sepsis C57BL/6 mice. It also signicantly decreased supernatant TNF-a
and IL-6 level, but did not affect IL-10 level in LPS-treated RAW264.7
macrophages. Further study showed IL-10 could markedly promote
a7nAChR protein expression, and anisodamine could further strengthen
the stimulatory role of IL-10 on a7nAChR expression in raw264.7 cells.
Spleen in IL10-/- mice showed decreased a7nAChR protein expression
compared with that in IL10+/+ mice. Anisodamine could markedly
increase spleen a7nAChR protein expression in LPS-treated IL10+/+
mice, but this effect was almost disappear in LPS-treated IL10-/- mice.
Therefore, we demonstrate here that IL10 can markedly promote
a7nAChR expression in vivo and in vitro. Anisodamine treatment do not
alter IL10 expression level in sepsis, but it can signicantly enhancing
stimulation role of IL10 on a7nAChR, and therefore augment its cholinergic anti-inammatory effect through IL-10.
Paper No.: 1214

DISCOVERY
QUANTIFICATION, PHARMACOKINETICS AND BRIAN
DISTRIBUTION KINETICS OF THIENCYNONATE AND ITS
OPTICAL ISOMERS IN RAT
results provided important information for developing a novel chiral

drug.
Key words: thiencynonate; isomer; LC-MS; pharmacokinetics; brain distrubution.
Paper No.: 1215

DISCOVERY
STRUCTURAL ELUCIDATION OF IN VIVO METABOLITES OF
PHENCYNONATE AND ITS ANALOGUE THIENCYNONATE IN
RATS BY LIQUID CHROMATOGRAPHY-TANDEM MASS
SPECTROMETRY
Y Liu(1), Ming Xue(1), Y Kou(1), Y Xu(1), J Ruan(2), K Liu(2)
(1) Capital Medical University School of Chemical Biology and Pharmaceutical Sciences, Department of Pharmacolgy, Beijing, PR China
(2) Beijing Institute of Pharmacology and Toxicology, Beijing, PR China
The elucidation of the in vivo metabolites of phencynonate, a novel anticholinergic drug, and its analogue thiencynonate in rats by the method of
liquid chromatography electrospray ionization mass spectrometry was
described. Structural identication of the metabolites were performed by
comparing their changes in molecular masses, retention-times and spectral patterns with those of these parent drugs. The results revealed that
ten metabolites of phencynonate and seven metabolites of thiencynonate
were identied in rat urine. These drugs were easily biotransformed by
the pathways of oxidative, hydroxylated and methoxylated to form several metabolites that retained some features of the parent molecules. The
described method has wide applicability to rapidly screen and provide
structural information of these metabolites. The investigation provides
the valuable information on phencynonate and thiencynonate metabolism
which are essential for understanding the safety and efcacy of these
drugs as well as for the development of a novel chiral drug.
Keywords: phencynonate; thiencynonate; metabolite; LC-MS
Paper No.: 1138

MULTIPLE THERAPEUTICAL TARGETS REVEALED BY
PROTEIN EXPRESSION PROFILES ANALYSIS IN THE
BRAINS OF BALB/C MICE SUBJECTED TO UNPREDICTABLE
CHRONIC MILD STRESS
Yanyong Liu, N Yang, C Ji, P Zuo
Y Liu(1), Y Xu(1), Ming Xue(1), J Ruan(2), K Liu(2)

(1) Capital Medical University School of Chemical Biology and Pharmaceutical Sciences, Department of Pharmacolgy, Beijing, PR China
Thiencynonate is a novel lead compound of the anticholinergic drug
phencynonate. Quantication, pharmacokinetics and brian distribution
kinetics of thiencynonate were investigated in rat by the method of highperformance liquid chromatographic assay with electro spray ionization
mass spectrometry detection. Simultaneous MS detection of thiencynonate and the internal standard of was performed at m/z 364.0 and m/z
358.4, and the selected reaction ion monitoring of the two compounds
were both 156. The linearity was obtained over the concentration range
of 1-100 ng/ml in blood. The lower limit of quantication was at 1 ng/
ml. The precision was obtained from 2.92 to 9.76%. Extraction recoveries were in the range of 69.6-79.1%. The main pharmacokinetic parameters of thiencynonate raceme were as follows: t 0.68h, t 3.98h, t Ka
0.013h, tmax 0.076h, Cmax 54.08 ng/ml, AUC 77.70 ng-h/l. There were
some differences for the blood and brain tissue concentration of the two
isomers after dosing the thiencynonate and the R and S isomers. The
(1) Institute of Basic Medical Sciences, Chinese Academy of Medical

Sciences & Peking Union Medical College, Department of Pharmacology, Beijing, PR China
Major depression is a highly prevalent and disabling condition affecting
more than 120 million people all over the world. However, the etiology
and pathophysiology of depression remain unknown. In present study,
the Balb/c mice were exposed to unpredictable chronic mild stress
(UCMS) for different periods. Brain tissue was separated and analyzed
by differential in-gel electrophoresis to explore the molecular alterations.
Proteomic results showed the abnormal expression of proteins involved
in the energy metabolism was rstly witnessed and run throughout the
whole 10 week period. Changes in expression of the mitochondrial complexes and enzymes relating to glycolysis, may reect the abnormal
energy mobilization coping with the stressful situation in mice. Calreticulin, one component of endoplasmic reticulum involves in calcium
homeostasis, declined throughout the whole experimental period. Except
for Glial brillary acidic protein, four cytoskeletal-related proteins continuously declined. The changes of dihydropyrimidine-related protein-2
almost synchronously occurred with the changes in proteins involved in
422
energy metabolism. The obtained data indicated that the insufciency of
ATP synthesis and overwhelming ROS subsequently contributed to the
cytoskeletal damage and inhibition to expression of some anti-oxidant
proteins, which might ultimately bring functional neuron to apoptosis or
death. We propose that oxidative stress and the ensuing cellular adaptations are linked to the development process of depression and combination therapy targeting to anti-oxidant and anti- endoplasmic reticulum
stress may achieve better efcacy in striding over the impediments of
remission and partial response in depression treatment compared with the
current treatment targeting to neurotransmitter deciency.
Paper No.: 946

DISEASES
EFFECTS OF ANTI-TCR ANTIBODY ON
COLLAGEN-INDUCED ARTHRITIS IN DBA/1 MICE
Yongxue Liu, C Han, Q Wang, Z Gao, M Peng, J Cheng
Beijing Institute of Radiation Medicine, Department of Pharmacology,
Beijing, PRChina
Chronic autoimmune diseases, including rheumatoid arthritis (RA), can
result from breakdown of regulation and subsequent activation of selfreactive T cells. We investigated the effects of anti-murine T cell receptor
(TCR) monoclonal antibody, H57-597, in DBA/1 mice with collageninduced arthritis (CIA). Subcutaneous administration of dexamethasone
(0.5 mg/kg per 2 days) could signicantly improve CIA by reducing
paw swelling and clinical scoring. H57-597, administrated subcutaneously the day (d-1) before the rst collagen injection (d0) and the day
(d22) after the second collagen injection (d21), respectively, also reduced
paw swelling and clinical scoring of established CIA. At day 41, the incidence of CIA in dexamethasone-treated and H57-597-treated groups was
respectively 33.3% and 16.7%, while that in the control group was
100%. At day 48, the incidence of CIA in the H57-597-rteated group
reached 83%, however, that in the other above two groups still kept
unchanged. These results suggest that self-reactive T cells play important
roles in the progression of CIA and that anti-TCR antibody might have
therapeutic effects. However, the times of the antibody usage should be
added during the CIA course.
Paper No.: 1188

CYTOCHROME P-450 ENZYME GENETIC POLYMORPHISMS
INFLUENCE ANTIDEPRESSANT ESCITALOPRAM
METABOLISM AND TREATMENT RESPONSE
Yu-Li Liu(1), K-M Lin(1), M-H Tsai(1), M-C Hsiao(2), WW Shen(3), M-L
Lu(3), H-S Tang(4), C-K Fang(5), C-S Wu(6), S-C Lu(7), SC Liu(1), C-Y
Chen(1)
(1) National Health Research Institute, Division of Mental Health and
Addiction Medicine, Zhunan, Miaoli County, Taiwan
(2) Chang-Gung Hospital and Chang-Gung University School of Medicine, Department of Psychiatry, Taipei, Taiwan
(3) Taipei Medical University, Wan Fang Medical Center, Department of
Psychiatry, Taipei, Taiwan
(4) Songde Branch, Taipei City Hospital, Department of Psychiatry,
Taipei, Taiwan
(5) Mackay Memorial Hospital, Department of Psychiatry, Taipei,
Taiwan
(6) Far Eastern Memorial Hospital, Department of Psychiatry, Taipei,
Taiwan
(7) National Taiwan University, Institute of Biochemistry and Molecular

Biology, College of Medicine, Taipei, Taiwan
The antidepressant escitalopram (S-CIT) is metabolized by the cytochrome-P450 (CYP) enzymes CYP 2D6, 2C19 and 3A4. This study
evaluated the impact of CYP 2D6, 2C19 and 3A4 genetic polymorphisms on plasma concentrations of S-CIT and patient treatment
response. One hundred patients diagnosed with major depressive disorder were recruited to the study and their depression symptoms were
assessed using the Hamilton Depression (HAM-D) Rating Scale. The
genetic polymorphisms *4, *5, and *10 on CYP2D6, *2, *3 and *17
on CYP2C19, and *18 on CYP3A4 were selected based on their function and respective allele frequencies in Asian populations and genotyped. The steady-state serum concentrations of S-CIT and its
metabolites S-desmethylcitalopram (S-DCIT) and S-didesmethylcitalopram (S-DDCIT) were analyzed. According to semi-quantitative gene
dose (SGD) and gene dose (GD) models for allele combinations of
these polymorphisms, CYP2D6 was clustered into intermediate (0.5, 1,
1.5 SGD) and extensive (2 SGD) metabolizers, while CYP2C19 was
clustered into poor (0 GD) and extensive (1, 2 GDs) metabolizers.
Patients with intermediate CYP2D6 metabolism (0.5 SGD) had signicantly more remitters from major depressive disorder during the 8week treatment (p=0.0001). Furthermore, CYP2C19 poor metabolizers
had signicantly higher S-CIT serum levels than did extensive metabolizers at weeks 2, 4 and 8 (p < 0.05). The allele frequencies in
CYP3A4*18 and CYP2C19*17 were too low to permit further subgroup analyses. Our results suggest that the genetic polymorphisms in
CYP2C19 may be inuencing S-CIT serum concentrations, and that
specic CYP2D6 polymorphisms may be predicting patient treatment
outcomes based on gene dosage analyses.
Paper No.: 1036

SAN-HUANG- XIE-XIN-TANG PROTECTS AGAINST
MPTP-INDUCED DOPAMINERGIC NEUROTOXICITY VIA
INHIBITION OF OXIDATIVE STRESS AND APOPTOSIS
Yi-Ching Lo, Y-T Shih
Kaohsiung Medical University, Department of Pharmacology,
Kaohsiung, Taiwan
Parkinsons disease is a neurodegenerative disorder characterized by a
loss of dopaminergic neurons in substantia nigra pars compacta and
can be modeled by the neurotoxin 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP). Oxidative stress plays an improtant role in
MPTP-related neurodegeneration. San-Huang-Xie-Xin-Tang (SHXT) is
a widely used traditional Chinese medicine which possesses antiinammatory and anti-oxidative effects and is composed of Coptidis
rhizoma, Scutellariae radix and Rhei rhizome. In this study, we investigated the effects of SHXT in MPTP-induced animal model of Parkinsonism in vivo and on the neuronal damage induced by 1-methyl4-phenylpyridinium (MPP+) in vitro. When administered prior to
MPTP, SHXT redueced behavior decits and cell death and led to a
marked increase of tyrosine hydroxylase (TH) expression relative to
mice treated with MPTP alone. In addition, pre-treatment of SHXT
signicantly protected aganist MPP+-induced damage in rat primary
mesencephalic neuron cultures by increasing the number of TH,
decreasing the ratio of Bax/Bcl-2 and inhibiting the expression of
NADPH oxidase, gp91phox, cytochrome c, caspase-9 and caspase-3.
Furthermore, SHXT atteuated apoptosis evaluated by annexin V staining and increased MPP+-induced decreases of reduced glutathione
(GSH) and superoxide dismutase (SOD). The present results suggest
that SHXT possesses benecial protection against MPTP-induced neurotoxicity in this model of Parkinsons disease via its anti-oxidative
and anti-apoptotic effects. Therefore, SHXT might be a potentialy
alternative and complementary medicine used in the treatment of
neurodegenerative disorders.
423
Paper No.: 2074
METALLIC SILVER IGNITES INFLAMMATION AND CAUSES
TOXIC CAVITY FORMATION IN THE BRAIN
LJ Locht(1), Agnete Larsen(1), M Pedersen(2), S Markholt(1),
BM Bibby(3), J Rungby(4), M Penkowa(2), M Stoltenberg(1)
(1) University of Aarhus, Institute of Anatomy, Aarhus, Denmark
(2) University of Copenhagen, Institute of Neuroscience & Pharmacology, Section of Neuroprotection, Copenhagen, Denmark
(3) University of Aarhus, Department of Biostatistics, Aarhus, Denmark
Silver is a popular anti-bacterial coating agent on medical devices, e.g.
catheters to be used in neurosurgery. Such catheters are in direct contact
with brain tissue and the impact of metallic silver on nervous tissue has
never been analyzed. We injected micro-sized silver particles suspended
in hyaluronic acid in the cerebral cortex of young female mice (n=34).
In situ histochemical analyses of silver ion uptake and tissue response to
the silver implants were performed after 7 and 14 days of exposure.
A huge up-take of silver ions was seen in the ipsilateral cortex and hippocampus and to a minor degree in the contralateral cortex and hippocampus. Also the ependymal cells and the plexus choroideus showed
silver up-take. Two-way ANOVA analysis showed that silver induces
statistically signicant astrogliosis, microgliosis, metallothionein (MT-1/
MT-2) response and acute phase cytokine TNF-a response in the brain
tissue surrounding the injected silver particles. All silver treated animals
developed a cavity formation in the brain tissue around the injected silver-micro particles. Apoptotic TUNEL positive cells were only found in
a few silver exposed animals at day 7, but not at day 14. Ultrastructurally
silver particles were found in lysosome-like structures. No systemic
spreading of silver ions to liver and kidney were seen. This knowledge
makes us recommend that metallic silver and silver-coated devices
should only be used with great care inside the brain.
Paper No.: 666

TRYPTOPHAN HYDROXYLASE 2 (TPH2) GENE IS
ASSOCIATED WITH MAJOR DEPRESSIVE DISORDER IN THE
THREE MAIN ETHNIC GROUPS OF THE MALAYSIAN
POPULATION
Ai Chin Loke, LH Lian, Z Mohamed, NZ Zainal
The tryptophan hydroxylase (TPH) gene, which encodes the rate-limiting
enzyme, TPH, is involved in the biosynthesis of serotonin (5-HT).
Hence, it has been considered a possible candidate gene in the etiology
of major depressive disorder (MDD). Two isoforms of the TPH gene has
been identied, i.e., TPH1 and TPH2 (Walther and Bader, 2003; 66:
1673-1680). TPH2, which is located on chromosome 12q21.1, is highly
expressed in the brainstem, the major locus of 5-HT-producing neurons
(Zill et al., 2007; 41: 168-173). The aim of this study was to examine
TPH2 gene association with MDD in the Malaysian population. Genotyping of four single nucleotide polymorphisms (SNPs), i.e., rs1386495,
rs1386494, rs17110563 and rs7305115, was carried out in 265 unrelated
patients (59 Malay, 129 Chinese and 77 Indian) and 332 unrelated
healthy subjects (110 Malay, 129 Chinese and 93 Indian). There were no
statistical differences in both the allele and genotype frequencies between
patient and control groups in the Malay ethnicity. In contrast, a signicant association was observed in the genotype frequency of SNP
rs17110563 in the Chinese (p = <0.001) and Indians (p = <0.0025). Similarly, in terms of the SNP rs7305115, a statistical signicance was also
found in the Chinese (genotype, p = 0.015; allele, p = 0.009) and Indians
(allele, p = 0.043). Haplotype analysis indicated that the TPH2 gene

maybe associated with MDD in the Chinese and Indian ethnicities, but
not in the Malays. These results however, need to be replicated in larger
sample sizes to conrm the data obtained.
Paper No.: 2955

DISCOVERY
ITH12233, A NEW MULTIFUNCTIONAL COMPOUND,
REDUCES INFARCT VOLUME AFTER FOCAL CEREBRAL
ISCHEMIA IN MICE
Silvia Lorrio(1,2), P Negredo(1,2), MI Rodrguez-Franco(3), S Conde(3),
MP Arce(3), M Villarroya(1), JM Roda(2), MG Lopez(1), AG Garca(1)
(1) Instituto Teolo Hernando-Universidad Autonoma de Madrid,
Madrid, Spain
(2) Unidad de Investigacion y Servicio de Ciruga Experimental-Hospital
Universitario La Paz, Madrid, Spain
(3) Instituto de Qumica Medica-Centro Superior de Investigaciones
Cientcas, Madrid, Spain
ITH12233 is a new L-glutamate acid derivative, chemically synthesized
as a possible treatment for Alzheimer and vascular dementia. Three pharmacophores have been merged in the compound: N-benzilpiperidine,
which binds to the catalytic site and inhibits acetylcholinesterase
(AChE); an N-benzoyl group which interacts with the peripheral site of
AChE, implicated in b-amyloid protein (A-b) aggregation; and a lipophilic ester to facilitate penetration through the blood-brain barrier
(BBB) (Arce MP et al, J Med Chem 2009;52:7249-7257). In human neuroblastoma cell cultures, the compound exerts protection against: calcium
overload, free radicals, s hyperphosphorilation and A-b 1-42. In rat hippocampal slices subjected to oxygen and glucose deprivation, the compound exerts 55% protection. These in vitro results encouraged us to test
whether ITH12233 would protect using in vivo models of brain damage. We performed experiments using photothrombotic focal cerebral
ischemia in mice. ITH12233 was injected i.p. (2.5, 5 and 10 mg/kg) 1 h
before ischemia and twice a day for the two following days. Mice were
sacriced 72 h after ischemia; fresh coronal brain sections were TTC
stained for infarct volume measurement. ITH12233 was able to reduce
infarct volume signicantly at 2.5 and 5 mg/kg (46 and 45%, respectively). Melatonin was used as a positive control (Zou LY et al, J Pineal
Res 2006;41:150-156), which reduced 28% infarct volume. It seems that
ITH12233 also protects against neuronal excitotoxic death after hippocampal kainate injection in rats. We believe these results show that
ITH12233 is an interesting compound with an evident neuroprotective
therapeutic potential in cerebrovascular disease.
Paper No.: 2016

LACTATE IS THE ENDOGENOUS LIGAND FOR GPR81 AND
SUPPRESSES LIPOLYSIS IN ADIPOCYTES
Timothy Lovenberg, C Liu, C Kuei, T Mirzadegan, J Shelton, J Yu, J Zhu
Johnson & Johnson Pharma, Department of R&D, San Diego, CA, USA
Lactate is a well recognized by-product of anaerobic metabolism as well
as a source of energy in mammals. Numerous studies have demonstrated
that lactate can also direct metabolic activity via a hypothesized action
on adipose tissue. We recently demonstrated that lactate, in fact, specically activates a heretofore orphan G-protein coupled receptor (GPR81)
which highly expressed in adipocytes. In contrast to many GPCRs which
activate in response to low concentrations of endogenous ligands,
424
GPR81 is activated by high concentrations of lactate (1-20 mM). However, this concentration range is highly relevant as it is the physiological
range of lactate concentrations experienced by animals and humans. We
also demonstrated that lactate suppresses lipolysis in mouse, rat, and
human adipocytes, as well as in differentiated 3T3-L1 cells. Adipocytes
from GPR81-decient mice lack an anti-lipolytic response to lactate but
are responsive to other anti-lipolytic agents. To further demonstrate a
specic interaction of lactate and GPR81, we have demonstrated internalization of GPR81 after receptor activation by lactate. Site-directed mutagenesis of GPR81 demonstrates that classically conserved key residues
in the transmembrane binding domain are responsible for interacting with
lactate. This poster will highlight additional new experiments designed to
elucidate the structure-function relationship between lactate and GPR81
and how this has been used to identify additional receptor agonists.
GPR81 represents attractive target for the treatment of dyslipidemia and
other metabolic disorders.
Paper No.: 764

GOLGI ACTIVATION OF CDC2 KINASE IN PACLITAXELINUCED APOPTOSIS IN HUMAN ANDROGEN-INDEPENDENT
PROSTATE CANCERS
Pin Hsuan Lu, JH Guh
National Taiwan University, College of Medicine, Taipei, Taiwan
Organelles such as mitochondria and endoplasmic reticulum (ER) play
an important role in mediating apoptosis. Recently, emerging evidence
shows that the localization and activation of several apoptotic factors
such as PKCd at Golgi complex reveal the indispensable role of Golgi
complex in apoptotic process. Microtubule-targeting agent, paclitaxel,
was reported to induce mitotic arrest followed by apoptosis through cdc2
activation. In this study, we investigated the role of Golgi complex in
paclitaxel -induced apoptosis. The exposure to paclitaxel promptly dispersed Golgi complex into small fragments in PC-3 cells. In untreated
cells, PKCd localized predominantly at cytoplasm, nucleoli and Golgi
complex. In the presence of paclitaxel, the nuclear localization of PKCd
was diminished. The Western blot showed that paclitaxel induced an
increase of Golgi complex-associated cdc2 phosphorylation at Thr161,
indicating the activation of this kinase. Both rottlerin (a PKCd inhibitor)
and roscovitine (a cdc2 inhibitor) signicantly rescued paclitaxel-induced
apoptosis. Moreover, rottlerin effectively blunted Golgi complex-associated cdc2 activation, indicating that PKCd served as an upstream effector. All together, the data suggest that the Golgi activation of cdc2 kinase
may contribute to paclitaxel-mediated apoptosis in PC-3 cells. The data
also provide evidence supporting that the combination use based on facilitating Golgi activation of cdc2 kinase may potentiate paclitaxel-mediated cancer chemotherapy.
Paper No.: 1710

PREPARATION OF MONOCLONAL ANTIBODIES BY IN VIVO
ELECTROPORATION-ASSISTED HE4 GENE IMMUNIZATION
IN MICE
H Luan(1,2), X Gao(1), J Fu(1), Z Ma(1), L Dong(1), L Fan(2), Haifeng
Song(1)
(1) Beijing Institute of Radiation Medicine, Department of Pharmacology
andToxicology, Beijing, PR China
(2) Anhui Medical University, Anhui, PR China
Aim: To prepare mouse monoclonal antibodies (mAb) specic against
human epididymis gene product 4 (HE4), an over-expressed biomar-
ker in human ovarian cancer, by the means of electroporation-assisted

DNA immunization in mice, and to identify the biological characteristics of the prepared mAbs. Methods: The cDNA encoding human
HE4 was obtained from ovarian cancer tissues in patients by RTPCR method. It was subcloned into an expression vector pPICZaA
and validated by sequencing. The reconstructed plasmid pPICZaAHE4 was used to immune BALB/c mice with the assistance of in
vivo electroporation. The hybridomas were achieved by fusing the
immunized spleen cells with Sp2/0 myeloma cell line. The positive
clones secreting anti-human HE4 antibodies were screened by further
subcloning, and the biological activities were determined by Western
blot, Ig subtyping and epitope analysis. Results: Two stable hybridoma cell lines, named 1-7-C and 3-12-C, continuously and stably
secreting specic anti-HE4 monoclonal antibodies were established.
The biological activity studies showed that the monoclonal antibodies
recognized the HE4 protein with natural conformation. With the Ig
subgroup belonged to IgM, and the light chains were type k. Conclusion: We successfully constructed pPICZaA-HE4 expression vector
and obtained two mouse anti-human HE4 hybridomas. The secreted
antibodies could specically recognize the HE4 protein, which laid
establishment for early diagnosis of ovarian cancer.
Paper No.: 2379

RELEVANCE OF MANGANESE SUPEROXIDE DISMUTASE
(SOD2 VAL16ALA) AND GLUTATHIONE PEROXIDASE1 (GPX1
PRO200LEU) FUNCTIONAL POLYMORPHISMS IN PATIENTS
WITH IDIOSYNCRATIC DRUG-INDUCED LIVER INJURY
(DILI)
Maribel Isabel Lucena(1,7), E Garcia-Martin(2,8), RJ Andrade(3,7),
C Martinez(4,8), C Stephens(1,7), JD Ruiz(4,8), E Ulzurrun(1,7),
MC Fernandez(5), JA Duran(6), A Melcon de Dios(6), Y Borraz(1,7),
JAG Agundez(4,8)
(1) University of Malaga, Hospital Universitario Virgen de la Victoria,
Department of Clinical Pharmacology, Malaga, Spain
(2) University of Extremadura Faculty of Science, Department of Biochemistry, Molecular Biology and Genetics, Badajoz, Spain
(3) Hospital Universitario Virgen de la Victoria, Faculty of Medicine,
Hepathology Unit, Malaga, Spain
(4) University of Extremadura Faculty of Medicine, Department of
Pharmacology, Badajoz, Spain
(5) Hospital Torrecardenas, Unit of Clinical Pharmacology, Almeria,
Spain
(6) Hospital Virgen de la Macarena, Department of Clinical Pharmacology, Sevilla,Spain
(7) Centro de Investigacion Biomedica en Red de Enfermedades Hepaticas y Digestivas (CIBERehd), Spain
(8) Red de Investigacion de Reacciones Adversas a Alergenos y Farmacos (RIRAAF)
Introduction: Drug-induced liver injury (DILI) susceptibility has a potential genetic basis. We have evaluated possible associations between the
risk of developing DILI and common genetic variants of the manganese
superoxide dismutase (SOD2 Val16Ala) and glutathione peroxidase
(GPX1 Pro200Leu) genes, which are involved in mitochondrial oxidative
stress management. Methods: Genomic DNA from 185 DILI patients
assessed by the CIOMS scale and 270 gender and age-matched controls
were analysed. The SOD2 and GPX1 genotyping was performed using
PCR-RFLP and TaqMan probed qPCR, respectively. Results: The SOD2
Ala/Ala genotype was associated with cholestatic/mixed damage
(OR=2.3; 95% CI=1.4-3.8; Pc=0.0058) while the GPX1 Leu/Leu genotype was associated with cholestatic injury (OR=5.1;1.6-16.0;
Pc=0.0112). The presence of 2 combined risk alleles (SOD2 Ala and
GPX1 Leu) was more frequent in DILI patients (OR=2.1; 1.4-3.0;
Pc=0.0006). Patients with cholestatic/mixed injury induced by mitochon-
425
dria hazardous drugs were more prone to have the SOD2 Ala/Ala genotype (OR=3.6;1.4-9.3; Pc=0.02). This genotype was also more frequent
in cholestatic/mixed DILI induced by pharmaceuticals producing
quinone-like or epoxide metabolites (OR=3.0; 1.7-5.5; Pc=0.0008) and
S-oxides, diazenes, nitroanion radicals or iminium ions (OR=16.0;
1.8-146.1; Pc=0.009). Conclusions: Patients homozygous for the SOD2
Ala allele and the GPX1 Leu allele are at higher risk of developing
cholestatic DILI. SOD2 Ala homozygotes may be more prone to suffer
DILI from drugs that are mitochondria hazardous or produce reactive
intermediates.
Funding: Spanish Medicine Agency, SAS PI-0082/2007 and FUNDESALUD. CIBERehd and RIFAAF are funded by ISC III.
Paper No.: 1257

COMPLEMENTARY AND ALTERNATIVE DRUGS USE
AMONG PREOPERATIVE PATIENTS: A CROSS-SECTIONAL
STUDY IN TUSCANY (ITALY)
Ersilia Lucenteforte(1,2,3), E Gallo(1,2), A Pugi(1,2), F Giommoni(1,2),
P Lupi(4), M La Torre(5), G Diddi(6), F Firenzuoli(4), A Mugelli(1,2), A
Vannacci(1,2), F Lapi(1,2,7)
(1) University of Florence, Department of Preclinical and Clinical
Pharmacology M. A. Mancini, Florence, Italy
(2) Tuscan Regional Centre of Pharmacovigilance, Florence, Italy
(3) University of Milan, Department of Labour Medicine L. Devoto,
Section of Medical Statistics and Biometry G.A. Maccacaro, Milan,
Italy
(4) ASL 11 Hospital, Anaesthesiology Unit, Empoli, Italy
(5) ASL 10 Hospital, Anaesthesiology Unit, Florence, Italy
(6) ASL 4 Hospital, Anaesthesiology Unit, Prato, Italy
(7) Regional Authority for Healthcare Services of Tuscany, Epidemiology Unit, Florence
Introduction: Patients in preoperative period are treated with different
classes of drugs for anesthesia and because of medical/surgical condition.
Herbal medications use is high and has been associated with signicant
adverse events [Ang-Lee MK et al, JAMA 2001; 286:208-16]: thus it
would be quite relevant to investigate the prevalence and the predictors
of complementary and alternative drugs (CADs) use among preoperative
patients. To our knowledge, no Italian studies in preoperative setting
have been conducted. Materials: A survey on 478 consecutive patients
was conducted in three Tuscan hospitals, using a structured questionnaire. Odds ratios (ORs) and corresponding 95% condence intervals
were estimated using logistic regression models. Results: The prevalence
of CAD suse was 49.8%, and 233 out of 238 used herbal products and/
or dietary supplements. Herbal remedies mainly reported were valeriana
ofcinalis, matricaria recutita, vaccinium myrtillus, aloe vera, and echinacea purpurea. Compared to patients with 18-47 years, the OR were 2.1
for patients with 48-69 years and 3.0 for those with 70-95 years. Direct
associations were also found with pensioners compared to homemakers
(OR=3.2) and with users of more than three conventional drugs compared to non users (OR=2.1). Adjustment for age, gender, education and
occupation did not change the univariate estimates, except for education:
the ORs were 2.6 and 3.5 for subjects with high school degree and university degree, respectively. Conclusion: Given the high prevalence of
use of CADs among patients undergoing surgery and the risk associated
with their use, a careful anamnesis of CADs consumption before surgery
should be recommended.
Paper No.: 3138

DISCOVERY
IN VITRO EVALUATION OF DRUG INTERACTION WITH
P-GLYCOPROTEIN AND PERMEABILITY OF F-80002-RR IN
EARLY STAGES OF DEVELOPMENT
Maria Luisa Lucero(1), A Gonzalo(1), I Soengas(2), M Rodriguez(2)
(1) FAES FARMA S.A., Department of Research & Development and
Innovation, Lejona, Vizcaya, Spain
(2) DynaKin S.L., Derio, Vizcaya, Spain
Introduction: F-80002-RR is a 5-(4-Methanesulphonyl-phenyl)-thiazole
derivate, a new drug family able to selectively inhibit both prototype
Th1 cytokine INF-c and TNF-a production. Early assessment of the ability of a drug candidate to penetrate into certain tissues is critical during
drug discovery, especially for therapeutic indications that require delivery
to a specic site of action. Transport determined using MDCK cell
monolayer approximates passive biological permeability. Moreover,
comparison of efux ratios between MDR1-MDCK and MDCK transwell assays can provide a measure of the specic human P-glycoprotein
(P-gp) mediated efux activity. Methods: The bidirectional transport of
F-80002-RR (10 microM) across MDR1-MDCK and MDCK parent cells
was examined for 2 hours. Reference compounds for low and high
permeability (atenolol and propranolol, respectively) and P-gp substrate
(digoxin) were also assayed. The transport was determined in duplicate.
Test and reference compounds were quantied by UPLC-MS/MS.
Apparent permeability (Papp) values in both directions and efux ratio
(RE=PB/A/PA/B) were calculated. Human P-gp mediated activity was
assessed by comparing the efux ratios between transfected and
wild type cells (RFinal=RET/REC). Results: Permeability for compound F-80002-RR was found to be high as Papp (A-B3
[28.210.92(meanSD)] and the RE<3 [0.860.06]. The RFinal was <3
[0.96] conrming that the compound is not a P-gp substrate. Conclusion:
An in vitro assay has been employed to early classify F-80002-RR as a
highly permeable drug which is not a P-gp substrate. As membrane
permeability and P-gp can be limiting factors for drug efcacy these
results have been used as criteria for further drug development.
Paper No.: 1009

INTRODUCING CLINICAL EVENTS CLASSIFICATION FOR
IMPROVING ANTIBIOTIC PRESCRIBING
Tatyana Lugovkina(1), B Richards(2)
(1) Clinical Hospital N40, Department of Clinical Pharmacology,
Yekaterinburg, Russian Federation
(2) University of Manchester, Medical informatics, UK
Introduction: The accurate assessment of a patients clinical status
according to the introducing clinical events classication has allowed the
creation of a data-base for evaluation the adherence of the real antibiotic
prescribing to the recommended prescribing protocols and the creation of
a model for calculating the drug cost. Materials and Methods: The database holds the 5 classes of clinical events. Every disease has its international (general) code. The record of every patient is reected in general
and special (class of clinical event) codes. The information of clinical
events includes two parts: the patients clinical status and concordant protocols of drug prescribing. The criteria for the determination of the
patients clinical status are based on the criteria of the inammatory reaction to infection (reected in international guidelines) with consideration
of additional risk factors with the inuence for making decision. The
rules for drugs-prescribing were built according to evidence based medicine data, or to consensus decisions made by the local specialists. Results
and Conclusions: The data-base built on the introduced clinical events
426
classication allowed to rise the adherence of the real antibiotic prescribing to the recommended protocols (by a factor of 6) and was used for
calculating the drug cost models of the denite diseases treatment.
Paper No.: 2391

MODELLING AND LITERATURE ANALYSIS TO EVALUATE
THE PHARMACOKINETICS OF NEW FAST RELEASE
IBUPROFEN BASED ON A COMPARISON OF STUDIES
PERFORMED IN INDIA AND EUROPE
CL/F to laboratory measures as predictors. A simple algorithm mimicking

clinician intervention to keep levels within the target range (5-15 ng/mL)
was linked to Dynapred. Results. The possibility of two modes in the
CL/F distribution lead to careful analysis of all relevant physiological variables. Eventually, the population pharmacokinetic model (used as a prior
for Bayes TDM in Dynapred) included two patient subgroups categorized by ALB and HCT status in addition to categories by GOT. Bayes
TDM was compared to the actual TDM outcomes. Conclusion. Due to PK
idiosyncrasies, population priors are best developed on patient populations
that are similar to the one treated. Bayes TDM using Dynapred, in this
example with TAC, shows signicant improvement (RMSE) in achieving
target levels early post transplant under the same random assay error levels. Within-patient variability was still large with the Bayes method.
John C Lukas(1), M Rodriguez(1), A Knapp(2), P Kraahs(2)

(1) Dynakin SL, Department of Drug Modelling & Simulation, Derio,
Vizcaya, Spain
(2) LOSAN Pharma GmbH, Germany
Introduction: Losan study BE-250-IBU-2009 (BE-250), performed with
Indian subjects, had higher than expected Cmax for the two fast absorption treatments, Ibuprofen Losan 400 mg Effervescent Tablet and Fast
release coated tablet at 400 mg p.o. single dose. The Cmax were 69.0
and 63.5 ug/mL, respectively versus a range of 40 to 50 mcg/mL
expected from the European BQE IBU-2008 (BQE) study and published
data. This difference raised safety concerns. Methods: The objective was
to explain the differences regarding Cmax in the fast-release formulations. The effervescent and lm-coated tablets tested in the BE-250 were
modeled independently. The coated forms from the BE-250 and the BQE
studies were modeled in combination. Parameter and demographic variable correlations were made. Results: WT was the only covariate in the
population PK model and it was crucial in predicting compartmental V2/
F. It completely explained the apparent kinetic difference between European (heavier) and Indian (lighter) subjects PK. Literature and other
study mean data (meta-analysis) were used to show a general trend of
Cmax, AUC and Tmax across fast and slow formulations and versus the
identied predictors. Finally, a literature review was performed showing
the achieved concentration levels lay well inside documented safety margins. Conclusions: Body weight explained the observed difference in
Cmax between the same fast formulations in the studied groups. The
AUC is unchanged across the two fast coated studies (BE-250 and BQE)
and across the weight range. The literature provides no support for a
safety concern with Ibuprofen Losan effervescent 400 mg p.o.
Paper No.: 2392

USEFULNESS OF BAYES TDM FOR MONITORING IN
IMMUNOSUPPRESSION USING THE DYNAPRED
PLATFORM: AN EXAMPLE WITH TACROLIMUS
John C Lukas(1), I Oteo(1), R Calvo(2), A Valdivieso(3), N Leal(1),
E Suarez(2)
(1) Dynakin SL, Department of Drug Modelling & Simulation, Derio,
Vizcaya, Spain
(2) Universidad del Pais Vasco, Spain
(3) Cruces Hospital, Spain
Introduction. The observed large variability in Cmin/Dose after therapeutic drug monitoring (TMD) in immunosuppression, in addition to intrinsic
variability in the pharmacokinetics, is indicative of dose adjustments outside steady state for a portion of patients. We use population pharmacokinetics and simulation to map that variability into drug exposures and
propose Bayes dose targeting as a rational manner of individualizing treatment. A web based user-friendly Bayes TDM application (Dynapred) is
presented using an example from tacrolimus (TAC) monitoring. Methods.
Retrospective monitoring data from 75 de novo liver transplant patients
under twice daily TAC (no other treatment) followed up to 15 days were
scrutinized and then modeled. Physiological reasoning was used to relate
Paper No.: 2800

HEALTH AND DISEASE
COX-1 OR COX-2 DELETION IS ASSOCIATED WITH
INCREASED NUCLEAR NF-JB IN THEENDOTHELIUM OF
THE AORTIC ARCH
Martina H Lundberg(1), LS Harrington(1), PDM Leadbeater(1), TD
Warner(2), JA Mitchell(1)
(1) Imperial College London, National Heart and Lung Institute, Cardiothoracic Pharmacology, London, UK
(2) The William Harvey Research Institute, Barts and the London School
of Medicine and Dentistry, London, UK
Nuclear translocation/activation of NF-jB in endothelial cells is associated with vascular inammation and increased risk of atherosclerosis.
Endothelial cells release the cardioprotective hormone prostacyclin
through the enzymatic actions of cyclo-oxygenase (COX) and prostacyclin synthase. COX is present in two isoforms: COX-1 which is expressed
constitutively and COX-2 which is induced at sites of inammation. We
have previously shown that COX-1 predominates over COX-2 in the
endothelium of the mouse aortic arch, and that deletion of COX-1, but not
COX-2, ablates prostacyclin production (Lundberg et al PA2 online 035P,
September 2009). In the current study, we hypothesised that loss of cardioprotective prostacyclin may be associated with increased activation of
NF-jB in aortic endothelium. On investigation, we found, using enface
confocal microscopy, that nuclear NF-jB is increased in the endothelium
of both the inner and outer curvature regions of the aortic arch, from
COX-1-/- and COX-2-/- mice in untreated and LPS (4mg/kg i.p.; 30min)
treated animals. Results: endothelial nuclear NF-jB/p65 (uorescence
intensity units; meanSEM n=11-15). (i) Control treated mice; outer curvature; wild type 2024451; COX-1-/- 5020602; COX-2-/- 5207773:
inner curvature; wild type 4031862; COX-1-/- 6411369; COX-2-/5398625. (ii) LPS treated mice; outer curvature; wild type 6564836;
COX-1-/- 87641094; COX-2-/- 136431285: inner curvature; wild type
10731766; COX-1-/-156341067; COX-2-/- 15988823. In non-LPS
treated mice, increased levels of nuclear NF-jB were not associated with
elevated serum levels of pro-inammatory cytokines (including TNF-a).
These data suggest that the functional effects of interactions between
COX and NF-jB in the endothelium remain local to the vessel wall.
Paper No.: 3432

MOLECULAR DETERMINANTS OF THE INTERACTION OF
SELECTIVE NEGATIVE ALLOSTERIC MODULATORS WITH
METABOTROPIC GLUTAMATE RECEPTOR 2
Linda Lundstrom, C Bissantz, J Beck, T Woltering, J Wichmann, JG
Wettstein, S Gatti
F.Hoffmann-La Roche Ltd., Discovery Research CNS and Chemistry,
Basel, Switzerland
427
Allosteric modulation of membrane receptors is the most important indirect mechanism for the control of receptor function and the allosteric
sites on G-protein coupled receptors (GPCRs) are of high interest for
their possible therapeutic applications. The present study is taking advantage of the presence of negative allosteric modulators (NAMs) with high
afnity for Group II metabotropic glutamate receptors (mGlu2 and
mGlu3). We show that interaction of NAMs with mGlu2 modulate the
afnity of the orthosteric agonist and inhibits signal transduction. A site
directed mutagenesis study, targeting the TM domain and extracellular
loops mGlu2, was combined with functional measurements to characterize the allosteric binding site. By comparing two chemically different
NAMs (NAM-1 and NAM-2) we identied a number of specic interactions in the binding site for each of the two modulators which were
conrmed with molecular Modelling using an mGlu2 homology model.
Furthermore, the ability of the NAMs to inhibit agonist binding at the
orthosteric site was particularly sensitive to mutagenesis in the extracellular side of the allosteric binding pocket. By utilizing a radiolabelled positive allosteric modulator (PAM) our study also conrm an overlapping
binding site of negative and positive allosteric modulators at mGlu2
while only the NAMs are active at mGlu3. This study is the rst molecular investigation of the allosteric binding pocket of the Group II metabotropic glutamate receptors and provides a better understanding for the
requirements to develop potent and specic mGlu2/3 NAMs as well as
giving some initial information for the interaction of PAMs at mGlu2.
Paper No.: 2248

LEPTIN INDUCES HYPERTROPHY VIA ACTIVATING PPARA
PATHWAY IN CULTURED NEONATAL RAT CARDIOMYOCYTES
Paper No.: 1408

EFFICACY OF PANAX NOTOGINSENG SAPONINS ON
PATIENTS WITH INTRACEREBRAL HEMORRHAGE
Yumin Luo, Q Liu, J Song, P Liu, L Gao
Xuanwu Hospital of Capital Medical University, Cerebrovascular
Diseases Institute, Beijing, PR China
The clinical efcacy of Panax notoginseng saponins on the patients with
acute intracerebral hemorrhage was determined in this study. The patients
were randomly assigned into control group (The patients were given regular without Panax notoginseng saponins) and treatment group (treated
with the regular therapy and the Panax notoginseng saponins, 175mg,
intravenous drip, once a day for 2 weeks). All patients were evaluated
with the basic health conditions, vital signs, history, and the National
Institutes of Health Stroke Scale (NIHSS). The blood regular test and Creactive protein were determined before and after treatment. NIHSS
scores and head CT were evaluated for all patients after treatment. The
two groups of patients showed no statistical signicant differences in
basic parameters when the patients were admitted for hospitalization, incaluding the NIHSS scores, hematoma volume, leukocytes, neutrophil
percentages, C-reactive protein levels. After 2 weeks of the therapy, the
treatment group showed signicant decreases, in NIHSS score and hematoma, compared to the control group. Moreover, the leukocytes, neutrophil percentage, and C-reactive protein value were signicantly lower in
the treatment group than that in the control group. In conclusion, Panax
notoginseng saponins can facilitate the absorption of hematoma and
improve the recovery of neurological function in patients with acute
intracerebral hemorrhage. Its effects may relate to inhibit inammatory
reaction.
Keywords: Panax notoginseng saponins; leukocyte; C-reactive protein;
platelet aggregation.
Jian-Dong Luo(1), N Hou(1), M-S Chen(2)

(1) Guangzhou Medical University, Department of Pharmacology,
Guangzhou, PR China
(2) Guangzhou Medical University, Guangzhou institute of
Cardiovascular Disease, Guangzhou, PR China
Our previous study has demonstrated that leptin induces hypertrophy.
However, the mechanisms by which leptin induces cardiomyocyte hypertrophy are poorly understood. Recent studies indicate that peroxisome
proliferator-activated receptor (PPARa) activation may be responsible for
pathologic reModelling and severe cardiomyopathy. Leptin, as PPARa
endogenous activator, regulates energy metabolism through activating
PPARa in many cells including cardiomyocytes. Therefore, we hypothesized that leptin induces cardiomyocyte hypertrophy through activating
cardiac PPARa pathway. Cultured neonatal rat cardiomyocytes were used
to evaluate the effects of PPARa on hypertrophy. The selective PPARa
antagonist GW6471 concentration-dependently decreased total RNA levels, ANF mRNA expression, protein synthesis, and cell surface areas, all
of which were elevated by leptin after treatment 72 hours in cardiomyocytes. The augmentation of reactive oxygen species (ROS) levels in cardiomyocytes treated with leptin was reversed by GW6471. After
treatment for 24 hours, leptin concentration-dependently enhanced
mRNA and protein expression of PPARa, upregulated its activity. Meanwhile, PPARa agonist fenobrate concentration-dependently increased
total RNA levels, ANF mRNA expression, and protein synthesis in cardiomyocytes treatment for 72 hours. ROS levels were also elevated in a
concentration-dependent manner compared to those cells without treatment. These ndings indicate that PPARa activation induces hypertrophy
and leptin induces hypertrophy through the PPARa pathway activation in
cultured neonatal rat cardiomyocytes. The PPARa antagonist therapy
strategy may provide another effective means in ameliorating cardiac
dysfunction in obese individuals.
Paper No.: 2959

DISEASES
LIPOPOLYSACCHARIDE INDUCED CHANGES OF LIVER
FUNCTION IN L-OPIOID-RECEPTOR-WILDTYPE
AND - KNOCKOUT MICE
Amelie Lupp, S Schulz
Friedrich Schiller University Jena, Institute of Pharmacology and Toxicology, Jena, Germany
Sepsis, septic shock and subsequent multiorgan failure remain an important cause of morbidity and mortality in critical care patients. Often, quite
early an impaired liver function can be observed. Opioids frequently are
used for analgesia in critical care despite several data indicate that morphine can aggravate inammatory reactions. Since the exact pathophysiology remains unclear, the aim of the present study was to elucidate if
the l-opioid receptor (mOP-R) is involved in this effect using an animal
model of bacterial endotoxinaemia. Adult male mOP-R-wildtype (wt)
and -knockout (ko) mice were treated either with saline or with 5 mg/kg
body weight lipopolysaccharide (LPS) i.p.. Animals were sacriced
16 hours thereafter and different parameters indicating liver function or
oxidative state were assessed. LPS treatment caused a decrease in liver
cytochrome P450 isoforms expression and activities, which was more
pronounced with wt than with mOP-R-ko mice. Hepatic bile acid excretion was reduced due to LPS administration with wt animals only. Endotoxin treatment further led to an increase in liver tissue levels of lipid
peroxidation products, whereas GSH values were diminished. Again,
effects were more distinct with wt than with mOP-R-ko animals. In summary, wt mice seem to be more susceptible to the inuence of LPS on
liver function than mOP-R-ko animals, pointing to an involvement of the
428
mOP-R in the modulation of systemic inammatory reactions. Thus, in
accordance with other data, also our results suggest that morphine and
other mOP-R agonists may be harmful in sepsis, while mOP-R antagonists may exert benecial effects.
Paper No.: 553

CHEMOENZYMATIC SYNTHESIS OF NEW DIAZABICYCLIC
AMIDES OF 12-PHENYLACETYLRICINOLEIC ACID AND IN
VITRO ANTINEOPLASTIC ACTIVITY IN MOUSE AND
HUMAN LEUKEMIC CELLS
Axel Luviano(1), R Tiburcio(1), I Aguiniga(1), P Demare(1), M Lopez(1),
J Cardoso(1), I Monsalvo(1), I Castillo(2), E Santiago(1), I Regla(1)
(1) Facultad de Estudios Superiores Zaragoza, Universidad Nacional
Autonoma deMexico, Laboratorio de Sntesis de Farmacos, Iztapalapa,
Mexico
(2) Instituto de Qumica, UNAM, Mexico
Introduction: Cancer is a major problem of public health. In the search
of new antitumoral drugs, a series of three new diazabicyclic amides
derived from12-phenylacetylricinoleic acid were synthesized using a
novel chemoenzymatic method. These compounds were provisionally
named PhAR-DMe, PhAR-DBn andPhAR-DEtOH. The antineoplastic
properties of this compounds were evaluated in mouse leukemic cell
lines P388, J774, WEHI-3, human leukemic cell line K562 and mouse
normal bone marrow cells. Materials: Reagents were obtained from
Sigma Co.P388, J774 and WEHI-3 cells were seeded on 96-well plates
in ISCOVES medium10% FBS and subjected to different concentrations
of the compounds and incubated in a 37 C humidied atmosphere of 5
% CO2 for 72 hours. After this time, the cytotoxic effect was evaluated
using the Sulforhodamine B assay. Results and conclusion: The synthetic
procedure involved base-catalyzed methanolysis of castor oil to afford
methyl ricinoleate, phenylacetylation (1) and regioselective enzymatic
hydrolysis of the methyl ester to yield phenylacetylricinoleic acid.
(1S,4S)-2-methyl-, (1S,4S)-2-hydroxyethyl- and (1S,4S)-2-bencyl-,5diazabicyclo[2.2.1] heptanes were synthesized according to the literature
(2). Finally, amides PhAR-DMe, PhAR-DEtOH and PhAR-DBn were
synthesized by reaction of phenylacetylricinoleic acid with (1S,4S)-2alkyl-2,5-diazabicyclo[2.2.1] heptanes by the mixed anhydride method.The sulforhodamine B assay for cytotoxicity screening showed significant high cytotoxic effects of PhAR-DMe and PhAR-DEtOH for
leukemic cells and low toxicity toward mouse bone marrow cells. This
remarkably selective cytotoxic effect highlights the antineoplastic potential of both new compounds. References: (1)Castillo et al, Synlett 2008,
2869-2873; (2) Regla I et al, Eur J Org Chem 2008,655-672.
Paper No.: 2840

CARDIAC SAFETY ASSESSMENT: BEYOND QT
S Lynagh, David Bunton
Biopta Ltd, Glasgow, UK
Since the conception of the ICH guidelines in 2000, the focus of cardiac
safety testing has been detection of QT prolongation and assessment of
torsades de pointes liability. There are multiple assays available to investigate such effects ranging from isolated ion channels to in vivo telemetry
many of which are a regulatory requirement. Despite rigorous testing a
reported 28% of drug withdrawals in the US are due to alterations in
heart muscle function, valve damage, fatal arrhythmias and coronary
artery effects in patients thus highlighting a continuing deciency in the
process which may be due to species differences in the case of animal
tests or simply the wrong end points being assessed. In recent years there
have been a number of high prole drug withdrawals related to cardiac
side effects, none of which were attributed to effects on the QRST complex. Attention is now turning towards the prediction of other potential
cardiac safety liabilities. Herein, we present data from our own laboratory utilising functional human cardiac tissue as well as a review of
already published investigations focussing on endpoints other than QT
prolongation. The data presented will highlight that while important,
developers should be looking beyond the QT effects of drugs when
choosing their drug candidates. By assessing effects in the models
described at an early stage one can truly de-risk a compound for cardiac effects and avoid costly late stage withdrawals and the litigation
mine-eld which has been observed in recent years.
Paper No.: 2136

DISCOVERY
IVERMECTIN ACTIVATES CYS-LOOP LIGAND-GATED
CHLORIDE CHANNELS BY INTERACTING WITH THE THIRD
TRANSMEMBRANE DOMAIN
Timothy Lynagh, T Webb, J Lynch
The University of Queensland, Queensland Brain Institute, St Lucia,
QLD, Australia
Ivermectin activates the inhibitory glycine receptor chloride channel
(GlyR), and the binding site and mechanism of action are unclear. Similarly, it is unclear how ivermectin activates its anthelmintic target, the
glutamate-gated chloride channel (GluCl). We generated a random
mutant library and screened approximately 1600 GlyR mutants for loss
of activation by ivermectin using a high through-put uorescence based
assay. Hits from the screenings, including A288T and Y279F, were characterised by sequencing and electrophysiology. When alanine 288 of
GlyR was substituted for larger amino acids, activation by ivermectin
was abolished. When tyrosine 279 was substituted for phenylalanine, the
ivermectin EC50 was right-shifted (WT EC50 = 2.70.4 microM,
Y279F EC50 > 10 microM, n=4). We investigated whether these substitutions also decreased sensitivity to potentiation by ivermectin of glycine-activated currents. Y279F actually showed higher than WT
sensitivity to potentiation by ivermectin (WT potentiation EC50 =
2.80.6 microM, Y279F potentiation EC50 = 0.50.1 microM, n=4),
whereas substitution of A288 abolished potentiation by most practical
concentrations of ivermectin in our experiments (A288F potentiation
EC50 >> 30 microM, n=6). Interestingly, substitution of GlyR alanine
288 with glycine increased sensitivity to ivermectin (A288G EC50 =
328 nM, n=4), and when we substituted the equivalent GluCl position
a glycine residue with larger residues, the GluCl ivermectin EC50
was right-shifted. These data indicate that ivermectin likely interacts with
A288 at the extracellular end of TM3 of cys-loop ligand-gated chloride
channels, and that the TM2-TM3 linker is involved in the subsequent
opening of the channel.
Paper No.: 1723

DISEASE AND THERAPY
DIFFERENT MUTATIONS IN THE ABCC8 GENE CAN EITHER
ENHANCE OR REDUCE SULFONYLUREA RECEPTOR 1
MEDIATED APOPTOSIS
Martina Losle, S Ackermann, S Hiller, A Hambrock
University of Tubingen, Institute of Experimental and Clinical Pharmacology and Toxicology, Department of Pharmacology and Experimental
Therapy, Tubingen,Germany
429
Sulfonylurea receptor 1 (SUR1) forms the regulatory subunit of ATPsensitive potassium channels (K-ATP channels) in pancreatic b-cells and
neuronal cells and plays a key role in triggering insulin secretion. Previously, we have shown that SUR1 can also be specically involved in the
modulation of b-cell apoptosis after treatment with glibenclamide, resveratrol, or 17b-estradiol, insulinotropic SUR ligands acting as K-ATP channel blockers. In this study, we investigate the effect of different
mutations in the SUR1 (ABCC8) gene on 17b-estradiol-induced apoptosis. Some of these mutations have been detected in patients with insulin
secretion disorders before. By analysis of different apoptotic parameters
in recombinant HEK293 cells, we show that the mutation M1289T in
transmembrane helix 17 (TM17) completely abolishes SUR1-specic
17b-estradiol-induced apoptosis whereas other mutations (K719R,
K1384M, R1379C, R1379L) located in the nucleotide binding folds
(NBFs) signicantly enhance the apoptotic effect. In conclusion, different
ABCC8-mutations can essentially inuence 17b-estradiol-induced apoptosis by either enhancing or abolishing the SUR1-specic effect. TM17
as well as both NBFs obviously are important structural elements for the
modulation of SUR1-mediated apoptosis. These results suggest that polymorphisms in the ABCC8 gene could not only confer an increased risk
for the development of insulin secretion disorders via affecting regulation
of K-ATP channel activity but possibly also via inducing pathophysiological changes in b-cell viability.
Paper No.: 841

BENZODIAZEPINES REVEALS THE ADDICTIVE
PROPERTIES OF BUPRENORPHINE IN A CONDITIONED
PLACE PREFERENCE IN MICE
Lin Lin Ma, V Lelong-Boulouard, A Coquerel
University Hospital Center, EA 3917 Department of Pharmacology and
Toxicology, Caen, France
Introduction: Buprenorphine, a partial l agonist (and d + j antagonist), binds to opioid receptors with high afnity. It is widely used
as substitution therapy for heroin addicts, but it is often abused and
misused specially in association with various benzodiazepines. This
type of association is implicated in deaths due to overdose after
injections and probably in drug craving. Previous study has shown
that benzodiazepine increase buprenorphines sedative effect but
decrease its anxiogenic properties. With the aim to evaluate the
addictive potential effects of this association, we studied the inuence
of different doses of three benzodiazepines on buprenorphine conditioned place preference (CPP) behavior in mice. Methods: Buprenorphine (1mg/kg) was injected subcutaneously in male Swiss mice (n
=10or12 per group) alone or in association with a benzodiazepine:
clorazepate (CRZ), at 1,4,10 and 16mg/kg; bromazepam (BMZ), at
0.5,1,3mg/kg; diazepam (DAZ), at 0.5,1,3mg/kg (injected intra-peritonal). Amphetamine (8mg/kg) was used as the reference drug.
Reward effects of buprenorphine and the association were measured
in CPP paradigm with an unbiased procedure. Results: Our results
showed that groups treated by buprenorphine associated with CRZ at
1, 4, 10mg/kg; with BMZ at 0.5, 1, 3mg/kg; with DAZ at 0.5, 1,
3mg/kg signicantly increased the time spent in the drug paired compartment compared to the group treated with buprenorphine alone.
Conclusions: Our results suggested that joint consumption of benzodiazepine might increase the addictive properties of buprenorphine
alone. This results could explain the need of BZDs for BPN substituted patients and sometimes the real craving induced by these associations.
Paper No.: 2934

EPAC INDUCES AIRWAY SMOOTH MUSCLE RELAXATION
BY SHIFTING THE RHOA/RAC1 BALANCE TOWARDS RAC1
Harm Maarsingh(1), SS Roscioni(1), CRS Elzinga(1), LJ Schuur(1),
MH Menzen(1), AJ Halayko(2), H Meurs(1), M Schmidt(1)
(1) University of Groningen, Department of Molecular Pharmacology,
Groningen, The Netherlands
(2) University of Manitoba, Department of Physiology and Internal
Medicine, Winnipeg, Canada
Dysfunctional regulation of airway smooth muscle tone is a feature of
obstructive airway diseases such as asthma and chronic obstructive pulmonary disease. Airway smooth muscle contraction is directly associated
with changes in the phosphorylation of myosin light chain (MLC), which
is increased by Rho and decreased by Rac. Although cAMP-elevating
agents are believed to relieve bronchoconstriction mainly via activation
of protein kinase A (PKA), here we addressed the role of the novel
cAMP-mediated exchange protein Epac in the regulation airway smooth
muscle tone. Isometric tension measurements showed that specic activation of Epac led to relaxation of guinea pig tracheal preparations pre-contracted with methacholine, independently of PKA. In airway smooth
muscle cells, Epac activation reduced methacholine-induced MLC phosphorylation. Moreover, when Epac was stimulated, we observed a
decreased methacholine-induced RhoA activation, measured by both
stress ber formation and pull-down assay. Furthermore, Epac activation
prevented methaholine-induced Rac1 inhibition measured by pull-down
assay. The Epac relaxant effect was signicantly attenuated by treatment
with the Rac1-inhibiting toxin B-1470 conrming the importance of
Rac1 in Epac-mediated relaxation. Importantly, we show that human airway smooth muscle tissue expresses Epac, and Epac activation relaxed
pre-contracted human tracheal preparations and decreased MLC phosphorylation. In conclusion, activation of Epac relaxes airway smooth
muscle by decreasing MLC phosphorylation by skewing the balance of
RhoA/Rac1 activation towards Rac1. Therefore, specic activation of
Epac may have therapeutical potential in the treatment of obstructive airway diseases.
Paper No.: 2275

SIX YEARS EXPERIENCE OF USING AN INTERACTIVE
VOTING DEVICE IN TEACHING BASIC PHARMACOLOGY
TO STUDENTS OF PHARMACY AND MEDICINE
Ewen MacDonald
University of Eastern Finland, Department of Pharmacy, Kuopio, Finland
The use of an interactive voting system (Interactive Presenter) is one
way to activate students in a large lecture theatre since it allows even the
shyest student to respond to a question without fear of embarrassment.
Recently, we have used the device in quiz-type settings. The quiz takes
elements from television counterparts, interspersing groups of questions
with musical interludes (we use music with a pharmacological theme,
but one could use the time for a revision of points raised by the questions
or videos). After the elimination round in which all students take part,
Interactive Presenter is used to identify the four students with the best
scores who are brought to the front to take part in the knock-out
rounds. During this phase, the participants can ask for help from the
audience, ensuring involvement of all students, not only those participating in the knock-out quiz. The majority of students (76%) stated that
they learned during the quiz and only 17% believed that traditional lectures were more conducive to learning, even fewer (7%) felt that the quiz
was a waste of time. All but one of the 108 respondents stated that
430
Interactive Presenter increased the attractiveness of this type of teaching. Their written comments conrmed their enthusiasm for this type of
teaching. In conclusion, teaching of basic pharmacology, a fact-lled
topic, can benet by inclusion of versatile lecture theatre feedback
devices such as Interactive Presenter.
Disclosure: The author has no nancial ties nor received any remuneration from the manufacturer of Interactive Presenter.
Paper No.: 1422

HEALTH AND DISEASE
5-HYDROXYTRYPTAMINE INDUCES CYCOLOOXYGENASE-2
THROUGH MECHANISM INVOLVING SRC, PKC AND MAPK
IN RAT VASCULAR SMOOTH MUSCLE CELLS
Takuji Machida, K Iizuka, M Hirafuji
Hokkaido Health Sciences University, Faculty of Pharmaceutical
Sciences, Department of Pharmacology, Hokkaido, Japan
The effects of 5-hydroxytryptamine (5-HT) on cyclooxygenase (COX)
expression in rat vascular smooth muscle cells (VSMCs) have been
investigated. VSMCs were enzymatically isolated from aortic media of
Wistar rats. When VSMCs were incubated with 5-HT for 24 h, 5-HT
stimulated prostaglandin I2 production, which was completely suppressed by NS-398, a selective COX-2 inhibitor. 5-HT induced transient
COX-2 protein and mRNA expression in a concentration- and timedependent manner, while it had no effect on COX-1 expression. This
effect of 5-HT was completely inhibited by sarpogrelate, a 5-HT2A
receptor antagonist. 5-HT-induced COX-2 expression was markedly
blunted by Ca2+-depletion; GF 109203X, a protein kinase C (PKC)
inhibitor; PP2, an inhibitor of Src-family tyrosine kinase; PD 98059, an
inhibitor of extracellular signal-regulated kinase (ERK) activation; SB
203580, an inhibitor of p38 mitogen-activated protein kinase (MAPK);
and SP 200125, an inhibitor of c-Jun N-terminal kinase (JNK). 5-HT
activated ERK and p38 MAPK, followed by JNK activation. PP2 inhibited these activations, while GF 109203X inhibited only JNK activation.
Furthermore, PD 98059 inhibited not only ERK activation but also JNK
activation. These results suggest that 5-HT induces COX-2 expression in
rat VSMCs, and that PKC, Src-family tyrosine kinase, and MAPK activation are each essential for the full expression of COX-2 pathways.
Paper No.: 2706

IMPAIRED FUNCTION OF CYP2C9 METABOLIC ENZYME
AND OATP1B1 TRANSPORTER AS A PREDISPOSITION FOR
HMGCOA REDUCTASE INHIBITORS INDUCED RHABDOMYOLYSIS:CASE REPORTS
V Macolic-Sarinic(1), N Mirosevic-Skvrce(1), Nada Bozina(2),
S Marusic(3), S Tomic(1)
(1) Agency for Medicinal Products and Medical Devices, Zagreb, Croatia
(2) Zagreb University School of Medicine and University Hospital Center,
Department of Laboratory Diagnostics, Zagreb, Croatia
(3) University Hospital Dubrava, Zagreb, Croatia
SEARCH study identied a strong association between myopathy and
variants in the SLCO1B1 gene which encodes an organic anion transporter OATP1B1 in patients who were treated with simvastatin (The
SEARCH Collaborative Group, N Engl J Med.;359(8):789-99). The
STRENGTH study expend these results with CK >3X normal to milder,
statin induced, muscle side effects in patients taking simvastatin, atorvastatin and pravastatin (Voora Det all, J Am Coll Cardiol. 2009;
54(17):1609-16). As statin-induced rhabdomyolisis is very rare adverse
drug reaction and data about genetic risk factors are lacking, we recognized importance of publishing cases reported to Croatian Agency for
Medicinal Products and Medical Devices. Case1. A 67-year-old woman
developed rhabdomyolysis after taking simvastatin in dose of 40 mg for

6 months. She has diabetes mellitus and renal impairment. Patient recovered after discontinuation of the drug. Pharmacogenetic analysis revealed
that she has low activity genotype SLCO1B1 388GG and 521TC, which
could predispose for adverse drug reaction. Case 2. A 58-year-old
woman developed rhabdomyolysis during treatment with uvastatin and
cyclosporine. After kidney transplantation she received 150 mg/day of
cyclosporine for two months. Than uvastatin was introduced (80 mg/
day). One month after she developed rhabdomyolisis and was admitted
to the hospital with muscle weakness and movement disorder. After discontinuation of uvastatin (CYP2C9 substrate) she recovered. She was
genotyped as CYP2C9*1/ *3 carrier. Before kidney transplantation she
was taking atorvastatin without adverse drug reactions. Impaired drug
metabolism and drug transporter capacity have impact on drug disposal
and interactions resulting in adverse drug reactions.
Paper No.: 1506
DISEASES
CEREBRAL ISCHEMIA INDUCES MICRO VASCULAR
PRO-INFLAMMATORY CYTOKINE EXPRESSION VIA THE
MEK/ERK PATHWAY
Aida Maddahi, L Edvinsson
Lund University, Vascular Research, department of medicine, Lund,
Sweden.
Cerebral ischemia results in increased expression of cerebrovascular endothelin and angiotensin receptors reduced cerebral blood ow (CBF) and
increased levels of pro-inammatory mediators. We hypothesise that inhibition with a specic MEK1/2 inhibitor (U0126) will block transcription
of these genes, reduce infarct size and improve neurology score. Rats
were subjected to 2 hours MCAO followed by reperfusion for 48 hours.
Two groups of treated animals were studied; (i) intraperitoneal administration of U0126 starting at 0, 6, or 12 hours after the occlusion, (ii) combination of the angiotensin AT1 and the endothelin ETA receptors inhibitor,
given immediately after occlusion. The expression of TNF-a, IL-1b, IL-6,
iNOS, pERK1/2, pp38 and pJNK were analyzed with immunohistochemistry. We observed an infarct volume of 25 +/) 2% and reduced neurological function. Immunohistochemistry revealed enhanced expression of
TNF-a, IL-1b, IL-6, iNOS and pERK1/2 in the SMC and in the associated
intracerebral microvessels. U0126 injection at 0 or 6 hours after the ischemic but not at 12 hours, reduced the infarct volume (11.7 +/) 2% and 15
+/) 3%, respectively), normalized pERK1/2 and prevented elevation of
expression of TNF-a -, IL-1b, IL-6 and iNOS. The combined inhibition
of angiotensin AT1 and endothelin ETA receptors decreased the brain
damaged (12.3 +/) 3%; P<0.05) but only slightly reduced the MCAO
induced enhanced expression of iNOS and cytokines The present study
shows that the elevated vascular expression of TNF-a, IL-1b, IL-6 and
iNOS is associated with the microvasculature following focal ischemia is
transcriptionally regulated via the MEK/ERK pathway.
Paper No.: 2791

THE PHARMACODYNAMICS OF SINGLE DOSE R- AND
S-WARFARIN WHEN ADMINISTERED ALONE AND IN
COMBINATION AS A FUNCTION OF VKORC1 GENOTYPE IN
HEALTHY VOLUNTEERS
John Maddison(1,2), A Somogyi(2), B Jensen(4), M Gentgall(3),
P Rolan(0)
(1) Royal Adelaide Hospital, Adelaide, SA, Australia
Australia
(3) University of Adelaide, Pain and Anaesthesia Research Clinic,
Adelaide, SA, Australia
431
(4) University of Otago, Department of Clinical Pharmacology,
Christchurch, NewZealand
Introduction: Despite taking into account known factors including age,
CYP2C9 and VKORC1 gene polymorphisms only 60% of interindividual dose variability with warfarin is explained. The contribution of R-warfarin and its relationship to VKORC1 genotype is unclear. Methods: We
are conducting a three-way crossover study of 80 mg of pure R-warfarin,
12.5 mg of pure S-warfarin and 25 mg of racemic warfarin in healthy
subjects stratied by VKORC1 genotype all CYP2C9 1*/1*. Results
from the rst 11 subjects (6 VKORC1 CC genotype, 2 CT, 3 TT genotype) show the mean AUCPT (AUC of change in prothrombin time from
baseline) for R-warfarin of 995 s.h (SD) 502), S-warfarin 198 s.h (SD
137) and for racemic warfarin 385 s.h (SD 284). The difference in AUCPT between S-warfarin and racemic warfarin is signicant (p < 0.01).
The mean ratio of AUCPT(R-warfarin)/AUCPT(S-warfarin) in the CC
group is 12.4 compared to 5.1 in the CT/TT group (p=0.13). Conclusion:
This is the rst human study in which unequivocally pure R-warfarin
clearly demonstrates therapeutic effect alone and with S-warfarin. In this
small sample there is a trend to difference in response to each enantiomer
based on genotype which needs to be conrmed with a larger cohort.
Paper No.: 3115
DRUG CONSUMPTION IN CROATIA: THE EFFECTS OF
PRICING POLICY CHANGES
Tomislav Madjarevic(1), T Buble(2), M Gantumur(3), M Vitezic(1,4),
B Sestan(1,4), J Mrsic Pelcic(4), D Vitezic(3,4)
(1) University Hospital for Orthopaedic Surgery Lovran, Lovran, Croatia
(2) Pharmadata, Zagreb, Croatia
(3) University Hospital Centre Rijeka, Rijeka, Croatia
(4) University of Rijeka, Faculty of Medicine, Rijeka, Croatia
The aim of this study is to detect changes in total drug usage and nancial
expenditure according to legal changes in Croatia, especially considering
pricing policy. The data were obtained from the Croatian National Health
Insurance Institute for the eight-year period (2001-2008). Financial expenditure data are presented in Euros, while drug utilization data are presented in dened daily doses/1000 inhabitants/day (DDD/1000). During
the investigated period drug usage increased 81.33%, while nancial
expenditure increased 77.23%. ATC C-group (cardiovascular drugs) represents approximately 30% of annual nancial expenditure and 50 % of
total DDD/1000. While total DDD/1000 increased for approximately 10%
every year, nancial expenditure increased 10-20% annually until 2006,
but since then there have been no signicant changes. After the introduction of the new pricing system, at the end of 2005, there was a decrease in
price which is calculated in Euro/DDD. This decrease differs from 2-20 %
relating to different ATC groups, i.e. for C-group is 15.85%. Drugs usage
and nancial expenditure increased during the investigated period. As a
result of the introduction of the new pricing system, which regulates
prices of the new original drugs and also stimulates generics prescription,
there has been a signicant decrease in total nancial expenditure.
Paper No.: 1091
DOPAMINE INHIBITS NO PRODUCTION BY LPS-ACTIVATED
MICROGLIA THROUGH THE FORMATION OF
QUINOPROTEIN
Sadaaki Maeda, A Tozawa, T Kitamoto, A Yamamuro, A Kasai,
Y Yoshioka
of dopaminergic neurons of the substantia nigra, which causes a severe

depletion in striatal dopamine. In this study, we investigated the effect of
dopamine on LPS-induced NO production in microglial cell line BV-2.
The production of NO was assessed as the accumulation of nitrite in the
culture supernatants, using a colorimetric reaction with Griess reagent,
and the level of quinoproteins was determined by using the DTNB/glycinate reagent. Pretreatment with dopamine (1-100 microM) for 24 h concentration-dependently attenuated the LPS-induced NO production in
BV-2 cells. Neither SCH23390 nor sulpiride, D1-like and D2-like dopamine receptor antagonists, respectively, affected the attenuation of NO
production by dopamine. In addition, Neither CY208-243 nor bromocriptine, D1-like and D2-like dopamine receptor agonists, respectively,
affected the LPS-induced NO production in BV-2 cells. On the other
hand, N-acetylcysteine (NAC, 10 mM), which scavenges hydrogen peroxide (H2O2) and hydroxy radical, inhibited the attenuation of NO production by dopamine. NAC has also been known to scavenge dopamine
quinone, which is a oxidized metabolite of dopamine and conjugates
with cysteine residues in proteins to form quinoproteins. Pretreatment
with H2O2 did not attenuate LPS-induced NO production. Dopamine
increased the level of quinoproteins in BV-2 cells. NAC inhibited the
increase in the level of quinoproteins by dopamine. These results suggest
that dopamine attenuates NO production by LPS-activated microglia
through the formation of quinoprotein.
Paper No.: 2169

ANALYSIS OF CELLULAR SIGNALLING FOR EPIDERMAL
GROWTH FACTOR-MEDIATED SUPPRESSION OF
ANDROGEN-INDUCED EXPRESSION OF PROSTATESPECIFIC ANTIGEN GENE IN HUMAN PROSTATE CANCER
LNCAP CELLS
Yuma Maeda, R Yamanaka, F Sekiguchi, A Kawabata
Kinki University School of Pharmacy, Division of Pharmacology and
Proliferation and progress of prostate cancer are initially dependent
on androgen in the majority of cases, whereas prostate cancer
patients often develop an androgen-refractory phenotype that leads to
poor disease prognosis and eventual death. Epidermal growth factor
(EGF) causes downregulation of androgen receptors at mRNA or
protein levels and prevention of neuroendocrine differentiation in
human prostate cancer LNCaP cells. There is evidence that EGFinduced downregulation of androgen receptors and prostate-specic
antigen (PSA) involves the phosphatidylinositide-3-OH kinase
(PI3K)/Akt pathway, but not MAP kinase pathways, in androgenindependent LNCaP C-81 cells (Hakariya et al, Biochem. Biophys.
Res. Commun. 2006;342:92). The present study thus aimed at clarifying mechanisms responsible for EGF suppression of androgen signaling in androgen-sensitive LNCaP cells. We rst conrmed EGFmediated suppression of dihydroteststerone (DHT)-induced elevation
of PSA mRNA levels in androgen-sensitive LNCaP cells. The inhibitory effect of EGF on DHT induction of PSA mRNA was abolished by inhibitors of EGF receptor (EGFR) tyrosine kinase and
PI3K. However, inhibitors of Src, NF-kappaB, phospholipase C,
MEK/ERK, p38 MAP kinase and JNK failed to affect the EGF
suppression of DHT-elevated expression of PSA mRNA. Thus, EGF
suppresses androgen-induced PSA expression via activation of EGFR
and subsequent activation of the PI3K pathway in androgen-sensitive
LNCaP cells, which resembles mechanisms for EGF-induced downregulation of androgen receptors and PSA in androgen-independent
LNCaP C-81 cells.
Setsunan University, Department of Pharmacotherapeutics, Osaka, Japan

The overproduction of nitric oxide (NO) by activated microglia has been
proposed to play a pathogenetic role in Parkinsons disease. The most
important neuropathological alteration in Parkinsons disease is the loss
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Paper No.: 2863
INHIBITION OF BASAL AND ULTRAVIOLET B INDUCED
MELANOGENESIS BY CANNABINOID RECEPTORS CB1
Soa Magina(1,2), C Esteves Pinto(1), E Moura(3), MP Serrao(1),
I Correia-de-Sa(1), D Moura(1), M-A Vieira-Coelho(1,3)
(1) Instituto de Farmacologia e Terapeutica, FMUP, Porto, Portugal
(2) Servico de Dermatologia e Venereologia, Hospital de S.Joao, Porto,
Portugal
(3) Instituto de Biologia Celular e Molecular, UP, Porto, Portugal
Introduction The melanocytes and keratinocytes are in close contact
forming the epidermal unit. It has been shown that keratinocytes synthesis endocannabinoids and that melanocytes express cannabinoid receptors. Since there is a cutaneous endocannabinoid system we have asked
whether it plays a role in melanogenesis. Objectives To study the effects
of cannabinoid drugs in basal and ultraviolet B (UVB) induced melanogenesis. Methods The expression of cannabinoid receptors was examined
by Western blot analysis. We utilized human melanoma cells (SK-mel-1)
in monoculture and in co-culture with human immortalized HaCaT keratinocytes and evaluated the melanin content in basal condition and after
UVB irradiation (3mJ) in three consecutive days. Cell cultures were treated for 120 hours, with a selective CB1 agonist ACEA (1 and 10
microM) and a selective CB1 antagonist AM251 (1 microM). At the end
of incubation period the melanin content of the cells was determined
spectrophotometrically. Cell viability was measured by the calcein assay.
Results The SK-mel-1 express CB1 and CB2 receptors but only in coculture experiments, activation of CB1 receptor signicantly decreased
basal and UVB induced melanogenesis. The selective CB1 agonist
(ACEA 10 microM) inhibited melanogenesis in co-cultures and this
effect was reverted by the AM251 1 microM. After the incubation, none
of the drugs used affected the viability of the two cell types. Conclusion
Our results suggest that CB1 receptor exert an inhibitory effect on melanogenesis, but this effect requires the modulation by keratinocytes. Cutaneous cannabinoid system may be a potential therapeutic target in
pigmentary disorders.
Paper No.: 1342

THE SEIZURES AND STATUS EPILEPTICUS IN EMERGENCY
DEPARTMENT OF PEDIATRICS.THE CLINICAL AND
PHARMACOLOGICAL IMPLICATIONS OF SOME
BENZODIAZEPINES
Ioan Magyar
Oradea University Faculty of Medicine and Pharmacy, Department of
Pharmacologyand Oradea Municipal Clinical Hospital, Emergency
Department of Paediatrics,Oradea, Romania
Benzodiazepines possess anxiolytic, sedative, hypnotic,central muscle
relaxant and anticonvulsant properties. Among benzodiazepines,
lorazepam,diazepam, midazolam and clonazepam are of utmost importance in the emergency treatment of seizures, the major epilepsy crisis
and status epilepticus.The authors of this article have analysed the pharmacological and clinical implications of some benzodiazepines, such as
diazepam and midazolam in the treatment of convulsive disorders in children in the pediatric emergency department. In children aged 0 to
9 years, the prevalence of seizures is 4.4 cases per 1000, and in those 10
to 19 years,the prevalence is 6.6 cases per 1000. We have studied 75
patients, all children,admitted into hospital with various convulsive disorders and who recieved treatment in the emergency department of pediatrics. In some cases of epilepsy we examined the blood levels of
anticonvulsant drugs such as carbamazepine and phenytoin. We did
laboratory tests such as blood glucose,plasma concentrations of sodium,
potasium, cloride, calcium,magnezium, creatinine, blood urea nitrogen

and arterial blood gases, as well as some imaging studies such as head
computed tomography. We also performed electroencephalogram in some
cases of nonconvulsive status. The conclusions of the study show that
diazepam and midazolam currently are and they will continue to be
amongst the most effective and most frequently used benzodiazepines in
the emergency treatment of seizures and status epilepticus in children.
Paper No.: 472

VENTRAL TEGMENTAL AREA NMDA RECEPTORS PLAY A
ROLE IN THE EFFECT OF SCOPOLAMINE ON INHIBITORY
AVOIDANCE MEMORY IN RATS
Gelavizh Mahmoudi(1), M-R Zarrindast(2), A Pourmotabbed(3)
(1) Azad University, Kermanshah Branch, Kermanshah, Iran
(2) Institute for Cognitive Science Studies,Tehran, Iran
(3) Kermanshah University of Medical Science, Department of Physiology, Kermanshah, Iran
Introduction: Ventral tegmental area (VTA) has an important role in
learning and memory, it is showed that neurons in the VTA receive cholinergic and glotamatergic innervations that regulate their ring activities.
Therefore, in the present study the effect of NMDA receptors in the VTA
on impairment of memory by scopolamine was investigated. Material
and method: 2 cannulae were stereotaxically implanted bilaterally into
VTA adult male rats, a step-through passive avoidance task,as a model
of learning and memory was used.After 1 week recovery, animals
received bilateral intra VTA injection of scopolamine (0.5 or 1 or 2 or
3 lg/rat), MK801 (0.5 or 0.75 or 1 or 1.5lg/rat), NMDA(0.0001 or
0.001 or 0.01 or 0.1lg/rat), scopolamine (0.25,0.5lg/rat)+
MK801(0.5lg/rat), scopolamine (2lg/rat)+ NMDA(0.001,.01lg/rat) after
training and 24h later memory parameter was tested. Results: Our results
showed that Post-training intra-VTA microinjection of muscarinic acetylcholine receptor antagonist scopolamine- (2,3lg/rat) and NMDA receptor antagonist -MK801- (1,1.5lg/rat) impaired memory. Interstingly
injection of ineffective doses of MK801(0.5lg/rat) and scopolamine
(0.25,0.5lg/rat),which had no effects alone, synergistically impaired
memory. On the other hand, administration of NMDA(0.001,0.01lg/rat),
in combination with an effective dose of scopolamine (2lg/rat) prevented
the impairing effect of scopolamine in memory. Conclusion: These
results suggest a possible role of NMDA receptors located in the VTA
on scopolamine-induced memory impairment.
Paper No.: 2055

DISEASES
PHARMACOLOGICAL EVALUATION OF CLINICALLY
ACTIVE COMPOUNDS IN A MODEL OF CYSTIC FIBROSIS
RELATED INFLAMMATION
Rangan Maitra, A Giddings, J Tajuba
RTI International, Department of Pharmacology & Toxicology, Research
Triangle Park, NC, USA
Cystic Fibrosis (CF) is a common genetic disorder caused by mutations
to the CFTR gene. Most CF patients lose lung function from chronic
inammatory infections involving pathogens like Pseudomonas aeruginosa (PA). There is no cure for CF and disease management is critical. A
key inammatory pathway in CF involves activation of NFjB through
stimulation of toll receptors by bacterial products and transcription of
pro-inammatory genes. We developed a CF-relevant image-based
screening assay to identify anti-inammatory agents for use in CF.
433
An immortalized CF airway cell line was engineered to express a green
uorescent protein (GFP)-based NFjB reporter. Filtrates of a PA strain isolated from the airways of a CF patient were used to stimulate NFjB reporter
activity. A collection of ~400 clinically approved compounds was screened.
Positive compounds identied included several steroids with known utility
in the treatment of CF and compounds with known NFjB inhibitory activity
but previously untested in the context of CF. Select compounds including
Fluticasone Propionate, an anti-inammatory drug that is utilized in the treatment of airway inammation, demonstrated pharmacologically relevant
IC50 values in nanomolar range. We are currently evaluating some of
these compounds in additional models of CF inammation. The developed model is also a useful tool to identify mediators and inhibitors of
inammation in CF and other diseases of the lung.
Paper No.: 2056

SYNTHESIS AND CHARACTERIZATION OF PERIPHERALLY
SELECTIVE CB1 RECEPTOR ANTAGONISTS
Rangan Maitra, Y Zhang, M Roche, J Tajuba, R Snyder, N Gaudette,
T Fennell, B Thomas, H Seltzman
RTI International, Department of Pharmacology & Toxicology, Research
Triangle Park, NC, USA
CB1 receptor (CB1R) antagonism is a promising therapy to treat liver
disease, diabetes, metabolic syndrome and other etiologies. CB1R is
expressed in the CNS and also in peripheral tissues. Currently available
antagonists of CB1R are unsuitable for use in disorders with peripheral
involvement of this receptor due to CNS-related adverse effects. Our goal
was to rationally design, synthesize and test peripherally selective CB1R
antagonists. Peripheral selectivity of a compound is dictated by its ability
to cross the blood-brain barrier (BBB). The BBB generally excludes passive penetration of compounds that are charged, highly water soluble or
large. With these factors in mind, several analogs of the known CB1R
inverse agonist SR141716 were synthesized and tested including
compounds bearing a quaternary ammonium group, analogs with unquaternized amide chains of various lengths, carboxy-substituted cyclohexylamide analogs and a pyridinium analog. Functional studies
indicated that many of the synthesized analogs of SR141716 were active
with pharmacologically relevant Ke values (<100 nM). Pharmacokinetic
analysis of select compounds in rats indicated that these compounds were
primarily restricted to the periphery with poor permeability across the
BBB. Our studies indicate that design of peripherally selective CB1R
antagonists is feasible and is a promising approach to treat various etiologies where peripherally expressed CB1R has been implicated.
Paper No.: 2054

FEATURES OF MELOXICAM ANTINOCICEPTIVE
POTENTIAL UNDER EXPERIMENTAL EQUIVALENT OF
PARKINSONS DISEASE ON A BACKGROUND OF
AMANTADINE THERAPY
Olga Makarenko
Dnepropetrovsk State Medical Academy, Department of Pharmacology,
Dnepropetrovsk, Ukraine
The pain syndrome, which occurs in 60% of patients, is particularly noteworthy among the sensory changes in the clinical picture of Parkinsons
disease/PD.This research is aimed at the study of antinociceptive potential of Meloxicam under experimental equivalent of PD (MPTP-induced
by PD) against the basic antiparkinsonian therapy by Amantadine.
Research was conducted on white male rats. MPTP-induced PD was
modeled through single intraperitoneal administration of neurotoxin in

30 mg/kg. Antiparkinsonian activity was assessed by changes in the values of both rigidity and tremor. Analgesic effect was evaluated by the
method of termoirritation of the tail in initial state and in a 90-minute of
the experiment. Control rigidity in the group was observed in 100% of
animals with the assessment of 1.62 points (with the maximum possible
2 points). Against the background of the administration of both Amantadine and Meloxicam the rigidity was recorded as 75% and 62,5% of the
animals with the assessment of 1 point, respectively, for the mentioned
groups. The higher expression of antitremor effect was observed at the
use of both Amantadine and Meloxicam with the evaluation of 0,87
points and was observed in 50% of rats. The analgesic potential of the
Amantadine-Meloxicam composition was 5.2-fold (p<0,05) higher compared with that of control group in 90 minutes of the experiment. Thus,
adequate antiparkinsonian effect with the analgetic activity of Meloxicam
is observed under condition of the MPTP-induced PD.
Paper No.: 2773

MALNUTRITION AND BIOMARKERS OF INFLAMATION IN
ELDERLY PATIENTS
Eva Mala(1), E Buresova(1), J Novosad(1), I Krcmova(1), J Krejsek(1),
M Pliskova(2), L Sobotka(2), I Urbanova(1)
(1) University Teaching Hospital, Department of Allergology and
Clinical Immunology,Hradec Kralove, Czech Republic
(2) University Teaching Hospital, Department of Gerontology and
Metabolism, Hradec Kralove, Czech Republic
Introduction: Geriatric patients of frailty is state with pow functional
reserve, unstable homeostatsis, number of comorbities and extensive dysfunction of systems. With aging is incrreasing the presence of comorbities together with their clinical manifestation. The aim of our trial was to
analyze biological parameters of inamation in undernutrished patients
as markers of mortality in selected 2 groups of elderly people. Materials/
Patients: Longitudinal study with examination of relationship between
inammatory and nutritonal biomarkers and early/late mortality (1-, 2-,
3-year mortality/5year mortality), stratied by gender. Our patients with
malnutrition were divided into 3 groups (A, B, C). Group A geriatric
patients with normal foof, gr.B - with peroral nutritional support (sipping), C geriatric pacients nutrished by PEG. For our study subjects
were excluded patients with following diseases: diabetes, cancer, asthma
and respiratory disease. Measures: biological measures (glucose, total
cholesterol, total protein, albumin, prealbumin, CRP, WBC), objective
measurement (systolic and diastolic preassure, weight, BMI, HR), anthropometric parameteres, MMSE, MNA, PNI, ADL, Depress Score, QoL
and phone contact. Results: Predictive value of theses indicators could
not be compared with examined biochemical parameters. Conclusion:
Regarding to biomarkers of malnutrition were highly signicantly associated with increased risk of early death in men (gr.A), the same for CRP
and Albumin in men in gr.B, C. Similarly hypocholesterolemia was associated with early death in women in gr.A, B and nearly signicantly
associated in men with PEG (gr.C).
Paper No.: 2774

UNDERNUTRISHED PATIENTS WITH HEAD-NECK CANCER
AND PEG IN ADJUVANT RADIOTERAPY
Eva Mala(1), E Buresova(1), I Krcmova(1), J Novosad(1), J Krejsek(1),
M Vosmik(2), R Cap(3), L Sobotka(4)
(1) University Teaching Hospital, Department of Allergology and
Clinical Immunology, Hradec Kralove, Czech Republic
434
(2) University Teaching Hospital, Deparment of Oncology and
Radiotherapy, Hradec Kralove, Czech Republic
(3) University of Defence, Department of Plastic Surgery, Hradec
Kralove, Czech Republic
(4) University Teaching Hospital, Departmentof Gerontology and
Metabolism, Hradec Kralove, Czech Republic
Introduction: Although adjuvant radioterapy (RT) is often recommended
for locally advanced squamous cell carcinoma of the head-neck cancer
(HNSCC), its effect on overall or cancer-specic survival has not been
clerly demonstrated. The aim of our study was to investigate survival in
undernutrished patients with resected lymph node-positive head and neck
cancer due to intervention both RT (+surgery) and nutritional support via
PEG. Materials/Patients: Patients were selected with node-positive
HNSCC who were treated with surgery alone or with surgery+RT. Each
of this group determined on the way of treatment, was divided into 2
subgroups with nutritional interventional via overfeeding/adequate nutrition via PEG. Results: Aduvant RT was utilizes in 76% patients. This
way of treatment improved the 5-year overall survival in 47.9%, for surgery+RT vs.36.7% for surgery alone P< .001. Cancer specic survival
(54.9% for surgery+RT vs.38.9% for surgery). Signicant benet was in
group with node-positive patients with PEG and adequate nutritional support via PEG. Conclusion: Adjuvant RT in patient with locally advanced
HNSCC resulted in approximately 11.2% absolute increase in 5-year
cancer-specic survival for patients with lymph node-positive HNSCC
with not overfeeding in the frame of nutritional support via PEG.
Paper No.: 1207

DISEASES
CLAVULANIC ACID REDUCES NEUTROPHIL INFILTRATION
AND OEDEMA RESPONSE IN CARRAGEENAN-INDUCED
ACUTE INFLAMMATION
Nasrin Maleki-Dizaji(1), A Banani(1), A Garjani(1), H Hamishehkar(2),
A Fakhrjou(3)
& Toxicology, Tabriz, Iran
(2) Tabriz University of Medical Sciences, Department of Clinical
Pharmacy, Tabriz, Iran
(3) Tabriz University of Medical Sciences, Department of Pathology,
Tabriz, Iran
Introduction: It is founded that b-lactam antibiotics, known for their
activity as inhibitors of bacterial peptidases or proteases, are also
inhibitors of N-acetylaspartylglutamate (NAAG) peptidase. Inhibition
of NAAG peptidase activity in the nervous system results in
increased extracellular levels of NAAG and decreased glutamate levels under conditions of elevated neuronal activity. Evidence suggests
that glutamate present at the inammation site can modulate nociception. Clavalunic acid is a suicide inhibitor that irreversibly binds
b-lactamases produced by a wide range of gram-positive and gramnegative microorganisms. In present study we aimed to investigate
the effect of clavulanic acid on acute inammation. Methods: Carrageenan-induced paw oedema model of acute inammation in rat was
used. Intraplantar carrageenan 1% and clavulanic acid (25-200
mcgkg-1) was administered simultaneously. The severity of inammation was assessed 4 h later using paw oedema volume, microscopic
changes of inamed paw and measurement of oxidative stress marker
myeloperoxidase (MPO) in paw tissues. Results and Conclusion: Clavulanic acid decreased paw oedema and microscopic inammation
scores over the dose range of 25-100 mcgkg-1. This was associated
with signicant reduction in neutrophil inltration indicated by
decreased paw MPO activity. These results, for the rst time, suggest
that clavulanic acid may be involved in modulating peripheral inammation and it is possible that exerts this effect by decreasing glutamate levels in paw tissue via inhibition of NAAG peptidase.
Paper No.: 2731

EFFECTS OF FESOTERODINE 8 MG ONCE DAILY ON THE
PHARMACOKINETICS AND PHARMACODYNAMICS OF A
SINGLE SUPRATHERAPEUTIC DOSE OF WARFARIN IN
HEALTHY SUBJECTS
Bimal Malhotra, G Duczynski, C Alvey, J Gong, X Li, K Gandelman
Pzer Inc, New York, USA
This was an open-label, randomized, 2-way crossover study in 14
healthy subjects (1855 y), with normal prothrombin time (PT) and International Normalized Ratio (INR), to demonstrate a lack of interaction of
fesoterodine with the pharmacokinetics (PK) and pharmacodynamics
(PD) of warfarin. Treatments were A) single dose of warfarin 25 mg
alone; B) fesoterodine 8 mg once daily on days 19, with a single dose
of warfarin 25 mg on day 3, with a 10-day washout between the 2 warfarin doses. Following warfarin administration, R- and S-warfarin plasma
concentrations were determined at 0, 0.5, 1, 2, 4, 6, 8, 12, 24, 36, 48, 72,
96, 120, 144, and 168 h; PT/INR was assessed at 0, 12, 24, 36, 48, 60,
72, 96, 120, 144, and 168 h. PK endpoints were area under the concentration-time curve to innity (AUC), maximum concentration (Cmax),
time to Cmax (tmax) and half-life (t). Primary PD endpoints were area
under the INR-time curve (AUCINR) and maximum INR (INRmax). For
warfarin alone vs warfarin + fesoterodine, the 90% CI were within the
bioequivalence acceptance range (80%, 125%) for ratios of AUC and
Cmax of R- and S-warfarin and for AUCINR and INRmax. PT was similar
and there were no clinically relevant changes in laboratory tests or vital
signs after administration of warfarin alone or with fesoterodine. The
PK, PD, safety, and tolerability of warfarin 25 mg in healthy adults are
unaffected by fesoterodine 8 mg. No dose adjustment is necessary for
warfarin when administered with fesoterodine.
Paper No.: 2908

IN SILICO APPROACH TO DEVELOP NEW SELECTIVE
PPARC MODULATORS (SPPARMS)
Francesca Mancini(1), M Vitiello(1), G Mangano(1), G Pochetti(2), MA
Alisi(1), R Ombrato(1), E Fioravanzo(3), N Cazzolla(1), L Polenzani(1)
(1) Angelini Research Center - ACRAF, Department of Pharmacology,
Rome, Italy
(2) CNR, Istituto di Cristallograa, Monterotondo, Rome, Italy
(3) S-IN Soluzioni Informatiche, Vicenza, Italy
Peroxisome Proliferator-Activated Receptor c (PPARc) is a clinically
validated target for treatment of insulin resistance. PPARc activation by
full agonists has shown potent therapeutic activity linked to safety and
tolerability issues such as weight gain, edema and congestive heart failure. There is a great interest in developing selective PPARc Modulators
(SPPARMs), to separate the effects of PPARc on adypogenesis and glucose metabolism, and to avoid side effects on the cardiovascular, gastrointestinal and immune systems. An in silico screening procedure was
performed to select new PPARc modulators. Virtual docking and ligandprotein binding afnities predictions were applied to 5000 compounds
from in-house and commercial libraries. In vitro afnity of selected compounds was evaluated by PPARc ligand-binding polarized uorescence
assay. Functional activity and selectivity were assessed in stable reporter
cells expressing PPARa, PPARd or PPARc, respectively. X-Ray crystal . 240 PPARc
lographic studies were performed at resolution of 2.20 A
virtual hits were selected by the above in silico screening procedure. In
vitro binding led to the identication of 39 compounds showing pIC50
ranging from 4.6 to 7.1. Following in vitro functional assay results two
compounds were characterized as partial agonists with selectivity
435
towards PPARc compared to PPARa and d isoforms. The X-Ray
co-crystal structures of these two compounds with PPARc showed that
the compounds interact with the receptor in an original double binding
mode. Further studies are needed to conrm differences in the ligand
binding sites afnities that could be associated with differential cell
cofactors recruitment and consequent modulation of in vivo biological
effects.
Paper No.: 3406

BUILDING THEMES TOGETHER: STUDENTS
INVOLVEMENT IN PHARMACOLOGY SUBJECTS
Victoria Maneu, J Formigos-Bolea, C Garca-Cabanes, MM Palmero
University of Alicante, Department of Optics, Pharmacology &
Anatomy, Alicante, Spain FARMAGITE
The Higher Education Space tries to transform the students traditional
passive attitude into a more active role in their learning process. The new
system emphasizes learning rather than teaching. Thus, the students must
be capable of building their training and have to be responsible of it in
an autonomous way. We have conducted an experience with students of
the subject Pharmacology and Sports from the Degree in Sport and
Physical Activity Sciences of the University of Alicante, Spain. In this
experience, students could voluntarily involve themselves in the development of Pharmacology themes. For all the themes, each student could
freely choose a scientic article related to a pharmacological group,
which is used in sports. The articles had to be selected and prepared
before the academic lecture. In class, the teacher started with a short
introduction, then students presented their articles and lecture nished
with a general discussion of the main points. The teacher ended the session pointing the highlights and made an exposition if necessary. Students who joined the experience openly expressed that it was very useful
for them in acquiring and xing knowledge. We noticed that students
progressively chose better articles and read them in a more critical way.
Moreover, these students achieved better academic results. So, this experience was very satisfactory for both, students and teachers, and we
strongly recommend it.
Paper No.: 3323

FOCUSED CONFERENCE GROUP: PW11 - NEW
OPPORTUNITIES IN THE PHARMACOLOGY OF NEUROIMMUNE INTERACTIONS
CANDIDA ALBICANS INCREASES TLR2 MRNA EXPRESSION
IN MOUSE RETINAL PROGENITOR CELLS
Victoria Maneu(1,2), C Murciano(3), ML Gil(3), A Yanez(3),
D Gozalbo(3)
(1) University of Alicante, Department of Optics, Pharmacology &
Anatomy, Alicante, Spain
(2) Instituto Teolo Hernando de I+D del Medicamento (UAM), Madrid,
Spain
(3) Universitat de Vale`ncia, Valencia, Spain
Toll-like receptors (TLRs) constitute a family of pattern-recognition
receptors that recognize molecular signatures of microbial pathogens and
induce the activation and development of the innate and adaptive
immune responses. TLR2 and TLR4 are involved in the host interaction
with Candida albicans and play a signicant role in the development of
host immune responses during candidiasis [1]. TLRs are expressed in
hematopoietic stem and progenitor cells, where they may play a role in
hematopoiesis in response to pathogens, including C. albicans, during
infection [2,3]. In addition, retinal progenitor cells (RPC) express mRNA
coding for several TLRs [4], although their role in ocular pathophysiology has not been fully studied. Therefore, the aim of this work was to
further investigate the role of TLRs in host responses during ocular
candidiasis. A RPC line from Muller cells, obtained from C57BL/6 mice,
was analyzed for TLR2, TLR4 and TLR6 expression by semiquantitative
RT-PCR after in vitro culture, either in the presence or absence of C.
albicans ATCC6555 and PCA2 strains, under different experimental conditions. In all conditions tested, C. albicans addition to the culture medium caused an increased expressio of TLR2 mRNA. These results
suggest that C. albicans may be sensed by TLR2 on RPC to promote the
host capability for defense against C. albicans.
[1] Gil ML and Gozalbo D. Front Biosci. 2009; 14:570-582.
[2] Yanez et al. Microbes Infect. 2009; 11:531-535.
[3] Yanez et al. Cell Microbiol. 2010; 12: 114-128.
[4] Shetcher R et al. J. Cell Biol. 2008; 183, 393400.
Paper No.: 3324

CANDIDA ALBICANS INDUCES PROLIFERATION AND
NEUROSPHERE FORMATION FROM MOUSE RETINAL
PROGENITOR CELLS
Victoria Maneu(1,2), C Murciano(3), ML Gil(3), A Yanez(3),
D Gozalbo(3)
(1) University of Alicante, Department of Optics, Pharmacology
& Anatomy, Alicante, Spain
(2) Instituto Teolo Hernando de I+D del Medicamento (UAM), Madrid,
Spain
(3) Universitat de Vale`ncia, Valencia, Spain
Neovascularization is a process that complicates ocular fungal keratitis
and may worsen visual prognosis. As this process involves to the expression of vascular endotelial growth factor (VEGF), it has been suggested
the use of anti-VEGF therapy to avoid neovascularization and visual loss
in ocular infections by Candida albicans [1, 2]. The objective of this
work was to characterize other factors potentially involved in neovascularization during fungal infections of the eye. For this purpose, the effect
of C. albicans in the proliferation and diffentiation of retinal progenitor
cells (RPC) was determined. Muller cells isolated from the retina of
C57BL/6 mice were cultured in a serum-replacement medium to induce
dedifferentiation into neurosphere-forming RPC. The ability to develop
neurospheres either in the presence or absence of inactivated yeast cells
of the high virulence C. albicans ATCC6555 strain was tested. Results
showed that the presence of C. albicans augmented both proliferation
and neurosphere diameter in a statistically signicant manner. Therefore,
the ability of C.albicans to induce proliferation and neurosphere formation from mouse RPC could be related with the neovascularization that
complicates Candida albicans ocular infections. It can be suggested that
this effect is mediated by the recognition of C. albicans by Toll-like
receptors (TLRs) [3], which are expressed in RPC [4].
[1] Yuan X and Wilhelmus KR. Mol Vis. 2009; 15:1988-1996.
[2] Schreiber W et al. Inves. Ophthalmol. Vis. Sci. 2003; 44:2634-2643.
[3] Yanez et al. Cell Microbiol. 2010; 12: 114-128.
[4] Shetcher R et al. J. Cell Biol. 2008; 183: 393400.
Paper No.: 3388

ASSOCIATION OF VKORC1 -1639 G>A POLYMORPHISM
WITH INTIMA-MEDIA THICKNESS OF COMMON CAROTID
ARTERY IN SUBJECTS WITH TYPE 2 DIABETES MELLITUS
Vangelis G Manolopoulos(1), I Petridis(1,2), G Ragia(1), M Vassileiadis(3), I Heliopoulos(4), D Christakidis(2), A Tavridou(1)
(1) Democritus University of Thrace, Medical School, Laboratory of
Pharmacology, Alexandroupolis, Greece
436
(2) Academic General Hospital of Alexandroupolis, Department of Internal Medicine, Alexandroupolis, Greece
(3) Hospital Papageorgiou, 1st Department of Interanl Medicine, Thessaloniki, Greece
(4) Academic General Hospital of Alexandroupolis, Department of Neurology, Alexandroupolis, Greece
Purpose: Coronary artery calcium (CAC) correlates with total atherosclerotic plaque. The extent of non-calcied atherosclerosis detected using
carotid artery intima-media thickness (CA-IMT) predicts progression of
calcied atherosclerosis. Vitamin K epoxide reductase complex subunit 1
(VKORC1) mediates recycling of vitamin K 2,3 epoxide to vitamin K
hydroquinone. Undercarboxylation of some vitamin K-dependent proteins due to genetic polymorphisms of VKORC1 can lead to arterial calcication. We investigated the association between VKORC1 -1639G>A
polymorphism and CA-IMT in subjects with type 2 diabetes mellitus
(T2DM). Methods: Genomic DNA was extracted from peripheral blood
of 118 subjects with T2DM and genotyped for VKORC1 -1639 G>A
polymorphism using PCR-RFLP analysis. CA-IMT measurements were
obtained with the use of a high-resolution ultrasound scanner. Results:
Mean CA-IMT was 0.068, 0.072 and 0.076 cm for genotypes GG, GA
A
respectively but this difference did not reach signicance. Statisand A
tically signicant difference (p=0.02) was observed in the maximum
value of CA-IMT among different genotypes (0.073, 0.077 eae 0.084 cm
A
respectively). Post-hoc analysis revealed
for genotypes GG, GA and A
that this was due to a signicant difference between subjects with GG
and AA genotypes (p=0.02). Linear regression analysis showed that
VKORC1 -1639 G>A polymorphism was an independent predictor of
CA-IMT (p=0.04) after adjusting for established risk factors (age, blood
pressure, duration of T2DM). Conclusions: T2DM subjects homozygous
for -1639 VKORC1 A allele had higher maximum value of CA-IMT
compared with homozygous subjects for the common allele (GG genotype). Moreover, VKORC1 -1639 G>A polymorphism was an independent predictor of CA-IMT.
Paper No.: 3389

DISEASES
ASSOCIATION OF GENETIC POLYMORPHISMS OF ENOS
AND INOS WITH IMMUNE SENSITIZATION IN ASTHMATIC
CHILDREN
Vangelis G Manolopoulos(1), M Iordanidou(1), A Tavridou(0),
E Paraskakis(2), A Chatzimichael(2)
(1) Democritus University of Thrace, Medical School, Laboratory of
Pharmacology, Alexandroupolis, Greec
(2) Academic General Hospital of Alexandroupolis, Medical School,
Department of Pediatrics, Alexandroupolis, Greece
Introduction: Nitric oxide (NO) plays a key role as a vasodilator and
inammatory mediator in the airways. Alterations in genetic loci that
encode nitric oxide synthase (NOS) enzymes that affect NO formation
could contribute to the pathophysiology of atopic diseases, such as
asthma. Aim: To evaluate the association of eNOS -786C>T and
894G>T polymorphisms and iNOS Ser608Leu (C/T) polymorphism with
atopic asthma in children and the sensitization of children with atopic
asthma for common aeroallergens with skin prick tests. Methods: 234
children were included in the study, 159 with atopic asthma and 75 controls. Genomic DNA was extracted from peripheral blood and analysed
for eNOS -786T>C and 894G>T polymorphisms and iNOS Ser608Leu
polymorphism using PCR-RFLP method. Results: There were no differences in genotype or allele frequencies of eNOS -786C>T and 894G>T
and iNOS Ser608Leu polymorphisms between atopic asthmatic children
and control group. However, the frequency of -786C allele carriers was
signicantly lower in asthmatic children with sensitization to wall pellitory and common grass (46.4% vs 69.4%, p=0.025; 50.0% vs 69.2%,
p=0.046, respectively). Among asthmatic children, those with Ser608Leu
polymorphism (CT and TT) had signicantly higher total serum IgE levels than those with CC genotype (497.87 IU/ml vs 275.35 IU/ml,p<0.05,
respectively). Conclusion: None of the three polymorphisms in NOS system

genes studied was associated with atopic asthma in children. However, in
asthmatic children, eNOS -786C>T polymorphism was associated with sensitization to wall pellitory and common grass, and iNOS Ser608Leu polymorphism was associated with total serum IgE levels.
Paper No.: 585

THE ROLE OF DOPAMINERGIC RECEPTORS IN THE
INHIBITION OF THE VASOPRESSOR SYMPATHETIC
OUTFLOW IN PITHED RATS
Guadalupe Manrique-Maldonado, A Gonzalez-Hernandez, BA MarichalCancino, A Sanchez-Lopez, D Centurion, CM Villalon
CINVESTAV-IPN, Department of Pharmacology, Mexico DF, Mexico
Dopamine is involved in the control of blood pressure and produces
complex effects on the cardiovascular system as a consequence of its
interaction with dopaminergic and adrenergic receptors. Dopamine receptors are divided into two families designated D1-like and D2-like. D1like receptors activate Gs proteins while D2-like receptors activate Gi
proteins. Thus, stimulation of D2-like receptors may inhibit noradrenaline release from sympathetic neurons. Results obtained in our laboratory
in male Wistar pithed rats have previously demonstrated that electrical
stimulation (0.03 3.0 Hz; 50 V; 2 mseg) of the vasopressor sympathetic
outow (at the level of T7-T9 of the spinal cord) induces frequency
dependent increases in diastolic blood pressure. The present study set out
to evaluate the pharmacological identication of the dopamine receptors
mediating systemic vascular sympatho-inhibition in pithed rats. Continuous intravenous (i.v.) infusions of dopamine (3, 10 30 and 100 lg/
kg.min) inhibited the vasopressor responses induced by either electrical
stimulation or i.v. bolus injections of exogenous noradrenaline (0.03, 0.1,
0.3, 1.0 and 3.0 lg/kg). In contrast, continuous i.v. infusions of quinpirole (0.1. 0.3, 1, 3, 10 and 30 lg/kg.min) inhibited the electrically
induced, but not the noradrenaline-induced, vasopressor responses. The
vascular sympatho-inhibition induced by i.v. quinpirole (1 lg/kg.min)
remained unaltered after i.v. treatment with saline or the antagonist
SCH23390 (300 lg/kg; D1-like), but was abolished by raclopride
(1000 lg/kg; D2-like). The above results, taken together, suggest that
quinpirole-induced inhibition of the vasopressor sympathetic outow is
primarily mediated by an action on prejunctional D2 like receptors.
Paper No.: 451

POTENTIATING ALPHA-LIPOIC ACID HEPATOPROTECTIVE
EFFECT BY L-CARNITINE AND METHYLSULFONYLMETHANE IN BROMOBENZENE- INDUCED HEPATOTOXICITY
IN RATS
Dina Mansour(1), SA Nada(1), ES El-denshary(2), AK Bahgat(2), EA
Omara(3)
(1) Department of Pharmacology, National Research Center, Dokki,
Egypt
(2) Department of Pharmacology and toxicology, Faculty of Pharmacy,
CairoUniversity, Egypt
(3) Department of Pathology, National Research Center, Dokki, Egypt
Bromobenzene (BrB), an inert halogenated aromatic hydrocarbon. It is
activated by liver CYP450 to reactive epoxide that covalently binds to
macromolecules causing severe centrilobuar necrosis in liver. BrB-treated
rats (10mmol/kg p.o) showed signicant depletion in reduced glutathione
level (GSH), elevation in tissue lipid peroxidation and hepatic nitric
437
oxide. Furthermore, serum isocitrate dehydrogenase (ICDH), ALT and
AST were signicantly increased 24-hours after Bromobenzene administration. The administration of a-lipoic acid (150mg/kg p.o) for seven
days exerted a signicant protection against BrB-induced acute heptotoxicity. The combination of a-lipoic acid either with L-carnitine (300mg/
kg) or Methylsulfonylmethane (300mg/kg) potentiated the hepatoprotective activity through inhibition of hepatic nitric oxide level compared
with a-lipoic acid alone. These results were conrmed by histopathological examination of the liver.
Key words: a-lipoic acid, L-carnitine, Methylsulfonylmethane, Bromobenzene, hepatotoxicity, rat.
Paper No.: 3362

ANTIDEPRESSANT USE AND SALIVARY CORTISOL: THE
NETHERLANDS STUDY OF DEPRESSION AND ANXIETY
(NESDA)
Leonie Manthey(1) and Caroline Leeds(1), EJ Giltay(1), T van Veen(1),
SA Vreeburg(2), BWJH Penninx(1,2,3), FG Zitman(1)
(1) Leiden University Medical Center, Department of Psychiatry, Leiden,
The Netherlands
(2) VU University Medical Center, EMGO Institute and Neuroscience
Campus Amsterdam, Department of Psychiatry, Amsterdam, The Netherlands
(3) University Medical Center Groningen, Department of Psychiatry,
Background: Antidepressants are clinically effective as treatment of depressive and anxiety disorders. Those disorders have frequently been found to be
accompanied by a hyperactive hypothalamic-pituitary-adrenal (HPA) axis.
Therefore, part of the antidepressant action might be mediated through putative effects on the HPA axis. Objectives: We explored the association
between antidepressant use and salivary cortisol levels to obtain a fuller
understanding of the biological and therapeutic effects of antidepressants.
Method: The association between antidepressant use and cortisol levels was
investigated in subjects of the Netherlands Study of Depression and Anxiety
with a lifetime diagnosis of depression and/or anxiety (n=1539). Subjects
were categorized as serotonin reuptake inhibitor (SSRI) users (n=321), tricyclic antidepressant (TCA) users (n=50), other antidepressant users (n
=100), and non-users (n=1068). Possible associations between dose and
duration of antidepressant use and salivary cortisol levels were also analyzed. Main outcome measure: Subjects provided 7 saliva samples, from
which 3 cortisol indicators were calculated: the cortisol awakening response
(CAR), evening cortisol, and cortisol suppression after ingestion of 0.5 mg
dexamethasone (DST). Results: As compared to non-users, TCA users had
attened CAR (effect size: d = 0.36), SSRI users had higher evening cortisol
levels (d = 0.17), and SSRI (d = 0.20) and other AD users (d = 0.21) showed
less cortisol suppression in the DST. Conclusions: These ndings suggest
that antidepressant subtypes have distinct effects on the HPA axis. TCA
users, who had a robustly attened cortisol awakening response, displayed
the most pronounced alterations of salivary cortisol levels.
Paper No.: 3363

CORRELATES OF (INADEQUATE) BENZODIAZEPINE USE:
THE NETHERLANDS STUDY OF DEPRESSION AND ANXIETY
(NESDA)
Leonie Manthey(1), T van Veen(1), EJ Giltay(1), JE Stoop(1),
A Knuistingh-Neven(2), BWJH Penninx(1,2,4), FG Zitman(1)
(1) Leiden University Medical Center, Department of Psychiatry, Leiden,
The Netherlands
(2) Leiden University Medical Center, Department Public Health &
Primary Care, Leiden, The Netherlands
(3) VU University Medical Center, EMGO Institute and Neuroscience

Campus Amsterdam, Department of Psychiatry, Amsterdam, The Netherlands
(4) University Medical Center Groningen, Department of Psychiatry,
Background: Results on determinants of benzodiazepine (BZD) use in
general and inadequate use are inconsistent and mostly univariate. The
relative importance of sociodemographic, psychological and physical
determinants has never been investigated in a comprehensive, multivariate model. Objective: We explored independent sociodemographic, psychological and physical determinants of BZD use in general and
inadequate BZD use in order to obtain a prole of the average BZD user.
Methods: Using 429 BZD users and 2423 non-users from the Netherlands Study of Depression and Anxiety (NESDA), sociodemographic,
psychological and physical determinants of BZD use and inadequate use
were investigated. Results: BZDs were used by a considerable proportion
of the 2852 NESDA participants (15.0 %). BZD use was independently
associated with older age, singleness, unemployment (OR=1.56), treatment in secondary care, higher medical consumption (OR=1.41), anxiety
(OR=1.95), depression, comorbidity, insomnia, and antidepressant use.
Overall, BZD use was rarely in accordance with all guidelines, mainly
because most users (82.5%) exceeded the recommended duration of safe
use. Inadequate use was independently associated with older age (b =
0.130) and chronic illnesses (b = 0.120). Higher scores on agreeableness
were associated with less inadequate use. Conclusions: Mentally or physically vulnerable subjects are most likely to use BZDs. The most vulnerable (i.e. old and physically ill) BZD users were at highest risk of
inadequate BZD use. Without further evidence of BZDs effectiveness in
long-term use, caution in initiating BZD prescriptions is recommended,
particularly when patients are chronically ill and old, as those are most
likely to display inadequate use.
Paper No.: 2877

ASSOCIATION OF B3-ADRENERGIC RECEPTOR
POLYMORPHISM WITH CONTRACTILE RESPONSES IN
HUMAN ATRIUM
Alina Marandykina, A Bogdelis, D Zablockaite, R Treinys, VA Skeberdis
Kaunas University School of Medicine, Institute of Cardiology, Kaunas,
Lithuania
In human atrium, b3-adrenergic receptors (b3-AR) stimulate L-type calcium current (I(Ca,L)) and force of contraction (P) (Skeberdis et al, J.
Clin. Invest. 2008; 118: 32193227), however the extent of the response
may depend on genetic polymorphism of the receptor. Three single
nucleotide polymorphisms, W64R, P251T and T265M, have been identied in b3-AR. By now, it has been shown only that W64R causes a
weaker cAMP accumulation upon b3-AR stimulation (Small KM et al,
Annu. Rev. Pharmacol. Toxicol. 2003; 43: 381411). Therefore, the aim
of our study was to examine the frequency of all three polymorphisms
and the role of W64R in b3-AR induced stimulation of I(Ca,L) and P of
isolated atrial myocytes and atrial trabeculae, respectively, obtained from
patients undergoing heart surgery in Kaunas University Hospital (KUH).
We identied genotypes of 65 control DNA and 93 DNA samples
obtained from KUH patients. The incidence of W64R genotype was
11% in each groups. In case of Trp64Trp, b3-AR agonists CGP12177 (1
microM) and BRL37344 (1 microM) stimulated I(Ca,L) by 5815%
(n=13) and 14925% (n=31) over control, respectively, and P by
133.9% (n=9) and 184.7% (n=12) over control, respectively. In case
of W64R, CGP12177 and BRL37344 stimulated I(Ca,L) by 347.1%
(n=7) and 8018% (n=4) over control, respectively, and P by 4.22.2%
(n=2) and 5.31.8% (n=2) over control, respectively. Our results indicate
that efcacy of b3-AR-dependent stimulation of I(Ca,L) and P may
438
depend on W64R polymorphism. More cases are needed to suggest the
role and frequency of P251T and T265M polymorphisms.
Paper No.: 3279

HEALTH AND DISEASE
INTERACTIONS BETWEEN CARBON MONOXIDE
RELEASING MOLECULES AND NITRIC OXIDE DONORS IN
VASCULAR TISSUE
Antonia Marazioti(1), P Baskaran(2), M Bucci(3), C Szabo(4), G Cirino(3), C Romao(5,6), AR Marques(5), A Beuve(2), A Papapetropoulos(1)
(1) University of Patras, Laboratory of Molecular Pharmacology, Department of Pharmacy, Patras, Greece
(2) University of Medicine and Dentistry, Department of Pharmacology,
Newark, New Jersey
(3) Department of Experimental Pharmacology,Faculty of Pharmacy,
University of NaplesFederico II, Naples, Italy
(4) Ikaria, Inc, Seattle, Washington
(5) Alfama, Inc, Lisbon, Portugal
(6) Instituto de Tecnologia Qumica e Biologica, Universidade Nova de
Lisboa, Oeiras, Portugal
Carbon monoxide (CO) is a weak sGC stimulator, leading to transient
increases in cGMP and vasodilation. Transition metal carbonyls liberate
CO in a controlled fashion and function as CO-releasing molecules
(CORMs). The aim of the present work was to test the ability of a number of CORMs to modulate basal and nitric oxide (NO)-induced cGMP
formation and vasorelaxation. cGMP accumulation was measured in rat
aortic smooth muscle cells in the presence of CORMs and/or nitric oxide
(NO) donors using ELISA. Basal and nitric oxide-stimulated sGC activity was determined using puried rat recombinant sGC. Vasodilation was
determined using pre-contracted rat aortic rings after incubation with a
CORM, in the presence or absence of S-nitroso N-acetylpenicillamine.
Incubation of cells with some, but not all of the CORMs caused a minor
increase in cGMP levels. Concentration-response curves were bellshaped for most of the CORMs studied. Although exposure of cells to
CORM-2 or ALF157 enhanced cGMP formation we observed that both
compounds inhibited NO-stimulated cGMP accumulation in cells and
NO-stimulated sGC activity that could be reversed by superoxide anion
scavengers. Superoxide anion generation from both CORM-2 and
ALF157 was conrmed using luminol-induced chemiluminescence. Furthermore, we observed that NO is scavenged by CORM-2. When used
alone CORM-2 relaxed vessels through a cGMP-mediated pathway, but
attenuates NO donor-stimulated vasorelaxation. We conclude that CORMs have variable, context-dependent effects on vessel tone as they can
directly dilate blood vessels and also block NO-induced vasorelaxation.
Paper No.: 3209

FOCUSED CONFERENCE GROUP: P01 - CLINICAL PHARMACOLOGY IN THE EMERGING COUNTRIES
EVALUATION OF THE EFFECT OF INCREASING HYDRATION TO PREMEDICATION TREATMENTS OF CHEMOTHERAPY WITH CISPLATIN IN DECREASING RENAL SIDE
EFFECTS OF IT IN PATIENTS OF AIR FORCE AND BUALI
HOSPITALS IN TEHRAN FROM 2007 TO 2009
Artin Mardirosian(1), MH Lashkari(2), FN Dadgar(1), H Molana(2),
M Fotouhi(2)
(1) Tehran Azad University Medical School, Department of Oncology,
Tehran, Iran
(2) Airforce Hospital, Tehran, Iran
Cancer is the second leading cause of death after cardiovascular disease.
One of the treatment modalities of cancer, is chemotherapy & one of the
widely used drugs is cisplatin. It has many side-effects, one important side
effect is renal toxicity. One of the very simple ways to reduce nephrotoxicity
is increasing hydration before each cisplatin dose.We chose 96 patients.

They were grouped into A & B groups randomly & equally. Before the rst
dose of cisplatin, we checked serum creatinine & estimated GFR. Then
patients in group A, were given 750 cc & group B, 1500 cc hydration before
each cisplatin dose. Chemotherapy was done for 4 cycles with intervals of
3 weeks. One month after 4th cycle, we checked serum creatinine & estimated GFR again.In group A:First creatinine=1.14 SD:0.28 FirstGFR=65.62 SD:30.14 Second creatinine= 1.4 SD:0.29,Second GFR=52.75
SD:23.01 In group B:First creatinine=1.16 SD:0.22,First GFR= 69.08
SD:26.08 Second creatinine=1.33 SD:0.23,Second GFR=59.57 SD:20.95
.We concluded that patients in group B, that had been given 1500cc hydration, had lesser increase of creatinine & decrease of GFR, so the renal toxicity was lesser. But because of P Value>0/05, It wasnt signicant
statistically. So we can conclude that increasing hydration can help to reduce
nephrotoxicity, but its not enough & we should use other ways as well.
Paper No.: 736

HEALTH AND DISEASE
SPINAL DIHYDROERGOTAMINE INHIBITS CAPSAICININDUCED CANINE EXTERNAL CAROTID VASODILATATION
VIA CENTRAL 5-HT1B/1D RECEPTORS AND A2-ADRENOCEPTORS
Bruno Marichal-Cancino, G Manrique-Maldonado, A Gonzalez-Hernandez, MT Villamil-Hernandez, O Alcantara-Vazquez del Mercado,
D Centurion, A Sanchez-Lopez, C Villalon
CINVESTAV-Coapa, Farmacobiologa, Mexico DF, Mexico
The aim of this study was to investigate the pharmacological prole and
nature of inhibition induced by intrathecal (i.t, C1-C3) administration of
DHE on the external carotid vasodilator responses to capsaicin. Intracarotid infusions (1 ml/min, for 1 min) of capsaicin (10, 18, 31 and 56 lg/
min), a-CGRP (0.1, 0.3, 1 and 3 lg/min) or acetylcholine (0.01, 0.03 and
0.1 lg/min) produced dose-dependent increases in external canine carotid
blood ow without affecting blood pressure or heart rate. The vasodilator
responses to capsaicin, but not those to a-CGRP or acetylcholine, were
signicantly attenuated after i.t. administration of DHE (10, 31 and
100 lg; accumulative dose). The inhibition DHE-induced (10 lg) was:
(i) unaffected by i.t. administration of saline (0.5 ml, twice), or the antagonists GR127935 (10 lg; 5-HT1B/1D) or rauwolscine (100 lg; a2 adrenoceptors); and (ii) signicantly blocked by GR127935 (30 lg) or
rauwolscine (300 lg). The inhibition response DHE-induced (100 lg)
was completely blocked by i.t. administration of GR127935 (10 lg) +
rauwolscine (100 lg). I.t. administration of the a2-adrenoceptors agonists,
B-HT933 (1000 lg) or clonidine (300 lg) inhibited the capsaicin-induced
vasodilatation. Lastly, i.t. bumetanide (10 and 100 lg), a NKCC1 inhibitor, inhibited the capsaicin-induced vasodilatation. Taken together, these
results suggest that spinal DHE inhibits the canine external carotid vasodilator responses to capsaicin via 5-HT1B/1D and a2-adrenoceptors. This
effect may involve the inhibition of CGRP release by a central mechanism
that could involve a dorsal root reex at C1-C3 level, because bumetanide
inhibited the capsaicin-induced vasodilatation.
Paper No.: 1819

ANALGESICS
ROLE OF PITUITARY ADENYLATE CYCLASE ACTIVATING
POLYPEPTIDE IN THE NITROGLYCERIN-INDUCED
MIGRAINE MODEL OF THE MOUSE
Adrienn Markovics(1), K Sandor(1), E Szoke(1), V Kormos(1), B Botz(1),
A Imreh(1), B Gaszner(2), D Reglodi(2), A Baba(3), J Tajti(4), J Szolcsanyi(1), Z Helyes(1)
(1) University of Pecs, Faculty of Medicine, Department of Pharmacology and Pharmacotherapy, Pecs, Hungary
439
(2) University of Pecs, Faculty of Medicine, Department of Anatomy,
Pecs, Hungary
(3) Osaka University, Graduate School of Pharmaceutical Sciences,
Osaka, Japan
(4) University of Szeged, Department of Neurology, Szeged, Hungary
Pituitary adenylate cyclase activating polypeptide (PACAP) exerts multiple actions via G-protein-coupled receptors (PAC1, VPAC1/2). We have
recently proved the excitatory function of PACAP in the central nervous
system in a variety of pain models. Migraine is a neurovascular disorder
characterized by severe unilateral head pain and associated symptoms,
such as photophobia. Therefore, our aim was to investigate the role of
PACAP in nitroglycerin (NG)-induced light aversion behaviour and cFos immunoreactivity in the trigeminal nucleus using PACAP genedeleted (PACAP-/-) mice and their wildtype (PACAP+/+) counterparts.
Migraine-like pathophysiological alterations were induced by ip. injection of nitroglycerin (10 mg/kg) as described in the literature. Mice were
individually observed in a custom made light-dark box for 2 hours and
the time spent in the light side were analyzed in each 300 s interval. At
the end of the experiment, C-Fos positivity was determined in the medullar trigeminal nucleus with immunohistochemistry and quantitative analysis was performed. Our results showed signicantly reduced light
aversive behaviour in PACAP-/- mice both in the early phase (0-30 min)
due to the acute vasodilating effect of NG and in the late phase (90120 min) when sensitization of the trigeminal system is supposed to be
involved. Furthermore, the number of c-Fos immunopositive neurons
was also signicantly smaller in the trigeminal nucleus of the knockouts
2 h after NG injection. These data suggest a pro-nociceptive role of PACAP in the trigeminal system involved in migaine-like pain. Identication of its targets might open interesting future perspectives in novel
anti-migraine therapy.
Paper No.: 3378

HEALTH AND DISEASE
PLASMA MELATONIN PRIMES FOR A LONG-TERM
ENDOTHELIAL CELL CULTURES
Regina Markus, E Tamura, P Fernandes, M Marcola, S Cruz-Machado
University of Sao Paulo, Laboratory Chronopharmacology - IB, Sao
Paulo, Brazil
Endothelial cells (EC) are responsible for the control of leukocyte migration, vascular tonus and are of great interest for cell therapy and tissue
engineering of vascular grafts. Here we tested whether the level of circulating melatonin in donor rats could interfere on the reactivity of cultivated endothelial cells. Circulating levels of melatonin were changed by
sacricing the animals at day/night-time, or by suppressing nocturnal
melatonin production with E.coli lipopolisaccharide (LPS). In order to
determine whether melatonin is the pre-conditioned factor, we evaluated
the effect of treating LPS-injected animals with melatonin. Male adult
rats (12/12h light/dark, lights on at 06h00) were injected or not (control)
with LPS (0.5 mg/Kg) or LPS+melatonin (3mg/Kg) two hours before
sacrice (12h00 or 24h00). EC obtained from cremaster muscle were cultivated till conuence (15 days). LPS treatment signicantly reduces
plasma melatonin in approximately 60% (control = 1108.41 pg/mL,
n=5, 24h00). Expression of iNOS, ICAM-1 and PECAM-1 and the
adherence of neutrophils to EC obtained from controls killed at day-time
or LPS-treated (night-time) was signicantly higher than that observed in
EC from night-time sacriced rats. Melatonin injected together with LPS
blocks the activation of EC, evaluated by its capability to adhering leukocytes and express iNOS, ICAM-1 and PECAM-1. Therefore, the cultures from animals with higher levels of plasma melatonin are in a more
quiescent state. Taking into account that melatonin is the hormone of
darkness, our results opens the possibility to equalize EC banks by collecting the cells at a given hour of the day.
Paper No.: 3184

CYSTAMINE AND SILDENAFIL TREATMENT OF RIGHT VENTRICULAR HYPERTROPHY IN RATS WITH PULMONARY
HYPERTENSION
Lars H Markvardsen, CU Andersen, M Aalling, JH Rnn, U Simonsen
Aarhus University, Department of Pharmacology, Aarhus, Denmark
Pulmonary hypertension is characterized by vasoconstriction and remodelling of pulmonary arteries and leads to right ventricular hypertrophy
(RVH) and failure. Tissue transglutaminase (tTG) that cross links matrix
proteins is associated to left ventricular hypertrophy and remodelling of
arteries in essential hypertension. The hypothesis of the present study
was that treatment with an inhibitor of tTG, cystamine, would attenuate
RVH and remodelling of pulmonary arteries in chronic hypoxic rats, and
that the effect would be enhanced by the pulmonary vasodilator, sildenal. Wistar rats were exposed to normoxia or hypobaric hypoxia and treated with vehicle, cystamine 40 mg/kg/day, sildenal 25 mg/kg/day or
both. Cardiac content of tTG, RVH, right ventricular systolic pressure
(RVSP), pulmonary content of tTG, muscularisation of pulmonary arteries, and the effect of cystamine and sildenal on isolated pulmonary
arteries were assessed. Hypoxic rats had a higher right ventricular tTG
content, RVH and increased muscularisation of pulmonary arteries. The
combination of cystamine and sildenal attenuated RVH, whereas the
two drugs alone had no signicant effect. Cystamine attenuated RVH
corrected for RVSP. In pulmonary arteries, the content of tTG was not
altered by hypoxia, and cystamine had no effect on pulmonary remodelling. High doses of cystamine induced relaxation in isolated pulmonary
arteries. There was a benecial effect on RVH by combining cystamine
and sildenal. Furthermore, cystamine made the right ventricle more
resilient for development of hypertrophy in response to increased afterload. We found no evidence of involvement of tTG in remodelling of
pulmonary arteries in hypoxic rats.
Paper No.: 3134

NEONATAL HYPERLEPTINAEMIA POSSIBLY MODULATES
CARDIAC FUNCTION
E Marques(1), F Toste(1), S Marques(1), J Raimundo(2), G Sudo(2),
R Sudo(2), A Vieyra(2), Christianne Scaramello(1)
(1) University Federal of Fluminense, Department of Physiology &
Pharmacology, Rio de Janeiro, Brazil
(2) University Federal of Rio de Janeiro, Brazil
Introduction: Previous studies showed cardiovascular diseases related to
altered leptin activity and leptin resistance in adult rats due to neonatal
leptin treatment. Our question is if neonatal leptin treatment programmes
cardiovascular function. Materials: During lactation injections of leptin
(Lgroup)/saline were perfomed. Rats (30, 90 and 150 days-old) were
submitted to maximal effort ergometer test. Rat hearts were removed for
morphological, biochemical and functional analysis. The use of animals
was in agreement to the Animal Care and Use Committee of Fluminense
Federal University (CEPA/UFF00123-09). Results: L group showed
higher body weight/food intake. Morphological assays showed no difference between groups. L group (30 days-old) had worse performance in
maximal effort ergometer test. Data suggest an increase of heart isometric
tension due 3lM isoproterenol except in 30 days-old rats of L group.
Biochemical assays showed a decrease of Na+/K+ATPase activity in
150 days-old rats but an increase of sarcoplasmatic reticulum (SR)
Ca2+ATPase activity in all ages of L group. Conclusions: Changes in body
weight/food intake are described due to hyperleptinaemia in early life
such as hormonal changesthat are related to an increase of b1-adrenergic
receptor expression in the heart (Toste et al. Br. J of Nutrition
440
2006,95,830-837; Trevenzoli et al. J.Physiol.,2007,580,629-637). Evidences indicate that Na+/K+ATPase activity are reduced in the failing
human heart (Barwe et al. J. Mol. Cell. Cardiol. 2009,47,552-560). SR
Ca2+ATPase activity increase is associated to Ca2+ overload and tachyarrhytmias (Gyorke & Carnes. Pharmacol. & Ther. 2008,119,340-354).
So our data suggest that neonatal leptin treatment may program cardiovascular function.
Paper No.: 1984

BRAIN REGION SPECIFICITY OF 5-HT1A RECEPTOR
ACTIVATION BY SELECTIVE AGONISTS DEMONSTRATED
BY FUNCTIONAL AUTORADIOGRAPHY
Jean-Claude Martel, N Leduc, A Newman-Tancredi
the constitutive internalization or to a defect in the maturation of the

receptor. The rst hypothesis was tested, pre-treating the cells with specic AT1R antagonists, DuP753 and [Sar1Leu8]-AngII in order to revert
the process of internalization. To evaluate the possible reversion, binding
assay using AngII, [Lys2]-AngII or [Sar1]-AngII as ligands and 3H-AngII and 125I-AngII as radioactive tracers were used. IP3 formation and
changes in intracellular Ca2+ were determined to evaluate the signaling
ability. The hypothesis of a defective maturation was tested, using calnexin, an endoplasmic reticulum marker. Cells expressing the mutant AT1
receptor conjugated with green uorescent protein (GFP) were targeted
to the anti-calnexin primary antibody and the Texas Red labeled anti-rabbit secondary antibody. Our data indicate that the low expression of the
C18S mutant on the plasma membrane was due to its internalization
since the pre-treatment with AT1 receptor antagonists was capable to
revert the binding properties and the IP3 formation and [Ca2+]i data. The
nding that the endoplasmic reticulum marker calnexin was poorly colocalized with C18S receptor ruled out the lack of maturation of the
receptor. It is concluded that the mutant receptor assumed a conformational structure similar to that of active mode of the AT1 receptor, favouring its internalization even in the absence of the agonist.
Institut de Recherche Pierre Fabre, Division de Neurobiologie 2, Castres,

France
The serotonin 5-HT1A receptor is an important therapeutic target in the
treatment of depression (Blier and Ward, Biol. Psychiat. 2003; 53: 193203), pain (Colpaert, Curr. Opin. Investig. Drugs 2006; 7: 40-47) and
schizophrenia (Jones and McCreary, Neuropharmacology 2008; 55:
1056-1065). 5-HT1A receptors exist both as presynaptic autoreceptors on
serotonergic neuron cell bodies and as post-synaptic heteroreceptors in
diverse brain regions with differing signal transduction and pharmacological properties. We evaluated concentration-response effects of chemically-related, highly selective, potent and efcacious 5-HT1A agonists
(F13640, F13714 and F15599; cf. Newman-Tancredi et al., Br.J.Pharmacol. 2009; 156: 338-353; Colpaert 2006, as above) and the prototypical
agonist (+)8-OH-DPAT using [35S]GTPcS functional autoradiography in
rat brain structures expressing 5-HT1A heteroreceptors (hippocampus, Hi;
frontal cortex, FrCx) or autoreceptors (dorsal raphe nucleus, DRN).
Serial rat brain sections were processed as described (Martel et al
Br.J.Pharmacol. 2009; 158: 232-242). Results are expressed as percentage of the activation elicited by 100 lM (+)8-OH-DPAT. F13640 exhibited maximal efcacy in all three brain structures, stimulating
[35S]GTPcS binding with pEC50/EMAX of 7.1/124%, 7.2/98% and 6.8/
112% in Hi, FrCx and DRN respectively. F13714 exhibited higher
potency in Hi and DRN than in FrCx (8.4/114%, 8.3/108%. and 7.9/69%
respectively), similarly to (+)8-OH-DPAT 6.6/111%, 6.6/71% and 5.0/
137%). In contrast F15599 showed higher potency in FrCx than in Hi or
DRN (6.8/67%, 6.5/96% and 6.4/63 respectively). These data indicate
that 5-HT1A agonists exhibit regionally-specic proles of 5-HT1A receptor activation, and suggest that the distinct signal transduction properties
of the present agonists translate to differential actions at 5-HT1A receptor
subpopulations.
Paper No.: 1947

CONSTITUTIVE ACTIVATION IS RESPONSIBLE FOR THE
INTRACELLULAR PREDOMINANCE OF THE C18S MUTANT
AT1 RECEPTOR OF ANGIOTENSIN II
Renan Martin, E Rodrigues, L Oliveira, S Shimuta
Paper No. 2914

FOCUS GROUP: P05 - TRANSLATIONAL SCIENCE IN THE
METABOLIC SYNDROME: BASIC AND CLINICAL
PHARMACOLOGY
LEVEL OF RESPONSE TO ANTIPLATELET THERAPY IN
PATIENTS WITH TYPE 2 DIABETES
E Martin-Aurioles(1), F Padin(1), MM Arrebola(2), JA Lopez-Villodres(3), Nuria Jebrouni(3), JP de la Cruz(3), JA Gonzalez-Correa(3)
(1) Health District Malaga, Clinical Management Unit La Roca,
Malaga, Spain
(2) University Hospital Carlos Haya, Service of Clinical Analysis,
Malaga, Sapin
(3) University of Malaga, School of Medicine, Department of Pharmacology, Malaga,Spain
Aim: To evaluate the prevalence and response to antiplatelet therapy in
patients with type 2 diabetes. Materials and Methods: Cross sectional
study conducted in an urban health district level. The study subjects are
patients with type 2 diabetes over the age of 30 who were not with anticoagulant treatment. The sample size (a risk of 0.05 to an accuracy of
0.035 percentage units in a two-sided for an estimated proportion of 0.25
and a replacement rate of 0.3) is 1100 patients, which are chosen by random sampling of 200 patients corresponding to 2 health centers. Collect
data related to demographic and anthropometric, metabolic control
(HbA1c), risk factors, cardiovascular events and drug therapy (focusing
on antiplatelet therapy). Assessment of response to antiplatelet therapy
with adherence and level of platelet inhibition (PFA-100). Univariate
analysis was performed with measures of central tendency and dispersion
(quantitative variables) and frequency and percentage (qualitative variables) and bivariate analysis using unpaired t student and chi square test.
Results: All patients (200) gave their informed consent. The studied population had a mean age of 67 11, predominantly male (52%). Metabolic control was poor (31% of patients had HbA1c less than 7). 63%
were antiagregation. The percentage of non-responders to antiplatelet
therapy was 47% (40% in patients on combination therapy as primary
prevention and 71% in those who received secondary prevention). Conclusion: the response to antiplatelet therapy should be considered poor,
of particular concern in patients receiving the drug for secondary prevention.
University Federal of Sao Paulo, Department of Biophysics, Sao Paulo,

Brazil
Previous studies reported that CHO cells expressing the AT1 receptor
(AT1R) bearing the C18S mutation which breaks the Cys18-Cys274
bridge, showed a high basal activity and intracellular localization. We
investigated whether this intracellular predominance was due either to
441
Paper No.: 1783
THERAPEUTIC TARGETS
ROLE OF NA+-K+ ATP-ASE IN THE NITRIC OXIDE
INDEPENDENT RELAXATION IN PIG URINARY BLADDER
NECK
Ana Martnez-Saenz(1), LM Orensanz(2), P Recio(1), S Bustamante(3), S
Benedito(1), A Garca-Sacristan(1), D Prieto(1), M Hernandez(1)
(1) Universidad Complutense de Madrid, Facultad de Farmacia, Departamento de Fisiologa, Madrid, Spain
(2) Hospital Universitario Ramon y Cajal, Departamento de Investigacion, Madrid, Spain
(3) Hospital Universitario Puerta de Hierro-Majadahonda, Departamento
de Urologa, Madrid, Spain
Nitric oxide (NO) is involved in inhibitory transmission of the pig bladder neck (Hernandez et al., Br J Pharmacol 2008; 153: 1251-1258). In
addition to NO, a large component of the nerve stimulation-evoked relaxation seems to be independent of the cAMP- or cGMP-dependent protein
kinase pathways or postjunctional K+ channels. The aim of the current
study was to investigate the mechanisms involved in non NO nerve
relaxations in such structure. Urothelium denuded strips 4-6 mm long
and 2-3 mm wide were suspended in 5 ml organ baths containing PSS at
37o C gassed with 95% O2 and 5% CO2. The signal was continuously
recorded on a polygraph. Passive tension of 2 g was applied to the strips
and they were allowed to equilibrate for 60 min. On 1 microM phenylephrine (PhE)-induced tone, relaxations to electrical eld stimulation
(EFS) was performed by delivering rectangular pulses (1 ms duration,
1-16 Hz), from a Cibertec CS20 stimulator. Noradrenergic neurotransmission, muscarinic receptors and NOS were blocked. Under these conditions, EFS evoked frequency-dependent relaxations which were
reduced by ouabain, a Na+-K+ ATPase inhibitor. However, the blockade
of barium-sensitive inward rectier K+ channels failed to modify the non
NO nerve responses. These results suggest that NO-independent nerve
relaxation in the pig bladder neck is produced, in part, via a mechanism
dependent of Na+-K+ ATP-ase activation. Blockers of the Na+-K+ ATPase pump may be useful for urinary incontinence treatment produced by
intrinsic sphincteric deciency.
Supported by grant PS09/00044 from Ministerio de Ciencia e Innovacion, Spain.
Paper No.: 1232

PHARMACOKINETICS AND OUTCOMES OF GENTAMICIN
THERAPY IN NEONATES CRITICALLY ILL DURING THE
FIRST WEEK OF LIFE: VOLUMES OF FLUID INFUSED OR
RETAINED AS POSSIBLE COVARIATES
assayed by a uorescence polarization immunoassay. Dosing was individualized to reach the target Cpeak (6-10 mg/L, 30min after infusion)
and Ctrough (< 2 mg/L, 30 min before next infusion). Additional
Ctrough,3 and Cpeak,4 concentrations related to infusion 4 were assayed.
Suspected uid retention (SFR) was calculated as the difference between
uid input and urine output. Cpeak,1 was variable (CV 29%) and <
6mg/L in 13% of neonates. Neither Cpeak,1 nor Vd were correlated with
input of uid within the interval -2 h - 0 h (P=0.4) or SFR from -6 h to
+1 h (P=0.8) relative to the start of infusion 1. CL decreased with SFR
within -6h - +1h (r= -0.31, p<0.005) and 0h-24h (r = - 0.37, P<0.001).
Cpeak,4 was over predicted (the mean prediction error 1.40 mg/L, 95%CI: 0.95-1.86) and < 6 mg/L in 45% of neonates. SFR between 0h and
infusion 4 correlated with the postnatal b. w. (r =0.58, P<0.001) and
negatively correlated with Cpeak,4 (r = -0.25, p=0.02).
Paper No.: 2088

DOES BUDESONIDE APPLIED IN PREVENTION OF CHILD
ASTHMA AFFECT VARIATIONS IN BODY WEIGHT IN
ASTHMATIC CHILDREN?
Milica Martinovic
Medical School Podgorica, Institute for Pathophysiology and Laboratory
Medicine, Montenegro
Introduction: Monitoring of variation (especially increase) in body
weight (BW) at asthmatic children treated with inchaled corticosteroids
(ICS) during several years is very important. But, this is less reliable
parameter than variations of body height. It is considered that budesonide
used in conventional doses doesnt cause systemic side effects such as
Sy Cushing and increase and redistribution of fat tissue. Patients and
methods: At 20 children, both sex, age 10-14 with moderate persistent
asthma (diagnosis was made using international criteria) budesonide was
applied in order to prevent asthma attacs. It was applied during 3 years
(9 months of budesonide therapy in conventional doses and 3 months
without budesonide). Body weight was measured once per month using
standard procedure. Results were recorded, analised and compared to
Montenegrin standards for healthy children (same body height, sex and
age). Daily prole of cortisol serum level was measured at the beginning
and at the end of each 9 months period of ICS therapy. Results: Measurement of body weight, done once per month, during three years, at
children with asthma treated with ICS has shown that there wasnt statistically signicant variation in BW in comparison to healthy children.
Daily prole of cortisol serum level was normal, too. Conclusion: It is
impotrant to carrefully monitor growth of asthmatic children during ICS
therapy. Budesonide has good safety prole and, applied in conventional
doses, during years did not cause deviation in body weight and did not
cause slowing of children growth.
Jirina Martinkova(1), P Pokorna(2), J Zahora(3), J Chladek(1),

V Vobruba(2), I Selke-Krulichova(3), J Chladkova(4)
(1) Charles University, Faculty of Medicine, Department of Pharmacology, Hradec Kralove, Czech Republic
(2) General Teaching Hospital, Department of Pediatrics and Adolescent
Medicine, Intensive Care Unit, Karlov, Prague, Czech Republic
(3) Charles University, Faculty of Medicine, Department of Biophysics,
Hradec Kralove, Czech Republic
(4) Charles University, Faculty of Medicine, Department of Paediatrics,
Hradec Kralove, Czech Republic
This study evaluated gentamicin (GE) pharmacokinetics (PK) in neonatescritically ill during the rst week of life. The main goal was to
investigate the impact of gestational age (GA) and uid input-output balance on GE volume of distribution (Vd) and clearance (CL). 84 neonates, 0.8 - 4.56 kg birth weight and 24-42 weeks GA were treated with
GE for suspected or proven sepsis. Individual PK parameters were estimated after dose 1 (4 mg/kg i.v., 30-mininfusion) using 4 concentrations
Paper No.: 2089

DOES BUDESONIDE APPLIED IN PREVENTION OF CHILD
ASTHMA DECREASE HEIGHT VELOCITY OF ASTHMATIC
CHILDREN?
Milica Martinovic, S Pantovic
Introduction: Uncontrolled, severe asthma itself can decrease children
growth. It is considered that budesonide (and other inchaled corticosteroids) has good safety prole and does not decrease height velocity at
treated children. Patients and methods: At 20 children, both sex, age
442
10-14 with moderate persistent asthma (diagnosis was made using international criteria) budesonide was applied in order to prevent asthma attacs. Budesonide was applied during 3 years (9 months in conventional
doses and 3 months without therapy). Body height was measured once
per month by standard procedure. Gained data was analysed and compared to Montenegrin standards for healthy children. Daily prole of cortisol serum level was measured at the beginning and at the end of each
9 month period of budesonide use. Stage of pubertal development was
assessed according to Tunner. Results: All children had asthma under
control, withouth or with very mild attacs. The curve of height velocity
was same at the children with asthma and healthy children. All children
with asthma were in the channel of height velocity in accordance with
their calculated genetic potential. Puberty acceleration in height velocity
(catch up) was seen at 5 girls during this periode. Pubertal development
was normal. Daily prole of cortisol serum levels were normal indicating
that children did not have supression of hypothalamo-pituitary-adrenal
axis. Conclusion: It is very important to monitor height velocity at children with persistent asthma especially in case of long term inhaled corticosteroids therapy. Budesonide showed good safety prole and didnt
decrease height velocity at asthmatic children.
Paper No.: 2090

SY CUSHING AT THE CHILD WITH ASTHMA, CASE REPORT
Milica Martinovic
Introduction: Boy, aged 11, from region of northern Montenegro, with
severe persistent asthma, was treated with oral corticosteroids (under
unclear circumstances) in order to establish control of asthma - prednisolon was applied during approximately 6 months. After few months of
this therapy, his parents were very satised, he had less asthma attacs,
attacs were milder and he had increase in body weight. But, after several
more months parents became concerned because they noticed that boy
was becoming owerweight and he had extraordinary type of obesity. At
the physical exam boy had hypertension, obesity and redistribution of fat
tussue tipical for Sy Cushing. Laboratory analysis showed high serum
cortisol level without physiological diurnal rhythm. Urinary excretion of
17-hidroxy-corticosteroids were high. Oral corticosteroids doses were
gradually reduced and inchaled corticosteroids were introduced. Unfortunatelly, contact with patient and his family was interrupted (they moved
to another country). Conslusion: This case was individual and with
unclear circumstances. Prevention of persistent asthma in Montenegro is
implemented with inhaled corticosteroids, in accordance to up to date
international standards, and oral corticosteroids are only used at the short
course in the treatment of severe asthma attacs.
Paper No.: 428

INVESTIGATION OF THE PHYTOCHEMISTRY, ANTIOXIDANT ACTIVITIES AND ANTICANCER EFFECTS OF TOTAL
EXTRACT AND SOME OF THE PURIFIED COMPOUNDS OF
FERULA SZOWITSIANA D.C. ON SEVERAL HUMAN CANCER
CELL LINES
erative activities of the organic extracts and fraction of Ferula szowitsiana on some human cancer cells. Materials and Methods: The essential
oils obtained from F. szowitsiana were analyzed by GC and GC/MS. We
also isolated persicausulde A, for the rst time, together with, Farnesiferol C, Umbelliprenin and Galbanic acid, as sesquiterpene coumarins
from F. szowitsiana roots. Organic extracts from F. szowitsiana were
evaluated for FRAP and DPPH radical scavenging in vitro. In vitro test,
samples was examined in rats in the prevention of plasma and liver lipid
peroxidation and activity of CAT and SOD. Results: The activities of
CAT and SOD also, increased in the liver. Total chloroform extract of F.
szowitsiana and its nanpolar fractions, prepared by column chromatography, substantially inhibited viable HT-29, HL-60 and K-562 cells number
in a dose dependent manner, whereas polar fractions resulted in the isolation of anticancer sesquiterpene coumarins, namely Farnesiferol C (FC)
and Umbelliprenin (UM). Extract, active fractions and FC induced apoptosis in HT-29 cells associated with nuclear DNA fragmentation, Caspase-3 and ROS generation, suggesting the apoptosis by these
compounds occurs through the mitochondria dependent pathway. Conclusion: Analyses of the data suggest that F. szowitsiana can be used for
cancer treatment because of its functions; as antioxidant and antiproliferative agent. Also FC isolated from this plant may represent novel therapeutic agents for treatment of colorectal cancer as it is capable of potent
cytotoxicity.
Paper No.: 1703

PROTECTIVE EFFECTS OF GLUTATHIONE AND THE
RELATED THIOLS AGAINST HEPATOTOXICITY OF
ACETAMINOPHEN AND FUROSEMIDE IN MICE
Yasuhiro Masubuchi, J Nakayama, Y Sadakata
Chiba Institute of Science, Faculty of Pharmaceutical Sciences, Laboratory of Clinical Pharmacy, Choshi, Chiba, Japan
An overdose of acetaminophen (APAP) causes liver injury in experimental animals and humans. N-Acetylcysteine (NAC) is clinically used as an
antidote for the APAP intoxication. It is thought that NAC provides cysteine (CYS) as a precursor of glutathione (GSH), which traps a reactive
metabolite of APAP, whereas other hepatoprotective mechanisms of
NAC have also been suggested. Here, we examined the effects of thiol
compounds with different abilities to restore hepatic GSH, on hepatotoxicity of APAP and furosemide in mice. Overnight-fasted male CD-1 mice
were given APAP or furosemide intraperitoneally. NAC, CYS, GSH or
glutathione monoethyl ester (GSH-EE) was administrated concomitantly
with APAP or furosemide. Hepatotoxicity was assessed by serum leakage
of alanine aminotransferase. Hepatic GSH contents were determined by
the dithionitrobenzoic acid-glutathione disulde reductase recycling
assay. Cytokine mRNA expression was assayed by real-time RT-PCR.
All of the thiols used in this study effectively protected mice against
APAP-induced liver injury. Only GSH-EE completely prevented the
APAP-induced early hepatic GSH depletion, while NAC and CYS produced partial GSH restoration. Exogenous GSH had no effect on the
hepatic GSH loss in the APAP-treated mice, but induced hepatic expression of IL-6, which may be involved in alleviation of the APAP hepatotoxicity. Furosemide-induced liver injury, which doses not accompany
hepatic GSH depletion, was also attenuated by NAC. In conclusion,
exogenous thiols could alleviate drug-induced liver injury without
increasing hepatic GSH, probably through cytokine-mediated and antiinammatory mechanisms.
Omid Mashinchian
Tabriz University (Medical Sciences), Research Center for PharmaceuticalNanotechnology (RCPN), Department of System Biology, Tabriz, Iran
Introduction: The objective of this study was the phytochemical analyses;
examine antioxidative and radical potentials, proapoptotic and antiprolifJournal compilation 2010 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 107 (Suppl. 1), 162692
443
Paper No.: 2113
DISEASES
PARADOXICAL INFLUENCES OF MTOR INHIBITOR
EVEROLIMUS ON PROGRESSIVE RENAL FAILURE
Satohiro Masuda, S Nakagawa, K Nishihara, K Inui
Kyoto University Hospital, Department of Pharmacy, Kyoto, Japan
To clarify the roles of mTOR pathway in the course of progressive
chronic renal failure (CRF), the 5/6 nephrectomized (Nx) rats were used
as a model of progressive CRF. At 1, 2, 4 and 8 weeks after Nx, the
proximal tubules from the remnant kidneys were isolated under microscopy. The microarray analysis with the isolated proximal tubules
revealed that the genes related cellular proliferation/cell cycle were markedly increased in the early-stage CRF, called hgcompensative CRFhh.
However, the genes related inammatory responses were much increased
in the end-stage CRF, called hguremiahh. Because the main proliferative cells were tubular epithelium and broblasts in the compensative
CRF and uremia, respectively, the pharmacological effects of everolimus
were compared between the two stages. In the compensative CRF,
immunouorescent analysis revealed that Ki-67-positive proximal tubular
epithelial cells were decreased to 20% in everolimus-treated Nx rats
compared to that in vehicle-treated Nx rats. Simultaneously, administration of everolimus markedly increased the BUN and plasma creatinine,
and decreased the creatinine clearance (Nishihara et al. Am J Physiol
Renal Physiol, 2010; in press). In contrast, the ameliorative effect of everolimus was observed in the uremia rats with the decreased expression
of smooth muscle a-actin and ED1-positive inltrated immune cells
(Nakagawa et al, Biochem Pharmacol 2010; 79: 67-76). Interestingly,
proximal tubular reabsorption of albumin was restored after the treatment
of everolimus in the uremia rats. These results suggest that mTOR inhibitor can paradoxically mediate both further deterioration and amelioration
of the remnant renal functions by the state of CRF.
Paper No.: 2342

DISCOVERY
A ROLE OF A2 ISOFORM OF THE NA,K-PUMP IN
MESENTERIC SMALL ARTERIES WALL
Vladimir Matchkov, NM Nielsen, C Aalkjr
Aarhus University, Institute of Physiology and Biophysics, Aarhus,
Denmark
I has been previously suggested that ouabain-sensitive a2 isoform of the
Na,K-pump regulates vascular tone by a spatial-restricted calcium signalling. We hypothesized also that this Na,K-pump interacts functionally
with gap junctions. We downregulated transiently the expression of the
Na,K-pump by transfecting mesenteric small arteries in vivo with specic
siRNA. siRNA directed against eGFP was used as a control. Micromoles
of ouabain reduced in a concentration-dependent manner an amplitude of
noradrenalin(NA)-induced oscillations in tone, vasomotion. Arteries
downregulated for a2 isoform initially had signicantly reduced vasomotion amplitude which was insensitive to ouabain. Interestingly, 10 lM
ouabain suppressed vasomotion in the control arteries to the level
observed in the arteries downregulated for a2 isoform. Neither ouabain
nor siRNA-induced downregulation affected frequency of vasomotion.
These results are in accordance with the suggested regulatory role of the
Na,K-pump for intercellular communications in the vascular wall. Surprisingly, the arteries downregulated for a2 isoform were less sensitive to
NA. This is in contrast to previously shown potentiating of agonistinduced contraction by ouabain. Although micromoles of ouabain
increased sensitivity in the control arteries, it had no effect on the a2
downregulated arteries. We demonstrate here the importance of a2 isofom of the Na,K-pump for the regulation of vascular tone.
Paper No.: 2343

INCREASED REUPTAKE MASKS ELEVATED SENSITIVITY
TO MONOAMINES IN PERIPHERAL BUT NOT CEREBRAL
RESISTIVE ARTERIES OF RATS DEPRESSED IN RESPONSE
TO THE CHRONIC MILD STRESS (CMS)
Vladimir Matchkov, E Bouzinova, T Brgger, NM Nielsen, K Henningsen, O Wiborg, C Aalkjr
Aarhus University, Institute of Physiology and Biophysics, Aarhus,
Denmark
Depression and cardiovascular disease are known to occur simultaneously but the reason for this is unclear. In CMS model of depression
only some rats develop depression-like symptoms, while others are
stress-resistant (resilient). CMS rats are shown to have a reduced cardiac
output and unchanged blood pressure suggesting increase in total peripheral resistance (TPR). To study the reason for elevated TPR after
8 weeks exposure to variable and unpredictable stress we have assessed
in vitro isometric force in the middle cerebral (CA), femoral (FA) artery
and mesenteric small arteries (MSA). MSA from all three groups developed similar maximal tension to noradrenaline (NA). MSA were also
similarly sensitive to NA under control conditions but cocaine unmasked
elevated NA sensitivity of MSA from depressed rats consistent with elevated neuronal reuptake of NA. FA from depressed rats also demonstrated increased sensitivity to NA which had been masked by elevated
NA reuptake. In addition, the maximal tension to NA of FA from
depressed rats was higher in comparison to the control and resilient
groups. CA from depressed rats were more sensitive to 5-HT in comparison to the resilient and non-stressed groups. This difference was not
affected by cocaine. The observed changes in the arterial responses to
monoamine correlated with changes in corticosterone which transiently
increased to stress and the transient lasted longer in the depressed rats.
Thus, our results indicate that depression is associated with changes in
the contractility and agonist sensitivity of resistance arteries.
Paper No. 3405

A NOVEL NANOSCALE HIGH THROUGHPUT ASSAY
SCREENING MEMBRANE BASED PROTEIN-PROTEIN
INTERACTIONS
S Mathiasen(1), Vikram Kjller Bhatia(1), S Pedersen(2),
K Strmgaard(2), D Stamou(1)
(1) University of Copenhagen, Institute of Neuroscience and Pharmacology & Nano-Science Center, Bio-Nanotechnology Laboratory, Copenhagen, Denmark
Department of Medicinal Chemistry, Copenahgen, Denmark
PDZ domains are important protein-interaction modules which function
to scaffold large protein complexes in cells. They recognize and bind
peptide stretches often located at the C-termini of various proteins. Interestingly many GPCRs and other transmembrane receptors (7TMs), carry
a PDZ recognition motif at their C-termini extremity. A broader understanding and insight to the molecular machinery of the scaffolding
protein is of great biological relevance. Thus, revealing the kinetic and
thermodynamic properties of PDZ interactions and molecular mechanisms would provide insight to the various cellular functions, the synap-
444
tic targeting, clustering and assembly of the larger protein domains and
aid pharmaceutical disease treatment. Traditional approaches to screen
such interactions rely on pull-down and Fluorescence Polarization (FP)
assays. However, all techniques are limited to address solution based protein-protein interactions. We present here a novel in-vitro surface based
assay to screen GPCR/7TM c-tail interaction with PDZ domains in a
membrane environment. Using bio-nanotechnology techniques, the assay
furthermore allows screening this interaction as a function of membrane
curvature (Hatzakis et al, Nat. Chem. Biol., 2009, 11: 835-41), a concept
recently shown to drastically affect biological reactions (Ramamurthi et
al, Science, 2009, 323: 1354-57). As a proof-of-concept we tested interactions between the NMDA receptor subunit NR2B and the PDZ2
domain of PSD-95. Comparison with FP literature results allowed us to
evaluate the effect of the membrane on kinetics and thermodynamic
parameters.
Paper No.: 3108

DEVELOPMENT AND CHARACTERIZATION OF A BETA2
ADRENERGIC RECEPTOR CAMP BIOSENSOR ASSAY
Jesper M Mathiesen(1), H Brauner-Osborn
University of Copenhagen, Department of Medicinal Chemistry, Faculty
of Pharmaceutical Sciences, Copenhagen, Denmark
cAMP is a common second messenger that mediate numerous biological
responses. Intracellular cAMP levels are increased by activation of Gascoupled seven-transmembrane receptors via the adenylyl cyclase (AC).
To functionally characterize ligands of the b2 adrenergic receptor (b2AR)
for Gas pathway activity, a uorescence resonance energy transfer
(FRET) based cAMP biosensor was generated. The cAMP FRET biosensor consisted of the cAMP binding protein EPac1 amino acids 149-881
anked by cyan and yellow uorescent proteins on either side. Upon
cAMP binding to the EPac1 part of the biosensor, conformational
changes increase the distance between the uorescent proteins, resulting
in decreased FRET. The construct was generated as described (Violin JD
et al, JBC 2008; 283: 2949-61) except that a more efcient mCerulean/
mCitrine FRET pair was used. The biosensor was stably expressed in
HEK293 cells that endogenously express b2AR. The b2AR agonist isoproterenol and forskolin (directly activating AC), produced clear reductions in the emission peak of mCitrine upon excitation of the mCerulean
FRET donor. For ligand screening and characterization a sustained signal
over time in a plate assay format is preferable. To this end the b2AR was
overexpressed in the EPac1 biosensor cell line. Isoproterenol dose dependently decreased the FRET ratio, which was stable for at least 2 hr when
measured in a 384-well plate Tecan Sare2 plate reader. The assay is
suitable for screening with a Z-factor of 0.6-0.8. The assay can be tuned
for inverse agonist detection by assaying in the presence of a phosphodiesterase inhibitor.
Paper No.: 937

HEALTH AND DISEASE
GLYCOSAMINOGLYCANS AND CANDESARTAN PREVENT
RENAL ALTERATION AND OXIDATIVE STRESS IN THE
KIDNEY OF DIABETIC RATS
(3) Central University of Venezuela, Anatomical Institute Jose Izquierdo, Caracas, Venezuela
Increased renin-angiotensin-aldosterone system (RAAS) and decreased
levels of glycosaminoglycans (GAGs) have been involved in the pathogenesis of diabetic nephropathy. We evaluated the effect of the candesartan (CAN), an angiotensin II type 1 receptor blocker, and the GAG
sulodexide (SUL) on the activity of antioxidant enzymes, catalase (CAT)
and superoxide dismutase (SOD) in the rat kidney, and on morphological
and functional renal abnormalities in diabetic rats. Diabetes was induced
in male Sprague-Dawley rats by i.v. administration of streptozotocin
(STZ). Animals were randomly allocated in ve groups: C=control;
STZ; STZ+SUL (pretreated with SUL, 15 mg/kg, s.c.); STZ+CAN (pretreated with CAN, 10 mg/Kg p.o.) and STZ+SUL+CAN. After three
months of follow up, blood and 24h-urine sample were obtained and
then animals were sacriced and the kidneys microdissected for morphometric analysis. CAT and SOD activity was assessed by UV-visible spectrophotometry. Urinary albumin excretion was markedly increased in
untreated diabetic rats, and all treatments partially prevent the albumin
rise. The activity of CAT and SOD were found to be signicantly diminished in the STZ group, and this activity was restored by SUL, CAN and
combined treatment. Light microscope observation revealed typical tubular lesions described in experimental diabetes (STZ group) and all treatment prevent the lost of thickness of tubular wall. Our result
demonstrated a role for GAGs and CAN on the regulation of kidney
function, antioxidant enzymes activity and preservation of renal cytoarchitecture and suggest a protective effect of these treatments in diabetic
nephropathy.
(Supported by CDCH PI09-00-5102-2007).
Paper No.: 3243

ON EXPRESSING DRUG DOSAGE
Mukul Mathur, L Sharma, KK Sharma
Jaipur Medical College, Department of Pharmacology, Jaipur, Rajasthan,
India
A common premise in pharmacology is that the intensity of the effect of
a drug is related to its plasma concentration and that its duration of action
is determined by the plasma concentration above the minimum effective
levels but lower than the maximum tolerated or toxic level such that the
toxicity does not become manifest. Therefore, the dose of a drug and its
frequency of dosing is recommended so as to keep the plasma concentrations within the minimum effective and maximal tolerated levels. For this
reason expression of the amount of a drug, frequency of administration,
route of administration and the duration for which it ought to be prescribed are important therapeutically. However, surprisingly it is not
uncommon to read, even in standard text and reference books statements
which lack clarity and precision. The dose of a drug is often mentioned
as xx mg per day or yy mg daily in divided doses leaving the
reader to ip the pages to scan the pharmacokinetic prole of the drug to
ascertain how frequently the drug should be given. Such statements on
drugs which show steep log-dose effect relationship, mixed order kinetics
or cumulative toxicity can be hazardous.. It is, therefore, reasonable to
suggest expressing the amount of a drug on hourly (like 6 hourly, 12
hourly etc) rather than on daily basis. This will help the student understand and appreciate better the principles of pharmacokinetic parameter
in rational therapeutics.
Yaira Mathison(1), C Ciangherotti(2), M Alvarez(3), H Fernandez(1),

A Israel(2)
(1) Central University of Venezuela, School of Medicine Jose Mara
Vargas, Universidad, Caracas, Venezuela
(2) Central University of Venezuela, School of Pharmacy, Laboratory of
Neuropeptides, Caracas, Venezuela
445
Paper No.: 1556
DISEASES
EFFECT OF VALSARTAN ON BONE LOSS IN PERIODONTAL
DISEASE INDUCED BY LIPOPOLYSACCHARIDES IN THE
RAT
maintenance dose, and our result was reasonable that M/W patients are
prone to range out to lower PT-INR, rather than higher side. It has been
shown that the genetic difference can cause the warfarin therapy inconsistency in some extent, though it will be just one of many contributing
factors.
Maria Gabriela Matos, L Perdomo, M Alvarez, M Varela, A Stern, M del

Rosario Garrido
Universidad Central de Venezuela, Schook of Pharmacy, Laboratory of
Neuropeptides, Caracas, Venezuela
Local administration of bacterial endotoxin (lipopolysaccharide, LPS) in
the gum of the rat produces an inammatory response that progress to
periodontitis. It is know that angiotensin II (AngII) acts like a pro-inammatory cytokinesince the blockade of T1 receptors (AT1R) produces a
decrease of the progression of inammatory pathologies such as rheumatoid arthritis and hypertension. The role of AngII/AT1R in the periodontal
disease is not known. The aim of this study was to assess the effect of
valsartan (VAL), an AT1R blocker, in the progression of the histological
changes, bone loss and leukocytes count in an experimental model of
periodontitis induced by LPS. Sprague-Dawley rats, weighing 280-300g,
were divided into four groups: Control, LPS, VAL (20 mg/kg,7 and
14 days), LPS+VAL. Animals were euthanized by decapitation, the maxilla dissected, xed and demineralized with Osteomoll, followed by dehydratation.The tissue was sectioned along the molars in a mesiodistal
plane for histological analysis. The photographic recordings were taken
with an opticalmicroscope (20 xs). LPS induced severe histological
changes, bone loose and increased leukocytes count. These effects were
signicantly reduced by VAL treatment. Our results suggest a role of the
Ang II and its AT1 receptors in the pathogenesis of periodontal disease
induced by LPS-induced in rats.
(MisionCiencia, Proyecto ECCV No. 2007001585).
Paper No.: 1090

INFLUENCE OF VKORC1 POLYMORPHISM ON PT-INR
ADHERENCE TO THE TARGET THERAPEUTIC RANGE IN
LONG-TERM WARFARIN THERAPY
Naoki Matsumoto(1), M Watanabe(1), T Kumai(1), M Tanaka(1),
Y Takeba(1), Y Harimoto(1), S Takenoshita(1), Y Kinoshita(1),
M Yamauchi(2), F Miyake(1), H Musha(2), S Kobayashi(1)
(1) St. Marianna University School of Medicine, Department of
Pharmacology,Kawasaki, Japan
(2) Yokohama-City Seibu Hospital, St. Marianna University, Kawasaki,
Japan
Warfarin plays a big role in anticoagulation therapy for atrial brillation,
but its inconsistent maintenance dose makes it difcult to use it casually.
It is recommended to analyze VKORC1 polymorphism when determining warfarin maintenance dose, but it is still unclear whether the analysis
helps predicting the tendency to be adherent or inconsistent in keeping
the PT-INR results within the appropriate therapeutic range. We analyzed
VKORC1 polymorphism of mostly elderly Japanese atrial brillation
patients under warfarin therapy (n=201), and their retrospective PT-INR
results basically on 6 out-patient follow-ups in 6 months (total tests =
1156). The average PT-INR showed no statistical difference between
mutant-homo type (M) (1.86, n=842), mutant-wild-hetero type (M/W)
(1.81, n=314) and wild-homo type (W) (1.91, n=12). Events those
showed their PT-INR within (A), above (H) or below (L) the therapeutic
range of the Japanese Guideline for elderly atrial brillation patients (1.6
to 2.6) were statistically different between M and M/W (M: A=64.7%,
H=6.2%, L=20.1%. M/W: A=56.1%, H=7.0%, L=36.9%) (p value
0.0245, Prob>ChiSq). M/W type usually requires slightly higher warfarin
Paper No.: 2876

INFLUENCE OF ESCHERICHIA COLI NISSLE 1917 ON
CYTOCHROME P450 PROTEIN EXPRESSION IN THE
INTESTINE OF MALE RATS
Zuzana Matuskova(1), A Tunkova(1), E Anzenbacherova(2), R Vecera(1),
H Tlaskalova-Hogenova(3), Z Zidek(4), P Anzenbacher(1)
(1) Palacky University, Faculty of Medicine, Department of Pharmacology, Olomouc, Czech Republic
(2) Palacky University, Faculty of Medicine, Department of Medinal
Chemistry and Biochemistry, Olomouc, Czech Republic
(3) Institute of Microbiology AS CR, Prague, Czech Republic
(4) Institute of Experimental Medicine AS CR, Prague, Czech Republic
Escherichia coli strain Nissle 1917 (serotype O6:K5:H1) is probiotic
agent with benecial effects on the gastrointestinal tract. However, the
possibility of interactions with concomitantly taken pharmacotherapeutic
agents has not been tested yet. The aim of the study was to nd whether
E. coli strain Nissle 1917 inuences the cytochromes P450 protein
expression in the rat intestine. Live bacterial suspension of E. coli strain
Nissle 1917 was applied to six Wistar rats (2.36 billion bacteria/ ml PBS/
kg, orally) daily for 7 days. Four rats were stressed by oral application of
the physiological solution daily for 7 days as well. This group was taken
as control. Sections of small intestine, colon and coecum were taken.
Intestinal homogenate was prepared and expression of selected cytochromes P450 (CYP) was followed by Western blotting. Expression of
CYP3A1, CYP1A1 and CYP2C6 proteins was detected in the small
intestine and coecum, only CYP3A1 and CYP1A1 were also detected in
the colon. The results show that there are no differences in CYP protein
expression between control samples and samples from E. coli treated
rats. Therefore probiotic E. coli strain Nissle 1917 does not inuence the
expression of selected cytochromes P450 in the intestine.
Financial support from Grant Agency of Czech Republic (305/08/0535
and 303/09/H048) is gratefully acknowledged.
Paper No.: 459

ROLE OF TERMINALIA ARJUNA AN INDIAN MEDICINAL
PLANT IN CARDIOVASCULAR DISEASES
Subir Kumar Maulik
New Delhi, India
Terminaia arjuna (Roxb.) has been mentioned in Ayurveda (Traditional
IndianMedicine) for its benecial effects in various cardiac disorders. It
is also avery commonly prescribed drug by Ayurvedic physicians in
India and also available in India as a herbal medicine. Several animal
and human studies have supported these benecial effects of the bark
powder and its various extractsin cardiovascular disorders, like ischemic
heart disease and heart failure. We have earlier reported two novel properties of the plant. We have shown that chronic administration of the bark
powder and some of its extracts caused augmentation of endogenous car-
446
diac antioxidants (SOD, catalase and glutathione) and induction of heat
shock protein in rats and rabbits. These properties rendered the heart of
the treated animals resistant to in vivo and in vitro cardiac ischemic reperfusion injury, in terms of prevention of oxidative stress, preservation of
hemodynamic functions and prevention of histopathological changes.
More recently, we have demonstrated that a standardised preparation of
the water extract of the bark powder of T. arjuna prevented the pathological features, associated with cardiac hypertrophy, like brosis and induction of fetal gene programme in rat. This effect was comparable with that
of an ACE inhibitor. Our studies, along with those reported by various
other investigators, strongly support the Ayurvedic concept of the plants
cardioprotective effect. However, further research on standardization of
the extracts and properly designed clinical trials are needed for authentication of this concept.
Paper No.: 2151

MOLECULAR ORGANIZATION AND DYNAMICS OF THE
MT1 MELATONIN RECEPTOR/RGS20/GAI PROTEIN
COMPLEX REVEAL ASYMMETRY OF RECEPTOR DIMERS
FOR RGS COUPLING
P Maurice(1), AM Daulat(1), R Turecek(2), K Ivankova-Susankova(2),
F Zamponi(3), JL Guillaume(1), B Bettler(2), P Delagrange(4),
Ralf Jockers(1)
(1) Universite Paris Descartes, Institut Cochin, INSERM U1016, CNRS
8104, Paris,France
(2) University of Basel, Institute of Physiology, Department of
Biomedicine, Basel,Switzerland
(3) Ecole Normale Superieure, CNRS UMR 8549, Laboratoire de
Physique Theorique,Paris, France
(4) Institut de Recherches SERVIER, Suresnes, France
Regulators of G protein signalling (RGS) are known to accelerate GTP
hydrolysis of heterotrimeric G proteins by binding to the activated Ga
subunit, thus ne-tuning signalling of G protein-coupled receptors. By
searching for protein complexes associated with the carboxyl-terminal
(Cter) domains of the two Gai protein-coupled human MT1 and MT2
melatonin receptors in brain lysates, we identied RGS20 as a specic
partner of MT1. Using biochemical, uorescence imaging and functional
approaches, we conrmed the interaction between MT1 and RGS20 in
transfected HEK293 cells and in the ovine pituitary pars tuberalis
expressing both proteins endogenously. Both RGS20 and Gai bind
directly and independently to the third intracellular loop and the Cter of
MT1 to form a pre-associated complex. Agonist activation of MT1 rearranges this complex as monitored by bioluminescence resonance energy
transfer (BRET) between probes inserted at multiples sites in the MT1/
Ga1/RGS20 complex. BRET data and molecular Modelling suggest formation of a complex composed of one Gi protein and one RGS each
binding to separate protomers of a receptor dimer. Formation of an asymmetric complex is supported by studies on heterodimers composed of
MT2 (which does not interact with RGS20) and MT1 in which specic
activation of MT2 induces the activatory switch between the RGS and
the Gai proteins. Collectively, these data support the concept of pre-associated GPCR/ Gi/RGS (replace by GPCR/Gi/RGS ternary complexes that
rearrange upon agonist-promoted receptor activation to explain receptor
specicity of RGS proteins and indicate asymmetric binding of interacting proteins to GPCR dimers.
Paper No.: 2737

HEALTH AND DISEASE
NITRIC OXIDE LEVELS IN THE RENAL CORTEX OF
STREPTOZOCIN-INDUCED DIABETIC RATS TREATED WITH
RAMIPRIL AND EPLERENONE
T Mavrakanas, Constantinos Tomos, M Mironidou-Tzouveleki
Aristotle University Medical School, 1st Laboratory of Pharmacology,
Thessaloniki, Greece
Introduction: Our study investigates the effect of ramipril and eplerenone
treatment on nitric oxide levels in the renal cortex in the streptozocininduced diabetic rat. Materials: Wistar rats were divided into four groups:
non diabetic controls, streptozocin-treated diabetic rats (50 mg/kg), diabetic rats receiving ramipril (1 mg/kg), and diabetic rats treated with the
combination of ramipril (1 mg/kg) and eplerenone (100 mg/kg) for
8 weeks. Nitric oxide was directly measured in renal cortex homogenates. Results: Nitric oxide concentration was lower in the renal cortex of
diabetic rats (63 6 lM) than in control rats (84 6 lM), although this
did not reach statistical signicance (p = 0.24). Ramipril treated rats
exhibited further decrease in NO levels (49 3 lM) (p = 0.02 versus
non-DM group). On the other hand, combination treatment with ramipril
and eplerenone restored NO levels in the renal cortex (81 13 lM) (p =
0.03 versus DM+RAM group). Conclusion: A decrease in nitric oxide
levels was detected in the cortex of diabetic rats that is reversed by combined treatment with eplerenone and ramipril but not with ramipril alone.
Paper No.: 2781

DISCOVERY
EXPRESSION AND FUNCTION OF THE ORGANIC CATION
TRANSPORTER 3 (OCT3) IN THE HUMAN PLACENTA
Karen May(1), V Rozehnal(1), M Grube(2), M Zygmunt(3), HK
Kroemer(2), W Siegmund(1)
(1) University of Greifswald, Department of Clinical Pharmacology,
Greifswald, Germany
(2) University of Greifswald, Department of Pharmacology, Greifswald,
Germany
(3) University of Greifswald, Department of and Gynecology and
Obstetrics, Greifswald, Germany
The organic cation transporter 3 (OCT3) is expressed in placental syncytiotrophoblasts and is involved in diaplacental transfer of monoamines.
So far, little is known about its functional relevance in human and
whether its expression is inuenced by maturation or diseases. Therefore,
we studied the inuence of pre-eclampsia (PE), pregnancy induced
hypertension (PIH) and gestational age on the placental expression of
OCT3. Furthermore, we evaluated the function of OCT3 using a human
placenta perfusion model. Expression of OCT3 was determined by realtime RT-PCR and Western blot in normotensive term (n=40), preterm
(n=45) and early preterm placentas (n=40), and in placentas from women
with PE (n=15) and PIH (n=10). Transport function of OCT3 was evaluated by measuring permeability of MPP+ (50 lM) in materno-fetal vs.
feto-maternal transfer direction without (n=10) and in the presence of the
selective OCT3 inhibitor disprocynium 24 (D24, 10 lM, n=6). There
was an age-dependent increase of placental OCT3 mRNA expression
normalized to expression of TATA binding protein (early preterm
1.901.46 preterm 4.238.09, term 53.1723.92; p<0.05). In placentas
of patients with PIH, mRNA content (33.4023.7 vs. 53.1723.92) and
protein level (0.210.17 vs. 5.256.77) of OCT3 were signicantly lower
compared to term placentas (both p<0.05). The permeability of MPP+
was higher in materno-fetal compared to feto-maternal direction
(0.620.05 vs. 0.500.12; p=0.12) and was signicantly decreased in the
presence of D24 (0.420.15 vs. 0.620.05; p<0.05). Conclusions:
447
Placental OCT3 expression is inuenced by gestational age and pregnancy related diseases. In man, OCT3 is involved in diaplacental transfer
of cationic substances.
Paper No.: 2782

INCIDENCE, GENETIC AND NON-GENETIC RISK FACTORS
FOR SEVERE PHENPROCOUMON ASSOCIATED GASTROINTESTINAL BLEEDING LEADING TO HOSPITALISATION
Karen May(1), K Wergin(1), S Muller(2), M Hippius(3), K Farker(4),
B Drewelow(2), J Szymanski(5), D Rosskopf(6), PA Thurmann(5),
W Siegmund(1)
Regional Pharmacovigilance Center Greifswald, Greifswald, Germany
(2) University of Rostock, Department of Clinical Pharmacology, Regional Pharmacovigilance Center Rostock, Rostock, Germany
(3) University of Jena, Department of Clinical Pharmacology, Regional
Pharmacovigilance Center Jena, Jena, Germany
(4) University of Jena, Department of Clinical Pharmacology, Regional
Pharmacovigilance Center Weimar, Weimar, Germany
(5) University of Witten/Herdecke, Philipp Klee-Institute for Clinical
Pharmacology, HELIOS Clinic Wuppertal, Department of Clinical
Pharmacology, Wuppertal, Germany
Germany
Safety and dose variability of anticoagulation with phenprocoumon is at
least in part inuenced by polymorphisms of the metabolizing enzyme
CYP2C9 and the target vitamin K epoxide reductase complex 1
(VKORC1). There is limited information whether further pharmacogenetic risk factors are involved. Genetic polymorphisms in metabolism
(CYP2C9, CYP4F2), transport (OATP1B1, OATP2B1, OATP3B1) and
target enzymes (VKORC1, factor II and VII) of phenprocoumon were
assessed in 171 patients with severe gastrointestinal bleeding (GIB) evaluated by the German Regional Pharmacovigilance Centers (GRPC) in
comparison to patients without GIB (n=107, Study of Health in Pomerania; SHIP) and matched control subjects without phenprocoumon treatment (n=173, SHIP). The incidence of GIB was assessed using the
prescription data in the service areas of the GRPC Rostock and Greifswald. The incidence of severe GIB was 0.7% (90%-CI 0.16-1.16; 6806
exposed subjects; 45 patients with GIB). The incidence was not signicantly inuenced by age or gender. Patients with GIB received more
frequently NSAIDs (26.9% vs. 9.3%, p<0.05). The phenprocoumon dose
in patients without GIB was signicantly inuenced by sex (male
18.78.0 mg vs. female 14.35.6 mg, p<0.05), age (75years
18.67.6 mg vs. >75years 11.94.6 mg) and VKORC1 (AA,
10.62.8 mg; AB, 15.46.1 mg; BB 20.88.5 mg, p<0.05). In GIB, the
dose was only signicantly inuenced by age. Risk factors for GIB were
polymorphisms of OATP1B1 and factor VII. Conclusions: The risk for
phenprocoumon associated bleeding is inuenced by age, comedication
of NSAIDs and polymorphisms of OATP1B1 and factor VII.
Supported by: BMBF Fo 01ET0721, BfArM Fo 2.1-68502-201
Paper No.: 1971

FOCUSED CONFERENCE GROUP: P09 - INFLAMMATION
AND IMMUNOPHARMACOLOGY: NEW TOOLS FOR OLD
DISEASES
INHIBITOR OF APOPTOSIS PROTEINS (IAPS) AS NOVEL
TARGETS IN INFLAMMATORY PROCESSES OF
ENDOTHELIAL CELLS
Bettina Mayer(1), C Reichel(2), F Krombach(2), S Zahler(1),
A Vollmar(1), R Furst(1)
(2) University of Munich, Walter-Brendel-Centre of Experimental Medicine, Munich, Germany

Besides suppressing apoptosis by interaction with caspases, the inhibitor
of apoptosis protein (IAP) family (XIAP, cIAP1/2) is also known to play
a role in TNF receptor and NFjaB signalling. Despite these insights and
the crucial role of the TNF/NFjB system in inammation, no data exist
about the involvement of IAPs in inammatory processes. We used the
XIAP antagonist A-410099.1 (Abbott GmbH & Co. KG, Ludwigshafen,
Germany) to investigate its role in inammation-activated endothelial
cells. The inhibitor did not induce endothelial apoptosis but diminished
the adhesion of leukocytes to TNFa-activated endothelial cells by abrogating the expression of the endothelial cell adhesion molecule ICAM-1.
In an antigen-induced arthritis mouse model, the administration of A410099.1 strongly reduced knee joint swelling. Thus, we demonstrate for
the rst time an anti-inammatory potential of an XIAP antagonist in vitro and in vivo. Mechanistically, we found that A-410099.1 does not
inuence endothelial NFjB signalling, but decreased the TNFa-induced
activation of MAP kinases (TAK1, p38 and JNK), that are engaged in
inammatory processes. A-410099.1 seems to inuence MAPK signalling by induction of the proteasomal degradation of cIAP1 and cIAP2.
The latter are known to participate in the TNF receptor 1-associated signalling complex and we showed that their absence affects signalling molecules (e.g. TRAF2) upstream of the MAP kinases. In summary we
evidenced IAPs as regulators in inammatory processes and, moreover,
discovered an anti-inammatory potential of XIAP inhibitors. The future
challenge consists of in-depth analysis of the underlying signalling mechanisms.
Paper No.: 1983

THE PHARMACOLOGY OF GLUN3 CONTAINING NMDA
RECEPTORS
David McClymont, I Mello
University of Nottingham, School of Biology, Nottingham, UK
The NMDA receptor requires both glutamate and glycine for activation,
is highly permeable to calcium and requires depolarisation to remove
block by magnesium. A functional NMDA receptor contains four subunits from three subtypes, GluN1 consists of eight splice variants, GluN2
has four subtypes NR2A to NR2D and GluN3 has GluN3A and
GluN3B. cRNA coding for GluN1-1a and GluN2A were injected
together into Xenopus oocytes alone and then with GluN3A or GluN3B
subunits after which responses were recorded using two electrode voltage
clamp. Receptors were characterised using the blockers magnesium, memantine, MK-801 and the polyamines philanthotoxin-343 (PhTX-343),
philanthotoxin-12 (PhTX-12) and methoctramine. IC50 values were calculated at GluN1-1a/2A, GluN1-1a/2A/3A and GluN1-1a/2A/3B receptors. These showed signicant increases in IC50 between GluN1-1a/2A
and the GluN3 containing receptors for magnesium (IC50 at GluN1-1/2A
1.7 lM, GluN3A 5.828 lM and GluN3B 15 lM at -100 mV), memantine, PhTX-343 and MK-801, with no differences for PhTX-12 or methoctramine. The fraction of the electric eld that the blocker experiences
(d) was calculated using the Woodhull equation and showed a signicant
decrease in d between GluN1-1a/2A and the GluN3 receptors for magnesium, memantine and PhTX-343, with no change for methoctramine,
PhTX-12 or MK-801. These data show that GluN3 impairs binding of
channel blockers and that this may be due to changes in exposed amino
acids in the channel pore region.
(1) University of Munich, Center for System-Based Drug Research,

Department of Pharmacy, Munich, Germany
448
Paper No.: 1390
SYNERGY BETWEEN A-1-ADRENOCEPTOR SUBTYPES AND
P2X RECEPTORS IN NERVE INDUCED VASCULAR
RESPONSES REVEALED BY SUCCESSIVE
A-1-ADRENOCEPTOR KNOCKOUTS AND ANTAGONISTS
John McGrath(1), C Daly(1), P Simpson(2), E Wallace(1), L Methven(1)
(1) University of Glasgow, Department of Integrative & Systems Biology, Glasgow, Scotland, UK
(2) University of California, San Francisco, USA
All 3 a-1-adrenoceptor (AR) subtypes could contribute to vasoconstrictor
responses to sympathetic nerve stimulation of resistance arteries and
therefore contribute to autonomic regulation of blood pressure. However,
analysis is beset by poor selectivity of antagonist drugs and the sheer
complexity of dissecting out the individual contributions from these
receptors plus other adrenoceptors and receptors to potential co-transmitters. Here, we employ double and triple knockouts of the a-1-AR to analyse responses to electrical eld stimulation of the wire-myographmounted mouse small mesenteric arteries. The results show that a-1AAR and a-1D-AR act synergistically, particularly at low frequencies, so
that antagonists of either receptor produced a powerful blockade. After
elimination of all 3 a-1-AR there remained a P2X-mediated response
blocked by a, b, methylene ATP; antagonist analysis of the double
knockouts revealed synergism between P2X and a-1A-AR but not between
P2X and a-1D-AR. This reveals a pattern of powerful synergism between
purinergic and adrenergic responses with a degree of adrenoceptor selectivity, pointing to signalling interactions.
Supported by British Heart Foundation (PG/05/140/20094 and FS/04/
035).
Paper No.: 2047

HEALTH AND DISEASE
EFFECTS OF CYCLOOXYGENASE INHIBITORS ON
PROTEASE-ACTIVATED RECEPTOR 2 ACTIVATING
PEPTIDE-INDUCED RELAXATIONS OF AORTA IN CHRONIC
ANGIOTENSIN II TREATED MICE
John J McGuire, KH Hughes
Memorial University Faculty of Medicine, Cardiovascular Research
Group, Division of BioMedical Sciences, St Johns, NL, Canada
Protease-activated receptor 2 (PAR-2) activating peptides cause vasodilation of arteries. In non-obese diabetic mice having endothelial dysfunction, PAR-2 mediated relaxations of their aortas were reported to involve
cyclooxygenase-2 and endothelial nitric oxide synthase. PAR-2 mediated
vasodilation of resistance arteries has been found to be resistant to endothelium dysfunction in rodent models of genetic hypertension. We
hypothesized that PAR-2 relaxations of aortas from hypertensive mice
with endothelial dysfunction, as characterized by reduced endothelial
nitric oxide synthase activity, would involve activation of cyclooxygenases. In this study we compared PAR-2 mediated vasodilation of aortas
from angiotensin II-treated mice (s.c. infusion 1 lg/kg/min, 2 weeks) to
saline-treated mice. Isometric tension measurements were recorded from
isolated rings of thoracic aortas while contracted submaximally by addition of thromboxane receptor agonist (U44619) and then exposed to
cumulative concentrations of PAR-2 activating peptide (2-furoyl-leu-ilegly-arg-leu-orn-amide, 2y 1 nM - 3 lM). 2y-induced relaxations of
aortas from angiotensin II-infused mice were 50% less than relaxations
from saline-infused mice. Nitric oxide synthase inhibitor (100 lM LNAME) blocked completely the residual relaxations of angiotensin IItreated mice. Interestingly, either cyclooxygenase inhibitor NS398
(3 lM) or SC560 (1 lM) increased 2y-mediated relaxations of aortas
from angiotensin II-infused mice, but not the relaxations of aortas from
saline-infused mice. We conclude that in the angiotensin II infusion of
mice leads to induction of cyclooxygenases, which opposes the vasodilation of aortas by PAR-2 activation of endothelial nitric oxide synthase. In
this model of endothelial dysfunction PAR-2 activation does not lead to
cyclooxygenase-mediated vasodilation of aortas.
Paper No.: 1518

REGULATION OF NORADRENALINE RELEASE BY
ADMINISTRATION OF STRESSIN I INTO THE LOCUS
COERULEUS: A BRAIN MICRODIALYSIS STUDY IN AWAKE
RAT
J Javier Meana, I Horrillo, JE Ortega
University of Basque Country, Department of Pharmacology, Leioa, Bizkaia, Spain and Centro de Investigacion Biomedica en Red de Salud
Mental (CIBERSAM), Leioa, Spain
The noradrenergic system has been implicated in behavioural and cognitiveaspects of stress response. Corticotropin releasing factor (CRF) plays
animportant role in the activation of locus coeruleus (LC) noradrenergic
neuronsin response to stress. The aim of this study was to evaluate the
effect of localadministration into the LC of the CRF1-receptor (CRF1R)
agonist stressin I onnoradrenaline (NA) release in LC and prefrontal cortex (PFC). The antagonistantalarmin was used to characterize the CRF
receptor subtype involved in thestressing I effect. For this purpose, extracellular NA concentrations weremeasured simultaneously in LC and PFC
by dual-probe brain microdialysis infreely awake rats. Increasing concentrations of stressin I were administered(0.01-10 lM) every 70 min by
reverse dialysis into the LC. Antalarmin wasadministered intraperitoneally before basal samples collection. Basal values ofNA were 3.741.02
nM in LC and 4.621.01 nM in PFC and did not shown differencesbetween groups. Stressin I increased (p<0.05) extracellular NA in LC(Emax=13439%)
(F[1,292]=41.16,p<0.0001;n=30)
and
PFC
(Emax=9245%)(F[1,204]=9.41,p=0.0025;n=24) when compared to control group (n=16).Concomitant administration of antalarmin and stressin
I did not induce anychange in NA nor in LC nor in PFC. The present
results demonstrate that localadministration of the CRF1R agonist stressin I into the LC area increase in vivoNA release in somatodendritic and
terminal noradrenergic areas. This effectoccurs through CRF1R because
it is blocked by the CRF1R antagonist antalarmin.Supported by SAF 04/
02784 and SAF 09/08460, the Basque Government (IT199-07) andThe
Saiotek Program.
Paper No.: 1144

HEALTH AND DISEASE
CALCITONIN GENE-RELATED PEPTIDE TERMINATES
ENDOTHELIN-1-INDUCED VASOCONSTRICTION AND
VASOPRESSOR RESPONSES
Merlijn JPMT Meens, NJA Mattheij, H van Essen, MG Compeer, BJA
Janssen, JGR De Mey
Maastricht University, Department of Pharmacology and Toxicology,
Introduction: Endothelin-1 (ET-1), an irreversible agonist, causes longlasting vasopressor effects. In rat mesenteric resistance arteries (MRA),
most vasodilators and ETA-antagonists transiently reduce contractile
effects initiated by ET-1, but calcitonin gene-related peptide (CGRP) terminates these effects (Hypertension, 2009; 54:1186). Here, we report
similar ndings in i) another species, ii) other vascular beds and iii) in
vivo. Methods: Mouse MRA and rat spermatic, epigastric, splenic, renal
449
and saphenous arteries were studied using wire-myography. Anesthetized
rats recieved ET-1 (1000 ng/kg i.v.) followed after 8 min. by i) nothing,
ii) sodium nitroprusside (SNP) (30 lg/kg i.v.) or iii) CGRP (3 lg/kg
i.v.). Results: In mouse MRA, ET-1 (16 nM) caused persistent contractions which were terminated by capsaicin (1 lM) and CGRP (0.1 lM),
but not by ACh (10 lM) or BQ123 (1 lM). In rat arteries, ET-1 (32
nM) induced contractions which were not reversed by exposure to
BQ123 (1 lM). Also, BQ123 did not terminate long-term ET-1 effects.
In contrast, CGRP (0.1 lM) irreversibly reduced persistent ET-1-initiated contractions. In anesthetized rats, the long-lasting ET-1-induced
pressor responses were transiently reversed by SNP but were terminated
by CGRP. Conclusions: While arterial vasoconstrictor responses to ET-1
are resistant to ETA-antagonism and endothelium derived vasodilators
such as NO, they are terminated by CGRP in several vascular beds and
in vivo. This study was performed within the framework of the Top Institute Pharma project: T2-108-1; Metalloproteases and Novel Targets in
Endothelial Dysfunction.
Paper No.: 517

THE NEUROPROTECTIVE EFFECTS OF A PURIFIED
COMPOUND EXTRACTED FROM CURCUMA LONGA
AGAINST 6-OHDA -INDUCED DOPAMINERGIC
NEUROTOXICITY IN SH-SY5Y CELLS
Benjawan Meesarapee, A Thampithak, Y Jaisin, Y Sanvarinda
Mahidol University, Department of Pharmacology, Bangkok, Thailand
Parkinson disease (PD) is a neurodegenerative disorder characterized by
a loss of the dopaminergic neurons of the substantia nigra pars compacta
(SNpc). Free radicals induced oxidative stress is a major cause of cell
death in PD. 6-Hydroxydopamine (6-OHDA), a neurotoxin to dopaminergic neuron, is usually applied to induce experimental Parkisonism.
The neurotoxic effect of 6-OHDA is known to result from the generation
of free radicals and subsequent formation of dopamine quinones (DAQ).
Curcumin I is a puried natural compound extracted from turmeric
(Curcuma longa) which is an indigenous plant of Thailand. Curcumin
has been shown to possess an anti-oxidant, anti-inammation, and anticarcinogenic activities. In this study, we investigated the effects of diferuloylmethane or curcumin I on the dopaminergic neurotoxicity in
6-OHDA-treated SH-SY5Y cells. It was found that pretreatment of
SH-SY5Y, dopaminergic neuronal cell line, with curcumin I, at concentrations of 1, 5, 10 and 20 lM signicantly decreased quinoprotein and
reactive oxygen species (ROS) productions in a dose-dependent manner.
Moreover, curcumin I treatment could also signicantly increase tyrosine
hydroxylase (TH) expression in a dose-dependent manner. These results
suggest that curcumin I possess neuroprotective effects and may have a
therapeutic potential for the treatment of Parkinson disease.
Keywords: Curcuma longa; quinoprotein; dopamine quinone; ROS; TH
Paper No.: 1577

GRK6 REGULATES THE DESENSITIZATION OF 5-HT4
RECEPTORS IN THE RAT OESOPHAGUS
Alzheimers disease, pain, anxiety, stress, depression, urinary incontinence and functional gastro-intestinal disorders including irritable bowel
syndrome. An homologous type of desensitization which involves G-protein coupled receptor kinases (GRKs) has been reported for 5-HT4 receptors in mouse colliculi neurons, rat oesophagus and colon upon
activation by agonist (Ansanay et al., Mol Pharmacol 1992,42:808-16;
Grider JR, Am J Physiol Gastrointest Liver Physiol 2006,290:319-27;
Ronde et al., J Pharmacol Exp Ther 1995,272:977-83). However, the
specic G-protein coupled receptor kinase that mediates the desensitization of 5-HT4 receptors in the in vivo system is unknown. We investigated the in situ expression and interaction of 5-HT4 receptors and the
G-protein coupled receptor kinases in the tunica muscularis mucosae of
the rat oesophagus using immunohistochemistry and coimmunoprecipitation. 5-HT4 receptor immunoreactivity was detected in the tunica muscularis mucosa of the oesophagus, along with GRK3 and GRK6 while
there were no cells immunoreactive for GRK2 and GRK5. Stimulation
of tunica muscularis mucosae of the oesophagus using the 5-HT4 receptor agonist, tegaserod, followed by western blot analysis of the 5-HT4
receptor antibody immunoprecipitate revealed the coimmunoprecipitation
of GRK6 with 5-HT4 receptors. There was no coimmunoprecipitation of
GRK2, GRK3 and GRK5 with 5-HT4 receptor immunoprecipitate. This
study shows that GRK6 is the G-protein coupled receptor kinase responsible for the regulation of 5-HT4 receptors desensitization in the rat
oesophagus.
Paper No.: 819

THE ATTITUDE OF MEDICAL STUDENTS TOWARDS
TRADITIONAL MEDICINE
Ortrun Meissner-Kirch, EN Kwizera
Walter Sisulu University, Department of Pharmacology, Mthatha, South
Africa
Traditional medicine is still widely practiced in South Africa. Herbal
medication is the most commonly used therapeutic method. Herbal remedies are accessible and affordable. Consumers regard them as natural and
therefore safe. However, they may have serious adverse effects and interactions with conventional drugs. In addition, traditional medicine uses a
holistic approach that deals with body, mind and spirit. Medical doctors
are trained in scientic medicine and are generally unable to advise their
patients with regard to alternative health care choices actually practiced
by their clients. The present study was undertaken to assess the knowledge, skills and attitudes of medical students towards traditional medicine. Data were gathered via questionnaires to rst, third and fth year
medical students at Walter Sisulu University between April and August
2008. The results indicated that the majority of students do not regard
traditional medicine as useless or mumbo-jumbo. Traditional healers are
seen as highly knowledgeable in plants and rituals. The majority of participants do not wish to receive training in traditional medicine or have
the subject included in the medical curriculum. Most students require scientic evidence as proof of efcacy. There is a trend to accept cooperation with traditional practitioners. The issue of knowledge and attitude
by scientic medicine towards traditional medicine needs further consideration. In the meantime, medical students should be instilled with
respect for the subject as part of African culture. Students should also be
encouraged to keep an open mind in their approach towards traditional
medicine and its remedies.
Teshome Nedi Megersa, H Irving, I Coupar, P White

Monash University, Monash Institute of Pharmaceutical Sciences,
Medicinal Chemistry and Drug Action, Parkville, VIC, Australia
5-HT4 receptors mediate many cellular functions, both in the central
nervous system and at the periphery. They are targets for the treatment of
450
Paper No.: 2845
DISEASES
ADMINISTRATION OF EDOXABAN, AN ORAL FACTOR XA
INHIBITOR, IS SAFE IN HEALTHY SUBJECTS POST
WARFARIN TREATMENT- ENCORE ABSTRACT
Jeanne Mendell(1), R Noveck(2), I Rubets(3), M Shi(1)
(1) Daiichi Sankyo Pharma Development, Edison, NJ, USA
(2) Duke University Medical Center, Durham, NC, USA
(3) MDS Pharma Services, USA
Introduction: Edoxaban (the free base of DU-176b) is an oral direct factor Xa inhibitor in development for thromboprophylaxis. This study evaluated the safety and pharmacokinetics (PK) of edoxaban in healthy
subjects bridging from warfarin therapy. Methods/Patients: Subjects
received open-label warfarin titrated until INR was 2.0-3.0 for 3 days.
Post warfarin discontinuation (24 hours), subjects were randomized to
receive edoxaban 60 mg QD or placebo for 5 days. Primary endpoint
was INR, prothrombin time [PT], activated partial thromboplastin time
[aPTT], and adverse events. Secondary endpoints included PK and other
pharmacodynamic parameters. Results: Sixty-three of 72 subjects
achieved a stable INR of 2.0-3.0 within 16 days of warfarin treatment.
Mean (SD) INR values at 24 hours post warfarin dose were similar for edoxaban and placebo: 2.31 (0.193) and 2.30 (0.199), respectively. INR
increased transiently in edoxaban-treated subjects to a maximum (mean)
of 3.84 at 2 hours after dosing, but returned to <3.0 by 8 hours and
remained <3.0 for the next 5 days (pre-dose INR values). INR values
were not signicantly different between edoxaban and placebo at
24 hours. Similar trends were observed for PT and aPTT. Linear correlation (correlation coefcient >0.8) was observed between plasma edoxaban concentrations and both PT and INR. Edoxaban was well tolerated;
positive fecal occult blood, the only bleeding event, occurred in 6.9%,
7.0%, and 4.8% of subjects treated with warfarin only, edoxaban, and
placebo, respectively. Conclusions: Edoxaban may be safely administered to healthy subjects 24 hours after the last dose of warfarin (INR 2.5).
Paper No.: 2846

EFFECT OF LOW-DOSE AND HIGH-DOSE ASPIRIN ON THE
EDOXABAN
Jeanne Mendell(1), S Chen(1), R Noveck(2), F Lee(3), V Petrushun(3),
M Shi(1)
Introduction: Edoxaban (the free base of DU-176b) is an oral direct factor Xa inhibitor under development for stroke prevention and treatment
of thromboembolic events. Aspirin (ASA) is widely used in cardiovascular prophylaxis and as an anti-inammatory/analgesic. This investigation
evaluated the inuence of aspirin on bleeding times, pharmacokinetics
and pharmacodynamics of edoxaban. Methods/Patients: Healthy subjects
aged 18-45 years were randomized to edoxaban, ASA 100 mg QD
(ASA100mg) (n=36) or 325 mg QD (ASA325mg) (n=56 as 2 cohorts),
+edoxaban or ASA alone for 5 days in 2 open-label crossover studies.
Bleeding times were measured on Day -1 and Hour 4 post-dose on Day
5. Edoxaban pharmacokinetics and pharmacodynamics were measured
serially on Day 1 and 5. Results: Mean baseline-corrected bleeding time
ratios were 1.79 for ASA100mg+edoxaban and 1.63 and 1.74 (per
cohort) for ASA325mg+edoxaban compared with values of 1.12 to 1.34
for edoxaban or ASA alone. Edoxaban pharmacokinetics for edox-
aban+ASA100mg were within bioequivalence limits on both Days 1 and

5 while geometric mean ratios (90% CI) for edoxaban+ASA325mg were
129.9 (122.6, 137.7) and 134.6 (123.8, 146.3) for AUC and Cmax,
respectively. Neither ASA treatment affected the edoxaban-induced
increases in PT, aPTT or anti-FXa levels with highest mean peak values
observed for any of the cohorts as 17.38 sec, 51.06 sec and 3.803 IU/
mL, respectively. Conclusion: Concomitant administration of edoxaban
and ASA increased bleeding time compared with either drug alone. The
dose of ASA in phase III trials of edoxaban will be limited to 100 mg.
Paper No.: 2847

EFFECT OF NAPROXEN ON THE PHARMACOKINETICS AND
PHARMACODYNAMICS OF EDOXABAN- ENCORE
ABSTRACT
Jeanne Mendell(1), S Chen(1), R Noveck(2), F Lee(3), V Petrushun(3),
M Shi(1)
Introduction: Edoxaban (the free base of DU-176b) is an oral factor Xa
inhibitor under development for treatment and prevention of thromboembolic events. Preclinical studies indicate that edoxaban is a substrate for
P glycoprotein (p gp) and CYP3A4/5. Quinidine and verapamil, used in
AF, inhibit p-gp and CYP3A4/5. These studies evaluated effects of concomitant administration of quinidine or verapamil on edoxaban pharmacokinetics and pharmacodynamics. Methods/Patients: Healthy subjects
aged 18-45 years received edoxaban 60 mg QD for 4 days with single
doses of quinidine 300 mg or verapamil SR 240 mg on Day 3, or a single 60-mg dose of edoxaban on Day 3 during daily quinidine or verapamil dosing for 4 days in 2 open-label, randomized, crossover phase I
studies (N=42 and 34, respectively). Edoxaban pharmacokinetics and
pharmacodynamics (PT, INR, aPTT) were compared for concomitant
dosing versus edoxaban alone. Results: Concomitant administration of
quinidine increased edoxaban Cmax and AUC024 by 85.4% and 76.7%,
respectively; concomitant verapamil increased these parameters by
53.3% and 52.7%, respectively. Edoxaban did not signicantly affect
plasma quinidine, verapamil, or norverapamil concentrations. Peak
increases from baseline in PT, INR, and aPTT with edoxaban were augmented by 73%, 70%, and 46%, respectively, by quinidine, and 41%,
44%, and 29%, respectively, by verapamil, consistent with increased edoxaban exposure. Administration of quinidine or verapamil with edoxaban appeared safe and well tolerated. Conclusion: Edoxaban exposure
and pharmacodynamic effects are increased by concomitant administration of quinidine or verapamil. The dose of edoxaban should be carefully
considered when coadministered with these drugs.
Paper No.: 2848

THE EFFECT OF QUINIDINE AND VERAPAMIL, P
GLYCOPROTEIN/CYP3A4/5 INHIBITORS, ON EDOXABAN
PHARMACOKINETICS AND PHARMACODYNAMICS
Jeanne Mendell(1), R Noveck(2), H Zahir(1), F Lee(3), V Petrushun(3),
I Rubets(3), G Zhang(1), M Shi(1), S Chen(1)
Introduction: Edoxaban (the free base of DU-176b) is an oral factor Xa
inhibitor under development for treatment and prevention of thromboembolic events. Preclinical studies indicate that edoxaban is a substrate for
451
P glycoprotein (p gp) and CYP3A4/5. Quinidine and verapamil, used in
AF, inhibit p-gp and CYP3A4/5. These studies evaluated effects of concomitant administration of quinidine or verapamil on edoxaban pharmacokinetics and pharmacodynamics. Methods/Patients: Healthy subjects
aged 18-45 years received edoxaban 60 mg QD for 4 days with single
doses of quinidine 300 mg or verapamil SR 240 mg on Day 3, or a single 60-mg dose of edoxaban on Day 3 during daily quinidine or verapamil dosing for 4 days in 2 open-label, randomized, crossover phase I
studies (N=42 and 34, respectively). Edoxaban pharmacokinetics and
pharmacodynamics (PT, INR, aPTT) were compared for concomitant
dosing versus edoxaban alone. Results: Concomitant administration of
quinidine increased edoxaban Cmax and AUC0 24 by 85.4% and
76.7%, respectively; concomitant verapamil increased these parameters
by 53.3% and 52.7%, respectively. Edoxaban did not signicantly affect
plasma quinidine, verapamil, or norverapamil concentrations. Peak
increases from baseline in PT, INR, and aPTT with edoxaban were augmented by 73%, 70%, and 46%, respectively, by quinidine, and 41%,
44%, and 29%, respectively, by verapamil, consistent with increased edoxaban exposure. Administration of quinidine or verapamil with edoxaban appeared safe and well tolerated. Conclusion: Edoxaban exposure
and pharmacodynamic effects are increased by concomitant administration of quinidine or verapamil. The dose of edoxaban should be carefully
considered when coadministered with these drugs.
Paper No.: 607

A COLLABORATIVE CAPACITY BUILDING PROJECT FOR
DEVELOPING HUMAN RESOURCES IN THE AREA OF CLINICAL
TRIALS IN PSYCHIATRY: A CUBAN CANADIAN EXPERIENCE
Yudexi Mendoza(1), M Fors(1), MA Pascual(1), B Horner(2), C Cash(2),
S Kutcher(2), R Valle(1), Y Cachimaille(1)
(1) National Coordinating Centre for Clinical Trials (CENCEC), Havana,
Cuba
(2) Dalhousie University, Halifax, NS, Canada
The National Center of Clinical trials of Cuba (CENCEC) worked with
Dalhousie University, Canada for 5 years in a project for capacity building of human resources for conducting clinical trials with psychopharmacological drugs. We developed a training program for clinical researchers
and other staff involved in the design and conduct of clinical trials in this
area that included topics about diagnostic and management of depression,
anxiety disorders and schizophrenia, ethics in psychiatry research, methods and tools to design clinical investigations including GCP and other
international regulations. 86 professionals were trained from 16 different
sites around the country. Main results were related with increasing the
capacity of CENCEC and its network to deliver training in Cuba and in
other countries, improvement of mental health care delivery by the
improvement of diagnosis and management of depression and the way to
implement effective strategies to bridge the gap between research and
medical care. Besides, people from both parts gained knowledge in English and Spanish, respectively, strengthening the cooperation between
Cuban and Canadian professionals. So now Cuba count with a network of
trained health care professionals that had been developed for future trials.
Paper No.: 608

PLACEBO RESPONSE IN DEPRESSION. RESULTS FROM
TWO CLINICAL TRIALS IN CUBA
Yudexi Mendoza, R Valle, MA Pascual
criteria exposed by the regulatory agencies from Europe and United

States. The possible reasons that could have inuenced the high placebo response (58% intention to treat analysis) observed in a clinical
trial in depression coordinated by the National Coordinating Centre
for Clinical Trials in Cuba (CENCEC) were also analyzed. Among
then: spontaneous remission of symptoms, severity of depression,
high expectancy from patients and raters, systematic evaluations,
among others. We also present the different results obtained in a
similar academic trial conducted after a training developed in the
context of a collaborative research project Psycho-pharmacological
Training and Research Capacity in Cuba with Dalhousie University,
Canada. As part of it, clinical investigators were trained in different
topics including strategies to diminish placebo response that is something that can reduce the power of clinical trials and might invalidate
them. In this academic trial we had a signicant reduction of placebo
response (31.6% intention to treat analysis) compare with the previous trial, reinforcing the impact of the delivery training program that
permitted to have better trained staff in the diagnosis, selection of
patients, scales application and response evaluation.
Paper No.: 647

SAFETY AND EFFICACY OF GRANULOCYTE
COLONY-STIMULATING FACTOR (IOR-LEUKOCIM) IN
ONCOHEMATOLOGY PATIENTS. PHASE IV
IC Mendoza Hernandez(1), EG Lopez(1), MW de Leon(2), AM Veitia(3),
JDF Aguila(4), EV Olazabal(5), MR Garcia(1), PP Sierra(6),
GS Martinez(6), Yinet Barrese Perez(1)
(1) National Coordinating Center of Clinical Trials, Havana, Cuba
(2) Medical Surgery Research Center, Havana, Cuba
(3) Hematology and Immunology Institute, Havana, Cuba
(4) Gustavo Aldereguia Lima Hospital, Cienfuegos, Cuba
(5) National Oncology and Radiology Institute, Havana, Cuba
(6) Center of Molecular Immunology, Havana, Cuba
Introduction: Neutropenia does not in itself cause symptoms, but it
predisposes patients to infection, especially if the neutrophil count
falls below 0.5 x 109/L and persists longer than 10 to 14 days. In
patients receiving chemotherapy, the administration of G-CSF can lessen the incidence and severity of neutropenia. Purpose: This is a
multicenter phase IV study in oncohematology patients under chemotherapy and/or radiotherapy treatment evaluating ior LeukoCIM, a
Cuban granulocyte colony-stimulating factor. Patients and Methods:
Nine hundred three episodes received ior LeukoCIM (300 micrograms/kg) as primary, secondary prophylaxis or neutropenia treatment,
to recovery the neutrophil count (1.5 x 109/L) and were assessed for
safety and efcacy. Ior LeukoCIM was administered subcutaneous, in
prophylaxis beginning 24-72 hours after chemotherapy and/or radiotherapy, and as treatment from neutropenia diagnosis. Results: The
48.2 % of adverse events were evaluated as drug-related adverse
events. The most frequently were leucocytosis (14.9 %; 161 episodes), neutrophilia (13.1 %; 142 episodes) and bone pain (10.5 %;
114 episodes). Others adverse events were fever (7.4 %), thrombocytopenia (7.0 %) and headache (5.2 %). Ior LeukoCIM was safe with
no serious adverse events. The 94.6 % (819 episodes) nished treatment with neutrophil count l1.5 x 109/L. Conclusions: Ior LeukoCIM showed safety and efcacy as primary, secondary prophylaxis
or neutropenia treatment in oncohematology patients under chemotherapy and/or radiotherapy treatment.
National Coordinating Centre for Clinical Trials (CENCEC), Havana, Cuba

Some aspects related with depression and the use of placebo in clinical trials, were reviewed considering the Declaration of Helsinki and
452
Paper No.: 467
DISEASES
A NOVEL MECHANISM FOR NEUTROPHIL TRAPPING IN
ENDOTOXEMIC HEPATICMICROVASCULATURE: DOWN
REGULATION OF B-2 INTEGRIN VIA IL-10
Gustavo Menezes(1), W-Y Lee(2), H Zhou(2), C Waterhouse(2),
D Cara(1), P Kubes(1)
(1) ICB/Universidade Federal de Minas Gerais, Department of
Biochemistry, BeloHorizonte, Minas Gerais, Brazil
(2) Snyder Institute - III / University of Calgary, Canada
Hepatic neutrophil adhesion during endotoxemia is an integrin-independent, CD44-dependent process. Since integrins function in other endotoxemic vasculatures, we used spinning disk confocal intravital
microscopy to assess whether LPS down-modulated integrin function in
sinusoids. First, we applied fMLP onto the liver surface, and compared it
to systemic LPS administration. Local fMLP caused neutrophil adhesion,
crawling and emigration for at least 2 hours. Surprisingly, the number of
adherent and crawling neutrophils was markedly reduced in Mac-1-/- and
ICAM-1-/- mice, but not in mice treated with anti-CD44 mAb. By contrast, systemic LPS injection induced a robust accumulation of neutrophils in sinusoids, which was dependent on CD44 but not on integrins.
Strikingly, local fMLP could not induce any integrin dependent adhesion
in endotoxemic mice treated with anti-CD44 mAb, indicating that Mac1-dependent neutrophil adhesion was inhibited by LPS. This response
was localized to the hepatic microvasculature as neutrophils still adhered
via integrins in brain microvasculature. ICAM-1/ICAM-2 levels were not
decreased, but following LPS treatment, Mac-1 was down-regulated in
neutrophils localized to liver but not in the circulation. Mac-1 down-regulation in neutrophils was not observed in IL-10-/- mice. In vitro neutrophil incubation with IL-10 induced direct decrease of Mac-1 expression
and adhesivity in LPS-stimulated neutrophils. Therefore, our data suggest
that Mac-1 is necessary for neutrophil adhesion and crawling during
local inammatory stimuli in sinusoids, but during systemic inammation, neutrophils are exposed to high concentrations of IL-10 leading to a
CD44-dependent, integrin-independent adhesion. This may be a mechanism to keep neutrophils in sinusoids for intravascular trapping.
Paper No.: 1620

FIVE KINDS OF ALKALOIDS FROM RHIZOMA COPTIS
IMPROVE GLUCOSE UPTAKE OF 3T3-L1 CELLS WITHOUT
ADIPOGENESIS ACTIVITY
Xianli Meng, J Li, X Fan, Y Zhang
Chengdu University of TCM, Department of Pharmaceutical Sciences,
China
Insulin resistance is the key link of metabolic syndrome, and Rhizoma
Coptidis has denite clinical effect on improving insulin resistance. We
separated ve alkaloids from Rhizoma Coptis (berberine, palmatine, coptisine, jatrorrhizineand epiberberine), and observed the effect of ve alkaloids on 3T3-L1 cell proliferation and induce differentiation and insulin
resistance, then explored the mechanism. The results showed that these
ve alkaloids had obviously promoted cell proliferation, inhibited differentiation, decreased the consumption of glucose in culture medium,
improved insulin resistance. These effects are similar to rosiglitazone
maleate, its possible mechanism might be attributable to high expression
of PPARamRNA. The coptisine showed the most signicant activity on
regulating lipid metabolism and improving insulin resistance at the concentration of 16.5 .moloL-1. Moreover, the activity was obviously
beyond berberine which has been studied more in the world at present.
Based on these results, Rhizoma Coptidis can obviously improve insulin

resistance, the effect attributed to the synergism of each compontent; and
suggested that coptisine has potential advantage on the new drugs development of improving insulin resistance, which is worth for further study.
Paper No.: 1621

SIMULTANEOUS DETERMINATION OF GALLIC ACID AND
PROTOCATECHUIC ACID IN RABBIT SERUM BY HPLC
AFTER ORAL ADMINISTRATION OF
FRUCTUS CHOEROSPONDIATIS EXTRACT
Xianli Meng, P Wang, Y Zhang, H Liu
China
Fructus choerospondiatis is the mature fruit of Choerospondias axillaries
(Rox.) Burtt et Hill. It is included in Chinese Pharmacopoeia. It has been
widely used as antimyocardial ischemic agent in Mongolia medicine and
Tibet medicine. Gallic acid (GA) and Protocatechuic acid (PC) are the
major constituent of Fructus choerospondiatis. GC and PC have signicant antioxidant, antithrombotic and antianoxic effect in vivo. A sensitive, simple, and accurate method for simultaneous determination of GA
and PC in rabbit serum was developed using RP-HPLC with UV detection. Sample preparations were carried out by protein precipitation with
methanol, followed by the evaporation of the methanol to dryness. The
resultant residue was then reconstituted in methanol and injected onto a
Diamond C18 (2504.6 mm I.D. 5lm) analytical column. The mobile
phase consisted of acetonitrile0.0025mol.L-1 hexadecyltrimethylam
monium bromideN, N-dimethylformamide phosphoric acid
((9:81:7.5:0.1, v/v/v/v). The calibration curves of GC and PC in serum
were linear from 18.25-204.40 and 12.75-142.80 ng.ml-1 at least, respectively (r=0.9992 and 0.9960, correspondingly). The detection limits of
the method were 18.25 ng.ml-1 for GC and 12.75 ng.ml-1 for PC. Mean
recoveries of method were 76.28% for GC and 68.20% for PC. Interand intra-day precisions were 4.03% RSD. The HPLC method developed has been applied to determine the pharmacokinetics of GC and PC
in rabbit serum after having taken Fructus Choerospondiatis extract.
Keywords: Gallic acid; Protocatechuic acid; Fructus Choerospondiatis;
pharmacokinetics
Paper No.: 2786

DISEASES
AGONIST-MEDIATED CHANGES IN INTRACELLULAR
CALCIUM IN HUMAN BRONCHIAL EPITHELIAL CELLS
P Menon, K Varanou, Ian McFadzean
Kings College London, Department of Pharmaceutical Sciences,
London, UK
There is signicant interest in the utilisation of stem cells to regenerate
airway epithelium damaged during inammatory airway disease (Sueblinvong & Weiss (Curr. Op. Pharmacol., 2009, 9:268-273). Elsewhere
in our laboratory experiments are directed towards developing protocols
for the transformation of human embryonic stem cells into a human
bronchial epithelial cell (HBEC) phenotype. This will require prior characterisation of HBECs using cell-specic markers and functional
responses. Here we report the results of a series of experiments characterising intracellular calcium responses as measured using single-cell,
Fura-2 microuorimetry - in HBECs following extracellular application
of ATP. HBECs (Clonetics; cell line 2F1688) were thawed from frozen
aliquots, seeded onto collagen IVcoated glass cover slips and cultured
453
in bronchial epithelial growth medium (Lonza) at 37 C). Cells were
incubated with Fura-2-AM ester (5lM; 40min at 37 C) in DMEM.
Changes in intracellular calcium ([Ca2+]i) were measured in cells bathed
in a physiological salt solution containing 1.3mM Ca2+. Cell responses
to agonists are expressed as a percentage (mean SEM) of the change in
F340/F380 ratio produced by ionomycin (10lM). 89% of cells tested
responded to ATP with a concentration-dependent increase in [Ca2+]i
(48.20.3 (n=51) and 61.60.5 (n=35) for 0.1 and 1mM ATP respectively, whilst 72% of cells tested responded with an increase in ([Ca2+]i
to ADP (1mM; 540.2% (n=57)). No cells tested (n=20) responded to ab-methylene ATP (0.1mM). These results suggest that Fura-2 responses
to ATP offer a straightforward functional response that can be used in
characterisation of an HBEC phenotype
Paper No.: 550

DISEASES
THE ENHANCEMENT OF PENTYLENETETRAZOLEINDUCED SEIZURE THRESHOLD IN MICE VIA
CYCLOOXYGENASE-2 INHIBITION BY NIMESULIDE
Azam Mesdaghinia(1,2), A Heydari(1,2), P Khazaee(1,2),
H Yazdanpanah(1,2)
(1) Kashan University of Medical Sciences, Department of Physiology
and Pharmacology, Kashan, Iran
(2) Kashan University of Medical Sciences, Physiology Research Center,
Kashan, Ira.
Considering the consequence of epilepsy and lack of accurate information, different surveys must be conducted for investigating its probable
basic mechanisms. Cyclooxygenase-2 (COX-2) is the isoenzyme induced
at Injury and expressed constitutively in some tissues such as CNS and
may play a role in the pathogenesis of epilepsy. This study was done to
explicate the effect of COX-2-inhibitors on seizure threshold in mice.
Animals were randomly divided to nine groups including two control
and seven experiment groups received nimesulide (2.5, 5 and 10 mg/kg),
diazepam (0.1, 0.5 and 5 mg/kg) and combination of subprotective dose
of diazepam (0.1 mg/kg) with minimum dose of nimesulide (2.5 mg/kg).
The animal was observed throughout constant rate intravenous infusion
of pentylenetetrazole (PTZ) and the time between the start of infusion
and onset of convulsion was recorded and converted to threshold dosage.
One way ANOVA and t-test was done on all data. Nimesulide (2.5 and
5 mg/kg), diazepam (0.5 and 5 mg/kg), and the combination of diazepam
with nimesulide increased threshold of PTZ-induced convulsions. Our
results exemplify the possible role of COX-2 in the pathophysiology of
epilepsy. Lower doses of nimesulide signicantly increased threshold
probably by specic inhibition of COX-2 while the higher dose of nimesulide (10 mg/kg) was not effective. This discrepancy can be
explained by declining specicity of this compound for COX-2 and
effect on COX-1 isoenzyme as well in such a high dose. Nimesulide in
combination with diazepam protected animal signicantly so utilization
of COX-2-inhibitors with classic antiepileptic drugs can be considered.
Paper No.: 825

HEALTH AND DISEASE
ASPIRIN MAY PREVENT PREECLAMPSIA AND ITS
COMPLICATIONS IN WOMEN WITH ABNORMAL UTERINE
ARTERY DOPPLER ULTRASOUND
Elaheh Mesdaghinia(1,2), H Talari(3), A Zolfagharpour(4),
M Abedzadeh(1), P Rezapourian(1)
(1) Kashan University of Medical Sciences, Shabihkhani Hospital,
Department of Gynecology, Kashan, Iran
(2) Kashan University of Medical Sciences, Trauma Research Center,
Kashan, Iran
(3) Kashan University of Medical Sciences, Department of Radiology,

Kashan, Iran
(4) Tehran University of Medical Sciences, School of Medicine, Tehran,
Iran
Preeclampsia is a pregnancyspecic syndrome with hypertension and
proteinuria that may cause serious complications like cardiopulmonary
failure, hepatic dysfunction, coagulation disorder, renal failure, cerebral
involvement, seizure and maternal death. Until now, there is not a specic method to prevent this syndrome. This study was designed to examine the effect of low-dose Aspirin (80 mg/day) in prevention of
preeclampsia and its complications in pregnant women. This randomized
clinical trial was done on women predisposed to preeclampsia in 1216 weeks of gestation with abnormal uterine artery doppler ultrasound;
they (80 persons) were randomly divided to one control and one experiment group and followed up until delivery for preeclampsia, IUGR, preterm labour, low Apgar and root of delivery. Our data was analysed by
T-test, chisquare test, Fisher exact test, KolmogorovSimirnov test. Mean
age in experimental group was 27.8 years and in control group was
26.2 years. The preeclamnpsia incidence in the group which taken aspirin was 2.5%, whereas in control group was 22.5% and the risk of preeclampsia was 11.3 fold more in control group (p=0.007). The incidence
of preeclampsia was signicantly higher in persons with daughter newborns (p=0.016). There was no signicant relationship between incidence
of preeclampsia and history of preeclampsia, hypertension or diabet.
There was no signicant relationship between use of aspirin and root of
delivery and also none of our persons had IUGR, preterm labour or low
Apgar. Low-dose of aspirin can decline incidence of preeclampsia and its
utilization to prevent preeclampsia can be considered.
Paper No.: 1961

EFFECTS OF A WATER-SOLUBLE CINNAMOMUM CASSIA
EXTRACT ON ISOLATED RAT PLATELETS
Zsoa Mezei(1), G Mester(1), Z Mecs(1), A Lepran(1), D Redei(2),
G Szabo(1)
(1) University of Szeged Faculty of Medicine, Department of
Pathophysiology, Szeged, Hungary
(2) University of Szeged Faculty of Pharmacy, Department of
Pharmacognosy, Szeged, Hungary
Platelet activation may contribute to initiation and development of atherosclerotic lesions. Eicosanoids and other mediators synthesized and
excreted during platelet activation may further enhance the stimulation of
leukocytes and platelets. These processes play a role in the development
of vascular damage, e.g. in diabetic patients. Cinnamomum species have
been shown to afford protection against elevation of the serum glucose
level. It is already known that cinnamaldehyde, the main constituent of
the volatile oil of cinnamon, is able to reduce the activity of platelets.
The aim of our experiments was to investigate the in vitro effects of a
water-soluble extract of Cinnamomum cassia bark on the eicosanoid synthesis of non-activated rat platelets, using [14C]-arachidonic acid, as a
tracer substrate. The eicosanoids were separated by chromatography and
determined by liquid scintillation. The synthesis of the cyclooxygenase
metabolites (i.e. TxA2, 12-HHT, PGD2, PGE2, PGF2a and PGI2) were
dose-dependently reduced (total amount was 582404303, 424823814,
373354403, and 278452272 dpm after control, 50-100-200 lg dry
extract/ml, respectively), while the synthesis of the lipoxygenase metabolites were not changed by the cinnamon extract. In vitro platelet aggregation, induced by 10 lM ADP, was dose dependently reduced with the
extract. Our results suggest that the water-soluble extract of cinnamon
might reduce the sensitivity of platelets to different activators by decreasing the cyclooxygenase activity, thereby slowing the progression of atherosclerosis.
veges
This work was supported by the grants from ETT 2009 and O
(HEF_06_1-MEZEI_ZS).
454
Paper No.: 624
DISCOVERY
EFFECT OF THE P-GLYCOPROTEIN ON THE INTESTINAL
ABSORPTION OF HERBAL PRODUCTS ASSOCIATED WITH
ANTIRETROVIRALS IN MICE
Joe Miantezila Basilua(1), G Mamadou(1), N Limas Nzouzi(1),
G Peytavin(2), B Eto(1)
(1) TransCell-Lab, Paris, France
(2) Laboratory of Pharmacokinetics of Bichat Hospital, France
Herbal drugs usually used in Africa as adjuvant against the HIV/AIDS
exhibit interaction with antiretroviral drugs in the transport through intestinal barriers. The aim of this study is to assess interaction of those
herbal products (e.g. Fagaricine) with antiretroviral during the transport
through intestinal membranes. Methods: Evaluation of the transport of
the antiretroviral in presence or without Fagaricine was performed using
the Ussing chambers in mice. The results of the study showed that, the
transport of the herbal product (Fagaricine) is not involved P-gp compared to that of antiretroviral, in addition, some less interactions was nd
with some antiretroviral drugs. In conclusion, we showed that, P-gp has
not effect on transportation of herbal product (Fagaricine) used in adjuvant against HIV/AIDS in Africa.
Paper No.: 2213

HEALTH AND DISEASE
NAMPT/VISFATIN AS A NOVEL THERAPEUTIC TARGET FOR
STROKE
Chao-Yu Miao(1), P Wang(1), T-Y Xu(1), W-W Tian(1), Y-F Guan(1),
D-F Su(1), Y-C Rui(1), B Viollet(2), Q-W Zhai(3)
(1) Second Military Medical University, Department of Pharmacology,
Shanghai, PRChina
(2) Institut Cochin, University Paris Descartes, CNRS (UMR8104),
Paris, France.
(3) Institute for Nutritional Sciences, Chinese Academy of Sciences,
Shanghai, PRChina
Stroke is a leading cause of mortality and disability. Both injury and
defense mechanisms determine outcome of stroke. Here, we identied
nicotinamide phosphoribosyltransferase (Nampt, also known as visfatin)
as a novel neuroprotector defending against ischemic stroke. Nampt inhibition accelerated fatal stroke occurrence in genetically stroke-prone rats
and aggravated brain infarction in experimentally cerebral ischemia rats,
while Nampt overexpression in local brain reduced ischemia-induced
cerebral injuries. Nampt overexpression and knockdown regulated neuron survival via adenosine monophosphate activated kinase (AMPK)
pathway. Neuroprotection of Nampt was abolished in AMPKa2 knockout
neurons. As in other cells, Nampt modulated NAD+ levels and thereby
controlled SIRT1 activity in neurons. SIRT1 co-precipitated with and deacetylated LKB1, the upstream kinase of AMPK. Nampt-induced LKB1
deacetylation and AMPK activation disappeared in SIRT1 knockout neurons. Anti-stroke effect of Nampt blunted in SIRT1+/) heterozygous
mice. Our ndings reveal Nampt protects against ischemic stroke through
regulating neuron survival via SIRT1-dependent AMPK pathway and
indicate Nampt is a new therapeutic target for stroke.
(Supported by grants 30525045, 2009CB521902, 10XD1405300,
200369 and 2009ZX09303-002)
Paper No.: 1938

EXTINCTION OF AVOIDANCE BEHAVIOR BY SAFETY
LEARNING DEPENDS ON INTRAHIPPOCAMPAL
ENDOCANNABINOID SIGNALLING
Vincenzo Micale(1,2), R Marsch(1), FA Pamplona(1), F Drago(2), CT
Wotjak(1)
(1) Max Planck Institute of Psychiatry, Laboratory of Neuronal Plasticity,
Munich, Germany
(2) University of Catania, Department of Experimental and Clinical
Pharmacology, Spain
Avoidance behavior is a defensive response, which results from operant
conditioning processes. It represents a key symptom of a variety of
human anxiety disorders and is largely responsible for the decrease in
quality of life of the patients. Animal research may help to develop more
efcient therapeutic interventions. The aim of the present study was,
therefore, to characterize the role of response extinction vs. safety learning in extinction of step-down inhibitory avoidance in C57BL/6N mice.
In this task, mice are placed on a platform and receive an electric shock
as soon as they step down onto the gridoor with all four paws. Memory
performance is assessed by an increase in step-down latencies on subsequent encounters. Repeated exposure to the grid (i.e. safety learning), but
not repeated step-down from the platform (i.e. response extinction), leads
to extinction of avoidance behavior. This process is context-dependent
and can be blocked by systemic and intrahippocampal injection of the
cannabinoid CB1 antagonist rimonabant. These results highlight the
importance of safety learning for extinction of avoidance behavior and
suggest the endocannabinoid system as a pharmacological target for
reducing avoidance behavior in human anxiety disorders.
Paper No.: 2450

HEALTH AND DISEASE
ON PHARMACO-PHYSIOLOGICAL PROBLEMS OF
HYPERTONIA IDIOPATHICA
Michael C Michailov(1,2,3), E Neu(1,2,3), U Welscher(1,2,3),
S Ivanova(4,1), N Senn(1), J Foltinova(5), W Seidenbusch(3,1)
Muenchen, Germany
Germany
Austria
(4) University of Muenster, Faculty of Medicine, Muenster, Germany
Introduction: High morbidity/mortality by hypertension, caused by insufcient angio-cardiac pharmaco-therapy is due to decient information
about pathology of cellular (Cl.Bernard), blood-pressure (W.Cannon), volume (O.-H.Gauer) homeostasis. Summary of recent&earlier observations will be considered (J.Physiol.Sci. IUPS-2009/Kyoto;59:168;
Acta-Physiol. FEPS-2007/Bratislava;191:49; Abstract-Book ISIM-2008/
Buenos-Aires:42). Method: Blood pressure of anaesthetized (pentobarbital 30mg/kg) normal&spinal rats (decapitated; articial respiration &
thermoregulation) (Experientia 1969/25:621; Strahlenther. 1976/
151:549). Results (n=150): 2-mercaptoethylguanidine (MEG:100-400mg/
kg, inhibitor of Ca-independent-NO-synthase) induced (a) transformation
of depressor (dR) into biphasic dR/pR (pressor) reaction. to electrical
central-vagal-stimulation (55Hz/5s/5V), (b) acetylcholine (ACH: 5lg/kg)
also after nicotine (5-170 lg/kg), (c) pR-potentiation of non- (AHR-602/
MCN-A343) & nicotine-like (nicotine/DMPP: 20-200 lg/kg) ganglionstimulating-agents, (d) Serotonin-dR-conversion (5-HT:5 lg/kg) (1stob-
455
servation:1964), antagonized by antihistamines, (e) potentiation of octa-/
nona-peptide reactions, dR (vasopressin: VP:5-30 mU/kg,) & pR (bradykinine: 2-20lg/kg) in normal&spinal rats, (f) inhibited ACH-& 5-HTcontractions of rat portal vein. (g) 5-HT (0.1-1lg/ml), VP (0.2-10 mU/
ml) induced slow tonic contractions (5-12/h) & minute rhythms (0.1-0.2/
min) in isolated rat-aorta. Discussion: 3 pathogenetic mechanisms of idiopathic hypertension (independently from renal) are suggested, caused
by sensitization of neuro-effector regulatory structures: A. Central adrenergic/cholinergic neurons (CNS:formatio reticularis/ hypothalamus), B.
Preganglionic sympathetic neurons (nicotinic-cholinergic-receptors:nACHRs), C. Vascular effector cells (myocytes/endothelial). Participation in
excitation of nACHRs (ionotropic: voltage gated Na-, K-, Ca-channels)
in CNS & preganglionic sympathetic neurons, i.e. of CNS-/ganglionreceptor-types (pentamers of subunits) (1), of G-protein-coupled 5-HT1-7
receptors/subtypes (2), importance of vasopressin cell receptors (VPR1A,
neural receptor VPR1B) (3), also antagonizing drugs (4) have to be claried. Conclusion: Pharmacological analysis of 3 sensitizing mechanisms
of pressor/vasomotor reactions (a-g) could open new dimension for antihypertonic therapy incl. counteraction to cardiac angiospasms & cerebral
apoplexy.
Paper No.: 3156

EFFECTS OF CYP4F2 GENETIC POLYMORPHISMS ON
WARFARIN DOSE REQUIREMENTS IN CAUCASIAN
PATIENTS INITIATING THERAPY
Veronique Michaud(1), M Frappier(1), M Champagne(2), D Brouillette(3), D Roy(3), I Taillon(4), G OHara(4), D Gossard(5), M Phillips(6), A Ajami(2), J Turgeon(1)
(1) Faculty of Pharmacy, University of Montreal and CRCHUM, Centre
Hospitalier de lUniversite de Montreal, Montreal, QUE, Canada
(2) Xanthus Life Sciences, Cambridge, MA, USA
(3) Montreal Heart Institute, Montreal, Canada
(4) Quebec Heart Institute, Quebec, Canada
(5) Haut-Richelieu Hospital, St-Jean sur Richelieu, Canada
(6) Genome Quebec and Montreal Heart Institute Pharmacogenomic
Centres and Facultyof medicine, Universite de Montreal, Montreal,
Canada
CYP4F2 appears involved in the vitamin K1 metabolism. CYP4F2 variants may contribute to variance in warfarin dose requirements. Our aim
was to study the association between CYP4F2 genotypes and warfarin
dose requirements, before and after adjusting for CYP2C9 or VKORC1
genotypes. Patients (n=143) initiating warfarin treatment were enrolled.
Demographics, doses and INR were recorded during a 14-day period.
CYP4F2 genotyping for the rs2108622 variant was performed by a PCR
allelic discrimination assay (Applied Biosystem). CYP2C9 and
VKORC1 genotyping were performed by gene chip analysis using Autogenomic Innity system. The allelic frequency of the CYP4F2 variant
allele (n=126) was 0.32. Patients homozygous for the CYP4F2 wild-type
allele required lower doses of warfarin than patients carrying one or two
variant alleles (dose/INR: 1.30.6 mg vs 1.81.2). We observed a signicant cumulative effect of CYP4F2 variant alleles after adjusting for
CYP2C9 and VKORC1 genotypes. Lowest doses of warfarin were
required in patients carrying CYP4F2 wild-type alleles and variant alleles
of VKORC1 (SNPs 3673, 6484 and 6853) (dose/INR; 0.70.46 mg). In
contrast, patients carrying one or two CYP4F2 variant alleles and homozygous wild-type for VKORC1 (SNPs 3673, 6484 and 6853) required
higher doses (dose/INR; 2.81.6mg). Finally, the wild-type patients for
CYP4F2 and VKORC1 9041 needed lower doses compared to patients
carrying at least one variant allele for these genes (dose/INR; 1.10.6 vs
2.21.6mg). In conclusion, we demonstrate an association between
CYP4F2 genotypes and warfarin dose requirements. The modulatory role
of CYP4F2 genotype is further revealed after adjusting for CYP2C9 and
VKORC1 genotypes.
Paper No.: 2336

DISEASES
CANNABINOID RECEPTOR ACTIVATION INHIBITS SMOOTH
MUSCLE CELL PROLIFERATION AND ENDOTHELIAL
CYTOKINE PRODUCTION
Lisa Michel, K Skene, S Teixeira, E Hector, H Vosper, C Wainwright
Robert Gordon University, School of Pharmacy and Life Sciences,
Institute forHealth & Welfare Research, Schoolhill, Aberdeen, UK.
Regaining control of the awry immune response key to the progression
ofatherosclerosis, a chronic inammatory condition, is a potential target
fortherapeutic intervention. Cannabinoid derivative ?9-THC was recently
shown toslow disease progression in the apoE-/- model of atherosclerosis.
It isrecognised that immune cells express CB1 and CB2 receptors, hence
the observedeffects of ?9-THC may be mediated through multiple receptor pathways. Thecurrent research aimed to characterise the role of both
receptor sub-types inthe key cell types involved in atherogenesis. Human
endothelial (HUVEC) andmonocyte (THP-1) cell lines and primary murine smooth muscle cells were utilisedto examine the role of selective
CB1 and CB2 agonists and antagonists, as wellas the endocannabinoids
anandamide (AEA) and 2-AG, in three key atherogenicevents: vascular
endothelial cytokine production, macrophage lipid uptake andsmooth
muscle cell proliferation and migration. Cytokine production incannabinoid treated THP-1 cells was also investigated. Respective to control,
a50% reduction (P<0.05) in MCP-1 production was observed in LPSstimulated(1ug/ml) HUVECs exposed to 10-5M AEA, suggesting an
anti-inammatory effect.In contrast, lipid accumulation in PMA-stimulated (5ng/ml) THP-1 cells wassignicantly (P<0.05) increased in the
presence of the selective CB1 agonistACEA (10-5M) compared to the
vehicle control. CB2 antagonist AM630 was shown todownregulate
DNA synthesis and hence proliferation in PDGF-stimulated smoothmuscle cells. The data highlights the complex role of cannabinoids and
theendocannabinoid system in plaque development: some effects appear
benecialwhile others conducive to the pathophysiology of the condition.
Paper No.: 422

USING ADAPT IN PAEDIATRIC PATIENTS:
PHARMACOKINETIC MODELLING FOR LAMOTRIGINE
Janet Mifsud, D Soler, H Shabbi
University of Malta, Department of Clinical Pharmacology, Msida, Malta
It is estimated that around 70 % of medicinal products used in paediatric
population have never been specically evaluated for use in that age
group. There is still a lack of models that will allow accurate predictions
of drug levels in these populations. This is of particular importance in a
chronic neurological condition such as epilepsy. In this study, a sparse
data model, using Adapt software was developed for lamotrigine in a
group of paediatric patients. Plasma lamotrigine levels in 20 paediatric
patients (mean S.D., age 8.85 3.47 years and weight, 32.22
20.81 kg) were measured. The pharmacokinetic parameters in the four
groups (lamotrigine alone, with valproate, with carbamazepine, and with
clonazepam) were estimated. The only signicant difference (P < 0.05)
was obtained between the four groups in the case of estimated volume of
distribution, predicated minimum plasma concentration, and area under
the curve. The data was also analysed using a modication of a one compartment rst order absorption model with an Adapt population simulation programme. A population mean of 0.64 ml/min/hr was estimated for
systemic clearance, 1500 ml/kg for central volume of distribution and
4.15 hours for Lag Time. Overall, the results thus obtained from the
studies in this research, indicate the important need to streamline pharma-
456
cokinetic data for the use of AEDs in children. Our studies have shown
that several variables could inuence plasma drug concentrations
obtained and there is still of lack of software packages that will allow
accurate predictions of drug levels.
Paper No.: 1943

PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR
(PPAR) AGONISTS PROTECT PODOCYTES FROM HUMAN
KIDNEY AGAINST CELL DEATH INDUCED BY OXYGEN/
GLUCOSE DEPRIVATION-REOXYGENATION
Gianluca Miglio, AC Rosa, L Rattazzi, R Fantozzi
University of Turin, Department of Anatomy, Pharmacology and
Forensic Medicine, Turin, Italy
PPAR are ligand-activated transcription factors and three subtypes (a, b/
d and c) have been identied. PPAR agonism has been demonstrated to
decrease glomerular injury in an in vivo model of renal ischema/reperfusion (Collino M et al, Kidney Int. 2005; 68: 529-36; Patel NS et al, J.
Pharmacol. Exp. Ther. 2009; 328: 635-43). Whether or not a direct effect
on podocytes, a cell type integral part of the glomerular ltration barrier,
contributes to the observed protection remains to be established. By
using oxygen/glucose deprivation (OGD)-reoxygenation as an in vitro
model that mimics in vivo ischemia/reperfusion, the effects of selective
PPAR agonists on podocyte death have been compared. Human immortalized podocytes have been pre-treated with gembrozil (PPARa)GW0742 (PPARb/d), pioglitazone or rosiglitazone (PPARc), either as
acute (single dose) or as a repeated (3 days exposure) challenge. Cell
death was measured as decrease in cell number, necrosis (loss of membrane integrity) and apoptosis (nuclear pyknosis/fragmentation). Only the
repeated pre-treatment with each agonist signicantly prevented cell
death, mainly by decreasing apoptosis. In comparison, in a model of
serum-deprivation (48 h)-induced apoptosis both pre-treatments were
effective, although the repeated one achieving the highest maximal
effects. The EC50s followed the rank order GW0742<rosiglitazone<pioglitazone<gembrozil, with two log less values after repeated
pretreatment. These results suggest that PPAR agonists exert protective
effects on podocytes mainly by decreasing apoptotic cell death. These
ndings contribute to clarify the pathophysiological role of PPARs in the
kidney and indicate them as pharmacological targets for kidney diseases.
Paper No.: 2138

EFFECT OF SUBCHRONIC PUERARIA RADIX EXTRACT
ADMINISTRATION ON MRNA EXPRESSION OF POMC, PENK,
PDYN, MOR, DOR AND KOR GENES IN BRAIN OF WHP AND
WLP ALCOHOL DRINKING RATS
Przemyslaw L. Mikolajczak(1,2), A Bogacz(1), M Karasiewicz(1),
I Okulicz-Kozaryn(2), E Kaminska(2), M Szulc(2), P Figura(2),
T Bobkiewicz-Kozlowska(2), W Dyr(3), W Kostowski(3),
PM Mrozikiewicz(1,4)
(1) Institute of Natural Fibers and Medicinal Plants, Department of
Pharmacology and Biotechnology, Poznan, Poland
(2) Poznan University of Medical Sciences, Department of Pharmacology, Poznan, Poland
(3) Institute of Psychiatry and Neurology, Department of Pharmacology
and Physiology of Nervous System, Warszawa, Poland
(4) Poznan University of Medical Sciences, Laboratory of Experimental
Pharmacogenetics, Department of Clinical Pharmacy and Biopharmacy,
Poznan, Poland
It is known that an extract from Puerariae radix (kudzu) is involved in
reducing alcohol intake in experimental animals, however the detailed
mechanism by which kudzu suppresses ethanol intake is unknown. The

paradigm is probably mediated centrally, therefore an assessment of inuence of kudzu on mRNA expression of opioid system genes in the brain
of ethanol drinking rats seems to be of scientic importance. Alcohol
Warsaw High Preferring (WHP, 6.8 g/kg/day) and Warsaw Low Preferring (WLP, 1.7 g/kg/day) male rats were treated with kudzu (500 mg/kg,
p.o.) for 28 consecutive days and mRNA expression of the POMC,
PENK, PDYN, MOR, DOR and KOR genes in cortex, striatum and hippocampus were measured. The RT-PCR analysis using Master SYBR
Green was carried out. GAPDH was used as a housekeeping gene for normalization. Generally, there were signicant relationships between PDYN
and KOR (cortex: r=0.37; striatum: r=0.42), POMC and MOR (cortex:
r=0.66; hippocampus: r=0.69) or PENK and DOR (cortex: r=0.32, striatum: r=0.60, hippocampus: r=0.53) mRNA expression for all of rats. It
was found that kudzu (75%) lowered alcohol intake in WHP rats. This
paradigm corresponded with a signicant increase in POMC, MOR and
KOR mRNA expression in hippocampus, whereas in striatum and cortex
the levels of transcripts were not affected. On the other hand, kudzu did
not change the alcohol intake in WLP rats, but lowering of PDYN, KOR
and DOR mRNA expression in cortex only was found. Our ndings suggest a possible role of opioid system in the antialcoholic action of kudzu.
Paper No.: 1935

DISTRIBUTION OF THE CYP2C9*2, CYP2C9*3 GENE
VARIANTS IN A ROMANIAN EPILEPTIC PATIENTS GROUP
Florentina Claudia Militaru, A Buzoianu, C Bocsan, A Trifa, R Popp,
LP Dumbrava, E Brusturean
University of Medicine and Pharmacy, Department of Clinical Pharmacology, Cluj Napoca, Romania
Introduction. Drug treatment of epilepsy is characterized by unpredictability of efcacy, adverse drug reactions, and optimal doses in individual patients, which, at least in part, is a consequence of genetic variation.
CYP2C9, a member of the cytochrome P450 enzymes family, metabolizes a range of clinically signicant drugs, such as valproic acid. Its
homologous gene has been shown to be polymorphic, two major alleles,
CYP2C19*2 and *3 being responsible for the poor metabolizer (PM)
phenotype. In rare cases, valproic acid can be metabolized to the reactive
(hepatotoxic) metabolite, 4-ene-valproic acid. It is not yet clear whether
genetic variants of the involved enzyme (CYP2C9) are responsible for
this problem. Objective. The aim of this study is to determine for the rst
time the genotype distribution and the allele frequency of CYP2C9 variants in an epileptic patients group and in a control group of Romanian
volunteers. Methods. Based on the PCR-RFLP technique, CYP2C9*1,
CYP2C9*2, CYP2 C9 *3 variants were studied in 64 epileptic patients
and 200 healthy Romanian volunteers. Results and conclusions: the
results obtained show a relatively high frequency of heterozygous
CYP2C9 (20% for CYP2C9 * 2 and 16.9% for CYP2C9 * 3), while
4.5% of patients had a genotype associated with a phenotype of poor metabolizer CYP2C9 (3% compound heterozygous CYP2C9 * 2 / * 3 and
1.5% homozygous CYP2C9 * 2).
Paper No.: 2416

EFFECTS OF PARACETAMOL DURING FIRST DAYS OF LIFE
Petra Millfors(1), KJS Anand(2), LL Bergqvist(1)
(1) Karolinska Institute, Department of Woman and Child Health, Astrid
Lindgren Childrens Hospital, Stockholm, Sweden
(2) University of Tennessee, Department of Pediatrics, Memphis, TN,
USA
457
Paracetamol (acetaminophen) is a commonly used analgesic during
childhood, although its mechanism of action is not completely understood. Neonates have a less developed pain system, which results in
increased pain sensitivity and even greater need for analgesia. Pain
increases sympathetic tone and stress responses. Heart rate (HR) and HR
variability (HRV) are frequently used physiological indicators of pain in
infants. Subdural or subgaleal hematoma after vacuum extractions and
clavicle fractures are described complications after deliveries. Physical
handling of these infants might result in breakthrough pain and a specic
need for analgesics. Our aim was to increase understanding of the action
and effects of paracetamol in the newborn infant. Full-term newborn
infants were studied the rst three days of life; 26 infants born with normal vaginal delivery, 17 infants with complications were included.
Infants were studied with or without 60 mg paracetamol in a cross-over
trial. Recordings of heart rate were registered during sleep, followed by
standardized handling of the infants head. All infants decreased their
basal HR after paracetamol (118 to 113 bpm, CI 0.0-9.8, p=0.05) and in
response to the standardized handling (127 to 113 bpm, p=0.02 and
P<0.01). Normal vaginal delivery results in high levels of stress hormone release. Our results suggest that paracetamol reduces breakthrough
pain in the term newborn infant, thereby decreasing activation of the
sympathetic system.
Paper No.: 2098

CLINICAL PHARMACOLOGY SERVICE RATIONALIZED USE
OF COSTLY DRUG IN ACADEMIC HOSPITAL OF A COUNTRY
IN SOCIO-ECONOMIC TRANSITION
Dragan Milovanovic, S Jankovic, D Ruzic Zecevic
Medical Faculty and Clinical Centre, Department of Clinical
Pharmacology, Kragujevac, Republic of Serbia
Objective. Analysis of impact of implementation of therapeutic hospital
guideline, concerned with irrational use of octreotide, caused largely by
prescriptions in unsuitable indications. Method. Study was designed and
implemented as interventions in health system. In year 2009, in Clinical
Center in Kragujevac, Serbia, local therapeutic guideline for use of octreotide was introduced intended to rationalize its use. Department of Clinical Pharmacology prepared guidelines, Commission for Quality Control
adopted it and hospital management nally approved it. Effect was analyzed using trends in prescription and costs for octreotide, before and
after implementation of intervention. Drug utilization was calculated
using method of ATC/DDD of World Health Organization. Collected
data were analyzed using descriptive statistics and regression (p=<0.05).
Results. During seven-year period there were growing trends of octreotide prescribing and costs for its acquisition, which corresponded to signicant power regression lines (p=0.004 and p=0.002). Annual use in
2002. was 0.1 DDD per 1000 bed days and in subsequent years
increased 8.0, 21.0, 8.0, 32.0, 100.0 and 43.0 times from baseline. Drug
expenses in 2002. were 0.7 Euros per 1000 bed days and in subsequent
years increased 11.7, 29.6, 11.7, 47.4, 166.0 and 151.1 times from baseline. In immediate period after intervention prescription rate and costs
dropped by 80.7%, each. Conclusion. Implementation of octreotide local
therapeutic guideline, prepared by Department of Clinical Pharmacology,
immediately reduces both prescriptions and costs. Denite effect of this
part of rationalization of pharmacotherapy could be established in studies
with longer follow-up.
Paper No.: 2871

DISEASES
EXPRESSION OF 5-HT RECEPTORS BY HUMAN IMMUNE
CELLS: IMPACT OF PHYSIOLOGICAL ACTIVATION
Sarah Milton(1,2), A Butler(1,2), J Curnow(3), J Gordon(1,3),
N Barnes(1,2)
(1) Celentyx Ltd, Edgbaston, Birmingham, UK
(2) University of Birmingham, School of Experimental and Clinical
Medicine, Collegeof Medical and Dental Sciences, Cellular and Molecular Neuropharmacology Research Group, Edgbaston, Birmingham, UK
(3) University of Birmingham, School of Immunity and Infection, College of Medical and Dental Sciences, Institute of Biomedical Research,
Edgbaston, Birmingham, UK
It is becoming increasingly apparent that bi-directional communication
exists between the nervous system and the immune system; however the
mechanisms through which this occurs remain largely unclear. The substantial differences between the rodent and human immune systems highlight the importance of human immune cell-based assays for the
generation of novel therapies and drug development. In humans, the availability of serotonin (5-hydroxytryptamine; 5-HT) to immune cells is
thought to come primarily from platelets, yet immune cells in the intestine
would be open to 5-HT released from enterochromafn cells and inuenced by sympathetic innervation of lymphoid organs. Using puried
immune cell subsets isolated from human buffy coats, the expression of 5HT receptor subtypes has been investigated; of particular interest is the differential expression of 5-HT receptor subtypes following activation. There
is differential expression of the 5-HT3 receptor between different human
immune cell subsets and variation with their physiological state. Our subsequent studies are attempting to elucidate the functional consequences of
the differential expression and whether these receptors offer therapeutic
targets to treat disorders impacted by the human immune system.
Paper No.: 1280

INHIBITION OF HUMAN LIVER MICROSOMAL OPIOID
GLUCURONIDATION BY KETAMINE - A POTENTIAL
PHARMACOKINETIC DRUG-DRUG INTERACTION
John Miners(1), V Uchipichat(1,2), P Ruangrut(1,3), D Elliot(1),
B Janchawee(3), A Evans(4)
(1) Flinders University, Department of Clinical Pharmacology, Adelaide,
SA, Australia
(2) Khon Kaen University, Khon Kaen, Thailand
(3) Prince of Songkla University, Songkhla, Thailand
(4) University of South Australia, Adelaide, SA, Australia
Ketamine at sub-anaesthetic doses is used as an adjuvant medication with
analgesics, particularly opioids. While it is generally believed that the
enhanced analgesic effect arises from a pharmacodynamic interaction, it
has recently been demonstrated that ketamine decreased the metabolic
clearance of morphine via 3-glucuronidation in the isolated perfused rat
liver preparation. To explore further a potential pharmacokinetic basis for
the ketamine-opioid interaction, this study characterised the effects of ketamine on the kinetics of human liver microsomal morphine- and
codeine- glucuronidation. Incubations contained pooled human liver
microsomes (HLM, 1 mg/ml), cofactor (UDP-glucuronic acid; 5mM),
alamethicin, phosphate buffer (0.1M, pH 7.4) and bovine serum albumin
(2% w/v, to sequester inhibitory long-chain fatty acids). The inhibitor
constant (Ki) was determined from experiments that included 4 ketamine
concentrations at each of 3 substrate (morphine or codeine) concentrations. Ki values were corrected for the binding of ketamine to HLM and
albumin. Concentrations of the individual morphine and codeine glucuronides were measured by specic HPLC methods. Ketamine competi-
458
tively inhibited both the 3-and 6-glucuronidation of morphine and the 6glucuronidation of codeine. Derived mean (se of parameter t) Ki values for ketamine inhibition of the three pathways ranged from
3.50.1 lM to 5.80.2 lM. Additional studies with recombinant UDPglucuronosyltransferases (UGT) as the enzyme source demonstrated that
ketamine inhibited UGT2B7, the isoform primarily responsible for opioid
glucuronidation in humans. Taken together, the results demonstrate that
ketamine is a potent inhibitor of human liver microsomal codeine- and
morphine- glucuronidation, and may inhibit the metabolism of other
drugs metabolised by UGT2B7.
Paper No.: 3433

PROPIONYL-L-CARNITINE PREVENT METABOLIC AND
CARDIOVASCULAR ALTERATIONS IN A MODEL OF
DIET-INDUCED OBESITY
Carmen Mingorance Gutierrez(1), M Chalopin(2), G Simard(3),
PH Ducluzeau(3), Y Malthery(3), MD Herrera(1),
M Alvarez de Sotomayor(1), R Andriantsitohaina(2)
(1) University of Sevilla, School of Pharmacy, Department of Pharmacology, Sevilla, Spain
(2) University of Angers, INSERM U771, CNRS 6214, Angers, France
(3) University of Angers, INSERM U694, Angers, France
Obesity is pointed out as a primary contributor to acquired insulin resistance leading to the development of type 2 diabetes and cardiovascular
alterations. The carnitine derivate, propionyl-L-carnitine (PLC), plays a
key role in energy control. Our aim was to evaluate metabolic and cardiovascular effects of oral treatment with PLC in an experimental model
of diet-induced obesity.C57BL/6 mice were fed with a high fat diet for
8 weeks. Next, animals were divided into two groups of n=10, receiving
either water (vehicle-HF) or PLC supplemented water (200 mg/kg/day,
PLC-HF) during 4 weeks. Ten animals receiving a standard diet and
water was used as a lean group (vehicle-ST). Body weight and food
intake were daily measured. A glucose tolerance test (GTT), an insulin
sensitivity test (IST) and an echocardiography were performed and plasmatic insulin and fasting glucose were measured. Endothelial function
was assessed by myography and concentration-response curves to cumulative addition of acetylcholine in aortic rings pre-contracted at 80% of
their maximal response. Vehicle-HF displayed a greater increase of body
weight (P<0.001) compared to vehicle-ST whereas PLC treatment
induced a decrease of body weight (P<0.001) without changing food
intake. PLC restored the impaired responses to glucose overload and
insulin administration developed by vehicle-HF (P<0.05 and P<0.001).
Vehicle-HF exhibited a reduced cardiac output/body weight ratio compared to vehicle-ST (P<0.001) that was reversed by PLC (P<0.05). PLC
treatment led to an amelioration of hyperinsulinemia and endothelial dysfunction shown by HF animals (P<0.001 and P<0.01). Use of PLC may
be a potential mechanism for prevention of cardiovascular and metabolic
alterations associated with obesity.
Paper No.: 2739

ANTIPROLIFERATIVE ACTIVITY OF SOME SOLANIDINE
ANALOGS ON HL-60 CELLS
Renata Minorics(1), T Szekeres(2), G Krupitza(3), P Saiko(2),
B Giessrigl(3), J Woling(4), E Frank(4), I Zupko(1)
(1) University of Szeged, Department of Pharmacodynamics and
Biopharmacy, Szeged, Hungary
(2) General Hospital of Vienna, Clinical Institute of Medicinal and
Chemica lLaboratory Diagnostics, Vienna, Austria
(3) General Hospital of Vienna, Institute of Clinical Pathology, Vienna,

Austria
(4) University of Szeged, Department of Organic Chemistry, Szeged,
Hungary
There is an increasing evidence for direct antiproliferative effect of various steroidal structures including cardenolides, steroidal alkaloids and
sexual hormones. The presently investigated compounds were selected
from a set of solanidine analogs (Frank et al, J Am Chem Soc 2009;
131: 3894-904). Our aim was to characterize the mechanism of action on
HL-60 cells. The compound most effective on cell growth-inhibition
assay (IC50 value 1.3 microM) was subjected to HOPI-staining and
FACS analysis. To analyze the effect of the test substance on the activity
of DNA synthesis, a ribonucleotide reductase assay was performed. The
antioxidant activity of our compounds was additionally tested using lipidperoxidation (LOX) and DPPH-assay. Incubation with the most effective compound resulted in a marked increase in cell membrane
permeability after 8 hours. Both apoptotic and necrotic patterns were
observed by HOPI-staining. FACS analysis exhibited a marked decrease
in G1 phase and a substantial increase in S as well as G2/M phases. The
compound decreased signicantly the activity of ribonucleotide reductase
at 6.0 microM and proved to be a potent antioxidant (IC50 values: 2.0
and 13.7 microM on LOX and DPPH assays, respectively). Our results
indicate that our representative solanidine analog exerts its cytostatic
effect by inhibiting the DNA synthesis resulting in cell cycle arrest.
The work was supported by Hungarian Research Fund (OTKA PD sterreich-Ungarn
72403) and Stiftung Aktion O
Paper No.: 3302

CHANGES IN THE EXPRESSION OF ENOS AND NNOS IN
HUMAN PERIPHERAL BLOOD LYMPHOCYTES AND HEART
FAILURE
Diana Vicente Miralles(1), F Monto(1), E Oliver(1), D Barettino(3),
J Rueda(2), J Aguero(2), L Almenar(2), P DOcon (1)
(1) University of Valencia, Department of Pharmacology, Burjassot,
Valencia, Spain
(2) University Hospital La Fe,Valencia, Spain
(3) Institute of Biomedicine, Valenica, Spain
Aims: to quantify the mRNA expression of eNOS and nNOS in the left
and right human ventricle obtained from explanted samples of patients
with heart failure (HF) in biopsies from non-failing hearts and in
peripheral blood lymphocytes; to correlate them with clinical variables.
Methods: human hearts explanted from 39 HF patients and cardiac
biopsies obtained from 17 transplanted patients. Lymphocytes were isolated from 10 healthy volunteers, 30 patients with HF and 17 patients
after cardiac transplantation. The mRNA encoding eNOS, nNOS and
GAPDH (housekeeping gene) was quantied by TaqMan real-time
PCR using TaqMan Gene Expression Assays (Applied Biosystems).
Results: there was a signicant correlation (P<0.05) between the
mRNA levels of eNOS, but not nNOS, in LV and RV. The eNOS and
nNOS expressions decreased in RV of patients with HF (P<0.001 and
P<0.01; respectively). In lymphocytes, only eNOs was signicantly
detected and also down-regulated in patients with HF (P<0.001) compared with healthy volunteers. The eNOS expression and clinical variables, e.g., left ventricular ejection fraction (direct correlation),
pulmonary arterial pressure or left ventricle end-systolic diameter
(inverse correlations), signicantly correlated. Higher mRNA levels of
eNOS in LV of patients receiving IECAs treatment were found. Conclusions: we observed a decreased eNOS expression in lymphocytes and
hearts of patients with HF, thus conrming the use of lymphocytes as
biomarkers. Changes in the mRNA levels of eNOS in LV correlated
with characteristic HF clinical variables, suggesting decreased NO levels
are involved in the genesis/maintenance of this disease. IECAs treatment favours the eNOS expression.
459
Paper No.: 2120
HEALTH AND DISEASE
VASODILATORY ACTIVITY OF A NEW QUINOXALINE
N-ACYLHYDRAZONE DERIVATIVE LASSBIO-1120: THE
PARTICIPATION OF NO/CGMP PATHWAY
Ana Luisa Miranda(1), L Louback(1), LM Lima(2), A Monge(1),
EJ Barreiro(1)
(1) University Federal of Rio de Janeiro Faculty of Pharmacy, LASSBio,
Rio de Janeiro, Brazil
(2) University of Navarra, CIFA, Spain
Cardiovascular diseases are the main cause of death worldwide, demonstrating the inefcacy of the currently available drugs. N-acylhydrazone
derivatives have being described as vasodilators agents (Silva et al, Brit J
Pharmacol 135, 293, 2002; Silva et al, Bioorg Med Chem 13, 3431,
2005). The aim of present study was to investigate the vasodilatory activity and the mechanism of action of a new quinoxaline N-acylhydrazone
derivative LASSBio-1020. The vasodilatory activity has been evaluated
using rat toraxic aorta rings, with and without functional endothelium,
contracted with phenylephrine (10lM) and KCl (80mM). We evaluated
the interference of ODQ, DDA, L-NAME and atropine on the effects of
LASSBio-1020 to investigate its mechanism of action. LASSBio-1020
(100lM) induced relaxation of aorta rings pre-contracted with phenylephrine and KCl by 65% and 71%, respectively, showing an IC50 of
11.9 lM. The effect was partially endothelium-dependent (maximum
effect with (72%) and without (30%) endothelium). The aortic rings with
endothelium pre-incubation with DDA (58%) didnt alter LASSBio-1020
effect. In contrast, ODQ (23%) e L-NAME (35%), have blocked the
endothelium-dependent vasodilatory activity of LASSBio-1020. The
muscarinic antagonist atropine did not reverse this effect. The effect of
LASSBio-1020 has been potentiated in the presence of zaprinast, a
cGMP-phosphodiesterase inhibitor. The vasodilatory effect presented by
the quinoxaline N-acylhydrazone derivative LASSBio-1020 involves
mechanisms related to the NO-GC-cGMP pathway. Therefore, these new
molecular archetype represent a prototype for application in a variety of
pathologies resulting from alterations in this signalling pathway.
Paper No.: 1112

EVALUATION OF CALCIUM CHANNEL ANTAGONIST
ACTIVITY OF SOME NEW 9,10 DIHYDRO-7H,8H-CHROMENO
[4,3-B] CHROMENE-6,11-DIONE
Mohammad Miri(1), M Shari(2), A Bagheri(2), R Motamedi(3),
A Shaee(3)
(1) Shiraz University of Medical Sciences, Faculty of Dentistry, Shirsa,
Iran
(2) Shiraz University of Medical Sciences, Medicinal & Natural Products
(3) Tehran University of Medical Sciences, Faculty of Pharmacy, Department of Medicial Chemistry, Tehran, Iran
1,4-dihydropyridines (DHP) compounds are one of the most important
classes of calcium channel blockers (CCBs) that widely use for the treatment of hypertention. In this study we ealuated the calcium channel
antagonist activity of some new 9,10 dihydro-7H,8H-chromeno [4,3-b]
chromene-6,11-dione by means of Guinea-Pig Ileal Smooth Muscle.
Male guinea-pigs weighting 300 - 400 g were killed by a blow on the
head. The animal was deprived from food 18 hours before sacrice but
had free access to water. As soon as they were killed the mid part of their
ileum was removed and cut into 10-15 mm segments. All the stages of
tissue study performed in physiological Tyrodes solution. The Calcium
Channel Antagonist Activity of compounds was evaluated by using high
K+ concentration.(40Mm) in Guinea-Pig ileal longitudinal smooth
muscle. This procedure was done by organ bath and Pan lab physiograph
and the IC50SEM of each compound compare with reference drug
nifedipine. IC50 determination and statistical calculation were done by
curve expert,sigma plot and spss software. Among this derivatives, compound RM-92-1(R1=OCH3,R2,R3=H) show the least [IC50 = 2.00
2.00 X 10-4 ] and compound RM-81-2(R1,R3=H,R2=Cl) show the most
Calcium Channel Antagonist effect [ IC50 = 5.99 4.00 X 10-7]. To
prove that DMSO as a solvent and the passage of time had no effect on
the ileal contraction, we applied dose response experiments, showing statistically non signicant differences.
Paper No.: 1108

SYNTHESIS AND CYTOTOXIC ACTIVITY OF NOVEL
BENZOPYRANO[3,2-C]CHROMENE-6,8-DIONE DERIVATIVES
Ramin Miri(1), O Firuzi(1), S Meili(1), AR Mehdipour(1),
R Motamedi(2), A Shaee(2)
(1) Shiraz University of Medical Sciences, Medicinal & Natural Products
(2) Tehran University of Medical Sciences, Faculty of Pharmacy,
Department of Medicial Chemistry, Tehran, Iran
Hydroxycoumarins constitute the structural nucleus of many natural
products, drugs and pesticides. Promising biological properties in new
families of synthetic coumarins were recently reported. Therefore efcient synthesis of new benzopyrano[3,2-c]chromene-6,8-dione was
undertaken and the structures of 15 compounds were conrmed by their
IR, Mass, 1H-NMR and C, H, N analysis. Then, the cytotoxic activities
of these compounds were assessed on 4 different human cancer cell lines
(Raji, HeLa, LS180 and MCF-7). The results showed that these compounds had weak to moderate antitumoral activities and their IC50
ranged from 49 lM to more than 100 lM. Among the compounds 9,10Dihydro-7-(3-methoxyphenyl)-7H,11H-benzopyrano[3,2-c]chromene-6,8dione demonstrated the highest activity. Furthermore, conformational
analysis revealed that ortho substituents were clearly different from meta
and para substituents.
Paper No.: 1838

THE RELATIONSHIP BETWEEN GRA AND NFJB RELATED
PROTEINS IN CHILDREN IDIOPATHIC NEPHROTIC
SYNDROME
Ladislav Mirossay, K Szilagyi, L Podracka, A Mirossay, J Mojzis
(1) University of P.J. Safarik, Medical School, Department of Pharmacology, Kosice, Slovak Republic
(2) University of P.J. Safarik, Medical School,1st Department of Paediatrics and Adolescent Medicine, Kosice, Slovak Republic
The aim of our study was to investigate levels of glucocorticoid receptor
a (GRa), nuclear factor kappa B (NFjB) p65/p50 and inhibitor of NFjB
a (IjBa) in the peripheral mononuclear blood cells (PMBC) of children
with idiopathic nephrotic syndrome (INS). The expression of GRa,
NFjBp65/p50 and IjBa was determined in 59 patients and 25 controls
(C). They were divided according to their clinical sensitivity to glucocorticoids (GCs) as responders (RE), partial responders (PR), and nonresponders (NR). Peripheral blood was collected in EDTA tubes and
processed within 1 hour. PMBCs were isolated from all patients and
healthy controls by density centrifugation using the Ficoll PM 400. Overall expression of both GRa and NFjB p65 subunit were signicantly
higher in a group of RE than in PR, NR and even C (p<0.01 and p<0.05
460
respectively). To investigate the possible role of studied proteins in relation to activity of disease, we correlated their expression in particular
clinical subgroups with relapse and remission of INS. No differences
were observed between all studied groups in the remission of disease,
However, signicantly higher expressions of both GRa and NFjB p65
in relapse were detected in RE in comparison to PR and NR as well as C
(p<0.01). Lower levels of both GRa and NFjBp65 are associated with
poor or no response to GCs and the difference is more pronounced in
patients experiencing relapse of INS.
Supported by a grant from Iceland, Liechtenstein and Norway through
the EEA Financial Mechanism and the Norwegian Financial Mechanism
SK0017.
Paper No.: 1318

DISEASES
AQUEOUS CIGARETTE TAR EXTRACT CAUSES MARKED
BRONCHIAL SMOOTH MUSCLE HYPERRESPONSIVENESS
IN RATS
Miwa Misawa(1), A Watanabe(1), A Fujita(1), H Sakai(1), J Kamei(2),
Y Chiba(1)
(1) Hoshi University School of Pharmacy, Department of Pharmacology,
Tokyo, Japan
(2) Hoshi University School of Pharmacy, Department of Pathophysiological Therapeutics, Tokyo, Japan
A main cause of chronic obstructive pulmonary disease (COPD) is
known to be cigarette smoking. COPD is characterized by airow limitation that is not fully reversible, and is expected to be the third leading
cause of death in the world by the year 2020 (WHO). The number of
patients with COPD is thus increasing, but there have still no excellent
therapeutic agents for COPD. Airway hyperresponsiveness (AHR) is a
characteristic feature of patients with not only bronchial asthma but also
COPD. Recent our studies using smoking rats revealed that the development of cigarette smoke-induced AHR depends on the constituents of
tar, not nicotine, in the cigarette. In the present study, the effects of aqueous cigarette tar (ACT) on the contractility of the isolated bronchial
smooth muscle were investigated in rats. The ACT was prepared by
extraction from a lter membrane through which the main stream cigarette smoke was passed. In the bronchial smooth muscles treated with
ACT (0.07 mg/mL for 24 hr), the contractile responsiveness levels to
acetylcholine and high potassium ion-depolarization were markedly and
signicantly augmented. In addition, the contraction induced by sodium
uoride (NaF), an activator of G proteins, was also signicantly augmented in the ACT-treated bronchial smooth muscles. Interestingly, the
contraction induced by calyculin-A, a potent protein phosphatase inhibitor, was not changed by the ACT treatment. These ndings suggest that
the ACT is one of the important inducers of bronchial smooth muscle
hyperresponsiveness in the cigarette smoke-induced AHR in rats.
Paper No.: 510

EFFECT OF GENETIC POLYMORPHISM ON
HYPOLIPIDEMIC RESPONSE TO ROSUVASTATIN IN
METABOLIC SYNDROME
Misbah Misbahuddin(1), F Ahmad(1), S Ziaurrahman(1), A Khan(2),
S Siddiqui(3)
(1) Aligarh Muslim University, Department of Pharmacology,
J N Medical College, Aligarh, India
(2) Aligarh Muslim University, Interdisciplinary Biotechnology Unit,

Aligarh, India
(3) Aligarh Muslim University, Department of Medicine, J N Medical
College, Aligarh, India
Metabolic syndrome predisposes to diabetes and atherosclerotic vascular
disease. Statins treat dyslipidemia, reduce cardiovascular mortality and
morbidity with beneffcial effects on inammation, platelet activity, and
endothelial function, so all metabolic syndrome patients should be evaluated for statin treatment. Response to statins is highly variable with
genetic variability as a contributing factor. The Sterol Regulatory Element-Binding Factor (SREBF) - SREBF Cleavage-Activating Protein
(SCAP) pathway regulates lipid homeostasis. We studied the effect of
single nucleotide polymorphism in SREBF Cleavage-Activating Protein
(SCAP) gene on plasma lipid levels, and lipid-lowering response to rosuvastatin in metabolic syndrome. Patients (n=95) of metabolic syndrome
(NCEP-ATP III), aged 30-70 years, with LDL-C 130 mg/dl, were prescribed rosuvastatin 5 mg/day for 3 months. 63 patients completed the
follow-up. Plasma lipids were measured before and throughout the study.
No signicant association was found between the baseline lipid values
and SCAP genotypes (p>0.05). Distributions of SCAP genotypes were
20 GG, 20 AG, and 23 AA respectively. Mean reduction of (TC) total
cholesterol (-23.8%) was used arbitrarily to divide the patients into two
groups. Mean percentage decrease in TC was more in patients carrying
SCAP 2386G allele compared with those homozygous for 2386A allele
(-27.811.6 vs -18.813.3%, P=0.008). About 79% of the 2386G carriers
were above average responders for TC levels (TC -23.8%), whereas only
23% of 2386A homozygous reached this reduction (P=0.007). SCAP
2386A>G gene polymorphism is a signicant predictor of TC response
to rosuvastatin treatment. Further studies are needed with larger sample
sizes and more SNPs.
Paper No.: 1593

IDENTIFICATION OF A NOVEL LIGAND FOR GLUTAMATE
RECEPTOR d2
Masayoshi Mishina, T Uemura, S-J Lee, M Yasumura, T Yoshida
University of Tokyo Graduate School of Medicine, Department of
Molecular Neurobiology and Pharmacology, Tokyo, Japan
Glutamate receptor (GluR) d2 selectively expressed in cerebellar Purkinje cells (PCs) plays an essential role in parallel ber (PF)-PC synapse
formation, long-term depression and motor learning. However, unlike
other members of GluR family, no ligand is known for GluRd2 and it
remains unsolved how GluRd2 regulates neural network formation and
cerebellar functions. Recently, we have shown that the synaptogenic
activity of GluRd2 can be reproduced in vitro using primary cultures of
cerebellar granule cells (GCs) and the N-terminal domain (NTD) of
GluRd2 is essential and sufcient for the synaptogenic activity. Here, we
identied Cbln1 as a GluRd2-NTD binding protein by systematic screening. GluRd2 expressed in HEK293T cells induced presynaptic differentiation of cerebellar GCs prepared from wild-type mice but failed to
induce presynaptic differentiation of cerebellar GCs prepared from Cbln1
knockout mice. The synaptogenic activity of GluRd2 was restored by
adding recombinant Cbln1 to cerebellar GC cultures prepared from
Cbln1 knockout mice. These results suggest that Cbln1 acts as a functional ligand for GluRd2 and Cbln1-GluRd2 interaction plays a crucial
role in PF-PC synapse formation. When soluble recombinant Cbln1 was
incubated with HEK293T cells transfected with GluRd2, robust signals
for Cbln1 were found on the surface of the transfected cells. On the other
hand, AMPA-type GluRa1 or GluRa2 failed to show the binding activity
for Cbln1. The dissociation constant (KD) of the interaction between
Cbln1 and GluRd2 estimated by surface plasmon resonance assay was
16.5 nM. These results suggest that Cbln1 is a novel and high-afnity
ligand for GluRd2.
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Paper No.: 2106
ESTIMATION OF CREATININE CLEARANCE AND
GENTAMICIN DOSE USING LEAN BODY WEIGHT
PREDICTION
Sarah Mitchell(1,2), S Hilmer(1,2), C Kirkpatrick(3), R Hansen(1,4),
MF Singh(5,6,7)
(1) University of Sydney Medical School, Sydney, NSW, Australia
(2) Royal North Shore Hospital, Departments of Clinical Pharmacology
and Aged Care,Sydney, NSW, Australia
(3) The University of Queensland School of Pharmacy, Brisbane, QLD,
Australia
(4) Royal North Shore Hospital, Gastrointestinal Investigation Unit and
Centre forIn vivo Body Composition, Sydney, NSW, Australia
(5) University of Sydney Faculty of Health Sciences, Exercise, Health
andPerformance Faculty Research Group, Lidcombe, NSW, Australia
(6) Jean Mayer USDA Human Nutrition Research Center on Aging,
Tufts University,Boston, Massachusetts USA
(7) Hebrew SeniorLife, Boston, Massachusetts USA
The dosing of drugs per kg of lean bodyweight (LBW) may be more
accurate thandosing per kg of total bodyweight (TBW) as clearance
increases linearly with LBW(Han, et al. Clin Pharmacokinet 2007). This
may be particularly important inolder people who have decreased LBW
which alters drug pharmacokinetics. Thisstudy aimed to compare the
starting gentamicin dose estimated using creatinineclearance (CrCl) calculated using TBW (CrClTBW), fat-free mass (FFM;CrClFFM) andLBW
(CrClLBW) to determine how the size descriptor affects gentamicin
dose.Baseline data was obtained from participants enrolled in the Sarcopenia and HipFracture Study (Fiatarone-Singh, et al. Gerontol A-Biol SciMed-Sci2009;64:568-74). LBW was estimated using a gender specic
LBW equation(Janmahasatian, et al. Clin Pharmacokinet 2005;44:105165) and compared to thereference method, dual energy x-ray absorptiometry derived FFM (FFMDXA). CrClwas estimated using the Cockroft-Gault formula and gentamicin dose wasdetermined according to Australian
Therapeutic Guidelines. The mean sd age ofparticipants was 839years
(n=27). CrClTBW was signicantly higher(5529mL/min) than
CrClFFM
(3615mL/min;p=0.009)
and
CrClLBW(3717mL/
min;p=0.01). Gentamicin dose based on CrClTBW resulted in higher doesfor 90% of participants (25/27) compared to dosing based on CrClFFM.
93% (25/27)of doses were the same when CrClFFM and CrClLBW were
used to determinegentamicin dose. Creatinine clearance and gentamicin
dose calculated using LBWwere comparable with FFMDXA estimates.
The LBW equation maybe useful forestimating LBW to determine drug
dose, however studies of larger more diversecohorts are needed to assess
the clinical utility of CrClLBW for of drug dosing.
Paper No.: 2107

ESTIMATION OF LEAN BODY WEIGHT IN OLDER BLACK
AND WHITE MEN AND WOMEN: THE HEALTH, AGING AND
BODY COMPOSITION STUDY
Sarah Mitchell(1,2), C Kirkpatrick(3), A Newman(4), S Sattereld(5),
A Schwartz(6), A Simonsick(7), S Hilmer(1,2)
(1) University of Sydney Medical School, Sydney, NSW, Australia
(2) Royal North Shore Hospital, Departments of Clinical Pharmacology
and Aged Care, Sydney, NSW, Australia
(3) The University of Queensland School of Pharmacy, Brisbane, QLD,
Australia
(4) University of Pittsburgh, Division of Geriatric Medicine, Pennsylvania, PA, USA
(5) University of Tennessee, Department of Preventive Medicine,
Memphis, TN, USA
(6) University of California at San Francisco Department of

Epidemiology and Biostatistics, San Francisco, CA, USA
(7) National Institute on Aging, Clinical Research Branch, Baltimore,
MD, USA
In older people, decreased lean bodyweight (LBW) and increased total
body fat affect pharmacokinetics. A semi-mechanistic equation to estimate LBW using height, weight and sex was recently developed (Janmahasatian et al. Clin Pharmacokinet 2005;44:1051-65). This study
examined the ability of the LBW model to predict dual energy x-ray
absorptiometry derived fat free mass (FFMDXA) in community dwelling
older people across different sex, race and body mass index (BMI) subgroups. De-identied data was analysed from baseline measurements on
participants in the Health, Aging and Body Composition Study (Newman
et al. JAGS 2003;51:323-30) with complete relevant data (n=2963/3075,
age 733years). The ability of the LBW equation to accurately predict
FFM was determined by agreement with FFMDXA using Bland-Altman
plots and precision and bias. Bland-Altman plots demonstrated good
agreement between LBW and FFMDXA with a mean difference (limits of
agreement) of -1.54 kg (-7.62kg,4.54kg) [white males -3.4kg (9.0,2.2kg), black males -1.7kg (-7.9,4.5kg), white females -1.1kg (5.5,3.3kg) and black females 0.5kg (-4.7,5.7kg)]. The LBW equation
over-estimated FFMDXA for white males, black males and white females
with mean errors (ME; 95% CI) of -3.4kg (-3.3,-3.6kg), -1.7kg (-2.0,1.6kg), and -1.1kg (-1.2,-1.0kg) respectively. The LBW equation underestimated FFMDXA for black females with a ME of 0.5kg (0.4,0.6kg).
Results were consistent across normal, overweight and obese BMI subgroups. FFMDXA can be estimated easily from the LBW equation using
weight, height and sex, with good agreement in this population of older
people. Further studies are needed to determine the clinical utility of this
equation for calculation of drug doses.
Paper No.: 410

EFFECT OF POLYGONUM BISTORTA AND TANNIC ACID
AGAINST CARBON TETRACHLORIDE INDUCED HEPATIC
DAMAGE IN ALBINO RATS
Deepak Kumar Mittal
Jiwaji University, School of Studies in Zoology, Gwalior, India
Introduction: Many herbal preparations have been recommended in
alternative systems of medicine for the treatment of hepatic disorders.
Protective action of plant extract (PB) and tannic acid (TA) was evaluated animal model of hepatotoxicity induced by carbon tetrachloride
(CCl4). Material and Methods: Rats were divided in to ve groups.
Group I was normal control group; Group II, the hepatotoxic group
was given CCl4 (0.15 ml/kg); Group III received plant extract
(100 mg/kg) dose with CCl4; Groups IV received tannic acid (25 mg/
kg) dose with CCl4 and Group V received silymarin (50 mg/kg) dose
with CCl4 as a positive control. Liver marker enzymes were assayed
in serum and antioxidant status was assessed in liver tissue. Results:
Levels of marker enzymes such as alanine transminase, aspartate transaminase, alkaline phosphatase, and lactate dehydrogenase were
increased signicantly in CCl4 treated rats. PB brought about a significant decrease in the activities of all these enzymes. Lipid peroxidation
was increased signicant and reduced glutathione was decreased in
liver tissue in the CCl4 treated rats while the activities of glutathione
reductase, glutathione peroxidase, glucose-6-phosphatase dehydrogenase, glutathione-S-transferase, catalase, superoxide dismutase, comet
assay and in vitro technique MTT assay were recovered. Histopathological observations also supported to above ndings. TA treatment
led to the more recovery of these levels to near normal when compared to the plant extract. Conclusion: The present observations suggested that the treatment with Tannic acid enhance the recovery from
CCl4 induced hepatic damage in comparison to plant extract due to
its antioxidant and hepatoprotective property.
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Paper No.: 2267
INFLUENCE OF THE VKORC1 GENETIC POLYMORPHISM
AND PATIENT CHARACTERISTICS UPON WARFARIN DOSE
IN JAPANESE ELDERLY PATIENTS
Jun Miura(1,2), H Nakashima(3), T Tateishi(2)
(1) Muroran Institute of Technology, Center for Health Administration,
Muroran, Hokkaido, Japan
(2) Hirosaki University, Japan
(3) Matsunaga Hospital, Japan
This study investigated the inuence of VKORC1, CYP2C9 and
CYP2C19 genotypes, and patient characteristics such as age, sex, weight
and height upon warfarin dose in Japanese elderly patients. One hundred
and four Japanese elderly patients (F45, M59, mean age 73.6 years)
under xed warfarin dose at least for three months were recruited at
Matsunaga Hospital, Oita, Japan. The VKORC1 (-1639G>A),
CYP2C9*3 and CYP2C19*2 and *3 genotypes were determined by the
PCR-RFLP method at Hirosaki University. Statistical analyses were conducted using PASW Statistics 18. This study was approved by the Ethics
Committee of the Hirosaki University and written informed consent was
obtained from all participants. Our main ndings were: 1 The mean warfarin dose was higher in men than women (2.70 vs 2.26 mg/d, respectively, p<0.05) although neither the mean age nor the INR was different.
2 The VKORC1 genotype inuences the mean warfarin dose (p<0.001)
while CYP2C19 genotype was not. The mean dose was 3.57 and
2.30 mg/d in the VKORC1 - 1639GA (n=17) and -1639AA (n=87)
genotype groups, respectively. 3 The mean dose prescribed for the
patients with CYP2C9*1/*1 (n=9) and CYP2C9*1/*3 (n=95) were 2.03
and 2.56 mg/d, respectively, which was not statistically signicant
(p=0.108). 4 Age was inversely correlated (r=-0.455) and height
(r=0.289) and weight (r=0.306) were positively correlated with the mean
warfarin dose. In conclusion, in order to improve the safety of warfarin
therapy, especially in elderly patients, the warfarin dosing regimen
should be modied by taking into account the VKORC1 genotype, sex,
age and body weight.
Paper No.: 1187

PRENATAL STRESS INDUCES ANXIETY-LIKE BEHAVIOR
TOGETHER WITH STRUCTURAL AND FUNCTIONAL
DISRUPTION OF CENTRAL SEROTONIN NEURONS IN MICE
found that prenatal stress induced up-regulation of 5-HT- possitive cells

in the rostral hindbrain in embryonic mice. These ndings indicate that
exposure to prenatal stress induces anxiety-like behavior together with
disruption of the development of 5-HT neurons in mice.
Paper No.: 1573

HEALTH AND DISEASE
DISTINCT EFFECTS OF TISSUE-TYPE PLASMINOGEN
ACTIVATOR AND CPD-7 ON CEREBROVASCULAR
INFLAMMATION DURING THROMBOLYSIS
Takuro Miyazaki(1), O Hisayuki(1), K Hasumi(2), K Honda(1)
(1) Showa University School of Pharmacy, Department of Pharmacology,
Tokyo, Japan
(2) Tokyo Noko University, Department of Applied Biological Science,
Tokyo, Japan
Thrombolysis therapy using tissue-type plasminogen activator (tPA) is
currently the standard of care for patients with cerebral thrombotic
stroke. In addition to tPA, we have reported that Cpd-7, an activator of
plasminogen, has an excellent therapeutic effect on cerebral infarction in
embolic stroke model in mice. However, little is known how these
thrombolytic agents inuence cerebrovascular inammation during the
thrombolysis. In this study, we determined the impact of tPA- and Cpd7-induced thrombolysis on leukocyte dynamics in vessels in ischemic
core employing intravital confocal microscopy in a photochemicallyinduced thrombotic middle cerebral artery occlusion (MCAo) model in
mice. Both tPA and Cpd-7 administration to MCAo mice (10 and
10 mg/kg, respectively) resulted in thrombolysis. The tPA administration
subsequently led to severe rolling and attachment of leukocyte to vascular wall in surrounding supercial middle cerebral vein with an increasing mRNA expression of pro-inammatory cytokines IL-1b and TNFa
and endothelial adhesion molecules (ICAM-1 and VCAM-1) within
3 hours in comparison to the Cpd-7 administration. In vitro study further
suggested that tPA at a concentration of 20 lg/mL, but not Cpd-7 at a
similar concentration, promotes IL-1b and TNF-a-induced reactive oxygen species (ROS) generation in cultured human umbilical vein endothelial cells, moreover that Cpd-7 suppressed IL-1b and TNFa-induced
VCAM-1 induction in the cells whereas tPA does not inuence the
induction. Therefore, it is considered that relatively mild cerebrovascular
inammation in the Cpd-7-administrated MCAo mice is, at least in part,
due to lack of ROS promotion and suppressed VCAM-1 induction in
endothelial cells.
Kazuya Miyagawa, M Tsuji, T Takeuchi, H Takeda

International University Health & Welfare School of Pharmacy, Division
of Pharmacology, Ohtawara, Tochigi, Japan
Most pregnant women are at risk of showing some emotional abnormality, since some biological functions may dramatically change in pregnancy. Some of them may be exposed to strong stress as hesitation of
positive drug therapies because of worries regarding adverse effects on
the embryo. A growing body of evidence suggests that prenatal stress
increases the vulnerability to neuropsychiatric disorders, including
depression and anxiety. However, the mechanisms involved are still
unknown. First, to clarify the inuence of exposure to prenatal stress on
emotional development, we examined behavioral responses in offspring
of the mice exposed to restraint stress in pregnancy. The offspring that
had been exposed to stress exhibited anxiety-like behavior as determined
by the elevated plus-maze test. It has been widely accepted that central
serotonin (5-HT) neurons play a critical role in emotional behaviors.
Immunohistochemical studies showed that exposure to prenatal stress
increased the expression of 5-HT- positive cells in the dorsal raphe nuclei
in mice. Moreover, under these conditions, tryptophan hydroxylase-like
immunoreactivities were also dramatically increased. In addition, we
Paper No.: 2761

AMITRIPTYLINE IN THE MANAGEMENT OF DISTAL
SENSORY NEUROPATHY IN SOUTH AFRICAN PATIENTS
WITH HIV
Shirra Moch(1), N Dinat(2), P Kamerman(3), E Marinda(4), N Hatta(2),
N Zwane(2)
(1) University of the Witwatersrand, School of Therapeutic Sciences,
Johanneburg, South Africa
(2) University of the Witwatersrand, Palliative Care, Department of Internal Medicine, Johanneburg, South Africa
(3) University of the Witwatersrand, School of Physiology, Johanneburg,
South Africa
(4) University of the Witwatersrand, School of Public Health, Johanneburg, South Africa
Background: The estimated prevalence of HIV infection in South Africans adults was 16.9% in 2008. Distal sensory neuropathy (DSN), neuro-
463
pathic pain particularly in the legs is a. common symptom in these
patients and many can be severely debilitated. In addition, current antiretroviral (ARV) therapy causes DSN in 40%-50% of patients, further
decreasing physical and social functioning. Amitriptyline is currently the
drug of choice in the management of pain associated with HIV or ARVrelated DSN, based on evidence of efcacy in neuropathic pain of other
origin. There was thus a need to validate this therapy in a suitable South
African cohort. Patients and Methods:The trial was a randomised, double
blind, placebo-controlled cross-over study of 19 weeks duration. Patients
were recruited from HIV Clinics and hospitals in the Johannesburg area.
A total of 124 participants were recruited, 62 of whom were not on ARV
therapy and 62 currently taking an ARV regimen. They were randomised
to receive either amitriptyline or placebo and, within the rst 2 weeks,
doses were titrated upward in 25mg gradations to a self-determined maximum (limited to 150mg). Patients continued with therapy at that dose
for 6 weeks and then completed a 3-week washout period before receiving the converse therapy. Pain was measured using self-report pain diaries and elicited in personal interviews at each study visit. Results:
Patients perceptions of pain decreased with varied doses of both amitriptyline and placebo. There were no signicant differences found between
the ARV and non-ARV groups. Implications of these results are discussed.
Paper No.: 2762

DEVELOPING RATIONAL PRESCRIBING COMPETENCE IN
MEDICAL SCHOOL: THE RELATION BETWEEN STUDENT
PERCEPTIONS AND EXAMINATION PERFORMANCE
Shirra Moch(1), D Naidoo(2), L Green-Thompson(3)
(1) University of the Witwatersrand, Faculty of Health Sciences, School
of Therapeutic Sciences, Johannesburg, South Africa
(2) University of the Witwatersrand, Faculty of Humanities, School of
Education, Johannesburg, South Africa
(3) University of the Witwatersrand, Faculty of Health Sciences, Centre
for Health Science Education, Johannesburg, South Africa
Introduction: Prescribing medicines is the primary intervention that doctors offer to inuence their patients health; however concerns have been
expressed about the extent to which graduates are prepared by medical
schools to assume prescribing responsibility. Aim: To explore prescribing
performance of nal-year students in the Graduate Entry Medical Programme (GEMP) at the University of the Witwatersrand, Johannesburg
and to compare this with students perceptions of their prescribing competence. Methods: Examination questions were selected via an adjudicative process to determine the prescribing mark. Question items were then
analysed according to Blooms Revised and the SOLO Taxonomies.
Using a Bernsteinian lens, the knowledge structures of the skills were
explored. Student perceptions were interrogated via an internet-delivered
questionnaire. Results:. Examination marks showed that 83.6% of students were competent to prescribe according to the graduating standards
of the University; however, questionnaire data revealed that 66% of students did not feel that their training had enabled them to prescribe rationally. Conclusions: Students scored well on questions which test recall
and application of knowledge, but struggled with questions involving
evaluation. Since prescribing is a complex skill that requires evaluative
competence, this may explain why, despite high examination scores, students remained insecure. Curricular components including problem-based
learning and horizontal integration constrained epistemic access to the
structure of rational prescribing knowledge.
Paper No.: 1586

RELATION BETWEEN ETHANOL-INDUCED OXIDATIVE
STRESS AND APOPTOSIS IN HUMAN HEPATOMA (SK-HEP-1)
CELLS
Yoshiya Mochizuki, M Tsuji, H Oyamada, M Ota, C Kurahashi,
K Oguchi
Showa University, School of Medicine, Department of Pharmacology,
Tokyo, Japan
Alcohol liver disease is linked to oxidative stress and increased production of reactive oxygen species (ROS). Oxidative stress is an early event
in the development of apoptosis. In general, it has been shown that alcohol-mediated apoptosis is a multifactorial process. It has been shown that
alcohol-mediated apoptosis is the two main pathways involving either a
death receptor-mediated route or/and a mitochondrial stress pathway.
However, how and when these factors elicit apoptosis is not completely
understood. The purpose of this study is to clarify whether ethanolinduced ROS production stimulates the death receptor pathway in alcohol dehydrogenase-containing human hepatoma (SK-Hep-1) cells, in
linking the ethanol-induced oxidative stress. We transfected a gene with
SK-Hep-1 cells to knockdown Fas-associated death domain protein (dn
FADD SK-Hep-1). Wild and dn FADD SK-Hep-1 cells were incubated
with ethanol (50 to 500mM), or 200mM ethanol with 20mM N-acethylcysteine (NAC; antioxidant) and/or 300mM apocynin (NADPH oxidase
(NOX) inhibitor) for 1 to 5 hours. In wild SK-Hep-1 cells with treated
ethanol, the expression levels of Fas and NOX4 mRNA increased. Furthermore, exposure of ethanol to dn FADD SK-Hep-1 cells and pretreatment with apocynin in wild type SK-Hep-1 cells signicantly suppressed
apoptosis and caspase-8 and caspase-3 activity compared with wild
SK-Hep-1 cells . It was suggested that ethanol leads to Fas-mediated
apoptosis pathway by NOX-dependent ROS in SK-Hep-1 cells.
Paper No.: 1166

EFFECT OF SOYBEAN ON MALE REPRODUCTIVE
PHYSIOLOGY
Mehrdad Modaresi(1), M Messripour(1), H Khorami(2)
(1) Islamic Azad University, Khorasgan Branch, Department of
Physiology, Isfahan, Iran
(2) Payam e Noor Universiry, Isfahan, Iran
Background: Soybean (Soja hispida Moench) is a member of (Fabaceae),it is a species of legume native to east Asia .It is an annual plant
that has been used in china for 5000 years as a food and a component of
drugs. Soy contains signicant amount of all the essential amino acids
for humans,and so is a good source of protein. Soy has an important role
in improvement & treatment of some cancers such as Colon, Prostate &
breast. Material & method: In this research a total of male mice with 3035 gram weight were bought from Razi Institute. At rst samples were
kept under adaptation condition for tow weeks & then randomly grouped
in to 4 experimental sections.1) Control group were fed with soy-free
basic diet. 2) Nutrition containing 20% soy diet. 3) Nutrition containing
30% soy diet. 4) Nutrition containing 50% soy diet. At the end of
8 weeks of treatment blood collected & serum was stored for hormonal
analysis. Result were consider with SPSS software & results compared
with control group. Results:In 20% group the level of testosterone have
meaningful decrease in comparison with control group,but in 50% group
the level of testosterone have meaningful increase.level of LH in 30% &
50% group have a meaningful increase but no signicant differences
were observed in FSH & weight of testicles.The number of sperms in all
of the treatments have a meaningful decrease. Conclusion: The result of
464
this research indicated that the 20,30 & 50 percent soy diet had negative
effect on male reproductive system in mice.
Paper No.: 2821

HEALTH AND DISEASE
EVALUATION OF THE ENDOPAT AS A TOOL TO ASSESS
ENDOTHELIAL FUNCTION
50%). All 5-HT infusions (1, 30 and 80 ng/kg/min) induced vasodilatation after SL65.0472-00 treatment (+25-60%). Sumatriptan dose-dependently decreased FBF (maximally -35%), this effect was not altered by
SL65.0472-00 treatment. 5-HT-induced platelet aggregation was effectively inhibited by 90% by SL65.0472-00 for at least 6 hours. In conclusion, SL65.0472-00 has potent antagonistic effect on 5-HT-induced
vasoconstriction and platelet aggregation but not on sumatriptan-induced
vasoconstriction. This suggests that in humans, SL65.0472-00 is a
5HT2a blocker without clear 5HT1b antagonistic activity.
Matthijs Moerland, A Kales, L Schrier, D Bradnock, J Burggraaf

Centre for Human Drug Research, Leiden, The Netherlands
Endothelial dysfunction is gaining interest as potential target for (pharmaceutical) intervention of several systemic pathological conditions. To
overcome the practical limitations of conventional techniques for noninvasive measurement of endothelial function, the EndoPAT was developed. As there is only limited information on the performance of the
EndoPAT for repeated measurements in a relatively short time frame, we
investigated the feasibility of the EndoPAT to evaluate acute changes in
endothelial function. Endothelial function, as determined by reactive
hyperemia index, was stable over a longer period of time in patients with
impaired renal function (CV 13%), whereas arterial stiffness, as determined by augmentation index, was rather variable (CV 37%). In renally
impaired patients (n=6; 3 male/3 female; 587 years; on maintenance
therapy for treatment of hypertension), and in a group of subjects with
diabetes mellitus type 2 (n=12; 10 male/2 female; 567 years; untreated
for 2 weeks), endothelial function was not impaired compared to healthy
volunteers (2.871.44 and 2.261.08 versus 1.840.53, respectively).
The EndoPAT was not useful to detect the effect of robust interventions
on endothelial function in healthy volunteers (glucose intervention: difference from baseline 0.080.50, 95%CI -0.44 to 0.60, at 30 minutes;
smoking intervention: difference from baseline 0.490.92, 95%CI -0.47
to 1.46, at 30 minutes). Our ndings suggest that the EndoPAT is at present not suitable to assess (changes in) endothelial function and arterial
stiffness in populations with sizes that are commonly employed in clinical pharmacology studies.
Paper No.: 2822

HEALTH AND DISEASE
MODULATION OF VASOACTIVITY AND PLATELET
AGGREGATION BY 5-HT ANTAGONISM
Matthijs Moerland(1), M Kemme(1), L Bergougnan(2), A Cohen(1),
J Burggraaf(1)
(2) Sano-Aventis, Montpellier, France
Distinct serotonin (5-HT) receptors are involved in platelet aggregation
and vasoconstriction, both implicated in various vascular disorders. Compounds that simultaneously and selectively inhibit the pertaining 5-HT
receptors may represent a therapeutic strategy for vascular diseases. The
vasoactive and antiplatelet effects of SL65.0472-00, a selective 5-HT1b
and 5-HT2a receptor antagonist, were investigated in humans. Twentyfour healthy male volunteers, divided into two groups of 12, received an
oral dose of 20 mg SL65.0472-00 and placebo in a randomized, doubleblind, crossover study. Before and at 2, 4 and 6 hours after dosing, intraarterial infusions of 5-HT1b agonist sumatriptan (n=12) or 5-HT (n=12)
were administered. Forearm blood ow (FBF) was measured using plethysmography and platelet aggregation was measured using whole blood
aggregometry induced by the combination of a threshold collagen concentration and an excess of 5-HT. Treatments were compared using
ANOVA. After placebo treatment, infusion of 1 ng/kg/min 5-HT induced
vasodilatation (FBF +80% change from baseline). In contrast, infusion of
30 and 80 ng/kg/min 5-HT resulted in vasoconstriction (-25% and -
Paper No.: 851

ANGIOTENSIN II TYPE 1 (AT1) RECEPTOR BLOCKER,
TELMISARTAN, PROTECTS BRAIN DAMAGE INDUCED BY
ISCHEMIA AND AMYLOID-BA INJECTION PARTLY DUE TO
PPAR-C ACTIVATION
Masaki Mogi, J Iwanami, K Tsukuda, M Horiuchi
Ehime University Graduate School of Medicine, Department of Molecular Cardiovascular Biology and Pharmacology, Tohon, Ehime, Japan
Introduction: Telmisartan, an angiotensin II type 1 (AT1) receptor blocker
(ARB), has an unique feature which activates peroxisome proliferatoractivated receptor c (PPAR-c) compared with other ARBs. PPAR-c is
expressed in brain and its activation lead to attenuate neural inammation
and induce clearance of amyloid-b (A-b) peptide. Here, we investigated
the effect of telmisartan on brain damage induced by stroke and A-b
focusing on its PPAR-c activation. Materials and Methods: Mice were
administrated non-hypotensive doses of telmisartan, losartan and PPAR-c
antagonist, GW9662. Male KKAy mice were subjected to middle cerebral artery (MCA) occlusion and evaluated ischemic size. Male ddY
mice were undergone i.c.v.-injection of A-b (1-40) and evaluated spatial
learning by the Morris water maze test. Results: Treatment with telmisartan signicantly reduced neurological decit and ischemic size; however,
this reduction was not observed in mice with GW9662 co-administration.
Reduction of cerebral blood ow in the peripheral region was improved
by telmisartan treatment. These effects were weaker in mice treated with
losartan without PPAR-c activation, and not inuenced by GW9662. Pretreatment with telmisartan improved A-b -injection-induced cognitive
impairment. Co-administration with GW9662 attenuated this telmisartanmediated improvement of cognition. Co-treatment with GW9662 markedly reduced these benecial effects. A-b concentration measured by
ELISA in the brain was signicantly decreased by treatment with telmisartan, while co-administration with GW9662 attenuated the telmisartanmediated A-b deposition. Conclusion: Telmisartan had a protective effect
on brain damage induced by ischemia and A-b i.c.v.-injection. These
benecial effects of telmisartan were induced by partial agonistic effect
of PPAR-c as well as AT1 receptor blockade.
Paper No.: 901

PREPARATION OF ARTIFICIAL RECEPTORS FOR
CLOZAPINE AND STUDY OF THEIR BINDING PROPERTIES
Seyed Ahmad Mohajeri(1), G Karimi(2), M Rajabnia(2)
(1) Mashhad University of Medical Sciences, Pharmaceutical Research
Center, Mashhad, Iran
(2) Mashhad University of Medical Sciences, School of Pharmacy,
Mashhad, Iran
Introduction: Molecular imprinting is a technique in which functional
monomers are arranged and xed in a polymeric structure around a
465
template molecule. After washing the polymer, a cavity with a memory
of template molecule remains in a xed structure. Such an imprinted
polymer shows a higher afnity and selectivity for binding template in
comparison with blank non-imprinted polymer. In this study, clozapine
imprinted polymers were synthesized and their binding characteristics
was studied in comparison with their blank polymers. Materials and
method: Imprinted polymers were prepared using methacrylic acid as the
functional monomer with different colzapine/monomer ratios in organic
solvents. Clozapine binding to polymers were studied using rebinding
test and Scatchard analysis in acetonitrile. Results: Our data showed that
all imprinted polymers had higher afnity than non-imprinted ones for
binding clozapine. The best imprinted polymer was prepared in chloroform with a clozapine/monomer ratio of 1/5. The Scatchard analysis of
the best polymer indicated that two kinds of receptors (high and low
afnity) were synthesized during polymerization. Conclusion: In this
work a number of imprinted polymers were synthesized in organic solvents and their binding properties were studied in comparison with their
blank polymers. The data showed that the best template/monomer ratio
was 1/5 and the best solvent was chloroform. The high afnity of the
best polymer for binding clozapine demonstrated that this polymer can
be used as a sorbent in extraction of clozapine from biologic liquids with
a high selectivity and recovery.
Paper No.: 1312

ANALGESICS
THE INFLUENCE OF INTRAVENOUS PROMETHAZINE ON
INTRAVENOUS MORPHINE FOR THE TREATMENT OF
ACUTE SEVERE PAIN: A RANDOMISED CLINICAL TRIAL
Nasir Mohamad(1), AH Saim(1), NH Abu Bakar(2), KA Baharuddin(1),
R Ismail(2)
(1) University Sains Malaysia School of Medical Sciences, Emergency
Department, Kubang Kerian, Kelantan, Malaysia
(2) University Sains Malaysia, Institute for Research in Molecular Medicine (INFORMM), Health Campus, Pharmacogenetic Research Group,
Kubang Kerian, Kelantan
Morphine is frequently used in severe pain management. A frequent
accompaniment of morphine is the unpleasant side effects such as nausea
and vomiting for which promethazine is frequently used. Promethazine
has also been used to potentiate opiates. The objectives of this study
were to determine the effect of promethazine on morphine as an analgesic as well as to investigate its usefulness to prevent nausea and vomiting
due to morphine in acute pain management. This study was approved by
the Ethical Committee of Universiti Sains Malaysia. Patients who fullled the inclusion criteria were randomized into 2 groups, morphine
alone group and morphine with promethazine group. A clinical assessment was done before patients left the Emergency Department. One hundred patients were enrolled. Sixty-ve received morphine without
promethazine, 45 received morphine with promethazine. Mean numeric
pain score (NRS) at baseline was 7.2 (SD 0.40) for both the morphine
alone group and the morphine promethazine group. At 60 minutes, the
scores were 1.9 (SD 0.71), and 1.8 (SD 0.68) respectively for the two
groups (p=0.960). There was also no signicant difference in the occurrence of nausea (9.1% versus 8.9%) and vomiting (5.5% versus 4.4%)
when the groups were compared (p=0.626, p=0.595) respectively. We
conclude that there were no statistically and clinically signicant inuence of promethazine on morphine both in terms of improved analgesia
and in the prevention of nausea and vomiting secondary to morphine
analgesia.
Paper No.: 2035

METHYLSUFONYLMETHANE (MSM) TREATMENT
IMPROVES HEMODYNAMICS IN MONOCROTALINEINDUCED PULMONARY HYPERTENSION
Sadollah Mohammadi(1,2,3), A Garjani(3), M Naja(3), H Hamzeiy(3),
N Maleki-Dizaji(3), M Darabi(4), K Hassanzadeh(3), M Asadi(3)
(1) Tabriz University of Medical Sciences, Student Research Committee,
Department of Pharmacology and Toxicology, Tabriz, Iran
(2) Tabriz University of Medical Sciences, Research Center for Pharmaceutical Nanotechnology, Tabriz, Iran
and Toxicology, Tabriz, Iran
(4) Tabriz University of Medical Sciences, Department of Biochemistry,
Tabriz, Iran
MSM is a naturally accruing organic sulphur that is known as a potent
anti-oxidant/anti-inammatory compound. The aim of this study was to
investigate the effect of MSM on hemodynamics functions in rats with
monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH).
Wistar rats were randomly assigned to 38-days pretreatment or 28-days
treatment. MSM was administered to rats at 100, 200, and 400 mg/kg/
day doses either 10 days before or after a single dose of 60mg/kg, IP,
MCT. Rats were anesthetized with pentobarbital and hemodynamics of
ventricles were determined by Powerlab AD instrument .Blood and heart
tissue samples were obtained to evaluate changes in the antioxidative
system and inammatory gene expression. Right ventricular systolic
pressure (RSVP) was signicantly reduced and the mean arterial pressure
(MAP) was signicantly increased in dose dependent manners in MCTinduced pulmonary arterial hypertensive rats. There was also a similar,
non-signicant trend for the treatment protocol. Our present results suggest that long term administration of the MSM attenuates MCT-induced
PAH in rats. Should similar effects of MSM be found on markers of oxidative stress and on a selective inammatory gene expression prole, this
may have promise in the treatment and prevention of PAH.
Keywords: Methylsufonylmethane; hemodynamics; monocrotalineinduced pulmonary hypertension
Paper No.: 441

CLINICAL EFFICACY OF BOVINE COLOSTRUM IN
NON-ORGANIC FAILURE TO THRIVE
Yashar Moharamzad(1), Y Panahi(1), G Falahi(2), M Falahpour(2)
(1) Baqiyatallah Medical Sciences University, Research Center, Tehran,
Iran
(2) Tehran University of Medical Sciences, Tehran, Iran
Introduction: Management of non-organic failure to thrive (FTT), as one
of the most common presentations to paediatric clinics, is usually challenging. There is no report currently in the literature regarding the role of
bovine colostrum in FTT. Patients: In this randomized clinical trial, 120
children with mild or moderate nonorganic FTT were divided into 2
groups. One group (control) received routine treatments of FTT and the
other group (case), besides routine treatments, received supplementary
bovine colostrum at the dose of 40mg/kg/day for a 3-month period.
Fresh milk of Waldenstrom cows was pasteurized and through liolization process, capsule forms containing 500 mg of colostrum (450 mg
effective dose and 50 mg lactose) were produced. For quantitative measurements, Waterlow I (height for age) and Gomez (weight for age) indices were used. Results: Mean value of Gomez index in case group
(81.72) was signicantly higher than control group (77.12) at the end of
the third month of supplementation (P = 0.003). Such a difference was
not reported based on Waterlow I index between case and control groups
466
(92.91 vs. 91.71; P = 0.094). According to Gomez index, 12 patients
(20%) who received colostrum became healthy at the end of the third
month, which was signicantly higher than control group (2 cases,
3.3%); P = 0.006. Conclusion: Bovine colostrum supplementation for a
3-month period is a useful strategy without any side effects, in addition
to known medical and psychological treatments, to increase the weight
of children with nonorganic FTT.
Paper No.: 574

CLINICAL EFFICACY OF A TOPICAL HERBAL CREAM
(ALOE VERA, GERANIUM ROBERTIANUM AND LAVANDULA
STOECHAS) IN SECOND-DEGREE BURN WOUNDS
Yashar Moharamzad(1), Y Panahi(1), F Beiraghdar(1), I Feizi(2)
(1) Baqiyatallah Medical Sciences University, Research Center of
Chemical Injuries, Tehran, Iran
(2) Ardabil University of Medical Sciences, Department of Surgery,
Ardabil, Iran
Introduction: Healing of burn wounds is still a matter of concern in
almost every clinical setting. The application of topical silver sulfadiazine (SSD) 1% has been reported to associate with adverse reactions. We
decided to compare the clinical efcacy of SSD 1% cream with a herbal
cream (Aloe vera, Geranium robertianum, and Lavandula stoechas) in
burn patients. Patients: In this double-blinded randomized clinical trial,
111 patients (55 in SSD and 56 in herbal cream) admitted to hospital less
than 24 hours after suffering second-degree burns, which included less
than 5% of the total body surface, were treated topically once daily for a
two- week period. After debridemenet and cleaning the wound, ve
grams of cream was applied for each 10 cm2 of the burn area and covered with sterile gauze. Mean (standard deviation) duration of wound
healing and patients severity of pain using a 10-score visual analogue
scale (VAS) were evaluated by an attending physician. Results: Mean
duration of wound healing in herbal group (9.7 3.5 days) was shorter
than SSD group (12.8 1.8 days); P < 0.05. Mean pain VAS decrease
from baseline to day 14 in herbal group (5.68 3.2) was signicantly
higher than SSD group (4.54 2.83); P < 0.05. Only one patient developed wound infection in herbal group at day 7 which was resolved with
continuation of the treatment. Conclusion: Herbal preparation used here
showed better clinical efcacy in reducing pain and healing of seconddegree burn wounds in comparison to SSD 1%.
Paper No.: 1012

EVALUATION OF THE REPRODUCTIVE AND
DEVELOPMENTAL TOXICITIES OF THE AQUEOUS
EXTRACT OF LABISIA PUMILA VAR. ALATA IN FEMALE
RATS
Wan Ezumi Mohd Fuad(1), SS Amrah(2), J Hasnan(3), SSJ Mohsin(1)
(1) University Sains Malaysia Health Campus, School of Health
Sciences, Kota Bharu, Malaysia
(2) UniversitiySains Malaysia Health Campus, School of Health
Sciences, Departmentof Pharmacology, Kota Bharu, Malaysia
(3) University Sains Malaysia Health Campus, School of Health
Sciences, Departmentof Pathology, Kota Bharu, Malaysia
Labisia pumila var. alata (LPA) or Kacip Fatimah has been listed by the
government of Malaysia as one of the priority species for a multi-disciplinary scientic study towards the development of scientic, evidencebased herbal medicine. The present study was conducted to assess the
effect of aqueous extract of LPA on the oestrous cycle, reproductive
organs and serum hormonal levels of female rats. Groups of forty Sprague Dawley rats with regular oestrous cycle were given distilled water
(as control) or LPA at 20, 200 or 1000mg/kg/day daily by gavaging starting on the rst day of diestrus. They were sacriced at the same stage of
oestrous cycle upon completion of three weeks regimen. Laporatomy
was performed and reproductive organs (vagina, uterus and ovaries) were
weighed and prepared for histopathology. Blood serum was also collected for hormonal analysis. Results obtained revealed that LPA did not
alter oestrous cycle and general health of all rats. Neither signicant macroscopic changes nor momentous differences in weights of the reproductive organs were observed. This observation was veried by
histopathological investigation. However, the presence of ovarian follicular cysts and an increment trend of serum progesterone and free testosterone levels that were observed in the LPA 1000 mg/kg/day dose group
raised some concern even though these observations were not statistically
signicant. The current ndings suggest that oral treatment of LPA of up
to 1000 mg/kg/day is not associated with statistical signicant deleterious
effects in female rats.
Paper No.: 1942

DISEASES
TRPA1 AGONISTS IODOACETATE AND ALLYL
ISOTHIOCYANATE INDUCE COX-2 EXPRESSION IN
CHONDROCYTE AND MACROPHAGE CELL LINES
Lauri Moilanen, R Nieminen, E Moilanen
University of Tampere Medical School,and Tampere University Hospital,
The Immunopharmacology Research Group, Tampere, Finland
Transient receptor potential A1 (TRPA1) is a non-selective ion channel
linked to nociceptive sensations. TRPA1 has been shown to mediate
inammatory pain in in vivo models. There are also preliminary ndings
to suggest that TRPA1 may mediate some inammatory responses. In
inammation, various cytokines induce cyclo-oxygenase-2 (COX-2)
expression and subsequent prostaglandin (PG) production. PGE2 plays a
key role in inammation, inammatory pain and hyperalgesia. In the
present study, we investigated the effect of TRPA1 agonists on COX-2
expression in chondrocyte and macrophage cell lines. TRPA1 agonists iodoacetate and allyl isothiocyanate induced COX-2 expression in a dosedependent manner in H4 murine chondrocytes and in J774 macrophages.
Interestingly, COX-2 expression was greatly enhanced when the agonist
was added together with a proinammatory cytokine interleukin-1b.
TRPA1 agonists showed no effect on iNOS expression, or on nitric oxide
or interleukin-6 production. Presence of TRPA1 in these cells was investigated by Western blot. The results suggest that TRPA1 may enhance
COX-2 expression and contribute to the production of proinammatory
and hyperalgesic prostaglandins. Traditionally TRPA1 has been considered as a neural ion channel but the present results in chondrocytes and
macrophages propose that TRPA1 has also a role in joint inammation.
Paper No.: 1847

HEALTH AND DISEASE
ANTIANGIOGENIC EFFECT OF 4-HYDROXYCHALCONE
Jan Mojzis(1), L Varinska(1), M van Wijhe(2), P Koolwijk(2),
L Mirossay(1), G Mojzisova(3)
(2) VU University Medical Center Amsterdam, Laboratory for Physiology, Institute for Cardiovascular Research, Amsterdam, The Netherlands
(3) University of P.J. Safarik, Medical School, Department of Experimental Medicine, Kosice, Slovak Republic
467
Angiogenesis, the growth of new blood vessels from the pre-existing
vasculature is associated with physiological as well as pathological conditions (e.g. tumour development). Several natural compounds are able
to exhibit antiangiogenic activities. Chalcones are precursors of avonoids and isoavonoids and many studies and investigations suggested
that certain chalcones can inhibit tumor initiation as well as tumor progression. On the other hand, antiangiogenic effect of chalcones have
been studied only marginally. In the present study, we evaluated the antiangiogenic activity of chalcone analogues. Among the tested compounds,
4-hydroxychalcone (4-H) was the most potent angiogenic inhibitor. At
the concentration of 100 lmol.l-1 it inhibited the VEGF-induced proliferation and migration of human endothelial cells as well as the differentiation into tube structures on Matrigel coatings. In addition, the formation
of capillary-like tubular structures of human microvascular endothelial
cells on 3D brin matrices was completely inhibited without any signs
of cytotoxicity. The inhibition was accompanied by a decrease in urokinase-type plasminogen activator accumulation in the conditioned medium. Furthermore, we observed that 4-H inhibited the phosphorylation of
Akt and ERK1/2 pathways in response to VEGF or bFGF. However, our
results show that 4-H did not suppress the translocation of NF-jB stimulated by TNF-a In conclusion, 4-hydroxychalcone affects endothelial
cells as a negative mediator of angiogenesis in vitro and is therefore a
promising candidate for future study focused on elucidating the more
detailed mechanism of action.
This work was supported by the Slovak Research and Development
Agency under the contract No. APVV-0325-07.
Paper No.: 1852

ANTICANCER EFFECT OF HORSE CHESTNUT EXTRACT
Gabriela Mojzisova(1), J Mojzis(2), L Varinska(2), M Pilatova(2),
A Bomba(1)
(1) University of P.J. Safarik, Medical School, Department of Experimental Medicine, Kosice, Slovak Republic
In the present work, we tested horse chestnut extrakt (HCE) for their antiproliferative, antiangiogenic and proapototic effects on cancer cell lines
or human umbilical vein endothelial cells (HUVECs). The cell proliferation was evaluated by MTT assay and anchorage-independent growth by
colony forming assay (CFA). To understand the growth inhibitory effects,
carcinoma cell lines (Jurkat, CEM, HeLa and MCF-7) were treated with
various concentrations of HCE. Incubation of Jurkat, CEM, HeLa and
MCF-7 cancer cells with HCE at 0.125 mg/mL for 72h caused 38.8,
86.2, 79.6 and 59.6 % reduction in cell survival. CFA also conrmed
growth-inhibitory effects of compound studied. Cell cycle was analyzed
by ow cytometry and apoptosis was analyzed by DNA fragmentation.
In HCE treated cells we found signicant increase in the fraction of cells
with a sub-G0/G1 DNA content, which is considered to be a marker of
cell death by apoptosis. Apoptosis was also conrmed by DNA fragmentation. Furthermore, we have also tested the effects of the studied compound on the growth, endothelial cell migration (ECM) and matrix
metalloproteinase activity (MMP) in HUVECs. The level of VEGF
secreted by the cancer cells in the medium was determined using ELISA
kit. Our results suggest that HCE suppress cell proliferation, inhibits
angiogenesis and activity of matrix metalloproteinase and induce apoptosis in a dose-dependent manner in cancer cell lines.
This work was supported by the Slovak Research and Development
Agency under the contract No. APVV-0325-07.
Paper No.: 2323

THERAPEUTIC TARGETS
OZONE ATTENUATES IMIPRAMINE-INDUCED ANTIIMMOBILITY EFFECTS IN THE RAT, WHILE IMIPRAMINE
PROTECTS AGAINST OZONE-INDUCED OXIDATIVE
DAMAGE IN THE RAT HIPPOCAMPUS
Mmalebuso Mokoena, CB Brink, BH Harvey, DW Oliver
North-West University (PUK), Division of Pharmacology,
Potchefstroom, South Africa
Oxidative stress is well-recognised to play a role in the neurobiology of
depression. The effect of ozone as environmental toxin has not been
studied in this regard. We aimed to investigate the effects of acute and
chronic ozone inhalation on rat behaviour (immobility) in the forced
swim test (FST) and on hippocampal oxidative status, as well as its
effects on the antidepressant-like effect of imipramine. We also investigated the effects of imipramine on ozone-induced oxidative stress. Sprague Dawley rats were exposed to 0, 0.25 or 0.7 ppm ozone per
inhalation for 4 hours acutely (once), or to 0 or 0.25 ppm ozone chronically (daily for 30 days). On the last day animals were exposed to a
15 minutes pre-swim 24 hours before the nal test-swim in the FST, followed by the 1st imipramine (10 mg/kg) or saline intraperitoneal injection and the last ozone exposure. The 2nd and 3rd imipramine/saline
injections were administered 5 and 1 hours before the 5 minute scoring
swim. Hippocampal superoxide anion levels and lipid peroxidation were
determined. Imipramine successfully displayed antidepressant-like activity in the FST in the absence and presence of ozone regardless of ozone
exposure concentration or duration; however the antidepressant-like
effects were attenuated by ozone. Ozone induced oxidative damage following chronic but not acute exposure was signicantly reduced by imipramine treatment. The data suggest that ozone may impair the ability of
imipramine to reduce immobility. It also suggests that, while chronic
ozone induces hippocampal oxidative damage, antidepressant treatment
may protect against this.
Paper No.: 948

NEW HTRF SOLUTIONS FOR CELL SURFACE RECEPTORS
STUDY AND SCREENING. APPLICATION TO GPCR
DIMERIZATION AND HIGHLY SELECTIVE LIGAND BINDING
ASSAYS
Hamed Mokrane(1), E Trinquet(1), T Roux(1), L Comps-Agrar(2),
J-P Pin(2), J-L Tardieu(1)
(1) Cisbio Bioassays, Bagnols-sur-Ce`ze, France
(2) IGF, Montpellier, France
Cisbio introduces a new technological concepts for the investigation of
cell surface receptors. Based on the combination of SNAP-tag and
HTRF technologies, this new platform can applies for a comprehensive
investigation of receptor mechanistic. The receptor of interest is fused in
its N-terminus to SNAP-tag (20 kDa) and expressed at the cell surface.
We describe in this poster applications to G-protein-coupled receptor
(GPCR), and development of cell-based assays with the new proprietary
uorescent SNAP-tag substrates. This new technology is streamlined for
both highly selective ligand binding and receptor dimerization assays.
This rapid and non-radioactive platform will meet needs for a comprehensive and specic investigation of cell-surface-targets, and preserves
the functionality of the receptor and the intracellular signalling pathway.
The poster describes the simultaneous dynamic localization of both
GPCRs hetero- and homodimers at the cell membrane surface. Based on
this new platform, ligand binding assays were also optimized, results are
presented using COS7 cells expressing the vasopressin V1A receptor.
468
Paper No.: 2474
THE CONSERVED TRP IV:10 (4.50) TOGETHER WITH SER/
ASN II:05 (2.45) ARE CRUCIAL FOR 7TM RECEPTORS
ACTIVATION AND MIGHT BE A PUTATIVE CHOLESTEROL
BINDING SITE
Jacek Mokrosinski(1), R Nygaard(1,2), MS Engelstoft(1),
L Valentin-Hansen(1), B Holst(1), TW Schwartz(1)
(1) Laboratory for Molecular Pharmacology, University of Copenhagen,
Copenhagen, Denmark
(2) 7TM Pharma, Hrsholm, Denmark
Tryptophan residue in TM-IV (TrpIV:10) is known to be highly conserved among 7TM receptors. Recent crystallographic data have shown
TrpIV:10 facing lipid bilayer and making a hydrogen bond with SerII:05.
X-ray structures of b-2-adrenergic receptor suggest that TrpIV:10 together
with polar residue in TM-II (Ser II:05) and basic residue at the intracellular end of TM IV (from IV:03 to IV:-01) interact with cholesterol. A
mutational analysis of putative cholesterol docking site was performed in
the ghrelin receptor, B2AR, B1AR, GPR39, GPR119, NK1 and AT1
receptors representing a broad range of constitutive signaling activities;
from none to approx. 50% of Emax. Mutation of TrpIV:10 into Ala
almost eliminated agonist induced signaling in all receptors and in the
cases where it was appropriate also affected constitutive signaling. Introduction of Phe in general resulted in an intermediate signaling phenotype.
TrpIV:10 mutations in general did not affect receptor cell surface expression. Ala-substitution of Ser/AsnII:05, i.e. the putative hydrogen-bond
partner for TrpIV:10, had no or almost no effect in certain receptors, for
example the ghrelin receptor and B2AR, but eliminated agonist induced
signaling in for example the NK1 receptor. Mutation of the basic residue
located in positions IV:01 or IV:03 in general had no effect on receptor
signaling and cell surface expression. It is concluded that the conserved
TrpIV:10 as well as in some cases its putative hydrogen-bond partner in
position II:05 but not basic residues at the intracellular pole of TM-IV is
very important for receptor signaling in Family A 7TM receptors.
Paper No.: 1616

THE PROBLEM OF MEDICINES SAFETY MONITORING IN
KYRGYZSTAN
Saltanat Moldoisaeva, AZ Zurdinov
Kyrgyz State Medical Academy, Department of Basic and Clinical Pharmacology, Bishkek, Kyrgyzstan
Kyrgyzstan has been a full participant of WHO Program for International
Drug Monitoring since 2003; however, gathering of spontaneous reporting is still not adequately implemented. Our research investigated doctors activity level in monitoring medicines safety. We distributed 495
questionnaires to doctors. The result showed that 2/3 of doctors observed
the presence of adverse drug reactions (ADRs) in their practice. Of these
doctors, only 49 % indicated regular registration of ADRs on medical
records; 51 % seldom register or do not register in general. In a question
regarding the submission of yellow cards upon the development of
ADRs, only 9% answered positively, 74% negatively, and 17% refrained
to answer. Principal causes of low level of registration of ADRs according to doctors are: rst, lack of information for detecting the association
between drug administration and event; second, the erroneous opinion
that all medical products registered in our state medical system are safe
and third, the fear of answering to hospital administration. Our conclusion shows that the problem of ADRs monitoring in Kyrgyzstan remains
unresolved. As our research indicates, doctors have a serious deciency
of knowledge about ADRs and it is a principal cause of the low level of
their registration. From this, it follows that the level of preparation of

medical workers in clinical pharmacology must be increased, workers in
public health must be motivated, and communications mechanisms must
be reformed.
Paper No.: 3304

SCREENING AND CHARACTERIZATION OF GPCR
C-TERMINAL TAIL INTERACTIONS WITH THE PSD-95 PDZ
DOMAINS
Thor Mller(1), P Jacky(2), A Bach(3), K Strmgaard(3), T Schwartz(2),
K Martinez(1)
(1) University of Copenhagen, Department of Neuroscience and Pharmacology & Nano-Science Center, Bio-Nanotechnology Laboratory, Copenhagen, Denmark
(2) University of Copenhagen, Laboratory for Molecular Pharmacology,
Department of Neuroscience and Pharmacology, Copenhagen, Denmark
(3) University of Copenhagen, Department of Medicinal Chemistry,
Copenhagen, Denmark
G protein-coupled receptor (GPCR) interacting proteins mediate and
modulate GPCR signalling and regulate GPCR trafcking. Some have
been known for many years, such as G proteins and arrestins, but a large
number of other proteins play a role; the largest family of these is PDZ
domain proteins. PDZ domain proteins often function as scaffolds by
simultaneously binding the C-terminus of GPCRs and other proteins
downstream in the signalling cascade, usually resulting in enhanced
GPCR signalling (Stifer MA et al, Science 2007; 317: 364-369, Ritter
SL & Hall RA, Nat. Rev. Mol. Cell Biol. 2009; 10: 819-830). The identication and characterization of novel interactions between GPCRs and
scaffolding proteins remains tedious due to the complexity of the cellular
composition. Here we present the quantitative screening of a library of
C-terminal tails of GPCRs (Heydorn A et al, J. Biol. Chem. 2004; 279:
54291-54303) for interactions with the scaffolding protein postsynaptic
density protein 95 (PSD-95). GST pull-down is used to identify potential
new interactions and uorescence polarization is used to determine the
afnity of the interactions with each of the three PDZ domains of PSD95. Several known PSD-95 interactors were identied in the screen, validating the approach, but also several potentially novel interactors were
identied. The afnities of the identied GPCRs for the PDZ domains of
PSD-95 were found to be of low afnity and were generally higher for
the second PDZ domain. The method can be further extended to other
GPCR interacting proteins.
Paper No.: 481

A COMPARATIVE STUDY OF MEDICINAL PLANTS USED
AGAINST GASTROINTESTINAL DISORDERS IN TWO
DISTRICTS OF BANGLADESH
Ariful Haque Mollik(1), R Faruque(1), R McField(1), A Chowdhury(2),
KK Thapa(3), T Islam(4)
(1) Peoples Integrated Alliance, Dhaka, Bangladesh
(2) State College of Health Sciences, Dhaka, Bangladesh
(3) Dinhata College, Dhaka, Bangladesh
(4) Biogene Life Care, Dhaka, Bangladesh
Gastrointestinal problems in the form of diarrhea, dysentery, helminthiasis, acidity, and peptic ulcer are fairly common in the population of Bangladesh. The whole country is divided into sixty-four districts. The rural
populations of the various districts rely primarily on traditional medicinal
469
practitioners for cure of gastrointestinal problems. These traditional
medicinal practitioners [locally known as Kaviraazes] have their individual formulations based on medicinal plants to treat ailments, which can
vary between districts. It was thus of interest to obtain information on
medicinal plants that are used to treat gastrointestinal problems in various
districts. We chose the districts of Kurigram and Gazipur for this purpose. The locations of these districts are respectively in north and central
Bangladesh. The Kaviraazes were interviewed and information on medicinal plants, formulations, dosages, and ailments were collected. Medicinal plant samples were collected and identied at the Bangladesh
National Herbarium. The Kaviraazes of Kurigram district use Centella
asiatica, Carica papaya, Corchorus capsularis, Cassia stula, Citrus
acida, Aegle marmelos, Brassica napus, Amaranthus viridis, Paederia
foetida, Musa sapientum, Cocos nucifera, Curcuma longa, Phyllanthus
emblica, Allium sativum, Mentha spicata, and Zingiber ofcinale to treat
dysentery, indigestion, acidity, diarrhea, stomach pain, and atulence.
The Kaviraazes of Gazipur district use whole plant or plant parts of
Cocos nucifera, Tamarindus indica, Citrus acida, Brassica napus, Allium
sativum, Paederia foetida, Musa sapientum, Aegle marmelos, and Plantago major as remedy for indigestion, intestinal bleeding, stomach pain,
constipation, dysentery, peptic ulcer, and helminthiasis. The use of multiple medicinal plants opens up possibilities of novel drug discoveries.
Paper No.: 482

ETHNOMEDICINAL USES OF SOME PLANT SPECIES BY
THE TRADITIONAL HEALERS IN GAIBANDHA DISTRICT OF
BANGLADESH
Ariful Haque Mollik(1), R Faruque(1), R McField(1), D Sen(2),
KK Thapa(3), AI Hassan(4)
(1) Peoples Integrated Alliance, Dhaka, Bangladesh
(2) State College of Health Sciences, Dhaka, Bangladesh
(3) Dinhata College, Dhaka, Bangladesh
(4) Biogene Life Care, Dhaka, Bangladesh
An ethnomedicinal survey of plant species of Bangladesh was carried
out in Gaibandha district. This is the area in Bangladesh whose inhabitants subsist: primarily on pastoralist & agriculture, and therefore still preserve the traditional knowledge on plant species. This study represents
rst comprehensive ethnomedicinal survey in Gaibandha district of Bangladesh and is also an attempt to sum up the preexisting ethnomedicinal
information. The traditional healers were familiar mostly with the plant
species dealing with common ailments particularly coughs/colds, digestive problems, fevers, headaches, and skin infections. Extensive interviews were conducted on the traditional healers of Gaibandha district.
Plant specimens as pointed out by the traditional healers were collected
and identied at the Bangladesh National Herbarium. A total of twentyeight plant species are being reported from the study area which were
used for the treatment of coughs/colds, digestive problems, fevers, headaches, and skin infections. These plant species included Amaranthus blitum, Chenopodium album, Typhonium giganteum, Euphorbia prostrata,
Syzygium aromaticum, Triticum aestivum, Oryza sativa, Piper nigrum,
Lens culinaris, Artocarpus heterophyllus, Amaranthus spinosus, Momordica charantia, Carica papaya, Mentha spicata, Diospyros peregrina,
Allium cepa, Cuminum cyminum, Allium sativum, Cocos nucifera, Citrus
acida, Ipomoea batatas, Nymphaea nouchali, Abrus precatorius, Cymbopogon citratus, Kalanchoe pinnata, Aegle marmelos, Andrographis paniculata, and Musa sapientum. The reliance on traditional medicines for
health care is associated with the lack of modern medicines and medication, poverty, and the traditional beliefs of its effectiveness. However,
pharmacological studies are carried out in order to conrm the validity
of properties attributed to these plant species.
Paper No.: 2476

GENOTYPIC DETERMINANTS OF CYP2D6 ACTIVITY AND
ITS CLINICAL IMPLICATIONS IN INDO-TRINIDADIANS
Lazara Montane Jaime(1), A Lalla(1)
The University of the West Indies, Department of Pharmacology, St
Augustine, Trinidad & Tobago
Introduction: Limited information is available on the frequency of
CYP2D6 alleles, its correlation with CYP2D6 phenotypes and its clinical
consequences in Indian populations. Identifying the alleles with the
greatest therapeutic impact in Indo-Trinidadians may improve therapeutic
outcome and safety of CYP2D6-based drug regimes. Methods: CYP2D6
metabolic activity of 174 healthy Indo-Trinidadian was determined by
measuring urinary dextromethorphan-dextrorphan metabolic ratio (MR).
Subjects were classied as poor metabolizers (pPMs) if MR > 0.3; efcient metabolizers (pEMs) if 0.03 > MR < 0.003, intermediate metabolizers (pIMs) if 0.3 > MR < 0.03, and ultrarapid metabolizers (pUMs) if
MR < 0.003. The distribution of the CYP2D6 *2; *3; *4; *5; *6; *7;
*8/14; *10; *41, *69 and the duplicated alleles was determined. Results:
The percentage of subjects with altered enzymatic activity was 41.8%.
CYP2D6*4 allele explained the majority of the pPM (4%) while
CYP2D6*41 and CYP2D6*10 alleles explained 20.6% of the pIM. However, genotypic IMs account for only 43.3% of pIMs and gPMs for only
57% of pPMs. The relatively large number of pUMs (17.2%) was not
fully explained by the low frequency of duplicated alleles (20%) which
also included duplication of PM alleles. Conclusions: The signicant
number of subjects with altered CYP2D6 enzymatic activity may
increase the possibility of adverse effects and treatment failure in IndoTrinidadians treated with CYP2D6-based drug regimes. When performing CYP2D6 genetic testing in Indo-Trinidadians CYP2D6*4, *5, *10
and *41 alleles must be considered. The lack of complete phenotypegenotype correlation implies that additional underlying factors need to be
explored in Indo-Trinidadians.
Paper No.: 3334

MAKING THERAPEUTIC DRUG MONITORING OF
EFAVIRENZ WORK: WHAT IS A SUBTHERAPEUTIC
CONCENTRATION?
Emilia C Monteiro(1,2), U Caixas(1,3), I Germano(3), S Gouveia(3),
A Carlos(3), F Lampreia(3), T Branco(3), SA Pereira(1,2)
(1) New University of Lisbon, Faculty of Medical Sciences, Department
of Pharmacology, Lisbon, Portugal
(2) CEDOC, New University of Lisbon, Faculty of Medical Sciences,
Lisbon, Portugal
(3) Centro Hospitalar de Lisboa - Zona Central, Lisbon, Portugal
Background: One of the issues to apply in therapeutic drug monitoring
of efavirenz (EFV) is the absence of a consensus about the reference
value to dene a subtherapeutic concentration. Methods: The relationship
between HIV viral load and EFV Cthrough was evaluated. The more discriminating efcacy cut-offs for EFV were studied by using the raw
Cthrough or time-corrected Cthrough, by the precise delay since the last
efavirenz intake. Results: Raw Ctrough includes samples (n=107) collected in a broad time interval after efavirenz intake (Median 11 h). Median (IQR) EFV raw Ctrough was 2.27 (1.59-2.95) mg/L. Adjusted
Ctrough was 1.69 (1.15-2.18) (p<0.0001). Among those with detectable
viral load, adjusted Ctrough was associated with viral load (Spearman
r=0.4258, p=0.0239) and CD4 cell count (Spearman r=0.4596,
p=0.0139). Patients with detectable viral (1.346 (0.651-1.912) mg/L)
load had lower EFV plasma raw Cthrough than those with undetectable
viral load (1.739 (0.65-1.92) mg/L) (p=0.0138). Detectable viral load
470
was observed in twelve out of 20 patients (60%) with adjusted EFV
Ctrough bellow 1 mg/L and in eighteen out of 87 adjusted EFV Ctrough
above 1 mg/L (17%). The number of patients with subtherapeutic concentrations is two fold higher when concentrations are time adjusted.
Conclusions: EFV Cmin can be extrapolated correcting raw Cthrough
with based on t1/2 and the correct time between blood sample collecting
and last drug intake. The value of 1 mg/L can be used as reference for
Cmin value but not a raw Cthrough at other time point after efavirenz
intake.
Paper No.: 2031

EFFECTS OF THE CROTALUS DURISSUS CASCAVELLA
VENOM ON RENAL TUBULAR CELLS
Janaina Monteiro, A Torres, R Menezes, F Monteiro, R Dantas,
A Martins, H Monteiro
Federal University of Ceara, Department of Pharmacology, Fortaleza,
Brazil
Snakebites involving Crotalus genus are commonly serious, occurring
several complications, such acute renal failure. Previously, it has been
demonstrated that Crotalus durissus cascavella (Cdcasca) venom cause
nefrotoxicity, possibly by direct action on tubules and glomeruli or /and
release of mediators from cells. The aim of work was to study the effect
of Cdcasca venom on tubular Mardin-Dabin Canine Kidney (MDCK)
cells. MDCK cell were cultured in RPMI 1640 medium supplemented
with 10% of fetal bovine serum, and maintained at 37ordm;C and 5%
CO2 atmosphere. The cells were dislocated using tripsine/EDTA, plated
at 1x106cells/mL in a 96-well plate and incubated with different concentrations of Cdcasca (100, 50, 25, 12.5, 6.25 and 3.125 lg/mL) for 24h,
and then the mitochondrial activity was measured by reduction of MTT
in tetrazolium salts. Also the supernatant of the culture was collected to
the measurement of lactate dehydrogenase (LDH) enzymatic activity, to
verify the release of cytoplasmatic material, using a spectrophotometer
method. Cdcasca reduced in cell viability of MDCK cells in all the concentrations studied, and there was not a signicant release of LDH when
compared with control group. In conclusion, Crotalus durissus cascavella
caused cytotoxicity on renal tubular cells.
Paper No.: 1925

CHANGES IN THE EXPRESSION OF A1 AND B-ADRENOCEPTORS OR OF GRKS IN HUMAN AND RAT HEARTS INDUCED
BY CHRONIC CARVEDILOL TREATMENT
Ferm Josep Monto Guillot(1), D Vicente(1), E Oliver(1), D Barettino(3),
J Rueda(2), J Aguero(2), L Almenar(2), P DOcon(1)
(1) University of Valencia, Department of Pharmacology, Valencia, Spain
(2) Hospital Universitario La Fe, Valencia, Spain
(3) Instituto de Biomedicina. CSIC, Valencia, Spain
Aim: to determine the changes induced by carvedilol treatment in the
expression of adrenoceptors a1A, a1B, a1D, b1, b2 and b3 and of the
GRKs modulating their activity (GRK2, GRK3, GRK5) in human and
rat hearts. Methods: we used human left ventricles of patients with
dilated cardiomyopathy or ischemic heart disease who either received or
did not carvedilol with other treatments. We also used left ventricles of
the Wistar rats either treated or not with carvedilol (20mg/kg day) for
3 weeks. Total RNA from the human and rat samples was isolated using
the Tripure Isolation Reagent (Roche Biochemicals). cDNA was obtained
from 1 lg RNA with (dT)16 as a primer and Improm II RT (Promega).
The mRNAs encoding the 9 genes and GAPDH (the housekeeping gene)
were quantied by TaqMan real-time PCR using the TaqMan Gene

Expression Assays (Applied Biosystems) pre-designed probes. Results:
in human heart failure, Carvedilol decreases the mRNA levels for both
b1-AR (38.5911.44, n= 11 vs. untreated patients 77.2513.65*, n=8)
and GRK5, the main GRK expressed in the human heart (54.247.33 vs.
82.349.52*), irrespectively of other treatments received or pathologies
(dilated cardiomyopathy or ischemic heart disease). We observed similar
results in carvedilol-treated rats for b1-AR (56.593.96, n= 4, vs.
98.2214.98**, n =5) and for GRK2, the main GRK expressed in the rat
heart (48.633.86 vs. 35.073.29**). Conclusions: Carvedilol decreases
mRNA for b1-AR and the main GRK in the heart. This paradoxical regulation may relate to the cardioprotective actions of Carvedilol in heart
failure.
Paper No.: 1919

INSULIN RESISTANCE IN THE OBESE ZUCKER RAT IS NOT
ASSOCIATED WITH PULMONARY ARTERIAL
HYPERTENSION
Javier Moral-Sanz, C Menendez, L Escolano, L Moreno, A Cogolludo,
F Perez-Vizcaino
University Complutense de Madrid, Department of Pharmacology,
Madrid, Spain
Insulin resistance and obesity have emerged as current pandemic diseases
tightly related to cardiovascular complications. Recent reports have
related non insulin dependent diabetes with pulmonary arterial hypertension (PAH) in human and animal models. The aim of the study was to
analyze a possible link between insulin resistance and PAH in the obese
Zucker rat (OZR). Right ventricular wall thickness was similar in OZR
and their lean counterparts (LZR). Lung BMPR2, KV1.5 and 5-HT2A
receptor expression measured by Western blot or RT-PCR were also similar in the two rat strains. Additionally, similar KV current density was
registered in isolated pulmonary artery smooth muscle cells using the
patch clamp technique. In conductance and resistance pulmonary arteries,
the similar relaxant responses to acetylcholine and nitroprusside and
unchanged lung eNOS expression revealed a preserved endothelial function. However, in resistance (but not in conductance) pulmonary arteries
from OZR a reduced response to several vasoconstrictor agents (hypoxia,
phenylephrine and 5-HT) was observed. Interestingly, the hyporesponsiveness to vasoconstrictors was prevented by the iNOS inhibitor
1400W. In conclusion, in contrast to animals models of type 1 diabetes
or other models of insulin resistance, the OZR did not show any of the
characteristic features of PAH but rather a reduced vasoconstrictor
response associated to iNOS.
Paper No.: 2831

HEALTH AND DISEASE
NEUTRAL SPHINGOMYELINASE-NADPH OXIDASE AS A
REDOX AMPLIFICATION PATHWAY FOR HYPOXIC
PULMONARY VASOCONSTRICTION
Laura Moreno(1), G Frazziano(1), C Menendez(1), J Moral-Sanz(1),
E Moreno(1), L Escolano(1), C Gonzalez(2), E Villamor(3), A Cogolludo(1), F Perez-Vizcaino(1)
(1) University Complutense of Madrid, School of Medicine, Department
of Pharmacology and CIBERES, Madrid, Spain
(2) University of Valladolid and CSIC, Department of Physiology and
IBGM, Spain
(3) Maastricht University Medical Center (MUMC+), School for Oncology and Developmental Biology (GROW), Maastricht, The Netherlands
471
Mitochondrial electron transport chain (ETC) and NADPH oxidase have
been proposed as possible oxygen sensors underlying hypoxic pulmonary vasoconstriction (HPV), with derived reactive oxygen species
(ROS) playing key roles in coupling the sensor(s) to the contractile
machinery. However, the true signicance of ROS as players in HPV is
controversial. We have recently reported that activation of neutral sphingomyelinase (nSMase) and protein kinase C zeta (PKCz) participate in
the signaling cascade of HPV (Cogolludo A et al., Cardiovasc Res 2009;
82: 296-302). Herein, we studied the relationship between nSMase and
ROS production in rat pulmonary artery (PA). Global tissue ROS production (analyzed by dichlorouorescein and dihydroethidium uorescence) was increased by hypoxia in endothelium-denuded distal PA
segments. Inhibitors of mitochondrial ETC (rotenone) and NADPH oxidase (apocynin) prevented hypoxia-induced ROS production and vasoconstriction. Further supporting a role for ROS production, hypoxia
induced p47phox phosphorylation and its interaction with caveolin-1.
Inhibition of nSMase (GW4869), PKCz or mitochondrial ETC (rotenone)
prevented p47phox phosphorylation and ROS production. nSMasederived ceramide (analyzed by immunocytochemistry) was inhibited by
rotenone. However, mithocondrial superoxide production (analyzed by
MitoSOX Red uorescence) was decreased by hypoxia in PA segments
suggesting a more complex regulation of ROS which may differ among
subcellular compartments (Waypa GB et al., Circ Res 2009; Epub.) We
propose an integrated signaling pathway for HPV which includes the
mitochondrial ETC as the sensor and nSMase-PKCz-NADPH oxidase as
a necessary redox amplication pathway required for ROS production
and vasoconstriction.
Paper No.: 905

HEALTH AND DISEASE
ANTIHYPERTENSIVE EFFICACY OF ALISKIREN/
HYDROCHLOROTHIAZIDE COMBINATION:
A META-ANALYTICAL APPROACH
Manuel Morgado(1), S Morgado(1), L Castanheira(2), A Macedo(2),
M Castelo-Branco(2)
(1) Hospital Centre of Cova da Beira, Pharmaceutical Services, Covilha,
Portugal
(2) University of Beira Interior, Health Sciences Research Centre,
Covilha, Portugal
Introduction: Aliskiren/hydrochlorothiazide (A/H) single-pill combinations havebeen approved by the European Medicines Agency on the
16th January 2009 for thetreatment of essential hypertension in adults.
The purpose of this study was toassess the antihypertensive efcacy of
A/H in hypertensive patients. Methods: Asearch in MEDLINE was performed (January 2000 - November 2009), to identifyrandomised clinical
trials (RCTs) using A/H for the treatment of hypertension.Studies were
included if they evaluated the antihypertensive efcacy of A/H inpatients
with mild or moderate essential hypertension and age 18 years. Twoauthors selected studies and extracted the data independently. The efcacy
oftreatment was calculated using the weighted average reductions of systolic anddiastolic BP for each daily dosage combination. Results: We
included 5 RCTstesting several combinations of A/H and containing data
on 5448 patients. In allstudies, BP was assessed at inclusion (baseline)
and after 8 weeks of therapy.Mean systolic and diastolic BP were 152.5/
98.0 mmHg at inclusion and weresignicantly reduced by the several
combinations of A/H: -15.8/-10.3 mmHg(150/25 mg); -15.9/11.8 mmHg (300/12.5 mg); -16.9/-11.6 mmHg (300/25 mg). BPcontrol
rates (%) for the above combinations were, respectively: 43.9; 50.1
and51.9. BP reductions and control rates were signicantly higher with
the A/Hcombinations than with the same dosages of aliskiren or HCTZ
monotherapy.Conclusion: A/H provides clinically signicant additional
BP reductions andimproved BP control rates over aliskiren or HCTZ
alone and the single-pillcombination offers the convenience of a singletablet treatment regimen.
Paper No.: 3336

EFFECT OF KAMPO, CHINESE TRADITIONAL MEDICINE
ON CLOCK- AND METABOLISM-RELATED GENE
EXPRESSION IN MICE
Takeshi Morishima, G Koiwa, Y Tahara, A Hirao, S Shibata
Background: circadian clock gene is expressed not only in the suprachiasmatic nucleus, a main oscillator, but brain and peripheral organs.
Clock system controls the physiological function through clock gene
expression spread over whole body. Recently, it is elucidated that expression of clock gene is controlled by AMPK, Ppar a and Sirt1 suggesting
interaction between circadian clock and nutrition metabolism. On the
other hand, Kampo drugs, Chinese traditional medicine have been used
for disease and for health promoting effect through whole body. Then,
we studied whether Kampo may affect clock- and metabolism-related
gene, and improve circadian rhythm disorders and obesity. We selected
Juzen-taiho-to from 12 Kampo drugs through preliminary screening
experiments. Method: mouse body weight changes were examined under
ad lib feeding of powdered normal or high-fat diet containing 3% Juzentaiho-to or 0.3% each Shyoyaku herb which is a component of Juzen-taiho-to. Then mice were sacriced and gene expression of Per2, PGC-1a,
Sirt1 in the liver and visceral fat were measured by RT-PCR. Results and
Discussion: Juzen-taiho-to reduced the gene expression of Per2 and
increased that of PGC-1aand Sirt1. Moreover, in the experiment of 0.3%
each Shyoyaku herb, Touki increased PGC-1, Sirt1 gene expression, and
Saiko reduced Per2 gene expression. The present results suggest that Juzen-taiho-to may affect the metabolic syndrome and circadian system
disorder through Shoyaku herbal components such as Touki and Saiko.
Paper No.: 1338

NOVEL PATHWAY OF SMOOTH MUSCLE CONTRACTION BY
UTP IN RAT AORTIC MYOCYTE
Hiromitsu Morita(1), M Sugihara(0), M Matsuda(2), S Kajioka(3),
Y Ito(4), K Abe(1), M Hirata(2)
(1) Kyushu University Hospital, Special Patient Oral Care Unit,
Fukuoka, Japan
(2) Kyushu University Faculty of Dental Sciences, Department of
Molecular andCellular Biochemistry, Fukuoka, Japan
(3) Kyushu University Graduate School of Medical Sciences,
Department of Urology,Fukuoka, Japan
(4) Kumamoto Health Science University, Department of Health Science,
Kumamoto,Japan
Introduction: UTP is one of important substances, released from cells
surrounding vascular smooth muscle, for regulation of vasoconstriction.
It is broadly known that UTP is an agonist selective to P2Y receptors,
such as P2Y2, 4and 6 receptors, but not P2X receptors. However, we
identied that UTP activated P2X1-like receptors and thus caused vasoconstriction by activation of L-type voltage dependent calcium channel
(VDCC). Materials: Male Sprague-Dawleyrats (10-11 weeks old) were
used. Tension measurement, patch-clamp method, RT-PCR and Western
blot analysis were used for evaluation. Results: UTP(10 lM) caused a
biphasic, phasic and tonic, contraction. The phasic contraction was
almost completely inhibited by removal of extracellular Ca2+, application
of nifedipine (10 lM) or TNP-ATP (30 lM), a selective P2Xreceptor
antagonist, while the tonic contraction was abolished by thapsigargin(2microM) in a Ca2+-free bath solution. Patch-clamp recording also
472
revealed that UTP (10 lM) activated P2X1-like inward current, which
was inhibited by TNP-ATP and PPADS and suramin, potent P2 receptor
antagonists. UTP and a,b-methylene ATP, which is a potent P2X1 channel agonist, were mutually competitive. Moreover, functional antibody of
P2X1 channels also reduced theUTP-induced current. Single channel analysis also revealed that UTP activated10.5pS channel similar to a, b-methylene ATP. Western blot and RT-PCR showed a highly-expression of P2X1
receptor in aorta. Conclusion: These results suggest that UTP play important roles in both phasic and tonic contraction through novel P2X1/L-type
VDCC and classical P2Y/intracellular Ca2+ release pathways.
Paper No.: 2210

THE TREATMENT OF SEXUAL DYSFUNCTION AND DISEASES OF THE LOWER URINARY TRACT
REGULATION OF UROTHELIAL SPONTANEOUS
CONTRACTILE ACTIVITY
Christian Moro, R Chess-Williams
spectrum antiprotozoal agent. The properties of these two groups of compounds were combined by synthesizing metronidazole thiosemicarbazone
analogues, wherein the thioamide moiety of the thiosemicarbazone backbone was substituted by different cyclic and aromatic amines. The sensitivity of the chloroquine-sensitive P. falciparum strain (3D7) to the
compounds was assessed using the tritiated hypoxanthine incorporation
assay. The compounds were evaluated for their ability to inhibit b-haematin formation as well as their haemolytic properties. All but one of the
compounds inhibited parasite growth, with IC50 values below 10lM.
Results from the b-haematin assay indicated that, with the exception of
two compounds, all compounds inhibited b-haematin formation as effectively as quinine. Red blood cell toxicity results indicated negligible
amounts of haemolysis and were comparable to those of quinine and
metronidazole. Overall, (1E)-1-(4-((E)-2-(1-(2-hydroxyethyl)-5-nitro-1Himidazol-2-yl)vinyl)benzylidene)-4-cyclooctylthiosemicarbazide (Y1) and
(1E)-4-(2-chlorobenzyl)-1-(4-((E)-2-(1-(2-hydroxyethyl)-5-nitro-1H-imidazol-2-yl)vinyl)benzylidene)thiosemi-carbazide (Y3) were the most
active, with IC50 values of 2.990.10lM and 2.890.09lM respectively,
as well as possessing the best safety prole. When combined with quinine Y3 exhibited an additive relationship. These results indicate that
metronidazole thiosemicarbazones show promising antimalarial activity
and necessatate further investigation.
Bond University, Department of Health Sciences and Medicine, gold

Coast, QLD,Australia
The bladder urothelium (urothelium plus suburothelium) plays an important role in regulating detrusor contractile activity but also exhibits a
spontaneous contractile activity of its own. The aim of this study was to
identify which receptors when activated, can cause changes in the frequency of spontaneous urothelial contractions. Isolated strips of urothelium (plus suburothelium) were mounted in gassed Krebs-bicarbonate
solution and frequencies of spontaneous contractions (cycles per minute)
were recorded in the absence and presence of the drugs. Data obtained
was analysed using paired Students t-tests. Urothelial regular phasic
activity was present throughout the course of the experiment and this
was altered following cholinergic, adrenergic or purinergic receptor stimulation. Stimulation of nicotinic receptors with 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP, 100lM, n=8), or a2-adrenoceptors with
clonidine (100nM, n=9) did not inuence urothelial contractile frequency
or basal tension. However, stimulation of a1-adrenoceptors with phenylephrinine (100lM, P<0.01 n=15) increased the frequency by 39 9%.
Stimulation of muscarinic receptors with carbachol (1lM, P<0.001, n=27)
increased the frequency by 122 27%, while stimulating purinergic receptors with a,b-methylene ATP (10lM, P<0.01, n=26), increased the frequency by 43 12%. In contrast the stimulation of b-adrenoceptors with
isoprenaline (10lM, P<0.05, n=8) reduced urothelial spontaneous rate
by -49 16%. The results demonstrate an independent urothelial activity
that may be regulated by both cholinergic and adrenergic nervous
systems, and the independent urothelial activity within this layer identies complex functions of the urothelium in regulating bladder activity.
Paper No.: 660

THE ANTIMALARIAL PROPERTIES OF METRONIDAZOLE
THIOSEMICARBAZONE ANALOGUES
Paper No.: 2812

THE DEVELOPMENT OF A NOVEL SYSTEM FOR THE
INVESTIGATION OF MICROVASCULAR DYSFUNCTION
E Moss, L Rankin, S Lynagh, David Bunton
Biopta Ltd, Glasgow, UK
Adverse drug reactions may be manifested through changes in microvascular function (e.g. angioedema) or by subtle modication of the mechanisms controlling vascular tone, such as ow-mediated dilatation. Until
now the early detection of such adverse drug reactions has been hampered by the lack of a relevant in vitro model. The PM-1, an automated
perfusion myograph, allows detection of the external and internal dimensions of tubular biological structures and regulates both the pressure and
intraluminal ow independently. Drugs can be infused intraluminally or
extraluminally to determine effects on constriction, relaxation or modulation of vascular tone. To assess effects on ow mediated-dilatation we
increased ow rate in pressurised human subcutaneous arteries
(<500 lm diameter) in the absence and presence of various drugs.
Increasing ow from 0.04 ml/min to 0.3 ml/min resulted in a 39+3%
relaxation of a U46619 pre-constriction (10-6 M). This was enhanced in
the presence of ivermectin and inhibited in the presence of 100 microM
L-NAME (316+169% and 16+1% respectively). To assess effects on vascular permeability we infused albumin-bound Evans blue dye through
the lumen of human subcutaneous arteries as a marker, in the absence
and presence of a modulatory drug. Infusion of thrombin (0.5 units/ml)
through the vessel lumen caused an 11.8% increase in vessel permeability compared to vehicle infusion. The development of the PM-1 allows
new drugs to be tested in relevant human or animal tissues at an early
stage giving a more complete picture of the overall effects of test compounds on vascular function.
Emma Moseley(1), R van Zyl(1), M Abid(2), I Havlik(1), A Azam(2)

(1) Pharmacology Dvision, Department of Pharmacy and Pharmacology,
University of the Witwatersrand, Johannesburg, South Africa
(2) Department of Chemistry, Jamia Millia Islamia, Delhi, India
Ninety percent of malaria cases reported in sub-Saharan Africa are due
to the Plasmodium falciparum parasite. For decades malaria chemotherapy has hinged on a limited range of drugs, with the parasite developing
resistance to some. This has initiated a search for novel compounds with
unique mechanisms of action against plasmodia species. Thiosemicarbazones are a small class of compounds with reported antifungal and antimalarial effects; while metronidazole is a commonly used broad
Paper No.: 615

THE CHEMOTHERAPEUTIC ACTION OF SYNTHETIC DYES
AGAINST PLASMODIUM FALCIPARUM
Harold Motau, R Van Zyl, L Harmse
University of the Witwatersrand, Faculty of Health Sciences, Pharmacology Division, Department of Pharmacy & Pharmacology, Johannesburg,
South Africa
473
Plasmodium falciparum is responsible for over 80% of the clinical
malaria cases that result in high mortality rates especially amongst children below 5 years old and pregnant women. With the increasing resistance of P. falciparum to classic antimalarial drugs and the absence of a
vaccine, the need for new chemotherapeutic agents is critical in controlling the disease. In previous studies, thiazine dyes such as methylene
blue have demonstrated antimalarial activity. The purpose of the study
was to investigate the effect of monoazo and heterocyclic dyes on the in
vitro growth of erythrocytic stages of P. falciparum. Synchronized, chloroquine-sensitive strain of P. falciparum (3D7) was maintained in culture.
The in vitro parasite growth inhibition was measured using the [3H]hypoxanthine incorporation assay after 48 hrs of compound exposure, to
identify lead compounds that inhibited in vitro growth by 50% at concentrations (IC50 values) <100 lM from log-dose response curves. Toxicity
against uninfected human red blood cells and the ability to inhibit bhaematin formation was determined spectrophotometrically. Of the 32
compounds tested, methylene blue (MB), safranine O and mercury
orange (MO) demonstrated antimalarial activity at IC50 values of
2.090.70, 4.122.21 and 16.132.28 lM, respectively; although were
less active than quinine (0.1040.009 lM). Only MO demonstrated
RBC toxicity at a concentration of 4.111.03 lM. The compounds did
not inhibit b-haematin formation at equimolar ratios of haem to drug.
Both MB and SO interacted in an antagonistic manner when combined
with quinine. The observed antimalarial activity of these colourants and
their structural derivatives warrants further investigation.
Paper No.: 1131

NORADRENALINE PROTECTS NEURONS AGAINST
HYDROGEN PEROXIDE TOXICITY BY INCREASING THE
RELEASE OF GLUTATHIONE FROM ASTROCYTES
Paper No.: 2882

BLOOD LEVEL OF CYCLOSPORINE-A IN IRANIAN RENAL
TRANSPLANT PATIENTS
Manijeh Motevalian(1), H Otukesh(2), RH Shamsabadi(2), M Chalian(1)
(1) Iran University of Medical Sciences, Department of Pharmacology
and Razi Institute for Drug Research, Tehran, Iran
(2) Iran University of Medical Sciences, Aliasghar Hospital, Department
of Nephrology, Tehran, Iran
Frequent blood level monitoring in transplant patients is necessary due to
variabilities in cyclosporine-A (CsA) pharmacokinetics and total blood
cyclosporine concentration used for dosage adjustment in these patients.
The purpose of this study was to use a suitable method for determination
of cyclosporine blood level in renal transplant children and to nd out
the best time for TDM sampling. In this study twenty-nine pediatric
transplant recipients with stable renal function, were taking part. The
clinical status of the patients were recorded. The CsA levels were determined by radioimmunoassay of blood samples on time 0, 0.5, 1, 1.5 and
2 hours after drug administration. Results are indicative of good precision and reproducibility of the method. Minimum detection limit of
cyclosporine-A was 5 ng/ml and percentage yield was 86-109%. Interand intra-day variability were 8% and 5.8% respectively. The mean
blood concentrations 0.5 and 1 hour after drug administration were 100
3.0 and 515 19.2 lg/dl respectively. The mean serum creatinin level
was 0.9 (0.1-1.9) mg/dl. There was a high correlation between CsA dose,
serum creatinin and C1.5 level. The method was a precise and sensitive
method suitable for TDM of cyclosporine-A. Using single point monitoring can help to improve the cooperation of patients during TDM procedure. The C1.5 CsA levels seems more accurate than C0 in pediatric
transplant patients and is the best indicator for TDM of cyclosporine-A
in Iranian children and has the best correlation with dose and creatinin
level.
Toshiki Motegi, Y Yoshioka, A Yamamuro, A Kasai, S Maeda

Setsunan University, Faculty of Pharmaceutical Science, Department of
Pharmacotherapeutics, Osaka, Japan
Hydrogen peroxide (H2O2) has been implicated in a variety of neurodegenerative disorders, such as Parkinsons disease. Astrocytes express
many types of functional neurotransmitter receptors, and play a signicant role in maintaining survival of neurons. Recently, we nd that pretreatment with noradrenaline (NA) attenuates H2O2-induced cell death of
astrocytes through the increase in the level of intracellular glutathione
(GSH). In this study we investigated the neuroprotective effect of NA by
using the single culture of human neuroblastoma SH-SY5Y cells or the
co-culture system of SH-SY5Y cells and human astrocytoma U-251 MG
cells. We established SH-SY5Y cells that constitutively express green
uorescence protein (GFP), and the cell viability was determined based
on the morphology of GFP-positive cells under a uorescence microscope. Pretreatment with NA (10-30 microM) for 24 h protected SHSY5Y cells from H2O2-induced death in co-culture, but not in single culture. DL-buthionine-[S,R]-sulfoximine, a glutathione (GSH) synthesis
inhibitor, negated the cytoprotective effect of NA in co-culture. Treatment with NA concentration dependently increased the GSH release
from U-251 MG cells. An MRP-1 inhibitor MK-571, which inhibits the
GSH release via MRP-1, prevented the cytoprotective effect of NA in
co-culture. These results indicate that NA protects SH-SY5Y cells from
H2O2-induced death by increasing the synthesis and release of GSH from
U-251 MG cells.
Paper No.: 2961

EFFECTS OF ROSIGLITAZONE ON THE PREJUNCTIONAL
FACILITATORY ACTIONS OF ANGIOTENSIN II AND
A-2-ANTAGONISTS
Daniel Moura(1), H Pinheiro(2), F Silva(1), L Pereira(1), R Bastos(1)
(1) University of Porto, Faculty of Medicine, Institute of Pharmacology
andTherapeutics, Porto, Portugal
(2) University of Porto, Faculty of Pharmacy, Porto, Portugal
AT-1 receptor antagonists block the angiotensin II-enhancing effect on
noradrenaline release from sympathetic neurons (Guimaraes et al, Naunyn-Schmiedebergs Arch Pharmacol 2001;363:509-14). Storka et al
(Eur J Clin Invest 2008;38:820-6) reported that in a cell-free assay the
binding afnity of the AT-1 receptor antagonists telmisartan and valsartan
to the c peroxisome proliferator-activated receptor (PPARc) is close to
that of the PPARc selective agonist rosiglitazone. We tested whether rosiglitazone would also modify the prejunctional facilitatory effect of
angiotensin II. Facilitation by a-2-autoreceptor antagonists was also
tested in the presence of rosiglitazone. Left ventricular slices of rats were
incubated with tritiated noradrenaline, perifused and electrically stimulated. The negative logarithm of the drug concentration that caused a
30% increase of control (pEC30%) was calculated. Angiotensin II caused
a concentration-dependent increase of tritium overow induced by electrical stimulation [pEC30%=8.60.2 (meansem, n=18); maximum
increase=1108%]. Rosiglitazone (0.3-3 lM) had no direct effect. The
concentration-response to angiotensin II in the presence of xed concentrations of rosiglitazone was shifted to the left with increase of the maxi-
474
mum (pEC30%=8.80.2, 9.20.2 and 9.30.3; maximum increase=
11814%, 14613% and 14816%, in the presence of 0.3, 1 and 3
microM of rosiglitazone, respectively, n=4-6, each). The release-enhancing effects of neither rauwolscine nor phentolamine (1-300 nM) were
changed by rosiglitazone. Results show that rosiglitazone potentiates the
noradrenaline-release enhancing effect of angiotensin II without changing
the release-enhancing effect of a-2-receptor antagonists suggesting that a
positive allosteric modulation of angiotensin II receptors by PPARc
agonists may occur in sympathetic terminals.
Paper No.: 2487

A2C-ADRENOCEPTOR BLOCKAGE INCREASES DOPA
UPTAKE AND DOPAMINE SYNTHESIS IN THE CENTRAL
NERVOUS SYSTEM OF MICE
The cytotoxic properties of total methanol extract of S. litwinowii and its

fractions were investigated on different cancer cell lines including AGS,
HeLa, MCF-7, PC12 and NIH 3T3. Meanwhile the role of apoptosis in
this toxicity was explored. Methods: The cells were cultured in DMEM
medium and incubated with different concentrations of herb plant
extracts. Cell viability was quantitated by MTT assay. Apoptotic cells
were determined using propidium iodide staining of DNA fragmentation
by ow cytometry (sub-G1 peak). Results: S. litwinowii inhibited the
growth of malignant cells in a dose dependent manner. Among solvent
fractions of S. litwinowii, the methylene chloride fraction was found to
be more toxic compared to other fractions. The IC50 values of this fraction against AGS, HeLa, MCF-7 and PC12 cell lines after 24 h were
determined, 121.2 3.1, 40.9 2.5, 115.9 3.5, 64.5 3.4 lg/mL
respectively. S. litwinowii induced a sub-G1 peak in the ow cytometry
histogram of treated cells compared to control cells indicating that apoptotic cell death is involved in S. litwinowii toxicity. Conclusion: S. litwinowii exerts cytotoxic and proapototic effects in a variety of malignant
cell lines and could be considered as a potential chemotherapeutic agent
in cancer treatment.
Eduardo Moura(1), C Esteves-Pinto(2), MP Serrao(2), MA Vieira-Coelho(2,3)

(1) Instituto de Biologia Celular e Molecular, UP, Porto, Portugal
(2) Instituto de Farmacologia e Terapeutica, FMUP. Porto, Portugal
(3) Departamento de Psiquiatria, Hospital de Sao Joao, Porto, Portugal
a2C-adrenoceptor antagonists have been proposed has having therapeutic
value in the treatment of neuropsychiatric disorders associated with
enhanced startle responses and sensorimotor gating decits, such as
schizophrenia and drug withdrawal. In this study we investigated the
effect of a2C-adrenoceptor blockade on 3,4-dihydroxyphenylalanine
(DOPA) and dopamine (DA) synthesis in the central nervous system of
mice. DOPA and DA levels and the activity of the enzyme DOPA decarboxylase (AADC) were evaluated in the brain of C5BL/6J wild type
(WT) and a2C-adrenoceptor knockout (a2CKO) mice. The same parameters were evaluated in mice treated with the selective a2C-antagonist JP1302 (3micromol/Kg) or vehicle (saline) as three intraperitoneal injections given with 12h intervals. The assay of catecholamines was carried
out by HPLC-ED. Results are given as arithmetic meansem. DOPA and
DA levels were signicantly higher in the a2CKO mice compared to WT
(WT: 11617, 2739509; a2CKO: 30013, 4648861 pmol/g tissue,
DOPA and DA, respectively). This increase was accompanied by a signicant increase in AADC activity (WT: 401; a2C: 501, Vmax in
nmol/mgprot/h). Treatment with the a2C-adrenoceptor antagonist also
produced a signicant increase in DOPA levels (WT: 10112; a2CKO:
20829 nmol/g tissue) accompanied by an increase in DA (WT:
2821302; a2CKO: 43261060 nmol/g tissue) but without changes in
AADC activity (WT: 425; a2C: 411, Vmax in nmol/mgprot/h). In conclusion, in mice deletion of a2C-adrenoceptor (a2CKO) or blockade with
a selective antagonist produces an increase in DOPA and DA brain tissue
levels. Higher L-DOPA tissue levels could be explained by an increase
in uptake.
Supported by grant PTDC/SAU-FCF/66502/2006.
Paper No. 857

FOCUS GROUP: P16 - NATURAL PRODUCTS: PAST AND
FUTURE?
ANALYZING CYTOTOXIC AND APOPTOGENIC PROPERTIES
OF SCUTELLARIA LITWINOWII ROOT EXTRACT ON
CANCER CELL LINES
Seyed Hadi Mousavi, SA Emami, Z Tayarani-Najaran, J Asili, A Mirzaei
Mashhad University of Medical Sciences, Department of Pharmacology,
Mashhad, Iran
Introduction: The Scutellaria species is used as a source of avonoids to
treat a variety of diseases in traditional medicine. Anti cancer researches
on one of the Iranian species S. litwinowii have not yet been conducted.
Paper No. 858

FOCUS GROUP: P19 - GENERAL SESSION ONCOLOGY
STUDY OF NOSCAPINE-INDUCED CELL DEATH IN
HEPATOCELLULAR CARCINOMA CELL LINE
Seyed Hadi Mousavi, H Parsaee, Z Tayarani-Najaran
Mashhad University of Medical Sciences, Department of Pharmacology,
Mashhad, Iran
Current therapies for cancer treatment are often limited by the emergence
of drug resistance and side effects. There is much interest in the identication of new agents for cancer chemotherapy. Noscapine is an isoqiunoline alkaloid found in opium. It is not sedative and has been used as
antitussive drug in different countries. Recently, noscapine has been
introduced as an anti-mitotic agent. In this study cytotoxic effect of noscapine was evaluated in hepatocellular carcinoma (HepG2) cell line.
Meanwhile role of apoptosis was explored. HepG2 and non-malignant
cells (L929) were cultured in DMEM medium and incubated with different concentrations of noscapine and paclitaxel for 24, 48 and 72 h. Cell
viability was quantitated by MTT assay. Apoptotic cells were determined
using Annexine-V PI staining method. The results showed noscapine
could decrease cell viability in HepG2 cells as a concentration and timedependent manner. The IC50 value against HepG2 was determined after
48 h . Apoptosis was involved in the cytotoxic effect of noscapine. In
addition, noscapine-induced toxicity was accompanied with generation
of intracellular ROS production. Thus, noscapine exerts cytotoxic effects
in HepG2 cells in which apoptosis or programmed cell death plays an
important role. Noscapine could be considered as a potential chemotherapeutic agent in hepatocellular carcinoma in future.
Paper No.: 926

BAICALEIN PROTECTS THE BRAIN AGAINST NEURON
IMPAIRMENTS INDUCED BY MPTP IN C57BL/6 MICE
Xin Mu(1), G He(2), X Li(2), G Du(1)
(1) Chinese Pharmacological Society, Institute of Materia Medica,
Chinese Academy of Medical Sciences, Beijing, PR China
(2) National Center for Pharmaceutical Screening, Beijing, PR China
Introduction: Many studies of Parkinsons disease suggest that oxidative
stress is involved in the neurodegenerative process. Baicalein, a avonoid obtained from the root of Chinese medicinal herb Scutellaria baicalensis, has been shown to have antioxidant effects. The present study
475
examines the effect of baicalein on 1-methyl-4-phenyl- 1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity in C57BL/6 mice. Experimental approach: Mice were injected with MPTP for 5 consecutive days
followed by treatment with baicalein for a week, and the subsequent
behavioral, biochemical and immunohistochemical manifestations were
determined. Results and Conclusion: MPTP treatment impaired spontaneous motor activity and rotarod performance, but baicalein improved
this decit. Moreover, baicalein at 280 and 560 mg/kg exhibited a
protective effect against the MPTP-induced decrease in tyrosine
hydroxylase-positive bers in the substantia nigra, demonstrated by the
immunohistological, morphological and behavioural outcomes. MPTP
treatment also decreased dopamine levels in the striatum. However,
treatment with baicalein attenuated these decreases in dopamine levels
by changing dopamine catabolism and inhibiting dopamine turnover.
In addition, baicalein could inhibit oxidative stress in the striatum after
MPTP treatment. The neuroprotective effect of baicalein on dopaminergic neurons may partly owe to its antioxidant properties. Therefore, baicalein might be a promising candidate for prevention or treatment of
oxidative stress-related neurodegenerative disorders such as Parkinsons
disease.
Paper No.: 426

CLINICAL TOXICITIES OF FIRST-LINE ANTIRETROVIRAL
THERAPY IN A RESOURCE LIMITED SETTING
Tinashe Mudzviti(1), C Maponga(1,2), S Khoza(3), Q Ma(2),
D Tagwireyi(1)
(1) University of Zimbabwe, School of Pharmacy, Avondale, Harare,
Zimbabwe
(2) University at Buffalo, SUNY, Center of Excellence in Bioinformatics
and Life Sciences, Buffalo, New York, NY, USA
(3) The University of Texas at Austin College of Pharmacy, Austin, TX,
USA
Dilemmas exist between balancing cost and toxicity of antiretroviral
therapy (ART) in resource limited settings (RLS). First line therapy in
Zimbabwe for treating HIV is dispensed as a triple xed dose combination of stavudine, lamivudine and nevirapine. Most pharmacovigilance programs in RLS are unable to monitor adverse drug reactions
(ADRs) due to inconsistent laboratory support systems. The study was
carried out to determine the rate and nature of toxicities associated
with ART in a RLS (Zimbabwe). 388 HIV positive adults stable on
rst line ART dispensed from Parirenyatwa Hospital, Harare, Zimbabwe were interviewed and their respective patient charts were consulted. 279 (72%) participants experienced at least one ADR. The
ADRs that were observed included peripheral neuropathy (42%), skin
rash (26%), lipodystrophy (3%), abdominal pain (8%), gastro-intestinal
symptoms (7%) and headache (3%). Peripheral neuropathy and skin
rash were mainly observed with stavudine (93%) and nevirapine
(88%) based regimens respectively. Lipodystrophy occurred only in
participants on stavudine based therapy. 161(58%) ADRs were grade
1 events (World Health Organizations ADR grading system), 96(34%)
were grade 2, 15(7%) were grade 3 and 1(0.3%) was grade 4. One
patient on a nevirapine containing regimen had grade 4 Stevens Johnson syndrome. 8 patients were switched from a stavudine containing regimen after developing grade 3 peripheral neuropathy. 5 patients
were switched from nevirapine based regimens after developing grade
3 rashes. Data derived from within the country may have greater relevance in decision making, formulating preventative strategies and
effective patient management.
Paper No. 1831

FOCUS GROUP: PW12 - EXPERIMENTAL PAIN METHODS IN
DEVELOPMENT AND VALIDATION OF ANALGESICS
ANTINOCICEPTIVE EFFECT OF ENDOMORPHIN 1
ADMINISTERED USING POLYBUTYLCYANOACRYLATE
NANOPARTICULATE DELIVERY SYSTEMS
Ostin Mungiu(1), D Geta(2), I Jaba(1), B Tamba(1), C Bohotin(1),
X Patras(3)
(1) UMF Gr.T.Popa, Centre for the Study and Therapy of Pain, Department ofPharmacology-Algesiology, Iasi, romania
(2) Gh. Asachi Technical University, Iasi, Romania
(3) Petre Andrei University, Iasi, Romania
Numerous synthetic, pharmacological and physicochemical studies have
focused on opioid peptides as possible substitutes for alkaloid opiate
drugs due to their biological importance as natural analgesics. Yet the
penetration of the blood-brain-barrier remains an insurmountable obstacle
for an efcient analgesic effect of opioid peptides delivered systemically.The aim of this study was to evaluate brain delivery of endomorphin 1, an antinociceptive opioid tetrapeptide that does not cross bloodbrain barrier (BBB) by itself, following the administration using polybutylcyanoacrylate nanoparticulate delivery systems (PBCA-NDSs),
coated with Tween 80. Two different formulations of loaded nanoparticles were prepared, depending on the moment of drug addition/inclusion
during or after polymerization.In vivo central antinociceptive effect of
endomorphin 1 was quantied by thermoalgesic tests (tail ick and hot
plate) following the intraperitoneal administration of PBCA-NDSs in
mice. Our results demonstrated a signicant analgesia following administration of endomorphin 1 with PBCA-NDSs. The Tween 80 coating
increases the BBB penetration and in consequence the antinociceptive
effect. Signicant differences were observed depending upon the type of
loading used for endomorphin 1.The success of coated PBCA-NDS can
be hypothesized due to the association of a number of factors as particle
size (<100nm) and their coating with Tween 80. Hence, it could be concluded that Tween 80 surface coated PBCA-NDS are a good candidate
for brain targeting of endomorphin 1 or other opioid peptides administered systemically. Acknowledgements: This work was supported by a
Romanian Education and Research Ministry grant, PNCDI IDEI (no.
1734/2008). Key words: endomorphin-1, nanoparticles, polybutylcyanoacrylate, Tween 80, analgesia.
Paper No.: 1944

THE EFFECTS OF THE AQUEOUS EXTRACT OF ASYSTASIA
GANGETICA ON THE BLOOD PRESSURE AND HEART RATE
IN SPONTANEOUSLY HYPERTENSIVE MALE WISTAR RATS
THE EFFECTS OF THE AQUEOUS EXTRACT OF ASYSTASIA
GANGETICA ON THE BLOOD PRESSURE AND HEART RATE
IN SPONTANEOUSLY HYPERTENSIVE MALE WISTAR RATS
Pierre Mugabo, J-A Amunjela, D Hanse, G Hikumwah, F Hoedemaker,
M Motsoaole, P Mwandingi, X Sambo
(1) University of the Western Cape, Department of Pharmacology, Cape
Town, South Africa
In this study, the effect of Asystasia gangetica (A. gangetica) alone and
in combination with Angiotensin I (Ang I) and Angiotensin II (Ang II)
on the blood pressure (BP) and heart rate (HR), was evaluated. Drugs
were administered intravenously. The HR and BP were measured via a
pressure transducer connecting the femoral artery to a powerlab and a
computer for recording. A. gangetica signicantly (P<0.05) reduced the
systolic blood pressure (SBP), diastolic blood pressure (DBP) and mean
arterial blood pressure (MABP) by 42.54%, 47.10% and 42.38% respectively. A. gangetica did not affect the HR. Ang I and Ang II signicantly
(P<0.05) increased the SBP, DBP and MAP. No signicant changes in
HR were recorded after administration of Ang I and Ang II. Co-adminis-
476
tration of A. gangetica with Ang I signicantly (P<0.05) decreased the
SBP, DBP, MAP and HR by 31.41%, 40.23%, 36.75% and 40.10%
respectively. Co-administered with Ang II, A. gangetica signicantly
(P<0.05) decreased the SBP, DBP, MAP and HR by 30.83%, 51.98%,
43.70% and 50.66% respectively. A.gangetica decreased the BP by
inhibiting ACE and Ang II receptors. After co-administration with Ang I
andAng II, the mechanism by which A.gangetica decreased the HR is
unknown at this stage. This is a motivation for further studies.
Paper No.: 697

MOST FREQUENT ADVERSE DRUG REACTIONS IN
CARDIOLOGY PATIENTS REGISTERED WITH INTENSIVE
MONITORING SYSTEM
Snezana Mugosa(1), Z Todorovic(2)
(1) University of Montenegro Medical School, Department for Pharmacology & Clinical Pharmacology, Podgorica, Montenegro
(2) University of Belgrade Medical School, Institute for Pharmacology,
Clinical Pharmacology and Toxicology, Belgrade, Republic of Serbia
Introduction: Adverse drug reactions (ADRs) occur often in hospitalized
patients. The aim was to establish intensive monitoring system and to
analyze adverse drug reactions (ADRs) in hospitalized patients at Cardiology Department. Materials/patients: This prospective study covered
200 patients hospitalized during a ve month period in the Cardiology
Department, Clinical Center of Montenegro. With intensive monitoring
system, ADRs information was collected in the form of interviews based
on the symptoms list and any signs that could point to eventual adverse
drug reactions. Apart from that, lab tests and other available parameters
were observed. Results: Interviewed patients had in total 1692 hospital
days, with average hospitalization duration of 8.463.03 days. At the
time when interviews took place, patients received on average 7.962.63
medicines. In total, 67 patients (34%) had 75 ADRs. The most frequent
ADRs were caused by streptokinase (19%), metoprolol (12%) and isosorbide dinitrate (8%). Fifty four ADRs have resulted in the recovery of
the patient (72%). Twenty one ADRs (28%) are classied as serious eight had as an outcome prolonged hospital stay (11%), another eight
were life threatening (11%), while ve were the cause of the hospitalization (6%). The most frequent ADRs which had as an outcome admission
to hospital were caused by digoxin (40%), prolonged hospital stay by
furosemide (38%), while the most frequent registered ADRs which were
life threatening were caused by streptokinase (50%). Conclusion: Our
data conrm that ADRs occur frequently in cardio patient population
during hospitalization. Additional educational efforts could afrm the
rationalization of pharmacotherapy.
Paper No.: 2104

FREQUENCY OF ADVERSE DRUG REACTIONS TYPE A AND
TYPE B REGISTERED WITH INTENSIVE MONITORING
SYSTEM
Snezana Mugosa(1), Z Todorovic(2), N Duborija-Kovacevic(1)
(1) University of Montenegro Medical School, Department for Pharmacology & Clinical Pharmacology, Podgorica, Montenegro
Clinical Pharmacology and Toxicology, Belgrade, Republic of Serbia
and characterized by high frequency, reverse character and low mortality

rate. Type B ADRs primarily depend on the specicity of human body;
they occur very rarely, they are not common and expected, not dose
dependent and are characterized by low frequency and worse outcome.
The aim was to analyse ADRs of type A and type B obtained by intensive monitoring system. Materials/patients: This prospective study covered 200 patients hospitalized in ve months period in the Cardiology
Department, Clinical Center of Montenegro. With intensive monitoring
system, ADRs information was collected in the form of interviews based
on the symptoms list and any signs that could point to eventual adverse
drug reactions. Apart from that, lab tests and other available parameters
were observed. Results: At the time when interviews took place, patients
received on average 7.962.63 medicines. In total, 67 patients (34%) had
75 ADRs. Sixty-nine ADRs were of type A (92%), and six type B (8%).
The most frequent ADRs of type A were caused by selective b blocator
metoprolol, resulted in unconsciousness, dizziness and headaches and
bradycardia. The most frequent ADR of type B were caused by streptokinase, resulted in itching, nettle rash, breathlessness, tremor, face redness
and fall in blood pressure. Conclusion: Frequency of ADRs at cardiological patients of type A is signicantly higher comparaed to type B, which
is in accordance with the date that exist in medical literature.
Paper No.: 2105

SPONTANEOUS REPORTING OF ADVERSE DRUG
REACTIONS AND LEVEL OF INTERVENTION
Snezana Mugosa(1), Z Todorovic(2), A Boskovic(3), N Duborija-Kovacevic(1)
(1) University of Montenegro Medical School, Department for
Pharmacology & ClinicalPharmacology, Podgorica, Montenegro
ClinicalPharmacology and Toxicology, Belgrade, Republic of Serbia
(3) Clinical Center of Montenegro, Cardiology Center, Podgorica,
Montenegro
Introduction: With spontaneous reporting of adverse drug reactions
(ADRs), doctors give their personal contribution in expanding knowledge in ADRs, which can prevent the serious consequences that carries
pharmacotherapy. Materials/patients: The prospective study covered 655
patients hospitalized in ve months period in Cardiology Center of Clinical Center of Montenegro. In this study we used a modied Form for
registration of ADRs of Agency for Medicines and Medical Devices
Montenegro. Results: With spontaneous reporting, 22 patients had 22
ADRs which were noted. For eight ADR (36%) level of intervention
was two (dose adjustment or drug stopped, no additional treatment
required), for nine ADRs (41%) level of intervention was three (dose
adjustment or drug stopped, other treatment required), while for ve
ADRs (23%) the level of intervention was four (transfer to intensive care
setting). The most frequent ADRs after which the drug dose was
adjusted, or drug stopped, while the additional treatment was not needed,
caused a selective b blocator metoprolol (25%). The most frequent
ADRs after which the drug dose was adjusted, or drug stopped, while
the additional treatment was needed, caused streptokinase (55%). The
most frequent ADRs after which the patient was transferred to the intensive care unit, also caused streptokinase (40%). Conclusion: Health
workers, through their everyday clinical practice, are in such a position
that can most easily identify and report suspected ADRs. Therefore, they
should understand reporting of ADRs as part of their professional and
moral responsibility.
Introduction: Adverse drug reactions (ADRs) type A are related to pharmacological drug features; the reactions are dose dependant, predictible
477
Paper No.: 1884
PHARMACOLOGICAL EVALUATION OF THE
ANTIDEPRESSANT EFFECTS OF NEW A2-ADRENOCEPTOR
ANTAGONISTS
Carolina Muguruza(1,2), F Rodriguez(3), JE Ortega(1,2),
L Uriguen(1,2), JJ Meana(1,2), I Rozas(3), LF Callado(1,2)
(1) University of the Basque Country (UPV/EHU), Department of
Pharmacology, Leioa,Bizkaia, Spain
(2) Centro de Investigacion Biomedica en Red de Salud Mental,
CIBERSAM, Spain
(3) University of Dublin, Trinity College, School of Chemistry, Centre
for Synthesisand Chemical Biology, Dublin, Ireland
Depression is associated with a selective increase in the high-afnity
conformation of the a2-adrenoceptors (a2-AR) in the human brain Thus,
the development of new selective a2-adrenoceptor antagonists can be
considered as an effective therapeutic approach for the treatment of
depressive disorders The aim of the present study was to evaluate the
antidepressant effect of three new synthesized guanidine and 2-aminoimidazoline derivatives with a2-AR antagonist properties First, we tested the
effects of these compounds on noradrenergic transmission in vivo by
microdialysis experiments in rats Systemic administration of 17b
(10 mg/kg ip) increased noradrenaline extracellular concentration by
37373% (F[1,33]= 9570, P< 00001, n= 6) Administration (10 mg/kg
ip) of 8b and 20b increased rat cortical noradrenaline basal values
16130% and 15635%, respectively Both increases were statistically
signicant when compared with control (saline 02 ml/kg ip) The tail suspension test was used in mice to assess the antidepressant activity of the
compounds The 8b compound signicantly reduced the immobility time
at 10 and 20 mg/kg doses (p<0005 vs controls) The 17b compound only
reduced the immobility time at 40 mg/kg (p<001 vs controls), whereas
the 20b showed a signicant effect at 20 mg/kg (p<001 vs controls) The
well-known antidepressant uoxetine induced a signicant effect at
40 mg/kg (p<001 vs controls) These results support that these new synthesized a2-ARs antagonists may be useful antidepressant drugs in the
future.
Paper No.: 740

A NOVEL POLYMORPHISM IN ABCB1 GENE, CYP2B6*6 AND
SEX PREDICT SINGLE DOSE EFAVIRENZ POPULATION
PHARMACOKINETICS IN UGANDANS
Jackson K Mukonzo(1,2), D Roshammar(3), P Waako(2),
M Andersson(2), T Fukasawa(2), L Milani(4), J Ogwal- Okeng(2),
L Gustafsson(1), E Aklillu(1)
(1) Karolinska Institute, Stockholm, Sweden
(2) Makerere University, Kampala, Uganda
(3) University of Gothenburg, Gothenburg, Sweden
(4) Uppsala University, Uppsala, Sweden
Efavirenz exhibits pharmacokinetic variability causing varied clinical
response. The aim was to develop an integrated population pharmacokinetic/pharmacogenetic model and investigate impact of genetic variations
and sex on single dose efavirenz pharmacokinetics using NONMEM
simulation approach among Ugandans. Efavirenz plasma concentrations
(n=402) from 121 healthy subjects were quantied by HPLC. Subjects
were genotyped for 30 SNPs, six of them novel SNPs in CYP2B6,
CYP3A5 and ABCB1. The efavirenz pharmacokinetics were described
by a two-compartment model with zero- followed by rst-order absorption. Apparent oral clearance (95% CI) was 4 (3.5, 4.5) L/h in extensive
metabolisers. In the nal model, statistical signicance were found for
CYP2B6*6 and CYP2B6*11 on apparent oral clearance and ABCB1
(rs3842) on the relative bioavailability. Subjects homozygous for

CYP2B6*6 (G516T, A785G) and *11 displayed 21 and 20% lower
apparent oral clearance, respectively. Efavirenz relative bioavailability
was 26% higher in subjects homozygous for ABCB1 (rs3842). The
apparent peripheral volume of distribution was two-fold higher in
females compared to males. Efavirenz t1/2 was 1.5-fold longer for homozygous mutants compared to wild-type subjects and 2-3 fold longer for
females than males. The model identied CYP2B6*6, CYP2B6*11, a
novel variant allele in ABCB1 (rs3842) and sex as major predictors of
efavirenz plasma exposure in the Ugandan population.
Paper No.: 741

GENETIC VARIATIONS IN ABCB1 AND CYP3A5 AS WELL AS
SEX INFLUENCE QUININE DISPOSITION AMONG
UGANDANS
Jackson K Mukonzo(1,2), P Waako(2), J Ogwal-Okeng(2),
L Gustafsson(1), E Aklillu(1)
(1) Makerere University, Kampala,Uganda
(2) Karolinska Institutet, Stockholm, Sweden
Quinine is one of most effective antimalarial drugs although its clinical
use is limited due to its narrow safety margin. Quinine is a substrate of
the polymorphic p-glycoprotein and CYP3A4/3A5. This study aimed to
examine the effects of genetic variations in ABCB1 and CYP3A5 genes
and sex on quinine disposition among Ugandans. Quinine (600mg) was
orally administered to 140 healthy volunteers. Quinine and its metabolite
3-hydroxyquinine concentrations were determined from 16-hour post
dose plasma by HPLC. CYP3A5 activity was measured using Quinine/3hydroxyquinine ratio (MR). Genotyping for a total of 20 SNPs in
ABCB1 (n=13) and CYP3A5 (n=7) was done using Taqman and minisequencing on microarray. There was a 30 and 13 fold variations in
plasma quinine concentrations (mean SD; 5.10 2.4 lM, range; 0.54 16.70) and quinine-to-3-hydroxyquinine metabolic ratio (mean sd;
7.68 3.3, range; 1.66 - 22.3) respectively. Plasma quinine concentration
was signicantly inuenced by sex and ABCB1 haplotype. There was a
signicant sex difference in quinine MR, women being fast metabolizers
than men (p=0.01). CYP3A5 genotype/haplotype signicantly (p=0.03)
inuenced quinine disposition with a clear CYP3A5*1 gene dose effect.
The result conrms that quinine disposition is inuenced mainly by sex
as well as by ABCB1 and CYP3A5 genotypes. This may have clinical
signicance in determining individuals susceptibility to quinine associated side effects such as cinchonism.
Paper No.: 2789

DISEASES
EFFECT OF HELICOBACTER PYLORI INFECTION ON THE
ADHERENS JUNCTIONS OF GASTRIC PARIETAL CELLS IN
MONGOLIAN GERBILS
Motonobu Murakami(1), Y Tamura(1), Y Kuroda(1), A Sugiyama(1),
A Nakagiri(1), K Tsuchida(1), K Amagase(2), K Takeuchi(2)
(1) Doshisha Womens College of Liberal Arts, Faculty of Pharmaceutical Sciences, Department of Pharmacotherapy, Kyotanabe, Japan
(2) Kyoto Pharmaceutical University, Department of Pharmacology and
Experimental Therapeutics, Kyoto, Japan
Helicobacter pylori is a pathogen of the gastric diseases, such as atrophic
gastritis, peptic ulcer, and gastric adenocartinoma, and MALT lymphoma
in humans. Parietal cells are highly differentiated to have several
important roles in the maintenance of normal structure and function of
478
the gastric gland. Accumulating evidence indicates that gastric atrophy is
more consistently associated with gastric cancer than intestinal metaplasia. However, how the parietal cells are lost is not clear. Adherens junctions play important roles not only in the adhesive functions but also in
gastric carcinogenesis through extracellular degradation of E-cadherin.
Therefore, we studied the relationship between E-cadherin and parietal
cell loss. Mongolian gerbils infected with Helicobacter pylori were used.
The number of parietal cells and the staining of E-cadherin in the stomach were investigated by immunohistochemistry with monoclonal antibody against H+/K+-ATPase and E-cadherin respectively at 4, 8, and
12 weeks after inoculation. Quantitative investigation veried loss of
parietal cells and decrease of intercellular staining of E-cadherin from
4 weeks. E-cadherin was decreased at the mucosa where parietal cells
were lost or damaged. Loss of E-cadherin lasted in the mucosa in which
parietal cells have been lost. Inltration of lymphocytes was observed at
lamina propria and submucosa where parietal cells were lost. These
results support the hypothesis that Helicobacter pylori targeted adherens
junctions of parietal cells to the shedding of parietal cells and resulting in
the destruction of normal gland lineage and development of glandular
atrophy.
Paper No.: 2393

THE RELATIONSHIP BETWEEN HYPERLIPIDEMIAINDUCED ERECTILE DYSFUNCTION AND SERUM ADMA
LEVELS IN A TIME DEPENDENT MANNER
N Murat(1), A Toylu(2), Atiye Sinem Evcim(3), BC Soner(3), O Demir(4),
B Irer(4), T Demir(5), N Atabey(2), S Gidener(3), AA Esen(4)
(1) Dokuz Eylul University Advanced Professional School of Health Sciences, Izmir,Turkey
(2) Dokuz Eylul University School of Medicine, Department of Medical
Biology and Genetics, Izmir, Turkey
(3) Dokuz Eylul University School of Medicine, Department of Pharmacology, Izmir, Turkey
(4) Dokuz Eylul University School of Medicine, Department of Urology,
Izmir, Turkey
(5) Dokuz Eylul University School of Medicine, Department of
Endocrinology, Izmir,Turkey
Major mechanism of endothelial dysfunction (ED) is impaired release of
nitric oxide (NO) which leads to vasculogenic erectile dysfunction. Oxidized LDL-induced increased serum concentrations of asymmetrical
dimethylarginine (ADMA), major endogenous inhibitor of endothelial
nitric oxide synthase (eNOS), causes ED which is known to be a reversible process. The aim of this study was to investigate the time related
effects of hyperlipidemia and the role of serum ADMA levels on erectile
functions. Wistar rats were randomized into control and experiment
groups. Hyperlipidemia was induced by a single dose of i.v. 4 mg/kg
native LDL (nLDL). Experiments were performed 72 hours, 2 and
8 weeks after the injection of nLDL. Endothelium-dependent relaxations
(by organ bath studies in thoracic aorta), in vivo erectile functions (by intracavernous pressure measurement), eNOS mRNA expressions in cavernous tissue and serum ADMA levels were evaluated. In vivo erectile
functions impaired signicantly in 2-week and 8-week groups when
compared to control group (P<0.01, P<0.001, respectively). Endothelium-dependent relaxations signicantly reduced in 8-week group
(P<0.05). The eNOS mRNA expression in cavernous tissue signicantly
decreased in 72-hour, 2-week, 8-week groups (P<0.05). The signicant
increase of serum ADMA levels were found in 8-week group. These
results suggest that hyperlipidemia-induced impairments in erectile functions during 8-week period are not reversible and may closely be related
to decrease of eNOS gene expression and increase of ADMA levels.
Further molecular studies are needed to clarify mechanism of ADMA in
hyperlipidemic corpus cavernosum tissue.
Paper No.: 2134

HEALTH AND DISEASE
BOLDINE PROTECTS VASCULAR ENDOTHELIAL FUNCTION
IN SPONTANEOUSLY HYPERTENSIVE RATS (SHR) BY
INHIBITING FREE RADICALS
Dharmani D Murugan, YS Lau, MR Mustafa
University of Malaya Faculty of Medicine, Department of Pharmacology,
There are growing evident associating enhanced oxidative stress with the
development of endothelial dysfunction in hypertension. Antioxidants
like avonoids have been reported to improve endothelial dysfunction in
Spontaneously Hypertensive Rats(SHR) (Ajay et al., 2006;55:385-391).
Boldine ((S)-2,9-Dihydroxy-1,10-dimethoxy-aporphine) is a major aporphine alkaloid found from the leaf and bark of the Chilean boldo tree
and has been shown to possess potent antioxidant activity (OBrien et
al., 2006;159:1-17). This study focuses on the effect of chronic boldine
treatment for 7 days (20mg/kg) on vascular endothelial function in SHR.
Endothelium-dependent (acetylcholine, ACh) and endothelium-independent (sodium nitroprusside, SNP) vasorelaxation were assessed at the
end boldine treatment. Vascular superoxide and peroxynitrite production
induced by NADPH oxidase (chemiluminescence assay) as well as protein kinase C (PKC) mediated superoxide production were also assessed.
Boldine treatment in SHR showed a signicant increase in ACh-dependent relaxation compared to the vehicle-treated group (Rmax boldine :
94.67 5.28, Rmax vehicle: 40.60 5.64) whereas SNP -independent
relaxation was unaffected. Vascular superoxide and peroxynitrite production were markedly increased in SHR. Boldine treatment signicantly
reduced the peroxynitrite (SHRvehicle: 345.9 77.95, SHRboldine:
111.5 76.04 unit/mg, p< 0.001) but had no effect on NADPH induced
superoxide production. Interestingly, boldine effectively inhibited PKC
mediated superoxide production (SHRvehicle: 284.3 34.95, SHRboldine: 124.1 19.88 unit/mg, p< 0.001). The present result suggests that
boldine as an antioxidant compound effectively protects vascular endothelial function in SHR by inhibiting PKC mediated superoxide production and scavenging peroxynitrites.
Paper No.: 2424

CHRONIC ANTIDEPRESSANTS DAMPEN THE INCREASE OF
DEPOLARIZATION-EVOKED GLUTAMATE RELEASE
INDUCED BY ACUTE BEHAVIOURAL STRESS IN
PREFRONTAL/FRONTAL CORTEX
Laura Musazzi(1), M Milanese(2), F Pasqualina(3), Z Simona(2),
B Tiziana(2), M Alessandra(1), B Pietro(3), R Giorgio(1,4), R Maurizio(2), B Fabio(3), B Giambattista(2), P Maurizio(1)
(1) Center of Neuropharmacology, Department of Pharmacological
Sciences and University of Milano, Centerof Excellence on Neurodegenerative Diseases, Milano, Italy
(2) University of Genova, Department of Experimental. Medicine, Section of Pharmacology and Toxicology and Center of Excellence for Biomedical Research and NationalInstitute of Neuroscience, Genova, Italy
(3) The Italian Institute ofTechnology, Department of Neuroscience and
Brain Technologies and University of Genova and National Institute of
Neuroscience, Department of Experimental Medicine, Section of Physiology, Genova, Italy
(4) Istituto di Ricovero e Cura a Carattere Scientico San Giovanni di
Dio, Fatebenefratelli, Brescia, Italy
Behavioural stress is recognized as a main risk factor for many diseases,
including neuropsychiatric diseases. Several neurochemical ndings have
suggested that an increase of excitatory amino acid transmission in the
forebrain is associated with stress. Aim of this work was to study the
479
effect of acute stress on glutamate release, to analyse the mechanism
whereby acute stress modies glutamate release and if chronic antidepressants dampen this effect of stress. Rats were chronically treated with
different antidepressants (uoxetine, desipramine, venlafaxine and agomelatine), or vehicle. Rats were subjected to unpredictable footshock
(FS)-stress, hippocampus and prefrontal/frontal cortex (P/FC) were dissected and synaptosomes were puried on Percoll gradients. Acute FSstress markedly increased depolarization-evoked glutamate release from
synaptosomes of P/FC and chronic antidepressant treatments prevented
the increase; GABA release was unchanged. FS-stress induced rapid
increase in the circulating levels of CORT in all rats (vehicle/drug treated) and glutamate release increase was dependent on the binding of
CORT to glucocorticoid receptors (GR). On the molecular level, FSstress induced accumulation of presynaptic SNARE complexes, whether
or not pre-treated with antidepressants. Patch-clamp recordings of pyramidal neurons in P/FC revealed that FS-stress induces dramatic changes
in paired-pulse facilitation and in its Ca2+-dependence that are suggestive
of an increase in release probability. Previous treatment with desipramine
prevented these changes. We suggest that the dampening action of antidepressant on glutamate release must be on pathways downstream of
these processes and/or on alternative pathways. This novel effect of antidepressant could be related to their therapeutic (antidepressant/anxiolytic)
action.
Paper No.: 2969

ANALGESICS
SYSTEMATIC REVIEW AND META-ANALYSIS OF TRYPTANS
IN THE ACUTE TREATMENT OF CLUSTER HEADACHE
C Mustata, Ricardo Pardo, E Papaseit, M Farre
IMIM-Hospital del Mar, UAB, Department of Human Pharmacology and
Neurosciences, Barcelona, Spain
Introduction: Cluster headache is a primary headache syndrome characterized by attacks of unilateral excruciating pain and cranial autonomic
symptoms. The only current licensed treatment for acute attacks are subcutaneous sumatriptan and intranasal zolmitriptan. The aim of this study
was to evaluate the efcacy and safety of different tryptans in the acute
treatment of cluster headache. Materials/ Patients: The study was conducted according to the Cochrane Collaboration methodology and the
QUOROM statement. A systematic search was performed in Medline
and Cochrane library (1966-2009) with the following terms: cluster headache, tryptans, name of tryptan and clinical trial in English and Spanish.
Jadad scale was used to evaluate the quality of included trials. Efcacy
was evaluated using some binary outcomes as pain relief, pain free and
necessity of rescue medication. Safety was evaluated using adverse
events reported. Review Manager 5.0 software was used to conduct the
meta-analysis procedures. Indirect comparison was measured by simple
adjustment. Results: Six randomized, double-blind, placebo-controlled,
cross-over clinical trials involving 592 patients were included. Drugs and
routes of administration were sumatriptan (6 mg s.c, 20 mg i.n) and
zolmitriptan (10 mg p.o, i.n). Tryptans as a group were more active (OR
[95% CI) than placebo in pain relief (OR 4.02 [2.50-6.45]) and pain free
(OR 4.11 [2.28-7.42]). Tryptans needed less rescue treatment than placebo (OR 0.43 [0.30-0.60], but produced more side effects (OR 2.43
[1.77-3.33]). Different tryptans showed similar efcacy. Conclusions:
Tryptans are more effective than placebo in the acute treatment of cluster
headache.
Paper No.: 1789

DECREASED MATRICELLULAR PROTEIN
THROMBOSPONDIN (TSP) -4 EXPRESSION IN HUMAN
CALCIFIED AORTIC VALVES
Erja Mustonen(1), V Pohjolainen(1), P Taskinen(2), J Napankangas(3),
T Peltonen(1), H Leskinen(1), T Juvonen(2), J Satta(2), H Ruskoaho(1),
J Rysa(1)
(1) University of Oulu, Institute of Biomedicine, Department of Pharmacology and Toxicology, Oulu, Finland
(2) University Hospital of Oulu, Division of Cardiothoracic Surgery,
Department of Surgery, Oulu, Finland
(3) University of Oulu, Insitute of Diagnostics, Department of Pathology,
Oulu, Finland
Extensive extracellular matrix (ECM) reModelling occurs during the
development of Aortic valve stenosis (AS), and is characterized by brotic thickening, neovascularization and calcication of the aortic valves.
Thrombospondin-4 (TSP-4) is a matricellular protein induced in various
cardiac pathologies and associated with cardiac remodeling. However,
the expression or the role of TSP-4 in AS is unknown. Patients were
divided into three groups. Control group consisted of patients with normal, non-calcied, smooth, and pliable aortic valve cusps (n= 8). Fibrosis
group (n=5) consisted of patients who were identied to have macroscopic thickenings of aortic valve cusps with microscopic signs of brosis but no calcication or signicant transvalvular pressure gradient. AS
group (n=18) consisted of stenosed, nonrheumatic valves with different
degrees of calcication. Demographically the groups were equal, except
that the AS group tended to be older than the others. As measured by
real time reverse-transcriptasepolymerase chain reaction, TSP-4 gene
expression was markedly downregulated (57%, (P<0.01) in stenotic
valves compared with controls, while no signicant change was observed
in the brosis group. When studied by immunohistochemistry, TSP-4
was localized in the ECM to the stromal area and the TSP-4 positive
staining decreased along with the increase in calcication of the stenotic
valves. Our study is the rst to report matricellular protein TSP-4 expression in human aortic valves. During aortic valve calcication, TSP-4 levels are downregulated, possibly due to the replacement of normal
valvular ECM by calcied lesions, suggesting an important role for TSP4 in the pathogenesis of aortic stenosis.
Paper No.: 1031

EFFECTS OF CLOZAPINE AND OLANZAPINE ON
ACQUISITION, CONSOLIDATION AND RETENTION OF
MEMORY IN ELEVATED PLUS MAZE TEST IN MICE
Oguz Mutlu, G Ulak, I Komsuoglu Celikyurt, F Erden, F Yyldyz Akar
Kocaeli University Medical Faculty, Department of Pharmacology,
Kocaeli, Turkey
Current pharmacotherapies improve positive symptoms of schizophrenia
although they dont have sufcient potency in relieving cognitive symptoms which seriously disturb quality of life. Controversial results exist
for the effects of antipsychotics on learning and memory. Clozapine and
olanzapine are antipsychotic drugs widely used in schizophrenia and psychosis. The aim of this study was to investigate the effects of clozapine
(0.5 and 1 mg/kg) and olanzapine (0.15 and 0.30 mg/kg) on acquisition,
consolidation and retention of memory in naive male Balb-c mice in the
modied elevated plus maze test (EPM). Kruskal-Wallis post hoc Dunns
test was used for the comparison of groups while Wilcoxon signed rank
test was used to compare the rst and the second sessions of each drug
group. Clozapine (0.5 mg/kg) and olanzapine (0.15 and 0.3 mg/kg) signicantly prolonged the second day latency (TL2) compared to control
480
group when administered before the acquisition session. Administration
of 1 mg/kg clozapine immediately after the acquisition session signicantly prolonged TL2 while olanzapine had no effect. Clozapine and olanzapine, at the doses used, signicantly prolonged TL2 when
administered before the retential trial. In comparison of TL1 and TL2 for
each drug group, TL2 signicantly decreased in the control group while
there was no signicant difference in drug groups. Our results showed
that both clozapine and olanzapine had disturbing effects on acquisition,
consolidation and retention of memory in the EPM test which could be
attributed to their anticholinergic and antihistaminergic effects.
Paper No.: 1145

ANALYSIS OF GLUCOCORTICOID RECEPTOR GENE
HAPLOTYPE STRUCTURE AND ITS INFLUENCE ON
DISEASE ACTIVITY AND STEROID THERAPY OUTCOME IN
INFLAMMATORY BOWEL DISEASE PATIENTS
in cold) and the effect of acute restraint stress in male mice (11-13 weeks
old) lacking main cardioinhibititve receptors M2 muscarinic receptors
(M2KO). In the left ventricles, there was decrease in all cardiostimulative
receptors (b 1-, b2-AR, a1-AR: to 52%, to 55 %, and to 64%, respecitvely) and increase in cardiodepressive receptors (b 3-AR were increased
to 205%) in intact KO animals in comparison to WT. The cold stress in
WT animals resulted in decrease in b1- and b2-AR (to 37%/35% after
one day in cold and to 27%/28% after 7 days in cold). On the other
hand, b3-AR were increased (to 216% of control) when 7 days cold was
applied. MR were reduced to 46% and to 58%, respectively. In KOs cold
stress had effects as follows: the reaction of cardiostimulative b1- and
b2-AR to cold was similar in KO animals as in WT animals. In contrast
to cardioinhibitive receptors in wild types, b3-AR in KO animals did not
changed in reaction to cold. The basal temperature was lower in KO animals than in WT but other parameters were not changed. The restraint
stress caused similar changes in the activity and temperature in WT and
KO animals, but the heart rate was higher in KO animals. On conclusion,
our results suggest compensatory role of b3-AR in KO animals.
Supported by grant GACR 309/09/0406.
Jessica Mwinyi, C Wenger, JJ Eloranta, GA Kullak-Ublick

University Hospital Zurich, Division of Clinical Pharmacology and
Toxicology, Zurich, Switzerland
The inammatory bowel diseases (IBD), Crohns disease (CD) and ulcerative colitis (UC), are of proposed autoimmune etiology and are characterized by chronic recurrent inammation of the gastrointestinal tract.
Single nucleotide polymorphisms (SNPs) in the NR3C1 gene encoding
the glucocorticoid receptor (GR) are known to inuence the response to
endogenous and exogenously administered glucocorticoids, as well as
the susceptibility to develop various autoimmune syndromes. We hypothesized that genetic variability in the NR3C1 gene may affect response to
steroid treatment in IBD patients. We sequenced all coding exons and
anking intronic sequences of the NR3C1 gene in 181 IBD patients,
determined the SNPs, and predicted the NR3C1 haplotypes. Furthermore,
we investigated whether certain NR3C1 haplotypes are associated with
steroid therapy outcomes or with the disease course. We detected 13
NR3C1 variants, which led to the formation of 17 different haplotypes
with a certainty of >95% in 173 individuals. The three most commonly
occurring haplotypes were included in the association analysis of the
inuence of haplotype on steroid therapy outcome, IBD activity, and age
of disease onset. None of the NR3C1 haplotypes showed signicant
association with glucocorticoid therapy success or disease activity. A
higher age of disease onset was observed for the haplotype GR_2 (mean
age 31.4612.01), in comparison with wildtype (GR_1) carriers (mean
age 25.7313.03). In conclusion, NR3C1 haplotypes are not related to
steroid therapy outcome, but may have an inuence on the age of disease
onset in IBD.
Paper No.: 3254

B3-ADRENOCEPTORS SUBSTITUTE THE ROLE M2
MUSCARINIC RECEPTORS IN THE HEART OF STRESSED
MICE
Jaromir Myslivecek(1), M Novakova(1), V Farar(1), R Kvetnansky(2)
(1) Charles University, 1st Faculty of Medicine, Institute of Physiology,
Prague, Czech Republic
(2) Slovak Academy of Sciences, Institute of Experimental Endocrionology, Bratislava, Slovakia
Paper No.: 2922

VARIABILITY OF TAMOXIFEN METABOLISM IN VIVO IS
INFLUENCED BY GENOTYPES OF DRUG METABOLIZING
ENZYMES
Thomas Murdter(1), W Schroth(1), H Brauch(1), M Eichelbaum(1),
M Schwab(0)
(1) Dr Margarete Fischer-Bosch Institute of Clinical Pharmacology,
Department of Analytics, Stuttgart, Germany
(2) University Hospital Tubingen, Department of Clinical Pharmacology,
Tubingen, Germany
Tamoxifen (tam) is standard therapy for the treatment of estrogen
receptor positive breast cancer. However, one third to one half of
patients do not benet. Tam is extensively metabolized in vivo.
Among numerous metabolites 4-hydroxy-tam and 4-hydroxy-N-desmethyl-tam (endoxifen) have highest afnity to the estrogen receptor
and therefore are primarily responsible for treatment efcacy. Variability of plasma concentrations of these metabolites may contribute to
variable patients outcome. Steady state concentrations of active
metabolites depend on both, formation rates and metabolic clearance.
We therefore analyzed steady state plasma concentrations of more
than 20 tam metabolites in 240 breast cancer patients adjuvantly treated with 20 mg tam daily. Plasma levels were correlated to genotypes
of polymorphic CYPs and UGTs involved in tam metabolism. Plasma
levels for parent drug, desmethyl-tam, (Z)-endoxifen, and (Z)-4hydroxy-tam were 16360, 282111, 11.15.6, and 2.30.9lg/L,
respectively. O-glucuronides of 4-hydroxylated metabolites were present only in low concentrations. Metabolic ratios of endoxifen were
signicantly correlated with 2D6 genotype (ANOVA, p<0.0001).
However, there was an overlap of endoxifen plasma concentrations
among predicted phenotypes. CYP 2C9 genotype had a signicant
impact on 4-hydroxy-tam levels (t-test, p<0.001). Plasma level of
tam-N+-glucuronide (1.5lg/L) were inuenced by genotype of UGT
1A4. Polymorphisms of CYP 2B6, 2C19, and 3A5 did not show a
signicant effect on steady state levels of active metabolites. Plasma
levels of active tam metabolites are inuenced by polymorphisms of
CYP 2D6 and 2C9. Further studies including outcome data are
needed to evaluate whether genotyping or therapeutic drug monitoring
is best suited for guiding therapy.
b3-adrenoceptors (b3-AR) were identied to produce cardioinhibition in

ventricles. We investigated the role of b3-AR in cold stress (1 or 7 days
481
Paper No.: 3271
KNOCKDOWN OF DISC1 BY IN UTERO GENE TRANSFER
IMPAIRED POSTNATAL DOPAMINERGIC MATURATION IN
THE FRONTAL CORTEX AND ADULT BEHAVIORS
Paper No.: 1047

EFFECTS OF EARLY INTERVENTION WITH FLUOXETINE
ON ANXIETY-RELATED BEHAVIORS INDUCED IN
PRENATALLY GLUCOCORTICOID EXPOSED RATS
Toshitaka Nabeshima(1), M Niwa(1,2,3), A Kamiya(2), K Kubo(4),

K Kohda(4), P ODonnell(5), Y Noda(1), K Nakajima(2), A Sawa(4)
Masatoshi Nagano(1), M Liu(1), H Ozawa(2), H Suzuki(1)
(1) Meijo University, Department of Chemical Pharmacology, Pharmacy,

Nagoya Aichi, Japan
(2) Johns Hopkins University School of Medicine, Baltimore, MD, USA
(3) Nagoya University Graduate School of Medicine, Nagoya, japan
(4) Keio University School of Medicine, Tokyo, Japan
(5) University of Maryland School of Medicine, Baltimore, MD, USA
Adult brain function and behavior are inuenced by neuronal network
formation during development. Genetic susceptibility factors for adult
psychiatric illnesses, such as Neuregulin-1 and Disrupted-in-Schizophrenia-1 (DISC1), inuence adult high brain functions, including cognition
and information processing. These factors have roles during neurodevelopment and are likely to cooperate, forming pathways or signalosomes. Here we report the potential to generate an animal model via in
utero gene transfer in order to address an important question of how nonlethal decits in early development may affect postnatal brain maturation
and high brain functions in adulthood, which are impaired in various
psychiatric illnesses such as schizophrenia. We show that transient
knockdown of DISC1 in the preand perinatal stages, specically in a
lineage of pyramidal neurons mainly in the prefrontal cortex, leads to
selective abnormalities in postnatal mesocortical dopaminergic maturation and behavioral abnormalities associated with disturbed cortical neurocircuitry after puberty.
Paper No.: 3192

FOCUSED CONFERENCE GROUP: P19 - GENERAL SESSION AIRWAY
THE EFFECT OF INHALED KP-496, A NOVELANTAGONIST
FOR CYSTENYL LEUKOTRIENE RECEPTOR AND
THROMBOXANE A2 RECEPTOR, ON EXPERIMENTAL
ALLERGIC ASTHMA IN GUINEA PIGS
Hiroichi Nagai(1), M Suda(2), T Okuda(2), M Ishimura(2),
S Kurokawa(2), S Tokuoka(2), T Nakamura(2), Y Takahasi(1),
H Tanaka(1)
(1) Gifu Pharmaceutical University, Department of Clinical
Pharmacology, Gifu, Japan
(2) Kaken Pharmaceutical Co., Kyoto, Japan
The aims of this study was to evaluate the effects of inhaled KP-496, a
novel dual antagonist for cytenyl leukotriene receptor and thromboxane
A2 receptor on experimental allergic asthma in guinea pigs. Actively sensitized animals were repeatedly exposed to antigen, and KP-496 (0.01
and 0.1%) was inhaled for 5 minutes before every antigen exposure. KP496 (0.1%) signicantly inhibited both antigen-induced late asthmatic
response and airway hyperresponsiveness to acetylcholine. Furthermore
KP-496 clearly suppressed eosinophilia around airway smooth muscle,
airway epithelial hypertrophy and mucus production in airway in histopathological examination. In conclusion, KP-496 inhibited both an asthmatic respiratory disorder and inammatory histological changes of
airway. From these broad ameliorative effects of KP-496 on asthmatic
responses are thought to result from the inhibition of multiple chemical
mediators, KP-496 will be a benecial agent in the treatment of bronchial
asthma.
(1) Nippon Medical School, Department of Pharmacology, Tokyo, Japan

(2) Nippon Medical School, Department of Anatomy, Tokyo, Japan
Development of therapeutic strategies against psychiatric disorders is a
critical issue in psychiatric practice. Among a variety of pathogenesis
factors, prenatal stress is thought to cause some of the psychiatric symptoms observed in anxiety disorders. We recently reported that prenatal
dexamethasone (DEX) exposure, which mimics prenatal stress, induced
anxiety-related behaviors in adulthood in association with precedent
abnormalities in the hypothalamic-pituitary-adrenal axis during postnatal
development in rats. These results suggested that early therapeutic intervention would be benecial to prevent late-emerging anxiety symptoms.
To verify this hypothesis, we treated rat pups born to DEX-administered
mothers with uoxetine (FLX), a commonly used anti-anxiety medication, via breast milk from postnatal day 2 to day 21. FLX treatment prevented the occurrence of anxiety-related behaviors, as examined at
13 weeks of age, in the rat offspring. Allied with the behavioral effects,
FLX normalized 5-HT1A receptor mRNA expression in the medial prefrontal cortex that was decreased by prenatal DEX exposure. Serotonergic function has long been thought to be implicated in CNS development
and various psychiatric disorders. Accordingly, early pharmacological
intervention to address serotonergic transmission may provide a novel
strategy to treat some aspects of anxiety disorders.
Paper No.: 1295

DISEASE AND THERAPY
DECREASED EXPRESSION OF CONNEXIN 26 IN THE
COCHLEAR SPIRAL LIGAMENT FIBROCYTE FOLLOWING
INTENSE NOISE EXPOSURE
Reiko Nagashima, T Yamaguchi, H Tanaka, A Matsushita, K Ogita
In cochlear spiral ligament, gap junctions play a critical role in hearing
systems. Mutations in connexin genes are known to cause a high incidence of human deafness. In particular, the mutation in the Gap Junction
b2-encoding gap junction protein connexin 26 (Cx26) is related to hereditary non-syndromic deafness and maturation of cochlear development
through aberrance of responsibility for the recycling of K+ in the cochlear endolymph. In addition, noise-induced hearing loss has alterations in
the K+ concentration in the endolymph. The purpose of this study was to
evaluate the expression of Cx26 during hearing loss induced by intense
noise. Adult male Std-ddY mice were exposed to 8 kHz octave band
noise of 110 dB SPL for 1 h. The noise exposure produced marked outer
hair cell death in the Cortis organ and a dramatic threshold shift at frequencies of 4, 12, and 20 kHz. Immunohistochemical study revealed that
Cx26 was located in the type I spiral ligament brocyte (SLF) of cochlear lateral wall in nahEe mice. Noise exposure decreased the level of
Cx26 in the SLF 2 at 4 h afterward. In addition, signicant decrease in
the expression of Cx26 mRNA was seen in the SLF 2 h after noise exposure. Taken together, our data suggest that intense noise exposure negatively regulate the expression of Cx26 in the SLF of the cochlea. The
downregulation of Cx26 in the SLF may be involved in hearing loss
induced by intense noise exposure.
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Paper No.: 2652
CHRONIC L-DOPA TREATMENT PREVENTS INCREASED
BINDING OF [11C] MDL100907 TO 5-HT2A RECEPTORS IN A
RAT MODEL OF PARKINSONS DISEASE
Adjmal Nahimi(1), A Landau(1), M Hltzermann(1), S Jakobsen(1),
S stergaard(1), A Mller(1), D Doudet(1), A Gjedde(1,2)
(1) Aarhus University, Aarhus University Hospital, PET-Centre, Aarhus,
Denmark
(2) University of Copenhagen, Department of Neuroscience and Pharmacology, Copenhagen, Denmark
Objective: 5-HT2A receptor mRNA is increased in the lesioned side of the
striatum of rats unilaterally injected with 6-hydroxydopamine (6-OHDA)
(Numan et al. 1995). Treatment with high doses of L 3,4 dihydroxyphelyalanine (L-DOPA) prevent the mRNA increase. Here we use [11C]
MDL100907, a highly selective 5-HT2A-receptor antagonist, in post-mortem autoradiography experiments to test if the increase in 5-HT2A receptor mRNA results in increased expression of the receptor itself and if the
predicted elevation of the receptors can be prevented by treatment with
low doses of L-DOPA. Method: Female Sprague Dawley rats received a
unilateral lesion with 6-OHDA and were then treated with either L-DOPA
(8mg/kg) and benzeraside (15mg/kg) or saline for 5 weeks. In-vitro autoradiography was performed with the tracer [11C] MDL100907. Result:
5-HT2A receptor expression was increased in the lesioned striatum. Daily
L-DOPA and benzeraside treatment over a period of ve weeks attenuated
this increase. There was no difference in cortical 5-HT2A receptor expression between any of the groups. Conclusion: The nding that 5-HT2A
receptor expression is increased in the dopamine depleted striatum is consistent with the nding of increased 5-HT2A receptor mRNA. The prevention of part of this increase by means of L-DOPA treatment suggest an
inhibitory role of dopamine in the expression 5-HT2A receptors, presumably by down regulation of 5-HT2A receptor mRNA synthesis. However,
the expression of cortical 5-HT2A receptors was not affected by the lesion
or the L-DOPA treatment, suggesting differential roles for dopamine in
the expression of 5-HT2A receptors.
Paper No.: 2653

DYSKINESIA INDUCING DOPAMINE ORIGINATES IN SEROTONINERGIC NEURONS
Adjmal Nahimi(1), M Hltzermann(1), A Landau(1), K Vang(1),
S Jacobsen(1), A Mller(1), G Wegener(1), D Doudet(1), A Gjedde(1,3)
(1) Aarhus University, Aarhus University Hospital, PET-Centre, Aarhus,
Denmark
(2) Aarhus University Hospital, Center of Psychiatric Research, Aarhus,
Denmark
(3) University of Copenhagen, Department of Neuroscience and
Pharmacology, Copenhagen, Denmark
Introduction: L-DOPA induced dyskinesias (LID), a severe complication
of Parkinsons Disease therapy, occur in response to administration of
therapeutic doses of L-DOPA and are modulated by rapid changes of
extracellular and synaptic DA. 5-HT agonists and antagonists are potential adjuncts to L-DOPA used to modulate extracellular DA without loss
of therapeutic benets. We used raclopride, a tracer of the D2/D3 receptors as a surrogate marker of DA-release to evaluate the effect in-vivo of
8-OH-DPAT, a 5-HT agonist, on the L-DOPA induced decrease of raclopride binding and LID in 6-OHDA lesioned rats. Methods: Rats with a
unilateral 6-OHDA lesion as a model of Parkinsons disease, received a
[11C] raclopride baseline scan and then two pharmacological challenge
PET scans. For challenge scans, rats were pretreated with either
L-DOPA+benzeraside (50/25 mg/kg S.C.) or L-DOPA+benzeraside+8-
OH-DPAT (0,6mg/kg/i.p.) 30-45 minutes prior to the raclopride injection.

LID intensity was evaluated on a separate day. Results: The baseline
increase in raclopride binding in the parkinsonian striata was eliminated
with L-DOPA challenge. Concurrent administration of 8-OH-DPAT prevented the L-DOPA induced decrease in raclopride binding. LID was
consistently decreased with the co-administration of 8-OHDPAT. Conclusion: 8-OHDPAT reverses the L-DOPA induced decrease of raclopride
binding as well as the intensity of dyskinesias. The reversal of this effect
by a serotonin agonist implies that the dopamine associated with dyskinesia is generated in serotoniergic neurons.
Paper No.: 886

USING PRESCRIBING DATA TO DETERMINE DRUG
PERSISTENCE FOR THE PURPOSE OF
PHARMACOVIGILANCE
Isa Naina Mohamed(1), P Helms(1), C Simpson(2), J McLay(1)
(1) University of Aberdeen, Aberdeen, UK
(2) University of Edinburgh, Edinburgh, UK
The Yellow Card spontaneous reporting system is the primary pharmacovigilance system used in the United Kingdom to report and monitor
adverse drug reactions (ADR). However it suffers from a low reporting
rate, long lag time, and lack of a denominator. The object of this study
was to develop an ADR detection and monitoring system using routinely
acquired primary care prescribing data, which could overcome these
problems. Methods: Prescribing data for Amlodipine (A) and Lercanidipine (L) was extracted from the Practice Team Information database
which contains data on over 250 000 patients. Patients newly prescribed
these drugs were identied and then tracked as a cohort to determine the
length of time for which a prescription was issued (persistence). Patients
discontinuing therapy within 1, 2-3, and 4-6 months were identied, and
the patients general practitioner contacted to determine reasons for discontinuation. Results: Between 01/03/2004 and 28/02/2007, 3646 were
prescribed A and 269 L. Within 1 month 16.15% had discontinued A
and 17.84% L. Within 6 months 27.29% had discontinued A and 25.28%
L. Discontinuations were signicantly greater in females (P<0.01) and
the elderly (P<0.01). Comparison with published data conrmed that the
discontinuation rate determined using primary care prescribing data represents a good surrogate for ADR/E rate. Analysis of responses from GPs
conrmed that 98% of discontinuations were due to ADR/Es. Conclusion: This system is a simple yet effective method for monitoring drug
safety and persistence for chronically prescribed medicines.
Paper No.: 1416

IMPACT OF CYP2D6 AND CYP3A5 GENE POLYMORPHISMS
ON PHARMACOKINETICS OF OXYCODONE AND ITS
DEMETHYLATES AND DOSE ESCALATION RATE IN
CANCER PATIENTS
Takafumi Naito(1), K Yamamoto(2), Y Takashina(1), M Tashiro(1),
Y Kagawa(2), K Ohnishi(3), J Kawakami(1)
(1) Hamamatsu University School of Medicine, Department of Hospital
Pharmacy, Hamamatsu, Japan
(2) University of Shizuoka, Graduate School of Pharmaceutical Sciences,
Department of Clinical Pharmaceutics and Pharmacy Practice, Shizuoka,
Japan
(3) Hamamatsu University School of Medicine, Oncology Center,
Hamamatsu, Japan
483
Introduction: Oxycodone is metabolized to active metabolite oxymorphone by O-demethylation via CYP2D6 and to inactive metabolite noroxycodone by N-demethylation via CYP3A. Clinical impact of gene
polymorphisms of their drug-metabolizing enzymes in cancer patients
treated with oxycodone has not been fully claried. The aim of this study
was to evaluate the plasma disposition of oxycodone and its demethylates and dose escalation rate based on CYP2D6 and CYP3A5 gene
polymorphisms in cancer patients. Patients: Sixty-two Japanese cancer
patients receiving oxycodone extended-release tablets were enrolled. Predose plasma concentration of oxycodone and its demethylates were
determined in the patients receiving the titrated dose without rescue medication for four days. Opioid escalation index (OEI) was calculated from
dose escalation per day after the rst titration. Results: CYP2D6 phenotypes and CYP3A5 genotypes did not affect the plasma concentration of
oxycodone. Plasma concentration of oxymorphone and its ratio to oxycodone were signicantly higher in CYP2D6 extensive metabolizer (EM)
than intermediate metabolizer (IM). In contrast, plasma concentration of
noroxycodone and its ratio to oxycodone were signicantly higher in
CYP3A5*1/*1+*1/*3 than *3/*3 group. No signicant differences were
observed in the occurrence of dose escalation and OEI between the
CYP2D6 EM and IM. With respect to CYP3A5, the occurrence of dose
escalation and OEI were signicantly higher in *3/*3 than *1/*1+*1/*3
group. Conclusion: Dependency of oxycodone metabolic pathway which
did not pass through CYP3A5 may inuence the dose escalation rate.
This nding indicates that CYP3A5*3 affects the resistance to oxycodone
in cancer patients.
Paper No.: 2079

METFORMIN AND LACTIC ACIDOSIS: DO WE PAY
ATTENTION TO THE RENAL FUNCTION IN THE ELDERLY?
Makiko Nakagawa, U Bergman
Karolinska Institute, Department of Clinical Pharmacology, Stockholm,
Sweden
Introduction: Metformin is an oral anit-diabetic drug from the biganide
class developed in 1950s. Metformin is dependent on renal function for
its elimination. Elderly patients in general have decreased kidney function caused by aging and concurrent disease such as diabetes. As metformin is the rst line drug in type 2 diabetes according to the American
Diabetes Association (2006) and the European Association for the Study
of Diabtes (2006). We were concerned about dosage recommendations
for metformin in the elderly. Materials: We analyzed age, sex, and dosage (not in abstract) of the prescribing of metformin in the Swedish
national prescribed drug register (9 million inhabitants) 2008 and compared it with the Summary of the Products Characteristics (SPC) and the
Swedish FASS. Results: In 2008, 199 750 patients (2.2% of the Swedish
population, 56% men and 44% women) received metformin, 55% were
65 and 25% 75 years of older. Dosage recommendations for adults were
500 to 3000mg. For the elderly patients, metformin dosage should be adjsuted based on renal function. Regular monitoring of renal function is
necessary. Renal failure or renal dysfuntion (creatinine clearance < 60ml/
min) is considered a contraindication. Conclusion: Serum creatinine often
presents unreliable normal values in elderly pateints because of the
rduced muscle mass. Creatinine clearance is rarely estimated in clinical
parctice. As most pateints 75 years or older has a creatinine clearance <
60ml/min, this should be the standard measurement of renal function to
avoid dose dependent ADRs such as lactic acidosis.
Paper No.: 1128

WRONG; STABILIZATION OF CARDIAC FUNCTION -CLINICAL
EFFECTS OF ADMINISTRATION OF STATINS FOR A CERTAIN TERM BEFORE UNDERGOING CORONARY ARTERY
BYPASS GRAFTING ON PLATELET ACTIVATION AND
INFLAMMATORY RESPONSES
Kazuo Nakamura(1), H Iwase(1), H Kariyazono(2), S Oiso(2),
R Ikeda(3), J Arima(3), K Takahama(4), R Sakata(5)
(1) Nihon Pharmaceutical University, Department of Biopharmaceutics,
Saitama, Japan
(2) Nagasaki International University, Nagasaki, Japan
(3) Kagoshima University, Kagoshima, Japan
(4) Kumamoto University, Kumamoto, Japan
(5) Kyoto University, Kyoto, Japan
The ability to adapt to stress is an important defensive function of a living body, and impairment of this ability may contribute to some stressrelated disorders. Thus, the examination of brain mechanisms involved
in stress adaptation could help to pave the way for new therapeutic strategies for stress-related psychiatric disorders. The present study was tried
to examine the behavioral characteristics of mice subjected to chronic
variable stress (CVS), and also characterize changes in protein expression
in the hippocampus of this stress model. The daily schedule of CVS was
as follows: Day 1, restraint stress; Day 2, foot shock stress; Day 3, water
deprivation; Day 4, cold swimming stress; Day 5, psychological stress;
Day 6, food deprivation; Day 7, tail-pinch stress. The exploratory behaviors of mice were measured by hole-board test 24 h after the nal CVS
exposure, and then brain samples were collected. Exposure to CVS for 1
and 2 weeks produced the increase in head-dipping behavior and the
decrease in locomotor activity, respectively. These results suggest that
CVS may induce different types of emotional abnormalities depend on
the duration of stress exposure. On the other hand, exposure to CVS for
4 weeks did not induce any behavioral changes, indicating the development of stress adaptation. Proteome analysis revealed a signicant
increase in synaptosomal-associated protein 25 in the hippocampus of
mice adapted to CVS for 4 weeks. The present ndings suggest that
plasticity of transmitter exocytosis in the brain might be involved in the
development of stress adaptation.
Paper No.: 838

PRESYNAPTIC BK CHANNELS ACTIVATED BY IP3-INDUCED
CA2+ RELEASE ARE INVOLVED IN PROSTANOID TP
RECEPTOR-MEDIATED INHIBITION OF NORADRENALINE
RELEASE FROM THE RAT GASTRIC SYMPATHETIC
NERVES
Kumiko Nakamura, K Yokotani
Kochi University School of Medicine, Department of Pharmacology,
Nankoku City, Kochi, Japan
Prostanoids have been shown to modulate noradrenaline release from
central and peripheral noradrenergic nerves (Starke et al, Physiol Rev
1989; 69: 864-989, Malik and Sehic, Ann NY Acad Sci 1990; 604: 222236). Previously, we reported that prostanoid TP receptor mediates the
inhibition of electrically-evoked noradrenaline release from gastric sympathetic nerves in rats (Yokotani et al, Eur J Pharmacol 2003; 459: 187193). Prostanoid TP receptor has been shown to activate phospholipase
C (PLC), which catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate to inositol 1,4,5-triphosphate (IP3) and diacylglycerol; IP3
triggers the release of Ca2+ from intracellular stores and diacylglycerol
activates protein kinase C. In the present study, therefore, we examined
484
whether these PLC-mediated mechanisms are involved in the TP receptor-mediated inhibition of gastric noradrenaline release using an isolated,
vascularly perfused rat stomach. U-46619 (a prostanoid TP receptor agonist)-induced inhibition of noradrenaline release from the stomach was
attenuated by U-73122 (a PLC inhibitor) and ET-18-OCH3 (a phosphatidylinositol-specic PLC inhibitor), respectively. 2-APB (a putative IP3
receptor antagonist) also abolished the U-46619-induced inhibition of
noradrenaline release, but Ro 31-8220 (a protein kinase C inhibitor) had
no effect. Furthermore, a small dose of tetraethylammonium and charybdotoxin [blockers of BK-type Ca2+-activated K+ channel] abolished
the U-46619-induced inhibition, but apamin (a blocker of SK-type Ca2+activated K+ channel) had no effect. These results suggest that BK channels activated by IP3-induced Ca2+ release are involved in prostanoid TP
receptor-mediated inhibition of electrically evoked noradrenaline release
from the gastric sympathetic nerve terminals in rats.
Paper No.: 599

HEALTH AND DISEASE
KALLIDINOGENASE INHIBITS THE RETINAL
NEOVASCULARIZATION VIA THE CLEAVAGE OF VEGF
Irons are shown to be accumulated in particular brain regions in patients

suffering from different neurodegenerative diseases such as Parkinsonhfs
disease. We thus aimed to evaluate expression proles of different genes
relevant to iron mobilization in neurons. Marked mRNA expression was
seen for ferritin, transferrin and transferrin receptor-1 (TfR1) in mouse
neocortical neurons. In neuronal Neuro2A cells cultured in the presence
of retinoic acid (RA), neurite outgrowth was seen together with increased
expression of both mRNA and corresponding protein for TfR1. On
immunocytochemistry numerous cells were immunoreactive for TfR1 in
Neuro2A cells treated with RA. In mouse embryonal carcinoma P19
cells, exposure to RA led to neurite outgrowth toward neuronal differentiation along with a signicant increase in TfR1 mRNA expression.
However, TfR1 mRNA expression was signicantly decreased in
response to subsequent differentiation into an astroglial lineage in P19
cells. TfR1 siRNA was transfected into Neuro2A cells for 24 h, followed
by exposure to RA and subsequent determination of neurite length under
a microscope for a period of up to 6 days. Transfection with TfR1 siRNA signicantly promoted neurite outgrowth in cells cultured for 1 to
4 days. These results suggest that TfR1 would be highly expressed by
neurons rather than astroglia with a pivotal role in neurite outgrowth in
the brain.
Shinsuke Nakamura, H Isuta, K Tsuruma, M Shimazawa, H Hara

Department of Biopharmaceutics, Nihon Pharmaceutical University,
Saitama, Japan
Purpose: Kallidinogenase is used as a vasodilator in the treatments of hypertention and peripheral circulatory disorder for releasing kinin, a potent
vasodilator peptide. Moreover it has been recently reported that kallidinogenase suppressed quantity of intraocular vascular endothelial growth
factor (VEGF) in streptozotocin-induced diabetic rats. Therefore, we
examined whether kallidinogenase has anti-angiogenic effects in vitro
and in vivo models. Methods: Human umbilical vein endothelial cells
(HUVECs) were co-cultured with human broblasts and incubated for
11 days with or without kallidinogenase, with the concominant addition
of VEGF (10 ng/ml). Tube formation was measured using an Angiogenesis Image Analyzer. Proliferation and migration of HUVECs were measured using the WST-8 assay and the wound-healing assay, respectively.
Retinal neovasuclarization was induced in neonatal mice by returning the
retina to normoxia after exposure to hyperoxia (75% O2) from postnatal
day 7 (P7) to P12. Kallidinogenase was subcutaneously administered at
20 and 50 lg/kg once a day from immediately after hyperoxia to P16.
At P17, at-mounted retinas were prepared and evaluated for pathological and physiological angiogenesis. Results: Kallidinogenase signicantly
suppressed in vitro HUVEC tube formation, proliferation, and migration.
Furthermore, kallidinogenase signicantly suppressed in vivo retinal neovascularization (versus vehicle treatment), but revascularization of the
capillary free area did not differ between vehicle and kallidinogenase
treatments. It also inhibited the VEGF-induced phosphorylation of
VEGF-receptor via cleavage of VEGF. Conclusions: Kallidinogenase has
anti-angiogenic effects in vitro and in vivo, and may be useful as an antiangiogenic agent in the treatment of retinal neovascularization diseases.
Paper No.: 1347

FUNCTIONAL ANALYSIS OF TRANSFERRIN RECEPTOR-1
EXPRESSED IN BRAIN
Paper No.: 1162

EFFECTS OF SERCA ACTIVATORS, ELLAGIC ACID AND
GINGEROL, ON DIABETES MELLITUS-INDUCED DIASTOLIC
DYSFUNCTION IN ISOLATED MURINE VENTRICULAR
MYOCARDIA
Iyuki Namekata(1), M Ohhara(1), Y Hirota(1), T Kawanishi(2),
A Takahara(1), H Tanaka(1)
(1) Toho University Faculty of Pharmaceutical Sciences, Department of
Pharmacology, Funabashi, Chiba, Japan
(2) National Institute of Health Sciences, Japan
Cardiac dysfunction is a frequently reported complication of clinical and
experimental diabetes mellitus. In this study, we compared the contractile
force and calcium transient parameters of the myocardium from streptozotocin-induced diabetic mice with those from the normal mice, and
examined the effects of SERCA activators, ellagic acid and gingerol.
Diabetes was induced in mice by single injection of streptozotocin
(200 mg/kg. i.v). Contractile force and calcium transient were measured
at 4-6 weeks after streptozotocin treatment. The time required for relaxation of right ventricular free wall was signicantly longer in the diabetic
mice than in the normal mice. Both 10 micromolar ellagic acid and 10
micromolar gingerol accelerated the relaxation: the effects were larger in
the diabetic mice. The acceleration of relaxation by these agents was
inhibited by 10 micromolar cyclopiazonic acid. The basal calcium concentration of isolated ventricular myocytes from diabetic mice was significantly higher than those from normal mice. The time required for
calcium transient decay was signicantly longer in diabetic myocytes.
Both 10 micromolar ellagic acid and 10 micromolar gingerol accelerated
the calcium transient decay: the effects were larger in the diabetic myocytes. The acceleration of calcium transient decay by these agents was
inhibited by 10 micromolar cyclopiazonic acid. These results suggest that
the diabetes mellitus-induced myocardial diastolic dysfunction is caused
by reduction of SERCA function and can be ameliorated by SERCA
activators.
Yukary Nakamura(1,2), T Takarada(1), Y Yoneda(1)

(1) Kanazawa University Graduate School of Natural Science and
Technology, Division of Pharmaceutical Sciences, Kanazawa, Japan
(2) Setsunan University of Faculty of Pharmaceutical Sciences, Osaka,
Japan
485
Paper No. 3242
FOCUSED CONFERENCE GROUP: PW23 - APPLYING PHARMACOGENOMICS (PGX) FROM RESEARCH INTO CLINICAL
PRACTICE: PRESENT AND FUTURE
DISTRIBUTION AND GENOTYPE FREQUENCY OF THE SNP
45T/G POLYMORPHISM IN IRANIAN POPULATION: PROGRESSION TO DIABETES AND RESPONSE TO PIOGLITAZONE
Fatemeh Namvaran(1), P Rahimi-Moghaddam(1), N Azarpira(2)
(1) Iran University of Medical Sciences, Razi Institute for Drug
Research, Department of Pharmacology, Tehran, Iran
(2) Shiraz University of Medical Sciences, Transplant Research Center,
Shiraz, Iran
Adiponectin is secreted by adipocytes. It is a hormone with anti-inammatory and anti-atherogenic properties and plays an important role in
insulin sensitivity. It also has been associated with measures of adiposity
and plays a causal role in the association of obesity and insulin sensitivity which has therefore a target of recent research. The genetic variations
in the adiponectin gene can affect the circulating adiponectin level and
may have associated with insulin resistance. The aim of the present study
is to evaluate the frequency of common SNP of adiponectin gene (+45
T/G) in normal population to correlate these data with diabetes progression. Hundred healthy volunteers were enrolled to identify the genotype
of adiponectin gene (+45T/G) which was performed by the polymerase
chain reaction-restriction fragment length polymorphism (PCR-RFLP). It
was observed that the T allele and TG/TT genotype occurred more frequently than the G allele and the GG genotype in normal population. Frequency of allele T was 0.855 and frequency of allele G was 0.145. It
should be mentioned that the T allele is considered protective against diabetes progression. It may also have a correlation with pioglitazone
response in diabetic patients. Currently, we are examining this correlation
in diabetic patients recieving pioglitazone to compare thier genotypes with
non-diabetic patients and to nd the correlation with response to the drug.
Paper No. 2464

FOCUS GROUP: P03 - ION CHANNELS IN ANALGESIA &
ANAESTHESIA
THE EFFECTS OF GABAPENTIN ON NOREPINEPHRINE,
SEROTONIN AND THEIR METABOLITES IN RAPHE MAGNUS
IN FORMALIN TEST BY THE METHOD OF MICRODIALYSIS
IN MALE RATS
Mohammad M Namvaran(1), A Jamshidzadeh(1), J Sajedianfard(2),
MA Mobasher(1)
(1) Faculty of Pharmacy, Shiraz University of Medical Sciences, Department of Pharmacology and Toxicology, Shiraz, Iran
(2) Faculty of veterinary, Shiraz University, Shiraz, Iran
Gabapentin is a second generation anticonvulsant that is effective in the
treatment of chronic neuropathic pain. Gabapentin use has more recently
extended into the management of more acute conditions, particularly in
the perioperative period. A number of mechanisms may be involved in
the actions of gabapentin. In this study, we measured the concentrations
of noradrenalin, serotonin and their metabolites in nucleus raphe magnus
(NRM) before and after the injection of formalin in order to gure out
the effect of gabapentin in NRM and pain. Four groups were designed
and each one include 8 samples: group I: control, group II: formalin,
group III: gabapentin, and group IV: gabapentin and formalin. The concentration of neurotransmitters were detected by HPLC-ECD every
15 minutes. Signicant variations observed 25 and 30 minutes after the
injection of formalin in group II and group IV (p= 0.038 & p= 0.039).
Reactions to pain and concentration of neurotransmitters in third samples
of group IV signicantly reduced in compare with their counterpart samples in group II, which shows the effect of gabapentin mainly in chronic
phase of pain and also in acute phase of pain. Consequently, it is con-
cluded that gabapentin doesnt have a direct effect on NRM and regulates the neurotransmitters in NRM by interfering periaqueductal gray
matter signals and functions of VGCCs, AMPA and NMDA receptors.
Paper No.: 2378

OUTCOME AMONG HEAD TRAUMA PATIENTS ON ANTICOAGULANTS OR PLATELET INHIBITORS PRIOR TO TRAUMA
Sigrid Narum, M Kringen, O Brrs, O Stokland
Oslo University Hospital, Ulleval, Department of Pharmacology, Oslo,
Norway
Anticoagulant therapy is increasingly common among older persons. As
a consequence, the incidence of anticoagulant-associated intracerebral
hemorrhage has increased. Whether the use of anticoagulants or platelet
inhibitors affects the mortality after head trauma is not so well studied.
The aim of the study was to describe the mortality among head trauma
patients on anticoagulants or antiplatelet medication by analyzing data
from the trauma registry at Oslo University Hospital, Ulleval. A total of
418 patients 55 years or older admitted to hospital between January
2004 and December 2006 and diagnosed with blunt head trauma were
included in the study. Logistic regression analysis was performed to evaluate the mortality between warfarin/platelet inhibitors-users and nonusers. Motor vehicle accidents (111; 26%) and falls (254; 61%) were the
most common trauma mechanisms. Of the patients, 57 used warfarin and
84 used acetylsalisylic acid and/or clopidogrel. The mean age of patients
was 71 years and 70 (16.7%) patients died within 30 days after injury;
17 (30%) on warfarin and 14 (16.7%) on acetylsalisylic acid and/or clopidogrel. Use of warfarin was associated with signicantly higher mortality (adjusted for gender and age) compared to no use of warfarin (OR
2.1, CI 1.0-4.0). Use of platelet inhibitors prior to trauma was not associated with increased mortality. Higher age was a signicant predictor of
mortality independent of medication use (p=0.002). In conclusion warfarin use prior to trauma was associated with increased mortality after head
trauma. No association was found for use of platelet inhibitors and mortality.
Paper No.: 1949

THE GTP-CYCLOHYDROLASE DEFICIENT MOUSE SHOWS
REDUCED ACUTE PAIN PERCEPTION
Arafat Nasser(1,2), OJ Bjerrum(1), A-M Heedaard(1), LB Mller(2)
(1) Copenhagen University Faculty of Pharmaceutical Sciences, Department of Pharmacology and Pharmacotherapy, Copenhagen, Denmark
(2) Kennedy Center, Glostrup, Copenhagen, Denmark
The hph-1 mouse is a model of GTP cyclohydrolase (GTP-CH) deciency, the rst and rate-limiting enzyme in the de novo synthesis of tetrahydrobiopterin (BH4). GTP-CH is encoded by the GCH1 gene. This
gene is one of few genes that have been shown to modify pain sensitivity
and persistence (Tegeder I et al., Nat Med 2006; 12: 1269-1277). The
aim is to investigate whether GTP-CH deciency reduce pain induced by
formalin as a result of reduced BH4 synthesis. Wild type (WT), hph-1
heterozygotes (+,-), and hph-1 (-,-) littermates are delivered by hph-1 heterozygote breeding. In the formalin test, mice are lightly restrained and a
20-ll solution of 2.5 % formalin is injected subcutaneously into the plantar surface of a hind paw. Licking/biting of the affected paw during
60 min is recorded in 5 min blocks. Preliminary data show that hph-1 (,-) littermates have signicantly lower licking/biting behaviour in the
acute nociceptive phase (0-10 min) of the formalin test compared to WT.
A tendency towards reduced pain-like behaviour in the tonic
486
inammatory phase (10-60 min) is seen. In addition, there was no difference between WT and hph-1 (+,-) mice. Further experiments are ongoing
to complete this study and to elucidate the role of GTP-CH deciency in
a model of inammatory pain (complete Freunds adjuvant).
Paper No.: 2709

DISCOVERY
IMPACT OF OATP1B1, OATP1B3 AND ABCC2 ON THE
HEPATIC UPTAKE AND PHARMACOKINETICS OF THE
MRI-CONTRAST AGENT GD-EOB-DTPA IN HEALTHY
VOLUNTEERS
A Nassif(1), S Oswald(1), J-P Kuhn(2), C Modess(1), N Hosten(2),
Werner Siegmund(1)
Germany
(2) University of Greifswald, Center of Radiology, Greifswald, Germany
Gadoxetate (Gd-EOB-DTPA, Primovist) is a liver-specic magnetic resonance imaging (MRI) contrast agent, which hepatic uptake mechanisms
remain unknown. Because there is evidence from own in-vitro data and
animal experiments that Gd-EOB-DTPA is a substrate of OATP1B1,
OATP1B3 and ABCC2, disposition and hepatic uptake of Gd-EOBDTPA were measured in healthy subjects genotyped for OATP1B1,
OATP1B3 and ABCC2. 23 healthy volunteers (14 males, 9 females)
genotyped for OATP1B1, OATP1B3 and ABCC2 participated in the
study. Hepatic enrichment of GD-EOB-DTPA was quantied according
to a standardized MRI protocol using a 1.5T system (Siemens Magnetom) from 0.25-480 min after bolus injection of Gd-EOB-DTPA
(0.25 mmol/mL) in a volume of 0.1 ml/kg/BW. In addition, serum concentrations-time curves, renal and fecal excretion of Gd-EOB-DTPA were
quantied by LC-MS/MS. Comparative statistical analyses of MRI measurements and pharmacokinetic data of Gd-EOB-DTPA were performed
in different genotype-groups. The hepatic contrast enhancement was signicantly lower in carriers of OATP1B1*15 or *5 as compared to carriers
of the *1a/*1a wild-type alleles (AUC3-480min 25.17.2 vs. 37.2 7.1
arbitrary units, P<0.05). Serum AUC0- and t of Gd-EOB-DTPA were
increased in carriers of *15/*15 (155 22 vs. 126 35 lgh/ml, n.s.,
16.6 2.9 vs. 13.4 5.6 h, P<0.01). The hepatic contrast enhancement
and systemic exposure to Gd-EOB-DTPA was not altered in carriers of
OATP1B1*1b/*1b. Polymorphisms of ABCC2 and OATP1B3 were without inuence on MRI contrast and serum pharmacokinetics of Gd-EOBDTPA. OATP1B1 seems to be major variable in hepatic uptake of the
liver-specic contrast agent Gd-EOB-DTPA in human.
Paper No.: 2318

ANTI-INFLAMMATORY EFFECTS OF BERGAMOT
ESSENTIAL OIL IN CARRAGEENAN-INDUCED PAW
OEDEMA IN RAT
Michele Navarra(1), C Mannucci(2), E Trapasso(1), S Pergolizzi(3),
MR Ursino(1), S Micali(1), N Miceli(1), G Calapai(2)
(1) University of Messina, Pharmaco-Biological Department, Messina,
Italy
(2) University of Messina, Department of Clinical and Experimental
Medicine and Pharmacology, Messina, Italy
(3) University of Messina, Department of Animal Biology, Messina,
Italy
bergamia (bergamot), a small tree belonging to the family Rutaceae, is

cultivated almost exclusively in the Southern costs of Calabria region
(Italy) for the extraction of its essential oil widely used in perfume industry. It comprises a volatile fraction (93-96%) containing monoterpene
and sesquiterpene hydrocarbons and a nonvolatile fraction (4-7%) consisting essentially of coumarins and psoralens (Costa et al., Flav Fragr J,
2010). Because the toxicity of bergapten is well known, our study has
been performed using the BEO fraction deprived of bergapten (BEOBF). As determinate by plethysmometer, treatment with BEO-BF (100
and 500 microL/Kg) led to a signicant inhibition of paw oedema
induced by a subplantar injection of carrageenan. In order to gain a better
insight into the mechanism(s) of action of BEO-BF, some mediators of
early phase of inammation were take in account. So, IL-6 and TNF-a
levels in the paw homogenate were measured by ELISA assays. BEOBF treatments decreased both the IL-6 and TNF-a levels with respect to
the carrageenan control group. Furthermore, in BEO-BF-treated rats we
observed also a reduction of nitrite/nitrate levels, as measured by Griess
reaction in exudates. The latter could be related to the free radicals scavenging properties of BEO-BF (DPPH test). Moreover, histological examination of paw biopsies showed a reduction of pathological changes
typically of oedema in BEO-BF treated rats.
Paper No.: 2148

MUTATIONAL ANALYSIS OF AN M4 MUSCARINIC
ACETYLCHOLINE RECEPTOR ALLOSTERIC SITE
Vindhya Nawaratne(1), K Leach(1), CC Felder(2), PM Sexton(1),
A Christopoulos(1)
(1) Monash University, Drug Discovery Biology Laboratory, Department
of Pharmacology, Parkville, Melbourne, VIC, Australia
(2) Eli Lilly, IN, USA
The M4 muscarinic acetylcholine (ACh) receptor (mAChR), a G proteincoupled receptor, is a potential target in the treatment of a number of
CNS disorders. Recently, we identied a novel class of small molecule
modulator that selectively targets this receptor via an allosteric site (Chan
W et al, Proc Nat Acad Sci (USA) 2008; 105: 10978-10983, Nawaratne
V et al, Mol Pharmacol 2008; 74: 11191131). The current project aimed
to identify residues in the M4 mAChR extracellular loops 1 and 2 (e1
and e2) and transmembrane 7 (tm7) that may be involved in allosteric
ligand activity. Radioligand binding and phosphorylation of extracellular
signal-regulated kinase 1/2 (pERK1/2) were used to determine the binding and function, respectively, of ligands at mutated and wild type (WT)
receptors expressed in a CHO cell line. I93T (e1), whilst having no effect
on the potency of ACh, enhanced both the potency of the allosteric agonist, LY2033298, and its ability to potentiate ACh binding. Although,
F186A (e2) had no effect on the activity of ACh, it completely abolished
the binding of LY2033298. Y439A (tm7), whilst being detrimental to the
activity of ACh, surprisingly abolished the efcacy of LY2033298 and
decreased its ability to potentiate ACh binding, with the afnity of
LY2033298 remaining unchanged. These ndings highlight new roles
for extracellular loops and tm7 in allosteric modulator activity.
Paper No.: 2421

PRE-EMPTIVE TREATMENT WITH HEMIN SUPPRESSES
HYPERGLYCEMIA AND IMPROVES GLUCOSE
METABOLISM AND DIABETIC NEPHROPATHY IN THE
FIRST GENERATION OFFSPRING OF GOTO-KAKIZAKI RATS
Joseph F Ndisang(1), N Lane(1), A Jadhav(1)
Renewed interest in natural products as potential source of anti-inammatory led us to investigate the anti-inammatory activity of bergamot
essential oil (BEO) in the carrageenan-induced paw oedema in rat. Citrus
University of Saskatchewan, College of Medicine, Saskatoon, Canada
487
We investigated whether pre-emptive upregulation of heme-oxygenase
(HO) in pregnant Goto-Kakizaki-rat (GK) would be benecial in F1-generation. GK is a non-obese type-2 diabetic model characterized by elevated endothelin-1 (ET-1)/oxidative stress, impaired insulin-signaling/
glucose metabolism and diabetic nephropathy. HO was induced with
hemin or blocked with stannous-mesoporphyrin (SnMP). In 4-month-old
F1-generation, fasting/postprandial hyperglygemia, gastrocnemius/plasma
ET-1, 8-isoproatane, pro-inammatory/oxidative mediators including NFjB, activating-protein, c-Jun-N-terminal-kinase, proteinuria, albuminuria
and albuminuria-to-creatinine index were reduced while adiponectin and
creatinine clearance increased. Correspondingly, adenosine monophosphate-activated-protein-kinase (AMPK), GLUT4, aldolase-B, glucose tolerance (IPGTT), IPITT and HOMA-IR index revealed enhanced insulin
sensitivity/glucose metabolism and improved renal function, whereas
SnMP abolished the hemin effects. The metabolic/renoprotection were
associated with enhanced gastrocnemius-muscle HO-1, HO-activity,
cGMP, cAMP, bilirubin, ferritin, superoxide dismutase with subsequent
potentiation of the total-antioxidant capacity. Since ET-1, oxidative stress
and adiponectin deciency are associated with renal dysfunction, the
concomitant suppression of ET-1 and oxidative/inammatory insults
alongside increased adiponectin may account for reduced proteinuria/
albuminuria, increased creatinine clearance and thus improved renal
function. The renoprotection in combination to improved IPGTT, IPITT
alongside increased AMPK and GLUT4 may account for the improved
glucose metabolism in F1-generation. The hemin-mediated remediation
of impaired insulin-signaling/glucose metabolism and renal deciency,
and its subsequent transcendence from parents to offspring is a novel
observation, suggesting that pre-emptive upregulation of HO in pregnant
females positively impact the health-status of offspring.
Paper No.: 1548

THE INFLUENCE OF MAGNESIUM, ZINC AND COPPER IN
REWARD SYSTEM IN RATS
Mihai Nechifor, D Chelarescu, D Ciubotariu
Gr. T. Popa University of Medicine and Pharmacy, Department of
Pharmacology, Iasi, Romania
We have studied the inuence of 3 bivalent cations :magnesium (as
MgCl2, 0.5 mM/kg/day i.p), zinc (as ZnCl2, 0.1 mM/kg/day i.p) and
copper (as copper acetate, 0.05 mM/kg/day i.p) on morphine-induced
place preference in naive Wistar rats. We have used place preference
boxes (Panlab) on a 12 days experiment schedule (4 pairings with the
studied substance).We followed the inuence of specic cation alone on
place preference and the values were compared with those in cation associated with morphine group and morphine alone group. We have compared also for naive animals, time spent in cation-associated
compartment before and after conditioning. Results were statistically
interpreted with t test. Our data show that MgCl2 alone has moderate
effects on increasing the time spent in associated conditioned compartment (327.75 14 s in Mg2+ group before conditioning vs. 285.2 11 s
in Mg2+ group after conditioning, P<0.05). Added to morphine (3 mg/
kg/day i.p), Mg2+ has decreased acquisition of morphine-induced place
reference (312.1 37 s in post conditioning morphine+Mg group vs
462.15 28.69s in post conditioning morphine group, p< 0.05). Administrated alone, zinc doesnt inuence signicantly place preference, but
decreased morphine-induced place preference (394.1 28 s in morphine+Zn group vs. morphine group, p< 0.05). Copper has no inuence
either alone or associated with morphine on place preference. Our data
have shown that some of bivalent cations tested had differentiated inuence on reward system. Some of them could modulate the effect of some
addictive substances (such as morphine).
Paper No.: 3425

THE INVOLVEMENT OF NITRIC OXIDE IN THE LOCAL
ANTINOCICEPTIVE EFFECTS OF JWH-015 AND
EXPRESSION OF CANNABINOID-2, L- AND D-OPIOID
RECEPTORS DURING CHRONIC INFLAMMATORY PAIN IN
MICE
R Negrete(1), J Martn-Campos(2), A Hervera(1), S Leanez(1), Olga
Pol(1)
(1) Grup de Neurofarmacologia Molecular. Institut de Recerca de lHospital de la Sta Creu i Sant Pau & Institut de Neurocie`ncies, Universitat
Auto`noma de Barcelona, Barcelona, Spain
(2) Grup de Bioqumica, Institut de Recerca de lHospital de la Sta Creu
i Sant Pau, Barcelona, Spain
Nitric oxide modulates the antinociceptive effects of l-(MOR) and d(DOR) opioid receptor agonists during peripheral inammation but its
role in the local antinociceptive effects of cannabinoid-2 receptor
(CBR2) agonists and the expression of CBR2, MOR and DOR during
chronic inammatory pain is not known. Our aim is to evaluate if nitric
oxide released during inammatory pain could affect the antinociceptive
effects of JWH-015 and modulate CBR2, MOR and DOR gene expression. Chronic inammatory pain was induced by the subplantar injection
of CFA. The antinociceptive effects of JWH-015 and expression of
CBR2, MOR and DOR mRNA levels in the dorsal root ganglia and
spinal cord of wild type (WT) and neuronal nitric oxide synthase knockout (NOS1-KO) mice were evaluated. NOS1 mRNA levels were also
assessed in WT mice by real-time PCR. The antinociceptive effects of
JWH-015 were measured by using von Frey laments and plantar test.
Inammatory pain increases NOS1 gene expression. The local antinociceptive effects of a CBR2 agonist and their dorsal root ganglia gene
expression in NOS1-KO mice with inammatory pain were signicantly
decreased as compared to WT. Moreover, a diminished gene transcription
of MOR and DOR in the dorsal root ganglia of NOS1-KO mice was also
demonstrated. Results suggest that nitric oxide derived from the
increased expression of NOS1 is implicated in the local antinociceptive
effects of JWH-015 and in the regulation of CB2R, MOR and DOR gene
transcription during inammatory pain.
This work was supported by grants from FIS (PS0900968) and Fundacio
La Marato de TV3 (070810).
Paper No.: 2399

HEALTH AND DISEASE
ANTIHYPERTENSIVE EFFICACY OF DUAL ECE/NEP
INHIBITION DURING THE ONSET OF SPONTANEOUS
HYPERTENSION IN RATS
Jelly Nelissen, BFJ Heijnen*, P Lemkens, H van Essen, JGR De Mey,
HAJ Struijker-Boudier, BJA Janssen
Maastricht University, Department of Pharmacology & Toxicology,
Maastricht, Th Netherlands
Introduction: Endothelin-1 (ET-1) is produced by the metalloproteases
endothelin converting enzyme (ECE) and neutral endopeptidase (NEP)
and its role in the development of hypertension is discussed. The dual
ECE/NEP inhibitor SOL-1 (an SLV 309 analogue) inhibits stimulated
ET-1 synthesis in isolated mesenteric resistance arteries. Hypothesis:
Dual ECE/NEP inhibition by SOL-1 will have have blood pressure lowering efcacy during the onset of hypertension. Methods: 4 weeks old
spontaneous hypertensive rats (SHR) were treated for 4 weeks with the
dual ECE/NEP inhibitor SOL-1 (50 mg/kg/d s.c.). At 8 weeks of age rats
were instrumented with an arterial cathether and Mean Arterial Pressure
(MAP) was measured in conscious unrestrained rats. Afterwards rats
were sacriced and organs were harvested for further analysis.
488
Results: MAP was signicantly reduced in SOL-1 (n=4) treated compared
to vehicle (n=14) treated SHR (125.8 mmHg vs 143.5 mmHg). Relative
heart and lung weights were signicantly lowered in the SOL-1 compared
to the vehicle treated group (0.355 g.100g BW-1 vs 0.396 g.100g BW-1)
and (0.450 g.100g BW-1vs 0.491 g.100g BW-1) respectively. Conclusion: Chronic ECE/NEP inhibition during the onset of hypertension in the
SHR has a blood pressure-lowering effect. These data suggest a role for
ET-1 during the onset of hypertension in the SHR.
*Shared rst authorship.This research was performed within the framework of project T2-108 of the Dutch Top Institute Pharma.
Paper No.: 2498

DESCRIPTION AND APPLICATION OF THE THRITTENE
RADIOIMMUNOASSAY
by ligands that bind todistinct sites from the orthosteric one. Given that
these allosteric sites canprovide for selective targeting of receptors, we
reasoned that bivalent ligandscould bind both to a region within the orthosteric site as well extending intoregions of the receptor that contribute
to an allosteric site, resulting intighter binding and greater subtype specicity than that obtained with classicagonists and antagonists. Equilibrium
binding assays were performed tocharacterize the afnity prole of a series of 20 newly synthesized moleculesin which two carbachol heads are
linked by a methylene chain of various length.Radioligand binding studies were performed at the ve human muscarinicreceptors stably
expressed in CHO cells using [3H]NMS as the radioligand. Themost
interesting compounds were studied with kinetic binding assays to ndevidence for a simultaneous allosteric/orthosteric binding and they were
testedin functional assays to evaluate their efcacy. The binding afnity
of thesecompounds increased with the length of the spacer moiety (Ki
values ranging from100 to 0.1 micromolar) accompanied by a shift from
agonist to antagonistbehaviour. Some of the tested compounds were able
to slow the dissociationkinetics of the radioligand, indicating a possible
allosteric mode onteraction with the receptor.
Jozsef Nemeth(1,2), G Furjes(1), A Toth(1), B Peitl(1,2), R Sari(1,2),

R Porszasz(1,2), Z Szilvassy(1,2)
(1) University of Debrecen, Department of Pharmacology and Pharmacotherapy, Debrecen, Hungary
(2) Pharmapolis Debrecen Ltd, Debrecen, Hungary
Paper No.: 2357

GENDER DIFFERENCES IN THE DISPOSITION OF DRUGS
The peptide hormone - Thrittene - has the same amino acid structure as
somatostain-28 N-terminal (1-13) amino acid sequence. It was detected
in mammalian gut and plasma by immunochemistry and subtraction
radioimmunoassay (RIA). Our research group working on diabetes and
obesity considered that it is important to develop direct Thrittene RIA
method that is specic and sensitive enough for Thrittene. The antiserum
(TH3), sensitive to the C-terminal region of peptide was produced by the
immunization of rabbits administering Thrittene-bovine serum albumin
antigen subcutaneously. To show the antibody attachment we used iodogen method labelled mono-125I-Tyr(0)-Thrittene. Tyr(0)-Thrittene was
used as standard for the RIA determination in a range of 0-1000 fm/ml.
After 10 measurements the D50 value of the calibration curves was
8.611.22 fmol/ml. The detection limit of method was 0.2 fmol/ml Thrittene. The cross-reactivity studies showed that the antiserum used in our
RIA method has limited cross-reactivity with other peptides with similar
structure. By determining Thrittene content of rat gastrointestinal tract
tissue, the highest concentration was measured in duodenum followed by
jejunum and ileum; however, all the examined tissues contained highly
enough Thrittene for measurement. Eventually, the receptor of Trittene in
rat brain tissue was identied using receptor binding assay (Bmax:
0,230,05 fmol/mg protein and Kd: 9,501,81 nmol). The Thrittene RIA
method developed in our laboratory has high sensitivity and peptide
specicity. It is suitable for determining tissue and plasma concentrations
and it helps to get further information about the biological role of the
peptide hormone.
C Neta, J Silva, F Martins, A Pedroso, T Macedo, Carlos Fontes Ribeiro
Paper No.: 1948

PHARMACOLOGICAL EVALUATION OF NEW BIVALENT
LIGANDS AIMED TO INTERACT WITH ORTHOSTERIC AND
ALLOSTERIC SITES OF MACHRS
Paper No.: 2451

PHARMACOLOGY IN UNO-AGENDA 21
AIBILI, Centre for Bioavailability Studies, Coimbra, Portugal

Gender is usually considered to be one of the factors inuencing disposition of drugs, but the evidence available is sometimes conicting and
information for a large number of frequently used drugs is lacking. An
evaluation of sex differences in the pharmacokinetics of several drugs
(omeprazole, lanzoprazole, piroxicam, acyclovir and deazacort) was
carried out during bioequivalence studies performed at our laboratory,
with male and female volunteers. Furthermore, the oral contraception
inuence was also assessed within women. Differences between gender
and concerning oral contraception were evaluated by the Students t test.
The results from our studies suggest that women have a better bioavailability prole (statistical signicant differences observed for piroxicam,
omeprazole and deazacort principally for higher doses). Regarding
the contraception, it seems that women who take oral contraception show
higher concentrations (statistical signicant differences observed for acyclovir, deazacort and omeprazole). In conclusion, further studies should
be carried out, with a large number of participants, in order to conrm
the current data. In addition, we believe that the ofcial workforces
should reect on the magnitude of inclusion of women on bioequivalence studies and, by other hand, it should be considered, prior to prescription of a drug (especially those of a narrow therapeutic range), the
clinical relevance of sex-dependent differences on drug bioavailability.
Eva Neu(1,2,3), U Welscher(1,2,3), B Schubert(1), MC Michailov(1,2,3),

M Holler(4), M Schratz(5), W Seidenbusch(3,1), G Weber(6),
I Ivanova(1), V Foltin(7), U Harlin(1), Y Zebuhr(1)
Marta Nesi
University of Florence, Department of Preclinical and Clinical Pharmacology, Florence, Italy
Most of the current drugs targeting muscarinic receptors bind to the
orthostericsite of the receptor mimicking the action of the endogenous
ligand. Muscarinicreceptors are known to be also subject to modulation

Muenchen, Germany
Germany
Austria
489
(4) University of Hamburg, Faculty of Economics/Dean, Hamburg,
Germany
(5) University of Innsbruck, Faculty of Education/Dean, Innsbruck,
Austria
(6) University of Luxembourg & Vienna, Faculty of Psychology/Dean,
Vienna, Austria
(7) Slovak Technical University, Bratislava, Slovakia
Introduction: New scientic and organizational models of education/
research incl. the fundamental medical science pharmacology are necessary to counteract enormous global health problems. Method: Contribution-analysis of international congresses (physiology, radio-oncology,
genito-urology, etc.) demonstrates large discrepancies between developing and industrial countries (Faseb-J. IUPS-2005/ San-Diego;19/
5:A1355; J.Physiol.Sci. IUPS-2009/Kyoto;59/1:247-8). Results: Intern.
IUPHAR-congresses: Copenhagen (World-Pharma-2010, lectures: n=24
=100%)/Beijing (Acta-Pharm.Sinica 2006;27/1: evaluated 2583, 5%
unclear, speaker-poster contr, n(s)=279=100%, i.e. n(p)=2165 without
China 1019(47.1%) =1146=100%)/San Francisco (Pharmacologist 2002;
44/2/1; n=1607=100%) reect a similar situation: Africa-0/0.7-2.5/2.6%;
America-20.8/29.7-27.5/38.6% (North-A.-20.8/29.4-20.5/-, USA-12.5/
24.7-13.0/27.9;
South-A.-0/0.4-2.4/-);
Asia-20.8/41.5-29.2/25.8%
(China-8.3/13.6-47.1/5.6, India-0/0.4-1.7/1.2, Japan-8.3/10.0-11.2/13.1);
Australia-0/2.1-4.9/3.4%; Europe-58.3/40.5/36.9% (West-E.-58.3/39.425.4/25.3: France-8.3/8.2-1.9/3.6, Germany-12.5/6.4-2.4/3.1, GB-20.8/
10.7-3.8/7.5, East-E.-0/1.1-11.5/4.1: Russia-0/0-2.0/1.1). Extremely high
West-European-USA domination (50-70%) is evident: Whole (sub-) continents are nearly or not presented. Proposals on future pharmacology: 1.
Common interdisciplinary IUPHAR-sessions about philosophy/FISP
(medical ethics, etc.), psychology/IUPsyS (psychopharmacology), experimental & clinical medicine (angiocardiology/ISIM, physiology/IUPS,
etc.). 2. New fundamentals of IUPHAR-Council: a. 1-3 honorary (incl.
Nobel Laureates*: moral support) & presidents; b. establishment of interdisciplinary section (see 1.). 3. Congress-Books replacement by Proceedings or IUPHAR journal, compare Int. J. Psychol./IUPsyS Berlin-2008,
Urology/SIU Paris-2007). 4. Creation of rst Int. Inst. for Pharmacology
by network of national ones: Common educational & research programmes, personnel, possibility for whole life work, etc. Conclusion:
Realization of proposals (1.-4.) could increase scientic/political IUPHAR-authority and serve for renovation model in medicine, supporting
UNO-Agenda 21 for better health, ecology, etc. in all countries.
Dedicated to moral ICSD-support (1989-2010; honorary members/*): Sir
J.Black*/UK, U.&K.Graf Goess-Enzenberg/Austria, Furst H.-A.&Prinz
A. von Liechtenstein, L.Pauling*/USA, A.Saccharov*/Russia, W.Scheel/
Germany, M.Schumann France, Lord A.Todd*/UK, Bishop Tutu*/SouthAfrica, B.Vogel/Germany
Paper No.: 2446

FUTURE APPROACHES TO PHARMACOLOGY IN CONTEXT
OF PHILOSOPHY AND ECONOMICS
Renate Neu(1,5), MC Michailov(1,2,3), M Holler(4), E Neu(1,2,3),
T Senn(1,5), RK Stappen(1), R Schinabek(1), G Schulz(1), D Martin(1),
S Molnar(1,5), M Schratz(5), G Weber(6)
Muenchen, Germany
Germany
Austria
(4) University of Hamburg, Faculty of Economics/Dean, Hamburg,
Germany
(5) University of Innsbruck, Faculty of Education/Dean, Innsbruck,
Austria

Austria
Introduction: According to epistemology, normal & pathological human
physiology, morphology and genetics are 3 leading bio-medical sciences
(from molecular via cellular up to psychic level), destining diagnostics/
therapy, whereby pharmaco-toxicology (drugs&chemicals=D&C) is a
fundamental discipline for treatment/prophylaxis. Limits between useful/
toxic effects are deciding for application. Regrettably there is till today
D&C-use sometimes contradictory to scientic theory following disastrous repercussions (a), also elementary principles of moral philosophy/
theology are not applied (b). The most difcult factor is pharmaceutical
economy: Consumption of more D&C supporting industrial prot is not
conforming with a.-b. and can lead to misuse of D&C-research (c). Conception: Harmonious future education & research in pharmacology/pharmacy needs elementary implication of moral philosophy, ecumenical
theology (Brahmanism, Buddhism, Christianism, etc.), psychology and
economics (1), related to foundation of interdisciplinary pharmacological/pharmaceutical institutes incl. departments for philosophy, economics,
etc. between universities & industry on local (2), continental (European,
etc.) and international level (3) in context of international universities
(proposed by the British *Nobel-Laureate B.Russell) reecting an integrative pharmacology, toxicology & pharmacy by network of national
departments (philosophy, psychology, economics, physiology, etc.) (4)
incl. help for developing countries. Acc. to E.Neu et al. (Fund.&Clin.
Pharm. EPHAR-2004; 18/1:119&Acta-Pharm.Sinica, IUPHAR-2006;27/
1:1-250): 50-70% of scientic contributions are from West-Europe/USA,
but Asia, India, South-America:0-2% (5). Conclusion: New scientic &
organizational models in pharmacology (1-5) could help essentially for
UNO-Agenda21 for better health, ecology, economy in all countries.
Dedicated to moral ICSD-support (1989-2010; Hon. members): M.Allais*/France, G.Debreu*/USA, J.Deisenhofer*/FRG-USA, U.&K.Graf
Goess-Enzenberg, J.Mittelstra/FRG, H.Rohrer*/Suisse, B.Schubert/Austria, Lord G.Porter*/UK, H.Simon*/USA, J.Tinbergen*/ Netherlands
Paper No.: 3119

CONDITIONAL MUTANT MOUSE WITH ENHANCED GA-I2
SIGNALLING DUE TO LOSS OFREGULATOR OF G PROTEIN
SIGNALLING (RGS) REGULATION
Richard Neubig(1), X Huang(1), S Wade(1), R Charbeneau(1)
(1) University of Michigan, Department of Pharmacology, Ann Arbor,
USA
G protein coupled receptors (GPCRs) which signal through the Gi/o family of G proteins are the target of many therapeutically important agonists
and neurotransmitters including morphine, dopamine, and serotonin. It
has been difcult, however, to determine which of the 5 Gi/o family G
proteins mediates which of their effects. The Gi/o proteins are subject to
strong negative regulation by the GTPase accelerating activity of RGS
proteins which have been proposed to be potentially interesting drug targets. We have recently developed mutant mice in which RGS-mediated
negative regulation is lost for Gao and Gai2 (G184S mutants). This
results in enhanced but receptor-dependent signalling through the modied Gi/o protein and also demonstrates a role of RGS proteins in these
processes. Gai2 G184S mutant mice have interesting phenotypes including resistance to high fat diet, antidepressant-like behaviour, and protection against cardiac ischemia-reperfusion injury. To better understand
these effects and to identify potential receptors and/or RGS proteins that
could be targeted to produce them therapeutically, we have developed a
novel conditional Gai2 G184S knock-in mouse model. This will permit
tissue-specic and/or temporally regulated expression of the enhanced
Gai2 signaling. For complex phenotypes like obesity-resistance and antidepressant effects, the conditional mutants will let us determine the tissue
locus and/or neuronal subtypes that underlie the effects. This will permit
490
mechanistic analysis and denition of the receptors and/or RGS proteins
involved and will facilitate novel therapeutics development.
(Supported by NIH R01-GM39561)
Paper No.: 2960

PHENOTYPING OF HETEROZYGOUS CALSEQUESTRIN
TARGETED MICE
Joachim Neumann(1), P Bock(1), M Fischer(1), S Hauptmann(2),
U Gergs(1)
(1) Martin-Luther-University Halle-Wittenberg, Medical Faculty, Institute
for Pharmacology and Toxicology, Halle/Saale, Germany
(2) Martin-Luther-University Halle-Wittenberg, Medical Faculty, Institute
of Pathology, Halle/Saale, Germany
Cardiac calsequestrin (CSQ2) is thought to be the essential Ca2+ storage
protein in the mammalian heart. However, mice with deletion of CSQ2
are viable (Knollmann et al., J Clin Invest 2006; 116: 25102520). To
further characterize the role of CSQ2 we generated our own heterozygous CSQ2 (CSQ2+/)) mice. In contrast to Knollmann et al., we did not
delete the promoter of CSQ2 but replaced exon-1 by a loxP site leading
to a nonsense mutation. Atria and ventricles of CSQ+/) mice displayed
about 50% less CSQ than wild type mice (WT). CSQ2+/) mice are viable and gross morphology of the heart as well as relative heart weights
(6.340.63 and 5.741.51, n=4-5) were not different compared to WT.
Surface ECG was recorded before and after injection of isoprenaline.
The PR interval was prolonged after injection of isoprenaline to
37.71.01ms in WT and 42.50.34ms in CSQ+/) (n=3, p<0.05). Contractile studies were performed in isolated spontaneously beating right
and electrically driven left atria. Protocols of post rest potentiation, caffeine induced Ca2+ release and increased beating rates (staircase phenomenon) were applied. We noted a negative staircase phenomenon in
WT but not in CSQ+/) atria. For instance, from 1Hz to 5Hz force was
reduced to 44.77.4% in WT and 91.04.8% in CSQ+/) (n=3). Probably, less Ca2+ is lost from the cytosol in CSQ2+/) than in WT after elevation of the beating rate. In summary, CSQ2+/) might be a useful
model to investigate the role of CSQ2 in force generation.
(Supported by Roux program and DFG)
Paper No.: 3424

DISEASES
UP-REGULATION OF SURVIVIN EXPRESSION IN
SIMVASTATIN TREATED RATS IN ENDOTOXIC SHOCK
Lana Nezic(1), L Amidzic(2), R Skrbic(1), SS Satara(1), N Stojakovic(1)
(1) University Banja Luka Medical Faculty, Department of Pharmacology, Toxicology & Clinical Pharmacology, Banja Luka, Bosnia and
Herzegovina
(2) University Banja Luka Medical Faculty, Department of pathology,
Laboratory for Immunohistochemistry, Banja Luka, Bosnia and
Herzegovina
Introduction: Studies have showed that statins have protective effects in
high grade acute inammation, reducing leukocyte inltration and dealing with pro- and anti-apoptotic mechanism in cells and leucocytes. This
study explores effect of simvastatin (SIMVA) on the expression of antiapoptotic molecule survivin in multiple organ damage induced by endotoxic shock. Methods: Wistar rats were randomized into control (sham),
lipopolysaccharide (LPS) and SIMVA+LPS groups. LPS groups received
single median lethal dose (LD50) of LPS intraperitoneally (i.p). Rats in
SIMVA+LPS groups received increasing doses of SIMVA (10-30 mg/kg

p.o, over 5 days) before single LD50 LPS. Survival rate was monitored
at intervals of 12 h during 24 h over one week. Liver, heart and kidney
tissues were taken for pathological examination (stained with H&E, histologically graded according to the severity of injury) and for their
expression of survivin (cytoplasmic and nuclear) by immunohistochemistry. Results: SIMVA completely prevents LPS-induced death in dosedependently, signicantly ameliorated the deleterious effects of LPS on
the liver and heart, respectively (score 2, p<0,05 vs. LPS group; score
system of inammation 0-4), and preserved renal tissue histology (score
1, p<0,05 vs. LPS group; scoring system 0-2). The expression of survivin and survivin mRNA were signicantly up-regulated in SIMVA
groups in dose-dependant manner. Survivin overexpression was detected
in following order: hepatocytes, macrophages, renal proximal tubular
cells, cardiomyocites. Conclusion: One of the potential mechanisms of
statin protective role in severe inammation ie endotoxic shock could be
explained by its anti-apoptotic effect mediated by up-regulated expression of cell protective molecul survivin.
Paper No.: 1739

PERTUSSIS TOXIN INHIBITS EP4 AND IP RECEPTORMEDIATED CAMP PRODUCTION IN ADULT RAT DORSAL
ROOT GANGLION CELLS
Kai-Yu Ng(1), Y-H Wong(2), H Wise(1)
Sciences, Hong Kong, PR China
(2) The Hong Kong University of Science and Technology, Department
of Biochemistry, Hong Kong, PR China
We have observed that prostanoid receptor agonists such as cicaprost and
prostaglandin E2 (PGE2) stimulate [3H]cAMP production in cultures of
glial cells, from adult rat dorsal root ganglia (DRG), in an IP and EP4dependent manner. Furthermore, this receptor-specic activity in glial
cells can be inhibited by the presence of DRG neurons. To investigate
the potential role of Gi/o proteins in mediating this inhibitory effect of
neurons, we compared the effect of pertussis toxin (PTx; 100 ng/ml overnight) on receptor-dependent adenylyl cyclase activity in DRG glial cells,
neurons and mixed cell cultures. PTx had no effect on responses in glial
cells or neurons but surprisingly attenuated responses to cicaprost (35%
inhibition at 100 nM) and PGE2 (55% inhibition at 100 nM) with no
effect on EC50 values. In conclusion, PTx failed to increase cAMP production in any of the three cell groups tested. However, we cannot yet
exclude the role of Gi/o proteins in mediating the interaction between
neurons and glial cells because PTx unexpectedly inhibited adenylyl
cyclase activity in the mixed DRG cell cultures.
[Acknowledgement: This work was supported by a grant from the
Research Grants Council of the Hong Kong Special Administrative
Region (CUHK4516/06M).]
Paper No.: 591

THYMOQUINONE FROM NIGELLA SATIVA INDUCES
APOPTOSIS IN SIHA CELLS VIA P53-DEPENDENT PATHWAY
Wei Keat Ng(1), Latifah Saiful Yazan(2), Maznah Ismail(3)
(1) University Putra Malaysia, Institute of Bioscience, Laboratory of
Molecular Biomedicine, Upm Serdang, Selangor, Malaysia
(2) University Putra Malaysia, Faculty of Medicine and Health Sciences,
Department of Biomedical Science, Upm Serdang, Selangor, Malaysia
491
(3) University Putra Malaysia, Faculty of Medicine and Health Sciences,
Department of Nutrition and Dietetics, Upm Serdang, Selangor, Malaysia
Introduction: Thymoquinone (TQ) from Nigella sativa has been shown
to exert anti-inammatory, anti-oxidant and anti-cancer properties both in
vitro and in vivo. This study investigated the cytotoxicity of TQ towards
human cervical squamous carcinoma cells (SiHa). Methods: Cytotoxicity
of TQ was determined by the MTT assay and trypan blue dye exclusion
test. The morphological changes were viewed under an inverted light
microscope. Cell cycle analysis was performed by owcytometer. The
mode of cell death was determined by AO/PI and Annexin V/PI staining.
Expression of p53 was evaluated by Human p53 ELISA kit. Results: TQ
exhibited cytotoxicity towards SiHa cells with IC50 value of 10.67
0.12 lg/ml and 9.33 0.19 lg/ml as determined by the MTT assay and
trypan blue dye exclusion test, respectively after 72 hours of incubations.
As compared to cisplatin, TQ was more cytotoxic towards SiHa cells.
Nevertheless, TQ was less cytotoxic towards normal cells as compared
to cisplatin. Cell cycle analysis showed that TQ induced cells accumulation at the sub-G1 phase. Both AO/PI and Annexin V/PI staining indicate that TQ induced apoptosis in the cells. Expression of p53 detected
by using the Human p53 ELISA kit showed that SiHa cells incubated
with TQ for 72 hours resulted in up-regulation of the expression of the
protein as compared to the control untreated sample. Conclusions: It is
concluded that TQ was cytotoxic towards SiHa cells in a dose-dependent
manner and induced apoptosis via p53-dependent pathway.
Keywords: Thymoquinone; Nigella sativa, cervical cancer, apoptosis,
p53
Paper No.: 938

DETERMINATION OF TRIMETAZIDINE IN HUMAN PLASMA
FOR ASSESSING BIOEQUIVALENCE STUDY
Paper No.: 1164

DEVELOPMENT AND VALIDATION OF A SENSITIVE LIQUID
CHROMATOGRAPHY/TANDEM MASS SPECTROMETRY
(LC-MS/MS) METHOD FOR QUANTIFICATION OF
OLANZAPINE IN HUMAN PLASMA
Suthatip Ngokpol, P Pongnarin, A Kongsiang, J Jantaka, U Panich,
S Chatsiricharoenkul, S Kongpatanakul
Siriraj Bioequivalence Center, Department of Pharmacology, Bangkok,
Thailand
A sensitive and specic liquid chromatographytandem mass spectrometric (LCMS/MS) method for the determination of olanzapine in human
plasma was developed. Olanzapine and loratadine (internal standard)
were isolated from human plasma using liquid-liquid extraction technique at basidic pH. Chromatographic separation was achieved on a C18
column (2.5 lm, 50 mm3.0 mm i.d.) with the mobile phase of acetonitrile and 10 mM ammonium acetate (60:40, v/v). Olanzapine and its
internal standard were detected using a mass spectrometer, equipped with
positive-ion electrospray ionization and set in the multiple-reaction-monitoring mode. The ion transitions monitored were m/z 313.20>256.27;
313.20>83.78 for olanzapine, and m/z 383.07>337.18; 383.07>267.20
for loratadine. The method was validated over the linearity range of
0.005 50.0 ng/mL (r2 = 0.9996). The intra-day and inter-day of precision and accuracy values were better than 5.53% and 106.88%. The
recoveries of olanzapine were above 95.99%, with that of loratadine
being 105.7%. The lower limit of quantication for olanzapine was
0.005 ng/mL using a 500 lL plasma sample. This method was successfully applied to a pharmacokinetic study of olanzapine after oral application of 10 mg in healthy Thai volunteers.
(Nirogi R V.S.R. et al, J. Pharm. Biomed. Anal. 2006; 41:935-942. and
Arrigo C. D. et al, Therapeutic Drug Monitoring, 2006; 28: 388-393)
Suthatip Ngokpol
Siriraj Bioequivalence Center, Department of Pharmacology, Bangkok,
Thailand
Introduction: Trimetazidine is well-known as potential antianginal drugs
in ischaemic heart disease. Although the pharmacokinetics and clinical
pharmacology of trimetazidine have been thoughly studies, there is inadequate data on Thai population. Therefore, a sensitive LC-MS/MS
method for the quantication of trimetazidine in plasma was developed
and carry out to studies of pharmacokinetic and bioequivalence for two
modied release trimetazidine formulations in healthy Thai volunteers.
Materials: Trimetazidine hydrochloride and lidocaine (internal standard)
were obtained from Unison Laboratory. All solvents of HPLC grade and
analytical grade were purchased from Merck, Germany. Results: Trimetazidine plasma concentrations were quantied by a validated method
employing LC-MS/MS technique. Plasma samples were isolated by
liquid-liquid extraction with acidic condition and a mixture of n-Hexane
: Methyl-t-butyl ether (1:1, v/v). The chromatographic technique led to
the isolation of trimetazidine and lidocaine with a mobile phase, which
comprised of acetonitrile and 10 mM ammonium acetate under a gradient condition. The detection exhibited the lower limit of quantication
(LLOQ) according to 0.25 ng/mL. A linearity in the range of 0.25 to
1,000 ng/mL was established. The intra-day and inter-day variation were
in an acceptable range (80%-120%). The study was successfully applied
to evaluate the bioequivalence of trimetazidine modied release tablets
between healthy Thai volunteers. The pharmacokinetic parameters
including Cmax, AUC0-24 and AUC0-(obs) of trimetazidine were
entirely contained in 80-125%. In conclusion, these ndings clearly indicate that the test formulation is bioequivalent to the reference formulation. (Y. Jiao et al, Journal of Pharmaceutical and Biomedical Analysis
2007; 43:1804-1807)
Paper No.: 1797

DISCOVERY
MITOCHONDRIAL SODIUM/CALCIUM EXCHANGER AS A
NEW TARGET TO DEVELOP NEUROPROTECTIVE DRUGS
Santos Nicolau(1), J Egea(1,2), M Lopez(1,2), A Garca(1,2,3)
(1) Instituto Teolo Hernando
(2) Departamento de Farmacologa y Terapeutica, Facultad de Medicina,
Universidad Autonoma de Madrid, Spain
(3) Servicio de Farmacologa Clnica, Hospital Universitario de la
Princesa Madrid, Spain.
Vulnerable neurons of neurodegenerative diseases and stroke are prone
to enter into apoptosis because of excess energy demand and reduced
mitochondrial ATP synthesis. Mitochondria play a key role in controlling
neuronal Ca2+ homeostasis; and viceversa, the regulation of mitochondrial Ca2+ uptake and its subsequent release into the cytosol by mitochondrial Na+/Ca2+ exchanger (mNCX), so called mitochondrial Ca2+
cycling (mCC), is critical to prolong the viability of vulnerable neurons.
Here we have tested the hypothesis that CGP37157 (CGP), a compound
that blocks the mNCX and consequently that slows down the mCC,
could afford neuroprotection against cell death induced by Na+ and Ca2+
overload. Veratridine produced death of bovine chromafn cells and
CGP protected them against this insult. This protection was likely due to
blockade by CGP of veratridine-elicited free radical production, mitochondrial depolarization, and cytochrome c release (Nicolau et al JPET
2009, 330:844-854). Veratridine also caused a 40% neuronal death in rat
hippocampal slices. Again, CGP protected against this lesion in a
concentration-dependent manner. Reported blockade by CGP of voltagedependent calcium channels is not the mechanism underlying neuro-
492
protection because selective blockers of L, N and PQ channels afforded
little or no protection. We conclude that regulation of mCC rate by
ligands targeting the mNCX may become a novel pharmacological strategy for neuroprotection. We are presently synthesizing such ligands.
Paper No.: 2659

DISEASES
ADENOSINE A2A AGONIST AND A2B ANTAGONIST MEDIATE
AN INHIBITION OF INFLAMMATION-INDUCED
CONTRACTILE DISTURBANCE OF A RAT
GASTROINTESTINAL PREPARATION
Karen Nieber(1), C Warstat(1), S Michael(1), L Yan(2), CE Muller(2)
(2) University Bonn, PharmaCenter Bonn, Pharmaceutical Institute,
Bonn, Germany
Adenosine can show anti-inammatory as well as pro-inammatory
activities, and the contribution of the specic adenosine receptor subtypes in various cells, tissues and organs is complex. In this study, we
examined the effect of the adenosine A2a receptor agonist CGS 21680
and the A2b antagonist PSB-1115 on acute inammation induced by
TNBS on rat ileum/jejunum preparations. Preincubation of the ileum/
jejunum segment with TNBS for 30 min resulted in a concentrationdependent inhibition of ACh-induced contractions. Pharmacological activation of the A2a receptor with CGS 21680 (0.1-10 lM) preincubated
simultaneously with TNBS (0.01M) restored concentration-dependently
the TNBS-induced inhibition of the ACh-contractions. Stimulation of
A2b receptors with the selective agonist BAY 60-6583 (10 lM) did neither result in an increase nor in a further decrease of ACh-induced contractions compared to the TNBS-induced inhibition. The simultaneous
preincubation of the ileum/jejunum segments with TNBS (0.01 M) and
the selective A2b antagonist PSB-1115 (100 lM) inhibited the contraction-decreasing effect of TNBS. A signicant amelioration of the TNBSdiminished contractility was found by the combination of the A2aR agonist CGS 21680 and the A2B antagonist PSB-1115 at subthreshold concentrations of both agents, which was in the same range as the effect
induced by 1 lM methotrexate. Our results demonstrate that the activation of A2a or the blockade of A2b receptors can decrease the inammation-induced disturbance of the ACh-induced contraction in TNBS
pretreated small intestinal preparations. The combination of both may be
useful for the treatment on inammation bowel diseases.
Paper No.: 2660

THE MULTI-HERBAL DRUG STW 5: POTENTIAL
MECHANISMS FOR THE TREATMENT OF IRRITABLE
BOWEL SYNDROME
Karen Nieber(1), M Michael(1), U Voss(1), D Weiser(2), O Kelber(2)
induced disturbances in the muscle wall and ACh-induced contraction.

In parallel, STW 5 and STW 6 dose-dependently normalized the disturbed ACh-induced contractility. The TNBS-induced inammation
was accompanied by an increased TNF-a expression. STW 5 but not
STW 6 inhibited the increased gene expression by activation of adenosine A2a receptors and reduced the TNF-a release in LPS-stimulated
human monocytes. Radioligand binding assays conrm the afnity of
STW 5 to the adenosine A2a receptor. In further experiments STW 6
was macerated and the anti-inammatory effect of the fractions was
tested. The fractions, which contained the cucurbitacins I and E also
prevented the TNBS-induced inhibition of the intestinal contractility.
The cucurbitacins E and I, which act anti-inammatory, stimulate the
IL-10 gene expression comparably to STW 6. In conclusion STW 5
reveals signicant anti-inammatory properties which contribute to the
reduction of TNBS-induced morphological and contractile disturbances.
Its mode of action seems to be twofold, inhibition of the TNF-a pathway by activation of adenosine A2a receptors and activation of the
IL-10 pathway initiated by cucurbitacins. These anti-inammatory
mechanisms appear to be involved in the multi-target action of STW
5 as a new therapeutic approach in IBS.
Paper No.: 2028

HEALTH AND DISEASE
ENDOTHELIN RECEPTOR ACTIVATION REVERSES
HYPOXIC VASODILATION IN PORCINE LARGE CORONARY
ARTERIES
Elise Nielsen(1), E Stankevicius(1), U Simonsen(1), O Frobert(2)
rebro University Hospital, Sweden
(2) O
The present study investigated the role of endothelin-1 (ET-1), the nitric
oxide (NO) pathway, and radical oxygen species in hypoxia-induced
vasodilation of large coronary arteries. Porcine coronary artery segments
were mounted for functional studies in wire and pressure myographs. We
measured tissue concentrations of ET-1 by ELISA assay and asymmetric
dimethylarginine (ADMA) by HPLC. NO concentrations were measured
with a microsensor. In prostaglandin F2a (PGF2a, 10 lM)-contracted segments with endothelium, gradual lowering of oxygen tension from 95 to
1% O2 resulted in vasodilation. The response to O2 lowering was rightward shifted in segments without endothelium, but not at 1% O2. The
endothelin receptor antagonist SB217242 (10 lM) markedly increased
hypoxic dilation and exogenous ET-1 reversed hypoxic vasodilation in
segments with and without endothelium. The free tissue ET-1 concentration in the arterial wall was reduced by 20% in 1% O2 versus 21% O2.
Without affecting basal NO, hypoxia increased NO concentration in
PGF2a-contracted arteries, and a NO synthase inhibitor, nitro-L-arginine
(300 lM), reduced hypoxic vasodilation. ADMA concentrations were
unchanged by hypoxia. The superoxide scavenger tiron (10 lM) and the
putative NADPH oxidase inhibitor apocynin (10 lM) had no effect on
1% O2 vasodilatation although a leftward shift in concentration-response
curves for O2 was noticed at 10%-20% O2. Endothelin receptor activation reverses while endothelin receptor antagonism markedly enhances
hypoxic vasodilation in coronary artery segments with and without endothelium. Endothelium-derived NO may counteract the effects of ET-1
during hypoxia.

(2) Steigerwald Arzneimittelwerk GmbH, Scientic Department, Darmstadt, Germany
To identify an anti-inammatory effect the efcacy of STW 5 (Iberogast) and its main component STW 6 (Iberis amara) was studied in
inamed rat small intestinal preparations. The inammation was
induced by intraluminal instillation of 2,4,6 trinitrobenzene sulfonic
acid (TNBS, 0.01 M). Preincubation of STW 5 or STW 6 together
with TNBS concentration-dependently prevented the inammationJournal compilation 2010 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 107 (Suppl. 1), 162692
493
Paper No.: 2938
HEALTH AND DISEASE
DEXAMETHASONE-RELEASING BIODEGRADABLE
VASCULAR STENTS IN VITRO AND IN VIVO RESULTS
R Nieminen(1), A Kotsar(2), I Uurto(3), Mari Hamalainen(1),
J Mikkonen(4), J Leppiniemi(4), T Isotalo(5), M Talja(5), J-P Salenius(3), M Kellomaki(4), T Tammela(2), E Moilanen(1)
(1) University of Tampere Medical School and Tampere University
Hospital, The Immunopharmacology Research Group, Tampere, Finland
(2) Tampere University Hospital, Department of Urology, Tampere,
Finland
(3) Tampere University Hospital, Department of Vascular Surgery,
Tampere, Finland
(4) Tampere University of Technology, Tampere, Finland
(5) Paijat-Hame Central Hospital, Department of Surgery, Finland
Restenosis is a major complication of percutaneous transluminal angioplasty (PTA). It is characterized by contraction of vessel wall (negative
remodelling) and intimal hyperplasia. Drug-releasing endovascular stents
are a recent innovation used to control those complications of PTA. Sirolimus and paclitaxel have been successfully used as bioactive compounds to attenuate restenosis. We investigated the effects of
dexamethasone and dexamethasone-releasing biodegradable poly-D/Llactic acid (PLA) stents/stent material in in vitro and in vivo models
related to restenosis. Dexamethasone and dexamethasone-releasing PLA
stents inhibited the production of mediators related to inammation,
brosis and endothelial activation, including TNF-a, IL-8, MCP-1, RANTES, TGF-b and VEGF in cell cultures. In addition, dexamethasone
inhibited cell proliferation in vitro. PLA stent material induced a lowlevel inammatory reaction (measured by antibody array method that
detects 79 inammatory factors) when introduced to macrophages in culture, and that was signicantly attenuated by dexamethasone. Biocompatible testing of the stent material was carried out in rabbit dorsal muscle.
In the bioactivity testing, intravascular dexamethasone releasing PLA
stents induced minimal intimal hyperplasia as compared to non-drugreleasing PLA stents implanted in iliac arteries in the pig. Dexamethasone-releasing biodegradable stents may offer a promising new treatment
option in PTA. However, further studies are needed to nd out optimal
concentrations and releasing proles of dexamethasone and composition
of biodegradable PLA material for the aimed therapeutic properties.
Paper No.: 2757

FOCUSED CONFERENCE GROUP: FC09 - INFLAMMATION
AND IMMUNOPHARMACOLOGY: ......
GOLD-CONTAINING DMARDS INHIBIT COX-2, MMP-3 AND
IL-6 EXPRESSION IN CHONDROCYTES BY INCREASING
MKP-1 EXPRESSION AND DECREASING P38
PHOSPHORYLATION
Riina Nieminen(1), R Korhonen(1), T Moilanen(0), AR Clark(3),
E Moilanen(1)
(1) University of Tampere Medical School and Tampere University
Hospital, Immunopharmacology Research Group, Tampere, Finland
(2) Coxa Hospital for Joint Replacement, Tampere, Finland
(3) The Kennedy Institute of Rheumatology Division, Imperial College
London, UK
Gold containing disease modifying antirheumatic drugs (DMARDs)
aurothiomalate and auranon suppress inammation and retard erosions
in arthritis. The molecular mechanisms of action of these drugs are not
known in detail. Mitogen-activated protein kinase (MAPK) pathways are
major signalling pathways in inammation, which regulate the production of many inammatory and destructive factors in arthritis. MAP
kinase phosphatase-1 (MKP-1) is an endogenous negative regulator of
p38 MAPK activity. In the present study, we investigated the effects of
aurothiomalate and auranon on the p38 MAPK activity and MKP-1,

COX-2, MMP-3 and IL-6 expression in a chondrocyte cell line and in
intact cartilage. Aurothiomalate and auranon inhibited IL-1b -induced
COX-2, MMP-3 and IL-6 expression dose-dependently. Interestingly,
both these drugs also increased the expression of MKP-1 and reduced
IL-1b -induced p38 MAPK phosphorylation. Knock-down of MKP-1 by
siRNA signicantly impaired the ability of aurothiomalate to inhibit the
p38 MAPK phosphorylation and COX-2, MMP-3 and IL-6 expression.
Likewise, aurothiomalate reduced COX-2, MMP-3 and IL-6 expression
in human RA cartilage and in articular cartilage from wild type mice but
not in cartilage from MKP-1 decient mice. The results provide a novel
mechanism for the anti-inammatory and anti-erosive action of gold containing DMARDs through increased MKP-1 expression and decreased
p38 activity and suppressed COX-2, MMP-3 and IL-6 expression. The
results suggest that increasing of MKP-1 levels is a promising novel
mechanism to be directed in the search and development of novel antiinammatory and anti-erosive compounds that have the good efcacy
but not the toxicity problems of gold compounds.
Paper No.: 1792

IBOGAIN IN VITRO EFFECTS ON ERYTHROCYTES
ANTIOXIDATIVE ENZYMES ACTIVITES
Aleksandra Nikolic-Kokic(1), Z Orescanin-Dusic(1), M Slavic(1),
P Jamnik(2), R Paskulin(3), M Spasic(1), D Blagojevic(1)
(1) University of Belgrade, Institute for Biological Research, Sinisa
Stankovic, Belgarde, Republic of Serbia
(2) University of Ljubljana, Biotechnical Faculty, Ljubljana, Slovenia
(3) OMI Institute, Ljubljana, Slovenia
Previous studies demonstrated that ibogaine, an anti-addictive agent,
induced the quantities of energy related enzymes in cells, the effect not
being mediated by receptors. To further explore the mechanisms of its
action and connection to energy metabolism, redox state and reactive oxygen species as accompanying products, cellular antioxidant equilibrium
was studied in this work through effects of therapeutic doses of ibogaine
(20 mg/kg) on human antioxidant enzymes in erythrocytes in vitro. Blood
samples were collected in heparinized tubes. Control and Ibogain treated
aliquots of whole blood were incubated one hour at 37 degrees Celsius.
After incubation aliquots were washed three times and separated erythrocytes were lysed. In lysates, the activity of catalase (CAT), glutathione
peroxidase (GSH-Px) and glutathione reductase (GR) were measured.
Haemoglobin was removed by the method of Tsuchihashi and supernatant
was used for determination of copper-zinc superoxide dismutase (CuZnSOD) activity. The activities were expressed per g of blood haemoglobin.
Results showed that ibogaine induced an increase in CuZnSOD activity,
and slighly decreased CAT actvity. Results showed that the effects of ibogaine seems to be through the energy metabolism, redox active molecules and discrete uctuations of individual reactive oxygen species.
Paper No.: 1490

IDENTIFICATION OF ADRENERGIC RECEPTOR
INTERACTING PROTEIN (ARIP), AS A NOVEL RECEPTORINTERACTING PROTEIN THAT MODULATES A1A- AND
A1L-ADRENOCEPTOR EXPRESSION
Atsushi Nishimune(1,2), F Suzuki(1,2), H Yoshiki(1), S Morishima(1),
I Muramatsu(1,2)
(1) University of Fukui, School of Medicine, Department of Pharmacology, Fukui, Japan
494
(2) University of Fukui, Organization for Life Science Advancement Programs, Fukui, Japan
Introduction: Prazosin has long been recognized as a prototypical highafnity antagonist selective to the a1 adrenergic receptors (ARs). After
the original discovery of a1-AR showing low-afnity for prazosin, a1LAR (L for Low-afnity) concept had been coined. Unfortunately, since
attempts to demonstrate a1L-AR in the isolated membrane fraction by the
lignad-binding experiment had been essentially failed, some researchers
regarded it as a so-called eputative receptor. From our viewpoint today, it
can be explained at least in part by the fact that a1L-phenotype in vivo is
extremely sensitive to the mechanical homogenization which leads to its
rapid conversion into the high-afnity a1A-AR. Moreover, our recent
knockout mouse study unequivocally demonstrated that both a1L-AR
and a1A-AR phenotype is originated from a single gene ADRA1A. This
highlighted the importance of yet unknown mechanism(s) underlying
a1L-AR phenotype expression. We hypothesized that unidentied auxiliary subunit(s) promotes the generation of the a1L-AR. Results & Conclusion: A novel a1A-AR interacting protein (designated ARIP) was
isolated from the yeast two-hybrid screening. When ARIP is introduced
into the CHO cells permanently expressing a1A-AR, it strikingly
repressed the expression of the a1A-AR. Among these a1A-AR and ARIP
co-expressing cell lines, we successfully established permanent cell lines
stably expressing a1L-AR phenotype. Binding and functional proles for
various drugs in the a1L-AR cell lines were well consistent with those
reported for native a1L-ARs. These results provide functional clues for
molecular mechanisms for phenotypic diversication of the adrenergic
receptors and contribute to the development of the therapeutic intervention using a1L-AR selective drugs.
Paper No.: 1121

POSSIBLE DRUG INTERACTION WITH OREN-GEDOKU-TO
BY THE INHIBITION OF CYP2D METABOLISM
Paper No.: 1581

ANTITUMOR EFFECT OF CORDYCEPS SINENSIS VIA
ADENOSINE RECEPTORS
Arisa Nishiuchi, N Yoshikawa, Y Takahashi, Y Kimoto, A Sato, M Kunitomo, S Kagota, K Shinozuka, K Nakamura
Mukogawa Womens University, School of Pharmacy and Pharmaceutical Sciences, Department of Pharmacology, Nishinomiya, Japan
Cordyceps sinensis, a parasitic fungus on the larva of Lapidoptera, has
been used as a traditional Chinese medicine. We previously reported that
water extracts of the fruiting bodies of cultured Cordyceps sinensis
(WECS) exhibited antitumor effects and deoxycoformycin (DCF), an
adenosine deaminase inhibitor, reinforced the antitumor activity of
WECS in vitro (Yoshikawa N et al. In Vivo 2007; 21: 291-296). This
result suggested that the active components in WECS with antitumor
effects should be adenosine-related substances. In this study, we investigated whether WECS inhibits the growth of B16-BL6 mouse melanoma
(B16-BL6) cells and HT1080 human brosarcoma (HT1080) cells via
their adenosine receptors. As a result, the growth of two cell lines was
potently inhibited by WECS (10 microg/mL) and the inhibitory effect of
WECS was signicantly antagonized by MRS1191 (1 microM), a selective adenosine a3 receptor (a3-R) antagonist and 8-(3-chlorostyryl) caffeine (1 microM), a selective adenosine a2a receptor (a2a-R) antagonist.
In conclusion, WECS inhibited the proliferation of B16-BL6 cells and
HT1080 cells by stimulation of both a3-R and a2a-R. In other words, the
components in WECS with antitumor action might be the a3-R stimulant
and a2a-R stimulant or a sole component possesses both a3-R- and a2aR-stimulating activity.
Yuki Nishimura, N Kurata, M Iwase, Y Saito, H Yasuhara
Paper No.: 500

STUDY ON THE PHYTOESTROGENIC EFFECTS OF FERULIC
ACID
Showa University School of Medicine, Department of Pharmacology,

Tokyo, Japan
Jianzhao Niu, J Wang, Q Hao, P Zhao
Oren-gedoku-to(OGT), a Kampo medicine, consists of four herbal

extracts including Coptidis Rhizoma(COR) that inhibits CYP2D activity
in vitro. The current study was conducted to investigate the inhibitory
effect of OGT on CYP2D-mediated debrisoquine (DEB) metabolism in
vivo and in vitro. Sprague-Dawley rats were treated with either water,
OGT, or one of its four constituents 1 hour prior to oral DEB administration. Blood samples were collected up to 24 hours and the serum DEB
concentrations were determined by HPLC. The effect of OGT and its
constituents on DEB 4-hydroxylation(DEB 4-OH) was also investigated
using rat liver microsomes. The treatment of COR and Gardeniae Fructus
(GAF) prolonged the elimination half-life of DEB by 40 and 70%,
respectively, suggesting the inhibition of DEB metabolism. COR and its
component, berberine inhibited DEB 4-OH in a concentration-dependent
manner. GAF and its major components including crocin did not affect
the activity, however, its aglycon crocetin showed inhibitory effect on
the activity with Ki value of 12.5 lM. In addition, crocin treatment also
prolonged the half-life of DEB by 40%. These data suggested that crocetin, transformed by intestinal bacteria, play a major role in the inhibition
of DEB metabolism by GAF in vivo. OGT did not inhibit DEB metabolism in vitro and in vivo. Although OGT include several inhibitory components in its formula, the drug-drug interaction mediated by CYP2D is
unlikely to occur during medication. However, another Kampo formula
or herbal supplements that highly contain GAF or crocin may cause drug
interaction by the inhibition of this enzyme.
Beijing University of Chinese Medicine, The Center for Research on

Womens Health, Beijing, PR China
To demonstrate the phytoestrogenic effects of ferulic acid on proliferation
and cell cycle and the levels of ERa and ERb protein and gene pS2
mRNA expression were measured on breast cancer cellsestrogen receptor positive T47D cells and estrogen receptor negative MDA-MB231cells. Result (1) The effects of ferulic acid on cell proliferation were
enhanced signicantly by treatment with ferulic acid in the range of 107M to10-5M for T47D cells by means of MTT assay. The proliferation
effects were inhibited by adding antiestrogen ICI182 780(10-8 M). (2)
There was no signicant difference on the proliferation of MDA-MB231 cells by treatment with tested concentration of ferulic acid as well as
by co-treatment with antiestrogen ICI182 780(10-8 M) compared with
solvent control group. (3) Ferulic acid up-regulated pS2 mRNA expression of T47D cells at tested concentration 10-7M or 10-6M. They
increased the level of T47D cells ERa protein expression as well. Ferulic
acid(10-7M or 10-6M) did not show remarkable effects to the level of
ERb protein expression in T47D cells. Conclusion: Ferulic acid possessed phytoestrogenic effects by up-regulating pS2 gene expression and
the receptor subtype of ERa and ERb expression. The phytoestrogenic
effects were mediated by regulating ER. The action of ferulic acid was
weaker than estradiol. There was a positive correlation between ER
expression and pS2 gene expression.
This work was supported by the 111 project B70007, PR China and by
the International Science and Technology Cooperation Program of China,
2008DFA31970 to Dr. Niu Jianzhao.
495
Paper No.: 1311
HEALTH AND DISEASE
ROLES OF VINCULIN AND RHO KINASE ON FIBROBLAST
FIBER CONTRACTION
Sen mouse. We also conrmed that calcium-dependent protein kinase C

(PKC) expression was altered in Sen mouse. Conclusion: Our ndings
suggested that alteration of Adip inuences CCh-induced prostate contraction via the intracellular calcium dependency. It might involve in dysfunction of calcium-dependent PKC.
Hiromi Nobe(1), K Nobe(2), R Paul(3), K Honda(2), Y Sakai(1)

(1) Bunkyo Gakuin University, Department of Physical Therapy,
Fujimino, Saitama, Japan
(2) Showa University, Tokyo, Japan
(3) University of Cincinnati, Cincinnati, OH, USA
Introduction: Fibroblast cell contraction is considered to play an important role in wound repair processes and tissue reorganization. The aim of
this study is to reveal an actin-stress ber (ASF) mediated regulatory
mechanisms of the broblast cell contraction. Materials: Contractile
responses in NIH 3T3 broblast cell were evaluated by force development in a three-dimensionally re-constituted broblast ber in collagen
matrix. Vinculin-knockout (Vin-KO) broblast bers were prepared as
similar as NIH 3T3 broblast bers. ASF formation was detected as uorescent images using a laser microscope. Results: Treatment of the NIH
3T3 broblast ber with 30 % calf serum (CS) induced time- and dosedependent contraction. This contractile response was not affected by an
addition of calcium channel inhibitor (nicardipine). In this calcium-independent broblast contraction, ASF formation was one of the key steps
in CS-induced NIH 3T3 broblast ber. Rho kinase inhibitor, Y27632
signicantly reduced not only in the contraction but also ASF formation.
These results indicated that the broblast ber contraction involves rho
kinase-mediated ASF formation. To understand target of the ASF, contractile responses in Vin-KO broblast ber was measured. CS-induced
maximal contraction in Vin-KO broblast ber was approximately 40 %
of the response in NIH 3T3 broblast ber and it was not affected by the
Y27632 treatment. Moreover, Vin-KO broblast ber contraction was
followed by an inhibition of ASF formation. Conclusion: Our ndings
suggested that the association of ASF formation with vinculin has a critical role in the broblast contraction.
Paper No.: 1130

THE TREATMENT OF SEXUAL DYSFUNCTION AND DISEASES OF THE LOWER URINARY TRACT
ALTERATION OF CARBACHOL-INDUCED PROSTATE
CONTRACTION IN ADIPONECTIN TRANSGENIC MICE
Koji Nobe(1), A Fujii(1), T Negoro(2), K Saito(3), Y Nakano(2),
T Hashimoto(1), K Honda(1)
(1) Showa University, Department Pharmacology, Tokyo, Japan
(2) Showa University, Department of Pharmacogenoics, Tokyo, Japan
(3) Showa University, Department of Clinical and Molocular PK/PD,
Tokyo, Japan
Introduction: Dysfunction of prostate contraction is involved in urological complications in diabetic patients. Adiponectin (Adip), which is
secreted specically by adipocytes, improves insulin sensitivity and glucose incorporation; however, no relationship between Adip and prostate
contraction is known. In this study, prostate contractions were evaluated
in Adip-sense (Sen) mice Materials: Blood Adip level in Sen mice (male;
body weight, 34.4 1.46 g) was 135 % of those of age-matched control
mice (C57Bl). Isolated prostate was mounted on organ bath system and
developed isometric force levels were assessed. Results: Both carbachol
(CCh) and phenylephrine (PE) induced time- and dose-dependent contractions in control and Sen mice. Responses were the similar (3.79
0.04 and 3.87 0.03 mN/mm2 following 30 lM CCh treatment, respectively; n=5), but only the CCh-induced contraction was strongly inhibited
under calcium-free conditions in Sen mouse relative to the control
mouse. CaCl2-induced dose-dependent contraction in the presence of
50 mM KCl under calcium-free conditions was signicantly enhanced in
Paper No.: 2235

GAS MEDIATOR HYDROGEN AS A TOOL FOR PROTECTION
OF PARKINSONS DISEASE
Mami Noda(1), K Fujita(2), M Kido(1), T Katafuchi(1), Y Nakabeppu(3)
(1) Kyushu University, Graduate School of Pharmaceutical Sciences,
Department of Pathophysiology, Fukuoka, Japan
(2) Kyushu University, Graduate School of Dental Sciences, Fukuoka,
Japan
(3) Kyushu University, Medical Institute of Bioregulation, Fukuoka,
Japan
Recently, it has been suggested that molecular hydrogen exerts a therapeutic antioxidant activity by selectively reducing hydroxyl radicals
(OH), the most cytotoxic reactive oxygen species (ROS), and effectively protects against organ damage induced by ischemia/reperfusion.
Though the precise mechanism of hydrogen remains to be elucidated,
now is considered to be as one of the gas mediators, like nitric oxide
(NO) and carbon monooxide (CO). Oxidative stress damage to the brain
is also induced in Parkinsons disease (PD). We used mice treated with
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) as an animal
model of PD. We showed that hydrogen in drinking water, before or
even after administration of MPTP, signicantly reduced loss of dopaminergic neurons in PD model mice, using both acute and chronic administration of MPTP (Fujita et al., PLoS One, 2009; 4:e7247). MPTPinduced production of super oxide anion radical detected by intravascular
injection of hydroethidine, formation of nitrite/nitrate in midbrain, and
accumulation of 8-oxoG in cellular DNA in striatum, were signicantly
decreased in mice drinking hydrogen-containing water. Thus drinking
hydrogen-containing water may be useful in daily life to prevent or minimize acute and chronic oxidative stress-related diseases. Support: Panasonic Electric Works Co., Ltd., Osaka, Japan.
Paper No.: 1473

SAFETY IN PAEDIATRIC DRUG TRIALS AN UPDATE FROM
RANDOMISED CONTROLLED TRIALS PUBLISHED IN 2007
KNB Nor Aripin, Helen M Sammons, I Choonara
University of Nottingham School of Graduate Entry Medicine and Health
Sciences, Academic Division of Child Health, Derby, UK
Recent changes in drug regulation are resulting in more paediatric clinical trials. This is essential to provide evidence, however participating
children are at risk of adverse drug reactions (ADRs). A previous review
(Sammons et al, Acta Paediatrica 2008;97(4):474-7) found only 2% of
paediatric clinical trials mention a safety monitoring committee/data
safety monitoring board (SMC/DSMB). We performed a systematic
review of all paediatric randomised drug trials (RCTs) published in 2007.
Medline, Embase and Cochrane Central were searched using validated
search strategies. Articles were reviewed by consensus of 2 paediatric
clinical pharmacologists. 582 RCTs were identied. 207(36%) did not
mention method of monitoring safety and 119(20%) neglected reporting
adverse events. 210(36%) reported serious adverse events (SAEs).
87(15%) reported mortality occurring within the trial. ADRs were judged
to occur in 305(52%) trials, many were mild ADRs (162,53%) but
496
79(26%) trials had moderate and 66(22%) severe ADRs. Severe ADRs
were most frequently seen in trials of cytotoxic drugs (17,26%), antibiotics (15,23%) and nervous system drugs (13,20%). Only 69(12%) RCTs
mentioned the presence of a SMC. SMCs terminated 3 RCTs due to toxicity and intervened in the protocol of one other. Signicantly more trials
that reported a serious AE occurring mentioned that a SMC/DSMB was
present, in comparison to trials that had no or non-serious AEs (55/210
vs 14/372 trials, P<0.05). SMCs are vital in ensuring safety and should
occur in all paediatric RCTs. Reporting of safety and ADRs is still inadequate in published work and needs to be improved.
Paper No.: 3148

THE EFFECT OF PREMEDICATION FOR INTUBATION ON
AMPLITUDE-INTEGRATED ELECTROENCEPHALOGRAPHY
IN A RANDOMIZED BLINDED INTERVENTION STUDY OF
PRETERM INFANTS
Elisabeth Norman(1), S Wikstrom(2), I Rosen(3), V Fellman(0),
L Hellstrom-Westas(2)
(1) Lund University and Lund University Hospital, Department of Pediatrics, Lund, Sweden
(2) Uppsala University, Department of Womens and Childrens Health,
Uppsala, Sweden
(3) Lund University and Lund University Hospital, Department of Neurophysiology, Lund,Sweden
(4) University of Helsinki, Department of Pediatrics, Helsinki, Finland
Introduction: There are no evidence based guidelines for premedication
before intubation in newborn infants. Our aim was to investigate, in a
blinded RCT, whether EEG recovery differs after premedication with
thiopental-remifentanil (A) compared to morphine (B). Materials/
Patients: 30 preterm infants, mean (SD) gestational age 27.2 (3.2) w and
birth weight 1050 (643) g, were randomized to either (A) glycopyrron 5
microg/kg, thiopental 2-3 mg/kg (2 mg/kg < 1000 g), suxamethonium
2 mg/kg and remifentanil 1 microg/kg, or (B) atropine 0.01 mg/kg and
morphine 0.3 mg/kg, before nasal intubation. Continuous two-channel
EEG was recorded during the rst 24 hours after intubation. The EEG
was visually scored (Burdjalov et al. Pediatrics 2003;112:855-861, max
score 13), and exported to MatLab for quantitative analysis of interburst
interval, suppression%, and amplitudes. Results: EEG recovery was signicantly faster in group A than in group B. The median (range) Burdjalov score at 1-3, 3-6, 6-9, 9-12,12-15,15-18,18-21 and 21-24 and hours
in group A was: 3 (0-8), 4 (0-8), 3 (0-9), 5.5 (0-10), 5 (0-10), 7 (0-10),7
(0-11) and 6.5 (0-11) and in group B: 0 (0-0), 0 (0-2), 0 (0-6), 1(0-5),
1(0-6), 1(0-8), 2(0-8) and 1.5 (0-6) (all comparisons between groups
P<0.05). Also scores for sleep wake cycling differed signicantly
between the two groups for all epoques. Quantitative analyses will be
presented. Conclusion: Premedication with morphine before intubation in
preterm infants is associated with sustained EEG depression, while the
combination of thiopental and remifentanil is associated with more rapid
recovery.
Paper No.: 3340

BIOCHEMICAL AND BLOOD PRESSURE ALTERATIONS OF
ELDERLY PATIENTS WITH COLORECTAL CANCER AFTER
OF DIETARY SUPPLEMENTATION WITH AGARICUS
SYLVATICUS FUNGI
Objective: To evaluate the considerable improvement in biochemical

parameters and blood pressure alterations in elderly patients with colorectal cancer presented in post-surgery phase after supplementation with
Agaricus sylvaticus. Methods: A randomized, placebo-controlled, double-blind clinical trial. Sample consisting of 22 patients, 54.4% women
and 45.6% men, stadium phases II (n = 10) and III (n = 12). The placebo
(n=11)group received starch only and the experimental group (n=11)was
supplemented with Agaricus sylvaticus, orally, twice daily (30mg/kg/day,
for six months. Results: The group supplemented with Agaricus sylvaticus presented signicant reduction of total cholesterol low density lipoprotein, urea, uric acid, total bilirubin, indirect bilirubin, systolic blood
pressure and diastolic blood pressure In the placebo group, it was
observed a signicant increase of total cholesterol, glucose, low density
lipoprotein, urea, uric acid, total bilirubin, indirect bilirubin, AST, and
systolic blood pressure. Conclusions:. The results suggest benets in
elderly patients with colorectal cancer.
Paper No. 3341

EFFECTS OF DIETARY SUPPLEMENTATION WITH
AGARICUS SYLVATICUS FUNGI ON THE METABOLIC AND
BLOOD PRESSURE EVALUATION OF PATIENTS WITH
COLORECTAL CANCER IN THE POSTSURGICAL PHASE
Maria Rita Novaes, R Fortes
University of Brasilia, Department of Pharmacology, Brasilia, Brazil
Objective: To evaluate the metabolic and blood pressure effects on
patients with colorectal cancer in the postsurgical phase after dietary supplementation with Agaricus sylvaticus fungi. Methods: A randomized,
double-blind, placebo-controlled clinical trial was conducted at the Base
Hospital of the Federal DistrictC Brazil for six months. Samples of ftysix patients with colorectal cancer, divided into two groups: supplemented with Agaricus sylvaticus (30 mg/kg/day) and placebo (starch).
Three serum evaluations of glucose, total cholesterol, triglycerides, uric
acid, urea, creatinine, alkaline phosphatase, total, direct and indirect bilirubins, aspartate aminotransferase and alanine aminotransferase, immunoglobulins A (IgA), G (IgG) and M (IgM), total and fraction proteins
were monitored throughout the treatment, blood pressure levels were also
measured during treatment. Results: In the Agaricus sylvaticus group it
was observed signicant reduction of fasting plasma glucose (p = 0.02),
total cholesterol (p = 0.01), creatinine (p = 0.05), aspartate aminotransferase (p = 0.05), alanine aminotransferase (p = 0.04), IgA (p = 0.0001),
IgM (p = 0.02), systolic blood pressure (p = 0.0001) and diastolic blood
pressure (p = 0.0001). Such alterations were not observed in the placebo
group. Conclusions: The results suggest that a dietary supplementation
with Agaricus sylvaticus fungi is capable of promoting metabolic benets
as well as in the biochemical, enzymatic and blood pressure parameters
of patients with colorectal cancer in the postsurgical phase.
Paper No. 3342

IMMUNOLOGICAL AND HEMATOLOGICAL EFFECTS OF A
DIETARY SUPPLEMENTATION WITH AGARICUS SYLVATICUS
IN BREAST CANCER PATIENTS UNDERGOING
CHEMOTHERAPY
Maria Rita Novaes, F Valadares
Maria Rita Novaes, R Fortes, LC Novaes
Aims: Evaluate the nutritional and hematological effects of a dietary supplementation with Agaricus sylvaticus fungi in breast cancer patients
497
undergoing chemotherapy. Methods: A randomized, placebo-controlled,
double-blind clinical trial. Sample of 46 women with breast cancer at
stages II (61.5%) and III (38.5%) during chemotherapy, average age
52.415.94 years, divided in two groups: placebo (n = 23) and experimental (n = 23). The placebo group received starch only, orally, for six
months. The experimental group was received Agaricus sylvaticus fungus (2,1g/day), orally, 3 times daily for six months. The trial consisted
of: evolution of the disease, gastrointestinal symptoms, response to chemotherapy, prognosis, tumor size (observed by mammography) and body
weight. Results: After six months of supplementation with Agaricus sylvaticus, it was observed a substantial improvement in clinical and nutritional status as well as reduction of vomiting (30%), nausea (20%),
diarrhea (10%) and constipation (10%) in the group supplemented with
mushroom when compared with the placebo group. In addition to that,
in the group supplemented with mushroom there were alterations in
hematocrit (from 35,32 4,73 to 39.02 5,80. P = 0.06), hemoglobin
(from 11.68 1,66 to 12:77 1.89, P = 0.09), platelets (from 4,1 0,56
to 4.81 0.83, P = 0.03), MCH (from 29,703,63 to 36,902.90, P =
0.05), MCV (from 84,80 7,81 to 86,504,19, P = 0.2) when compared
with the placebo group. Conclusions: The patients with breast cancer can
experience signicant improvement in their nutritional and hematological
status if supplemented with Agaricus sylvaticus fungi.
Paper No.: 3343

QUALITY OF LIFE OF COLORECTAL CANCER PATIENTS IN
POST-SURGERY RECOVERY PERIOD AFTER BEING SUPPLEMENTED WITH AGARICUS SYLVATICUS FUNGUS: A RANDOMIZED, DOUBLE-BLIND, PLACEBO-CONTROLLED
CLINICAL STUDY
Maria Rita Novaes, R Fortes
Introduction: Colorectal cancer jeopardizes quality of life through metabolic, physiological and psychological alterations. Therapeutical alternatives directed to improve the quality of life of oncological patients
have been the purpose of recent studies which try to prove the immunomodulator effects of Agaricaceae fungi. Objective: To evaluate the
impact upon the quality of life of post-surgical patients with colorectal
cancer after being supplemented with Agaricus sylvaticus fungus,
Agaricaceae family, cultivated in Brazil. Methods: Randomized, double-blind, placebo-controlled clinical trial carried out at the Federal
District Base Hospital Brazil, for six months. Samples of 56
enrolled patients (24 men and 32 women), stadium phases I, II and
III, separated as placebo (starch) and Agaricus sylvaticus (30mg/kg/
day) supplemented groups. Form-standard and direct anamnesis-standard were used to evaluate indicators for quality of life. The method
of analysis was qualitative and descriptive, processed with Microsoft
Excel 2003 and Epi Info 2004 programs. The protocol was approved
by the Ethics Research Committee - Health Department - Federal District. Results: After six months of treatment, the supplemented group
had increased adhesion to physical activity; improved wellbeing and
good mood; reduced complaints of pains and sleep alterations such as
insomnia and restless sleep; presenting more appetite, reduced constipation, diarrhea, alternate diarrhea/constipation, atulence, atus retention, pyrosis, postprandial fullness, nausea, abdominal distention and
abdominal pain, facts not observed in the placebo group. Conclusions:
The results suggest that a dietary supplementation with Agaricus sylvaticus fungi is capable of improving the quality of life of patients
with colorectal cancer in post-surgical phase.
Paper No.: 678

CARDIOPROTECTIVE EFFECT OF RESVERATROL
Aleksandra Novakovic(1), LG Bukarica(2), D Nezic(3), M Peric(3),
V Kanjuh(4)
(1) School of Pharmacy, Belgrade, Republic of Serbia
(2) School of Medicine, Belgrade, Republic of Serbia
(3) Institute of Cardiovascular Diseases Dedinje, Belgrade, Republic
of Serbia
(4) Academy of Sciences and Arts, Belgrade, Republic of Serbia
Resveratrol is a stilbene polyphenol present in grapes and red wine.
Interest in this compound has expanded in recent years, when a numerous epidemiological studies showed an inverse correlation between red
wine consumption and incidence of cardiovascular diseases. It seems that
resveratrol might be partly responsible for cardiovascular benets associated with wine consumption. The mechanism of cardiovascular benets
probably includes vasorelaxation, antioxidant and anti-platelet effects of
resveratrol. The mechanisms by which resveratrol causes vasodilatation
are uncertain. Aim: The aim of this study was to investigate the mechanism (s) of resveratrol-induced vasorelaxation in human internal mammary artery (HIMA) with endothelium and without endothelium.
Methods: HIMA were precontracted with phenylephrine (10 microM).
Results: Resveratrol induced a concentration-dependent relaxation of the
rings with endothelium and without endothelium. Highly selective
blocker of ATP-sensitive K+ channels, glibenclamide, as well as nonselective blockers of Ca-sensitive K+ channels, tetraethylammonium, did
not block resveratrol-induced relaxation of HIMA rings. Charybdotoxin,
a blocker of calcium-sensitive K+ channels, did not affect the resveratrolinduced relaxation. 4-Aminopyridine, non selective blocker of voltagegated K+ (KV) channels, and margatoxin that inhibits KV1 channels,
abolished relaxation of HIMA rings induced by resveratrol. Conclusions:
In conclusion, we have shown that resveratrol can induce relaxation of
HIMA with endothelium and without endothelium. It seems that 4-aminopyridine and margatoxinsensitive K+ channels located in vascular
smooth muscle mediated relaxation of HIMA produced by resveratrol.
Paper No.: 2810

HEALTH AND DISEASE
AGING HAS A NEGATIVE EFFECT ON ESTROGEN MODULATION OF NITRIC OXIDE (NO) PRODUCTION IN MICE AORTA
Laura Novensa(1), S Novella(2,3), G Segarra(2), P Medina(2),
N Castillo(1), C Hermenegildo(2.3), AP Dantas(1)
(1) Institut dInvestigacions Biome`diques August Pi i Sunyer (IDIBAPS),
Department of Experimental Cardiology, Barcelona, Spain
(2) University of Valencia, Department of Physiology, Valencia, Spain
(3) Hospital Clnico Universitario, Research Foundation, Valencia, Spain
Aging is among the major causes for the lack of cardiovascular protection by estrogen (E2) during postmenopause. Our study aims to determine the mechanism whereby aging changes E2 effects on vascular
function in model of accelerated senescence (SAM). Relaxations to acetylcholine (ACh) were determined in aortas from young (SAMR, resistant) and aged (SAMP, prone), intact (OE), ovariectomized (OVX), and
OVX treated with E2. NO production was measured in aortic sections by
DAF-2 uorescence and eNOS expression was determined by immunouorescence. E2 is also thought to increase NO by decreasing its catabolism by superoxide (O2-), therefore O2- production and NAD(P)Hoxidase activity were also determined by hydoethidine and lucigenin
respectively. Impaired sensitivity to ACh was observed in all SAMP
groups compared to SAMR. These responses were mostly associated to
NO, as L-NAME [NO synthase (NOS) inhibitor] blunted ACh vasodilation. Corroborating vascular reactivity studies, we observed a marked
498
decrease on NO production in SAMP-OE compared to SAMR-OE.
OVX reduced NO production in SAMR, but had a smaller effect on
SAMP. E2 treatment had no effect on SAMP while restored NO production on SAMR. No signicant effect on NOS expression was observed,
suggesting that another mechanism rather than diminished NOS expression is involved. Interestingly, we observed that E2 inhibits OVXinduced increase of both O2- production and NAD(P)H-oxidase activity
in young mice, while increases O2- on SAMP-OVX. Our studies suggest
that aging has detrimental effects on E2-mediated effects on NO production, partially by up-regulation of O2- and its negative effects on NO
metabolism.
Paper No.: 3316

INHIBITION OF PROSTATE CELL GROWTH AND
ANGIOGENESIS INDUCED BY DIFFERENT PHYTOSTEROLS
OF MARINE ORIGIN
was to focus on mutual interactions between Francisella tularensis LVS

and murine macrophage-like cell line J774.2 due to in vitro macrophages
stimulation by interferon c (IFNa), lipopolysaccharide (LPS) separatelly
both with combination before or after infection. Materials: Murine macrophage-like cells (J774.2) were incubated in cultivation asks (2x106
cells/10ml of medium Dulbecco Ls MEM with Glutamax-1 with 10%
BSA) and activated with LPS (10 or 50 ng / 1ml of medium) or with
IFNa (100 or 1 000 I.U. / 1ml of medium) separately or in combination
and infected by F. tularensis LVS (multiplication of infection 1:100) in
particular time schemes.We followed up the F.t. proliferation by colony
forming units (CFU) counts after 24-hours cultivation of cell supernatant
on McLeod agar. F.t. proliferation was correlated with NO production,
meassured by Griess method. Results: Separate LPS or IFNa stimulation
does not statistically signicantly affects the F.t. survival. In contrast to
this observation, stimulation of macrophages with the combination of
IFNa and LPS leads to elimination of incection. This result correlates
with highest NO production in this case and supports the theory of essential role of NO in infection control. Conclusion: IFNa and LPS stimulation of host cells only in combination but not separately leads to
infection elimination.
Silvia Novio, M Freire-Garabal, G Amigo, MJ Nunez-Iglesias

Stress and Neuroimmunology Laboratory. Department of Pharmacology.
School of Medicine - Faculty of Medicine, Dentistry and Nursing, Santiago de Compostela, Spain
Modulation of angiogenesis is now a well-known strategy for the prevention and treatment of cancer. Being prostate cancer the most common
invasive malignancy and the second cause of cancer-related death, the
purpose of this study was to investigate the activity of phytosterols of
marine microalgae (PMM), on angiogenesis by using in vitro and in vivo
model systems. PMM extracted of six species of microalgae (Tetraselmis
suecica, Dunaliella tertiolecta, Ellipsoidon sp, Phaeodactylum tricornutum, Chlorella autotroca, Nannochloris atomus) were used to treat prostate cancer PC3 cells. The antiproliferative, apoptotic and antiangiogenic
activity of PMM was determined by the MTT assay, the acridine orangeethidium bromide differential staining method and the enzyme-linked
immunosorbent assay, respectively. Further, athymic mice bearing subcutaneous xenografts were treated with the PMM which showed antiangiogenic and antiproliferative properties in vitro. PMM extracted from the
microalgae Nannochloris atomus and Tetraselmis suecica showed antiangiogenic activity without reducing cell viability or inducing apoptosis,
but only the treatment with PMM from the microalga Nannochloris atomus produced tumors that were less well vascularized than tumors
formed in mice treated only with vehicle. In conclusion, these results
highlight the potential use of PMM for both chemopreventive and chemotherapeutic purposes in the future and, they open a new eld to reduce
or avoid the secondary toxic effects associated with other chemotherapic
agents utilized nowadays.
Paper No.: 2775

DISEASES
STIMULATION OF MACROPHAGES BY INFA IN
COMBINATION WITH LPS LEADS TO EFFECTIVE
ELIMINATION OF F. TULARENSIS EXPERIMENTAL
INFECTION
Paper No.: 2418

INFLUENCE OF RIFAMPICIN ON NEVIRAPINE LEVEL IN
HIV PATIENT WITH TB
Agus W Nugroho(1), XXX Nafrialdi(1), A Simatupang(2)
(1) Faculty of Medicine, Universitas Indonesia, Department of Pharmacology, Jakarta,Indonesia
(2) Faculty of Medicine, Universitas Kristen Indonesia, Jakarta, Indonesia
The incidence of HIV-TB coinfection in Indonesia is increasing. This
leads to unavoidable use of antiretroviral and TB drug. It has been
known that TB drug, rifampicin is a potent enzyme inducer of CYP. The
previous studies showed the difference is a decrease in the plasma level
of nevirapine because of rifampicin. Until this studies has not been
reported the inuence of rifampicinto nevirapine on patient HIV with TB
in Indonesia. To evaluate the magnitude of inuence of rifampicin on nevirapine plasma level in patients with HIV-TB coinfection. Analytical
cross-sectional studies with sample of 40 patient consist of 24 patient of
HIV without TB and 16 patient HIV with TB who meet the inclusion
criteria and came to Pokdisus AIDS RSUPN-CM Jakarta. This study
found that nevirapine plasma level in HIV patient without TB was 7.5
2.2 lg/ml, whereas in patient with TB it was 5.5 2.7 lg/ml, with T test
for 2 independent groups found that differences are signicance with
value of p =0.018. There are signicance differences between plasma
level of nevirapine of HIVpatients without TB and plasma level of nevirapine of HIV patient with TB.
Paper No.: 3158

PEDIATRIC OUTCOMES FOLLOWING IN UTERO EXPOSURE
TO PSYCHOTROPIC MEDICATIONS: AN UPDATE
Irena Nulman
J Novosad, Eva Mala, M Holicka, M Novosadova, I Krcmova, J Krejsek

University Teaching Hospital, Department of Allergology and Clinical
Immunology, Hradec Kralove, Czech Republic
Introduction: Francisella tularensis (F.t.) is a facultative intracellular bacteria, enrolled at the list of Centre for Disease Control (CDC) as a high
risk bioterrorism agent, category A. There is a long-term effort to understand to the immunopathogenesis of F.t. infection. The aim of our trial
University of Toronto, Hospital for Sick Children, Division of Clinical

Pharmacology and Toxicology, Toronto, Ontario, Canada
Psychotropic medications are widely used in pregnancy. When left
untreated, maternal psychiatric disorders are associated with stress and
dysregulation of hypothalamic-pituitary-adrenal and placental axes leading to increased risk of adverse fetal development, miscarriages, preterm
labor, obstetric complications, future psychopathology and cognitive
499
impairments. Pharmacotherapy during pregnancy involves weighing the
potential risks of fetal teratogenic effects, neonatal toxicity and/or withdrawal, and long-term cognitive and behavioral effects against potential
adverse effects of untreated maternal mental disorder to both mother and
child. An association between psychotropic medications and increased
rates of immediate and long-term teratology domains above the general
population baseline has not been substantiated due to methodological
challenges. In the majority of studies, effects of maternal disorder have
not been separated from effects of psychotropic medication rendering
interpretation of results challenging. The presenter will provide an update
of the current knowledge regarding the reproductive risk/safety of psychotropic medications. Appropriate criteria for treatment algorithms,
proper guidelines for optimal management of mental illness before conception, during pregnancy and postpartum will lead to more favorable
outcomes for both mother and child.
Paper No.: 804

INTERACTIVE 3D ENCYCLOPEDIA FOR THE TEACHING OF
PHARMACOLOGY TO MEDICAL STUDENTS
Evelyn P Obando, MP Usta, T Escobar, R Escobar, J Estupinan, DA
Ospina
Stretching of the artery at a rate of 1 mm/sec from the initial length

(Li) to 1.5 Li produced a contraction. Keeping in the stretched state
for 15 min (15-min stretch) produced triphosphorylation of 20-kDa
myosin light chain (MLC20) at serine-19, threonine-18, and threonine9, and gradual reduction of the contraction, both of which were
reversed by Go6976, an inhibitor of conventional protein kinase C
(PKC) (Obara et al, J Vasc Res 2010; 47: 115-127). The 15-min
stretch increased PKCa activity, whereas it decreased protein phosphatase 2A (PP2A) activity. Stretching increased phosphorylation of 17kDa PKC-potentiated inhibitory phosphoprotein (CPI-17), which was
inhibited by Go6976. Low concentration of okadaic acid (OA), acting
as an inhibitor of PP2A, relaxed KCl (80 mM)-induced contraction of
the artery, and this relaxant effect was partially inhibited by Go6976.
KCl increased phosphorylation of MLC20 at serine-19. OA additionally increased MLC20 phosphorylation at threonine-18 and threonine-9,
resulting in triphosphorylation of MLC20, which was inhibited by
Go6976 (Obara et al, Eur J Pharmacol 2008; 598: 87-93). OA stimulated phosphorylation of PKCa and CPI-17, and Go6976 inhibited
these phosphorylations. The stretch- and OA-induced triphosphorylation of MLC20 was partially inhibited by ML-9, an inhibitor of myosin light chain kinase. These results suggest that activation of PKCa
mediated by an inhibition of PP2A is involved in the stretch- and
OA-induced triphosphorylation of MLC20. Of the triphosphorylated
amino acid residues of MLC20, the phosphorylated threonine-9 via
PKCa has an inhibitory action on the contraction.
Andes University, Faculty of Medicine, Bogota, Colombia

Education in pharmacology. Introduction: the growing number of available medications, the ongoing advances in knowledge around pharmacodynamics, pharmacokinetics, and interactions in the molecular and
genomic eld make pharmacology a science of continuous growth. It is
because of this, that its teachings represent a challenge for both students
and teachers, who are always in search for motivation and long lasting
learning. Objective: to develop the rst 3D interactive encyclopedia (in
Spanish and English) for the different therapeutic types of medications,
where principles of pharmacodynamics, pharmacokinetics, efcacy, security, pharmacoeconomics, drug interactions and posology are found.
Materials: six senior medical students from Universidad de los Andes
(Bogota, Colombia) who will develop the rst pilot with the therapeutic
type anticoagulants, under the coordination of the pharmacology professor. The following steps were included: 1) systematic information
research. 2). Development of a monograph and visual material. 3).
Arrangements with a 3D animation vendor. Results: during August and
November of 2009 the monograph and visual material has been done for
six different anticoagulants. In the mean time work is being done with
the 3D animation vendor in order to have the rst version of the interactive anticoagulant encyclopedia in April 2010. Conclusions: the education in pharmacology has its limitations because of the lack of available
teaching material that facilitate understanding. The use of technological
tools and the condensation of information in an interactive way will
allow students a better understanding in pharmacology as well as educational tools for professors.
Paper No.: 1565

HEALTH AND DISEASE
INHIBITORY ROLE OF PHOSPHORYLATION OF MYOSIN
LIGHT CHAIN AT THREONINE-9 IN THE MECHANICAL
ACTIVITY OF CANINE BASILAR ARTERY
Kazuo Obara(1), T Ishikawa(1), K Nakayama(2)
Paper No.: 1481

EVALUATING THE ANTI-DIABETIC PROPERTIES OF
PASPALUM VAGINATUM
Nkechi Veronica Ofah, CD Ezeokoli
University of West Indies, School of Veterinary Medicine, St Augustine,
Trinidad & Tobago
The study aimed at evaluating the anti-diabetic effects of extract of
Paspalum vaginatum Sw., a major component of Shanda a herbal
supplement used locally for the treatment of diabetes. Aqueous extract
of P. vaginatum was prepared using the dried, oven baked (120oC for
30 min) and ground leaves and stem of P. vaginatum. The hypoglycaemic effect was studied using in vivo models of normal, glucose-hyperglycaemic and streptozotocin-induced diabetic rats. The effects of
20 mg - 160 mg/kg Paspalum vaginatum extract were compared with
those produced by 200 mg/kg extract of commercial Gymnema sylvestre leaf (herbal hypoglycaemic supplement), 3 mg/kg glibenclamide
(oral hypoglycaemic agent) and 15 ml/kg distilled water as controls.
Intraperitoneal acute toxicity effect of P. vaginatum was determined in
mice. Single oral doses of P. vaginatum extract caused no signicant
reduction in the blood-glucose levels of the treatment models. The
effects of P. vaginatum were similar to those produced by G. sylvestre
but different from that produced by glibenclamide, a standard oral
hypoglycaemic agent. Acute toxicity test following intraperitoneal
administration in mice indicated that Paspalum vaginatum extract up
to a dose of 702 mg/kg was not toxic. The study suggests that a single oral dose of the aqueous extract of dried, baked Paspalum vaginatum alone has no signicant anti-diabetic activity in healthy and
experimental diabetic rats. Further work to evaluate the chronic effect
of the extract and the effect of the herbal supplement Shanda,
which contains Paspalum vaginatum, Solanum and Soy as these could
potentiate the effect of P. vaginatum is on going.
(1) University of Shizuoka School of Pharmaceutical Sciences, Department of Pharmacology, Shizuoka, Japan
(2) Iwate Medical University Faculty of Pharmaceutical Sciences,
Department of Molecular and Cellular Pharmacology, Iwate, Japan
500
Paper No.: 1765
RENAL SECRETION CLEARANCE OF
N1-METHYLNICOTINAMIDE AS AN ENDOGENOUS
BIOMARKER FOR TUBULAR ORGANIC CATION
TRANSPORTER TYPE 2 ACTIVITY IN MAN
Ryuichi Ogawa, Y Nobuoka, H Echizen
Meiji Pharmaceutical University, Department of Pharmacotherapy,
Tokyo, Japan
Introduction: Metformin and other cationic drugs are considered eliminated via renal tubular transporter(s). While intrinsic creatinine clearance
is a well established biomarker for GFR in man, none is available for
renal tubular transports of xenobiotics. Here, we report that renal secretion clearance of endogenous N1-methylnicotinamide (MNA) would be a
biomarker of renal organic cation transporter type 2 (OCT2) activity.
Methods: Twenty-four healthy Japanese volunteers (14 males and 10
females) were recruited for the study. Three-hour timed-urine and midpoint blood samples were collected. Plasma and urine levels of MNA
and creatinine were measured by LC/MS system. A loss-of-function
SNP (Ala270Ser) of OCT2 gene was genotyped by bidirectional
sequencing of PCR products. The study protocol was approved by IRB
and written informed consent was obtained from each participant.
Results: The mean (+/) SD) total renal clearance (ltration plus secretion) and secretion clearance of MNA were 275 +/) 85 and 156 +/)
81 mL/min/1.73m2, respectively. No gender differences were observed
in the clearances. Those having homozygous (n=1; 17 mL/min/1.73m2)
and heterozygous (n=8; 126 +/) 31 mL/min/1.73m2) Ala270Ser mutation showed approximately 90% and 30% lower renal secretion clearance
of MNA than those having wild-type genotype (n=15; 166 +/) 74 mL/
min/1.73m2), respectively. Conclusion: Since the Ala270Ser mutation
was associated with a reduction of MNA clearance in a gene-dose manner, this variant may be a clinically signicant covariate of renal elimination of OCT2 substrates. We consider that renal secretion clearance of
MNA may be a novel endogenous biomarker for renal OCT2 activity.
Paper No.: 2039

DETERMINANTS OF ANTIBIOTICS PRESCRIPTION AMONG
DOCTORS IN A NIGERIAN TEACHING HOSPITAL
Olayinka O Ogunleye(1,2), SO Ogundele(0), AF Yinka-Ogunleye(3)
(1) Lagos State University College of Medicine, Department of Pharmacology, Ikeja,Lagos
(2) Lagos State University Teaching Hospital, Department of Medicine,
Ikeja, Lagos, Nigeria
(3) Lagos University Teaching Hospital, Department of Preventive
Dentistry, Idi-Araba, Lagos, Nigeria
The problem of antimicrobial drug resistances is highly prevalent in
many parts of the world with overwhelming magnitude in the resource
poor nations like Nigeria. Irrational uses of currently available agents are
major contributory factors. It therefore becomes necessary to investigate
the factors determining antibiotic prescription in these settings with a
view of planning appropriate intervention strategies. This was a cross
sectional survey of the factors determining antibiotics prescription among
doctors in the Lagos State University Teaching Hospital, Ikeja, Lagos, a
major referral centre in Lagos metropolis serving an estimated catchments population of over 19 million people. Through a structured questionnaire, information was obtained about the doctors and factors
determining their uses of antibiotics. Statistical tests were carried out
with SPSS 15.0; continuous variables were expressed as means (standard
deviation), categorical variables as proportions. 98 respondents were
studied with mean age of 36.24(9.01) years, mean duration of practice of

10.68(9.25) years, males; 64.3%. Respondents cuts across all clinical
subspecialties. About 97% prescribe antibiotics frequently, mostly based
on clinical judgments (93.9%), experience (87.8%) and senior colleagues decisions (70.4%) with infrequent laboratory supports. No evidence for existence of institutional policies regarding antimicrobial
therapies. Factors of cost, drug availability and drug promotion activities
of pharmaceutical representatives evidently inuence doctors prescription.There is an urgent need to institute measures to promote rational
antimicrobial uses in the studied population.
Paper No.: 3345

FLURBIPROFEN PHARMACOKINETICS IN RELATION TO
CYP2C9 GENOTYPE
Da-Hee Oh, J-W Bae, M-J Kim, C-G Jang, S-Y Lee
Flurbiprofen is one of the phenylalkanoic acid derivatives of non-steroidal anti-inammatory drug (NSAIDs) used for the management of pain
and inammation in patients with arthritis. The major metabolic pathway
of urbiprofen is 4-hydroxylation, and in vitro studies have demonstrated that CYP2C9 is plays an important role in the metabolism of urbiprofen to 4-hydroxy-urbiprofen. We evaluated the effect of CYP2C9
genetic polymorphisms, especially focused on CYP2C9*3 variant allele,
on the pharmacokinetics of urbiprofen and 4-hydroxy-urbiprofen in
healthy Korean subjects. Eighteen Korean volunteers were selected and
they were grouped according to CYP2C9 genotype; group1 (CYP2C9*1/
*1, n=12) and group 2 (CYP2C9*1/*3, n=6). Each subject was received
a single oral dose of 40 mg urbiprofen the plasma concentrations of
urbiprofen and 4-hydroxy-urbiprofen were measured by using HPLC
system with UV and uorescence detection, respectively. There were statistically signicant differences in AUC0- of urbiprofen between
group1 and group2 (29.3 3.9 `ghr/mL vs. 47.1 12.2 `ghr/mL, P =
0.0002). Elimination half-life (t1/2) of urbiprofen in group2 was also signicantly longer than that in group1 (4.2 0.5 vs. 3.2 0.4, P =
0.0002). Differences in Cmax and t1/2 of 4-hydroxy-urbiprofen
between two groups were also statistically signicant (P < 0.01 and P <
0.001, respectively). The metabolic ratio of urbiprofen to 4-hydroxyurbiprofen in the group1 was nearly two-fold lower than in group2 (6.3
1.8 vs. 12.4 2.6, P < 0.0001). We concluded that CYP2C9 genotype,
particularly CYP2C9*3 allele, can affect the pharmacokinetics of urbiprofen in Koreans.
Paper No.: 3346

DISCOVERY
INFLUENCE OF ABCB1 C3435T POLYMORPHISM ON THE
PHARMACOKINETICS OF EZETIMIBE AND ITS
GLUCURONIDE IN HEALTHY SUBJECTS
Da-Hee Oh, C-I Choi, J-W Bae, M-J Kim, C-G Jang, S-Y Lee
Ezetimibe is an anti-hyperlipidemic agent, suppressing intestinal absorption of cholesterol via inhibition of Niemann-Pick C1-Like 1 (NPC1L1)
protein. It is reported that the interindividual changes of ezetimibe
plasma concentration and drug response are highly variable. MDR1, an
energy-dependent efux transporter, is encoded by the ABCB1 gene and
plays a signicant role in the pharmacokinetic processes of a large spectrum of drugs. So we investigated whether ABCB1 genetic polymor-
501
phism, especially ABCB1 C3435T genetic variant, could contribute to
the pharmacokinetics of ezetimibe. Forty-six healthy Korean subjects
participated in this study and they were divided into three groups according to ABCB1 C3435T genotype; ABCB1 3435CC (CC, n=21), ABCB1
3435CT (CT, n=20) and ABCB1 3435TT (TT, n=5). Each subject was
administered a single oral dose of 10 mg ezetimibe and plasma concentration of ezetimibe and its glucuronide metabolite was determined by
using LC-MS/MS system. Overall pharmacokinetic parameters (Cmax,
tmax, AUC, t1/2, CL/F and Vd) of ezetimibe between three genotype
groups were not signicantly different. However, maximum plasma concentration (Cmax) and the area under the plasma concentration-time
curve (AUC) values of ezetimibe glucuronide were signicantly different
between ABCB1 3435T allele carriers (CT + TT genotype) and non-carriers (CC genotype). From these results, functional change of MDR1
cannot affect the pharmacokinetic of ezetimibe, but it partially affects the
disposition of ezetimibe glucuronide, the major metabolite of ezetimibe.
Paper No.: 3347

DISCOVERY
THE ROLE OF OATP1B1 GENETIC VARIANTS IN THE
DISPOSITION OF PITAVASTATIN IN HEALTHY KOREANS
the treatment of hypertension. Metoprolol is rapidly absorbed and is

primarily metabolized via a-hydroxylation mediated by CYP2D6, the
highly polymorphic drug metabolizing enzyme. CYP2D6*10 allele is
frequently distributed among Asian population, including Koreans, and
is known to be associated with decreased CYP2D6 enzymatic activity.
We investigated the effects of CYP2D6*10 allele on the pharmacokinetics of metoprolol and a-hydroxymetoprolol. After genotyping for
CYP2D6, 35 healthy Korean volunteers were selected. They were
divided into three groups according to the number of CYP2D6*10
allele, group1 (CYP2D6*1/*1, n=15), group2 (CYP2D6*1/*10, n=10),
and group3 (CYP2D6*10/*10, n=10). A single oral dose of 100 mg
of metoprolol was administered to each subject and plasma concentrations of metoprolol and its metabolite were determined using HPLC
system with uorescence detection. Cmax and AUC0- of metoprolol
in group3 were signicantly higher than those in group1 and group2
(P < 0.0001, respectively), and elimination half-life of metoprolol in
group3 was signicantly longer than that in group1 and group2 (P <
0.0001, respectively). Oral clearance of metoprolol in group3 was signicantly lower than that in other groups (P < 0.0001, respectively).
Pharmacokinetic parameters of a-hydroxymetoprolol in each group
were also signicantly different. AUC ratio between metoprolol and
a-hydroxymetoprolol in each group were 3.3 1.4, 2.2 1.1 and 0.4
0.2, respectively (P < 0.0001). From these results, CYP2D6*10
allele is found to affect the pharmacokinetics of metoprolol and its
metabolite.
Pitavastatin is a HMG-CoA reductase inhibitor used for the treatment of
hypercholesterolemia. Hepatic uptake of pitavastatin is mediated mainly
by OATP1B1 drug transporter. Many studies show that OATP1B1*15, a
relatively uncommon variant allele of OATP1B1, is associated with
decreased transporting activity of this hepatic inux transporter. In this
study, we investigated the effects of OATP1B1*15 allele on the pharmacokinetics of pitavastatin and its lactone form in healthy Korean subjects.
After genotyping for OATP1B1, 27 healthy Korean volunteers were
selected for this study. They were divided into three groups according to
the number of OATP1B1*15 allele, group1 (n=14, OATP1B1*1/*1),
group2 (n=10, OATP1B1*1/*15), group3 (n=3, OATP1B1*15/*15). A
single oral dose of 2 mg pitavastatin was administered to each subject,
and plasma concentration of pitavastatin and pitavastatin lactone were
determined by using LC-MS/MS system. Cmax of pitavastatin in each
group were signicantly different (44.3 - 17.1 ng/mL vs. 66.7 - 24.8 ng/
mL vs. 103.1 - 19.0 ng/mL, P = 0.0004) AUC0- of pitavastatin in
group1, group2 and group3 was 153.7 - 56.4 nghr/mL, 168.8 56.1 nghr/mL and 289.6 - 18.8 nghr/mL, respectively, and these
changes were also statistically signicant (P = 0.0023). Other parameters
were not statistically signicant between each group. Furthermore, pharmacokinetics of pitavastatin lactone was also not signicantly different
between three genotype groups. OATP1B1*15 variant allele is found to
affect the pharmacokinetics of pitavastatin.
Paper No.: 3376

CONTRIBUTION OF CYP2D6*10 ALLELE TO THE INTERINDIVIDUAL VARIABILITY IN THE PHARMACOKINETIC OF
METOPROLOL AND ITS B-HYDROXY METABOLITE IN
KOREANS
Paper No.: 1073

GENERATION OF GUT-LIKE TISSUE FROM INDUCED
PLURIPOTENT STEM CELLS THAT CAN BE APPLIED FOR
FUNCTIONAL CHARACTERIZATION
Kazuhiko Oishi, Y Ogawa
Meiji Pharmaceutical University, Department of Pharmacology, Tokyo,
Japan
Induced pluripotent stem (iPS) cells are prepared by transducing several genes into somatic cells. Their features are similar to those of
embryonic stem (ES) cells, and it is possible to differentiate iPS cells
into various organ/tissue cells. Therefore, these cells may replace ES
cells as a cell resource that can be applied for regenerative therapy.
On the other hand, iPS cells may be useful for clarifying the pathogeneses of disorders and conducting pharmacological studies for the
development of pharmaceutical preparations in addition to their clinical
application including cell transplantation. If disease-specic iPS cells
prepared using cells from patients are compared with normal iPS cells,
the etiologies of refractory diseases may be claried, contributing to
the detection of new pharmaceutical targets. We have established the
formation of functional gut-like tissue form iPS cells that reect their
mechanical activity, such as gastrointestinal motility, and can be
applied for the quantitative measurement of spontaneous contraction.
Mouse iPS cells were cocultured with PA6 stromal cell line for 7 days
to induce differentiation into spheres. The cells in the spheres were
three-dimentionally cultured in a collagen gel and grew into gut-like
tissue after two weeks of culturing. The tissues showed rhythmic
motility and stained positive for the specic marker proteins of neurons and ICC, PGP9.5, and c-Kit, respectively. When the contractility
of these tissue was evaluated isometrically, they exhibited spontaneous
contraction that reect their mechanical activity. Therefore, this iPS
cell-derived gut-like tissue may be of use in elucidating the developmental mechanisms of gastrointestinal motility.

Metoprolol is a selective b1-adrenergic receptor blocking agent, which
is used alone or in combination with other antihypertensive agents for
502
Paper No.: 2264
HEALTH AND DISEASE
INTRAVENOUS ADMINISTRATION OF SODIUM NITRITE
ALLEVIATES ISCHEMIA/REPERFUSION-INDUCED
CEREBRAL INJURY IN NONDIABETIC RATS BUT NOT IN
DIABETIC RATS
Mari Okazaki, N Iwata, E Takamura, S Kamiuchi, Y Hibino
Josai University Faculty of Pharmaceutical Sciences, Laboratory of Immunobiochemistry, Sokado, Japan
Nitrite has been reported to be a reservoir of nitric oxide (NO) exerting
cytoprotective effects against liver, heart, or brain ischemia-reperfusion
injury. However, other evidence has indicated that NO shows both protective and toxic effects depending on its level and location. Previously,
we revealed that the diabetic (DM) state increases oxidative stress and
aggravates ischemic-reperfusion injury in rat brain. In the present study,
we investigated whether nitrite exerts neuroprotective effects against
cerebral injury induced by middle cerebral artery occlusion followed by
reperfusion (MCAO/Re) in DM rats. Type1-diabetes was induced by a
single injection of streptozotocin in male Sprague Dawley rats. Cerebral
injury was induced by MCAO (2 hrs) following reperfusion (24 hrs) in
the rats. Saline solutions of sodium nitrite (1.25, 12.5, or 25 micro mol/
kg) were intravenously infused at the time of MCAO. We found that
infract volume, edema, and the number of apoptotic cells in the non-DM
rat brain determined after the reperfusion were reduced by nitrite at a
dose of 12.5 micro mol/kg, but not at the other doses or at any doses of
nitrite when nitrite was administered at the start of reperfusion. On the
other hand, no signicant improvement in the injury was observed in the
DM rats at any doses or infusion timings of nitrite. These results indicate
that early treatment with nitrite relieves the cerebral ischemia-reperfusion
injury presumably by NO-mediated restoration of the cerebral blood
ow. Augmented oxidative stress in DM state might disrupt the protection.
Paper No.: 2788

THE STUDY OF ANTIDIABETIC EFFECTS OF PARTIALLY
PURIFIED EXTRACT FROM THE ROOTS OF THOTTEA
GRANDIFLORA ROTTB
Patrick Okechukwu(1), G Marimuthu(1), GA Akuwoah(2)
(1) UCSI University Faculty of Applied Sciences, Biotechnology Programme, Kuala Lumpur, Malaysia
(2) UCSI University Faculty of Pharmaceutical Sciences, Biotechnology
Programme, Kuala Lumpur, Malaysia
Thottea grandiora Rottb., locally known as hempedu beruang is used
as a traditional remedy to treat diabetes and wounds caused by the complication of this disease. Preliminary pharmacological investigation
revealed that the dichloromethane root extracts and partially puried fraction E possessed anti-diabetic activity. This present study was aimed at
further-purifying the fraction E and evaluates its antidiabetic effects using
streptozotocin-induced diabetic rats. Three sub-fractions E1, E2 and E3
were obtained, and their antidiabetic effects were evaluated using a dose
of 100 mg/kg for 15 days. The blood glucose levels and the bodyweights
of the diabetic rats were determined before and at day 1, 10 and 15 of
treatment. Sub-fraction E1 signicantly (p < 0.05) reduced the blood glucose levels of the diabetic rats by an average of 45.9 % and increased
the bodyweights of the diabetic rats by an average of 5.56 % which corelates positive control tolbutamide, while sub-fraction E2 and sub-fraction E3 did not show any positive effect. Phytochemical tests of sub-fraction E1 revealed the presence of alkaloids, avonoids, saponins and
terpenoids all of which have been reported to possess anti-diabetic activity. Hence, the anti-diabetic effects of sub-fraction E1 could be due to the
presence of these phytochemicals. Ongoing research is being performed

to investigate the characteristics of the compound(s) involved in the antidiabetic effects of sub-fraction E1 to deduce its mechanism of action.
Paper No.: 1566

TOPICAL APPLICATION OF DISODIUM
ISOSTEARYL 2-O-L-ASCORBYL PHOSPHATE, A SKINPERMEABLE AMPHIPHILIC ASCORBIC DERIVATIVE,
ATTENUATES CHEMOTHERAPY-INDUCED NEUROPATHIC
HYPERALGESIA IN RATS
Kazumasa Okubo(1), T Takahashi(1), M Matsunami(1), H Shibayama(2),
A Kawabata(1)
(1) Kinki University School of Pharmacy, Division of Pharmacology &
(2) HBC Science Laboratory Co. Ltd., Japan
Cav3.2, an isoform of T-type calcium channels, that is targeted by hydrogen sulde (H2S), a gasotransmitter (Maeda et al, Pain 2009;142:127),
participates in the maintenance of neuropathic pain (Messinger et al, Pain
2009;145:184). Given recent evidence that ascorbic acid (VC) selectively
inhibits Cav3.2, but not Cav3.1 or Cav3.3 (Nelson et al, J. Neurosci.
2007;27:12577), in the present study, we examined and compared effects
of intraplantar (i.pl.) injection or topical application of VC and a skinpermeable amphiphilic VC derivative, disodium isostearyl 2-O-L-ascorbyl phosphate (DI-VCP), on the hyperalgesia induced by local administration of NaHS, an H2S donor, and on the neuropathic hyperalgesia
following repeated systemic administration of paclitaxel, an anticancer
drug, in rats. NaHS, administered i.pl., caused prompt and transient hyperalgesia, while paclitaxel, administered i.p. repetitively, produced
delayed and persistent hyperalgesia. Either hyperalgesia was reversed by
i.pl. administration of VC as well as mibefradil, a pan-T-type calcium
channel inhibitor. VC, when applied topically as an ointment, failed to
block the NaHS-evoked and paclitaxel-induced hyperalgesia. In contrast,
topical application of DI-VCP ointment prevented the NaHS-evoked hyperalgesia in naïve rats, and caused slowly developing anti-hyperalgesic effect in rats with the paclitaxel-induced neuropathy. These data
suggest that topical application of DI-VCP, but not VC, as an ointment
may be available for treatment of chemotherapy-induced neuropathic
pain, and the underlying mechanisms might involve inhibition of Cav3.2,
a target for H2S.
Paper No.: 3412

DISEASES
EVALUATION OF ANTIOXIDANT AND ANTIINFLAMMATORY EFFECTS OF FLAVONOIDS AND METALFLAVONOIDS COMPLEXES
Cristina Okuyama, D de Oliveira, C Miyashiro, R Pereira
Bandeirante University of Sao Paulo, Department of Pharmacy, Sao
Paulo, Brazil
Introduction: Flavonoids have high antioxidant and anti-inammatory
activities. Besides, recent researches demonstrate that the antioxidant
activity of avonoids is believed to increase when they are coordinated
with transition metal ions. Methods: The antioxidant effect of the compounds was measured by activity of radical superoxide scavenging, dispersed in the environment. For the anti-inammatory activity evaluation,
the peritonitis model in mice was used. Results: The results showed that
the percentage of superoxide radicals scavenging by Rutin, Cu2-Rutin,
Ni2-Rutin, Fe-Rutin, complexes are, respectively: 8,66%, 1,95%, 1,21%,
503
0,53%, at the concentration of 1uM; 6,95%, 46,42%, 21,69%, 11,01% at
the concentration of 10uM; and 51,80%, 71,32%, 53,57%, 52,59% at the
concentration of 100uM. In case of peritonitis, the cell migration inhibition caused by dexamethasone, rutin 10 and 100mg/kg, Cu2-rutin 10 and
100mg/kg groups were, respectively: 48%, 0%, 9%, 26% and 38% when
peritonitis was induced by carrageenin; 54%, 15%, 25%, 0% and 0%
when induced by histamine; 28%, 0%, 9%, 26% and 38% when induced
by bradikynin; 43%, 21%, 42%, 49% and 57%, when induced by PGE2;
19%, 31%, 15%, 20% and 15% when induced by substance P. Discussion: The results demonstrated that the Cu-Rutin complex presented the
highest superoxide radicals scavenging effect, on the proposed assay,
when compared with rutin alone. Furthermore, the Cu2-rutin reduced signicantly the cell migration in case of peritonitis induced by carrageenin,
bradikynin and PGE2, in mice.
Financial Support: UNIBAN and FAPESP.
Paper No.: 2676

G PROTEIN-COUPLED ESTROGEN RECEPTOR 1 FUNCTION
IN VIVO AND IN VITRO
release of pro-inammatory cytokines, e.g. IL-8, from various cells,

including airway smooth muscle cells. This IL-8 release is associated
with activation of the nuclear factor-j B (NF-jB) pathway. Here we
investigated the modulatory role of cAMP-regulated effectors Epac and
PKA on cigarette smoke extract (CSE)-induced IL-8 release in cultured
airway smooth muscle cells. CSE was applied to immortalized human
airway smooth muscle cells with or without the ?2-adrenoceptor agonist
fenoterol - to stimulate both cAMP targets simultaneously - the Epacselective agonist 8-pCPT-2-O-Me-cAMP or the PKA-selective agonist
6-Bnz-cAMP. IL-8 release in culture media was measured by ELISA.
IjBa-degradation, as a measure for NF-jB activation, and ERK phosphorylation were evaluated via western analysis. Treatment with CSE
induced an increase in IL-8 production in airway smooth muscle cells,
which was reduced by fenoterol, 8-pCPT-2-O-Me-cAMP as well as by
6-Bnz-cAMP. PKA activation inhibited CSE-induced IL-8 release more
strongly than Epac activation. CSE also induced IjBa-degradation - indicating activation of NF-jB - in airway smooth muscle cells, a process
being reversed following Epac activation. Moreover, CSE increased the
phosphorylation of ERK, which was reduced by the PKA activator. Collectively, our data indicate that cAMP exerts anti-inammatory effects by
inhibiting CSE-induced IL-8 release in human airway smooth muscle via
activation of both Epac and PKA. The inhibitory effect by Epac is primarily caused by inhibiting NF-jB activation, whereas ERK inhibition is
the main action of PKA.
B Olde, C Sanden, S Broselid, U Martensson, Fredrik Leeb-Lundberg

Lund University, Department of Experimental Medical Science, Lund,
Sweden
GPR30, or G protein-coupled estrogen receptor 1 (GPER1), has been
proposed to be a membrane estrogen receptor in part responsible for
non-genomic estrogen responses. We developed model systems to study
the function of GPR30 in vivo and in vitro. Mice lacking the GPR30
gene (GPR30(-/-) mice) had decreased insulin expression, glucose-stimulated insulin release, and longitudinal bone growth, and increased blood
pressure. 17 b-Estradiol (E2)-stimulated insulin release and closure of
the bone growth plate was impaired in ovariectomized adult GPR30(-/-)
mice. On the other hand, E2 responses on several known estrogen receptor a-mediated parameters including uterine weight, thymus atrophy, fat
mass, and trabecular bone mineral density were normal in these mice.
GPER1 is core N-glycosylated and localized primarily intracellular, but a
limited amount of receptors reach the plasma membrane where they are
subject to rapid and constitutive endocytosis apparently via a recycling
pathway. In recombinant cells, GPER is co-localized and partially associated with the endoplasmatic reticulum chaperone calnexin, whereas in
native cells it is not. E2 and G-1, a GPR30 agonist, do not stimulate
cAMP production in recombinant cells. On the other hand, both agonists
stimulate cAMP production in a GPER1-dependent manner in native
cells. However, the maximal response of E2 is only about 10% of that of
G-1 suggesting that it acts as a partial agonist.
Paper No.: 2218

DISEASES
EPAC AND PKA INHIBIT CIGARETTE SMOKE-INDUCED
PRODUCTION OF IL-8 IN AIRWAY SMOOTH MUSCLE CELLS
Anouk Oldenburger(1), SS Roscioni(1), E Jansen(1), MH Menzen(1),
AJ Halayko(2), H Meurs(1), M Schmidt(1)
(1) University of Groningen, Department of Molecular Pharmacology,
Groningen, TheNetherlands
(2) University of Manitoba, Department of Physiology, Winnipeg, MB,
Canada
Paper No.: 2341

USE OF CONFIDENCE INTERVALS IN THE FRACTIONAL
ANALYSIS OF THE INTERACTION BETWEEN
NON-DEPOLARIZING NEUROMUSCULAR BLOCKERS
Antonio Carlos Oliveira, CR Souza, WL Silva, CSM Serra
University of Sao Paulo, Institute of Biomedical Sciences, Department
ofPharmacology, Sao Paulo, Brazil
Use of condence intervals in the fractional analysis of the interaction
between non-depolarizing neuromuscular blockers. Oliveira, AC; Souza,
CR; Silva, WL; Serra, CSM. Department of Pharmacology, Institute of
Biomedical Sciences, University of Sao Paulo, Sao Paulo, SP, Brazil.
The interaction between the non-depolarizing neuromuscular blockers:
atracurium(A), cisatracurium(C), mivacurium(M), pancuronium(P), rocuronium(R) and vecuronium(V) was studied in vitro, in the sciatic nerveextensor digitorum longus preparation of the rat. Dose-response curves
for the block of indirect twitches furnished IC50 for each blocker individually and for all their 2x2 combinations. The blockers were mixed
proportionally to their individual IC50. In each group experiments were
replicated from 5 to 8 times. Fractional analysis was performed for each
combination of blockers. Condence intervals (95%) were used to evaluate whether the fractions of individual IC50 that summated to constitute
the IC50 of the combination was equal, smaller or larger than 1. These
situations correspond, respectively, to additivity, supra-additivity or subadditivity of effects of the combined blockers. The individual IC50 (micromol/L) were (mean SE): A(2.97 0.19), C(0.61 0.02), M(1.22
0.26), P(0.80 0.01), R(1.77 0.16) and V(0.88 0.05). The condence
intervals for the sum of the fractions of IC50 were: A+C(1.0-1.3);
A+M(0.7-0.8); A+P(0.7-1.0); A+R(1.0-1.1), A+V(1.2-1.3); C+M(0.50.6), C+P(0.8-1.1); C+R(0.9-1.2), C+V(0.9-1.3), M+P(0.9-1.1),
M+R(0.8-1.1), M+V(0.7-1.2); P+R(0.9-1.1); P+V(0.9-1.1); R+V(1.01.2). It can be concluded that supra-additivity of blocking effects was
present in A+M and C+M combinations; sub-additivity in A+V combination and simple additivity in all of the other combinations. Supported
by FAPESP, CNPq and CAPES.
Cigarette smoke is the main risk factor for development of chronic

obstructive pulmonary disease (COPD). Cigarette smoke stimulates the
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Paper No.: 868
DISEASES
LIPOPOLYSACCHARIDE STIMULATES DIABETIC GINGIVAL
FIBROBLAST TO EXPRESS TGF-BETA VIA MIP-2. ROLE OF
PI3K
marked increase in ability to inhibit NO formation in the homogenate

when compared to tryptamine itself. In vitro evaluation of the new inhibitors ability to inactivate NOS activity revealed an improved potency
brought about by the pentacyloundecane substituent. This study clearly
demonstrates the potential of the pentacyloundecane cage structure to
enhance the activities of compounds acting at NOS.
Sandra HP Oliveira, A Tamae, MA Machado

State University of Sao Paulo, School of Dentistry, Department of Basic
Sciences,Aracatuba, Sao Paulo, Brazil
Introduction: Our aim was to investigate the mechanism involved in the
TGF-b expression on gingival broblasts of diabetic mice (FgD) stimulated by lipopolysaccharides (LPS). Materials: Balb/c mice was treated
with streptozotocin (STZ) (80 mg/g, ip) for diabetes induction. The gum
of the diabeticsanimals had been removed and kept in culture for the
growth of the FgD. The cells were stimulated by LPS (0.1, 1, 10 and
100 lg/mL) and after 1, 6 and 24 hours was evaluated the expression of
TGF-b for RT-PCR and MIP-2 for ELISA. LPS-stimulated cells were
pre-treated with antibody anti-MIP-2 or p42/44 MAPK inhibitor,
PD98059 (30 lM), p38 MAPK inhibitor, SB203580 (30 lg/mL) and
PI3K inhibitor Wortmannin, (0,1 lM) 30 min before LPS (0,1 lg/ml)
stimulation. Results: LPS-stimulated FgD (0,1 lg/mL) expressed TGF-b
already in the rst hour. When the LPS concentration was increased (1,
10 and 100 lg/ml) this expression was inhibited. LPS-stimulated FgD
produced MIP-2 after 1 hour and its production peaked 6 hours after the
LPS-stimulation. Antibody anti-MIP-2 was able to inhibit the TGF-b
expression. TGF-b expression was partially inhibited by PI3K but not by
p42/44 MAPK neither p38 MAPK via. Conclusion: LPS-stimulated FgD
(0,1 lg/mL) to express TGF-b. It expression is mediated by MIP-2 production. The PI3K pathway seems to be partially involved in the LPSinduced TGF-b expression by FgD and p38 and PI3K is mediating MIP2 production. Development of inhibitors targeting at MAPK could be an
attractive strategy for the treatment of gingival inammatory process.
Financial supported: FAPESP
Paper No.: 1849

PENTACYCLOUNDECANE CONJUGATES OF
AMINOGUANIDINE AND TRYPTAMINE: NITRIC OXIDE
SYNTHASE INHIBITION
Douglas W Oliver, SF Malan, DK Wilkes, A de Vries
North-West University School of Pharmacy, Department of Pharmacology, Potchefstroom, South Africa
Selective inhibition of the isoforms of nitric oxide synthase (NOS) has
an important therapeutic potential in the treatment of certain diseases
arising from pathologically elevated synthesis of nitric oxide (NO).
Focusing on neurodegeneration, the neuronal isoform of nitric oxide synthase (nNOS) in the central nervous system represents an attractive target
for the treatment of various disorders such as stroke, Parkinsons and
Alzheimers disease. This rationale prompted the selection of aminoguanidine and tryptamine moieties as pharmacophores owing to their selective inhibition of nNOS and iNOS isoforms. Both compounds are
however weak inhibitors of the NOS enzyme and complete inhibition
only occur at high millimolar concentrations. The present study aimed to
evaluate the effect of the pentacycloundecane cage moiety as an alkyl
substituent on the in vitro NOS inhibition of aminoguanidine and tryptamine compounds. The compounds were designed from aminoguanidine
and tryptamine analogues and were substituted at the N-1 position with
the pentacyloundecane cage structure. The NOS activity of rat brain
homogenate was determined by the in vitro conversion of oxyhemoglobin to methemoglobin following the reaction with NO. The series of
indole derivatives containing the pentacyclic cage moieties showed a
Paper No.: 1945

THE EXPRESSION OF CARDIAC AND LYMPHOCYTIC GRKS
IN HUMAN CARDIOVASCULAR DISEASES CORRELATED TO
THE EXPRESSION LEVELS OF B-ARS
Eduardo Oliver(1), F Monto(1), D Vicente(1), J Aguero(2),
EE Rovira(3), L Almenar(2), P DOcon(1)
(1) University of Valencia, Department of Pharmacology, Burjassot,
Spain
(2) Hospital Universitario La Fe, Heart Failure and Transplant Unit,
Valencia, Spain
(3) Hospital de la Ribera, Hypertension Unit, Alzira, Spain.
Introduction: it is well-known that G-protein coupled receptor kinases
(GRKs) regulate b-Adrenoceptors (ARs), and the changes in their
expression and functionality are implicated in cardiovascular diseases
(Penela P et al, Cardiovascular Research 2006; 69:46-56). This work
aims to determine whether the changes observed in the expression of
GRKs could relate to changes in the b-AR expression, or if they constitute an independent process. Materials/Patients: we quantied the mRNA
expression of b1- and b2-AR, GRK2, GRK3 and GRK5, and Gapdh
(housekeeping gene) by TaqMan real-time RT-PCR in lymphocytes
(from healthy volunteers, hypertensive or heart failure (HF) patients and
transplanted patients, n=72), and in the cardiac biopsies (n=17) from
transplanted hearts, and in left and right ventricles of HF patients (n=35).
Results: irrespectively of the changes found in the expression of each
gene in the different pathologies, linear regressions were established
between the mRNA levels of b-ARs and GRKs. In lymphocytes, the correlations between b1-AR/GRK5, and b2-AR/GRK2 and GRK3, were
signicant. We found correlations between b1-AR and b2-AR/GRK2
and GRK5 in biopsies from nonfailing hearts and right ventricles of HF
patients. Each b-AR correlated with all three GRKs in the left ventricles
of HF patients. Conclusions: the relationship observed between the
expression of b-ARs and GRKs in a given tissue and pathology suggest
that changes in the GRKs expression relate to changes in b-ARs. Therefore, it is necessary to carefully interpret isolated GRKs determinations
that are not accompanied by a similar b-AR analysis.
Paper No.: 2122

THE EFFECT OF TESTOSTERONE ON INTRACELLULAR
CALCIUM AND CAMP IN HUMAN CULTURED PROSTATIC
STROMAL CELLS
Victoria Oliver, C Anderson, J Haynes
Monash University, Department of Medicianl Chemistry and Drug
Action, Parkville, Melbourne, VIC, Australia
Increased smooth muscle tone is a signicant component of benign prostatic hyperplasia, the onset of which correlates with age and declining
serum testosterone levels. This study investigates the effects of testosterone on key regulators of smooth muscle tone: intracellular calcium
([Ca2+]i) and cyclic adenosine monophosphate (cAMP) in human cultured prostatic stromal cells (HCPSC). HCPSC were cultured in the
505
absence or presence of testosterone (300 pM 300 nM). [Ca2+]i was
quantied in FURA-2AM (10 lM) loaded cells. Changes in cAMP were
determined by Alpha Screen assay. Some cells (26 3 % of 324 cells
from six individuals) exhibited spontaneous elevations of [Ca2+]i which
were abolished by the L-type calcium channel blocker, nifedipine
(10 lM), the sarcoplasmic reticulum calcium release inhibitor, ryanodine
(1 lM) and the adenylate cyclase inhibitor MDL 12,330A (20 lM). The
adenylate cyclase activator, forskolin (20 lM) had no effect. Testosterone
had no effect on either the proportion of cells exhibiting this activity or
the frequency of elevations. However, the magnitude of spontaneous elevations were signicantly (P<0.01) increased at a physiologically low
concentration of testosterone (3 nM). Under these conditions, resting levels of both [Ca2+]i and cAMP were also signicantly (P<0.001) elevated
in all cells when compared to higher testosterone concentrations. These
ndings demonstrate that some HCPSC have the ability to spontaneously
and transiently elevate calcium. These elevations, along with resting levels of calcium and cAMP appear to be regulated by testosterone.
Paper No.: 703

ADVERSE REACTIONS TO MEDICINES EXPERIENCED IN A
NIGERIAN HYPERTENSION CLINIC
Abimbola Olowofela, AO Isah
University of Benin Teaching Hospital, Department of Medicine, Clinical
Pharmacology and Therapeutics Unit, Benin City, Nigeria
There has been a considerable increase in the arsenal of antihypertensive
medicines in the last few decades. However, there are no comprehensive
data on the prole of adverse reactions to these medicines in the Nigerian
setting. This study characterizes the adverse reactions experienced in this
black African population. The study was carried out at the University of
Benin Teaching Hospital, Benin-City Nigeria, in eligible hypertensive
patients 18 years who agreed to participate. Adverse reactions were
sought using patient self report and a medicine-induced symptom checklist. A total of 514 patients (340 F) aged 22-97 years were studied.
13.0%, 27.6%, 26.7%, 22.0% and 10.7% were on 1,2,3,4 and 5 medicines respectively with the frequency of adverse effects increasing proportionately up to 4 medicines. CCB were the most prescribed group(70%).
Adverse reactions to antihypertensive medicines were reported by a total
of 93(18.1%) patients. Diuretics (27.9%), CCB(26.8%) and ACEI(26.8%)
accounted for most of the adverse reactions seen. Notable adverse reactions encountered included frequent micturition and headaches (CCB);
excessive micturition and dizziness (Diuretics); dry irritating cough
(ACEI). Notable complaints for all patients using the checklist were
increased frequency of micturition, reduction in sexual urge, headaches,
and fatigue. The adverse reactions resulted in 49.5% of the patients experiencing adverse effects discontinuing therapy. Frequent micturition is a
neglected complaint in antihypertensive therapy. Adverse reactions
deserve due consideration in antihypertensive therapy.
Paper No.: 2482

HEALTH AND DISEASE
UII PLAYS A PROATHEROGENIC ROLE IN THE
HYPERGLYCEMIC MILIEU CHARACTERISTIC OF
DIABETES
Murat Olukman(1), A Watson(2), TJ Allen(2), ME Cooper(2)
(1) Ege University, Faculty of Medicine, Department of Pharmacology
and Clinical Pharmacology, Izmir, Turkey
(2) Diabetes and Atherosclerosis Laboratory, BakerIDI, Melbourne,
Australia
Background and Aims: Urotensin II (UII), the most potent vasoactive
peptide known to date, is upregulated in diabetes and atherosclerosis. In
the present study we evaluated the effect of UII and SB-657510 (UII
receptor antagonist) on the gene expressions of pro-atherosclerosis associated molecules MCP-1, ICAM-1,VCAM-1, TGF-b1 and NF-j-B in
human aortic endothelial cells (HAEC) by real time-PCR. Materials and
Methods: HAECs were exposed to either low glucose (5mM) or high(25
mM) glucose for 72 hours. UII treatment (10-8- 10-11M) was performed
in the last 24 hours of incubation. Another group was treated with SB657510(10-8-10-10 M) for 24 hours after 30 minutes pre-incubation
with UII 10-8 M. Results: In normal glucose concentrations UII caused a
signicant reduction in the gene expression of MCP-1, ICAM-1,
VCAM-1, TGF-b1 and NF-j-B. However, UII signicantly enhanced
the gene expression of MCP-1, ICAM-1,NF-j-B and TGF-b signicantly
in high glucose concentrations. VCAM-1 expression was not affected
from high glucose concentrations. SB-657510 treatment caused a signicant reduction in MCP-1 and NF-j-B gene expressions both in normal
and high glucose concentrations. On the other hand it reduced ICAM-1
and VCAM-1 expressions in high glucose concentrations. Conglusions:
These results suggest that UII may be protective and anti-atherosclerotic
within normal glucose concentrations but it may act as a proatherosclerotic agent under high glucose concentrations. SB-657510 shows an antiatherosclerotic effect in vitro regardless of glucose concentrations. In vivo
studies are now warranted to determine if blockade of UIIs actions in
the vasculature can have a cardiovascular benet in diabetes.
Paper No.: 1218

ARTEMETHER MAY AFFECT THE ANALGESIC ACTIVITY
OF PIROXICAM IN MALARIA
Temidayo Olurishe, S Ado, I Adamu
Ahmadu Bello University, Department of Pharmacology and Therapeutics, Kaduna,Nigeria
Malaria in spite of all the attempts made by governments and organizations inAfrica continues to be responsible for millions of deaths annually.
In addition,sickle cell anaemia has also contributed to the mortality and
morbidityassociated with the disease. The use of artemether a derivative
of artemisininin treatment of malaria as a drug of choice against chloroquine resistantmalaria has become common. Pain management also forms
a major problem oftenassociated with malaria and sickle cell anaemia
(SCA) sometimes requiring theuse of non steroidal anti inammatory
drugs like piroxicam. The current studywas designed to investigate the
possible effect of artemether on analgesiceffect of piroxicam in mice and
rats. The acetic acid induced writhe model wasused to evaluate the effect
of two doses of artemether (15mg/kg and 30 mg/kg)against a single dose
of piroxicam (5mg/kg). The result of the study shows thatartemether
affects the analgesic effect of piroxicam signicantly (P<0.05).Artemether
also shows some analgesic effects on its own at the doses tested.However
Artemether at both doses failed to show centrally mediated analgesiawhen
evaluated against the mechanical pain model. The preliminary data from
thisstudy shows there may be need for caution in the concurrent use of artemetherand piroxicam in malaria therapy as pain is often a critical aspect
of malariaespecially when severe in SCA. Further studies are intended to
evaluate theeffect this interaction may have in the presence of parasitemia.
Paper No.: 1405

EFFECT OF LAMIVUDINE-ARTESUNATE
CO-ADMINISTRATION ON SOME HEMATOLOGICAL,
HEPATIC AND RENAL BIOMARKERS IN RATS
Temidayo Olurishe(1), H Kwanashie(1), J Anuka(1), H Muktar(2)
(1) Ahmadu Bello University, Department of Pharmacology & Therapeutics, Zaria, Nigeria
506
(2) Ahmadu Bello University Teaching Hospital, Department of Haematology, Zaria, Nigeria
With HIV-malaria co-morbidity being a common clinical presentation in
Africa,polypharmacy presents challenges of interactions and adverse
drug events.Lamivudine (3TC) and Artesunate (AS) are key components
of drug combinationsemployed in chemotherapy of these diseases. The
effect of 3TC-ASco-administration on selected biomarkers was investigated in cyclophosphamide(CYC) immunosupressed and Plasmodium
berghei (MP) infected wistar rats, a modelfor immunosuppression as in
HIV and parasitemia in malaria. The experimentalprotocol with n=5, had
a group each receiving 3TC (20 mgkg1) and AS (10 mgkg1),while two
other groups received 3TC and AS alone respectively, and a controlgroup
was also added. A control group that received neither CYC nor MP wasincluded. Weights of animals were monitored weekly. At the end of a
21 day drugtreatment, animals were anaesthetized and blood was collected for hematologicalindices, liver enzymes, urea and electrolytes
determination using standardlaboratory analytical protocols. Relative
organ weights were also determined.RBC was signicantly (P<0.05,
ANOVA) decreased in 3TC-AS and AS alone groupcompared against
both controls, while other hematological indices except WBC in3TC-AS
group (P<0.05) were not signicantly altered. Liver function, renalfunction, body weights as well as relative weights of the liver, lung pancreasand kidney were largely unchanged. The results show varying extents
ofalteration in parameters investigated and it is recommended that cautionarymeasures and monitoring of these parameters in clinical situations.
Paper No.: 1462

ANTIMALARIA AND ANTICANCER PROPERTIES OF LABISIA
PUMILA
Maizatul Hasyima Omar, A Crozier, NR Abdullah, Z Ismail
University of Glasgow, Faculty of Biomedical and Life Sciences, Institute of Medical Research, Plant Products and Human Nutrition Group,
Glasgow, Scotland, UK
Labisia pumila (Myrsinaceae) commonly known as Kacip Fatimah is
a small herbaceous plant and traditionally used by Malay practitioners as herbal preparation for post-natal care and was reported to
have estrogenic properties (Fazliana et al., 2009). Herbal preparations
of L. pumila are made by boiling the root of the plant and drinking
the resultant infusion. In the present study we investigated the cytotoxicity and antiplasmodial properties of the methanol extract of root
of L. pumila. The cytotoxicity test (Sulforhodamine B assay) showed
L. pumila extract at single dose at 25 lg/ml tested on HepG2 cell
showed growth inhibition less than 50%. The extract was also tested
for anti plasmodial activity using 3D7 and K1 strain and exhibited
IC 50 values of 5.12 and 4.93 lg/mL respectively. Activity of the
extract with normal cell line MDBK showed an IC 50 of 22.1 lg/mL
which gives a selectivity index of 0.23 and 0.22 that are considered
slight harmful to the cells. However these results indicate that root
extract of L. pumila has promising anti plasmodial effect suggest that
further study should be carried out in analysing the bioactive chemicals that are responsible for the observed in vitro activities.
stenson,
Reference: Fazliana, M., Wan Nazaimoon, W. M., Gu, H. F., O
C.-G., 2009. Labisia pumila extract regulates body weight and adipokines in ovariectomized rats. Maturitas 62, 91-97.
Paper No.: 1393

CNS EFFECTS OF CB2 CANNABINOID RECEPTORS
Emmanuel Onaivi(1,2), H Ishiguro(2,3), Q-R Liu(2), J-P Gong(2),
P Tagliaferro(4), A Brusco(4), G Uhl(2)
(1) William Paterson University, Department of Biology, Wayne, NJ,
USA
(2) IRP-NIDA-NIH, Baltimore, MD, USA
(3) Keda Hospital, Happoukai Medical Corporation, Ryugasaki, Japan
(4) University of Buenos Aires, Buenos Aires, Argentina.
Cannabinoids, endocannabinoids and marijuana use activate two well
characterized cannabinoid receptors (CBRs), CB1-Rs and CB2-Rs. The
expression of CB1-Rs in the brain and peripheral tissues has been well
studied but neuronal CB2-Rs have received much less attention than
CB1-Rs. We used immunoblotting, genotyping, immunoelectron microscopy, mouse behavioral assessment and quantied human and rodent
CB2-R specic isoforms in different tissues and brain regions as well as
in mice treated with CBR ligands. Association studies were performed
between CB2-R SNPs in schizophrenia and depression in human population. We discovered the peripheral and CNS CB2-R subtype specic
expression patterns in human and rodents that resolved the ambiguity
and controversy over the neuronal expression of CB2-Rs that are localized mainly in post-synaptic elements of rat hippocampus and substantia
nigra. Species comparison found that the CB2-R gene of human, rat and
mouse genomes deviated in their gene structures and isoform expression
patterns. Naive BTBR mice that have been reported to have autism-like
behavioral phenotypes have an upregulated high level of CB2A gene
expression in the cerebellum. There is an increased risk of schizophrenia
for people with low CB2 receptor function. Indeed, our studies provide
the rst evidence for the neuronal CNS effects of CB2-Rs and its possible involvement in drug addiction and neuropsychiatric disorders. Thus
the results provides much improved information about CB2 gene structure and its human and rodent variants that should be considered in
developing CB2-R-based therapeutic agents.
Support NIDA-NIH, WPUNJ.
Paper No.: 933

DISEASES
COX IMAGING BY PET WITH 11C-LABELED NONSTEROIDAL
ANTI-INFLAMMATORY DRUGS (NSAIDS)
Hirotaka Onoe, M Shukuri, M Takashima-Hirano, M Goto, T Takashima,
T Keiko, Y Watanabe, H Doi, M Suzuki
RIKEN, Center for Molecular Imaging Science, Functional Probe
Research Laboratory, Koube, Japan
Recently, neuroinammation that play an important role in the host
defense mechanism is known to be involved in various neurological and
neurodegenerative diseases including Alzheimaers disease, Parkinsons
disease, amyotrophic lateral sclerosis, multiple sclerosis, traumatic brain
injury, HIV dementia, and prion diseases. In order to develop the imaging agent for the in vivo monitoring of cyclooxygenase (COX) activity
and/or expression, which is critically involved in a cascade of inammatory process, by positron emission tomography (PET), a series of 2-arylpropionic acid derivatives of nonsteroidal anti-inammatory drugs
(NSAIDs), COX inhibitors, were labeled with short-lived positron emitter (11C; T1/2 = 20.4 min). 11C radiolabeling of 2-arylpropionic acid
derivatives was performed by a novel rapid C-[11C]methylation of the
corresponding enolate intermediate, and accumulation to the inamatory
area in the brain, which was induced by microinjection of LPS into the
striatum of the rat, was examined to estimate the usefulness of labeled
compounds as a COX selective PET imaging probe. As the result, we
507
have succeeded to prepare 11C-radiolabeled NSAIDs such as [11C]Flurbiprofen, [11C]Ketoprofen, [11C]Fenoprofen, [11C]Loxoprofen, as well as
their methyl esters, and have obtained the pharmacokinetic characteristics
of these PET imaging probes in rat with LPS-induced neuroinammation.
Paper No.: 3223

THE ROLE OF ENDOGENOUS ADENOSINE ON MECHANISM
OF CITALOPRAM-INDUCED CARDIOVASCULAR TOXIC
EFFECTS
K Oransay, N Hocaoglu, Mujgan Buyukdeligoz, Y Tuncok, S Kalkan
Dokuz Eylul University School of Medicine, Department of Pharmacology, Izmir, Turkey
This study was designed to clarify the role of endogenous adenosine on
mechanism of citalopraminduced cardiovascular toxic effects. Rats
(n=24) were randomized into three groups. First group received 5% dextrose i.p one hour ago before citalopram infusion (4 mg/kg/min/60 minutes). Other rats pretreated one hour prior to experimental protocol with
EHNA (10 mg/kg i.p, an inhibitor of adenosine deaminase) and NBTI
(1mg/kg i.p, an inhibitor of facilitated adenosine transport). After EHNA/
NBTI administration, Group 2 received 5% dextrose, while Group 3
received citalopram for 60 minutes. Mean arterial pressure (MAP), heart
rate (HR), QRS and QT durations were recorded. Plasma adenosine concentrations were measured by HPLC. Statistical analysis was evaluated
by ANOVA. Plasma adenosine concentrations did not show any signicant change after citalopram (p>0.05). In EHNA/NBTI administered
groups plasma adenosine concentrations showed a signicant increase
(P<0.01). Citalopram caused a signicant decrease in the MAP, HR and
caused a signicant prolongation in the QT and QRS duration (after
10 minutes, P<0.001). In EHNA/NBTI groups, citalopram infusion
caused a signicant decrease in the MAP and HR (after 30 minutes,
P<0.05) and caused a signicant prolongation in the QRS duration (at
50.min, P<0.05). In EHNA/NBTI administered groups, citalopraminduced MAP and HR reductions, QRS and QT prolongations were more
signicant than the dextrose-induced MAP and HR reductions, QRS and
QT prolongations (P<0.05). It can be suggested according to these
results, citalopram in overdose without any increase in the adenosine
plasma concentrations, may cause to cardiovascular toxic effects by
increasing the sensitivity to endogenous adenosine.
Paper No.: 715

COMPARATIVE STUDY OF ADVERSE DRUG REACTIONS
BETWEEN A PUBLIC AND A PRIVATE SPECIALIST HOSPITAL IN LAGOS, SOUTH WEST NIGERIA
three drugs having rates as high as 86% (LUTH) and 91% (HSH). The
effect of age on ADR occurrence was also similar in both hospitals with
most patients being between ages 30-39 years of age. (LUTH, 28%;
HSH, 45%). The class of drugs and severity of reaction were however
different in both settings with LUTH having antiretrovirals (54%) and
HSH having analgesics (30%) while moderate reactions were the most
frequent in LUTH as against severe (58%) in HSH. There is no signicant difference in the patterns of ADRs occurrence between patients in a
public health institution and a private health institution.
Paper No.: 1794

EFFECTS OF IBOGAINE ON RAT UTERUS CONTRACTION
AND ANTIOXIDATIVE ENZYMES ACTIVITY
Zorana Orescanin-Dusic(1), M Slavic(1), R Paskulin(2), P Jamnik(3),
S Milovanovic(4), M Spasic(1), D Blagojevic(1)
(1) University of Belgrade, Institute for biological research Sinisa
Stankovic, Belgrade, Republic of Serbia
(2) OMI Institute, Ljubljana, Slovenia
(3) University of Ljubljana, Biotechnical Faculty, Slovenia
(4) University of Eastern Sarajevo, Faculty of Medicine, Foca, Bosnia
and Herzegovina
Ibogaine has been extensively studied in relation to its anti-addictive
properties that have been reported as being addiction interruptive and
craving eliminative. Ibogaine binds to several types of receptors, but
recent studies demonstrated that induction of energy related enzymes in
cells after ibogaine treatment was not mediated by binding to receptors.
Here we studied the effect of therapeutic doses of ibogaine (20 mg/kg)
on isolated rat uterus contraction and the activity of antioxidant enzymes:
Copper-Zinc superoxide dismutase (CuZnSOD), manganese superoxide
dismutase (MnSOD), catalase (CAT), glutathione peroxidase (GSH-Px)
and glutathione reductase (GR). Ibogaine slightly increased strength of
contraction of two hours spontaneous active rat uterus. However, there
were no changes in contractility in Ca-induced rat uteri for the same period and treatment. In spontaneous active uterus, ibogaine treatment elevated the activities of CAT and GR. Ibogaine increased the activities of
the all measured antioxidant enzymes in Ca-induced active uterus. Our
results suggest that ibogaine through the induction of energy metabolism
related enzymes and redox mediated processes induce cellular antioxidative defense, but the extension of this induction is different in spontaneous and Ca-induced active uterus. In spontaneous active uteri, ibogainemediated increased strength of contraction suggests subtle metabolic differences of individual ROS.
Paper No.: 3107

IN VIVO EVALUATION OF THE ANTITUMOR ACTIVITY OF
DAPHNETIN IN MURINE MELANOMA MODEL
Ibrahim Oreagba, I Ishola, A Akinyandenu

University of Lagos, Department of Pharmacology, Lagos, Nigeria
This study was carried out to compare the prole of Adverse Drug Reactions between a Private Tertiary Health Institution and a Public Tertiary
Health Institution and to determine the role of concomitant medicines in
predisposing a patient to ADRs. A retrospective study was carried out.
Medical records between 2002- 2009 were examined for ADRs. Out of
3,300 medical records examined, 7% had experienced an ADR. Female
patients had an incidence rate of 52% while male patients had 48%
showing that ADRs are more prevalent in the female population than in
the male population. The proles of ADRs were comparable between the
two hospitals. The skin (36%) and the central nervous system (26%)
were the most implicated systems in this study. Multiple drug therapy as
a risk factor was similar in both hospitals with patients on more than
Fausto AJ Orozco(1), MJ Garca Mondragon(1), A Maldonado Espinza(1), MA Herrera Henriquez(2), N Mendoza Patino(1), JJ Mandoki
Weitzner(1)
(1) Departamento de Farmacologa, Facutad Medicina, UNAM, Department of Pharmagolgy, Mexico
(2) Departamento de biologa celular y tisular, Mexico
Daphnetin (6, 7-dihydroxycoumarin) is a secondary metabolite in several
plants used in folk medicine for inammatory and allergic diseases. This
coumarin has in vitro pleiotropic actions including free radical scavenger
activity, inhibitory kinase activity, antiproliferative activity, induction of
cell differentiation and apoptosis. On the present work we evaluated the
in vivo antitumor activity of daphnetin in a murine melanoma model.
Male C57BL/6 mice 6-7 weeks of age were injected subcutaneously with
508
100000 B16-F10 murine melanoma cells in order to induce a primary
tumor. The animals were distributed among groups according to a balanced design based on body weight (n = 6 animals per group in each
experiment) and randomly assigned to treatment groups. Daphnetin was
administrated via oral (40 or 80 mg/kg/day). After two weeks, the tumor
growth was estimated and analyzed by Kaplan-Meier graphs. Results and
discussion. Only the pretreatment of 80 mg/kg of daphnetin signicantly
increase the survival time respect to the control group. The mean of tumor
size of de correspondent groups was not different, but inammation was
observed only in the tumors of the control group. Daphnetin has low antitumor activity per se, but it diminished the inammatory process; for this
reason it cut be considered as adjuvant on melanoma treatment.
Supported by PAPIIT/UNAM IN209010
Paper No.: 2710

DISCOVERY
INFLUENCE OF EFAVIRENZ ON THE DISPOSITION AND
LIPID-LOWERING EFFECTS OF EZETIMIBE IN HEALTHY
SUBJECTS
Stefan Oswald(1), H Meyer zu Schwabedissen(1), A Nassif(1), C Modess(1), D Lutjohann(2), Z Desta(3), H Kroemer(1), W Siegmund(1)
(1) University of Greifswald, Department of Clinical Pharmacology, Greifswald, Germany
(2) University of Bonn, Germany
(3) University of Indiana, USA
Hypercholesterolemia frequently occurs in HIV-infected patients treated
with antiretroviral drugs as efavirenz (EFA). These patients may benet
from combination with ezetimibe (EZE). Disposition and lipid-lowering
effects of EZE are dependent on intestinal ABCB1, ABCC2 and
UGT1A1. These genes are known targets of the nuclear receptor CAR,
which was shown to be activated by EFA. This study investigated the
inuence of EFA on disposition and effects of EZE. Steady state pharmacokinetics of EZE (10 mg, po) alone and in the presence of single and
multiple doses of EFA (400 mg, po) was studied in 12 healthy male subjects. Intestinal mRNA and protein content of known CAR targets was
quantied in duodenal biopsies taken before and after EFA treatment (8
d). EZE and EZE glucuronide (GLUC) in serum, urine and feces were
quantied by LC-MS/MS. Intestinal expression of ABCB1, ABCC1,
ABCC2, ABCG2 and UGTs was not affected by chronic EFA treatment.
Single-dose EFA increased AUC(0-6h) (18.58.1 vs. 13.65.5 ngh/ml,
p=0.005) and Cmax (5.52.7 vs. 4.82.7 ng/ml, p=0.049) of EZE but
markedly decreased serum AUC(0-24h) of GLUC (411195 vs.
466223 ngh/ml, p=0.019). Chronic EFA treatment had no effect on
the serum levels of EZE but signicantly decreased the GLUC exposure
(to 325152 ngh/ml, p=0.005) and its renal excretion (406210 vs.
620194 lg, P<0.05). Sterol-lowering effects of EZE were not altered
by EFA. EFA did not induce important intestinal determinants in EZE
pharmacokinetics. The observed interactions between EFA and EZE are
most likely caused by inhibition of intestinal ABCB1 and UGTs.
Paper No.: 616

GENERATION AND CHARACTERIZATION OF CONDITIONAL
HEPARIN-BINDING EGF-LIKE GROWTH FACTOR
KNOCKOUT MICE
Atsushi Oyagi(1), Y Oida(1), K Kakefuda(1), M Shimazawa(1),
N Shioda(2), S Moriguchi(2), K Kitaichi(3), D Nanba(4), Y Furuta(5),
K Fukunaga(2), S Higashiyama(4), H Hara(1)
(1) Gifu Pharmaceutical University, Department of Biofunctional Evaluation &, Molecular Pharmacology, Gifu, Japan
(2) Tohoku University, Graduate School of Pharmaceutical Sciences,

Department of Pharmacology, Tohoku, Japan
(3) Nagasaki International University, Faculty of Pharmaceutical Sciences, Department of Pharmacology, Nagasaki, Japan
(4) Ehime University Graduate School of Medicine, Department of Biochemistry and Molecular Genetics, Japan
(5) The University of Texas, Department of Biochemistry and Molecular
Biology, USA
Introduction: Heparin-binding epidermal growth factor-like growth factor
(HB-EGF), a member of epidermal growth factor (EGF) family, is a
potent mitogenic peptide for various types of cells. In the brain, it serves
as a neurotrophic molecular and plays a signicant role in cell proliferation, neural differentiation, and synaptic plasticity. Recently, neurotrophic
factors and cytokines have been shown to be associated in psychiatric
disorders. However, little is known about the relationship of HB-EGF
and the disorders. Materials/Patients: To investigate the role of HB-EGF
in brain, we created mice in which HB-EGF activity is disrupted specically in ventral forebrain. We performed some kinds of neurobehavioral
tasks and measured monoamine and their metabolites in various region
of the brain. We also analyzed the morphology of pyramidal neurons in
cortical layer III in the prefrontal cortex. Results: These knockout mice
showed (a) behavioral abnormalities similar to those described in psychiatric disorders, which were ameliorated by typical or atypical antipsychotics, (b) altered dopamine and serotonin levels in the brain, (c)
decreases in spine density in neurons of the prefrontal cortex, (d) reductions in the protein levels of the NR1 subunit of the N-methyl-D-aspartate (NMDA) receptor and post-synaptic protein-95 (PSD-95), (e)
decreases in the EGF receptor, and in the calcium/calmodulin-dependent
protein kinase II (CaMK II) signal cascade. Conclusion: These results
suggest the alterations affecting HB-EGF signalling could comprise a
contributing factor in psychiatric disorder.
Paper No.: 2196

CHINESE HERBAL MEDICINE WU LING SAN HAD
ANTI-INFLAMMATORY ACTIVITY IN CRYSTAL INDUCED
NEPHRITIS IN RATS
Yoko Oyama(1), S Tancharoen(2), T Ito(1), T Hashiguchi(1), T Uchimura(1), K Kawahara(1), I Maruyama(1)
(1) Kagoshima University Graduate School of Medical and Dental Sciences, Department of Laboratory and Vascular Medicine, Kagoshima,
Japan
(2) Mahidol University Faculty of Dentistry, Department of Pharmacology, Japan
Monocyte chemoattractant protein-1 (MCP-1) participates in a wide
range of renal impairments from acute renal failure, obstructive nephropathy, diabetic nephropathy and glomerular damage to chronic failure.
Therefore, interventions favoring the scavenging of this inammatory
cytokine should attenuate the subsequent renal damage. Traditional herbal medicine, Wu Ling San (WLS) is widely used in Asia to coordinate
water balance in patients suffering from nephritis and nephritic syndrome. However, the mode of WLS in the regulation of MCP-1 still
remains unknown. In the previous study, we found that taking WLS in
powdered diet (350mg/kg/day, oral intake;14 days) reduced the increase
of MCP-1, which also mediates initiation and persistent of granulomatous inammation, in both serum and urine in nephritis rat models in
which the deposition of crystals of the abnormal adenine metabolite 2,8dihydroxyadenine (DHA) in the kidney induced granulomatous nephritis.. In the present study, we tried to reveal how WLS decreased MCP-1
release, in vitro. We found that DHA induced MCP-1 release in rat renal
tubular epithelial cells (RTEC). Furthermore, WLS decreased MCP-1
release signicantly in DHA stimulated RTEC. We conclude that DHA
triggers the granulomatous nephritis with inducing the release of MCP-1.
Then WLS decreased the nephritis with suppressing the MCP-1 and it
509
might have an effect to protect renal impairment that would be caused by
excessive inammatory reactions. This conservative treatment with traditional herbal medicine that used for renal disease experientially; WLS
may be a potential alternative novel treatment for various kidney diseases
as well as other granulomatous diseases.
Paper No.: 2068

HEALTH AND DISEASE
THE MECHANISM OF CONTRACTIONS INDUCED BY
ELECTRICAL FIELD STIMULATION OF RAT MESENTERIC
ARTERY IN CALCIUM-FREE SOLUTION
Melike Hacer Ozkan, S Uma
Ankara,Turkey
In rat superior mesenteric artery electrical eld stimulation (EFS)-induced
relaxations turn into contractions upon removal of extracellular calcium.
Our aim was to establish the underlying mechanism of this response to
EFS in precontracted rings of rat mesenteric artery in the presence of
atropine and guanethidine. After precontraction with phenylephrine EFS
(30 V, 300 ms, 5 s, 0.5-8 Hz) was applied to endothelium-intact or denuded arterial rings bathed in calcium-free Krebs-Henseleit solution
(with EGTA). In the absence of calcium EFS elicited frequency-dependent contractions which were blocked by nifedipine or 2-APB, blocker
of inositol triphosphate receptors, but not by tetrodotoxin or indomethacin. Removal of the endothelium did not alter the amplitude of the contractions. U-73122, inhibitor of phospholipase C, signicantly attenuated
the contractile response. These data indicate that EFS-induced contractions in calcium-free solution are not mediated by factor(s) released from
nerves or endothelium. Voltage-gated L-type calcium channels are likely
to be responsible for the enhanced release of calcium from intracellular
stores during the electrical stimulation. A similar mechanism has been
reported to play role in contractions evoked by depolarizing potassium
solutions or drugs which stimulate voltage-gated L-type calcium channels in rat, pig, and human isolated coronary arteries incubated with calcium-free solution (Calderon-Sanchez E. et al., Cardiovasc Res 2009; 82:
115-24). Phospholipase C pathyway may play a crucial role in the signal
transmission between voltage-gated L-type calcium channels and intracellular calcium stores.
All experiments were conducted in accordance with the approval of Animal Ethics Committee of Hacettepe University (2007/7-7).
Paper No.: 566

ANTIOXIDANT EFFECT OF A METHANOL EXTRACT OF
GARCINIA KOLA BIFLAVONOID AGBILU
Jones Chukwudi Ozokwere(1), F Udoh(2), B Lawal(2), E Olivier(1)
(1) Tshwane University of Technology (TUT), Department of Pharmaceutical Sciences, Pretoria, South Africa
(2) University of Calabar (Unical), Calabar, Cross River State, Nigeria
Garcinia kola is used in treatment of various diseases such as diarrhea,
hepatitis, asthma, dysmenorrheal and dysentery. Spasmolytic interaction
between G. kola and oxytoxin in the estrogen primed virgin rat uterus
markedly diminished toxic spasms. Antioxidants such as vitamin C
appear to limit the damage caused by free radicals. The effect of a methanolic extract of G. kola on oxidation was evaluated. A titration method
using 2,6-dichlorophenol-indophenol was used to evaluate the antioxidant properties. The effect of CuSO4, NaCl, cysteine and a methanolic
G. Kola extract was evaluated in the presence of ascorbic acid. The titration values at 20 minutes were 5.95 ml for ascorbic acidalone, 14.81 ml
for NaCl with ascorbic acid, 21.51 ml with cysteine, 2.62 ml with
CuSO4 and 35.09 ml with the G. kola extract in the presence of ascorbic
acid. The G. kola methanolic extract inhibited vitamin C oxidation. It
might, therefore, be considered as exhibiting antioxidant or anti-oxidant
synergist properties.
Keywords: Garcinia kola, antioxidant, extract.
Paper No.: 2067

HEALTH AND DISEASE
EFFECTS OF ATORVASTATIN AND L-ARGININE PRETREATMENTS ON ELECTRICAL FIELD STIMULATION-MEDIATED
RELAXATIONS IN MONOCROTALINE-INDUCED
PULMONARY HYPERTENSIVE RATS
Elif Inci Ozturk, S Uma
Ankara,Turkey
Endothelium-dependent nitric oxide (NO)-mediated vasodilation in pulmonary circulation is impaired in monocrotaline (MCT)-induced pulmonary hypertensive rats. This study aimed to examine the effect of MCTinduced pulmonary hypertension on electrical eld stimulation (EFS)induced relaxation of rat main pulmonary artery and to see whether pretreatment with atorvastatin or L-arginine would prevent the action of
MCT. Male Sprague-Dawley rats were killed 21 days after MCT injection and the main pulmonary arteries were isolated. EFS (40 V, 0.2 ms,
5 s, 10 Hz) was applied to the arterial rings precontracted with phenylephrine (10-6-3x10-6M). Relaxations induced by EFS were abolished in
MCT-injected group. Treatment of the MCT group with atorvastatin
completely, whereas treatment with L-arginine, partially but signicantly
prevented the inhibition. Acetylcholine (10-9-10-5M)-evoked relaxations
that were markedly inhibited in MCT-group were also protected from
inhibition following pretreatment with atorvastatin or L-arginine. However, calcium ionophore ionomycine (10-10-10-7M)-induced relaxations
were unchanged in MCT group. Similarly, responses to endotheliumindependent relaxants sodium nitroprusside (10-9-10-5M), pinacidil (1010-10-4M) and papaverine (10-8-10-4M) were also unaltered in pulmonary hypertensive rats. The present ndings suggest that MCT-induced
pulmonary hypertension inhibits the EFS-mediated non-adrenergic noncholinergic relaxation through suppression of endothelial NO production.
Reversal of this inhibition by atorvastatin treatment presumably results
from stimulation of endothelial nitric oxide synthase expression. Relatively weak protection elicited by L-arginine might be secondary to
depletion of the NO substrate caused by MCT-induced pulmonary hypertension. All experiments were conducted in accordance with the approval
of Animal Ethics Committee of Hacettepe University (2008/59-3).
Paper No.: 565

CONDITIONAL FUNCTIONAL SELECTIVITY OF B2-ADRENOCEPTOR LIGANDS IN STIMULATING ADENYLYL CYCLASE
AND ERK PHOSPHORYLATION IN HEK 293 CELLS
Gulnihal Ozcan, A Kaya, O Onaran, O Ugur
Ankara University Faculty of Medicine, Department of Pharmacology
and Clinical Pharmacology, Ankara, Turkey
Here, we investigated functional selectivity of a set of b2-adrenoceptor
ligands in terms of their ability to activate adenylyl cyclase and induce
ERK (extracellular signal regulated kinase) phosphorylation in HEK cells
expressing human b2-adrenoceptor heterologously. We obtained the following results: 1) Although the efcacy of the tested ligands in stimulat-
510
ing cAMP and ERK phosphorylation responses were generally well correlated, four agonists (namely, terbutalin, clenbuterol, cimaterol and
procaterol) were unable to stimulate ERK phosphorylation despite their
high efcacy in activating adenylylcyclase, 2) ERK phosphorylation
responses to catecholamines (namely adrenalin, isoproterenol and dobutamine) were sensitive to pertussis toxin treatment suggesting the
involvement of Gi/Go in this response, 3) functional selectivity of the
four ligands mentioned above was dependent on the conuence state of
the cell culture: their functional selectivity disappeared as the conuence
of the culture (or cell-to-cell contact) decreased gradually, 4) when the
cell-to-cell contacts were minimized by bringing the cells to the suspension, cAMP itself gained the ability to induce ERK phosphorylation,
which explains the results obtained in point 3 above, 5) consistent with
the latter results, ERK phosphorylation induced by the four outlier agonists in cell suspensions was insensitive to pertussis toxin treatment. These
results suggest the four outlier agonists possess a clear functional selectivity in b2-adrenoceptor-cAMP-ERK system. The latter phenomenon is
observable only when the cell-to-cell contacts are intact, in which case
the cAMP-induced ERK phosphorylation is inhibited. To the best of our
knowledge, this is the rst example of conditional functional selectivity
in a GPCR system.
Paper No.: 2856

INFLIXIMAB PHARMACOKINETICS IN RHEUMATOID
ARTHRITIS IS INFLUENCED BY BODY SURFACE AREA,
ERYTHROCYTE SEDIMENTATION RATE, METHOTREXATE
AND ANTIBODIES TOWARDS INFLIXIMAB
Gilles Paintaud(1), E Ducourau(1), T David(1), C Anca(2),
LG Benot(3), P Aleth(4), D Valerie(5), S-G Elisabeth(6), G Philippe(1),
M Denis(1)
(1) Tours University Hospital, Department of Pharmacology and Toxicology, Tours,France
(2) Orleans Hospital, France
(3) Nantes University Hospital, France
(4) Rennes University Hospital, France
(5) Brest University Hospital, France
(6) Poitiers University Hospital, France
Introduction. Iniximab, a chimeric monoclonal antibody targeting
tumour necrosis factor a, is used in rheumatoid arthritis (RA) but around
1/3 of patients are non responders. Our objective was to describe iniximab pharmacokinetics (PK) in RA and to quantify the inuence of its
sources of interindividual variability. Materials/Patients. Forty-three RA
patients treated with iniximab for at least 14 weeks were studied. They
had a stable dose of iniximab, prednisone and methotrexate (MTX)
since the last infusion. Iniximab concentrations were measured by
enzyme-linked immunosorbent assay before the infusion, 2 hours,
1 week, 4 weeks after the infusion and immediately before the next infusion and analysed by population approach. Inuences of sex, body surface area (BSA), age, cotreatment, disease activity score on 28 joints
(DAS 28), erythrocyte sedimentation rate (ESR), and presence of antibodies toward iniximab (ATI) were studied. Results. A conventional
two-compartment model allowed a satisfactory description of iniximab
PK. Population estimates were: clearance 0.01 L/h, volume of the central
compartment 2.1 L, intercompartment clearance 0.02 L/h, and volume of
the peripheral compartment 1.7 L. Volume of distribution of the central
compartment increased with BSA. Clearance was lower in presence of
MTX and higher in the presence of ATI and increased with ESR. Mean
elimination half-life was 9 days without MTX, 12 days with MTX and
2.6 days with MTX and ATI. Conclusion. For a given dose, iniximab
concentrations were higher during MTX cotreatment and lower in the
presence of ATI. Exposure to iniximab decreased when BSA and ESR
increased.
Paper No.: 1936

DISEASES
THE EFFECT OF TERPENES ON SKIN ABSORPTION OF
TIAPROFENIC ACID GEL EX VIVO AND IN VIVO IN RATS
Zeliha Pala Kara, A Okyar, M Nuriyev, A Yyldyz, N Ozturk
Istanbul University, Faculty of Pharmacy, Department of Pharmacology,
Istanbul, Turkey
Introduction: Non-steroidal antiinammatory drugs (NSAIDs) have been
frequently prescribed drug groups and are employed for rheumatoid
arthritis and osteoarthritis. In systemic administration tiaprofenic acid
(TPA), a NSAI drug, causes gastrointestinal side effects e.g., gastrointestinal bleeding. In order to eliminate these side effects and obtain high
drug concentration at the application side, percutaneus application of
TPA seems to be an ideal route for administration. Materials and Methods: In this study, gel formulation of TPA(%1) was prepared with Carbopol 940 and the inuence of different type terpenes (d-limonene,
menthol and nerolidol) was also investigated using Franz diffusion cells
by using excised abdominal rat skin. The enhancement effect of terpenes
on absorption of TPA was evaluated by in vivo in rats. Plasma samples
were removed from 1 to 48 hours and area under the curve (AUC0-48h)
was calculated. Results and Conclusion: Amongst the used terpenes in
the gel formulations, d-limonene was found to be most effective penetration enhancer for TPA in ex vivo studies. The calculated ux value is
1.42 0.16 and 0.230.04 for d-limonene and control group, respectively
(P<0.05). In vivo penetration study shows that the AUC0-48h was
increased by about 10 fold by the addition of 5% d-limonene to the formulation. Since TPA creates gastrointestinal disturbances, topical formulations of TPA in gel form including %5 d-limonenecould be suggested
as an alternative.
Paper No.: 2899

COMPARISON OF THE EFFECTS OF ADTX1 AND
TAMSULOSIN ON THE INCREASE IN INTRA-URETHRAL
AND ARTERIAL PRESSURES INDUCED BY PHENYLEPHRINE
IN ANESTHETIZED MALE RATS
Stefano Palea(1), N Gilles(2), V Guilloteau(1), M Guerard(1), P Lluel(1)
(1) UPS Faculte des Sciences Pharmaceutiques, UROsphere, Toulouse,
France
(2) CEA, Service dIngenierie Moleculaire des Proteines, Gif sur Yvette,
France
AdTx1 is a new peptidic selective a1A-adrenoceptor antagonist from
green mamba venoms. This study aimed to compare the effects of tamsulosin and AdTx1 on phenylephrine (PHE)-induced intra-urethral (IUP)
and arterial pressure (AP) increases in anesthetized male rats. Under
anesthesia, catheters were inserted into the jugular vein, carotid artery
and in the urethra for drug administrations, AP and IUP recordings
respectively. Vehicle, AdTx1 and tamsulosin were intravenously administered 30min, 1h and 1h30 before PHE-induced increases in IUP and AP.
Increasing doses of PHE (10 to 3000lg/kg) were intravenously administered every 3 min. In each animal and for each PHE dose, dIUP and
dAP were calculated. Statistical analysis was performed using one-way
Anova followed by Newman-Keuls test. When administered 30 min
before PHE, AdTx1 (0.3 and 1mg/kg) and tamsulosin (0.1mg/kg) signicantly decreased the effects of PHE at 150 and 300lg/kg on IUP. On AP,
AdTx1 (0.3mg/kg) reduced the effect of PHE at 10lg/kg only, whereas
tamsulosin signicantly reduced AP for PHE doses between 10 to
150lg/kg. AdTx1 (0.3mg/kg) administered 1h before PHE reduced also
the increase in IUP. When administered 1h30 before PHE, AdTx1 and
511
tamsulosin did not reduced the effects of PHE on IUP. In conclusion,
AdTx1 by intravenous route is able to antagonize a1-adrenoceptors
located in urethra. Importantly, AdTx1 (0.3mg/kg) exhibited a relevant
effect on IUP and a small effect on AP. In contrast, tamsulosin reduced
both PHE-induced increases in IUP and AP. In conclusion, AdTx1 could
be a good candidate for the treatment of BPH.
Paper No.: 1753

MODULATION OF COGNITION AND NEURONAL FUNCTION
IN THE AGE-ACCELERATED MOUSE (SAMP8) BY PTEROSTILBENE AND RESVERATROL
Merce Pallas(1), J Chang(2,3), K Bryan(3), J Reeves(3), MA Smith(4),
A Camins(1), A Rimando(5), B Shukitt-Hale(6), J Joseph(6), G Casadesus(3)
(1) University of Barcelona, Department of Pharmacology and Medical
(2) Case Western Reserve University, Department of Biochemistry,
Cleveland, OH, USA
(3) Case Western Reserve University, Department of Neuroscience,
Cleveland, OH, USA
(4) Case Western Reserve University, Department of Pathology, Cleveland, OH, USA
(5) United States Department of Agriculture (USDA), Natural Products
Utilization Research, Oxford, MS, USA
(6) United States Department of Agriculture (USDA), Agriculture
Research Service, Tufts University, Human Nutrition Research Center on
Ageing, Boston. MA, USA
To date there are no mouse lines that model late onset/age-related Akzheimers Disease (AD), the feature which accounts for the vast majority
of AD cases. The senescence-accelerated mouse (SAMP8), a model of
aging, displays many features that are known to occur early in the pathogenesis of AD. Recent studies have implicated resveratrol and pterostilbene in the protection against age-related diseases, such as cancer,
diabetes, and neurodegenerative diseases in addition to age-related conditions such as cognitive decline, a risk factor for AD. The purpose of this
study was to compare the effectiveness of resveratrol to that of pterostilbene when given in achievable dietary doses to reverse cognitive decits
and the high oxidative stress prole observed in SAMP8. Two months of
diet signicantly improved memory function in the diet-fed SAMP8
compared to control-fed animals. Importantly pterostilbene but not resveratrol achieved signicance in the radial arm water maze test (P<0.05).
In addition, markers associated with the aging process and oxidative
stress were also modulated by pterostilbene. As such, while both resveratrol and pterostilbene were able to increase Sirt1 expression only pterostilbene improved free radical protection mechanism (MnSOD)
(P<0.01). Our results indicate that at identical doses, pterostilbene is a
more powerful regulator of cognitive function and free radical protection
in the SAMP8 mouse. Because in pterostilbene there is an increase in
lipophilicity, we suggest that the better response in cognition and oxidative stress may be associated with better bioavalability of pterostilbene.
These studies have been supported by Alzheimer Association (NIRG07-595) and SAF2009-13093.
Paper No.: 2279

DESCRIPTION OF A NEW METHOD OF PRIMARY CULTURE
OF BOVINE RETINAL GANGLION CELLS
The retinal ganglion cells (RGC) are the primary cell type injured in a
variety of diseases of the optic nerve, including glaucoma and optic neuritis. In order to study cellular and/or molecular pathways culminating in
the death of RGCs, it is essential to establish the culture of RGC. Different methods have been described in rat retinal ganglion cells in order to
increase survival time in culture, increase the number of RGC in a neural
retina culture or extensive neurite outgrowth (Barres BA and Chun LLY.
Neuroprotocols. 1993. Vol 2: 201-204.; Shoge K et al. Neuroscience Letters. 1999. Vol 259: 111-114). We have developed a new technique to
culture retinal ganglion cells from bovine retinas. In contrast to other
techniques, this is a direct and less aggressive method. We have obtained
a stable culture that allows the survival of the cells in culture for 15 days
and extensive neuritis outgrowth. This cell culture system may be used
for future studies of survival or degeneration of bovine RGC and the
effect of different drugs, under normal culture conditions and under
adverse conditions, such as those that mimic glaucoma o neuronal degeneration.
Paper No.: 3422

PROBLEM-BASEDLEARNING IN PHARMACOLOGY
SUBJECTS: PRONS AND SOME CONS
Mercedes Palmero, MC Garca-Cabanes, J Formigos-Bolea, V Maneu
Problem-basedlearning (PBL) is a learning model focused in the students, which self-conduct their learning while working in groups and discuss about a given problem. This system can help them to acquire a
long-lasting knowledge and has been widely used in Medical disciplines
[1,2]. In the Pharmacology Area of the University of Alicante we have
been implementing PBL as a learning model in Pharmacology subjects
for 3 years. We have applied this method in some/all of the practical
and/or theoretical classes of the subjects: Care of patients with chronic
pain, Pharmacovigilance and Pathways in Drugs Administration
(Nursing Degree) and Pharmacology and Metabolic Alterations
(Human Nutrition and Dietetics Degree). When the students were asked
to evaluate the PBL sessions, they had always shown a good attitude
towards this learning methodology. Our evaluation after this time is satisfactory, although we nd some extra effort must be made to improve
some logistic aspects. When PBL was applied in groups with small number of students, sessions could be conducted in the General Library of
the University, with plenty of resources, librarys bibliography and Internet. But with larger groups of students, troubles with tables disposition
or bibliography resources were some of the aspects that could not be
properly solved. [1] Banos JE. Methods Find Exp Clin Pharmacol. 2006;
28: 57-58. [2] Branda LA. DPM 2009; vol. 2 no 4: 15-21.
Paper No.: 1570

QUALITY OF LIFE OF ARTHRITIS RHEUMATOLOGY
PATIENTS EVALUATION
PCR Palmiere, WA Souza, OMGC Vilas-Boas, EB Ferreira, Marcia
HMC Podesta
Mercedes Palmero, MA Company, V Maneu, JA Formigos
Federal University of Alfenas, Department of Pharmacy, Alfenas, Minas

Gerais, Brazil
University of Alicante Faculty of Science, Department of Optic, Pharmacology & Anatomy, San Vicente del Raspeig, Alicante, Spain
The arthritis rheumatoid (AR) is a chronic disease witch needs medicines

doses to control pain and synovial inammation. The AR can evolutes
with joint deformities and functional incapacity, leading alterations in
512
physical and emotional aspects since it interrupts daily activities of the
patient. To measure the Quality of Life of the patients in medicine treatment of AR, we used the Arthritis Impact Measurement Scales (Brandao,
CM et al, Journal of Rheumatology, 1998; 25: 1499-1501). The 16 patients
from both genders were interviewed in the city of Alfenas, Brazil. Some relevant questions were selected and established crossing of interest and evaluated the independence between variables with the x2, followed by Fishers
test (p < 0,05). About the pain severity, 75% of the patients considered it as a
strong pain, 69% feel it every day and 19% feel constantly depressed. However, the intensity and frequency of the pain dont interfere on the frequency
that the patient goes out of home or with the depressive cases. We concluded that the patients, despite the medicine use, feel hard pain every
day but can still practice all the daily activities.
Paper No.: 429

EFFECT OF TOPIRAMATE ON TUMOR ANGIOGENESIS AND
SERUM PROTEOME OF MICE BEARING LEWIS LUNG
CARCINOMA
conditions such as schizophrenia, Huntingtons disease and Parkinsons

disease. In vitro studies have shown that ACR16 was mainly metabolised
to the inactive metabolite ACR30 by the hepatic CYP2D6 enzyme. A
clinical phase 1 study showed that renal clearance was around 50% of
plasma clearance. Thus, other drugs metabolised by CYP2D6 may inuence the metabolism of ACR16. To further investigate the impact of
CYP2D6 activity we performed a pharmacokinetic single dose study to
evaluate the elimination of ACR16 in extensive and poor metabolisers of
debrisoquine (CYP2D6). Methods: Six poor and six extensive metabolisers of debrisoquine were included in the study. Twenty-ve mg of
ACR16 was given to poor metabolisers and 50 mg to extensive metabolisers. Serum samples were taken 0-48 hours after dose and also
fractionated urine samples. Cmax, AUCt, t1/2, AUC ratio, urine recovery and renal clearance were calculated. Results: t1/2, AUC0-t, urinary
excretion and plasma clearance differed signicantly between the two
groups, but Cmax and Tmax did not. Mean plasma clearance of ACR16
was 542259 ml/min in extensive metabolisers and 13729 mL/min in
poor metabolisers. Mean renal clearance was about 100 mL/min in
both groups. Conclusion: The metabolism of ACR16 is mainly catalysed by CYP2D6. The clinical signicance of this needs further investigations.
Yan Pan, B Ma, X Yang, H-M Yu, X-J Li

Department of Pharmacology, School of Basic Medical Sciences and
State Key Laboratory of Natural and Biomimetic Drugs,Peking University, Beijing, PR China
Topiramate has been used in epilepsy patients with brain tumor. In our
previous research we found topiramate could inhibit tumor growth and
metastasis of Lewis lung carcinoma in C57/BL/6 mice. At present, we
conrmed the anti-tumor and anti-angiogenesis activity of topiramate on
Lewis lung carcinoma by detecting the expression of VEGF in tumor tissue by immunohistochemistry and primary angiogenesis by chicken
embryo chorioallantoic membrane angiogenesis experiment. Then by
using two-dimensional electrophoresis (2-DE) combined with matrix
assisted laser desorption ionization time of ight mass spectrometry
(MALDI-TOF-MS), we analyzed and compared the protein prole of
neoplasm serum before and after treated with topiramate (50 mg/ kg/ d,
for 21 days, p.o.) to try to discover its possible targets. The results
showed that the topiramate could dramatically reduce the primary tumor
growth (P < 0.05) and damage of lung alveolar caused by metastasic
tumor deposits. The 2-DE and software analysis results revealed 401 protein spots in control gel and 396 protein spots in topiramate treated
(60 mg.kg-1.d-1, 20 d) gel. In which, four obviously down regulated
protein spots after topiramate treatment were identied: Tropomyosin,
Osteopontin, Transthyretin, Serum amyloid A-1. Then we reconrmed
the expression of osteopontin and its receptor integrin v 3 by western
blotting method in tumor tissue. The results suggest that topiramate has
an anti-tumor and anti-metastasis effect on Lewis-lung-carcinoma. The
mechanism may be related to its inhibition on angiogenesis by down-regulation of osteopontin and VEGF.
Paper No.: 2408

THE METABOLISM OF THE NOVEL DOPAMINE STABILISER
ACR16 (PRIDOPIDINE)IS MAINLY MEDIATED BY CYP2D6
Georgios Panagiotidis(1), A Helldén(1), J Tedroff(2),
N Waters(2), S Waters(2), L Bertilsson(1)
(1) Karolinska Institutet, Department of Laboratory Medicine, Stockholm, Sweden
(2) Carlson Research AB, Gothenburg, Sweden
Paper No.: 3404

ALISKIREN REDUCES ALBUMINURIA AND AFFECTS RENAL
TNF-A AND BCL-2 EXPRESSION IN A MODEL OF DIABETIC
NEPHROPATHY IN RATS
P Panagopoulos(1), Stergios Tsartsalis(1), T Georgopoulou-Karanikola(2), C Tomos(1), T Mavrakanas(1), M Mironidou-Tzouveleki(1)
(1) Aristotle University of Thessaloniki, School of Medicine, A Laboratory of Pharmacology, Thessaloniki,Greece
(2) Aristotle University of Thessaloniki, School of Dentistry, Department
of Alveolar Surgery, Implantology and Radiology, Thessaloniki, Greece
Introduction: Diabetic nephropathy is one of the most common causes of
end-stage renal disease. Renin-angiotensin system (RAS) plays an important role in the pathogenesis of diabetic nephropathy and hypertension
worsens the prognosis of this disease. Aliskiren is a direct renin inhibitor
that blocks RAS and lowers blood pressure. The aim of this study is to
demonstrate the effects of aliskiren in the kidneys of streptozotocin-diabetic rats. Materials: 15 male Wistar rats were randomised into three
groups: DN, DS and DSA. DS and DSA rats received a single intraperitoneal injection of streptozotocin (55 mg/kg) for the induction of diabetes. DSA rats received aliskiren 30mg/kg/day, incorporated in their chow.
DN rats received no treatment. Eight weeks later urinary albumin excretion was measured and rats were sacriced. RNA was isolated from the
renal cortex and reverse transcription followed. TNF-a and Bcl-2 gene
expression was determined with real-time PCR, with b-actin as internal
control. Results: Urinary albumin excretion was increased in diabetic rats
and was lowered by aliskiren (DN: 17.54.7 mg/day, DS: 58.34.3 mg/
day, DSA: 25.75 mg/day) (P<0.05). Moreover, aliskiren reduced TNFa expression (2.05-fold) (P<0.05) and increased Bcl-2 expression (7.23fold) (P<0.01). Conclusion: Aliskiren mitigates albuminuria, an important marker of the progression of diabetic nephropathy, in diabetic rats.
Furthemore, aliskiren reduces TNF-a expression and increases Bcl-2
expression in the renal cortex of diabetic rats. Thus, this drug attenuates
inammation and reduces apoptosis in the renal cortex of diabetic rats.
These results demonstrate the renoprotective effects of aliskiren in this
model of diabetic nephropathy.
Introduction: ACR16 (Pridopidine) is a dopamine stabiliser and belong

to compounds that can either enhance or counteract dopaminergic effects
in the brain. Dopamine stabilisers like ACR16 are suggested in several
513
Paper No.: 1340
HEALTH AND DISEASE
CIRCULATING MOLECULES LIPOPROTEIN - HDL, LDL AND
VLDL: POSSIBLE RISK FACTOR WITH FUNCTION OF TIME
IN RESTENOSIS?
Snezana Pantovic(1), M Martinovic(2)
(1) University of Montenegro Medical Faculty, Department of Clinical
Biochemistry, Podgorica, Montenegro, Republic of Serbia
(2) University of Montenegro Medical Faculty, Department of Pathophysiology, Podgorica, Montenegro, Republic of Serbia
Background: Reastenosis is a serious clinical problem caused by a complex ekstracellular and intracellular events.In the fasting state, most of
circulating triglycerides lokalize in VLDL may impair endthotelium dependent vasodilatation and activiti inammatory responses in endothelial cells.The structural heterogeneity of HDL particles implies functional
heterogeneity, and many of these functions can contribute to protection
against atherosclerosis. Methods and Results: Patients who underwent revascularisation by PCI were analyzed,divided in 2 groups: group 1- without restenosis; group 2 - with restenosis, six month after PCI. Assessed
the value of biochemical lipoproteins parameters - from blood samples
taken at specied time intervals (6 intervals) after PCI. From studies have
excluded patients with hepatic or renal insufency, mental illness, the
existence of metabolic syndrome and women in over menopause. LDL
lipoprotein values ranged from 2.78 - 3.99 mmol / L, VLDL from 0.71 0.91 mmol / L while not found a statistically signicant increase in lipoprotein concentrations measured within the group 1 and 2 HDL measured values ranged from 0.71 - 1:13 mmol / L, and has not found
statistically amended value in any group for this parameter. Conclusion:
Our results are to conrm that lipids and their products have an inuence
on intracellular and extracellular events in the genesis of atherosclerosis
and restenosis makes them diagnostic biomarkers of risk factors.
Paper No.: 3318

HEALTH AND DISEASE
THE GROWTH FACTOR PLEIOTROPHIN INDUCES MIGRATION OF ENDOTHELIAL AND TUMOUR CELLS THAT
IA
3 INTEGRIN AND NUCLEOLIN ON THEIR
EXPRESS A
CELL SURFACE
ence of at least three different cell surface receptors that form a functional complex in order to transduce PTNs signal for migration..
Paper No.: 2455

HISTOLOGICAL STUDY OF BONE HEALING IN RATS AFTER
TREATMENT WITH PARECOXIB
Paraskevi Papaioannidou(1), P Akritopoulos(1), M Dermentzopoulou(1),
H Hatzokos(1), E Iosidou(1), M Kotoula(1), V Mirtsou(1)
(1) Aristotle University of Thessaloniki, Medical Faculty, Department of
Pharmacology, Thessaloniki, Greece
Cyclo-oxygenase inhibitors (COX inhibitors) impair bone healing by
inhibiting prostaglandin synthesis, angiogenesis and bone formation (16). The purpose of this study was to study histologically the effect of
parecoxib, a selective COX inhibitor, on bone healing in rats. Closed
non-displaced mid-diaphyseal fractures of the left femoral shaft were
generated in each animal. In the study group, parecoxib was administered
intra-peritoneally every day for seven days, immediately after fracture. In
the control group, normal saline was administered intraperitoneally every
day for seven days, immediately after fracture. The dose of parecoxib
was selected to be sufcient enough to produce inhibition of COX, while
it was quite close to doses used in clinical practice: slightly higher than
the average recommended therapeutic dose in humans, but less than twofold the highest recommended dose. In both groups bone healing was
evaluated histologically 28 days after fracture. In both groups we
observed osteoblasts, accumulation of chondrocytes, initiation of neoangiogenesis and Haversian canals. In some sections we observed chondrocyte residues, ossication of cartilage tissue and change into osseous
tissue with the presence of osseous cells in both groups.
References : 1. Akritopoulos P et al. Aristotle University Medical Journal 2007; 34:S113
2. Akritopoulos P et al. Helliniki Iatriki 2008; 74:S35
3. Akritopoulos P et al. Arch Orthop Trauma Surg 2009; 129:1427-1432
4. Akritopoulos P et al. Bas Clinic Pharmacol Toxicol 2009; 105:89-90
5. Akritopoulos P et al. Aristotle University Medical Journal 2009;
36:S29
6. Akritopoulos P et al. Epitheorese Klinikes Farmakologias kai Farmakokinetikes 2009; 27:12-14
Evangelia Papadimitriou, M Koutsioumpa, C Mikelis

University of Patras, Laboratory of Molecular Pharmacology, Department
of Pharmacy, Patras, Greece
Pleiotrophin (PTN) is a heparin binding growth factor with diverse biological activities, among which a direct role on tumor growth and angiogenesis. It has been shown that PTN acts on tumor cells through its
receptors anaplastic lymphoma kinase and receptor protein tyrosine phosphatase b/f (RPTPb/f). We have previously shown that RPTPb/f is
expressed on human endothelial cells and that PTN induces their migration through RPTPb/f. Similarly, RPTPb/f mediates PTN-induced migration of tumor cells. We found that RPTPb/f forms a functional complex
with amb3 integrin on the cell surface and that amb3 is required for PTNinduced migration of endothelial and tumor cells. After inhibition or in
the absence of amb3, PTN inhibits cell migration. Interestingly, expression of amb3 on the cell plasma membrane results in cell surface expression of nucleolin, a multifunctional protein that is increased on the
surface of angiogenic endothelial cells, as well as tumor cells and binds a
variety of ligands that play critical role(s) in tumorigenesis and angiogenesis. Nucleolin interacts with both amb3 an RPTPbf, and is also required
for PTN-induced cell migration. Pharmacological blockage of cell surface nucleolin or down-regulation of its expression by siRNA results in
abolishment of PTN-induced cell migration. These data suggest that
PTN-induced migration of endothelial and tumor cells requires the pres-
Paper No.: 2910

3,4-METHYLENEDIOXYMETHAMPHETAMINE (MDMA,
ECSTASY) IN HUMANS: GENDER DIFFERENCES IN
PHARMACODYNAMICS AND PHARMACOKINETICS
Ricardo Pardo(1), M Farre(1), S Yubero(2), B OMahony(2),
M Torrens(3), C Mustata(1), C Perez-Mna(1), E Menoyo(4), M Perez(4),
S Martin(4), R de la Torre(2)
(1) IMIM-Hospital del Mar, UAB, Department of Human Pharmacology
and Neurosciences,Barcelona, Spain
(2) IMIM-Hospital del Mar, UPF, Barcelona, Spain
(3) Hospital del Mar, UAB, Baecelona, Spain
(4) IMIM-Hospital del Mar, Barcelona, Spain
Introduction: MDMA is the third illegal substance most consumed in
Europe. There are limited data about the pharmacokinetics and acute
pharmacological effects of MDMA in women. In this study we compared
pharmacological effects and concentrations of MDMA and metabolites
between genders. Material/Patients: Healthy recreational users of MDMA
(12 females and 15 males), extensive metabolizers for CYP2D6 (dextro-
514
methorphan probe) were selected. A single oral dose of MDMA
(1,4 mg/kg; mean dose for males and females: 95 and 80 mg, respectively) was administered. Females were administered during follicular
phase. Pharmacodynamic evaluations included physiological parameters
(BP, HR, T, PD) and subjective effects using VAS, ARCI and VESSPA
questionnaires. Blood samples were obtained at different times until
72 hours for determination of MDMA and metabolites with GC/MS.
Results: Both genders presented similar effects on BP and positive and
euphoria-related scales, although women received lower doses of
MDMA. In addition females showed higher effects on HR and T, and in
negative-related symptoms as dizziness, depression/sadness (VAS), sedation (VESSPA, ARCI) and psychotic symptoms (VESSPA). Plasma concentrations of MDMA (Cmax and AUC) were signicantly higher in
men, but female presented signicantly higher concentrations of MDA
(Cmax) than men. Conclusions: Females present higher physiological
and negative effects of MDMA than males with lower plasma concentrations of MDMA. Our results indicate that females could be more sensitive to the effects of MDMA than males.
Acknowledgements. NIDA Grant 5R01BA017987-01, FIS RTA RD06/
0001/1009, 2009 SGR 718, FIS-CAIBER CAI08/01/0024, MICINN
FI09/00355 and. R. Pardo is fellowship Rio Hortega, FIS no. CM08/
00051.
Paper No.: 3338

DISCOVERY
SLC26 TRANSPORTERS ARE REGULATED VIA THE
[CL-]I-SENSITIVE STE20P KINASES
Hyun Woo Park, JH Nam, MG Lee
Yonsei University College of Medicine, Brain Korea 21 Project for Medical Sciences, Department of Pharmacology, Seoul, South Korea
WNK1 and SPAK/OSR1 are recently identied serine-threonine protein
kinases that are known to be the Cl- sensor. SLC26 transporters are multifunctional anion exchangers that include the Cl-/HCO3- exchange activity. Transporters that interact with SPAK/OSR1 kinases, the SLC12
transporters, have a specic interaction motif, [RFxV/I]. In addition we
newly found that two members of the SLC26 transporters, SLC26A3
and SLC26A6, also have the SPAK/OSR1 binding motif. In the present
study, we investigated the regulation of SLC26 transporters by WNK1SPAK/OSR1 signal pathway. 293T cells were used for transient expression. For intracellular pH measurement, BCECF uorescence dye were
loaded and 490/440 ratio was measured. Activation of WNK1 and
SPAK/OSR1 were induced by incubating cells in Cl--free buffer. The
Cl-/HCO3- exchange activity of SLC26A3 and SLC26A6 was inhibited
by the WNK1- SPAK/OSR1 signal pathway. However, SLC26A4 which
does not have the binding motif was not negatively regulated by the signal pathway. These ndings demonstrate that SLC26A3 and SLC26A6
are dynamically regulated by the WNK1- SPAK/OSR1signal pathway
via the binding motif.
Paper No.: 3371

ACETYLATION OF P53 BY HISTONE DEACETYLASE
INHIBITORS SENSITIZES LUNG CANCER CELLS TO
IONIZING RADIATION INDUCED CELL DEATH
(2) Kon-Kuk University, Department of Microbiological Engineering,

Seoul, South Korea
Histone deacetylase inhibitors (HDACI) are currently being developed
and tested as anticancer agents and may be useful to increase the therapeutic efcacy of established anticancer treatments. In addition to their
intrinsic anticancer properties, some studies have demonstrated that
HDACIs can modulate cellular responses to ionizing radiation (IR).
However, its action mechanisms are not fully understood. In the present
study, we show evidence that acetylation of p53 plays an important role
in radiosensitization of HDACI, including suberoylanilide hydroxamic
acid (SAHA), trichostatin A (TSA) in human non-small cell lung cancer
cells. Cell viability and clonogenic survivial were markedly decreased in
cells co-treated with IR and HDACIs. Acetyl p53 levels were increased
in cells co-treated with IR and HDACs. siRNA-mediated knockdown of
p53 inhibited cell death induced by co-treated with IR and HDACIs.
Transient overexpression of p53 enhances the cell death induced by IR
and HDACs, whereas overexpression arginine mutation of p53 at lysine
residues did not show the effect of radiosensitization in p53 null human
lung cancer cells. These results indicate that the radiosensitization by
HDACIs is mediated by acetylation of p53 in lung cancer.
Paper No.: 1540

DISCOVERY
MODIFICATION OF EQUILIBRATIVE NUCLEOSIDE
TRANSPORTER 1 (ENT1) FUNCTION BY SULFHYDRYL
REAGENTS INVOLVES CYSTEINE RESIDUES IN
TRANSMEMBRANE REGIONS 6 AND 7
Jamie Park, J Hammond
The University of Western Ontario, Department of Physiology and Pharmacology,London, Ontario, Canada
Equilibrative nucleoside transporter 1 (ENT1) is an eleven transmembrane domain protein involved in cellular uptake of endogenous nucleosides and their cytotoxic analogues. ENT1 function was shown to be
sensitive to sulfhydryl modiers. To establish which cysteine residues
contribute to this sensitivity, we individually mutated each of the 10 cysteines in ENT1 to serines followed by stable expression of recombinant
transporters in PK15 cells. The mutant ENT1 proteins were assessed for
their ability to bind the ENT1-specic inhibitor [3H]nitrobenzylthioinosine (NBMPR) and transport [3H]2-chloroadenosine (2-CADO), treatment
with
the
membrane-permeable
cysteine
modier
methylmethanethiosulfonate (MMTS). Each cysteine mutant, with the
exception of C416S and C439S, were transport-capable. Mutation of
C87 and C414 had no signicant effect on the ENT1 characteristics measured. The C193S mutant had signicantly lower afnity (Kd = 1.18nM)
for NBMPR versus wild-type ENT1 (Kd = 0.43 nM), while the C213S
and C222S mutants had signicantly lower afnities (Km = 85 and 77
uM, respectively) for 2-CADO transport compared with wild-type (Km
= 51 uM). MMTS enhanced NBMPR binding and inhibited 2-CADO
uptake in cells expressing wild-type ENT1. This effect of MMTS on
NBMPR binding was eliminated upon mutation of C193, C213, or
C297, while the effect on 2-CADO uptake was lost in the C297, C333,
or C414 mutants. Results suggest that the regions involved in inhibitor
binding are distinct from, but likely overlap with, the regions mediating
substrate translocation. Cysteines implicated in both ligand binding and
substrate transport include C213 and C297 located in transmembrane
regions 6 and 7 respectively.
In-Chul Park(1), S-K Seo(1,2), H-O Jin(1), T-B Choe(2)

(1) Korea Institute of Radiological and Medical Sciences, Division of
Radiation Cancer Research, Seoul, South Korea
515
Paper No.: 3308
EXCRETION OF THE ANTIPARASITIC MOXIDECTIN INTO
THE BREAST MILK OF LACTATING NON-BREASTFEEDING
WOMEN
Virginia Parks(1), J Korth-Bradley(2), S Chalon(1), I Gourley(2),
K Matschke(2), L Fleckenstein(3), P Bryson(4), S Gossart(1)
(1) Wyeth Pharmaceuticals France, a Company within the Pzer Group,
Paris-La Defense, France
(2) Pzer, Collegeville, PA, United-States
(3) University of Iowa, Iowa City, IA, United States
(4) Veeda, Plymouth, United Kingdom
Moxidectin (MOX) is registered worldwide as a veterinary antiparasitic
agent. It is currently under development for humans in collaboration with
the WHO for the treatment of onchocerciasis. The objective of this openlabel, inpatient/outpatient, single 8-mg-dose study was to assess the
extent of MOX transfer into the breast milk of lactating non-breastfeeding women. Data are presented for 12 women (aged 23-38 years, weight
54-79 kg, all > 6 months post-partum). All subjects provided milk samples for >20 days; plasma samples for 90 days. Noncompartmental pharmacokinetic (PK) methods were used. 0.7010.299 % of MOX dose, or
0.0560.024 mg of MOX (the absolute infant dose) was excreted in
milk. The relative infant dose was 8.733.17 % of the maternal dose.
The ratio of AUC for MOX in breast milk to AUC plasma was
1.770.66. Milk excretion parameters were qualitatively similar to what
has been observed in animals. Plasma PK parameters were similar to
what has been observed in healthy volunteers. The 8-mg single dose of
MOX was well tolerated; there were no serious or severe adverse events
(AEs), and the most commonly reported AEs (headache, nausea, rhinitis,
viral pharyngitis, and viral upper respiratory tract infection) were
reported during the long outpatient phase >90 days. Safety and PK information from young children receiving MOX will allow better understanding of the implications of these data
Paper No.: 1323

HEALTH AND DISEASE
THE EFFECT OF RAPAMYCIN ON ENDOTHELIAL
FUNCTION
Ayse Parlar(1), A Erol(2), C Can(2)
(1) Manisa Mental Health Hospital, Laboratory of Toxicology, Manisa,
Turkey
(2) Ege University Faculty of Medicine, Department of Pharmacology,
Izmir, Turkey
We investigated the potential effects of therapeutic doses of the immunosuppressive agent rapamycin on endothelial function with respect to
nitric oxide (NO) synthesis in rat thoracic aorta in vivo and in rat coronary endothelial cells in vitro. Wistar rats were injected with rapamycin
(1.5 mg/kg/d, i.p) for 14 days. Thoracic aortas were suspended in organ
chambers and were evaluated in terms of endothelium dependent and
independent vascular responses. Rapamycin administration resulted in
increased relaxant responses to L-arginine and to the higher concentrations of calcium ionophore A23187 in the aortas; however, KCl, acetylcholine, sodium nitroprusside, and NG-nitro-L-arginine methyl ester
responses remained unchanged. In addition, phenylephrine-induced contractions signicantly decreased in the aortas regardless of the presence
of functional endothelium. In the in vitro series of experiments, isolated
rat coronary endothelial cells were incubated with therapeutic concentrations of rapamycin (10 nM). Nitrite accumulation in the supernatants
revealed that rapamycin decreased nitrite release induced by interleukin1b; whereas it did not affect basal or stimulated (calcium ionophore,
A23187) nitrite levels. Western blot analysis of inducible nitric oxide
synthase (iNOS) demonstrated that rapamycin decreased iNOS protein

expression in coronary endothelial cells. These data suggest that rapamycin in posttransplant therapeutic concentrations not only preserve vascular endothelial function mediated by NO synthesis, but possibly interacts
in vivo with adrenergic receptors in favor of vasodilatory mechanisms.
Paper No.: 2855

ROLE OF GENETIC FACTORS ON BETWEEN-SUBJECT
VARIABILITY IN CYTARABINE CYTOTOXICITY
Sumit Parmar, A Seeringer, K Pitterle, D Denich, F Gartner,
E Lebedeva, J Kirchheiner
University of Ulm, Institute of Pharmacology of Natural Products and
Clinical Pharmacology, Ulm, Germany
Background: Recent whole genome approaches on cytosine arabinoside
(AraC) cytotoxicity utilized human lymphoblastoid cell lines (LCL) to
evaluate the role of genes within the metabolic pathway of AraC. Since
LCLs might vary from native cells in apoptosis behavior and gene
expression proles, we intended to study variability of AraC toxicity in
an ex-vivo-assay using native peripheral blood mononuclear cells
(PBMCs) from hundred healthy donors. Methods: PBMCs were incubated for 48h with 3lM AraC and double stained for annexin-V-FITC
and propidium iodide to determine cell death via ow cytometry. AraC
induced toxicity (AIT) was calculated as: AIT = 100X ((death cells (treated) [%] - death cells (control) [%]) / (100- death cells (control) [%])).
Human equilibrative nucleoside transporter 1 (hENT1) and cytidine
deaminase (CDA) mRNA-expression was determined by Real-time-PCR
and candidate polymorphisms were selected from literature. Results:
Within-subject variability in AraC induced toxicity was 1.3-fold whereas
between-subject variability was 21-fold. The CDA haplotype *2 and the
hENT1 promoter variant -706 G>C were associated with elevated
mRNA expression levels of the respective gene (P<0.05). Mean AraC
induced toxicity was elevated among individuals carrying CDA *2 compared to wild type (P<0.05). Additionally the T Allele of CDA 435C>T
was related to higher AraC induced toxicity. Further differentiation of *2
haplotype via CDA 435 C>T genotype into *2A and *2B Alleles,
revealed for *2B no signicant effect on mean AIT, whereas *2A
remained related with elevated AIT (P=0.01). Conclusion: Individual
AraC toxicity on native cells shows remarkable variability and is inuenced by CDA haplotypes.
Paper No.: 802

ROLE OF AT1 RECEPTOR ON THE CNS INSULIN
SIGNALLING IN ANIMAL MODEL OF TYPE 2 DIABETES
Mariella Pastorello, A Israel
Central University of Venezuela, School of Pharmacy, Caracas,
Venezuela
Hypothalamic hormonal signals related with nutritional condition, such
as insulin, are required for the maintenance of energy homeostasis. The
activation of the hypothalamic insulin receptor (IR) induces a decrease in
food intake and hepatic glucose release. It is unknown whether these
mechanisms are altered in type 2 diabetes (DM2). Additionally, both
angiotensin-II AT1-receptor and the IR, are co-located in key regions for
the control of energy balance. To assess the possible involvement of AT1
receptor on insulin signalling in the hypothalamus in diabetic condition,
we developed an animal model of DM2 and determined the activation of
key proteins in insulin signalling in rats treated chronically, or not, with
516
valsartan (AT1 antagonist). Sprague-Dawley rats (10020g) were divided
into: Control (standard diet), Control + VAL (standard diet + valsartan
20 mg/kg, po), HFD (High-fat diet) and HFD + VAL. After ten weeks,
groups HFD and HFD+VAL received a single dose of streptozotocin
(35 mg/kg, ip). Two weeks later, the state of insulin resistance was
assessed by the glucose tolerance test. The animals were sacriced by
decapitation and hypothalamus removed. Analysis was made by Western
blot using anti-pIRS/anti-IRS, anti-pAkt/Akt and anti-ERK/ERK as primary antibodies. Our results demonstrated an increase in the phosphorylation of IRS and ERK, without signicant changes in Akt, in animals
with HFD compared with control. These effects were blunted by valsartan treatment. Our results indicate the existence of alterations in insulin
signalling in the hypothalamus of animals with DM2. The AT1-receptor
seems to play an important role in these changes.
Paper No.: 1973

NEPHROTOXIC MEDICATIONS IN ACUTE KIDNEY INJURY:
OPPORTUNITIES FOR LEARNING
Peysh Patel, A Sengupta, O Martin, F Razik, O Johnson
Mid-Yorkshire Hospitals NHS Trust, Pontefract General Inrmary,
Department of Medicine, Pontefract, UK
Introduction: Acute kidney injury (AKI) is a prevalent issue amongst
hospitalised patients. The NCEPOD (National Condential Enquiry into
Patient Outcome and Death) has recently published recommendations on
the management of AKI, including the need for a robust assessment of
contributory risk factors. This would include the omission of nephrotoxic medications, and those likely to worsen complications such as hyperkalaemia and acidosis. Materials/Patients: We performed an audit of
patients with AKI (creatinine > 200) who had presented to the Medical
Assessment Unit at a UK district general hospital. Patients with chronic
kidney disease were only included if an acute deterioration had occurred
in their renal function. Various aspects of presentation and management
were considered, including observation frequency and time to ultrasonography. A qualitative assessment of the identication of contributory factors, such as nephrotoxic medications, was also conducted. Results: Of
the 15 cases analysed, various shortcomings were identied in management. Of particular concern, in 30% of patients, nephrotoxic drugs were
not appropriately discontinued: bumetanide, bendroumethiazide, coamilofruse and lisinopril. In the remainder, however, the drug list was felt
to have been appropriately rationalised on admission. Discussion: This
audit suggests that simple measures to avoid nephrotoxicity may not be
universally considered in patients with AKI. We propose improved education for junior doctors to enhance awareness of the contribution of prescribed medications to renal injury. We also suggest that wider audit may
highlight deciencies in patient management and provide an impetus to
scrutinise pharmacological risk factors more rigorously.
Paper No.: 2872

DISEASES
ANTI-INFLAMMATORY PROPERTIES OF A DUAL PPARC/A
AGONIST MURAGLITAZAR ININ VITRO AND IN VIVO
MODELS
Erja-Leena Paukkeri(1), M Lindholm(1), T Leppanen(1),
PH Aulaskari(2), MF Yam(0), MZ Asmawi(4), E Moilanen(1)
(3) Universitii Putra Malaysia, Faculty of Medicine and Health Science,

Department of Human Anatomy, Serdang, Selangor, Malaysia
(4) Universiti Sains Malaysia, School of Pharmaceutical Sciences, Penang, Malaysia
Peroxisome proliferator-activated receptor (PPAR) agonists are widely
used drugs in the treatment of diabetes and dyslipidemia. In addition to
their metabolic effects, PPAR isoforms PPARa and PPARc are also
involved in the regulation of immune and inammatory responses.
Because the immunoregulatory effects of PPARa and PPARc are partly
different, we hypothetized that a dual PPARc/a agonist would have more
anti-inammatory properties than a PPARc or PPARa agonist alone. In
the present study, we investigated the effects of a dual PPARc/a agonist
muraglitazar on some key parameters of inammation in activated macrophages and on carrageenan-induced inammation in mice. Muraglitazar inhibited lipopolysaccharide (LPS) -induced iNOS expression and
NO production in a dose-dependent manner in J774 macrophages. Muraglitazar and PPARc agonists also reduced the levels of iNOS mRNA
when measured by quantitative RT-PCR, although this effect was not
shared by PPARa agonists. Interestingly, PPARa agonists and muraglitazar were able to reduce iNOS protein levels at post-transcriptional time
points. LPS-induced IL-6 production was also inhibited by PPARa and
PPARc agonists and by muraglitazar, but the agonists had no effects on
COX-2 expression. In addition, TNFa production was reduced by muraglitazar and PPARc agonists, but not by PPARa agonists. Finally, muraglitazar inhibited carrageenan-induced paw inammation in a dosedependent manner in mice as did iNOS inhibitor L-NIL and anti-inammatory steroid dexamethasone. These results show that muraglitazar has
anti-inammatory effects both in vitro and in vivo and these effects
reect the agonistic action through both PPARa and PPARc.
Paper No.: 1993

EARLY POSTNATAL KETAMINE ANESTHESIA CAUSES
PERSISTENT DEFICITS IN RHESUS MONKEY COGNITION
Merle G Paule(1), M Li(1), X Zou(1), C Hotchkiss(2), JP Hanig(3),
TA Patterson(1), W Slikker(1), C Wang(1)
(1) National Center for Toxicological Research USFDA, Division of
Neurotoxicology, Jefferson, AR, USA
(2) Bionetics Corporation, USA
(3) Center for Drug Evaluation and Research USFDA, Jefferson, AR,
USA
Twenty-four hours of ketamine-induced general anesthesia during periods
of rapid brain development in monkeys causes signicant increases in
neuronal cell death. Here, six monkeys were anesthetized at age 5 or
6 days to via intravenous ketamine (light surgical plane) for 24 hrs; six
controls were unexposed. At 7 months of age animals began performing
cognitive function tasks [National Center for Toxicological Research
Operant Test Battery (OTB)] whlich assessed learning, motivation, color
discrimination, and short-term memory. Training scores were assigned
based upon task performance. Beginning around 10 months of age, control animals had higher training scores than ketamine-exposed animals
over about a 6-month period after which the ketamine animals caught
up. Here we report that for animals now over 2 years of age, cognitive
impairments continue to manifest in the ketamine group as poorer performance in learning and visual discrimination tasks, primarily as decits in
task accuracy and speed. Differences in motivation between the 2 groups
may be contributing to these effects.Thus, a single 24-hr neonatal episode of ketamine anesthesia results in very long-lasting decits in brain
function in primates that are not just a result of developmental delay.
Supported by NICHD, CDER/FDA and NCTR/FDA.
(1) University of Tampere Medical School and Tampere University Hospital, The Immunopharmacology Research Group, Tampere, Finland
(2) University of Joensuu, Department of Chemistry, Joensuu, Finland
517
Paper No.: 541
STUDY OF ANTIDEPRESSANT ACTIVITY OF PORTULACA
OLERACEA LINN IN MICE
activity is not a consequence of the cytotoxic activity; but, may be linked

to the antiangiogenic effect.
Supported by FAPEMIG, CNPq and CAPES.
Vinod Pawar(1), S Gujar(1), S Rayate(1), RN Patil(1), S Hugar(2)

(1) SVPMs College of Pharmacy, Malegaon BK, Department of Pharmacology, Baramati, India
(2) SCS College of Pharmacy, Department of Pharmacology, Harapanahalli, Karnataka, India
A large number of herbal drugs have been introduced during the recent
past which can elevate mood in depressive illness. The present study
describes the investigation on the antidepressant effect of Portulaca oleracea Linn leaves in mice using the forced swimming and tail suspension
methods. The methods for animal experimentations were approved by
the Institutional Animal Ethics Committee (Ref No. IAEC 697/2009).
The dose range of test extract was selected as 100,200 and 400 mg/kg
i.p. In the present study imipramine (5 mg/kg i.p.) was used as standard
antidepressant. As an indicator of antidepressant effect, it was shown that
the immobility time of animals in the forced swimming and tail suspension experiment was shorter i.e. the activity of the animals was higher.
With the successive pretreatment of the test extract for 7 days caused
dose dependent and signicant antidepressant effect as indicated by
decrease in immobility time in mice, compared with control group. The
observed result may be due to inuence of N-transferuloyl tyramine,
dopamine and high concentration of noradrenalin constituent present in
it. Both models have proved to be equally valuable for demonstration of
substances with a potential antidepressant activity.
Keywords: Antidepressant, TST, FST, Imipramine
Paper No.: 3249

ANTITUMORAL AND ANTIMETASTATIC ACTIVITIES OF A
PROTEOLYTIC FRACTION FROM CARICA
CANDAMARCENSIS LATEX
Miriam Teresa Paz Lopes(1), D Dittz(1), C Figueiredo(1), CTR Viana(1),
CE Salas(2)
(1) Universidade Federal de Minas Gerais, Department of Pharmacology,
Belo Horizonte, Brazil
(2) Universidade Federal de Minas Gerais, Department of Biochemistry
and Immunology, Belo Horizonte, Brazil
The proteolytic fraction, P1G10, obtained from latex of Carica candamarcensis is rich in cysteine proteinases and shows different pharmacological activities, such as wound cutaneous and gastric healing. Based on
literature data showing that some proteases have antitumor activity and
on preliminary results obtained with P1G10, we evaluated now the mice
antitumor and antimetastatic effects by P1G10 (1 10 mg/kg). P1G10
(5 mg/kg) exhibits antitumor activity, evidenced by ~ 90% decrease in
tumor mass of colon carcinoma (CT26.WT) and melanoma (B16-F1)
models (P<0.001 and P<0.05, respectively, ANOVA Bonferroni test).
It was also observed a survival increase (P<0.01, Logrank test) in B16F1 model. The antimetastatic action was assessed by the survival
increase (p<0.03, Logrank test), decrease in frequency (83%) and number of metastatic points (82%, P<0.05, ANOVA Dunnet test) in the
mice carrying metastatic melanoma (B16-F10). These effects are unlikely
due to cytotoxic activity, since P1G10 displays IC50 of 10-5 g/ml on
tumor or normal cell lines. Furthermore, the fraction exerts antiangiogenic activity on B16F1 tumor mass, as measured by 73% decrease in
hemoglobin content (P<0.01), 55% decrease in VEGF (P<0.01) and
47% increase in NAG activity (0.05). The TNF-a and TGF-b levels are
unaltered (Student t test). We conclude that the antitumoral/antimetastatic
Paper No.: 2488

HIPPOCAMPAL NEUROPLASTICITY PATHWAYS IN AN
ANIMAL MODEL OF DEPRESSION: INFLUENCE OF
CHRONIC FLUOXETINE AND
D-9-TETRAHYDROCANNABINOL ADMINISTRATION
Angel Pazos, F Pilar-Cuillar, V Valdizin, R Madureira, A Diaz,
A Rodriguez-Gaztelumendi, E Castro
University of Cantabria, IBBTEC, Department of Physiology and
Pharmacology,Santander, Spain
Several intracellular pathways involved in cell proliferation in adult hippocampus, including the endocannabinoid (EC) system, are modulated
by antidepressants. To extend our knowledge on the regulation of neuroplasticity by antidepressants and the implication of EC in these
responses, we have evaluated the effects of chronic in vivo exposure to
the SSRI drug uoxetine (F) alone or in combination with d-9- tetrahydrocannabinol (T) on the expression of several neuroplasticity-related
systems (b-catenin, AKT, pCREB and BDNF), in an animal model of
depression. We measured protein levels by western blot in male Sprague
Dawley bulbectomized rats, treated for 21 days with vehicle (saline), T
(10 mg/kg/day, i.p.), F (10 mg/kg/day, p.o.) and T plus F. Olfactory
bulbectomy resulted in a signicant motor hyperactivity (open eld test),
reversed by chronic F. Bulbectomized animals exhibited a clear decrease
(p< 0.05) in the levels of expression of b-catenin, AKT, BDNF and
pCREB. Both F and T treatments produced an increase of hippocampal
b-catenin (F= 15917%, T= 14270%, p<0.05), AKT, pCREB and
BDNF (F= 31897% T= 32990%, p<0.05) levels in bulbectomized
rats, with respect to the untreated group. A signicantly higher effect
(P<0.01) was observed following T+F association. Our results suggest
the existence, in depressive disorders, of a reduced activity of proliferative pathways in the hippocampus, that could be reversed by both F and
T treatments, indicating the possible implication of the EC system in the
therapeutic antidepressant responses.
Supported by Ministerio de Ciencia e Innovacion (SAF07-61862) and
Plan Nacional de Drogas (2006).
Paper No. 2371

DISEASES
METALLIC GOLD IMPROVES CLINICAL OUTCOME,
INDUCES NEUROPROTECTIVE ASTROCYTOSISAND
EVOKES STEM CELL RESPONSE IN AN EAE RAT MODEL OF
MULTIPLE SCLEROSIS
Dan Sonne Pedersen(1), A Larsen(1), PM Fredericia(2),
M Pedersen(2), M Stoltenberg(1), J Rungby(3), M Penkowa(2)
(1) University of Aarhus, Institute of Anatomy, Department of Neurobiology, Aarhus, Denmark
(2) University of Aarhus, Institute of Pharmacology, Aarhus, Denmark
(3) University of Copenhagen Faculty of Health Sciences, Panum Institute, Section of Neuroprotection, Department of Neuroscience and Pharmacology, Copenhagen, Denmark
Multiple sclerosis (MS) is the most common neurodegenerative disease
in the Western world. MS mainly affects younger, healthy individuals
518
and as of today no curative treatment exists. MS patients experience
recurring attacks resulting in demyelination due to an underlying neuroinammation, ultimately leading to loss of neurons. Anti-inammatory
drugs has thus gained increased interest in the treatment of MS. Gold
ions are well know for their anti-inammatory properties but systemic
spread of gold from traditional gold compounds causes side effects like
nephrotoxicity. Alternatively, gold implants can be used as permanent
gold ion donors well suited for the treatment of a chronic inammation.
The present study is the rst to investigate whether metallic gold
implants ameliorates the MS-like disease seen in Experimental Autoimmune Encephalomyelitis (EAE), a rodent model of MS. Metallic gold
particles (20-45 lm) in hyaluronic acid were injected bilaterally in the
lateral ventricles of young Lewis rats prior to EAE induction. Comparing
gold-treated animals to vehicle-treated ones, statistically signicant
improvement of clinical outcome was seen after gold treatment. Histologically we saw statistically signicant up-regulation of GFAP-positive astrocytes in periventricular areas of the lateral and fourth ventricle and an
up-regulation of NSCs migrating from SVZ into the surrounding area.
Furthermore, Metallothionein 1+2 expression was up-regulated. Conclusion: Gold implants improve the clinical outcome, induce astrogliosis
throughout the brain and elicit a NSC response in EAE. Cerebral
immuno-modulation by gold may prove benecial in future treatments of
MS.
Paper No.: 1224

HEALTH AND DISEASE
CYSTAMINE REDUCES BLOOD PRESSURE IN SHR AND
ATTENUATES INWARD EUTROPHIC REMODELLING
IN VITRO
Morten Engholm Pedersen, A Eftekhari, MJ Mulvany
Aarhus University, Institute of Pharmacology, Aarhus, Denmark
Tissue transglutaminase (t-TG) is involved in small artery remodelling
and the organic disulde cystamine (CYS) has been demonstrated to
inhibit t-TG competitively. We aimed to study if inhibition of t-TG could
reduce blood pressure and reverse eutrophic inward remodelling of small
arteries in SHR. Methods: Organ culture and wire-myograph setups were
used to study in vitro inhibition of t-TG, with CYS, in SHR and WKY
small mesenteric arteries. In vivo treatment with CYS (80 mg/kg/day) or
amlodipine (10 mg/kg/day) was performed with osmotic pumps in adult
SHR. Hemodynamic parameters were determined with telemetry (DSI)
and small artery structural and functional characteristics determined. [pCYS] was determined with a liquid chromatography setup. Results:
Small artery lumen diameter was reduced by 7.21.9% in SHR* and
3.40.4 % in WKY**, indicating signicant (*p<0.05, **P<0.01)
inward remodelling following in vitro activation with ET-1. In presence
of CYS, activation caused a 1.20.9 reduction in SHR passive lumen
diameter, compared to 2.51.0 % increase in WKY, indicating signicant
(p<0.05) attenuation of inward remodelling by CYS, with a reduced
potency in SHR. In vivo administration of CYS caused a 62 mmHg
(p<0.05) reduction in blood pressure, with no concomitant alterations in
small artery structure. [p-CYS] was 2.47 0.036 nmol/ml and conrmed
appropriate presence of CYS. Conclusion: CYS reduces blood pressure
in SHR, but does not reverse vascular remodelling after 3 weeks administration. t-TG is involved in inward remodelling of SHR and WKY
small arteries and CYS attenuates remodelling in vitro, with a reduced
potency in hypertensive rats.
Paper No.: 3359

LINKAGE DISEQUILIBRIUM BETWEEN THE RAPID
CYP2C19*17 ALLELE AND WILDTYPE CYP2C8 AND CYP2C9
ALLELES: IDENTIFICATION OF CYP2C HAPLOTYPES IN
HEALTHY NORDIC POPULATIONS
Rasmus S Pedersen(1,2), C Brasch-Andersen(1), SC Sim(2), TK Bergmann(1), J Halling(3), MS Petersen(3), P Weihe(3), A-L BorresenDale(4,5), VN Kristensen(4,5), K Brsen(1), M Ingelman-Sundberg(2)
(1) University of Southern Denmark, Department of Clinical Pharmacology, Odense, Denmark
(2) Karolinska Institute, Section of Pharmacogenetics, Stockholm, Sweden
(3) The Faroese Hospital System, Department of Occupational Medicines
and Public Health, Torshavn, Faroe Islands
(4) Oslo University Hospital, Department of Genetics, Oslo, Norway
(5) University of Oslo, Institute of Clinical Medicine, Oslo, Norway
The distribution of clinically important CYP2C genotypes and allele frequencies in healthy Nordic populations was determined with special
focus on linkage disequilibrium. 896 healthy subjects from three Nordic
populations (Faroese, Danish and Norwegian) were genotyped for ve
frequent and clinically important CYP2C allelic variants: the defective
CYP2C8*3, CYP2C9*2, CYP2C9*3 and CYP2C19*2 alleles, and the
CYP2C19*17 allele that causes rapid drug metabolism. Linkage disequilibrium was evaluated and CYP2C haplotypes were inferred in the entire
population. Ten CYP2C haplotypes were inferred of which the most frequent (49%) was the CYP2C wildtype haplotype carrying CYP2C8*1,
CYP2C9*1 and CYP2C19*1. The second most frequent haplotype (19%)
is composed by CYP2C19*17, CYP2C8*1 and CYP2C9*1. This haplotype accounts for 99.7% of the CYP2C19*17 alleles found in the 896
subjects. CYP2C19*17 is a frequent genetic variant in Nordic populations that exists in strong linkage disequilibrium with wild type
CYP2C8*1 and CYP2C9*1 alleles which effectively makes it a determinant for a haplotype exhibiting efcient CYP2C substrate metabolism.
Paper No.: 2771

THE CYCLIC GMP-INDEPENDENT SELECTIVE INSULIN
SENSITIZING EFFECT OF ORAL ISOSORBIDE
MONONITRATE IN CONSCIOUS RABBITS
Barna Peitl, R Sari, Z Szilvassy
University of Debrecen, Department of Pharmacology and Pharmacotherapy and Pharmapolis Debrecen Ltd, Debrecen, Hungary
Background: We have found that transdermal nitroglycerin induces an
insulin sensitizing effect in healthy volunteers at a dose used for angina
prophylaxis. Aim: We studied the effect of single oral isosorbide mononitrate (ISMN) doses of 1, 2, 4 and 8 mg/kg on insulin sensitivity and global myocardial ischemia induced by rapid ventricular pacing (RP, 500
b.p.m. over 10 minutes) in chronically instrumented conscious rabbits
made insulin resistant (IR) by a preceding 4-week exposure to 1.5% cholesterol-enriched diet. Methods: Insulin sensitivity was measured by
hyperinsulinaemic euglycaemic glucose clamping. Average glucose infusion rate (GIR, mg/kg/min) to mainain euglycaemia (5.5 mmol/l) at
clamped hyperinsulinaemia (100 mikroU/ml) was used to assess insulin
sensitivity. Post-pacing intracavitary ST-segment elevation (mV) and an
increase in left ventricular end-diastolic pressure (LVEDPI, mmHg) were
used to estimate the severity of pacing-induced myocardial ischemia.
Post-ischemic cardiac cyclic GMP level (pmol/mg wet wt) was determined by in situ left ventricular samples from open-chest articially ventilated animals during the last 5 seconds of the 10-min pacing period.
519
Results: The table shows that ISMN attenuated RP-induced changes at
an oral dose of 8 mg/kg, however, a signicant insulin sensitizing effect
appeared in a dose range of 2-8 mg/kg. Conclusion: ISMN applied
orally, produces an insulin sensitizing effect at doses much lower than
those required to produce an anti-ischemic effect against RP-induced
myocardial ischemia in insulin resistant conscious rabbits. This insulin
sensitizing effect seems to be independent of the cyclic GMP system.
Paper No.: 3260

ACUTE AND SUBACUTE TOXICITY OF VIRGIN COCONUT
OIL IN RODENTS
Ryan Pekson(1), I Sia(2)
(1) Adamson University, College of Pharmacy, Manila, The Philippines
(2) University of the Philippines, College of Medicine, Department of
Pharmacology, Manila, The Philippines
This research determined the acute and subacute toxicity of virgin coconut oil (VCO) in Swiss-Webster mice and Sprague-Dawley rats, respectively. The procedures were adopted from the established protocols of
the National Integrated Research Program for Medicinal Plants, of the
Department of Science and Technologyt. In the acute phase, 50 adult
Swiss-Webster mice were block randomized into four VCO treatment
groups (36.7 g/Kg, 14.6 g/Kg, 5.8 g/Kg and 2.3 g/Kg) and control (NSS
40.7g/Kg). Evaluation of toxidromes was done for 14 days using Irwins
multidimensional observation sheet. In the subacute toxicity study, 48
Sprague-Dawley rats were block randomized, three groups received
VCO (6.3 g/Kg, 4.14 g/Kg, and 2.7 g/Kg) and one control (NSS 7 g/
Kg), administered by gavage tube feeding for 21 days. Rats were
observed for toxidromes including hematologic, blood chemistry, urine,
and fecal analyses. Gross pathologic and histopathologic examination of
the liver, heart, kidney, lungs, spleen, thymus, and thoracic aorta were
done on the 0th, 10th, and 21st day. The LD50 of VCO was found
greater than 36.7 g/Kg in mice and the NOAEL at 2.3 g/Kg. Reversible
acute toxidrome consists of oily stool and blood-streaked feces. Subacute
administration of VCO is associated with dose-dependent elevation of
serum triglycerides, BUN, creatinine, AST, and ALT. Fasting blood sugar
(FBS) was reduced at low to medium doses of VCO, while an increase
in FBS was observed at high dose. Histopathological examination
revealed that VCO is potentially hepatotoxic and nephrotoxic at 4.14 g/
Kg and 6.3 g/Kg.
Paper No.: 1077

PROLYL OLIGOPEPTIDASE IS NOT LOCATED IN THE LONG
PROJECTION NEURONS OF THE RAT BRAIN
Iida Peltonen, TT Myohanen, PT Mannisto
Prolyl oligopeptidase (POP; prolyl endopeptidase; EC 3.4.21.26), is an
80-kDa serine protease which cleaves peptides smaller than 30-mer at
the carboxyl side of a proline-residue. POP is widely distributed in the
body and the cytosolic form is abundantly expressed also in the brain.
The substrates of POP include a number of neuropeptides claimed to be
involved in learning, memory and mood. More recently POP has been
colocalized with GABAergic, glutamatergic and cholinergic neurons.
The purpose of this study was to investigate further the neuronal localization of POP by producing lesions to major origin nuclei of acetylcholine (ACh), serotonin (5-HT), noradrenaline (NA) or dopamine (DA)
neurons in the rat brain by neurotoxins and then measuring the expres-
sion and activity of POP in the respective projection areas. The lesioned
nuclei were chosen based on their projections to the areas known to contain POP protein, POP mRNA or high POP activity. The lesioned nuclei
were medial septum (ACh), Meynert nucleus (ACh), dorsal raphe (5-HT)
and locus coeruleus (NA). For dopaminegic neurons, the lesion was
made to the medial forebrain bundle which contains the axons ascending
from substantia nigra. Neurotransmitter lesions were successful since
only minor immunoreactivity of respective neurotransmitters was seen in
the lesioned side. However, they did not affect the expression level of
immunoreactive POP protein or the activity of POP in the corresponding
projecting areas of the lesioned nuclei. Our results suggest that POP is
not present in the long projection neurons but rather in short interneurons.
Paper No.: 1643

L-3-N-BUTYLPHTHALIDE SUPPRESSED
LIPOPOLYSACCHARIDE-INDUCED INFLAMMATION
THROUGH NRF2 DEPENDENT PATHWAY
Ying Peng, Y Hu, L Lu, S Xu, L Wang, X Wang
China
Increased oxidative stress and neuroinammation have been considered
to play primary roles in the pathogenesis of neurodegenerative disease,
such as Alzheimers disease (AD) and Parkinsons disease (PD). Transcription factor Nrf2 is the guardian of redox homeostasis, and activates
a battery of antioxidant and cytoprotective genes. L-3-n-butylphthalide
(L-NBP), an extract from seeds of Apium graveolens Linn (Chinese celery), has been demonstrated to have neuroprotective effects on cerebral
ischemic, vascular dementia and AD animal models. In the current study,
we examined the effect of L-NBP on lipopolysaccharide (LPS)-induced
neuroinammatory reaction in adult C57BL6 mice. IL-1b and TNF-a
levels of plasma were determined by ELISA. Microglia and astrocyte
activation were examined by immunohistochemistry and Real-time PCR.
Nrf2, heme oxygenase-1 (HO-1), NADPH: quinine oxidoreductase-1
(NQO-1) and inducible nitric oxide synthase (iNOS) expressions were
measured by PCR and western-blot. The results showed that L-NBP signicantly reduced LPS-induced cytokine release and glia activation. At
the meantime, the elevation of expression of Nrf2, HO-1 and NQO-1
were observed. L-NBP was found to enhance the elevation of Nrf2, HO1 and NQO-1. The data suggested Nrf2 might be a therapeutic target of
L-NBP against brain inammation. In conclusion, L-NBP may be an
effective agent in modulating neuroinammatory process in neurodegenerative diseases.
Paper No.: 1472

THE CANNABINOID, WIN55,212, REDUCES PACEMAKER
FREQUENCY IN THE GASTRIC ANTRUM OF CONSCIOUS
FERRETS; A POTENTIAL MECHANISM FOR
CANNABINOID-INDUCED GASTROPARESIS
N Percie du Sert(1), Wing Sze Vanessa Ho(1), JA Rudd(2),
PLR Andrews(1)
(1) St Georges University of London, Department of Basic Medical
Sciences, London, UK
(2) The Chinese University of Hong Kong, Hong Kong, PR China
Gastric motility arises from a pacesetter potential termed gastric myoelectric activity (GMA), which is generated by the interstitial cells of Cajal
(ICCs). In rodents, cannabinoids have been shown to inhibit gastrointes-
520
tinal motility but cannabinoid (CB) receptors have not been identied on
the ICCs and the effect of cannabinoids on GMA is unknown. This study
used telemetry to identify the effect of the CB1/CB2 receptor agonist,
WIN 55,212-2, in freely moving, conscious ferrets. Ferrets were surgically implanted with telemetry transmitters; 2 biopotential electrodes
were sutured in the antral serosa. Spectral analysis was carried out on the
GMA recordings to identify the dominant frequency. Following WIN
55,212-2 (1 mg/kg, i.p.) the frequency of the GMA was reduced (8.2
0.4 cpm) compared to vehicle (9.6 0.1 cpm). WIN 55,212-2 also
decreased body temperature by 2.2 0.6 C and the heart rate by 19.4
7.7% (P<0.05, two-way analysis of variance, n=5-6). The results suggest
that, in the ferret, modulation of the ICC network could contribute to the
inhibition of gastric motility commonly associated with cannabinoids.
The mechanism could be concomitant to previously identied mechanisms such as CB1 receptor-mediated inhibition of acetylcholine release
from nerve terminals of the autonomic nervous system. The observed
hypothermia and bradycardia are also consistent with CB1 receptor activation, as reported in rodents. Further investigation is warranted to identify how the effect of cannabinoids on the ICCs is mediated; possible
mechanisms include the release of an inhibitory neurotransmitter or a
direct effect on the ICC via CB receptors.
Paper No.: 1644

DISCOVERY
THE EXPRESSION OF ORGANIC CATION TRANSPORTER
MRNA IN CULTURED ADULT AND NEONATAL RAT
ASTROCYTES AND THEIR POSSIBLE FUNCTIONAL ROLE
Paper No.: 2795

EFFECTS OF VERAPAMIL, OVER MATERNO-FETAL
PHARMACOKINETICS AND TRANSPLACENTAL EXCHANGE
OF IVERMECTIN, IN PREGNANT SHEEP
Ruben Perez, C Palma, MJ Nunez, P Urrutia, L Morales, D Vera, J Cox
University of Concepcion, Faculty of Veterinary Sciences, Chillan, Chile
In pregnant sheep, a study was performed to know the effect of the coadministration of verapamil and ivermectin (IVM) on materno-fetal disposition kinetics of IVM after its IV administration. Ten pregnant Suffolk
Down sheep (63.5 6.6 kg, bw) were surgically prepared to insert catheters into fetal femoral artery and vein. The ewes were randomly assigned
to two experimental groups: Group 1 (control), 5 ewes were treated with
0.2 mg IVM/kg bw, IV. Group 2 (verapamil-IVM), 5 ewes were treated
with 2.5 mg/kg verapamil (3 doses at 12 h intervals) and 0.2 mg/kg
IVM 30 min after verapamil. Maternal and fetal blood samples were
taken before and after IVM administration during 144 h. Samples were
analyzed by HPLC. A non-compartmental pharmacokinetic analysis was
performed and statistical differences were determined using the MannWhitney test. The main maternal pharmacokinetic changes observed in
the group of verapamil/IVM were decreases in the volume of distribution
and in the half-life of elimination (ta). A signicantly faster and higher
(P <0.05) increase in fetal plasma IVM concentration was observed in
the group of fetuses of Group 2. Higher mean values of Cmax (P <0.05)
associated to a decrease in Tmax and the fetal half life of absorption (P
<0.05) were observed in Group 2 compared to IVM alone group. The
results demonstrated that the pharmacological blockage of P-gp with
verapamil can increase the transfer of IVM to fetal circulation.
Supported by Grant FONDECYT 1060896, CONICYT CHILE.
Katja Perdan-Pirkmajer(1), S Pirkmajer(2), Z Grubic(2), M Krzan(1)

(1) University of Ljubljana Faculty of Medicine, Department of Pharmacology and Experimental Toxicology, Ljubljana, Slovenia
(2) University of Ljubljana Faculty of Medicine, Institute of Pathophysiology, Ljubljana, Slovenia
Astrocytes have been shown to play an important role in the inactivation
of various neurotransmitters, including histamine. Organic cation transporters (OCT1, 2 and 3) are often mentioned as possible participants in
histamine transport, but their expression pattern and functional role in astrocytes remain to be elucidated. The goal of our present work was to
establish whether OCTs might participate in histamine uptake in cultured
rat astrocytes. We rst assessed the expression pattern of OCT mRNA
(real-time PCR) in cultured rat astrocytes and rat cortical tissue. We then
assessed the possible functional role of OCTs in histamine transport with
a [3H]-histamine uptake study, using a specic OCT inhibitor Decynium
22. OCTs were expressed both in cultures and cortical tissue. The OCT3
was most prominently expressed in neonatal and adult cortical tissue
samples, but it was barely detectable in cultured neonatal astrocytes. Its
expression levels were more than 20-fold lower than OCT2 in adult
astrocyte cultures. The level of OCT1 mRNA was almost undetectable in
cortical tissue (more than 1000-fold lower than OCT3), but was
expressed at a level comparable to OCT2 in astrocyte cultures. The
OCT2/OCT1 ratio reached 2 in adult astrocytes (N=4, P<0.05) and 30 in
neonatal astrocytes (N=4, P<0.001). Decynium 22 lowered histamine
uptake into neonatal an adult astrocyte cultures, however the observed
effect was statistically signicant only in the adult astrocyte cultures
(P<0.001, ANOVA). Our preliminary results imply that astrocytes not
only express mRNA for OCTs, but are also possible candidates for histamine uptake into adult astrocytes.
Paper No.: 2042

HEALTH AND DISEASE
DUAL CHANGES IN PULMONARY VASCULAR REACTIVITY
IN RATS WITH VENTILATOR-INDUCED LUNG INJURY
Francisco Perez Vizcaino(1), C Menedez(1), L Martinez-Caro(2),
J Moral-Sanz(1), E Moreno(2), JA Lorente(3), N Nin(3), A Esteban(3)
(1) Universidad Complutense, Department of Pharmacology, Madrid,
Spain
(2) Ciber Enfermedades Respiratorias, Spain
(3) Hospital Universitario de Getafe, Spain
Ventilator-induced lung injury (VILI) is characterized by pulmonary
inammation, lung edema and it is associated with systemic vascular
dysfunction and hypoxemia. We aimed to analyze the changes induced
by VILI in ex vivo pulmonary vascular function. Male Sprague-Dawley
rats (325-375 g) were subjected to low (VT=9 mL/kg, PEEP=5 cmH2O)
or high (VILI, VT=25 mL/kg, zero PEEP) mechanical ventilation for
2 h. Pulmonary resistance arteries (PA) were mounted on a myograph to
examine ex-vivo vascular reactivity. Potassium currents were analyzed
by patch-clamp in isolated PA myocytes. Contractile responses to
hypoxia were signicantly enhanced in rats with VILI. Sphingomyelinase, an enzyme involved in hypoxic pulmonary vasoconstriction, produced a similar response in the two groups. The density of Kv currents
and its inhibition by hypoxia was also similar in both groups. The contractile responses to serotonin (5-HT) were similar in both groups. In the
presence of the inhibitor of the inducible NO synthase (iNOS) 1400W,
the contractile responses to 5-HT were strongly increased in rats with
VILI but with minimal effects in the low VT ventilated rats. This effect
was associated with an increased lung iNOS expression in the VILI
group. In conclusion, ventilation-induced lung injury produced dual
effects in pulmonary vascular reactivity, an inhibitory effect via iNOS
induction and a potentiation of the vasoconstrictor response via a yet
521
unidentied mechanism. These results are in agreement with those previously reported for sepsis- or endotoxin- induced lung injury.
Paper No.: 1745

THE THREE A1-AR SUBTYPES SHOW DIFFERENT
SPATIO-TEMPORAL PATTERNS OF ERK1/2
PHOSPHORYLATION AND INTERNALIZATION
Miguel Perez-Aso, V Segura, M-A Noguera, P DOcon
University of Valencia, Department of Pharmacology, Valencia, Spain
Introduction: we studied the spatio-temporal patterns of receptor-internalization and ERK1/2 phosphorylation after the activation of each a1-AR
subtype in transfected-HEK293 cells. Materials: ERK phosphorylation
was determined by Western blot and confocal microscopy was used to
visualize p-ERK cellular distribution and VSV-tagged receptor internalization. Results: activation of a1A- and a1B-AR by phenylephrine elicited
an early ERK phosphorylation directed to the nucleus and inhibited by
the PKC-inhibitor Ro-31-8425. Phenylephrine promoted receptor internalization, which was abolished in a1A-, but not in a1B-AR, by ConcanavalinA (ConA), an inhibitor of b-arrestin-mediated receptor
internalization. Concomitantly to receptor internalization, a1A, but not
a1B-AR, showed late ERK phosphorylation which was restricted to the
cytosol, PKC-independent and blocked by ConA. a1D-AR showed constitutive ERK phosphorylation, which was dependent on receptor internalization as it was reduced by incubation with the selective a1D
antagonist BMY7378 or with ConcanavalinA. After activation by phenylephrine, a1D-AR elicited a fast and transient ERK phosphorylation that
was PKC-independent and restricted to the cytosol. Conclusions: the
three a1-AR subtypes present different spatio-temporal patterns of ERK
phosphorylation and receptor-internalization. a1D-AR constitutively
phosphorylates ERK in a receptor-internalization manner. Only a1A-AR
elicits a late and sustained ERK phosphorylation that is dependent on barrestin-mediated internalization and restricted to the cytosol. This late
ERK activation could relate to the antiapoptotic-cardioprotective effect of
a1A-AR. (Huang Y et al. Circulation 2007; 115:763-772).
Paper No.: 1954

ROLE OF THE DOSE OF ETHANOL ON THE GENERATION
OF ENDOGENOUS HYDROXYTYROSOL DOPET
Clara Perez-Mana(1), M Farre(1), R Pardo(1), C Mustata(1),
E Papaseit(1), A Pastor(1), E Orrtiz(3), E Menoyo(2), M Perez(2),
S Martin(2), R de la Torre
(1) University Auto`noma of Barcelona, IMIM-Hospital del Mar,
Department of Pharmacology and Neurosciences, Barcelona, Spain
(2) IMIM-Hospital del Mar, Barcelona, Spain
(3) IMIM-Hospital del Mar, UPF, Barcelona, Spain
Introduction: Previous studies in animals have shown an increase of hydroxytyrosol (3,4.dihydrohyphenylethanol, DOPET), a potent antioxidant
and a minor metabolite of dopamine, after ethanol intake. The interaction
of ethanol with the metabolism of dopamine to produce hydroxytyrosolDOPET could explain the human benecial health effects of low doses
of ethanol. The aim of this pilot study was to demonstrate an increased
endogenous generation of hydroxytyrosol-DOPET after a controlled
administration of ethanol in humans. Methods/Patients. A group of 18
healthy male volunteers were selected. Subjects were distributed in three
different cohorts (n=6) and each volunteer received two doses of ethanol
or placebo. Doses of ethanol administered were 6, 12, 18, 24, 30 and
42 g. Study design was double-blind, randomized, cross-over and controlled. Hydroxytyrosol concentrations in blood and urine, ethanol con-
centrations in blood and subjective drunkenness were evaluated along a

6 hour period. Urine were collected at different time intervals along
24h. Results. A dose-dependent increase in blood ethanol concentrations
and their subjective effects was observed. Urinary concentrations of hydroxytyrosol-DOPET, analyzed to date, showed an increase related to
ethanol dose (urinary concentration in the group of 18 g increased by
180% compared to placebo and up to 342% compared to placebo in the
group of 30g). Conclusions. This pilot study is the rst evidence in
humans of signicant increase of hydroxytyrosol-DOPET endogenous
production in relation to ethanol dose.
Acknowledgements. FIS PI081913, FIS RTA RD06/0001/1009, 2009
SGR 718, FIS-CAIBER CAI08/01/0024. R. Pardo is recipient of a fellowship Rio Hortega, FIS no. CM08/00051 fellowship.
Paper No.: 2010

DETERMINING TOXICOLOGICAL PARAMETERS OF
ATROPINE/SCOPOLAMINE MIXTURE AS FOOD
CONTAMINANT
L Perharic(1), M Zidaric(2), Lovro Stanovnik(3)
(1) National Institute of Public Health, Department of Toxicology,
Ljubljana,Slovenia
(2) Institute of Public Health, Department for Water, Food and Consumer
Products,Maribor, Slovenia
(3) University of Ljubljana, Faculty of Medicine, Department of Pharmacology andExperimental Toxicology, Ljubljana, Slovenia
Following mass poisoning with Datura alkaloids, present in contaminated
buckwheat our, we performed preliminary risk assessment, based on
clinical doses of atropine and scopolamine. We proposed acute reference
dose (ARfD) of atropine/scopolamine mixture: 0.04/0.03 lg/kg of body
mass. Due to several uncertainties in our risk assessment, we performed
a study in human volunteers to verify our assumptions. We ran a randomised double blind crossover study in 20 healthy male volunteers aged
18-28 years. They ingested a traditional Slovenian dish, zganci, made
of boiled buckwheat our to which 5 doses of atropine/scopolamine mixture in 2:1 ratio were added prior to cooking. The breakdown of alkaloids during cooking, determined by liquid chromatography - tandem
mass spectrometry analysis of zganci, was dose dependent. The precooking dose was adjusted accordingly. The volunteers ingested the following doses of atropine/scopolamine mixture: 0, 0.22/0.14, 0.56/0.48, 1.39/
1.12, 3.71/2.92 and 12.04/9.45 lg/kg of body mass. The volunteers were
observed for four hours after ingestion. Changes in heart rate and saliva
secretion were checked hourly. Dose response curve for heart rate at 2h
after ingestion was J shaped with a signicant decrease at the dose level
1.39/1.12 lg/kg of body mass and a signicant increase at the highest
dose (P < 0.001). Saliva secretion at 3.5 h after ingestion showed a
decreasing trend. No signicant change in these two effects was noted at
the lowest two doses, suggesting that ARfD, used in preliminary risk
assessment was appropriate.
Paper No.: 1083

HEALTH AND DISEASE
PARTICIPATION OF ENDOTHELIAL SUPEROXIDE ANION
ON ALTERATION OF ANGIOTENSIN II-INDUCED
CONTRACTION IN CONTRALATERAL CAROTID OF
DIABETIC RATS AFTER BALLOON CATHETER INJURY
Larissa Pernomian(1), M Santos Gomes(2), AM de Oliveira(2)
(1) Faculdade de Medicina de Ribeirao Preto / Universidade de Sao
Paulo (FMRP/USP), Department of Pharmacology, Ribeirao Preto, Brazil
(2) Faculdade de Ciencias Farmaceuticas de Ribeirao Preto / Universidade
de SaoPaulo (FCFRP / USP), Ribeirao Preto, Brazil
522
Diabetes mellitus I (DMI) increases superoxide anion production, and
balloon-catheter injury (BCI) increases rat contralateral carotid responsivity to Ang II (Accorsi-Mendonca D et al., Br J Pharmacol 2004; 142:
79-88). So, we characterized the participation of superoxide anion on
Ang II contraction in contralateral carotid of diabetic rats. Methods:
induction of DMI in male Wistar rats (300g) by streptozotocin (55mg/
kg) and balloon catheter injury 28 days after induction. Curves for Ang
II (10pmol/L 1lmol/L) were performed 15 days after lesion in control
(CO), diabetic (DM), contralateral (CL) and diabetic contralateral (DCL)
carotids, with or without endothelium, in absence or presence of tempol
(superoxide scavenger, 1mmol/L). Flow cytometry in endothelial cells
was performed with dihydroethidine to quantify reactive oxygen species
(ROS) in absence or presence of tiron (superoxide scavenger, 1mmol/L).
Results: Ang II contraction (Emax) is increased in DM (1.65 0.08g/
mg), CL (0.92 0.05g/mg) and DCL (0.86 0.05g/mg). Endothelium
removal normalized Emax in DM (0.69 0.05g/mg) but increased Emax
in DCL (1.58 0.04g/mg). Tempol normalized Emax in DM (0.59
0.03g/mg), CL (0.50 0.01g/mg) and DCL (0.58 0.02g/mg). Endothelial ROS bioavailability is increased in DM (28,181.00 1,555.00U),
CL (17,940.40 564.51U) and DCL (32,104.40 2,350.71U). Tiron
reduced ROS bioavailability in DM (1,564.80 346.22U), CL (9,002.20
369.09U) and DCL (12,090.80 300.06U). Conclusion: DMI and BCI
increases endothelial superoxide anion bioavailability in DM, CL and
DCL carotids, involved in alterations of Ang II contraction in these arteries.
Paper No.: 2740

DISCOVERY
INFLUENCE OF RIFAMPICIN ON PULMONARY EXPRESSION
OF ABCB1 AND ABCC2 AND ON DISTRIBUTION OF
CLARITHROMYCIN INTO EPITHELIAL LINING FLUID AND
BRONCHO-ALVEOLAR CELLS OF FOALS
Jette Peters(1), J Freyer(1), W Block(2), S Oswald(1), M Venner(2),
W Siegmund(1)
Greifswald, Germany
(2) University of Veterinary Medicine Hannover, Clinic for Horses,
Hannover, Germany
First line therapy of pneumonia as frequently caused by Rhodococcus
equi in foals consists of a combination of rifampicin (RIF) and a macrolide antibiotic, e.g. clarithromycin (CLA). With RIF being a strong inducer of drug metabolism and transport, this combination might bare the
risk of signicant pharmacokinetic drug interactions. Effects of RIF on
distribution of CLA and pulmonary expression of ABCB1 and ABCC2
were studied in 9 healthy foals (4 females, 5 males, age 6 -8 weeks) after
oral administration of CLA alone (7.5 mg/kg, twice daily for 7 days)
and after RIF comedication (10 mg/kg, twice daily for 11 days). Concentrations of CLA and its active metabolite 14-OH-clarithromycin (14-OHCLA) were measured in plasma, epithelial lining uid (ELF) and broncho-alveolare cells (BAC) using LC-MS/MS. Gene expression in BAC
and bronchial epithelial cells (BEC) was quantied using real-time RTPCR. RIF co-medication signicantly decreased AUC(0-12h) and concentration of CLA in ELF(12h) and BAC(12h): 5.544.42 vs.
0.370.31* lgh/ml, 9.496.12 vs. 0.690.66* lg/ml and 263.8375.4
vs. 10.1710.17* lg/ml, respectively (all P<0.05). The ratio CLA:14OH-CLA in BAC decreased from 3.5 to 0.5 but overall exposure with
CLA and 14-OH-CLA decreased likewise. Pulmonary expression of
ABCB1 and ABCC2 in BAC and BEC was not inuenced. Conclusions:
As we observed no changes in gene expression of pulmonary efux
transporters but remarkable differences in pharmacokinetics of CLA and
14-OH-CLA, up-regulation of intestinal efux transporters rather than of
CLA metabolism might be the rationale behind signicantly lower accumulation of the drug in ELF and BAC after RIF comedication.
Paper No.: 2383

MODELS FOR SCREENING IN RATS THE EFFECTS OF
TRPV1 RECEPTOR ANTAGONISTS ON THERMAL
HYPERALGESIA AND THERMOREGULATION
G Petho(1), K Bolcskei(2), V Tekus(2), A Kis-Varga(3), L Dezsi(2),
E Szentirmay(4), A Visegrady(5), C Horvath(3), Janos Szolcsanyi(1)
(2) University of Pecs, Analgesic Research Laboratory and Gedeon
Richter Plc., Budapest, Hungary
(3) Gedeon Richter Plc., Laboratory of Neuropharmacology,
Pharmacological and Drug Safety Research; Budapest, Hungary
(4) Gedeon Richter Plc., Medicinal Chemistry II., Budapest, Hungary
(5) Gedeon Richter Plc., Lead Discovery Unit, Budapest, Hungary
The search for novel analgesics targeting the noxious heat-sensitive transient receptor potential vanilloid 1 (TRPV1) receptor expressed by nociceptors has been hampered by hyperthermia commonly observed in both
animals and humans. Therefore the effects of some recently described
TRPV1 receptor antagonists were compared in models of heat hyperalgesia and thermoregulation in rats. Thermal hyperalgesia induced by intraplantar injection of resiniferatoxin, mild heat injury (51 C, 20 s) or
plantar incision was measured with a novel increasing-temperature water
bath suitable for determination of the behavioral noxious heat threshold
in rats. Escape from a heat chamber (35 C) was assessed during the
effect of drugs on body temperature. The drop of the behavioral noxious
heat threshold i.e. heat hyperalgesia evoked by the TRPV1 receptor agonist resiniferatoxin was dose-dependently inhibited by oral pre-treatment
with the TRPV1 antagonist AMG9810, BCTC or SB705498 with a minimum effective dose of 1 mg/kg. The mild heat injury- or plantar incision-evoked heat threshold drop was also reduced by AMG9810, BCTC
and SB705498 with minimum effective doses in the range of 110 mg/
kg (i.p. post-treatment after heat injury and oral post-treatment after incision). AMG9810, AMG517 and BCTC increased the percentage of time
spent at 35 C whereas SB705498 did not impair thermopreference. Rectal temperature measured at the end of the experiment was increased by
AMG517 and BCTC but not by AMG9810 and SB705498. In conclusion, potent thermal antihyperalgesic effects of TRPV1 receptor antagonists are not necessarily coupled to disturbance of thermoregulation.
Paper No.: 2051

HEALTH AND DISEASE
ENDOTHELIAL TREK-1 POTASSIUM CHANNELS MEDIATE
ACETYLCHOLINE-INDUCED LONGITUDINAL
HYPERPOLARIZATION TRANSFER TO SYNCHRONIZE THE
DIAMETER CHANGES ALONG CEREBRAL ARTERIES
Olivier Petrault(1), C Widmann(2), C Misioreck(1), M De Bock(3),
V Berezovski(1), L Leybaert(3), R Ceccelli(1), C Heurteaux(2),
M Borsotto(2)
(1) Universite dArtois, Laboratoire de Physiologie de la Barrie`re
Hemato-Encephalique (LBHE), Lens, France
(2) Sophia Antipolis University, Institut de Pharmacologie Moleculaire et
Cellulaire, UMR6097 CNRS Nice Paul Hamel Institut, Valbonne, France
(3) Ghent University, Faculty of Medicine and Health Sciences, Basic
Medical Sciences Department, Physiology Group, Gent, Belgium
The endothelial dysfunction, evidenced by impairment in endotheliumdependent relaxation in response to acetylcholine (ACh) or bradykinin is
often associated with cardiovascular and cerebral diseases. Recently, evidences emerged that the TWIK-related K+ channel (TREK-1) of the
two-pore domain potassium channel gene family, may be involved in the
regulation of vascular tone. In this study, we explored the mechanism
523
involving TREK-1 channels on cerebral vascular function, particularly in
vasodilation phenomenon. Segments of cerebral basilar artery from wild
type (WT) or TREK-1 knock out (KO) mice were mounted in a myograph system to explore vascular function. Smooth muscle cells (SMCs)
were obtained from basilar arteries enzymatic dispersion, endothelial
cells (ECs) from primary cultures in both mice phenotypes. Real time
quantitative RT-PCR and immunouorescence techniques show the
prominent expression of TREK-1 mRNA and protein in the ECs but not
in the SMCs. Using different patch-clamp congurations, a native
TREK-1-like current was regularly recorded on endothelial but not on
smooth muscle cells, in presence of classical potassium channels blockers. The TREK-1 deletion led to a major alteration of ACh-induced
endothelial relaxing response while endothelial hyperpolarization capability remained intact in both phenotypes. Cell calcium imaging experiments using fura2 did not show any difference between WT and KO
TREK-1 endothelial cells neither in the Ca2+ baseline levels nor AChinduced Ca2+ responses. These ndings highlight a new mechanism
involving TREK-1 potassium channels as the main mediator of longitudinal endothelial-endothelial hyperpolarization transfer to synchronize
the diameter changes along the vessel in response to ACh.
Paper No.: 1371

DISEASES
IN VITRO AND IN VIVO EFFECTS OF WIN55,212 ON
EXOCRINE PANCREAS IN PHYSIOLOGICAL CONDITION
AND DURING ACUTE PANCREATITIS
Carla Petrella(1), S Alema`(1), S Agostini(2), M Broccardo(1),
P Casolini(1), C Giuli(1), F Carpino(3), G Improta(1)
(1) Sapienza University of Rome, Department of Physiology and
Pharmacology, Rome, Italy
(2) INRA- Neuro-Gastroenterology and Nutrition Unit, Toulose, France
(3) Sapienza University of Rome, Department of Experimental Medicine
and Biopathology, Rome, Italy
Cannabinoids (CBs) exert an anti-inammatory effect in disparate
inammatory diseases by activating cannabinoid 1- (CB1-r) and cannabinoid 2- receptors (CB2-r). CB1-r and CB2-r are expressed on rat exocrine pancreatic acinar cells and are not involved in the modulation of
digestive enzyme release. Rodent pancreatic acini produce and release
cytokines and chemokines. Acute pancreatitis is an inammatory condition characterized by digestive enzyme activation and release of inammatory mediators. Aim of this study was to investigate the role of the
pancreatic acinar cell- CB1/CB2-r system in rats, by evaluating in vitro
and in vivo effects of WIN55,212 on different parameters (IL-6 and
MCP-1 acinar release and content, morphological pancreatic examination, serum amylase and pancreatic edema) in physiological condition
and during caerulein (CK)-induced acute pancreatitis. In vitro results
showed that WIN 55,212 (10-6M) inhibits the release of IL-6 and MCP1 from acinar cells both in basal condition and after pancreatitis. In vivo
treatment with WIN 55,212 (2mg/Kg, i.p.) before inducing acute pancreatitis, reduces serum amylase, pancreatic edema, IL-6 and MCP-1 acinar
content, as well as histological changes, putting in evidence an improvement of pancreatitis. In contrast, treatment with the same dose of WIN
55,212 after inducing pancreatitis, increasing all these parameters, worsens pancreatitis. This study demonstrates that CB1/CB2-r on pancreatic
acinar cells modulate pro-inammatory cytokine and chemokine levels.
The dual, time-dependent in vivo effect of WIN 55,212 supports the idea
that the CB endogenous system plays a different role in acute pancreatitis
in rats, depending on the status of inammation.
Paper No.: 3227

SAFETY OF HERBAL PRODUCTS: DATA FROM THE SERBIAN PHARMACOVIGILANCE DATABASE
Marija Petronijevic(1,2), K Ilic(1), I Kovacevic(2)
(1) University of Belgrade School of Pharmacy, Department of Pharmacology, Belgrade, Republic of Serbia
(2) Medicines and Medical Devices Agency of Serbia, Belgrade, Republic of Serbia
Herbal products (HP) are increasingly marketed and commonly available
to patients over the counter. The aim of this study was to assess the
potential safety risks of HP by analyzing the Serbian pharmacovigilance
database (SPD). Retrieval of the SPD was performed in order to identify
reports of adverse events (AEs) associated with the use of HP submitted
from January 2005 to December 2009. Reported reactions were classied
according to Medical Dictionary for Regulatory Activities (MedDRA).
Data were observed for causative agents and patients characteristics
(age, sex, systemic diseases, and concomitant therapy). Among the 1,705
individual case reports submitted to the SPD during 2005-2009, 16 (<
1%) indicated 52 reactions to HP. The majority of these cases (10) referenced hepatotoxicity. Of the 6 different reported HP, two were Chinese
traditional products (CTP). Ten cases were reported for Dolorexa, a CTP
intended for headache treatment; eight of which presented hepatic AEs.
Besides Dolorexa, two products used for the treatment of prostate disorders were reported for hepatotoxicity. Furthermore, 3 cases of widely
used ginko biloba preparations were found. The 16 patients (9 females, 7
males) ranged in age from 31 to 87 years. No fatality associated with HP
use was reported, but 6 cases of hepatotoxicity required hospitalization.
HP were rarely reported for AEs in the SPD. Signicant polypharmacy
amongst elderly patients highlights the potential for herb-drug interactions. The primary safety concern associated with HP use in Serbia was
CTP induced hepatotoxicity.
Paper No.: 982

INFLUENCE OF LHRH ON SEX HORMONE RECEPTORS IN
THE CA3 OF HIPPOCAMPI OF THE MALE RAT
Bojana Petrovic(1), Z Zoric(1), D Drekic(2)
(1) University of Kragujevac Medical Faculty, Kragujevac, Republic of
Serbia
(2) Faculty of Veterinary Medicine, Department of Anatomy, Belgrade,
Republic of Serbia
Introduction: Autoradiography was used to localize estrogen accumulating cells in the CA3 of hippocampi of male rats with LHRH hormones
of seven adult male rats. Seven mail rats were each treated with an injection of luteinizing hormone-releasing hormone at 83 days old and 3 days
later with 250 lCi 3H-estradiol. A control group of male rats was also
treated with 250 lCi 3H-E2, two hours before sacrice. Both groups
were sacriced at 86 days old. Materials/Patients: The presumptive distribution of estrogen receptors in the CA3 of hippocampi of adult male rats
was determined using autoradiography and uorescentn methods.
Results: In the control group, radioactivity was concentrated in the cell
nuclei and to a lesser extent in the cytoplasm of the neurons of the CA3
of hippocampi of male rats injected with estradiol. The intensity of
nuclear labeling varied among individual neurons and between neuronal
populations of CA3 of hippocampi. Conclusions: Our opinion is that
LHRH affects the inuence of oestrogen on the CA3 of hippocampi neurons by altering the distribution and number of receptors in the nuclei of
different types of neurons studied with autoradiographyc and uorescents
methods. These results indicate that developing LHRH neurons express
the transcript for ERb and suggest that continued expression of ERb is
either a characteristic of LHRH neurons that may require cues from the
524
central nervous system and/or periphery or predetermined to be maintained in a subpopulation of LHRH neurons.
Paper No.: 983

SILVERGOLD-CYANKALI METHODS FOR STANED GLIAL
CELLS
Bcrp, MRP2, Oatp1a4, Ntcp and Cyp3a2 mRNA was observed in poly
I:C-treated rats. Hepatic Mdr1b and Mrp3 were signicantly induced.
Bile acids in maternal plasma were signicantly increased with the
higher dose of poly I:C. Bile acids in fetal pools, as well as maternal
plasma bilirubin levels were increased, but results were not statistically
signicant. In summary, viral infection imposes signicant changes in
the expression of key drug transporters in hepatic and placental tissues of
pregnant rats. Since many clinically important drugs are their substrates,
inammation-mediated changes in transporter expression could affect
maternal and/or fetal drug disposition.
Bojana Petrovic(1), Z Zoric(1), D Drekic(2)

(1) University of Kragujevac Medical Faculty, Kragujevac, Republic of
Serbia
(2) Faculty of Veterinary Medicine, Department of Anatomy, Belgrade,
Republic of Serbia
Introduction: Tine specicities a silvergold-cyanka1i (SGC) markers for
different glial cell populations in pigs, macaw and equine brain were
studied. Materials/Patients: In this study its used method by Drekic/Malobabic with exception that after conc.alcohol, specimen is treated with
goldcyankali solution. Results: The most obvious staining with SGC was
seen in the white matter studied at low magnication. However, at higher
magnication, the strangest impregnated was observed in small, rounder
or oval neuronal cells in the gray matter whereas a large population of
similar cells in the white matter stained with varying intensity. The SGC
staining products in each positive cell were restricted to the cytoplasm,
especially to the perikaryon, and the origin of the few processes. The
subpial layer exhibited a diffuse, specic staining of glial cells. In lover
cortical layers the SGC positive cells were fairly. In the white natter the
SGC positive glial cells were scattered through out a diffuse, granular or
lamentous staining, which was found to be scanty in the grey matter.
Many of the labeled cells were arranged in rows. Based on their morphology and distribution, the SGC-positive celIs were identied as oligodendrocytes and the granular or lamentous staining SGC in myelin
sheaths. Conclusion: By using SGC methods, large numbers of glial cells
can be visualized over large areas of gray and white matter of CNS. At
the cellular level this SGC was localized excessively in cerebral, glial
cells.
Paper No.: 2952

DISCOVERY
EFFECT OF VIRAL INFECTION ON HEPATIC AND PLACENTAL DRUG TRANSPORTERS AND ENDOGENOUS
SUBSTRATES IN PREGNANT RATS
Paper No.: 1795

PROTEIN KINASE G REGULATES MITOCHONDRIAL BIOGENESIS AND DIFFERENTIATION OF BROWN ADIPOCYTES
VIA INHIBITION OF THE RHOA/ROCK PATHWAY
Alexander Pfeifer(1), B Haas(1), K Jennissen(1), P Mayer(2)
(1) University of Bonn, Institute of Pharmacology and Toxicology, Bonn,
Germany
(2) Federal Institute for Drugs and Medical Devices, Bonn, Germany
Here, we analyzed the role of cGMP/cGMP-dependent protein kinase
(PKG) signalling in brown fat. PKG-decient brown fat cells displayed
reduced mitochondrial contents and reduced expression of fat cell specic markers - including UCP-1. Further analysis revealed that RhoA
and ROCKII activity is elevated in PKG-decient cells, due to a loss of
PKG-dependent inhibitory RhoA Ser188 phosphorylation. Increased
ROCKII activity led to reduced insulin signalling via ROCKII-dependent
phosphorylation of IRS-1 and subsequently to reduced IRS-1-PI3K association, Akt and GSK-3 phosphorylation. Histological analysis of PKGdecient brown adipose tissue (BAT) revealed a marked defect in BAT
differentiation and BAT wet weight to body weight of PKG-/- mice was
signicantly lower. Additionally, BAT of PKG-/- mice displayed reduced
mitochondrial content. PPAR and UCP-1 protein expression were
reduced as compared to their wt littermates. We found strongly reduced
phosphorylation of Akt in BAT of the mutant mice, showing that PKG
seems to play a permissive role in insulin signalling also in vivo. To
assess the consequences of PKG ablation on BAT function in vivo, we
measured thermogenic activity using infrared thermographic imaging in
neonates. PKG-/- mice exhibited signicantly reduced surface temperatures as compared to their wt littermates demonstrating that interscapular
BAT thermogenic activity is reduced in these mice (Haas et al., Sci Signal 2009). Taken together, these data show a so far unknown role for
PKG in mitochondrial biogenesis and brown adipocyte differentiation.
Vanja Petrovic, M Piquette-Miller

University of Toronto, Department of Pharmaceutical Sciences, Toronto,
Ontario, Canada
While inammation is known to impose changes in the expression and
activity of drug transporters, little is known about the impact of inammatory stimuli on drug transporter expression during pregnancy. Our
objective was to study the effect of virally-induced inammation on key
maternal hepatic and placental drug transporters and their endogenous
substrates. Acute inammation was induced in pregnant Sprague-Dawley
rats (G17-18, n=5-6/group) by single i.p. doses of polyinosinic:polycytidylic acid (2.5 or 5.0 mg/kg) with saline as control. Tissues were harvested 24 hours later. Expression of transporters was measured via realtime PCR and Western blotting. Plasma cytokine levels were measured
via ELISA. Maternal levels of plasma bile acids and bilirubin, as well as
fetal bile acids were examined. Plasma concentrations of IFN-g, TNFa
and IL-6 were signicantly induced in poly I:C-treated rats, as compared
to controls (P<0.05). Signicant downregulation of placental Mdr1a/b,
Mrp1, Mrp3, Bcrp, Oatp1a4 and Oatp4a1 mRNA, as well as hepatic
Paper No.: 2704

OPTIMIZATION OF ARISTOLOCHIC ACID I DETECTION IN
ARISTOLOCHIA TAGALA CHAM AND THAI TRADITIONAL
RECIPE USING TLC
Rattana Phadungrakwittaya(1), P Akarasereenont(1,2), A Lumlerdkij(2),
S Chaisri(2), S Chotewuttakorn(1), T Laohapand(2)
(1) Mahidol University, Siriraj Hospital Faculty of Medicine, Department
(2) Mahidol University, Siriraj Hospital Faculty of Medicine, Center of
Applied Thai Traditional Medicine, Bangkok, Thailand
Aristolochic acid (AA) is a mixture of nitrophenanthrene carboxylic
acids found in Aristolochiaceae family. Some dried roots of this family
were commonly used as the component of Thai traditional recipe. Due to
525
the toxicity of AA, the simple and rapid method for sensitive detection
of AA in the plant specimens is needed. The objective of this study is to
develop a method for AA detection in Aristolochia tagala Cham and
Thai traditional herbal recipe. Three mobile phases were tried with
HPTLC to obtain the best separation. RP-TLC was used to conrm the
identication. Chloroform: methanol: acetic acid (65:20:1, v/v) was the
optimal mobile phase for HPTLC and acetonitrile: methanol: water
(3:0.5:1, v/v) for that of RP-TLC. The lower LOD for AA-I was 8 ng
using HPTLC and 15 ng using RP-TLC. Using the above system, AA-I
could not be clearly resoluted from Thai herbal recipe due to the interference from other moieties over the same Rf. In summary, the optimized
HPTLC and RP-TLC analyses are suitable for detecting AA-I in a single
herb (A. tagala Cham), but not in the recipe. HPTLC has greater sensitivity than has RP-TLC.
Paper No.: 1454

MEMBRANE WITH A VEGETATIVE EXTRACT BASED ON
THE COLLAGEN FOR TISSUE REGENERATION
Guram Pichkhadze, G Ustenova, I Kaulambaeva, D Assylbekova
` GS- kaskain, LAS -141

nogalactan and a-cyclodekstrine (LAS -137 A
TsD- kaskain ) with local anaesthetic agents, which are widely applied
in clinical practice. Screening and profound researches of activity at inltration, conduction analgesia and acute toxicity were studied according
to methodical recommendations. In all series of experiences the greatest
activity at inltration and conduction analgesia have shown connections
of kaskains homolog - LAS -134, LAS -137 and LAS -141. At inltration analgesia the specied connections exceeded indicators of all preparations of comparison by duration of total anaesthesia in 2,4 - 6,8 times,
their advantages were traced by indicator of the general duration of
action. At conduction analgesia the above-stated connections caused a
total anaesthesia,which is exceeding indicators of reference preparations
in 2,1 - 6,3 times, and on the general duration - in 1,9 - 4,0 times. All
investigated connections in 1,5-2 times are less toxic, than lidocaine and
trimecain, and they are comparable with novocain and kaskain. Thus,
expressed activity at piperidines derivatives with benzoiloxygroup aethoxyaethylic, radical and immobilized forms of kaskain.
Paper No.: 2290

ACUTE BLOCK OF KV1.5 CHANNELS BY DHA INCREASES
RABBIT ATRIAL REFRACTORY PERIOD
Kazakh National Medical University, Department of Pharmacology,

Almaty, Kazakhstan
Christophe Pignier, R Letienne, N Cabriol, Y Calmettes, B Le Grand
Recently uses the vegetative extracts, which are containing the wide
spectrum of biologically active substances, causing their expressed pharmacological action. The preparation of the prolonged action is created, it
was based on extract, which is received by a method supercritical carbonic acid extraction. Collagen is used as auxiliary substance. The membrane is received with 0,5 % dry extract of the camel prickle. Processes
of reparative regenerations studied on model of a plane wound of a skin
with the area 300 ``2 on not purebred rats-males: 1 group a collagen
membrane, 2- collagen membrane with 0,5 % dry extract of the camel
prickle, 3 ointment Laevomecolum, 4 group without treatment.
Intensive reduction of the wound area observed mainly at the animals
which wounds have been covered by a membrane with a vegetative
extract. So, full healing of wounds occurred for ninth day from the
moment of application on a wound and initiation of treatment, that was
for fourth day earlier, than in 1 and 3 groups, for six days earlier, than in
4 group. Also it is shown that the membrane with extract promotes more
expressed increasing quantity of DNA, RNA, and parities of RNA/DNA.
Considering that if the factor RNA/DNA is higher, metabolism in the
wound is intensive and the synthesis of albumines is more active, also
taking into account the data about collagen (stimulates growth of granulation tissue) and extract of the camel prickle (inuences on epithelialization), the offered membrane provides earlier healing of wounds.
Institut de Recherche Pierre Fabre, Castres, France
Paper No.: 1476

LOCAL ANAESTHETIC ACTIVITY OF NEW PIPERIDINES
DERIVATIVES AND THEIR IMMOBILIZED FORMS
Kv1.5 is considered as a target for treatment of atrial brillation or utter.

Polyunsaturated fatty acids exert anti-arrhythmic activity in-vivo as well
as in human. Thus by means of patch clamp technique we investigated a
potential regulation by DHA on hKv1.5 channels transfected in HEK
293 cells. Among PUFAs, monounsaturated and saturated fatty acids,
DHA was the most effective in reducing IKv1.5 with an IC50 of
6.6 lM. DHA fastened the apparent slow inactivation in a concentrationdependent manner leaving peak current nearby unaffected and slowed
the deactivating tail current leading to a cross-over phenomenom, consistently with an open channel block mechanism. For a simple channel
open-state block model, derived values of Kon and Koff are 3.1 lM-1 s1 and 8.4 s-1, respectively leading to a Kd of 2.7 lM. Fit of recovery
from inactivation showed that time constants were signicantly increased
with fast and slow of 36.51.2 msec and 1173.6270.0 msec (control)
and 89.421.5 msec and 4134.9450.5 msec (10lM DHA). To test
whether the inhibition of IKv1.5 may have physiological implication, we
developed a rabbit model of left atrial effective refractory period (AERP)
using a conventional pacing protocol through epicardial. Direct injection
of ecainide(1.25 mg/kg) in atrium increased AERP by 15.02.1 %
(n=6, P<0.05). DHA (20 mg/kg) signicantly increased AERP by
10.72.3% (n=6, P<0.05). As a conclusion, DHA exerted the most powerful inhibition of IKv1.5 that appears to be sufcient to signicantly
increase AERP in rabbit. Such a lengthening of AERP may be of therapeutic benet in atrial brillation.
Guram Pichkhadze(1), K Praliev, D Kadyrova, S Nasyrova, S Imashova,

M Amyrkulova, G Muhamedzhanova, D Assylbekova
Paper No.: 2291

CARDIOPROTECTION AGAINST ISCHEMIA AFFORDED BY
F 16916, A BLOCKER OF PERSISTENT SODIUM CURRENT
Kazakh National Medical University, Department of Pharmacology,

Almaty,Kazakhstan
Christophe Pignier, R Letienne, P Pajeau, L Bel, A-M Bessac, B Vacher,

B Le Grand
Searching the new anaesthetics, which are economical advantageous,

minimum toxic and especially have long action, is important problem in
modern medicine. In the work led experimental studying of 61 piperidines derivatives (under the code numbers LAS) and comparison of
combined (LAS-135, 136) and immobilized forms of kaskain (it was created and studied earlier) on nanostructural biopolymers sulfated by arabi-

Persistent sodium current (INap) is a major source of Na+ entry which
leads to Ca2+ overload and cell death. Here, we investigated the mechanism of action of F 16916, a new drug targeting INap and a potentate car-
526
dioprotective action against myocardial infarction. Experiments were performed with patch-clamp technique on HEK293 cells transfected with
hNav1.5. INap was induced with 40 lM veratridine. F 16916, reversibly
reduced veratridine-induced INap (holding potential (HP) of -110 mV)
with an IC50 of 6.4 lM. When HP was depolarized to -90 mV, IC50
obtained was of 4.3 lM, suggesting that F 16916 was not voltage-dependent. F 16916 at 10 lM shifted the steady-state inactivation V0.5 by 5.02.7 mV (NS, n=6). Inhibition of INap by F 16916 (5 lM) was not
frequency-dependent with mean inhibition values of 53.38.6 % (3) and
60.66.7 % (n=5) obtained at 0.2 and 1.0 Hz frequency, respectively. In
addition, F 16916 exhibited almost a pure tonic block. The cardioprotective activity of F 16916 was assessed in a pig model of myocardial
infarction with 1h coronary occlusion and 48h reperfusion. Infarct size
was signicantly reduced by 4314% (n=7) and 872% (n=6) with 0.63
and 2.5 mg/kg F 16916, respectively. This was paralleled by a reduction
in plasma levels of troponin I. F 16916 was devoid of major hemodynamic effects. As a conclusion, F 16916 is a potent specic blocker of
INap that may provide a new opportunity in the treatment of conditions
like myocardial infarction, cardiac surgery, cardiac insufciency, and
heart failure.
Paper No.: 2292

SELECTIVE INHIBITION OF PERSISTENT SODIUM
CURRENT BY F 15845 PREVENTS ISCHEMIA-INDUCED
ARRHYTHMIAS
Christophe Pignier, R Letienne, B Vie, C Culie, B Vacher, B Le Grand
Mutations in the gene coding for a-synuclein have been found in families affected by autosomal dominant Parkinsons disease. Several mouse
lines with changes in the expression or structure of a-synuclein have
been created to elucidate the physiological and pathophysiological roles
of the protein. We have measured amphetamine induced (1 or 2.5 mg/
kg) locomotor activity in 6 months old, and spontaneous 24 h locomotor
activity in aged, 14-17 months old transgenic mice that overexpress
mutated human A30P a-synuclein (A30P +/+). We observed signicantly
decreased locomotor activity induced by 1 mg/kg of D-amphetamine in
A30P +/+ mice than their wild-type littermates. This may be a consequence of a lower capacity of dopamine storage pool, which has earlier
been described for this mouse strain. Higher dose of D-amphetamine did
not produce signicant differences in total locomotor activity between
genotypes, although there were differences at certain time points. In the
aged A30P +/+ mice, we observed a signicant decrease both in locomotor activity and rearing. These changes were especially noticeable during
activity peaks, ie. in exploratory behavior at the start of the experiment
or the beginning of lights-out period. This is in line with earlier results
showing impaired rearing in a shorter activity test. Our results support
the view that human type a-synuclein mutation disturbs the brain dopaminergic system regulating motoric activity.
Paper No.: 648

ANALYZING THE KEY ENZYMES OF TRICARBOXYLIC
ACID CIRCULATION AND RESEARCHING METABONOMICS
IN DIABETES AND DIABETIC SUSCEPTIBLE GROUPS

Jing Ping(1), X Guan(2), L Wu(3), Y Zhao(3), K Bi(1)
Electrophysiology of cardiomyocytes is altered under myocardial ischemia and persistent sodium current (INap) appears as one of the major contributor to ischemic arrhythmias. We investigated the effects of F 15845,
a new anti-anginal drug on INap and in models of INap-induced arrhythmias. Using patch-clamp technique on hNav1.5 channels transfected in
HEK293 cells we demonstrated that F 15845 is a potent voltage-dependent blocker of INap (IC50=1.8 lM). F 15845 concentration-dependently
shifted steady-state inactivation V0.5 (-9.11.1 mV, <0.05, n=6, 10 lM).
Moreover blockade arised from extracellular side of the channel and was
characterized by an almost pure tonic block. F 15845 reduced action
potential duration at 50% and 90% repolarization from 0.1 to 10 lM
(maximal change: -133.811.4 msec, n=6, P<0.001 and 97.310.8 msec, n=6, P<0.001, respectively) from rabbit Purkinje bers.
F15845 (10 lM) counteracted the veratridine-induced lengthening of AP
with mean change of +27.1 ms (APD50, n=6, NS). In-vivo anti-arrhythmic activities of F 15845 were studied in rat models of INap-induced arrhythmias. Firstly, F15845 dose-dependently reduced the incidence of
VT and brillation in ischemia-induced arrhythmias in rats in a dose
related manner from 43 14 sec (vehicle, n=25) to 209 sec (n=12, P
0.4<0.05) and 52 sec ((n=16, P<0.05) with 2.5 and 5 mg/kg F 15845,
respectively. Secondly, 2.5 mg/kg F 15845 increased the dose threshold
of aconitine required to induce VPB, VT and VF when compared to
vehicle group. F 15845, a novel anti-anginal drug targeting INap, shows
anti-arrhythmic properties which may be of therapeutic benet against
ischemia-induced arrhythmias.
Paper No.: 1079

SPONTANEOUS AND AMPHETAMINE INDUCED
LOCOMOTOR ACTIVITY IN A30P -TRANSGENIC MICE
Marjo Piltonen, S Patrikainen, PT Mannisto
(1) Department of Pharmaceutical Analysis, Shenyang Pharmaceutical

University,Shenyang, PR China
(2) Department of Pharmacy, Shenzhou Hospital, Shenyang Medical
College, Shenyang, PR China
(3) Department of Clinical Laboratory, Shenzhou Hospital, Shenyang
Medical College, Shenyang, PR China
Introduction: This study focuses on inevitable connection of diabetes
mellitus (DM) physiology, that is, the metabolic enzymes levels and biochemical metabolic patterns on the key rate-limiting steps of tricarboxylic
acid circulation (TAC), a necessary metabolism pathway of glucose aerobic oxidation in the body. Patients: 56 cases were selected and divided
into 5 groups: I. diabetic susceptibility, II. impaired glucose regulation
(IGR), III. earlier period DM, IV. mid and later period DM and V.
healthy control. The blood citrate synthase (CS), isocitrate dehydrogenase (ICD), a-ketoglutaric dehydrogenase complex (a-KGDHC) by Elisa
method. The lactate dehydrogenase (LDH) and its isozyme-1 (LDH-1),
creatine kinase (CK) and its isozyme-MB (CK-MB) were determinated.
The blood glucose, Insulin, C-peptide, glycosylated hemoglobin, fasting
blood lipid, renal and liver function were also analyzed by biochemical
methods. Results: CS activities in DM and diabetic susceptible groups
were higher than those in healthy subjects, there were signicant differences in comparison with V, I and II (P<0.05), III and IV (P<0.01), relationships of CS and myocardial enzymes (LDH and LDH-1, CK and
CK-MB) showed a positive correlation. Statistically signicant decrease
of ICD activity was found in diabetic patients, I, II, III (P<0.05), IV
(P<0.01). The activity of a-KGDHC was inversely correlated with
kinase activity. Conclusions: Mitochondrial dysfunction is a major contributor to diabetes. The activities of CS, ICD and a-KGDHC affected
the metabolism of TAC velocity of ow.
Key words: Tricarboxylic Acid Circulation; Diabetes mellitus; Citrate
synthase; Isocitrate dehydrogenase; a-Ketoglutaric dehydrogenase complex.

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Paper No.: 2783
DISEASES
INVESTIGATION OF ANTI-INFLAMMATORY AND
ANTI-NOCICEPTIVE EFFECTS OF A NON-PEPTIDE
SOMATOSTATIN 4 RECEPTOR AGONIST TEZ-321
Erika Pinter(1), Z Helyes(1), T Szuts(2), G Keri(2), A Kemeny(1),
K Sandor(1), K Elekes(1), E Szoke(1), T Bagoly(1), Z Hegedus(2),
J Szolcsanyi(1)
(1) University of Pecs, Department of Pharmacology and Pharmacotherapy, Pecs, Hungary
(2) Biostatin Ltd. Budapest, Hungary
Original basic studies have established that somatostatin released from the
sensory nerve endings exerts anti-inammatory and anti-nociceptive
effects acting on somatostatin 4 (sst4) receptors. Since somatostatin itself
is not suitable for drug development because of its wide spectrum and
short duration of action we have developed orally active non-peptide sst4
receptor selective compounds as new candidates of anti-inammatory and
anti-nociceptive drugs. Replacement binding studies on sst4 receptor
expressing validated CHO cells did not support the binding of TEZ-321
on somatostatin binding site, thus allosteric activation was supposed.
Since sst4 receptor belongs to G-protein coupled receptor family, G-protein activation assay was also performed. TEZ-321 was ableto activate
protein Gi in each applied concentration. TEZ-321 (100 ug/kg p.o.) significantly inhibited the ear oedema induced by 1% mustard oil in C57BL/
6wild-type mice, but it was ineffective in sst4 gene decient knockout animals. This compound (50-500 ug/kg p.o.) showed signicant, but non
dose-dependent inhibition in the decrease of mechano-nociceptive threshold (hyperalgesia) and touch sensitivity (allodynia) induced by partial
nerve ligature in trauma ticmononeuropathy model of Wistar rats and
evoked signicant decrease of mechanical hyperalgesia and oedema formation in Freunds adjuvant-induced chronic arthritis model in Lewis rats.
TEZ-321 in 0.1-100 pg/ml concentrations did not cause considerable inhibition of interleukin-1b production induced by lipopolysaccharide or
phorbol-myristate-13-acetate induced from isolated peritoneal macrophages of CD1 mice. On the basis of the present study we conclude that TEZ321 is effective in different acute and chronic inammation and pain models and also inhibits the neurogenic components of inammation.
Paper No.: 3317

DISEASES
IS SEVERITY OF CLINICAL MANIFESTATIONS FOLLOWING
H LEPTURUS ENVENOMATION RELATES TO SERUM
TNF-ALPHA LEVEL?
Mohammad Hassan Pipelzadeh, A Jalali, A Ahmadzadehd,
A Khodadadi, M Makvandi, M Taraz
Ahvaz University of Medical Sciences, Department of Pharmacology,
Ahvaz, Iran
In the present study 36 patients, with varying degrees of severity of
envenomation, from Khuzestan province in the southwest of Iran, who
were admitted to hospital because of envenomation by Hemiscorpius
lepturus(H. leptuturs) scorpion, were systematically investigated. The
serum levels, measured by double-ligand ELIZA kit, of interlukin-1 (IL1), interlukin-6 (IL-6), interlukin-8 (IL-8) and tumor necrosis factor-a
(TNF-a) were compared with 30 healthy controls and 10 age-matched
patients envenomed by Mesobuthus eupeus (M. eupeus) scorpion,
another less dangerous scorpion in this area. Blood samples from M.
eupeus and H. lepturusvictims were taken on admission and once more
after 6 hr after serotherapy with multivalent antiscorpion serum from H.
lepturusenvenomed subjects. Compared with healthy volunteers, with
exception of TNF-a, signicantly higher cytokines serum levels were

recorded for M eupeus stung patients. However, all three graded H.
lepturusvictims showed signicantly, in a severity-related manner, higher
mean values for all the measured interleukins as well as TNF-a on
admission compared with the M eupeus envenomed cases. Six hours
after serotherapy, greater percentage of reduction in cytokine and TNF-a
levels were observed among mild H lepturusenvenomed patients compared with moderate and severe cases. The results of the present study
suggest that, unlike M eupeus, the toxic manifestations observed following H lepturusmay be associated with increase in serum TNF-a level and
are positively correlated with its severity. Furthermore, serotherapy is
only effective when administered to mild cases of envenomation due to
H lepturusscorpion
Paper No.: 1099

INHIBITION OF LEUKOCYTE ENDOTHELIAL CELLS
INTERACTIONS BY ACTIVATION OF RETINOID X
RECEPTOR
Laura Piqueras(1), E Morcillo(2), C Company(2), J Cortijo(2),
MJ Sanz(2)
(1) Fundacion Hospital Clinico Universitario, Department of Pharmacology, Valencia, Spain
(2) University of Valencia, Department of Pharmacology, Valencia, Spain
Background: The inltration of polymorphonuclear leukocytes (PMNs)
into tissues is a prominent feature in inammation. The mechanism
underlying PMNs recruitment depends on the release of chemotactic
mediators and cell adhesion molecule expression on endothelial cells.
Retinoid X receptor (RXR), a member of the nuclear receptor superfamily, forms heterodimers with several nuclear receptors and mediates many
biological effects. Objetive: The nuclear receptor RXR is widely
expressed in many tissues including the vascular endothelium, however
its role in inammation remains unclear. Methods: For these experiments
we used human vascular endothelial cells (HUAECS) and mice (C57BL/
6/129Sv) between 6-10 weeks of age. Single unbranched cremasteric
arterioles (20-40 um in diameter) were selected for study and the number
of adherent leukocytes was determined. Results: Using intravital microscopy in the mouse cremasteric microcirculation, we have shown that activation of RXR by its selective ligand bexarotene inhibits TNFa-induced
arteriolar adhesion. Furthemore, in the ow chamber asssay the RXR agonists inhibited monocyte rolling and adhesion induced by TNFa in a
concentration dependent manner. However in cells transfected with siRNA RXRa this inhibition was absent. These inhibitory responses were
accompanied by a reduction in TNFa-induced endothelial MCP-1
release. Conclusions: Taken together, our results show that RXR modulates inammation in vivo and in vitro under ow by targeting the monocyte-endothelial cell interaction.
(This work has been funded by Instituto de Salud Carlos III- Fondo Investigacion Sanitaria (CP07/00179 and PI081875).
Paper No.: 990

ANTIDIABETIC ACTIVITY OF BIOTIN, SODIUM SELENATE
AND FOLIC ACID IN DIABETIC RATS
Shweta Pitre, R Ghosh, P Ganpathy, V Kadam
Bharati Vidyapeeths College of Pharmacy, Department of Pharmacology, Navi-Mumbai, India
The objective of the study was to elucidate the effectiveness of combining select nutritional supplements in the treatment of type II diabetes
528
mellitus. The pharmacological screening model used was a high fat diet
in combination with a single injection of low dose streptozotocin, a
model which simulates the metabolic characteristics of human type II
diabetes. Male Sprague-Dawley rats were divided into two groups with
one fed the normal pellet diet and the other group fed a 40% olive oil
based high fat diet for a period of 2 weeks. On day 15 the animals were
subjected to low dose streptozotocin (45mg/kg,i.p). The treatment group
received a combination of biotin, sodium selenate and folic acid. This invivo animal study concludes that the combination shows statistically signicant (P<0.05) improvement in the serums levels of glucose, triglycerides, total cholesterol, HOMA-IR and glycosylated hemoglobin (GlyHb) in the treatment groups compared to the disease control. Although,
anti-diabetic drugs occupy a major market share, there still exist enormous market potentials for agents with fewer side effects and those
which can be used for extended periods. The promising in-vivo results of
the above suggested combination opens avenues for similar therapies.
Paper No.: 3187

ROLE OF ACE AND MTHFR GENES POLYMORPHISMS IN
MIGRAINE
V Pizza(1), A Bisogno(2), E Lamaida(3), A Agresta(1), G Bandieramonte(4), A Volpe(4), R Galasso(4), L Galasso(4), Anna Capasso(2)
(1) S. Luca Hospital, Department of Neurophysiopathology, Vallo della
Lucania ((SA), Italy
(2) Maria SS Addolorata Hospital, Department of Neurology, Eboli
(SA), Italy
(3) S. Luca Hospital, Department of Neuroradiology, Vallo della Lucania
((SA), Italy
(4) University of Salerno, Department of Pharmaceutical Science, Fisciano (SA), Italy
Angiotensin Converting Enzyme gene deletion polymorphism (ACEDD) and Methylentetrahydropholate reductase genes polymorphism
(MTHFR C677T and A1298C) increase migraine susceptibility, with a
greater effect in migraine with aura. The TT polymorphism is also
associated to an increate risk of migraine with aura, independently of
other cardiovascular risk factors. The aim of our study was to evaluate
the incidence of ACE and MTHFR genes polymorphisms in a consecutive series of migrainous patients and of patients affected by myocardial infarction. We studied a series of 103 migrainous patients, whose
age was between 13 and 75 years (81 suffering from migraine without
aura, MwA, 9 from migraine with aura, MWA, 13 from mixed forms
MwA-MWA, according to ICHD-II 2004 criteria) and of 336 patients
suffering from ischaemic cardiopathy (myocardial infarction, MI). The
analysis, based on Polymerase Chain Reaction (PCR) and on reversehybridization, showed as follows: MTHFR (C677T): 60 patients
(58%) (1) and 186 (56%) (2) were heterozygous; 9 patients (9%) (1)
and 54 (16%) (2) were mutated. The result of 1patient (2) was
unknown. MTHFR (A1298C): 54 patients (52%) [1] and 146 (44%)
[2] were heterozygous, 7 patients (7%) (1) and 33 (10%) (2) were
mutated. The result of 1 patient (2) was unknown. ACE (evaluated on
101 patients (1) and 245 (2)): 45patients (43%) (1) and 133 (54%) (2)
had an ID genotype; 42 (41%) (1) and 87 (36%) (2) had a DD genotype. The results of our study indicate the high incidence of ACE and
MTHFR genetic polymorphism in migraineurs.
Paper No.: 3188

ELDERLY PATIENTS WITH MIGRAINE:
AN OPEN-LABEL STUDY ON PROPHYLAXIS THERAPY
WITH LEVETIRACETAM
V Pizza(1), V Busillo(2), A Agresta(1), A Bisogno(3), Anna Capasso(3)
(2) Maria SS Addolorata Hospital, Department of Neurology, Eboli
(SA), Italy
In the last years, the hypothesis that cortical hyperexcitability may play a
role in the physiopatology of migraine led to the therapeutic use of some
antiepileptic drugs. To evaluate the efcacy of levetiracetam as prophylactic treatment for migraine without aura in elderly patients. We performed a small open-label trial treating 13 elderly patients(8F 5M) mean
age 64.7 years (SD 3.4), range 60-72 years affected by migraine without
aura (ICDH 04 criteria). The mean age of disease was 21.3 years
(SD13.4) range 2-45 years. At baseline: the frequency of attacks was
12.2/month (SD 5.9), range 6-25; the mean number of drugs for acute
attacks was 12.6 (SD 6.5) tablets/month. All patients took concomitant
medication for other cronic diseases. After recruitment Levetiracetam
500 mg/die was administered for 1 week and 1000 mg/die for six
months. The basal frequency of attack was 12,2 (SD 5.9) and 8,3 (SD
4.9), 4,1 (SD2.6), 1,3 (SD1.4) after 1, 3 and 6 months respectively
[P=0.079; P<0.0001; P<0.0001].The basal value of intaking drugs for
acute attacks was 12,6 (SD 6.5) and 6,7 (SD 4.3), 2,8 (SD 2.2), 1,4
(SD1.7) after 1, 3 and six months respectively [P=0.012; P<0.0001;
P<0.0001](T-test analysis). Levetiracetam was well tolerated (7 patients
complained somnolence, lack of concentration and gastralgia but none
patient withdrew the study). In our study levetiracetam showed a good
efcacy in frequency and intensity reduction of headache attack and
showed a very good tolerability despite all elderly patients took drugs for
concomitant diseases.
Paper No.: 3189

POLYMORPHISM GENE APOLIPOPROTEIN E IN MIGRAINE
AND CARDIOVASCULAR DISEASE
V Pizza(1), F Infante(2), G Schiavo(2), A Agresta(1), C Colucci
dAmato(3), A Bisogno(4), Anna Capasso(4)
(2) S. Luca Hospital, Department of Molecular Biology, Vallo della
(3) University of Naples, Naples, Italy
(4) University of Salerno, Department of Pharmaceutical Science,
Fisciano (SA), Italy
Nitric oxide plays an important role in the pathogenesis of migraine.
Studies suggest that the expression of molecules involved in the pathogenesis of headache (i.e., nitric oxide-interleukin) is inuenced by apolipoprotein E (APOE) and is gene specic. Hence, we hypothesized that
APOE polymorphism may be associated with migraine. Our study analysed the incidence of genetic polymorphism Apoliporotein E in a sample
of migraineurs and a control group of the patients with ischemic cardiopaty. In this study 100 consecutive patients aged 18-64 years (mean age
33.7 years), suffering from migraine [1] (78 migraine without aura, 22
migraine with aura, ICHD-II criteria) and 90 patients aged 36-74 years
(mean age 46.5 years), with ischemic cardiopathy [2] were studied with
Polymerase Chain Reaction (PCR) for genetic polymorphism Apoliporo-
529
tein E. ApoE: 71 patients (71%) [1] and 63 (70%) [2] had an E3/E3
genotype; 15 (15%) [1] and 14 (15,5%) [2] had a E3/E4 genotype; 6
(6%) [1] and 9 (10%) [2] had a E2/E3 genotype; 2 (2%) [1] and 4
(4.4%) [2] had a E4/E4 genotype; 3 (3%) [1] and 0 [2] had a E2/E4
genotype; 0 [1] and 0 [2] had a E2/E2 genotype. Our results highlighted
a more or less equivalent prevalence of apoliprotein E gene polymorphisms in migraineurs and in subjects suffering from ischemic cardiopathy. Further research is required to conrm the ndings of the present
study in a larger sample and to elucidate the role of APOE polymorphism in headache.
Paper No.: 3190

FACTOR XIII VAL 34 LEU POLYMORPHISM IN MIGRAINE
AND CARDIOVASCULAR DISEASE
cellular solutions containing rosuvastatin (19.6-979 nM). From this rst

series of experiments, IC50 for block of IKr was estimated at 195 nM
(95% CI: [62;612]). These effects are thought to be clinically-relevant
since peak plasma concentrations of rosuvastatin can reach 200 nM after
a 40 mg dose. The inhibitory effects of rosuvastatin on IKr were further
evaluated on cells transiently transfected with transporters involved in
the disposition and cardiac distribution of the drug. Coexpression of the
BCRP efux transporter attenuated block of IKr by rosuvastatin 156.8
nM (174% (n=6) vs 428% (n=5); P<0.05); extent of block returned to
control values when transfection was conducted with three non-functional mutants of BCRP (Q141K, F208S, S441N). Transfection with the
inux transporter OATP2B1 was associated with an increased block
(483% (n=6) vs 428% (n=5)) while an intermediate phenotype was
observed with MDR1 (276% of block (n=6; P<0.05)), a weak efux
transporter of rosuvastatin. In conclusion, rosuvastatin blocks IKr at clinically relevant concentrations of the drug. Block of IKr by rosuvastatin
was modulated by the coexpression of transporters involved in the disposition of the drug in humans. Rosuvastatin could modulate cardiac repolarization, especially under conditions of decreased clearance.
V Pizza(1), F Infante(2), G Schiavo(2), E Lamaida(1), A Agresta(1), C

Colucci dAmato(3), A Bisogno(4), Anna Capasso(4)
(2) S. Luca Hospital, Department of Molecular Biology, Vallo della Lucania ((SA), Italy
(3) University of Naples, Naples, Italy
At present, it is contradictory to determine if the combination of certain
prothrombotic polymorphisms and migraine and also the risk to develop
ischaemic vascular disease. Recently, the common Val34Leu polymorphism of the A-chain factor XIII gene, associated with variations in factor XIII activity, has been suggested to play a signicant role in the
development of arterial and venous thrombotic disorders. Our study analysed the incidence of genetic polymorphism Factor XIII (V34L) in a
sample of migraineurs and a control group of the patients with ischemic
cardiopaty. In this study 100 consecutive patients aged 18-64 years
(mean age 33.7 years), suffering from migraine [1] (78 migraine without
aura, 22 migraine with aura, ICHD-II criteria) and 90 patients aged 3674 years (mean age 46.5 years), with ischemic cardiopathy [2] were
studied with Polymerase Chain Reaction (PCR) for genetic polymorphism Factor XIII (V34L). Factor XIII (V34L): 62 subjects (62%) [1]
and 40 (44.4%) [2] were heterozygous; 6 subjects (6%) [1] and 5 (5.5%)
[2] were mutated. These data evidenced that the incidence the factor XIII
Leu 34 allele in two population studied not evidenced meaningful differences. Therefore a role in the pathogenesis of such disturbances is hypothetical and deserves ulterior deepenings in more important casuistries.
Paper No.: 3162

TRANSPORT: PERSPECTIVES FOR DISEASE AND DRUG DISCOVERY
BLOCK OF THE IKR CURRENT BY THE HMG-COA REDUCTASE INHIBITOR ROSUVASTATIN IS MODULATED BY BCRP,
MDR1 AND OATP2B1 MEMBRANE DRUG-TRANSPORTER ACTIVITES
I Plante, M Frappier, F Belanger, Jacques Turgeon
CRCHUM, University of Montreal Faculty of Pharmacy and University
Central Hospital, Montreal, Canada
Block of the potassium current IKr (hERG) cause an increased risk of
drug-induced Long QT syndrome. Rosuvastatin is an orally effective
HMG-CoA reductase inhibitor that shows structural similarities (methanesulfonamide) with IKr blockers. The objective of our studies was to
assess block of IKr by rosuvastatin. Patch-clamp experiments were conducted, on hERG-stably transfected HEK293 cells superfused with extra-
Paper No.: 2663

DUAL EFFECT OF ANTAGONIST K-14585 ON PAR-2 PROINFLAMMATORY SIGNALLING
Robin Plevin, F Goon Goh, PY Ng
University of Strathclyde, SIPBS - Strathclyde institute for Pharmacy
and Biomedical Sciences, Glasgow, UK
Proteinase-activated receptor -2 (PAR-2) is a member of a family of
GPCRs activated by serine protease cleavage. PAR-2 is expressed cell
types such as neutrophils and keratinocytes, which are implicated in
inammatory diseases. In this study we examined the potential of using
a novel antagonist peptide, K-14585, assessing effects upon cell signalling pathways linked to inammation. In keratinocytes exogenously
expressing PAR-2, the activating peptide (AP) SLIGKV-OH stimulated a
six fold increase in inositol phosphate accumulation. This effect was
reduced by 60% following pretreatment with K-14585 (5 micromolar).
K-14585 (5 micromolar) pretreatment also reduced AP-stimulated p38
MAP kinase however, at 30 micromolar alone, K-14585 stimulated p38
MAP kinase by 4 fold. The Gq/11 inhibitor, YM-254890, reduced APstimulated p38 MAP kinase phosphorylation but not the K-14585
response. K-14585 (5 micromolar) also reduced AP-stimulated NFjB
phosphorylation and reporter activity, however 30 micromolar K-14585
stimulated a strong activation of NFjB reporter activity. K-14585 at low
concentrations reduced AP-induced IL-8 production however, at higher
concentrations increased IL-8. AP stimulated IL-8 production was
reduced by YM-254890 however, the response to K-14585 was unaffected. We conclude that K-14585 has dual effects on signalling functioning as both an antagonist and agonist. This indicates partial agonist
activity or the possibility of agonist-directed signalling. It also suggests
that PAR-2 may regulate p38 MAP kinase signalling by Gq/11-dependent and independent routes.
Paper No. 3354

PSAMMOMYS OBESUS A PROMISING ANIMAL MODEL FOR
TYPE 2 DIABETES
Rasmus Pold(1), C Godfredsen(2), S Lund(1), T Bodvarsdottir(2)
(2) Novo Nordisk, Mlv, Denmark
530
Animal models of type 2 diabetes is a valuable tool when developing
new pharmaceutical agents aimed at treating or preventing the disease.
Psammomys Obesus is a gerbil that in its natural environment feeds on a
low-calorie diet. If the gerbil is introduced to regular rat chow it develops
diabetes-like features within 3-5 days. However, the underlying mechanisms for this reaction to the change of diet are unknown. The aim of
this study was to investigate the pathological process underlying the diabetes-like features in Psammomys Obesus fed regular rat chow. This was
done by measuring the insulin sensitivity using a hyperinsulineamic euglycemic clamp, and by measuring b-cell mass using histological sections of the pancreas. Gerbils fed a regular rat chow diet for two weeks
had a decrease in glucose infusion rate during the clamp, clearly demonstrating insulin resistance. In addition, animals fed regular rat chow
increased b-cell mass when compared with gerbils on a low energy diet.
Most likely as a compensation for the insulin resistance. The Psammomys Obesus is a promising new animal model of type 2 diabetes. It
develops marked insulin resistance and compromised b-cell function
when introduced to regular rat chow for only 2 weeks, both key aspects
of type 2 diabetes. The animal model is therefore an obvious model to
consider when testing pharmaceutical treatments aimed at preventing the
disease.
Paper No.: 3339

A STUDY OF THE LONGITUDINAL UTILIZATION OF
TRADITIONAL NON-STEROIDAL ANTI-INFLAMMATORY
DRUGS AND COX-2 SELECTION INHIBITORS USING A
PHARMACY BASED APPROACH
Emilian Damian Popovici(1), MG Popovici(2), C Trandarescu(3),
M Kolliali(2), IM Malita(2), LM Baditoiu(1)
(1) Victor Babes University of Medicine and Pharmacy, Department of
Epidemiology, Timisoara, Romania
Pharmacology, Timisoara, Romania
Pharmacology Chemistry, Timisoara, Romania
The extent of heterogeneity of drug utilization among NSAIDs users is
less studied according to the literature. In our investigation we aimed to
explore tendencies of prescribing traditional NSAIDs and selective
COX-2 inhibitors. We studied the longitudinal prescribing of NSAIDs
regarding age groups, gender, accutely and chronicaly ill patients, application form, amount and strength of the drug, for a period of one year, in
a Romanian pharmacy. We retrospectively analysed the pharmacy electronic databases which covered the period from November 1, 2008 to
October 31, 2009. The selected data includs NSAIDs and COX-2 inhibitors prescribed by general practitioners, in a Romanian city (Timisoara)
in the study period. The study population consisted of 1150 persons
(56.5% women) with at least one NSAID prescription recorded. A total
of 1318 prescriptions related to 10 different NSAIDs were analysed. Ibuprofen was the most frequently prescribed NSAID, followed by ketoprofen, diclofenac and celecoxib. Almost half of patients (48.4%), aged
18 years or younger, named incidental users, were exposed to a NSAID
30 days or less. Only ibuprofen has been liberated for the use in the
pediatric age group. In conclusion, this study shows that NSAID users
cannot be viewed as an homogenous group of patients and also indicates
that pharmacies can be considered as an important source of exposure
information and that the longitudinal prescribing patterns of NSAIDs use
are rather similar to those in other EU countries.
Paper No.: 2801

HEALTH AND DISEASE
EFFECT OF CHRONIC SHEAR STRESS ON ENDOTHELIAL
CELL MORPHOLOGY IN VITRO AND IN VIVO: RELEVANCE
TO AREAS AT RISK OF ATHEROSCLEROSIS IN THE AORTA
Claire MF Potter, MH Lundberg, HH Gashaw, J Gorelik, AV Moshkov,
LS Harrington, PD Weinberg, JA Mitchell
Imperial College London, National Heart and Lung Institute, Department
of Cardiothoracic Pharmacology, London, UK
The haemodynamic wall shear stress experienced by endothelial cells
varies from region to region within the vasculature. These differences are
exemplied by two regions of the aortic arch. Endothelial cells of the
arch outer curvature are thought to experience high laminar shear stress,
are elongated, aligned and protected from inammation and atherosclerosis. Endothelial cells of the inner curvature are thought to experience low
oscillatory shear stress, are randomly orientated and susceptible to
inammation and atherosclerosis. Endothelial cells in culture are routinely studied under static conditions where they appear non-aligned with
cobblestone morphology. We used an orbital shaker to model high
(HS; 5dyn/cm2) and low (LS; 2dyn/cm2) shear stress in vitro and confocal and scanning ion conductance microscopy to image resultant effects
on endothelial cell morphology. Porcine aortic endothelial cells were cultured in 6 well plates under static conditions or were sheared, as shown
previously (Potter et al., PA2online P018 December 2009). Alignment
was assessed by blinded scoring of 385lm2 areas of the well exposed to
high, low or no shear stress, using an arbitrary scale where 0 represents
no alignment and 3 represents most cells in a frame being aligned in one
direction. The rank ordering of alignment scores of endothelial cells
grown for 7 days was HS(1.460.21)>LS(0.690.14)>static(0.180.09)
(n=12), which is consistent with our observations of endothelial cell morphology in the predicted high and low shear stress regions of the mouse
aortic arch in vivo. Other parameters including cell number, CD31
expression, cell volume and topographical changes were also measured.
Paper No.: 3391

CHARACTERIZATION OF PHILANTOTOXIN ANALOGUES AS
SUBTYPE-SELECTIVE AMPA RECEPTOR ANTAGONISTS
Mette Poulsen, S Lucas, K Strmgaard, AS Kristensen
University of Copenhagen, Department of Medicinal Chemistry,
Copenhagen, Denmark
Fast excitatory transmission in the brain relies almost entirely on activation of ionotropic glutamate receptors (iGluRs). iGluRs are ligand-gated
ion channels which are involved in many brain functions. Overstimulation of iGluRs triggers neuronal death and is thought to contribute to a
range of neurological disorders for which iGluRs are considered important drug targets. iGluRs exists as homo- or heteromeric-assemblies of
four subunits from a pool of 17 subunits. The resulting iGluRs subtypes
can be divided into three main subfamilies: Kainate-, NMDA- and
AMPA-receptors. Several compounds exist with excellent subfamilyselectivity. However, pharmacological separation of individual subtypes
is difcult to achieve with currently available drugs and at present no
existing antagonists exhibit more than 5‐fold selectivity between
AMPA-receptor subtypes. Therefore, identication of novel subtypeselective ligands could is of importance as valuable pharmacological
tools as well as potential new therapeutic agents. Polyamine toxins are a
group of small molecules found in spiders and wasps. The natural polyamine toxin PhTX-433 is a non-selective use- and voltage-dependent
channel blocker of iGluRs. PhTX-433 contains an aromatic head-group
and a polyamine-tail. Recent work has proposed that substitution of one
of the secondary amino groups within the polyamine-tail with a methy-
531
lene group generates AMPA-receptor selectivity. Furthermore, systematic
variation of the remaining secondary amino group resulted in changes of
potency. In this study, we explore the effect of the systematic variation of
the polyamine-tail in relation to subtype-selectivity among the most prevalent AMPA-receptor subtypes.
Paper No.: 1063

MEK1/2 INHIBITION IMPROVES NEUROLOGICAL OUTCOME AND ABOLISHES CEREBROVASCULAR ETB AND 5HT1B RECEPTOR UPREGULATION AFTER SUBARACHNOID
HEMORRHAGE IN THE RAT
Gro Klitgaard Povlsen(1), C Larsen(2), M Rasmussen(1), L Edvinsson(1)
(1) Glostrup Research Institute, Glostrup Hospital, Department of Clinical Experimental Research, Glostrup, Copenhagen, Denmark
(2) Glostrup Hospital, Department of Neurosurgery, Glostrup, Copenhagen, Denmark
Delayed cerebral ischemia remains a major cause of death and disability
after subarachnoid hemorrhage (SAH). Upregulation of endothelin B
(ETB) and 5-hydroxytryptamine 1B (5-HT1B) receptors have been demonstrated in smooth muscles of cerebral arteries after experimental SAH,
and it has been hypothesized that vasoconstrictor receptor upregulation
contributes to delayed cerebral ischemia. The receptor upregulation is
mediated by the MEK-ERK1/2 signalling pathway. We here investigated
whether treatment with the specic MEK1/2 inhibitor U0126 could prevent cerebrovascular receptor upregulation and improve functional outcome after experimental SAH in rats. SAH was induced by injection of
autologous blood into the basal cisterns. U0126 treatment was administered intracisternally at 6, 12, 24 and 36 hours after SAH. Smooth muscle ETB and 5-HT1B receptor expression and functionality was studied
in isolated cerebral artery segments by immunohistochemistry and myograph contractility studies. Gross sensorimotor function of the rats was
assessed by a rotating pole test. We demonstrate that SAH induces
upregulation of ETB and 5-HT1B receptors in cerebrovascular smooth
muscles and treatment with U0126 abolishes this process. Furthermore,
SAH resulted in sensorimotor decits that were strongly alleviated by
U0126 treatment. These ndings suggest that MEK-ERK1/2-mediated
cerebrovascular vasoconstrictor receptor upregulation is critically
involved in delayed cerebral ischemia after SAH, and MEK1/2 inhibition
may be a promising novel SAH treatment strategy.
Paper No.: 1755

HEALTH AND DISEASE
IMPORTANCE OF CEREBROVASCULAR VASOCONSTRICTOR
RECEPTOR UPREGULATION FOR THE LONG-TERM OUTCOME OF EXPERIMENTAL SUBARACHNOID HEMORRHAGE IN RATS
tors during the rst 4 days after experimental SAH in rats and the correlation in time between SAH-induced neurological decits and
cerebrovascular receptor upregulation. SAH was induced by intracisternal
injection of autologous blood. At 2, 3 and 4 days after induction of SAH
smooth muscle ETB and 5-HT1B receptor expression and functionality
was studied in isolated cerebral artery segments by immunohistochemistry and myograph contractility studies. Neurologica decits were
assessed daily by general behaviour observation, a rotating pole sensorimotor test and an established neurology scoring paradigm. We demonstrate that SAH induces upregulation of ETB and 5-HT1B receptors in
cerebrovascular smooth muscles with the highest receptor expression and
functionality levels observed at day 3 after SAH. We also demonstrate
that development of SAH-induced neurological decits correlates in time
with the degree of cerebrovascular receptor upregulation. In conclusion,
cerebrovascular receptor upregulation after experimental SAH correlates
in time with the development of neurological decits, and is of major
importance for the severity of the functional outcome of an SAH.
Paper No.: 1969

PARENTERAL ALANYL-GLUTAMMINE IN CRITICALLY ILL
PATIENTS: A BAYESIAN META-ANALYSIS OF PUBLISHED
TRIALS
Lorenzo Pradelli(1), O Zaniolo(1), S Iannazzo(1), M Eandi(2)
(1) Adres srl, Outcomes Research, Torino, Italy
(2) University of Turin, Department of Clinical Pharmacology, Turin,
Italy
Glutamine, although abundant in human tissue, can become conditionally
essential in clinical conditions with hyper-catabolism and glutathione
depletion (burns, pancreatic necrosis, surgical complications), but has not
been added to parenteral nutrition solutions for a long time, for its
alleged non-essentiality and the low solubility and stability in aqueous
solutions, which have been solved by conjugation with alanine. In 2002
a meta-analysis of available trials conducted with alanylglutamine dipeptide revealed signicant reductions of mortality, infections and ICU
length of stay. Since then, data from other trials have become available.
Aim of the present study is to update the treatment effect estimates by
means of a series of Bayesian random effects models. We searched EMBASE and Medline for clinical trials of standard total parenteral nutrition
(TPN) vs. TPN + parenteral alanylglutamine in critically ill patients
reporting hospital mortality, relative ICU-incident infection rate, and relative hospital length of stay. Outcomes from 15 trials and 781 patients
were retrieved. For each outcome, a Bayesian random effects model was
specied, in which the treatment effect observed in the individual trials is
assumed to be drawn from a common distribution and expressed as a relative risk or duration. The models estimate a relative mortality of 0.70
(95% CrI: 0.46-0.97), a relative infection rate of 0.71 (95% CrI: 0.490.97), and a relative length of stay of 0.91 (95% CrI: 0.76-1.00). In conclusion, the available evidence supports a highly credible benecial effect
of alanylglutamine on mortality, infections and hospital length of stay in
ICU-admitted critically ill patients.
Gro Klitgaard Povlsen, S Johansson, C Larsen, L Edvinsson

Glostrup Research Institute, Glostrup Hospital, Department of Clinical
Experimental Research, Copenhagen, Denmark
Delayed cerebral ischemia is a major cause of death and disability after
subarachnoid hemorrhage (SAH). Upregulation of vasoconstrictor endothelin-B (ETB) and 5-hydroxytryptamine-1B (5-HT1B) receptors have
been demonstrated in smooth muscles of cerebral arteries after SAH, and
it has been hypothesized that this contributes to delayed cerebral ischemia. However, long-term development of cerebrovascular receptor
changes and the importance of these changes for the long-term functional
outcome of SAH have not been studied. The aim of the present study
was to investigate upregulation of cerebrovascular vasoconstrictor recep-
Paper No.: 3355

PROVOCATIVE POTASSIUM HANDLING TO PREDICT
CHANGES IN AMBULATORY SERUM POTASSIUM WITH
DUAL RENIN-ANGIOTENSIN-ALDOSTERONE (RAAS)
BLOCKADE
Richard Preston, D Afshartous
University of Florida, Department of Medicine, Division of Clinical
Pharmacology, Miamai, FL, USA
532
Dual blockade of the RAAS with an aldosterone-receptor antagonist
added to an angiotensin-converting-enzyme inhibitor effectively reduces
cardiovascular and renal events. Unfortunately, dual RAAS blockade can
produce unpredictable hyperkalemia in patients with chronic kidney disease (CKD). We sought to determine whether our dynamic potassium
handling protocol could predict changes in ambulatory serum potassium
with dual RAAS blockade. We conducted a 4-week crossover trial in 18
CKD patients with GFR 25-65 mL/min. At baseline we measured hourly
potassium excretion (UkV, mmol/h) and serum potassium (K, mmol/L)
following 35 mmol oral potassium. We determined ambulatory serum
potassium (aK, mmol/L) during and after 4 weeks of treatment with
40 mg lisinopril/25 mg spironolactone or placebo. Our preliminary analyses suggested dynamic potassium handling could possibly predict aK.
Here we employ a series of models to elucidate the utility of dynamic
potassium handling in predicting changes in ambulatory K: 1)-Linear
regressions of summary measures of ambulatory potassium versus summary measures dynamic potassium handling, 2)- Mixed models with aK
as dependent variable and summary measures from dynamic potassium
handling as covariates, and 3)-multivariate mixed models of the joint trajectories of weekly aK and hourly potassium handling variables. Model
t was assessed via residual plots, likelihood ratio tests and AIC. Across
the statistical methods we found that dynamic potassium handling predicts changes in aK in response to dual RAAS blockade. Our results
present the possibility of an ofce test to predict hyperkalemia and to
augment individualized prescription of dual RAAS blockade.
Paper No.: 2963

PHARMACOLOGICAL PATTERNS OF TREATMENT IN
ACUTE MANIC EPISODE: A SPANISH EPIDEMIOLOGICAL
MULTICENTRIC STUDY
Esther Prieto, J Galan
AstraZeneca Spain, Department of Neurosciences, Madrid, Spain
Introduction: Despite having extensive information from clinical trials
about the efcacy of treatments for the acute manic episode of bipolar
disorder, there are few data about the true management in clinical
practice. To our knowledge, there is only one important study on how
Spanish psychiatrists treat manic patients (Montoya A, Actas Esp
Psiquiatr 2007; 35: 315-322). Our objective is to provide more data
about pharmacological patterns of treatment of acute manic episode in
clinical practice. Patients: Descriptive, non-interventional, transversal
study including bipolar patients hospitalized within previous 24 hours
due to a manic episode with a YMRS score >20. Results: 44 centers
participated (242 patients involved, 235 analyzable). There were 133
(56.6%) females and 102 (43.4%) males. The polarity of the rst episode was manic in 153 patients (65.1%) and depressive in 75
(31.9%). 42 (17.87%) patients had no previous manic episodes. Mean
YMRS score was 33.276.95. 180 (76.59%) patients were treated with
any second generation antipsychotics (SGA), 148 (62.97%) with any
mood stabilizers, 116 (49.36%) with any benzodiazepines and 39
(16.59%) with any rst generation antipsychotics (FGA). 16 (6.80%)
included any antidepressants in their treatment. Among patients treated
with SGA, 164 (91.11%) patients were in combined treatment. The
SGA most frequently used was quetiapine followed by olanzapine and
risperidone. 38 out of 39 patients with FGA were in combined treatment. Conclusion: There is an important heterogeneity in the treatment
of acute mania as well as a high level of polypharmacy, in many
cases not supported by recommendations of clinical guidelines.
Paper No.: 2887

EMOTIONAL STATE DEPENDENCE OF THE PRESSOR
RESPONSE IN NORMAL DOGS
Joseph Prinsen(1), NS Archambault(2), AJ Lange(2), NB Olivier(2)
(1) Michigan State University, Department of Pharmacology &
Toxicology, East Lansing, MI, USA
(2) Michigan State University, College of Veterinary Medicine, East
Lansing, MI, USA
It is widely accepted that blood pressure is partially modulated by physiologically relevant emotional responses in humans. Moreover, clinicians
are acutely aware of transient white coat hypertension elicited by the
emotional state of the patient as a complicating factor in the diagnosis of
chronic hypertension and the risks arising from treatment based on these
numbers. We sought out to rst, determine if an emotionally driven
increase in blood pressure is seen in dogs (pressor response), and second,
to better understand the mechanism mediating this response. Dogs were
instrumented with devices that would allow their unfettered movement
and simultaneously record a dynamic electrocardiogram and arterial pressure. Separate cohorts of dogs were additionally instrumented with xedrate His-bundle pacers to provide a control for heart rate related pressure
changes. Isolated dogs were treated with human interaction, both verbally and through physical restraint. This was repeated with atenolol or
prazosin treatment. Verbal interaction resulted in a higher change in
mean systolic pressure (32.9 mmHg) compared to physical restraint
(7.3 mmHg). Atenolol attenuated the peak pressor response while prazosin delayed the development of a peak response. We conclude that the
pressor response is at least in part sympathetically mediated, is sufcient
to cloud the diagnosis of hypertension and should be considered prior to
the institution of antihypertensive therapy.
Paper No.: 1622

ADULT NEUROGENESIS AND NOVELTY-INDUCED C-FOS
EXPRESSION IN THE HIPPOCAMPUS OF GLUA1
AMPA/GLUTAMATE RECEPTOR KNOCKOUT MICE
Chiara Procaccini(1), T Aitta-aho(1), K Jaako-Movits(2), A Zarkovski(2),
A-M Linden(1), E Korpi(1)
(1) University of Helsinki, Biomedicum Helsinki, Institute ofBiomedicine/Pharmacology, Helsinki, Finland
(2) University of Tartu, Department of Pharmacology, Turku, Finland
In the central nervous system the fast excitatory glutamatergic transmission is mediated by AMPA, NMDA and kainate ionotropic receptors.
AMPA receptors are homo- and hetero-tetrameric ion channels formed
by four different subunits, GluA1-4. Expression of AMPA receptors is
dominant in the hippocampus, a region related to memory processing,
learning, novelty comparison and emotions. The adult hippocampal dentate gyrus retains active neural progenitor cells which eventually integrate
in the existing neural circuitries. Adult neurogenesis is linked to several
cognition-dependent operations and may represent a measure of hippocampal functionality. We employed a knockout mouse strain (GluA1-/-)
in which the GluA1 subunit is inactivated and as controls the correspondent wildtype littermates (GluA1+/+). We evaluated the dynamics of adult
neurogenesis in the dentate gyrus of GluA1-/- mice, employing BrdU and
Ki-67 as mitotic markers and analyzed which hippocampal sub-areas are
activated when GluA1-/- mice are exposed to novelty, using c-Fos protein
as a marker of neuronal activity. We observed in dentate gyrus of
GluA1-/- mice reduced proliferation and increased survival of newly born
cells but no changes in the differentiation into neuronal or glial phenotype. We also found increased c-Fos expression in the dorsal dentate
gyrus and CA3 and in the caudal CA1 and CA3 in the GluA1-/- mouse
533
hippocampal formation. Our ndings indicate that the lack of the GluA1
subunit impacts hippocampus neuronal activation and adult neurogenesis.
These alterations might relate to the abnormal behavioural phenotypes of
GluA1-/- mice in models of various neuropsychiatric diseases.
Paper No.: 1228

TEACHING CLINICAL PHARMACOLOGY AND
PHARMACEUTICAL MEDICINE IN SERBIA:
POSTGRADUATE STUDIES
Milica Prostran-Veselinovic, Z Todorovic, R Stojanovic
University of Belgrade School of Medicine, Department of Pharmacology, Clinical Pharmacology & Toxicology, Belgrade, Republic of Serbia
Fentanyl is the prototype of the 4-anilidopiperidine class of synthetic opioid analgesics. From the aspect of structure-activity relationship (SAR)
studies, it was interesting to introduce methyl, ethyl, propyl, iso-propyl,
butyl, benzyl, phenethyl, as well as carbomethoxy group in the position
3 of the piperidine ring and test the newly synthetized fentanyl analogues
(all in the racemic form) for the antinociceptive activity by using tailimmersion test in rats. It has been revealed that the presence of an alkyl
group substituent in position 3 of the piperidine ring generally decreases
or completely inhibits the analgesic activity compared to fentanyl. The
exceptions are the (+/))cis-3-Me fentanyl (about 8 x fentanyl) and (+/
))cis-3-Et fentanyl (about 1.5 x fentanyl). With increasing voluminosity
of the alkyl group, the potency decreases rapidly. The relative cis/trans
stereochemistry is important since the cis isomers are 1.5-V6 times more
active than the trans isomers. Also, it was found that the introduction of
a carbomethoxy group in position 3 of the piperidine ring of fentanyl
decreased antinociceptive activity by a factor of 2 and 10 in (+/))cis and
(+/))trans 3-carbomethoxy fentanyl, respectively. (+/))cis 3-carbomethoxy fentanyl seems to be equipotent to the (+/))cis 3-propyl fentanyl.
The observed similarity in antinociceptive potency could result from the
similar voluminosity of the 3-carbomethoxy and 3-propyl group. In conclusion, it seems that a steric factor (voluminosity of the group and the
cis/trans isomerism) has a predominant role in the antinociceptive
potency of 3-substituted fentanyl analogues, while the nature of the substituent is probably irrelevant.
Paper No.: 1751

IN VIVO UPREGULATION OF CENTRAL NICOTINIC RECEPTORS INDUCED BY METHYLENEDIOXYMETHAMPHETAMINE (MDMA) AND ITS ASSOCIATION WITH NICOTINE
David Pubill, S Garcia-Rates, E Escubedo, J Camarasa
University of Barcelona Faculty of Pharmacy, Department of Pharmacology and Therapeutic Chemistry, Barcelona, Spain
We had demonstrated that MDMA (ecstasy) has afnity for nicotinic acetylcholine receptors (nAChRs) and can induce their upregulation in PC
12 cells as nicotine does. Here we assessed whether such upregulation
takes place in vivo and the consequences of its association with nicotine
(NIC). Male adult Sprague-Dawley rats were treated either with nicotine
bitartrate dihydrate alone (2 mg/kg, s.c., b.i.d.) for 10 days or associated
with MDMA (20 mg/kg, s.c, b.i.d) during the last four days. Also,
another two groups received MDMA alone or saline following the same
schedule. All were killed on day 11. Binding experiments with
[3H]epibatidine (EB) and [3H]methyllycaconitine (MLA) were performed in homogenates of brain areas to quantify heteromeric and homomeric a7 nAChRs, respectively. MDMA did not modify the regulatory
effects of NIC in the striatum. In prefrontal cortex NIC and MDMA
induced each signicant increases in [3H]EB binding (around 30%) with
respect to saline-treated rats, that were signicantly potentiated (up to

70%) when both drugs were associated. Also in this area, [3H]MLA
binding was increased about 40% with NIC+MDMA but not when they
were given alone. In the hippocampus MDMA potentiated the a7 regulatory effects of NIC (25% to 52% increase) but alone was devoid of
effect. Specic immunoprecipitation of solubilised receptors suggests that
the upregulated heteromeric nAChRs contain a4 and b2 subunits. Western blots with specic a4 and a7 antibodies showed no signicant differences between the groups, pointing that upregulation is due to
postranslational events rather than increased receptor synthesis.
Paper No.: 1497

PHARMACOEPIDEMIOLOGY AS AN OPPORTUNITY FOR
PROGNOSTIC STUDIES IN RARE DISEASES: THE EXAMPLE
OF GIANT CELL ARTERITIS AND THE FRENCH APOGEE
RITE EN POPULATION GENE
RALE)
COHORT (ARTE
Gregory Pugnet(1), L Sailler(1), R Bourrel(2), A Sommet(1), JL Montastruc(1), M Lapeyre-Mestre(1)
(1) Universite de Toulouse, EA3696, Faculte de Medecine, Toulouse,
France
(2) Caisse Nationale dAssurance Maladie, Echelon Midi-Pyrenees, Toulouse, France
Long term cohort studies are very costly and time consuming when
based on patient cases. Pharmacoepidemiologic studies in automated databases may be an opportunity to answer to major questions at a low
cost. We illustrate this possibility in a population based cohort of giant
cell arteritis patients (GCA). Incident GCA cases (ICD-10 code M31.5
or M.31.6) occurring between January 2005 and December 2008 were
extracted from the French Health Insurance System (FHIS) database of
the Midi-Pyrenees county, South of France. The database covers about
1.6 millions patients. The characteristics of each reimbursed medical care
or drug are automatically and prospectively registered. The primary endpoint was corticosteroids (CS) withdrawal attested by the reimbursement
of a less than 0.5 prednisone delivered daily doses (1 pDDD =
10 mg)monthly during at least 6 months. We identied 103 incident
cases of GCA (80 women and 23 men) aged 77 years (range: 51-91). CS
were withdrawn in 27.2% (8.6) of the cases at month 42 (Kaplan-Meier
estimation). In the multivariate Cox analysis, male gender was associated
with CS withdrawal (OR: 2.2 [1.01-4.7]; P<0.05). There was no inuence of age, location, the initial pDDD or exposure to various drugs on
the outcome. The frequency of CS withdrawal seemed lower in the general population than usually reported in hospital-based cohorts and the
inuence of female gender has yet not been reported. Using further data
extractions yearly, this pharmacoepidemiologic approach will offer major
perspectives for the long-term follow-up of a rare disease in the general
population.
Paper No.: 1788

DISEASES
MILDRONATE REGULATES INOS AND MYELIN
EXPRESSION IN THE RAT MODEL OF EXPERIMENTAL
AUTOIMMUNE ENCEPHALOMYELITIS
Jolanta Pupure, J Rumaks, N Karajeva, S Isajevs, B Jansone, S Svirskis,
V Klusa
University of Latvia, Department of Pharmacology, Riga, Latvia
Multiple sclerosis (MS) is the most prevalent autoimmune disorder of the
central nervous system with neurological symptoms caused by inamma-
534
tion and demyelination. Besides immune-mediated cytotoxicity, the
involvement of mitochondria in MS pathology is strongly suggested
(Kalman, Curr Neurol Neurosci Rep 2006;3:244-52). The aim of the
present study was to investigate the effects of mildronate, which has been
shown previously as a regulator of mitochondrial functions (Pupure et al,
Cell Biochem Func 2008;26:620-31). Experimental autoimmune encephalomyelitis (EAE) was induced in Lewis female rats by myelin basic protein (MBP) fragment (p68-86) administered subcutaneously (100ug).
Mildronate (100 mg/kg) was administered intraperitoneally once a day
for 2 weeks before induction of EAE. The onset and progression of EAE
signs were monitored daily using clinical scale described elsewhere (SaYoon Kang et al, J Clin Neurol 2009;5:39-45). 14 days after immunization rats were sacriced, lumbal spinal cord and optic nerve were dissected from each rat. Samples were processed for immunohistochemistry
to evaluate iNOS and myelin expression. MBP induced iNOS overexpression in spinal cord gray matter, as well as in Schwann cells of the
optic nerve, compared to saline. Mildronate coadministration with MBP
signicantly reduced iNOS expression in the spinal cord, whereas in
Schwann cells it showed only a tendency. MBP decreased myelin expression in the optic nerve compared to saline; mildronate restored partially
this decrease. Our preliminary data demonstrate that mildronate exhibits
a protective action against MBP-induced inammatory and degenerative
processes in EAE model, probably via mitochondria-protecting mechanisms.
Acknowledgements: ESF Nr.2009/0217/1DP/1.1.1.2.0/09/APIA/VIAA/
031.
Paper No.: 3396

CHIMAERING OREXIN RECEPTORS REVEAL
SELECTIVITY-CONFERRING AREAS FOR OREXINS
Jaana Putula(1), P Turunen(1), L Johansson(2), R Ra(1), J Nasman(3),
J Kukkonen(1,2,4)
(1) University of Helsinki, Department of Veterinary Biosciences,
Helsinki, Finland
(2) Uppsala University, Department of Neuroscience, Uppsala, Sweden
bo Akademi University, Department of Biosciences, Turku, Finland
(3) A
(4) Minerva Medical Research Institute, Helsinki, Finland
Orexin A and B (OxA and OxB, respectively) are neuropeptides that
were rst found as regulators of feeding behavior in rats. Later they were
also found to regulate arousal state and stress response. Orexin receptors
1 and 2 (OX1R and OX2R, respectively) belong to the G protein-coupled
receptor family. While orexin-A is non-selective, orexin-B is believed to
display some preference for the OX2R.We have here designed six orexin
receptor chimeras to study the receptor sites conferring selectivity for orexin peptides. Three cutting points were designed in OX1R and OX2R
by PCR. The cutting points were located approximately one third, one
half and two thirds of the OX1R and OX2R peptide lengths. N-terminals,
cut from different cutting point, were switched with each other to create
two-piece receptor chimeras.HEK-293 cells were transiently transfected
with the orexin receptor chimeras and wild type OX1R and OX2R, and
the potency of OxA, OxB and the synthetic agonist ala11,d-leu15-OxB
to elevate intracellular Ca2+ was measured using uo-4. The results point
out the areas of importance for the receptor subtype selectivity. The Nterminal half of the receptor appears clearly more important than the Cterminal.
Paper No.: 1399

BEHAVIORAL INHIBITION IN RATS: A MODEL TO EXAMINE
MECHANISMS UNDERLYING THE RISK TO DEVELOP
ANXIETY AND DEPRESSION
Chao Qi(1,2), P Roseboom(1), S Nanda(1), J Lane(1,3), J Speers(1), N
Kalin(1,3)
(1) University of Wisconsin, Department of Psychiatry, Madison, WI,
USA
(2) University of Wisconsin, Molecular & Cellular Pharmacology Training Program, Madison, WI, USA
(3) University of Wisconsin, Department of Psychology, Madison, WI,
USA
Behavioral inhibition (BI) is an adaptive defensive response to threat that
has been evolutionarily conserved. Extreme BI in childhood is a temperamental disposition for the development of psychopathology. Our goal
was to develop a rodent model of BI that is elicited by predator exposure. The ferret exposure test consists of placing a rat in a housing cage
in the ferret colony room for 15 min and rating behavior for BI. We
show that predator-induced BI is stable across development when measured in rats (n = 37) in adolescence and again in adulthood (r = 0.475,
p < 0.01). BI is reduced by the anxiolytic diazepam (1 mg/kg, 30 min
pretreatment; n = 8/group; p < 0.05), and BI is associated with heightened paraventricular nucleus of the hypothalamus (PVN) and CA3 activity as measured by expression of the immediate early gene homer1a (r =
0.379, p < 0.05; r = 0.309, p < 0.05 respectively). Finally, we show that
rats with high levels of BI display disruptions in appetitive behavior as a
consequence of prior threat exposure compared to rats with low levels of
BI (p < 0.001, n = 7 and 8). Taken together, these ndings show that
rodent BI is a stable, trait-like characteristic that is associated with maladaptive responding. This model can be useful in identifying the molecular underpinnings for the childhood risk to develop psychiatric illness.
This work was supported by NIH grant MH43454, Meriter Hospital and
UW HealthEmotions Research Institute.
Paper No.: 968

THE MYOCARDIAL PROTECTIVE AND ANTIOXIDATIVE
EFFECTS OF L-CARNITINE ON PATIENTS WITH CHD AFTER
PCI
Haijun Qu(1), F Jing(1), Z Sui(1), Y Cao(1), Z Han(1), L Zhu(1),
C Wang(2)
(1) Qingdao University, Medical College, The Afliated Hospital,
Qingdao, PR China
(2) Qingdao University, Medical College, Department of Pharmacology,
Qingdao, PRChina
To investigate the myocardial protective effects of L-carnitine (L-C) on
patients with coronary heart disease (CHD) after primary percutaneous
coronary intervention (PCI), 20 cases of patients with non-ST-segment
elevation acute coronary syndrome (NSTE-ACS) undergoing interventional therapy were randomly divided into two groups: the control group
(10 cases) and the treatment group (10 cases). The treatment group was
given L-C 5 g intravenous injection 30 min before and during PCI, and
then L-C 10 g/d for three days after PCI. The control group was given
the same dose of normal saline intravenous infusion. The serum concentration of creatine kinase isoenzyme (CK-MB), troponin I (c-TnI), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione
peroxidase (GSH-PX), catalase (CAT) and total antioxidant capacity (TAOC) were detected before and after PCI. The results showed that L-C
inhibited the increase of the serum concentration of CK-MB, c-TnI and
535
MDA after PCI signicantly. Meanwhile, the decrease of serum concerntration of SOD, GSH-PX, CAT and T-AOC after PCI were abrogated in
the presence of L-C. These results suggested that L-C could increased
the activity of antioxidant enzymes and might be benecial for the treatment of reduce myocardial injury on patients with CHD after coronary
intervention therapy.
Q2008C04.)
Key Words: L-carnitine (L-C), coronary heart disease, percutaneous coronary intervention (PCI), antioxidant
Paper No.: 2843

PHARMACOKINETICS, PHARMACODYNAMICS AND
BIOEQUIVALENCE OF DEXAMETHASONE LIQUID AND
TABLET FORMULATIONS
Christian Queckenberg(1), B Wachall(2), P Di Gion(1), D TomalikScharte(1), M Abdel-Tawab(3), U Fuhr(1)
(1) University of Cologne Hospital Department of Pharmacology,
Cologne, Germany
(2) InfectoPharm GmbH, Heppenheim, Germany
(3) Central Laboratory of German Pharmacists, Germany
Dexamethasone is a synthetic glucocorticoid being used extensively
worldwide, e.g. for anti-allergic, anti-inammatory and immunosuppressive treatment, and for treatment of croup in children. For the latter,
liquid preparations are especially suitable, because both exact dosing and
proper intake are facilitated compared to other dosage forms. In a randomized controlled cross-over trial in 24 healthy volunteers we evaluated
pharmacokinetics, bioavailability and safety of a liquid oral formulation
relative to a tablet. As a pharmacodynamic variable, we measured plasma
concentrations of endogenous cortisol (pre-/post-dose). Both preparations
were administered as single doses, containing 2 mg of dexamethasone
each. Blood samples were taken up to 24 hours post-dose. Quantication
was carried out by a validated specic and sensitive HPLC method.
Non-compartmental pharmacokinetic parameters were compared between
treatments using standard bioequivalence methods. Both preparations
were well tolerated and showed similar pharmacokinetic and pharmacodynamic proles. Mean AUC0-t, AUC0-8, and Cmax were 37.8 ngh/ml,
46.0 ngh/ml and 9.35 ng/ml, respectively for the liquid and 41.3 ngh/
ml, 48.1 ngh/ml and 9.17 ng/ml, respectively for the tablet formulation.
The point estimates and 90% condence intervals for AUC0-t, AUC0-8,
and Cmax ratios (liquid vs. tablet formulation) were 91.42% (82.05101.86%), 95.72% (84.46-108.5%) and 102.04% (86.94-119.76%),
respectively. Thus, point estimates and 90% condence intervals were
within the bioequivalence range of 80-125% for all relevant parameters,
including the pharmacodynamic parameter AUEC (area under the effect
curve). The liquid test formulation fulls the bioequivalence criteria of
the European Committee for Proprietary Medicinal Products and therefore may be used to replace tablets for paediatric use.
Paper No.: 436

CARBAMAZEPINE INDUCED LEUCOPENIA IN 20-YEAR-OLD
MALE PATIENT
Vesna Radovic
STADA Hemofarm A.D., Institute Hemofarm A.D., Belgrade, Republic
of Serbia
This report was received from a physician via the Medicines and Medical
Devices Agency of Republic of Serbia (ALIMS) on 2009-Sep-19. This
patient started treatment with Tegretol CR (INN: carbamazepine) for

fronto-temporal epilepsy, in 2009. In Aug 2009, he experienced leucopenia
(values not provided), and the physician reduced the dose of Tegretol. When
the leucopenia was diagnosed, the patient was taking 1000 mg/day, and then
it was reduced to 800 mg/day, with the leukocyte count monitored. Concomitant treatment included phenobarbitone, Melleril (INN: thioridazine)and
Zoloft (INN: sertraline). The outcome of the event was not provided (continue). Patient prolonged hospitalisation.
Key words: carbamazepine, leucopenia, prolonged inpatient hospitalisation
Paper No.: 3267

EFFECT OF CITRULLUS COLOCYNTHIS ON SERUM
GLUCOSE, LIPIDEMIC PROFILE AND C-PEPTIDE CHANGES
IN PATIENTS WITH TYPE II DIABETES MELLITUS
Alireza Rahbar, I Nabipoor
Bushehr University of Medical Sciences, Department of Nutrition,
Bushehr, Iran
Introduction: It is believed that there is ineffective glucose metabolism in
diabetic patients. Traditional medicine has introduced Citrullus colocynthis L. Schrad for improving insulin action. The peel of the fruit contains
colesentin, colesentinin, Glycosidic phytosterols, gum, pectin and albominoeds. The aim of this study was to determine the in vivo hypoglycemic effects of Citrullus colocynthis L. Schrad in human being. Materials
and Methods: Fifty diabetic patients were randomly divided (n=25) to
treated by the Citrullus colocynthis dried powder (300mg) daily and placebo group for 12 weeks. At rst and at the last, the serum FBS, TG, Chol,
LDL-C, HDL-C, SGOT, SGPT and C-Peptide were measured with enzymatic methods (pars Azemoon Company). Results: The signicance of differences within groups was calculated by Paired T-test, and between them by
analysis of covariance. There was no signicant differences within and
between treated and placebo groups during our treatment in FBS
(167.4766.77 to 160.3349.54 versus 134.8258.86 to 151.2175) in spite
of signicant differences in TG (203.2375.93 to 171.7158.77 versus
192.70 77.23 to 193.9282.09) and CHOL(204.7143.62 to 187.14
39.58 versus 198.4142.32 to 193.9247.14). Conclusion: prescription
of Citrullus colocynthis L. Schrad in diabetic patients can reduce TG and
CHOL with no effects on their serum glucose and C-peptide.
Keywords: Diabetes, Citrullus colocynthis,Triglyceride,cholesterol,Fasting blood sugar
Paper No.: 1619

EFFECTS OF GENETIC POLYMORPHISMS OF ANGIOTENSIN
CONVERTING ENZYME IN IRANIAN CHILDREN WITH
IDIOPATHIC NEPHROTIC SYNDROME
Parvaneh Rahimi-Moghaddam
Iran University of Medical Sciences, Razi Institute for Drug Research &
Department of Pharmacology, Tehran, Iran
The angiotensin converting enzyme (ACE) has been considered to be
responsible for the pathogenesis or progression of many kidney diseases.
Contraversies exist about a correlation of ACE gene polymorphisms to
the outcome of idiopathic nephrotic syndrome (INS) in children. We
investigated the genetic polymorphism of ACE insertion/deletion (I/D) in
Iranian children with INS and its relationship with clinical outcome especially response to steroids. The ACE gene polymorphism I/D (I, insertion; D, deletion) was assessed in 56 children 14 with steroid-sensitive
INS, 22 with steroid-resistant INS and 20 in control group. Children with
no history of kidney disease and negative proteinuria were enrolled as a
control group. Polymerase chain reaction (PCR) amplication was performed on genomic DNA isolated from blood leucocytes. Results were
536
correlated to clinical course. The genotypes for ACE I/D polymorphism,
including DD, ID, and II, were analyzed. The ACE genotype was D/D
and non-D/D in 5 (35.7%) and 9 (64.3%) patients, respectively, with steroid-sensitive INS, and 16 (72.7%) and 6 (27.3%) patients, respectively,
with steroid resistant INS. There was a signicant correlation between D/
D ACE genotype and steroid resistance (P<0.05). In this study, the D/D
ACE genotype was found to be related with steroid resistance in idiopathic nephrotic syndrome.
Paper No.: 2250

ASSOCIATION BETWEEN POLYMORPHISMS OF IL-4 RECEPTOR ALPHA CHAIN GENE AND PLASMA IGE LEVELS IN
MALAYSIAN ASTHMATICS
Vijaya Lechimi Raj(1), R Naidu(2), AK Radhakrishnan(3), CK Liam(4),
MR Mustafa(1)
(1) University of Malaya, Department of Pharmacology, Kuala Lumpur,
Malaysia
(2) Monash University Sunway Campus, School of Medicine and Health
Sciences, Selangor, Malaysia
(3) International Medical University, Faculty of Medical and Health, Dicision of Pathology, Kuala Lumpur, Malaysia
(4) University of Malaya, Faculty of Medicine, Department of Medicine,
The IL-4 receptor is a cell-surface, heterodimeric complex consisting of a
specic high-afnity a chain (IL-4Ra) and the common c chain. The activation of the a chain by circulating IL-4 will cause activation of transcription of target genes inducing IgE switching. The IL-4Ra chain gene, a
candidate gene in asthma, has been shown to have several single nucleotide polymorphisms (SNPs) that affect the receptor function and levels of
IgE. Two of these SNPs are Ile50Val and Glu375Ala. These SNPs have
been shown to affect the IgE levels and thus increase the bronchial inammatory response which may lead to a more severe form of asthma. This is
a pilot study to determine the frequency of these SNPs and their effect on
the plasma IgE levels in Malaysian asthmatics. Peripheral blood was
obtained from 147 asthmatics and 131 healthy volunteers. Plasma IgE levels were quantied by ELISA and Taqman Genotyping assay was performed to identify the SNPs. The plasma IgE levels were signicantly
(P<0.05) higher for asthmatics (460.3232.64 ng/mL) compared to the
volunteers (330.7044.56 ng/mL). Allelic and genotype frequencies of
the SNPs were not signicantly different between the two groups. The haplotyping results indicated that Val50Val/Ala375Ala combination did not
exist in the Malaysian population. The asthmatic patients with Ile50Ileu/
Glu375Glu combination and the Ile50Ileu/Glu375Ala combination had
signicantly higher plasma IgE levels (P<0.05) compared to volunteers,
indicating that these combinations predisposed the patients to more
inammation. Our observations suggest that these polymorphisms of IL4Ra chain gene affected the plasma IgE levels in Malaysian asthmatics.
Paper No.: 3259

PRESENTATION PREFERENCE: POSTER PRESENTATION
DISCOVERY
A LIPOSOMAL FORMULATION OF AZITHROMYCIN FOR
TREATMENT OF CUTANEOUS LEISHMANIA
Liposomes are an important colloidal carrier system for controlled

drug delivery. In this study Azithromycin was entrapped inside small
liposomes composed of egg lecithin, cholesterol, and glucose (Wight
ratio 3:1:4) and 0.1% oleic acid were prepared by dehydration-rehydration method. Liposomes were characterized by particle size analyzer, and the mean structure diameter was 100 nm. Drug entrapment
efciency of liposomes was 80%. The efcacy of liposomal azithromycin on the treatment of BALB/c mice experimentally infected by
Leishmania major was evaluated. BALB/c mice were infected with
106 promastigotes of Leishmania major and, starting on day 51, mice
were treated topically liposomal azithromycin (40 mg/ kg), only liposome (without azithromycin) and without treatment (control). Lesion
size and swelling were weekly monitored for 10 weeks after the
beginning of the treatment. Mean lesion size of the mice receiving
liposomal azithromycin compared with 2 other groups showed signicant difference. In the other study 25 patients with cutaneous Leishmania resistant to common treatments such as antimony compounds
was selected. Liposomal azithromycin (~7.5 mg) was administrated 3
times a day topically. More than 90 percent of patients were treated
in 4 weeks and all of them were treated in 8 weeks. The patients
were followed for 6 months after treatment. Based on the results of
this study, liposomal azithromycin is a suitable substitute instead of
common drugs which are used for treatment of cutaneous Leishmania.
Paper No.: 503

TREATMENT OF SHEEP BY HYPERTONIC DEXTROSE
AFTER EXPERIMENTAL SALINOMYCIN POISONING
Hamid Rajaian, S Nazi, R Hosseini, A Hajmohammadi, J Jalaei,
K Nayeri
Shiraz University School of Veterinary Medicine, Department of Basic
Sciences,Shiraz, Iran
Salinomycin is an ionophore used as growth promoter for ruminants and
as coccidiostat in chickens. The mechanism of action of ionophores is to
selectively bind ions creating cellular biochemical disturbances. This
interferes with potassium transport across mitochondrial membranes,
resulting in low intracellular energy. Signs of ionophore intoxication
include tachycardia, muscle tremor, restlessness, incoordination and continual panting. The aim of the present study was to evaluate the effect of
hypertonic dextrose in reversing the toxic signs induced by salinomycin
in sheep. Acute toxicity with salinomycin (0.5mg/kg; intravenously) was
induced in 9 anesthetized (sodium thiopental; 20mg/kg) mixed breed
female sheep (BW=33.1 3.4kg). Blood samples were collected before
and at various time intervals after the administration of salinomycin and
their sera were kept at -12 ordm;C to be analyzed for ALT, AST, CPK
and LDH. After the onset of clinical signs, six of the animals were treated with hypertonic dextrose (50%; 2ml/kg) administered intravenously
for 2 days. Toxic signs were alleviated 2 hrs after treatment. Two apparently healthy sheep suddenly died while being approached for bleeding
on days 2 and 3 after poisoning. All untreated sheep also died within
3 hours after poisoning. Postmortem examination revealed no major
changes except congestion of cardiac muscle and petechial hemorrhage
in fat tissues of heart base. Clinical chemistry tests indicate a signicant
(P<0.05) increase in CPK in both treated and untreated groups. In conclusion, hypertonic dextrose proves to be effective in the treatment of salinomycin toxicity.
Omid Rajabi(1), P Layegh(2), M khoddami(2), R Salari(1)

(1) Mashad University of Medical Sciences, School of Pharmacy,
Department of Medicinal Chemistry, Mashad, Iran
(2) Mashad University of Medical Sciences, School of Medicine, Department of Dermatology, Mashad, Iran
537
Paper No.: 1447
COMPARATIVE ANALYSIS OF GENE REGULATION BY THE
BETWEEN MOUSE AND
TRANSCRIPTION FACTOR PPARA
HUMAN
Maryam Rakhshandehroo(1,2), G Hooiveld(1,2), M Muller(1.2),
S Kersten(1,2)
(1) Nutrigenomics Consortium, TI Food and Nutrition, Wageningen, The
Netherlands
(2) Wageningen University, Nutrition, Metabolism and Genomics Group,
Division of Human Nutrition, Wageningen, The Netherlands
Introduction: Studies in mice have shown that PPARa is an important
regulator of hepatic lipid metabolism and acute phase response. However, little information is available on the role of PPARa in human liver.
Here we compare the function of PPARa in mouse and human hepatocytes via analysis of target gene regulation. Materials: Primary hepatocytes from 6 human and 6 mouse donors were treated with PPARa
agonist Wy14643 and gene expression proling was performed using Affymetrix GeneChips followed by a systems biology analysis. Results:
Baseline PPARa expression was similar in human and mouse hepatocytes. Depending on species and time of exposure, Wy14643 signicantly induced the expression of 362-672 genes. Surprisingly minor
overlap was observed between the Wy14643-regulated genes from
mouse and human, although more substantial overlap was observed at
the pathway level. Most of the genes commonly regulated in mouse and
human were involved in lipid metabolism and many represented known
PPARa targets, including CPT1A, HMGCS2, FABP1, ACSL, and
ADFP. Several genes were identied that were specically induced by
PPARa in human (ALAS1, CYP1A1, TSKU) or mouse (Fbp2, lgals4,
Cd36). Furthermore, several putative novel PPARa targets were identied that were commonly regulated in both species, including CREB3L3,
KLF10, KLF11 and MAP3K8. Conclusion: Our results suggest that
PPARa activation has a major impact on gene regulation in human hepatocytes. Importantly, the role of PPARa as master regulator of hepatic
lipid metabolism is generally well-conserved between mouse and human.
However, PPARa regulates a mostly divergent set of genes in mouse and
human hepatocytes.
Paper No.: 469

POPULATION PHARMACOKINETICS OF HYDROCORTISONE
IN EXTREMELY PREMATURE INFANTS
Hesty Ramadaniati(1), B Charles(2), H Liley(3)
(1) Faculty of Pharmacy, Pancasila University, Indonesia
(2) School of Pharmacy, The University of Queensland, Australia
(3) Neonatology Unit, Mater Mother Hospital, Brisbane, Australia
Introduction: extremely premature infants show adrenal insufciency in
early post natal life. Cortisol plays a key role in normal gestation for
maturation and adaptation of fetal organs in preparation for birth.
Patients: the eligible infants were randomized and blinded to receive
either placebo or hydrocortisone sodium succinate 1 mg/kg body weight/
day intravenously in two divided dose for 14 days. Blood sampling for
cortisol levels were performed prior to the rst dose for measuring baseline cortisol levels. Blood samples were also collected on day 3, 4, or 5
and day 12, 13, or 14 before the rst dose of hydrocortisone/placebo on
that day. The sampling was then repeated at 4 time points after dosing.
The time points for blood sampling followed pre-assigned, randomly
generated schedule. Cortisol concentrations were determined by radioimmunoassay (RIA). Population pharmacokinetic modelling was performed
using NONMEM. Results: population typical values, together with the
interindividual variability (expressed as coefcient of variation) were as
follows : clearance (Cl), 0.0627 L/h/kg (32%); volume of distribution

(V), 0.201 L/kg (45%); baseline cortisol concentration (BAS), 204 nmol/
L (37%). The half-life alimination was 2.19 hours. An additive residual
unexplained variability (RUV), expresses as the standard deviation (SD),
was 128 nmol/L. Conclusion: there was no discernible biological rhythm
over 24 hours with respect to cortisol concentrations in these infants.
The results of this study have increased knowledge of the pharmacokinetic disposition of this drug and have potential for improving its use in
premature infants.
Paper No.: 2895

CANNABIDIOL INHIBITS HUMAN CANCER CELL INVASION
VIA UPREGULATION OF TISSUE INHIBITOR OF MATRIX
METALLOPROTEINASES-1
Robert Ramer, J Merkord, B Hinz
University of Rostock, Institute of Toxicology and Pharmacoloy,
Rostock, Germany
Although cannabinoids exhibit a broad variety of anticarcinogenic
actions, their potential use in cancer therapy is limited by their psychoactive effects. Here we evaluated the impact of cannabidiol, a plant-derived
non-psychoactive cannabinoid, on cancer cell invasion. Using Matrigel
invasion assays we found a cannabidiol-driven impaired invasion of
human lung cancer cells (A549) that was reversed by antagonists to both
CB1 and CB2 receptors as well as to transient receptor potential vanilloid 1 (TRPV1). The decrease of invasion by cannabidiol appeared concomitantly with upregulation of tissue inhibitor of matrix
metalloproteinases-1 (TIMP-1) and downregulation of plasminogen activator inhibitor 1 (PAI-1). Knockdown of cannabidiol-induced TIMP-1
expression by siRNA led to a reversal of the cannabidiol-elicited
decrease in tumor cell invasiveness, implying a causal link between the
TIMP-1-upregulating and anti-invasive action of cannabidiol. P38 and
p42/44 mitogen-activated protein kinases were identied as upstream targets conferring TIMP-1 induction and decreased invasiveness. Moreover,
a possible link between cannabidiols effects on PAI-1 release and invasion was provided by experiments showing a complete reversal of its
anti-invasive action by addition of recombinant PAI-1. In line with a proinvasive function of PAI-1, transfection of cells with PAI-1 siRNA led to
a concentration-dependent down-regulation of invasiveness. In vivo studies in thymic-aplastic nude mice revealed a signicant inhibition of
A549 lung metastasis in cannabidiol-treated animals as compared to
vehicle-treated controls. Altogether, these ndings provide insight into
mechanisms underlying the anti-invasive action of cannabidiol and imply
its use as a therapeutic option for the treatment of highly invasive cancers.
Paper No.: 3409

INCIDENCE OF SEVERE HYPONATREMIA DRUG-INDUCED
AND OTHER CAUSES IN A TERCIARY CARE HOSPITAL
Elena Ramirez, AM Borobia, SL Lei, R Munoz, A Campos, C Zegarra,
AJ Carcas, J Frias
La Paz University Hospital, School of Medicine, Universidad Autonoma
de Madrid, Clinical Pharmacology Service, Madrid, Spain
Purpose: Severe hyponatremia (SH:serum-sodium-level<116 mEq/L) is
life-threatening condition. The aim was to evaluate the incidence of its
aetiologies and differences in characteristics of patients from a Pharmacovigilance Program from Laboratory Signals at hospital(PPLSH). Methods: During 30 months periods (July2007-December2009) all admissions
538
to all wards were monitored by PPLSH. Patients who died in the emergency were also included. The program was conducted according to the
Spanish Personal Data Protection Law. The incidence rate of SH and
other aetiologies were calculated (two-sided Poisson95%CI) and differences in age and sex were evaluated by t-students test and chi-squared
test respectively. Results: In this period the number of hospitalizations
was 125,278. During this same period 548,915 routine serum-sodiumlab-tests were performed in inpatients and emergency wards patients.
There were 790 HS-lab-results, corresponding to 307 patients, including
patients who died in emergency ward. The incidence of SH was
24.51x10,000 patients(Poisson 95%CI 16.18-35.71). Excluding analytical
error, drugs-induced SH(DISH) was the third most frequent cause of SH
with an incidence of 2.71x10,000 patients(Poisson95%CI:0.62-7.23)
behind of bleeding/CNS masses(2.95,Poisson95% CI:0.62-7.23) and neoplasia(2.87,Poisson95%CI:0.62-7.23). The most frequent drugs involved
were thiazide diuretics, SSRI and antiepileptics. Mortality of DISH was
9%(3/34). Patients with DISH were signicantly older (76 vs 56 years of
age,p=1.8e-9) and female sex(p=0.036) than patients of other etiologies.
The relative-risk of having a DISH in women over 76years was
3.3(95%CI:1.74-5.98). Conclusions: Due to the incidence of DISH and
the fact that thiazide diuretics, SSRI and antiepileptics are frequently prescribed, a careful monitoring of susceptible patients (elderly women)
must be warranted.
Paper No.: 1355

INTERETHNIC AND GENDER DEPENDENT DIFFERENCES IN
CYP2C19 ACTIVITY. A COMPARISON BETWEEN SWEDES
AND KOREANS
Margareta Ramsjo(1), E Aklillu(1), L Bohman(1),
M Ingelman-Sundberg(2), H-K Roh(3), L Bertilsson(1)
(1) Karolinska Institute, Department of Laboratory Medicine, Division of
Clinical Pharmacology C1:68, Stockholm, Sweden
(2) Karolinska Institute, Department of Physiology and Pharmacology,
Stockholm,Sweden
(3) Gachon University Gil Hospital, Korea
Objectives: To investigate the CYP2C19 geno-phenotype relationship
and the effect of smoking and oral contraceptives (OC) on enzyme activity in Swedes and Koreans. Methods: CYP2C19 activity was determined
in 185 healthy Swedish and 150 Korean subjects as omeprazole/5-hydroxyomeprazole ratio (metabolic ratio; MR) determined using high-performance liquid chromatography. Genotyping was performed by PCR
using Taqman assay. Results: As expected, a higher incidence of poor
metabolizers (PM) was found in Koreans (14%) compared to Swedes
(3.8%) and the frequency of the CYP2C19*17 allele was very low in
Koreans 0.3%. Among subjects homozygous for CYP2C19*1 Koreans
were slower than Swedes (p<0.000001) and interestingly in Koreans a
pronounced gender difference was apparent: females (n=24) had signicantly lower MR than males (N=30) (p<0.0001) but such a gender difference was not seen among Swedes. Swedish OC users had a higher MR
than non-users (p<0.00001), whereas OC was only used by one Korean.
No effect of smoking was observed. Conclusions: We nd specic gender dependent effects of CYP2C19 activity in Koreans but not in
Swedes. The genetic, epigenetic or environmental basis for this difference has to be identied.
Paper No.: 1829

INTRAPERITONEAL AND ORAL LD50 OF AN ACTIVE
PRINCIPLE OF NIGELLA SATIVA, THYMOQUINONE, IN MICE
AND RATS
Muhammad Akram Randhawa(1), A Al-Ali(2), AA Alkhawajah(3),
NA Shaikh(4)
(1) University of Dammam College of Medicine, Department of
Pharmacology, Dammam, Saudi Arabia
Biochemistry, Dammam, Saudi Arabia
Pharmacology, Dammam, Saudi Arabia
Pathology, Dammam, Saudi Arabia
Nigella sativa oil and seed are commonly used as a natural remedy for
many ailments. Thymoquinone is the main active principle of N. sativa
and constitutes about 30% of its volatile oil. Many pharmacological
actions of N. sativa are reported in literature, including immunostimulant,
antiinammatory, anticancer, antioxidant, antihistaminic, antiasthmatic,
hypoglycemic, antimicrobial and antiparasitic. However, there are only
few controversial reports about the toxicity of thymoquinone. Present
study aimed to determine LD50 of thymoquinone in mice and rats that
had been given thymoquinone intraperitoneally or orally, by method
described by Miller and Tainter. In addition, histopathological studies of
liver, kidney, heart and lungs were carried out. After intraperitoneal injection, the LD50 in mice was determined to be 104.7mg/kg (89.7-119.7,
95% condence intervals), while after oral ingestion it was 870.9mg/kg
(647.1-1094.8, 95% CI). The LD50 in rats after intraperitoneal injection
was found to be 57.5mg/kg (45.6-69.4, 95% CI) while after oral ingestion it was 794.3mg/kg (469.8-1118.8, 95% CI). On autopsy of mice and
rats recieving lethal doses, visceral organs and peritoneum were congested. The cause of death was possibly hypotension. Histopathologically, no abnormalities were found in liver, kidney, heart and lungs of
control and test animals. The LD50 values presented here after intraperitoneal injection and oral gavage are 10-15 times and 100-150 times
greater than doses of thymoquinone reported for its anti-inammatory,
anticancer and other effects in small animals. This study shows that thymoquinone is a relatively safe compound in doses reported to possess
useful pharmacological activities, particularly when given orally.
Paper No.: 3222

PROVOCATION-BASED LEARNING (PBL) IN LARGE
CLASSES: PROMISING?
Patangi Rangachari, S Nastos
McMaster University, BHSc (Honours) Program/Medicine, Hamilton,
Ontario, Canada
Active learning works, but is not easy to put into practice, particularly in
large classes. Faculty need to move beyond being information dispensers
and become agent provocateurs. We have used Provocation-Based Learning (PBL) in large classes (160-220 students) with some success. Cell
signalling was the focus of an undergraduate rst year, two term Biology
course. In Term 1, interactive didactic sessions gave students a broad
overview of cellular signalling (gene expression, synthesis, storage,
release, effects and termination of responses to specic signalling molecules: histamine, acetylcholine, catecholamines, prostaglandins, nitric
oxide and steroids). In Term 2, students were expected to go beyond the
mere facts to explore the construction of scientic facts, demonstrate creativity in transferring learning to novel situations as well as exibility in
responding to challenges. Both group and individual exercises were
developed. (i) A jig-saw approach got students recognising key elements
539
of peer-reviewed publications and help them frame and write abstracts.
(ii) Students were given a list of Nobel laureates, asked to explore the
Nobel archives and read their primary publications. (iii) Groups were
also required to frame projects to explore the molecular basis of specic
sins (greed, lust, anger and sloth). (iv) Another group project required
students to design problem-solving exercises and frame acceptable solutions and leave these as legacies for future classes. Throughout the
course, the instructors were active - provoking, annoying and challenging
students.
Paper No.: 1248

EFFECTS OF ROUTE OF ADMINISTRATION ON
DEXMEDETOMIDINE PHARMACOKINETICS IN C57BL/6J
AND A2A-ADRENOCEPTOR KNOCK-OUT MICE
noid haemorrhage and transient middle cerebral artery (MCA) occlusion.

Here we investigate whether a small vascular insult leads to a locally
altered receptor expression prole of segments located up- and downstream to an occlusion. Focal permanent occlusion by distal MCA ligation is performed on male Wistar rats, were right MCA is accessed by
craniotomy and occlusion is veried by laser-Doppler. Controls consist
of un-ligated left MCA and sham operated rats. After 24 and 48 hours,
MCA segments are investigated for expressional changes of ETB, AT1 and
5-HT1B receptors at functional (myograph) level. Ischemic damage is
examined by TTC staining. After 24 hours the contractile responses to S6c
(selective ETB agonist) and 5-CT (5-HT1 agonist) are signicantly stronger
in the downstream occluded MCA segments (101.3 19.6 and 35.2 5.2,
mean and s.e.m in % of 125 mM K+) compared to left MCA (15.6 3.8%
and 10.7 2.4%), and most interestingly to right upstream segments (21.1
5.7 and 11.4 2.7). This is repeated at 48 hours for ETB and 5HT1B,
accompanied by increased contractillity of AT1 after 48 hours occlusion
when comparing up- and downstream segments and with 24 hours. A small
focal vascular occlusion with limited ischemia leads to locally increased
contractile function of ETB and 5-HT1B receptors after 24 hours and additionally increased contration of AT1 receptors after 48 hours.
Ville Ranta-Panula(1,2), M Lahtinen(1), S Tikka(2), T Streng(2),

M Scheinin(1,2)
(1) Clinical Research Services Turku CRST, Turku, Finland
Dexmedetomidine is a potent b2-adrenoceptor (b2-AR) agonist. b2-AR
activation decreases blood pressure and heart rate, inhibits sympathetic
nerve activity, locomotor activity and pain transmission. Activation of
vascular b2-ARs also contracts blood vessels and may increase blood
pressure. The effects of dexmedetomidine on the circulatory system are
thus complex and dose-dependent.The aim of this study was to explore
the effects of three different routes of administration (i.v., i.p. and s.c.)
on dexmedetomidines pharmacokinetics inC57Bl/6J and b2A-KO mice.
Some of dexmedetomidines hemodynamic effects on the circulatory system were assumed to be lacking in b2A-KO mice. This was predicted to
inuence the drugs pharmacokinetics, as has been observed in dogs (SalonenJS et al., J Vet Pharmacol Ther., 1995;18: 328-32), but not in
humans after concomitant b2A-antagonist administration (Scheinin H et
al., Anesthesiology,1998; 89: 574-84.). Especially absorption from the
place of administration, distribution into tissues, hepatic metabolism and
renal excretion were assumed to be more rapid in b2A-KO mice. Six
mice per administration route per strain were taken into the study. Each
mouse received dexmedetomidine 30 lg/kg once,either i.v., i.p. or s.c.
and six 40 ll blood samples were collected from each.The analysis was
with HPLC-MS/MS. The results indicated that the elimination half-life
of dexmedetomidine is about 60 min in mice. There were no major differences between the groups in AUC or half-life. The starting hypothesis
could thus not be conrmed.
Paper No.: 1916

LOCAL ENHANCED CONTRACTILE RESPONSE OF ETB,
5-HT1B AND AT1 RECEPTORS FOLLOWING DISTAL FOCAL
PERMANENT OCCLUSION
Marianne NP Rasmussen(1), M Hornbak(1), L Edvinsson(0)
Paper No.: 874

PHARMACOKINETICS OF FLORFENICOL FOLLOWING A
SINGLE SC INJECTION IN CALVES
Ali Rassouli(1), P Lees(2), P Sidhu(2)
(1) University of Tehran Faculty of Veterinary Medicine, Department of
Pharmacology, Tehran, Iran
(2) Royal Veterinary College, University of London, London, UK
This study was a part of a larger project using PK-PD modelling to evaluate
antimicrobial drugs in calf pneumonia. Ten healthy animals received
40 mg/kg orfenicol by a single SC injection and at pre-determined time
points (0-120 h) serum, exudate and transudate samples collected using a
tissue cage model. The samples were analysed by a validated HPLC
method and then PK proles of orfenicol in calves were generated using
HPLC data of samples through Excel and WinNonlin 5.2 softwares. Florfenicol PK data of serum, exudate and transudate samples were used for PKPD integration and for determination of average concentrations/MIC
(Cave/MIC) ratios on M. hemolytica and P. multocida in broth and serum
in 24 h periods following injection. In PK modelling, a two-compartmental
model, 1st order kinetics for serum and a one-compartmental model for
exudate and transudate were used. Comparing the PK parameters of three
biological uids indicated that mean serum Cmax (5.91 micg/ml) was
higher and mean serum Tmax (2.28 h) was much shorter than those of exudate (3.39 micg/ml, 17.2 h) and transudate (2.84 micg/ml, 17.9 h). These
results suggest that orfenicol readily penetrates and distributes in interstitial uids and achieves concentrations about half of the serum levels in tissue cage uids. PK- PD integration of orfenicol in serum of calves
showed desireable values for AUC 0-24 h/MIC in M. hemolytica and P.
multocida, 151.8 and 183.3 h, respectively and T>MIC, at least 72 h.
These assumed positive values should be conrmed in clinical eld trials.
Paper No.: 1859
DISCOVERY
SMALL-MOLECULE DRUG DESIGN TARGETING PICK1
USING A C-TERMINAL DOPAMINE TRANSPORTER PEPTIDE
(1) Glostrup University Hospital, Glostrup Research Institute,

Department of Clinical Experimental Research, Copenhagen, Denmark
(2) Lund University, Institute of Clinical Sciences, Department of
Medicine, Lund,Sweden.
Mette Rathje(1), MV Andersen(2), SE Andersen(2), PO Heidarsson(3), S

Erlendsson(3), A Bach(2), FM Poulsen(3), K Strmgaard(2), U Gether(1)
There has previously been demonstrated a time-dependent up-regulation

of the contractile endothelin-B (ETB), 5-hydroxytryptamin-1B (5-HT1B)
and angiotensin-1 (AT1) receptors in cerebral arteries following subarach-
(1) University of Copenhagen, INF, Copenhagen, Denmark

(2) Univrsity of Copenhagen, Department of Medical Chemistry, Copenhagen, Denmark
540
(3) University of Copenhagen, Department of Biology, Copenhagen,
Denmark
The dopamine transporter (DAT) promotes the clearance of dopamine
from the synaptic cleft. It belongs to the family of solute carrier 6 transporter proteins, which is characterized by Na+/CL- coupled transport
function, and includes transporters for other neurotransmitters, such as
serotonin and norepinephrine. The C-terminal of DAT has the ability to
bind intracellular proteins, such as the PSD95/Disc-large/ZO-1 homology
(PDZ)-domain proteins, which play important roles in scaffolding membrane-bound transporters and receptors in the synaptic density. Protein
interacting with C kinase (PICK)-1 is a PDZ-domain protein that binds
the C-terminal of DAT. The function of PICK1 has mainly been studied
in relation to the AMPA receptor, where it has been shown to be essential for the induction of long term depression. Recently, it has been suggested an interesting candidate drug target in treatment of neuropathic
pain. In this study we used a C-terminal DAT peptide as a tool for structurally characterizing the PICK1-DAT interaction. NMR spectroscopy
showed that the overall structure of the PICK1 PDZ domain was relatively unaltered compared to previously published PICK1 PDZ interaction studies but that there may be ligand specic modulation of the
binding pocket. Using a uorescence polarization binding assay we
found that the three extreme C-terminal amino acid residues of DAT are
sufcient for PICK1-binding making it a suitable template for small-molecule drug design targeting PICK1. Ala-scan modied peptides showed
that the P0 Alanine and the P-2 Leucine residues are crucial for high
afnity binding, and dimeric peptide modications had signicantly
improved afnity for PICK1.
Paper No.: 1894

FOCUSED CONFERENCE GROUP: PW33 - SYNAPTIC EVENTS
IN BRAIN FUNCTION
ENDOMORPHIN-1 STIMULATES NOREPINEPHRINE
RELEASE IN RAT HYPOTHALAMUS
Lucia Recinella, L Brunetti, G Orlando, A Mollica, C Ferrante, A Chiavaroli, S Leone, C Di Nisio, R Shohreh, P Di Michele, F Pinnen, M Vacca
Endomorphin-1 (Tyr-Pro-Trp-Phe-NH2) is an opioid peptide, with high
afnity and remarkable selectivity for mu-opioid receptor. It is widely
distributed throughout the brain and upper brainstem. Central administration of endomorphin-1 stimulates appetite and this effect is blunted by
mu-opioid receptor antagonists. Endomorphin neurons are widely distributed in the hypothalamus, in direct association with mu-opioid receptors.
The hypothalamus plays a pivotal role in regulating feeding behavior and
we have previously found that neuropeptides and hormones involved in
feeding control are able to modify catecholamine and serotonin release in
the hypothalamus. In order to further evaluate the mechanisms of endomorphin-1 in the feeding regulatory network, we have investigated its
effects on dopamine, norepinephrine and serotonin release from rat hypothalamic neuronal endings (synaptosomes) in vitro. Hypothalamic synaptosomes were obtained as previously described, suspended in KrebsRinger buffer and incubated at 37C with either [3H]dopamine, [3H]norepinephrine or [3H]serotonin, for 15 min. Then synaptosomes were layered onto 0.8 microm Millipore lters, placed into 37C water-jacketed
superfusion chambers, and perfused with graded concentrations (0.1-100
nM) of endomorphin-1, both basally and during a mild depolarization
stimulus (K+ 15 mM). Perfusate was collected (2 min fractions for dopamine and norepinephrine, and 1 min fractions for serotonin release) to
detect released [3H] by liquid scintillation scanning. We have found that
endomorphin-1 stimulates basal norepinephrine release, in a dose-dependent manner, leaving unaffected either basal or depolarization-induced
dopamine and serotonin release. These data suggest that the orexigenic
effects of endomorphin-1 could be partially related to increased norepinephrine release in the hypothalamus.
Paper No.: 2889

HEALTH AND DISEASE
ENDOTHELIAL PROGENITOR CELL DEPLETION IN
ATHEROSCLEROTIC PATIENTS IS NOT RELATED TO
INCREASED APOPTOSIS BY PLASMATIC TGF-B
Santiago Redondo(1,2), A Gonzalez-Rocafort(3), J Navarro-Dorado(1),
M Ramajo(1), M Hristov(4), A Gordillo-Moscoso(5), F Reguillo(3),
M Carnero(3), E Rodriguez(3), C Weber(4), T Tejerina(1)
(1) Universitu Complutense, School of Medicine,Department of Pharmacology, Madrid, Spain
(2) Hospital Clinico San Carlos, Service of Hematology and Hemotherapy, Madrid, Spain
(3) Hospital Clinico San Carlos, Service of Cardiac Surgery, Madrid,
Spain
(4) University Hospital, Institute for Molecular Cardiovascular Research,
Aachen, Germany
(5) Universidad Autonoma de San Luis Potosi, Department of Clinical
Research, Mexico
Introduction: A growing body of evidence links atherosclerosis with
endothelial progenitor cell (EPC) depletion and functional impairment,
which can be measured as a high apoptotic rate and decreased adhesion.
We aimed to assess the number of EPCs in coronary artery bypass grafting (CABG) patients, compared to valvular patients, and the apoptotic
effect of the plasma from these two groups on cultured EPCs from
healthy donors. Material: CD34+/CD144+ cells were assessed by ow
cytometry. EPCs were cultured from peripheral blood mononuclear cells
obtained from healthy donors. CD144 and CD105 expression were
assessed by Western blot. Apoptosis was assessed by DNA fragmentation ELISA. Results: We found a lower number of CD34+/CD144+ cells
in CABG patients. In cultured EPCs from healthy donors, plasma from
CABG patients induced a higher expression of CD144 and CD105 at
only 3 h time, this effect being independent of the Smad3 and ALK-4
TGF-b1 pathways; long-term incubation for 24 h with TGF-b1 was able
to decrease CD14+ cells. Incubation of cultured EPCs from healthy
donors with plasma from CABG patients was able to inhibit apoptosis,
this effect being abrogated by SIS3 and SB-431542. Conclusions: Plasma
from CABG patients is an antiapoptotic stimulus for EPCs from healthy
donors by involving the TGF-b1 pathway. Thus, the observed lower
number of CD34+/CD144+ in vivo may be mediated by a primary bone
marrow defect rather than a deleterious role of their plasma.
Paper No.: 1520

HEALTH AND DISEASE
TLR3 AND TLR4 LIGANDS INDUCE INTRACELLULAR IL-1B
IMMUNOREACTIVITY IN HUMAN AORTIC ENDOTHELIAL
CELLS (HAEC) AND LUNG MICROVASCULAR
ENDOTHELIAL CELLS (HLMVECS)
Daniel M Reed, WR Wright, CMF Potter, HH Gashaw, LK Bailey,
M Paul-Clark, JA Mitchell
Imperial College London, Department of Cardiothoracic Pharmacology,
London, UK
The endothelium is an important innate immune tissue and expresses
pattern recognition Toll-like receptors (TLR). TLR4 is the receptor for
bacterial LPS whilst TLR3 is the receptor for viral double-stranded RNA
(mimicked by Poly(I:C)). LPS acts together with ATP, via P2X7 receptors, to activate the IL-1b inammasome leading to IL-1b release in leukocytes. The role of endothelial cells in the synthesis of IL-1b is less
clear. Here we investigated the effects of LPS and Poly(I:C) on IL-1b in
primary cultures of HAEC and HLMVECs. Cells were stimulated with
LPS or Poly(I:C). After 24h medium was replaced and cells were stimulated with/without ATP (5mM; 30 minutes). Cells were lysed by freeze
541
thawing. IL-1b was measured by ELISA. Neither LPS nor Poly(I:C)
induced IL-1b release from HAECs or HLMVECs stimulated with or
without ATP (p>0.05, n=6; one-way ANOVA followed by Bonferronis
post test). In contrast, ATP-pulsed THP-1 monocytes released large
amounts of IL-1b when stimulated with LPS, but not with Poly(I:C)
(basal 21.46.2, plus LPS 386.1 69.1, plus Poly(I:C), 26.29.3 pg/ml).
However, increased levels of retained IL-1b were detected in lysates
from endothelial cells stimulated with LPS (1lg/ml) or Poly(I:C) (10lg/
ml) (HAEC; basal 1.30.5; LPS 5.52.1; Poly(I:C) 34.47.7 pg/ml:
HLMVECs; basal 1.30.6; LPS 6.51.8; Poly(I:C) 12.42.6 pg/ml).
These ndings that IL-1b is retained and not released by the cells conrm other observations elsewhere. Our observations are the rst to implicate a role of TLR3 in IL-1b pathways in endothelial cells showing
differences in how endothelial cells and leukocytes participate in IL-1b
pathways/processing.
Paper No.: 2870

FEXOFENADINE IS NOT A SUBSTRATE FOR OATP2B1
properties, may be benecial in reducing the side effects of phenytoin

due to favorable pharmacodynamic and pharmacokinetic interaction. This
study investigates the interaction of phenytoin with melatonin and ALA
in maximal electroshock seizures (MES) in male Wistar rats (150-200 g).
Animals were divided into ve groups - control, phenytoin (20 &
40 mg/kg, i.p.), melatonin (50 mg/kg, p.o.) with phenytoin (20 mg/kg)
& ALA (100 mg/kg, p.o) with phenytoin (20 mg/kg). MES (70 mA,
9 ms pulse width, 0.2 s) was used to induce seizures. Melatonin &
ALA were administered 30 min prior to phenytoin which was administered 2 h before MES. Latency and duration of tonic hind limb
extension (THLE) were noted. Behavioral parameters were assessed
before & after MES. Serum phenytoin levels were estimated at , 1
and 2 h after phenytoin administration. Co-administration of melatonin
& ALA with the sub-anticonvulsant dose of phenytoin (20 mg/kg,
i.p.) showed 75% & 100% protection, respectively against THLE as
compared to 20% protection in phenytoin alone (20 mg/kg, i.p.) group
and signicantly decreased the duration of THLE. Learning and memory behavior did not show signicant changes before & after MES.
Melatonin and ALA did not cause any signicant change in serum
concentration of phenytoin. Co-administration of melatonin and ALA
showed enhanced protective effect of phenytoin against MES. The results
suggest the potential of these compounds as adjuvant to phenytoin therapy in epilepsy.
Gregory Reed, C Flynn, B Hagenbuch

University of Kansas Medical Center, Department of Pharmacology,
Kansas City, KS, USA
Fexofenadine is used as a probe drug for measuring transporter function.
Interpretation of these studies is complicated by reports that fexofenadine
may be a substrate for several different uptake transporters. In order to
dene the utility of fexofenadine as a probe we are characterizing its
transport by human OATPs. We have examined interactions of fexofenadine with OATP2B1 in a stably-transfected CHO cell model system.
Uptake of the radiolabeled model substrate estrone-3-sulfate was measured by scintillation counting and uptake of fexofenadine was quantied
using a validated LC-MS/MS assay. All studies used CHO cells transfected with the empty pcDNA5/FRT vector as a control and were performed at both pH 7.4 and 5.5. Estrone-3-sulfate uptake by OATP2B1expressing cells at pH 7.4 was not affected by fexofenadine at concentrations up to 100 lM and was inhibited by less than 35% by 100 lM
fexofenadine at pH 5.5. Direct examination of fexofenadine uptake by
CHO cells showed no signicant uptake at pH 7.4, but did show concentration-dependent uptake at pH 5.5. This apparent uptake, however,
exhibited no linear time dependence, was not saturable, and uptake by
OATP2B1-expressing cells and control cells was comparable. These ndings support a pH-sensitive uptake of fexofenadine by CHO cells, but
rule out transport of fexofenadine by OATP2B1. This differs markedly
from cells expressing OATP1A2, which do transport fexofenadine.
Removing OATP2B1 from consideration limits the number of transporters involved in fexofenadine pharmacokinetics, and thus claries its
function as a probe drug.
Supported by NIH grants AT002907, RR021940, and ES007079.
Paper No.: 1431

INTERACTION OF PHENYTOIN WITH MELATONIN AND
ALPHA-LIPOIC ACID IN EXPERIMENTAL SEIZURES IN
RATS
KH Reeta, J Mehla, YK Gupta
New Delhi, India
Phenytoin is known to have dose-related side effects. Melatonin and a-lipoic acid (ALA), known to have potential antiepileptic and antioxidant
Paper No.: 2839

STIMULATION OF B3-ADRENOCEPTORS INHIBITS
EFS-INDUCED CONTRACTIONS OF HUMAN ISOLATED
URINARY BLADDER
M Rekik(1), C Rouget(1), P Camparo(2), H Botto(3), P Lluel(1),
T Westfall(1), Stefano Palea(1)
(1) UPS. Faculte des Sciences Pharmaceutiques, UROsphere, Toulouse,
France
(2) Hopital Foch, Service dAnatomie et Cytologie Pathologiques, Suresnes, France
(3) Hopital Foch, Service Service dUrologie, Suresnes, France
The aim of the present study was to investigate the effect of b-adrenoceptor (b-AR) agonism on neurogenic contractions of human isolated
urinary bladder and the nature of the subtypes involved. Detrusor smooth
muscle strips obtained from 11 patients undergoing cystectomy for bladder cancer (723 years old) were mounted in organ bath and electrical
eld stimulation (EFS) was applied (maximal current, 10 Hz, pulses of
0.1 ms, trains of 5 s every 60 sec). b1, b2 or b3-AR antagonists or vehicle were added followed by a cumulative concentration-response curve
to non selective b-AR agonist isoproterenol or selective b3-AR agonists,
L755,507 and CL316,243. Responses were expressed as % variation
from basal EFS-induced contractions. Isoproterenol concentration-dependently inhibited TTX-sensitive EFS-induced contractions of human bladder smooth muscle strips with an EC50 value of 0.190.11 lM and an
Emax value of 66.63.9% at 30 lM. In contrast L755,507 and
CL316,243 decreased EFS-induced contractions by only 30.08.9% at
10 lM and 20.83.5% at 30 lM, respectively. The b1 and b2-AR antagonists, CGP20712 and ICI118,551, respectively had no effect on the
response to isoproterenol. However, the b3-AR antagonist L748,337
induced a concentration-dependent rightward shift of the isoproterenol
response without affecting the maximal effect. A pA2 value of 7.37 and
a slope of 1.070.19 indicated competitive antagonism. We conclude that
isoproterenol is able to potently inhibit EFS-induced contractions of
human detrusor muscle through activation of b3-ARs. These results suggest that in addition to producing relaxation of detrusor muscle during
urine storage, b3-AR agonism might inhibit neuronally-mediated voiding
contractions.
542
Paper No.: 484
FROM PHARMACOGENETICS TO PERSONALIZED
MEDICINE: A CUBAN REGULATORY PERSPECTIVE
Diadelis Remirez-Figueredo
National Center for State Quality Control of Drugs, Havana, Cuba
The science of pharmacogenomics has advanced signicantly in the last
ve years, but it is still in infancy and is mostly used on research basis.
The Pharmacogenomics helps identify interindividual variabilities in drug
response (both toxicity and effectiveness). This information will make it
possible to individualize therapy with the intent of maximizing effectiveness and minimizing risk. The aims of this work are to present the bases
of pharmacogenetic, the advantage and challenges of this specialty, the
main enzymes characterized for the genetic polimorphism and the world
and cuban regulatory perspective about this subject. We will show the
main biomarkes for pharmacogenetics studies and a general guidance for
submission of this type of research. The hope for the future is that
through personalized medicine, doctors and patients will be able to make
better-informed choices about treatment. This treatment will avoid the
adverse drug reaction to the medication and will improve the diagnosis
diseases as well as the prevention and treatment of diseases.
Paper No.: 485

REGULATORY STATUS OF HERBAL MEDICINES.
CONSIDERATIONS ABOUT CUBA
bility network of phamarcologic action of TCM formula on account of

network theory. Methods: Chinese herb be viewed as node and compatibility relationship of herb-herb viewed as edge according to network theory. Compatibility network of phamarcologic action of TCM formula
was constructed after the compatibility relationship of herb-herb was
analysed by two-way analysis of variance (ANOVA) through phamarcology experiment, then compatibility principle of TCM formula was
analysed with network efciency (NE) and NE related parameters.
Results: The network approach was applied for Studying compatibility
principle of jiawei shengmaisan on its anti-myocardial ischemia reperfusion injury action.the results indicated that rhizoma corydalis was
the main herb in jiawei shengmaisan, and in turn was radix ophiopogonois, radix salvia miltiorrhiza, radix ginseng and fructus schizandrae; radix ginseng and radix salvia was clustered rst,and in turn was
radix ophiopogonois and fructus schizandrae, radix ginseng and radix
salvia and radix ophiopogonois and fructus schizandrae;radix ginseng,
radix salvia miltiorrhiza and rhizoma corydalis was the most effective
formula among all formulaes. Above results generaly was consistent with
validation experiments. Conclusions: Studying compatibility principle of
traditional chinese medicie formula by network theory is a new and feasible method.
Paper No.: 1325

HYPERTENSION IN GENERAL PRACTICE - AN APO-AUDIT
Lene Reuther(1), P Schultz-Larsen(2), J Damsgaard(3),
D Gilsa Hansen(4), A Munck(4), J Sndergaard(5),
M Skov Paulsen(6), M Andersen(6)
Diadelis Remirez-Figueredo
National Center for State Quality Control of Drugs, Havana, Cuba
In the last decade there has been a global upsurge in the use of traditional
medicine and complementary and alternative medicine in both developed
and developing countries. This is one of the main reasons for reinforcing
the surveillance of the safety, efcacy and quality control of traditional
medicine, complementary and alternative medicines. This work describes
important aspects about the art state of the regulatory status of herbal
medicines as well as the main requirements for registering of herbal
medicinal products. Besides that, data related with the countries involved
in the WHO program for traditional medicine will be showed. The market and the main challenges are analysed in the investigation of the phytomedicines as well as the tendencies in the growth of this attractive
sector. Moreover, the main requirements for the registering of herbal
medicinal products in Cuba will be showed. The strategies for the development of herbal medicinal products are showed as well as some of the
interactions between natural and synthetic drugs. The natural health products are considered a very important source for the health.
Paper No.: 3244

ANALYSING ON COMPATIBILITY PRINCIPLE OF
PHARMACOLOGIC ACTION OF TRADITIONAL CHINESE
MEDICINE FORMULA BY NETWORK APPROACH
Junguo Ren, X Ma, C Lin, H Li, M Wang, J Li, Y Wang, J Liu
Chinese Academy of Chinese Medical Science, Xiyuan Hospital, Center
for Experimental Research, Beijing, PR China
(1) University of Copenhagen Bispebjerg Hospital,

Department of Clinical Pharmacology, Copenhagen, Denmark
(2) General Practice, Virum, Denmark
(3) General Practice, Hvals, Denmark
(4) University of Southern Denmark, Audit Project Odense, Research
Unit for General Practice, Odense, Denmark
Denmark
(6) University of Southern Denmark, Research Unit for General Practice,
Odense, Denmark
Introduction. The study aims to elucidate whether different levels of
intervention and registration of data from own patients can improve the
quality of treatment of hypertensive patients. Materials/Patient. 184 Danish practices in 3 regions of Denmark registered 7342 hypertensive
patients on an APO registration chart during November 2007. Duration
of hypertension, actual level of blood pressure, risk factors, complications and treatment were registered. In the biggest region (The Capital
Region) 82 practices registered 3082 hypertensive patients and here the
participating general practitioners were randomised into three groups.
One group attended courses, a second additionally had access to support
by a hospital cardiologist, an electronic treatment support and feedback
from a colleague, especially interested in hypertension, and the third was
a control group. In the 2 other regions doctors were randomised into two
groups; one attended courses, the second was a control group. A second
registration had been carried out during October 2009 from all practices.
A questionnaire to the patients was also included from the beginning of
the project. Results and Conclusion. Main results from the rst registrations showed that 50% of all patients included reached the target value.
More than 50% of the patients had duration of hypertension of more than
5 years. The pattern of treatment showed that 1/3 received one drug, 1/3
two drugs and 25% received 3 or more drugs. Eighty percent of the
patients returned the questionnaire. Results from the second registration
are awaited.
Objective: To provide a new approach that Study compatibility principle

of traditional chinese medicine (TCM) formula by constructing compatiJournal compilation 2010 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 107 (Suppl. 1), 162692
543
Paper No.: 1717
RATIONAL DRUG USE AT HOSPITALS AND IN THE
PRIMARY-SECONDARY CARE INTERFACE
Lene Reuther(1), H Thomsen(1), HR Christensen(1), B Kirkeby(2)
(2) Medicinfunktionen, Koncern Praksis, Hillerd Denmark
Introduction. Drug Committees at hospitals in the Capital Region of
Denmark work on a rational drug use at hospitals. They also coordinate
the choice of medication with primary care (general practice) to obtain
rational drug use in both sectors and to avoid a spill over effect of irrational drug use between hospital and general practice and vice versa. Materials/Patient. Recently The Main Drug Committee in the Capital Region
and a subgroup from this, the Sector group, and a group from primary
care, Medicinfunktionen contacted all Local Drug Committees at the hospitals in the region, all Hospital Managers and -through Medicinfunktionen- general practitioners in the region regarding inappropriate use of
medication in both sectors where cheaper, therapeutic equal, recommended analogues could be used. Data on drug use were collected form
the pharmacy databases on drug use. Results. In an uncontrolled setting
we demonstrated after intervention at the most consuming hospital a signicantly and persistently decrease of an un-recommended, low-molecular-weight heparin (LMWH) and a simultaneously increase in
consumption of the recommended LMWHs. Likewise, use of an expensive, but not better nonsteroidal anti-inammatory drug (NSAID), etodolac at the most consuming hospital persistently was replaced by a
cheaper and recommended NSAID, ibuprofen, a trend that was also
reected in the surrounding municipalities. Conclusion. Although uncontrolled it seems to be possible to change clinicians prescription patterns
in a cost-effective manner.
Paper No.: 1089

THE EFFECTS OF 8- OH-DPAT ON 6-OH DOPAMINEINDUCED MOTOR DEFICIT IN RAT
Siamak Reyhani Rad(1), A Mohajel Nayebi(2), J Mahmoudi(3)
(1) Azad University of Marand, Department of Biology, Tabriz, Iran
(2) Medical University of Tabriz, Faculty of Pharmcy, Tabriz, Iran
(3) University of Tehran, Department of Sciences and Researches,
Tehran, Iran
Parkinsons disease(PD) is a neurodegenerative disorder that is associated
with motor symptoms like tremor, rigidity and bradykinesia. The cause
of the PD is decrease of dopamine level in nigrostriatal pathway. Studies
have shown that other neurotransmitter systems such as serotonergic neurons are also involved in pathogenesis of PD. This study was aimed to
investigate the effect of 8-OH-DPAT (5-HT1A receptor agonist) in motor
decit induced by unilateral injection of 6-OH-dopamine (6-OHDA) to
the substania nigra pars compact (SNc) region. Catalepsy was induced
by intra-SNc injection of 6-OHDA (8 lg/2ll/rat) and was assessed by
using bar test method in male rats. The effect of 8 OH-DPAT (0.25, 0.5
and 1mg/kg, i.p.) and NAN-190 (0.1, 0.5 and 1 mg/kg, i.p.) on 6OHDA- induced catalepsy was studied in parkinsonian rats. Results
obtained from this study showed that 8 OH-DPAT attenuates signicantly
6-OHDA induced catalepsy at the dose of 1 mg/kg. NAN-190 (0.1, 0.5
and 1 mg/kg, imp.) did not produce any signicant effect of bar test
when compared with control group. The anti-cataleptic effect of 8-OHDPAT (1 mg/kg) was reversed by simultaneous administration with
NAN-190 (0.5 mg/kg). In conclusion we suggest that 8 OH-DPAT could
markedly reduce catalepsy of PD by affecting on 5HT1A receptor. Also,
we suggest doing further studies to test usefulness of co-administration
of 5-HT1A receptor agonist with antiparkinsonian drugs on catalepsy of

PD.
Paper No.: 483

CANNABINOID-INDUCED TOLERANCE REDUCTION BY
LITHIUM CHLORIDE IN GUINEA-PIG SMALL INTESTINE
Fatemeh Rezania, S Ejtemaei Mehr, AR. Dehpour
Department of Pharmacology, School of Medicine, Tehran University of
Medical Sciences, Tehran, Iran
Cannabinoid-induced tolerance that has been reported for most of effects
of cannabinoids is attributed to pharmacodynamic events, including
down-regulation/desensitization of cannabinoid receptors (Gonzalez et
al., 2005; Pharmacol. Biochem. Behav. 81: 300-318). The effect of lithium was examined on tolerance to WIN 55,212-2, a synthetic cannabinoid, in guinea pig ileum, a functional model to examine the mode of
action of cannabinoids (Pertwee et al., 2001; Gut 48: 859 867). Ileum
strips were mounted in bath containing 37C Tyrode, under 0.5 g resting
tension. The pulses were delivered, via platinum electrode, to the tissue
and subsequent twitches were recorded using PowerLab system. Graphpad Prism was applied to analyse the data. Electrically evoked contractions were inhibited in a dose dependent manner by WIN 55,212-2
(pD2= 8.560.41). Tolerance to this effect could be induced by incubating isolated ileum for 4h with WIN 55,212-2 (3IC50) (pD2= 6.360.26,
degree of tolerance: 159.32) (P<0.01). Lithium (1mM) could interfere
with the development of this cannabinoid-induced tolerance and restores
the sensitivity of ileum to inhibitory action of WIN 55,212-2 either when
strips were exposed to lithium chloride during 4h incubation period
(pD2=7.260.39) (P>0.05 versus non-tolerant group) (degree of tolerance: 20.28), or when lithium was added immediately before examining
the inhibitory action of WIN 55,212-2 (pD2=8.090.33) (P<0.05 versus
corresponding tolerant group) (degree of tolerance: 2.96). Upon these
results, it appears reasonable to consider an interventional role for lithium
which may be able to break the chain of development and/or expression
of tolerance to WIN 55,212-2 in this experimental model.
Paper No.: 1535

SALIVA EFAVIRENZ LEVELS AS A NON-INVASIVE
ALTERNATIVE TO DETERMINE ADHERENCE IN CHILDREN
Malie Rheeders(1), M Viljoen(1), A Theron(1), T Meyers(2)
(1) North -West University, Department of Pharmacology, Potchefstroom,
South Africa
(2) ECHO, Chris Hani Baragwanath Hospital, Soweto, South Africa
Efavirenz is a non-nucleoside reverse transcriptase inhibitor used in combination therapy for HIV-1-infected children. Subtherapeutic levels (<
1000 lg/L) can lead to resistance and toxic levels (> 4000 lg/L) to
severe side-effects. Monitoring of plasma levels may improve therapy
outcome and adherence, however drawing blood from children can be
traumatic. In this study the possibility of saliva levels as a non-invasive
alternative was investigated. Seventy-two paired plasma (1-1.5ml) and
saliva (0.5ml) samples were collected from 21 children (4 13 years) of
both genders. This pilot study was conducted at the Chris Hani Baragwanath Hospital, South Africa. The samples were taken at mid-dose interval, 2 hours apart, on 2 different study visits. A validated LC-MS/MS
method for the determination of salivary efavirenz levels was developed.
The limit of detection (LOD) and limit of quantication (LOQ) were
1.84 and 6.11 lg/L, respectively. Salivary efavirenz and total plasma
concentrations correlated at three of the four sampling times:
544
(p<0.005**, r =0.8; p<0.005**, r=0.6; p>0.005, r=0.5 and p<0.005**,
r=0.8). Although a correlation did exist, no regression equation to calculate plasma efavirenz levels from saliva, could be determined. It seems
that saliva levels can be employed to monitor adherence but more data is
required to investigate the correlation between salivary and plasma efavirenz levels.
Paper No.: 2406

DISEASES
IRINOTECAN-INDUCED INTESTINAL MUCOSITIS: ROLE OF
CYTOKINES ON INDUCIBLE NITRIC OXIDE SYNTHASE
ACTIVATION
Ronaldo A Ribeiro(1), MHLP Souza(1), GAC Brito(2), RB Oria(2), FQ
Cunha(3), DVT Wong(1), RCP Lima-Junior(1)
(1) Federal University of Ceara, Faculty of Medicine, Department of
Physiology and Pharmacology, Fortaleza-Ceara, Brazil
(2) Federal University of Ceara, Faculty of Medicine, Department of
Morphology, Fortaleza-Ceara, Brazil
(3), University of Sao Paulo, School of Medicine of Ribeirao Preto, Sao
Paulo, Brazil
Intestinal mucositis (IM) and diarrhea are common costly side effects of
irinotecan. Cytokine modulators are capable of attenuating IM. Nitric
oxide (NO) seems to be a key mediator of anticancer drug toxicity,
observed in other experimental models. Then, we aimed to investigate
the role of NO on the pathogenesis of IM, and the participation of cytokines on iNOS (inducible nitric oxide synthase) activation. iNOS knockout (iNOS-/-) and C57BL/6 animals (n=5-6) were given either saline or
irinotecan (60 mg/kg/4 days), with/without aminoguanidine (50 mg/kg),
thalidomide (specic inhibitor of TNFa, 60 mg/kg), or pentoxifylline
(non-specic inhibitor of cytokines, 1.7 mg/kg) daily. On day 5, diarrhea
was assessed, and following sacrice, duodenal samples were obtained
for myeloperoxydase (MPO, neutrophils/mg tissue), morphometric analyses (villus height, lm), western blot (iNOS/B-actin ratio), immunohistochemistry to iNOS, and for in vitro evaluation of duodenal contractility
(Acetylcholine/KCl60mM % ratio). Data were analyzed with ANOVA/
Student Newman Keul or Kruskal Wallis/Dunns test. P<0.05 was
accepted. Irinotecan induced severe diarrhea (3[3-3]), intestinal smooth
muscle over-contractility (176.154.7), histopathological changes
(15614), and increased MPO activity (3319407) compared with saline
group (0[0-0], 96.410.9, 382.28.3, 3012618, respectively). These
effects were abrogated in aminoguanidine-treated (1.5[1-2], 89.39.6,
2848, 1814327, respectively) and iNOS-/- mice (1[1-1], 69.47.1,
24119, 231103 respectively) compared with irinotecan group. Moreover, pentoxifylline, but thalidomide did not, inhibited iNOS expression
(0.40.1) and immunostaining (1.5[1-2]), compared with irinotecan
(1.30.4, 4[3-4]). This study suggests the pivotal role of NO in the pathogenesis of irinotecan-induced IM, and provides evidence for the participation of cytokines on iNOS activation cascade.
Support: CAPES/CNPq.
Paper No.: 2429

HEALTH AND DISEASE
SOLUBLE EPOXIDE HYDROLASE INHIBITION PREVENTS
CORONARY ENDOTHELIAL DYSFUNCTION IN
RENOVASCULAR HYPERTENSION
BWe have shown previously that cytochrome epoxygenases derived

metabolites (presumably epoxyeicosatrienoic acids, EETs) are endothelium-dependent hyperpolarizing factors both in humans and in some
mouse models. We tested whether experimental arterial hypertension
alters EETs-mediated coronary relaxations and whether this can be
prevented by inhibition of soluble epoxide hydrolase (sEH) which
degrades EETs. Hypertension was induced in FVB mice by chronic
renal artery stenosis (two kidney-one clip, 2K1C). After 7 weeks, NOindependent coronary relaxations to acetylcholine (assessed in the presence of a NO synthase inhibitor) were decreased in 2K1C mice, without modication in the responses to NS309 and NS1619, the openers
of calcium-activated potassium channels involved in the hyperpolarizing effect of EETs. The cytochrome epoxygenases inhibitors uconazole and MSPPOH impaired the coronary relaxations to acetylcholine
in control mice but not in 2K1C mice. The sEH inhibitor AUDA
administered for 2 weeks starting 5 weeks after surgery in 2K1C mice
(25 mg/L in drinking water) prevented the increase in blood pressure
and cardiac hypertrophy, improved the NO-independent coronary
relaxations to acetylcholine and restored the inhibitory effect of uconazole and MSPPOH on acetylcholine-induced relaxations, without
modifying the relaxations to NS309 and NS1619. Finally, sEH expression was increased in 2K1C mice.: These results thus demonstrate that
reduced EETs-mediated relaxations contributes to coronary endothelial
dysfunction in hypertensive mice, mediated at least in part by an
increased degradation by sEH. Furthermore, inhibition of sEH appears
to be a novel treatment of endothelial dysfunction in hypertension,
acting through restoration of EETs availability.
Paper No.: 2850

NEW APPROACH FOR FITTING OF DATA OF
FLUORESCENCE ANISOTROPY AND INTENSITY FOR
CHARACTERIZATION OF LIGAND BINDING TO
MELANOCORTIN 4 RECEPTORS
Ago Rinken, S Veiksina, S Kopanchuk
University of Tartu, Institute of Chemistry, Tartu, Estonia
Implication of uorescence methods in studies of ligand binding to their
receptors opens new possibilities to characterize these processes. We
have worked out a methodology where data of changes in uorescence
intensity and uorescence anisotropy by global numerical analysis can
be used for estimation of kinetic parameter of 7TM receptor-ligand interactions. The complex formation, activation and signal transduction of
7TMR is complex process which cannot be characterized by conventional, approximated analytical models and global numerical approach in
data analysis based on mechanistic models allowed to achieve better
solutions. We could integrate into our models also problems of the experiments, like receptor or ligand degradation, tracer photobleaching, drift
of baseline, etc., and get a working model. On-line registration of twoway uorescence data gave multivariate massive, which allowed to avoid
the overparameterisation in the analysis. We have validated this approach
for characterization of Cy3B-NDP-MSH binding to human melanocortin
4 receptors expressed in Sf9 cells by baculovirus system. In addition to
uoroligand binding parameters allowed uorescence intensity/anisotropy read-outs from multiwell plate together with the presented data
analysis method also estimation of kinetic properties of competitive
ligand.
Vincent Richard, J Gao, E Gomez, B Dautreaux, F Bounoure,

M Skiba, C Thuillez, J Bellien
Inserm U644, Department of Pharmacology, Rouen, France
545
Paper No.: 2893
HEALTH AND DISEASE
IMPROVED VASCULAR FUNCTION AND ALTERED CARDIAC
PHENOTYPE IN MICE OVEREXPRESSING MELANOCYTESTIMULATING HORMONE
Petteri Rinne(1,2), I Heinonen(3), A Saraste(4), ST Ruohonen(1),
S Ruohonen(1), E Savontaus(1,5)
(2) University of Turku, Drug Discovery Graduate School, Turku,
Finland
(3) University of Turku, Turku PET Centre, Turku, Finland
(4) Turku University Hospital, Department of Medicine, Turku, Finland
(5) Health Care District of Southwest Finland, TYKSLAB, Department
of Clinical Pharmacology, Turku, Finland
The central melanocortin system plays a crucial role in the regulation of
energy homeostasis and cardiovascular functions. Here we identify that
long-term activation of the melanocortin system affects autonomic nervous system function, vascular reactivity and cardiac growth. We applied
echocardiography, blood pressure telemetry and isometric tension recordings to examine changes in cardiac and vascular function of transgenic,
a- and c-MSH overexpressing (MSH-OE) mice. Experiments were conducted in young and aged (3- and 6-month-old) MSH-OE mice and the
observed phenotype was assessed against age-matched wild type (WT)
mice. MSH-OE mice did not differ in terms of arterial blood pressure,
but displayed reduced heart rate in both age groups. Elevated cardiac
vagal activity was observed especially in young MSH-OE mice. The
echocardiographic examination revealed that aged MSH-OE mice had
increased left ventricular (LV) dimensions. Despite the evidence of
enlarged LV chamber size, aged MSH-OE mice showed normal myocardial performance and no signs of cardiac hypertrophy. Interestingly, aged
MSH-OE mice had lower heart weights than WT mice. Furthermore, we
observed a decreased vasoconstrictory prole and improved endothelium-dependent vasodilatation in the large conduit arteries of MSH-OE
mice. These inter-genotype differences were completely abolished by
inhibiting endothelial nitric oxide synthase. The concept of improved
vascular phenotype was further strengthened when we applied Doppler
echocardiography to assess coronary microvascular function, whereby
MSH-OE mice showed enhanced vasodilatory response to hyperaemic
stimuli. In conclusion, MSH overexpression improves vascular function
and may provide cardioprotective regulation by increasing parasympathetic activity and nitric oxide bioavailability, thereby restraining the ageassociated cardiac hypertrophy.
Paper No.: 1982

INTERACTION BETWEEN PEROXISOME PROLIFERATORACTIVATED RECEPTOR-A AND NADPH OXIDASES PLAYS
AN IMPORTANT ROLE IN THE DEVELOPMENT OF
PRESSURE-OVERLOAD CARDIAC HYPERTROPHY
Emma Robinson, DJ Grieve
Queens University Belfast, Centre for Vision and Vascular Science, Belfast, UK
Interaction between peroxisome proliferator-activated receptor-a and
NADPH oxidases plays an important role in the development of pressure-overload cardiac hypertrophy. Emma Robinson and David J
Grieve.Centre for Vision and Vascular Science, Queens University Belfast, UK. Peroxisome proliferator-activated receptor-a (PPAR- a) deactivation and NADPH oxidase activation play essential roles in the
development of left ventricular hypertrophy (LVH). Here, we investigated potential interaction between the two as an important component
in overall regulation of LVH. Nox2-/-, PPAR-a-/- and matched wild-type

(WT) mice (n 8) were subjected to thoracic aortic constriction (TAC)
or sham surgery and studied 7 days later. No differences in basal contractile function (echocardiography) or LVH were observed between groups.
However, increased mRNA expression (real-time RT-PCR) of PPAR-a in
Nox2-/- (1.49 0.08 vs WT, 1.04 0.11 arbitrary units; P < 0.05) and
of Nox2 in PPAR-a-/- mice (3.61 1.21 vs WT, 0.95 0.12 arbitrary
units; P < 0.05), together with increases in NADPH oxidase activity
(lucigenin-enhanced chemiluminescence: PPAR-a-/-, 6.81 0.94 vs WT,
3.49 0.35 RLU; P < 0.05), indicated co-regulation of myocardial
Nox2/PPAR-a. As expected, TAC-induced LVH was signicantly
increased in PPAR-a-/- versus WT mice, but similar in Nox2-/- mice.
Decreases in fractional shortening in WT mice (-16 3%) were augmented in PPAR-a-/- and attenuated in Nox2-/- mice after TAC (-28 4
and -10 3%; P < 0.05 vs WT). PPAR-a mRNA expression was
increased in WT, but not Nox2-/- mice (52 11 vs -17 11%;
P < 0.05), whilst Nox2 mRNA expression remained elevated
(1.33 0.22 vs 0.69 0.16 arbitrary units; P < 0.05) in PPAR-a-/- versus WT mice after TAC. NADPH oxidase activity was signicantly
increased in TAC WT, but not PPAR-a-/- or Nox2-/- mice, although absolute levels in PPAR-a-/- remained elevated compared to WT sham. These
results suggest co-dependence of PPAR-a and NADPH oxidases during
pressure-overload LVH, although the underlying mechanisms are clearly
complex.
Paper No.: 2375

FUNCTIONAL AND MORPHOLOGICAL
CHARACTERIZATION OF PRESYNAPTIC A1 AND A3
ADENOSINE RECEPTORS IN THE SUPERIOR MESENTERIC
ARTERY OF SPONTANEOUSLY HYPERTENSIVE RATS
C Rocha-Pereira(1), P Fresco(1), S Arribas(2), MC Gonzalez(2),
MV Conde(2), J Goncalves(1), Carmen Diniz(1)
(1) University of Porto Faculty of Pharmacy, Department of Pharmacology, Porto, Portugal
(2) University Autonoma of Madrid Faculty of Medicine, Department of
Introduction: Adenosine may play a role in the development of hypertension by changing sympathetic neurotransmission in vessels (Karoon P et
al, BJP 1995;116:1918-22). The aim of this study is to characterize the
function and location of A1/A3-adenosine receptors in mesenteric artery
(MA) from Wistar Kyoto (WKY) and Spontaneously Hypertensive Rats
(SHR) and to evaluate their putative contribution to hypertension. Materials-Functional study: WKY and SHR MA-segments, pre-incubated with
0.1microM [3H]-NA, were submitted to four-identical periods of stimulation (2Hz, 200pulses, 1ms, 80mA) every 20min (S1-S4). Effects of selective A1/A3-agonists and/or antagonists on NA release (estimated as
tritium overow) were studied; Morphological study: MA-segments were
longitudinally opened, paraformaldehyde-xed and incubated overnight
at 4C with primary anti-A1 or anti-A3 and anti-dopamine b-hydroxilase
(DBH) and uorescent secondary antibodies, mounted on a slide exposing the adventitia and visualized by confocal microscopy. Results: A1agonist CPA (10 and 100nM) inhibited NA release in MA, the inhibition
being lower in SHR (89.09 3.59%; 74.36 2.85%; n = 14) than in
WKY (76.55 2.75%; 61.68 2.74%; n = 14; p < 0.05). A3-agonist
IB-MECA (1 and 10nM) inhibited NA release in WKY MA
(91.71 1.72%; 91.39 0.95%; n = 12; p < 0.05), an effect not
observed in SHR MA. Confocal microscopy showed that A1- and A3receptors were located on the same structure as DBH-staining. Conclusion: the less efcient A1 receptor-mediated inhibition (receptor reduced
function) and the absence of A3 receptor-mediated inhibition (suggesting
the presence of non-functional receptors) of NA release may contribute
to the described increased NA spillover observed in SHR.
546
Acknowledgements: FCT (SFRH/BD/32161/2006); SAF2007-31134-E;
Acciones Integradas Hispano-Lusas 2010-2012.
Paper No.: 2806

A2 ADRENOCEPTOR DISTRIBUTION IN PIG BRAIN, MAPPED
WITH RADIOLABELED YOHIMBINE BY MEANS OF
POSITRON EMISSION TOMOGRAPHY
caused by ischemic heart disease in 65 and dilated cardiomyopathy in 13

patients. All patients including in the study were randomized into 2
groups, control received standard therapy and the main one additionally
received adenocin. Results. All biological markers of the intensity of endotoxemia and oxidative stress, immune inammatory status, redoxpotential, considerably improved in patients of the main group. The signs
of SIRS disappeared in main group, but persisted in patients of control
group. The improvement of the symptoms of SIRS in main group was
coupled with the increasing of ejection fraction by 12%, and NYHA
functional class by the 37% (only 10% in the control group). Conclusion.
The treatment with adenocin cessates the systemic hypoxic and SIRS,
improves cardiac hemodynamics and symptoms of CHF.
A Rodell(1), S Jakobsen(1), Albert Gjedde(2)

(1) Aarhus University Hospital, Aarhus PET Center, Aarhus, Denmark
(2) University of Copenhagen, Faculty of Health Sciences, Department
of Neuroscience and Pharmacology, Copenhagen, Denmark
We introduce a new method for measuring the population-based displacement of noradrenergic receptor binding in pig brains without a common non-binding reference region and with no need for spatial blurring
of individual data. Six Danish landrace pigs were anesthetized and PETscanned, rst during the circulation of 500 MBq 11C-yohimbine, a selective a2 adrenoceptor antagonist, and subsequently twice during circulation of 500 MBq 11C-yohimbine with pharmacological doses of
unlabeled yohimbine (0.075-2 mg) or RX821002 (0.15-0.75 mg), a
selective a2 adrenoceptor antagonist. The pigs were PET-scanned in the
ECAT HR47 tomograph (CTI/ Siemens), and we sampled arterial blood
throughout the scanning sessions of 1.5 hours. To reduce noise in the
images without loss of resolution by spatial ltering, we normalized the
dynamic images to the AUC of the blood curves, and then averaged the
six normalized images. We calculated the distribution volumes in the tissue (Vd) by integration of the average normalized dynamic image. We
determined the non-displaceable distribution volume by inhibition plotting of values of Vd after inhibition against baseline Vd, The inhibition
plot showed specic binding in all brain regions, and the plot indicated a
partition coefcient (Ve) of close to 1.2 ml plasma/cc tissue for labeled
yohimbine. We calculated the binding potentials (receptor availability) as
BP = Vd/Ve -1 for the baseline and inhibition images. Frequency analysis of each state suggested a bimodal distribution of BP which cleanly
separated intra- and extracerebral a2 adrenoceptors.
Paper No.: 911

TARGETING THERAPY OF THE SYSTEMIC
INFLAMMATORY RESPONSE SYNDROME IN PATIENTS
WITH CONGESTIVE HEART FAILURE
Mamanti Rogava(1), T Bochorishvili(1), N Dolidze(2), M Mirziashvili(3)
(1) N.Kipshidze National Centre of Therapy, Department of Cardiomyopathy, Tbilisi, Georgia
(2) GPI Clinic, Department of Endocrinology, Tbilisi, Georgia
(3) Tbilisi state Medical University, Department of Pharmacology, Tbilisi, Georgia
Introduction. The systemic inammatory response syndrome (SIRS)
which accompanied severe form of congestive heart failure (CHF) are
associated with activation of a number of complex and interrelated pathways that include the endotoxemia, tissue hyperoxia and activity of the
intrinsic defense system. The purpose of the present study was to test the
hypothesis that inotropic drug with pronounced antihypoxic/antiischemic
and antioxidant properties, adenocin, would signicantly reduce inammatory signalling and cardiac dysfunction. Materials/Patients. Plasma
levels of cytokines (interleukine-6, interleukine -8, TNF-a) and soluble
adhesion molecules, redox potential NAD/NADH, superoxide anion production, activities of superoxide dismutase and catalase were determined
in samples obtained from 78 patients with CHF NYHA classes II-VIV,
Paper No.: 3247

LIQUID FRUCTOSE MODIFIES INSULIN SIGNALLING
PATHWAY INDEPENDENTLY OF AMPK ACTIVATION IN
FEMALE SPRAGUE-DAWLEY RATS
Nuria Roglans(1), A Rebollo(2), A Padrosa(2), L Vila`(2), M Alegret(1),
JC Laguna(1)
(1) University of Barcelona. IBUB and CIBERDEM, Faculty of
Pharmacy, Department of Pharmacology, Barcelona, Spain
(2) University of Barcelona, Faculty of Pharmacy, Department of
Pharmacology, Barcelona, Spain
Women are affected differently than men by fructose ingestion, showing
an association between fructose consumption and an increased risk of
type 2 diabetes mellitus. Rats are considered a suitable model for human
fructose metabolism. Fructose administration (10% w/v) in the drinking
water to female Sprague-Dawley rats induces hypertriglyceridemia, hepatic steatosis and clear signs of glucose intolerance and hepatic insulin
resistance. To elucidate the molecular switches involved in these effects,
we performed a temporal study with control and fructose-fed rats (7 and
14 days of treatment). Plasma analytes, liver triglycerides, liver enzyme
activities and expression of enzymes and transcription factors related to
fatty acid metabolism and insulin signalling were determined. At day 14
of treatment, fructose-fed female rats showed hyperinsulinemia, an
altered glucose tolerance test, less liver insulin receptor substrate 2 (IRS2) and a marked decrease in the hepatic expression of SIRT-1. These
alterations are not observed at 7 days of fructose consumption, indicating
that insulin resistance manifestations appear later than hepatic steatosis.
It has been reported that SIRT-1 and IRS-2 expression may be regulated
by the AMP-activated protein kinase (AMPK). However, liver AMPK
expression was not modied in our study, suggesting that the impairment
of insulin signalling observed in fructose-fed female rats is independent
of the AMPK pathway.
Paper No.: 2768

VISFATIN/PBEF/NAMPT: A NEW THERAPEUTICAL TARGET
IN VASCULAR INFLAMMATION
Tanya Romacho(1), V Azcutia(1), N Matesanz(1), E Cercas(1), R Carraro(2), L Rodriguez-Manas(3), CF Sanchez-Ferrer(1), C Peiro(1)
(1) University Autonoma of Madrid, Faculty of Medicine, Department of
Pharmacology and Therapeutics, Madrid, Spain
(2) University Hospital de La Princesa, Department of Endocrinology
and University Autonoma of Madrid, Madrid, Spain
(3) University Hospital of Getafe, Department of Geriatrics, Diagnosis
Unit, Getafe, Spain
547
Visfatin/pre B-cell colony enhancing factor/nicotinamide phosphorybosil
transferase (visfatin) is an adipokine whose levels are enhanced in metabolic disorders exhibiting high cardiovascular risk, like type 2 diabetes
and obesity. In cultured human aortic smooth muscle cells (HASMC),
visfatin (100 ng/ml) triggered the consecutive activation of ERK 1/2,
NF-jB and iNOS, as determined by Western blot and EMSA. Such activation was not affected by insulin receptor blockade with specic antibodies, but was blunted by APO866 (100 nM), a nicotinamide
phosphorybosil transferase (Nampt) inhibitor. Indeed, nicotinamide
mononucleotide (NMN, 1mM), the product of Nampt, mimicked iNOS
induction and NF-jB activation by visfatin. On the other hand, visfatin was constitutively detected in cultured human umbilical vein endothelial cells (HUVEC) by Western blot and immunouorescence,
mainly in cell nuclei. Under a pro-inammatory stimulus such as IL1b, visfatin RNA expression and protein content in HUVEC was upregulated. IL-1b also favoured visfatin localization in the cell cytoplasm, where it co-localized with F-actin bers, suggesting a secretory
pathway. Visfatin induction by IL-1b was abolished by the poly-ADPribose polymerase (PARP)-1 inhibitor, PJ34 (10 lM). We conclude
that visfatin, either released from adipose tissue or locally synthesized in
the vascular wall, arises as a new vascular pro-inammatory factor, as
well as a potential therapeutical target to treat vascular inammation and
related atherothrombotic diseases.
Paper No.: 1890

THE FAT-INDUCED SATIETY FACTOR OEA SUPPRESSES
FEEDING THROUGH CENTRAL RELEASE OF OXYTOCIN
Adele Romano(1), S Gaetani(1), J Fu(2), T Cassano(3), P Dipasquale(1), L Righetti(1), S Cianci(1), L Laconca(3), E Giannini(1),
S Scaccianoce(1), D Piomelli(2), V Cuomo(1)
(1) Sapienza University of Rome, Department of Pharmacology and
Physiology, Rome,Italy
(2) University of California, Department of Pharmacology, Irvine, CA,
USA, and Italian Institute of Technology, Genoa, Italy
(3) University of Foggia, Department of Biomedical Sciences, Foggia,
Italy
Oleoylethanolamide (OEA) is a biologically active lipid amide that is
released by small-intestinal enterocytes during the absorption of dietary
fat and inhibits feeding by engaging the nuclear receptor, PPAR-a. Previous studies have shown that the anorexic effects of systemically administered OEA require the activation of sensory afferents of the vagus nerve.
The central circuits involved in mediating OEA-induced hypophagia
remain unknown. Here, we report the results of in situhybridization and
immunohistochemistry experiments in rats and mice, which show that
systemic injections of OEA enhance expression of the neuropeptideoxytocin in magnocellular neurons of the paraventricular andsupraopticnuclei of the hypothalamus. No effect is observed with other
hypothalamic neuropeptides, including vasopressin, thyrotropin-releasing
hormone and pro-opiomelanocortin. The increase in oxytocin expression
elicited by OEA was accompanied by enhanced oxytocin release in the
paraventricular, as detected by in vivomicrodialysis, and was absent in
PPAR-a null-mice. Pharmacological blockade of oxytocin receptors in
the brain by intracerebroventricular infusion of the selective oxytocin
antagonist, L-368,899, prevented the anorexic effects of OEA. The
results suggest that OEA suppresses feeding by activating central oxytocin transmission.
Paper No.: 613

STATE OF ENDOTHELIAL AND HUMORAL REGULATION IN
HEART FAILURE OF THE AGED PATIENTS ON ESSENTIAL
HYPERTENSION WITH COMBINED IHD DURING THE
TREATMENT
Olesya Romashenko
The Belgorod State University, Faculty of Medicine, Belgorod, Russian
Federation
The research is dedicated to learning of endothelial and humoral regulation state in chronic heart failure (CHF) of the 105 aged patients on
essential hypertension with combined IHD from the adaptation mechanisms point of view. The regulatory systems state was learned during different variants of pharmacotherapy with the aim of working out the
recommendations on individual choice of optimal therapy scheme in
order to improve the CHF treatment effectiveness. According to the
results of the research the disturbance of endothelial and humoral regulation state was revealed during CHF in the aged patients on essential
hypertension with combined IHD. By the age of patients the regulatory
systems state changes were revealed according to the phases of stressage-syndrom. Basic therapy of CHF (by ACE inhibitors, b-adrenoblokers, diuretics) showed moderate clinical effectiveness and excessive
activation of adaptation process, that appeared in unbalanced changes of
endothelial and humoral regulation. Additional prescribtion of mildronat
improved clinical effectiveness of treatment thanks to normalisation
action on regulatory systems state of patients with safe adaptation
reserves. The recommendations on individual choice of optimal variant
of pharmacotherapy, based on research of patients initial clinical state,
were proposed with high reliability in prognosis of advanced clinical
effectiveness of CHF treatment.
Paper No.: 521

THE EFFECTS OF URB597, FATTY ACID AMIDE HYDROLASE
(FAAH) INHIBITOR, IN THE BASOLATERAL AMYGDALA OF
RATS ON ANXIETY-LIKE BEHAVIORS
Ali Roohbakhsh(1), A Hajizadeh Moghaddam(2), J Damchi Jelodar(3)
(1) Rafsanjan University of Medical Sciences, Department of Physiology
& Pharmacology, Rafsanjan, Iran
(2) University of Mazandaran, School of Basic Science, Department of
Biology, Babolsar, Iran
(3) Islamic Azad University, School of Basic Science, Department of
Biology, North Tehran Branch, Tehran, Iran
There are reports indicating that endocannabinoid system may be
involved in anxiety-related behaviors. Cannabinoid CB1 receptors are
particularly concentrated in limbic structures such as the hippocampus
and amygdala suggesting that endocannabinoids may contribute to learning, memory and anxiety. Amygdala complex is an important brain site
in the modulation of fear- or anxiety-like behaviors. The effects of bilateral intra-basolateral amygdala (Intra-BLA) injection of URB597, a
FAAH inhibitor, and AM251, a selective CB1 receptor antagonist, on
anxiety-related behaviors using elevated plus-maze test of anxiety were
evaluated in the present study. Injection of URB597 at the doses of
0.001, 0.005 and 0.01`g/rat showed a signicant anxiolytic effect at
0.005 and 0.01`g/rat. Moreover, intra-BLA injection of AM251 at the
doses of 0.001, 0.01and 0.1`g/rat produced anxiogenic effect in the elevated plus-maze at dose of 0.1 `g/rat. The ineffective doses of AM251
(0.01 lg/rat) on anxiety-related behaviors was injected with different
doses of URB597. The obtained data showed that AM251could reverse
the anxiolytic effect of URB597. Our results indicated that endocannabi-
548
noid system in the basolateral amygdala may modulate anxiety-like
behaviors through activation of CB1 receptors.
Paper No.: 1940

DISEASES
ANGIOTENSIN II PROMOTES CYTOKINE PRODUCTION IN
HUMAN PODOCYTES
Arianna Carolina Rosa, L Rattazzi, G Miglio, R Fantozzi
University of Turin, Department of Anatomy, Pharmacology and Forensic Medicine, Turin, Italy
Angiotensin II (AngII) hemodynamic and nonhemodynamic effects are
critical for glomerular functioning. Its ability to induce proinammatory
cytokines (e.g. TNF-a and IL-6) production in the rat kidney (Ruiz-Ortega et al, Kidney Int Suppl 2002; 82: S12-22) suggests an AngII role in
the kidney pathophysiology. Whether podocytes, glomerular ltration
barrier pivotal cell components, are responsive to AngII and a source of
these cytokines remains poorly understood. For this purpose human
immortalized podocytes were exposed to AngII (0.1-100 nM; 1-6 days)
and proinammatory (TNF-a, IL-6 and IL-18)/antiinammatory (IL-10)
cytokine production was evaluated by RT-PCR and ELISA assays. RTPCR and western-blot analysis revealed that podocytes express both AngII receptors (AT1R and AT2R) that are overexpressed by exposure to
AngII, with the maximum achieved at 10 nM at the 4th day. The AngII
stimulation signicantly reduced IL-10 expression since the 1st day and
throughout the experimental period. TNF-a and IL-6 production displayed a biphasic pattern, with a rst peak at the 3th and a second at the
5th day. IL-18 kinetics showed a single peak at the 4th day. The
observed effects are concentration-dependent with the maximum at 10
nM AngII. The coadministration of AngII with receptor antagonists, losartan (AT1R) and PD123,319 (AT2R), both at 100nM, abolished the
production of TNF-a, IL-6 and IL-18, whilst that of IL-10 was increased.
In conclusion, our data support the hypothesis that AngII, interacting
with its receptors, might contributes to an inammatory pathway leading
to podocytes dysfunction and the following associated glomerular ltration barrier damage.
Paper No.: 1388

DISEASES
A-PINENE INHIBITS INTERLEUKIN-1-INDUCED NITRIC
OXIDE PRODUCTION BY NF-JB-DEPENDENT AND
INDEPENDENT MECHANISMS IN HUMAN CHONDROCYTES
Susana Rosa(1,2), J Goncalves(2), F Judas(3,4), L Salgueiro(5),
C Cavaleiro(5), C Lopes(1,2), AF Mendes(1,2)
(2) Center for Neurosciences and Cell Biology, Coimbra, Portugal
(3) University Hospital of Coimbra, Orthopedic Department, Coimbra,
Portugal
(4) University of Coimbra, Faculty of Medicine, Coimbra, Portugal
(5) University of Coimbra, Laboratory of Pharmacognosy/CEF, Faculty
of Pharmacy, Coimbra, Portugal
Nuclear Factor-jB (NF-jB) plays a major role in arthritic diseases by
promoting the expression of inammatory and catabolic mediators,
namely nitric oxide (NO). Our previous study showed that a fraction
(Fa-p) of the essential oil from the leaves of Juniperus oxycedrus, mainly
composed of a racemic mixture of a-pinene [2,6,6-trimethyl-bicyclo(3.1.1)hept-2-ene], decreases IL-1-induced NF-jB activation and NO
production in human chondrocytes [Neves A et al. Planta Medica (in
press)]. This study aimed at further elucidating the mechanism of action
of a-pinene. NF-jB activation was evaluated in the human chondrocytic

cell line, C-28/I2, by western blot for cytoplasmic phospho-IkB-a levels
and by ELISA for DNA binding activity. To determine the ability of the
test compounds to directly inhibit NO production, regardless of their
effect on NF-jB-dependent iNOS expression, nitrite levels were measured by the Griess reaction in human chondrocytes treated with the test
compounds after pretreatment with IL-1 for 18h. Fa-p decreased phospho-IkB-a, NF-jB DNA binding and NO production to 22.1% 7.8,
46.4% 13.2 and 11.3% 0.4 of the levels induced by IL-1 alone,
respectively. When added after IL-1, Fa-p decreased NO levels to 42.1%
7.3. The commercial racemic and (+) enantiomer were less effective
while the (-) enantiomer had no signicant effects. (+)a-pinene probably
accounts for most of the ability of Fa-p to inhibit IL-1-induced NF-jB
activation and NO production. The later seems to involve NF-jB dependent and independent mechanisms, probably through direct inhibition of
iNOS activity.
Supported by grants PTDC/SAU-OSM/67936/2006 and SFRH/BD/
19763/2004 from FCT.
Paper No.: 1608

ATTENUATED CORONARY VASODILATOR RESPONSES TO
ADENOSINE RECEPTOR AGONISTS IN HEARTS FROM
ZUCKER DIABETIC RATS
Roselyn RoseMeyer, D Wang
Grifth University, School of Medical Sciences, Southport, QLD, Australia
Adenosine induces coronary vasodilation through multiple adenosine
receptor subtypes coupled to signalling pathways that include nitric oxide
and potassium channels. Chronic hyperglycaemia caused by diabetes
mellitus is well established to cause endothelial damage through several
mechanisms including injury caused by excessive ROS production. We
investigated the vasodilator actions of the NO donor sodium nitroprusside (SNP) and adenosine receptor analogue 5-(N-ethylcarboxyamido)
adenosine (NECA) on coronary resistance vessels in hearts from diabetic
obese Zucker rats. Rats were anaesthetised with pentobarbitone, 60 mg/
kg IP and hearts were dissected and perfused in Langendorff mode with
Krebs-Henseleit solution. Following 30 mins equilibration, concentration-response curves to SNP and NECA were then obtained. At
14 weeks of age, obese Zucker rat blood glucose levels were
14.83 1.97 mmol/L compared to age matched Zucker lean rat (control)
values of 6.72 0.52 mmol/L, n = 6 per group, (P < 0.05). Maximal
coronary vasodilator responses to both SNP and NECA were attenuated
in hearts from Zucker obese rats when compared to control tissues
(P < 0.05), however the agonist potencies remained the same with
NECA pEC50 CI values in Zucker obese rats; 8.45 0.08 vs control
data; 8.35 0.09, P > 0.05. In conclusion, at 14 weeks of age the Zucker obese rats were hyperglycaemic in contrast to age matched Zucker
lean rats. Vasodilator responses to NECA and SNP were reduced in
hearts from the Zucker obese rats indicating a possible change occuring
in NO signalling pathway function in the coronary resistance vessels
with chronically elevated blood glucose levels.
Paper No.: 2814

INVOLVEMENT OF BOTH B2- AND B3-ADRENOCEPTORS IN
THE INHIBITION OF NEUROGENIC CONTRACTIONS OF
MOUSE ISOLATED URINARY BLADDER
C Rouget, T Westfall, M Rekik, P Lluel, Stefano Palea
UPS, Faculty of Pharmaceutical Sciences, UROsphere, Department of
549
In the present study, we evaluated the functional presence of b2- and b3adrenoceptors (AR) in mouse urinary bladder by investigating the effects
of isoproterenol as well as the selective b2- and b3-AR agonists fenoterol
and CL316,243 respectively on neurogenic contractions in the absence
and presence of b2- and b3-AR antagonists. Bladder strips obtained from
C57/Bl6 mice were placed in organ baths lled with a Krebs solution
containing 1 lM prazosin. After addition of ICI118,551 or L748,337
(selective b2- or b3-AR antagonists, respectively) strips were subjected to
electrical eld stimulation (EFS: 800mA, 2.5Hz, pulses of 0.3ms; train
of 2s every 60s). Then, cumulative concentration-response curves to
CL316,243, fenoterol or isoproterenol were constructed. CL316,243 concentration-dependently inhibited EFS-induced contractions (Emax=36%,
pEC50 = 7.48). L748,337, but not ICI118,551, signicantly blocked the
inhibitory effects of CL316,243 (pKB=7.00). Fenoterol inhibited EFSevoked contractions (Emax=68%, pEC50 = 6.85) in a concentrationdependent manner. ICI118,551 potently blocked the inhibitory effects of
fenoterol (pKB=8.80) while L748,337 inhibited the relaxing effects of
fenoterol only at relatively high concentrations (pKB=5.79). Similarly,
isoproterenol concentration-dependently inhibited EFS-induced contractions (Emax=65%, pEC50 = 6.98). The inhibitory effect of isoproterenol
was potently blocked (pKB=8.53) by ICI118,551 while L748,337 only
inhibited the relaxing effects of isoproterenol at high concentrations
(pKB=5.49). These results demonstrate that activation of both b3- and
b2-ARs produces inhibition of neuronally-mediated contractions in
mouse urinary bladder. In addition, these results suggest that stimulation
of b2-ARs produces a greater degree of inhibition than b3-ARs.
Paper No.: 2815

STIMULATION OF BOTH B2- AND B3-ADRENOCEPTORS
PRODUCES RELAXATION OF KCL-INDUCED
CONTRACTIONS OF MOUSE ISOLATED URINARY BLADDER
Paper No.: 1508

HEALTH AND DISEASE
NON-INVASIVE LASER SPECKLE CONTRAST IMAGING TO
ASSESS SKIN MICROVASCULAR REACTIVITY IN HUMANS
Matthieu Roustit, C Millet, S Blaise, B Dufournet, J-L Cracowski,
Grenoble University Hospital, Clinical Pharmacology Unit - Inserm
CIC03, Grenoble, France
Post-occlusive reactive hyperemia (PORH) and local thermal hyperemia
(LTH) assessed with laser-Doppler owmetry (LDF) are functional markers of microvascular function. However, LDF reproducibility on the forearm is poor due to spatial variability. Recently developed laser speckle
contrast imaging (LSCI) allows continuous recording over wide areas.
However, reproducibility data and correlation with LDF are lacking. We
performed PORH and LTH assessed with LDF and LSCI on the forearm
of fourteen healthy volunteers, at day 0 and day 7. PORH was expressed
as peak cutaneous vascular conductance (CVC), peak-baseline (CVCPKCVCBL), percentage of baseline (%CVCBL), and percentage of 43C
vasodilation (%CVC43). LTH peak and plateau were expressed as CVC,
%CVC43 and %CVCBL. Reproducibility was expressed as intra-subject
coefcients of variation (CV) and intra-class coefcients of correlation
(ICC). Inter-day reproducibility of PORH assessed with LSCI was very
good whether expressed as peak CVC (CV=8%; ICC=0.76), CVCPKCVCBL (CV=11%; ICC=0.75) or %CVCBL (CV=14%; ICC=0.52).
Inter-day reproducibility of LTH peak and plateau assessed with LSCI
was good whatever the way of expressing data (CV<25%; ICC>0.47).
Spatial reproducibility of PORH peak CVC was better with LSCI compared to LDF (CV=12 and 47%, respectively). Finally, LDF and LSCI
measurements were correlated for PORH peak CVC (r = 0.46;
p = 0.001), CVCPK-CVCBL (r = 0.47; P < 0.001) and %CVCBL
(r = 0.34; p = 0.01). Reproducibility of PORH and LTH assessed with
LSCI is very good on the forearm, probably due to low spatial variability. Moreover, LSCI and LDF measurements of PORH are correlated. In
conclusion, LSCI is a promising tool to assess human skin blood ow.
C Rouget, T Westfall, M Rekik, P Lluel, Stefano Palea

UPS, Faculty of Pharmaceutical Sciences, UROsphere, Department of
We studied the relative roles of b2- and b3-adrenoceptors (AR) in mouse
urinary bladder by investigating the effects of isoproterenol as well as
the selective b2- and b3-AR agonists fenoterol and CL316,243 respectively on myogenic contractions in the presence or absence of urothelium. Bladder strips obtained from C57/Bl6 mice were placed in organ
baths lled with a Krebs solution containing 1 lM prazosin. After addition of CGP20712, ICI118,551 or L748,337, selective b1-, b2- or b3-AR
antagonists, respectively, strips were contracted with 30 mM KCl. Cumulative concentration-response curves to fenoterol, CL316,243 or isoproterenol were constructed. CL316,243 concentration-dependently inhibited
KCl-induced contractions in a similar manner in the presence (pEC50 =
7.17) or absence of urothelium (pEC50 = 6.99). L748,337 but not
CGP20712 or ICI118,551 signicantly inhibited the relaxing effects of
CL316,243 (pA2 = 6.38). Fenoterol concentration-dependently inhibited
KCl-induced contractions in the presence (pEC50 = 6.05) or absence of
urothelium (pEC50 = 6.21). ICI118,551 but not L748,337 signicantly
inhibited the relaxing effects of fenoterol. Isoproterenol inhibited KClinduced contractions in a concentration-dependent manner in the presence (pEC50 = 6.71) or absence of urothelium (pEC50 = 7.02).
L748,337 inhibited the relaxing effects of isoproterenol with low potency
(pKB=4.80 and 5.45 in presence and absence of urothelium, respectively), while ICI118,551 potently inhibited the relaxing effects of isoproterenol (pKB=7.80 and 8.35 in presence and absence of urothelium,
respectively). The activation of both b3- and b2-ARs located in the
smooth muscle layer produces relaxation of KCl pre-contracted mouse
urinary bladder. While CL316,243 and fenoterol act through b3- and b2ARs, respectively, isoproterenol appears to produce relaxation primarily
through b2-ARs.
Paper No.: 1515

HEALTH AND DISEASE
CATHODAL IONTOPHORESIS OF TREPROSTINIL AND
ILOPROST INDUCE A SUSTAINED INCREASE IN SKIN
BLOOD FLOW IN RATS
Matthieu Roustit, S Blaise, C Ribuot, C Millet, J-L Cracowski
Grenoble Medical School, Inserm ERI17, Grenoble, France
The treatment of scleroderma related digital ulcers remains a difcult
therapeutic challenge. The most effective drugs are prostacyclin analogues. However, their use is limited by the intravenous route and their
frequent vascular side effects. The objective of this study was to test
whether treprostinil, iloprost and epoprostenol may induce a sustained
vasodilation in rat skin when delivered locally through a iontophoretic
device. Treprostinil, iloprost and epoprostenol were delivered through
cathodal and anodal iontophoresis on anaesthesized rat hindquarters. Skin
blood ow was quantied using laser Doppler Imaging and cutaneous
tolerance was assessed from day 0 to day 3. Cathodal but not anodal iontophoresis of treprostinil (6.4 mM), iloprost (200 lM), and epoprostenol
(1.4 mM) induced a signicant and sustained increase in cutaneous blood
ow when compared to NaCl. Treprostinil and iloprost effects were concentration dependent. Weak effects were observed when both drugs were
applied locally without current. Skin resistance was similar in area treated with prostacyclin analogues and NaCl. Lastly, skin tolerance was
good, with no evidence of epidermal damage. In conclusion, treprostinil
and iloprost cathodal iontophoresis should be investigated as a new local
therapy for digital ulcers in patients with scleroderma.
550
Paper No.: 2933
DISRUPTIVE EFFECTS OF SYSTEMICALLY ADMINISTERED
ANGIOTENSIN II ON CEREBRAL PERFUSION AND
LEARNING IN THE RAT
Michael Rowan, W Cullen, B Ryan
Trinity College Dublin, Department of Pharmacology and Therapeutics,
Dublin, Ireland
There is great interest in the role of vascular risk factors including hypertension in age-related cognitive decline but relatively few pharmacological models are available to explore possible mechanisms. Hypertensive
doses of angiotensin can reduce cerebral blood ow, potentially causing
impaired cognition. Here we examined the ability of systemically administered Angiotensin II to selectively affect learning at doses that caused
mild-to-moderate cerebral hypoperfusion in rats. Acute injection of adult
male Wistar rats with angiotensin II dose-dependently (0.05-1 mg/kg,
s.c.) lowered cortical perfusion by up to one third in freely behaving animals, as measured using continuous transcranial laser doppler methods.
Pretreatment with 0.2 mg/kg, but not 0.1 mg/kg, angiotensin II prior to
acquisition of a contextual fear conditioning task impaired 24 h recall as
measured by freezing behaviour, and disrupted delayed spontaneous
alternation behaviour in a T maze. In contrast the same dose of angiotensin II had no signicant effect on general spontaneous motor activity in a
novel arena (hole board). Furthermore, continuous s.c. infusion of angiotensin II via an osmotic minipump (0.35 ug/kg/day for 2 weeks) selectively impaired contextual fear conditioning without signicantly
affecting behavioural activity in the hole board test. These results indicate that relatively low doses of angiotensin II administered systemically
acutely or repeatedly can selectively impair learning, possibly as a result
of reduced cerebral perfusion.
Paper No.: 2738

BAK FOONG PILLS, A TRADITIONAL REMEDY FOR
DYSMENORRHOEA, INHIBITS NOCICEPTION VIA THE
SOMATOSTATIN PATHWAY
Dewi K Rowlands(1,2), YG Cui(1), SC So(1), LL Tsang(1),
YW Chung(1), HC Chan(1)
stimuli, was able to inhibit BFPs analgesic effect. The present study thus
demonstrated that sub-chronic treatment of BFP has anti-nociceptive
qualities mediated via the somatostatin pathway.
Paper No.: 3364

IMPACT OF CYP2D6 GENETICS ON SERUM
CONCENTRATION OF ESCITALOPRAM IN RELATION TO
CYP2C19 GENOTYPE
Ida Rudberg(1), M Hermann(1), H Refsum(1), E Molden(1,2)
(1) Diakonhjemmet Hospital, Department of Psychopharmacology, Oslo,
Norway
(2) University of Oslo, School of Pharmacy, Oslo, Norway
In a recent study we reported that mean serum concentration of escitalopram ranged 10-fold between different CYP2C19 genotypes (Rudberg I et
al. Clin Pharmacol Ther 2008;83:322-7). However, considerable variability in serum concentrations also existed among subjects carrying the same
CYP2C19 genotype. Assessed by covariate analysis in the previous study,
a fraction of this variability was ascribed to the presence of defective
CYP2D6 alleles. This is apparently in contrast to other studies reporting
that CYP2D6 genetics did not affect the pharmacokinetics of escitalopram
(Herrlin K et al. Br J Clin Pharmacol 2003;56:415-21, Sogaard B et al. J
Clin Pharmacol 2005;45:1400-6). It is however possible that CYP2D6mediated metabolism of escitalopram is of importance primarily in subjects with impaired CYP2C19 activity. To investigate this, >500 doseadjusted serum concentration measurements of escitalopram from >300
CYP-genotyped patients were grouped according to CYP2C19 genotype
as either CYP2C19 homozygous extensive metabolisers (CYP2C19 EMs)
or CYP2C19 heterozygous extensive metabolisers (CYP2C19 HEMs).
The two CYP2C19 genotype groups were subdivided in patients homozygous for functional CYP2D6 alleles or patients carrying defective
CYP2D6 allele(s). Whereas mean serum concentration of escitalopram
appeared unaffected by CYP2D6 genetics within CYP2C19EMs (+6%,
p = 0.5), a 36% higher mean serum concentration was observed in carriers of defective CYP2D6 allele(s) within CYP2C19 HEMs (p = 0.019).
Thus, defective CYP2D6 allele(s) imply elevated serum concentration of
escitalopram in patients with impaired CYP2C19 activity.
(1) The Chinese University of Hong Kong Faculty of Medicine, Li Ka

Shing Institute of Health Sciences, Epithelial Cell Biology Research Centre, Hong Kong, PR China
(2) The Chinese University of Hong Kong Faculty of Medicine Laboratory Animal Services Centre - Research Unit, Hong Kong, PR China
Paper No.: 3208

CLINICAL TRIAL OF EPIGALLOCATECHIN-3-GALLATE IN
DUCHENNE MUSCULAR DYSTROPHY
Dysmenorrhoea, dened as cramping pain in the lower abdomen occurring before or during menstruation, effects up to 90% of women of childbearing age to varying degrees. The present study investigates whether
Bak Foong Pills (BFP), a traditional Chinese medicine treatment for
dysmenorrhoea, possesses analgesic properties. Results showed that BFP
was able so signicantly reduce pain responses following sub chronic
treatment for 3 days, but not following acute (1 hour) treatment in
response to acetic acid-induced writhing challenge. The analgesic effect
was not due to inhibition of cyclooxygenase activity as evidenced by the
lack of inhibition of prostacyclin and prostaglandin E2 production by
BFP. Molecular analysis revealed that BFP treatment was able to modulate the expression prole of a number of genes in the spinal cord of
mice subjected to acetic acid writhing. RT-PCR analysis of spinal cord
samples demonstrated that both somatostatin receptor 4 and 2 (sst4 and
sst2) receptor mRNA, but not l opioid receptor and neurokinin 1 receptor mRNA, were downregulated following BFP treatment, thus implicating somatostatin involvement in BFP-induced analgesia. Administration
of cyclo-somatostatin, a somatostatin antagonist, prior to nociceptive
Urs T Ruegg(1), A von Moers(2), F Paul(2), Y Nakae(1), O Dorchies(1)

(1) University of Geneva, Geneva, Switzerland
(2) NeuroCure Clinical Research Center, Charite, Berlin, Germany
We reported that epigallocatechin-3-gallate (EGCG), a major polyphenol
in green tea, given to mdx mice via their diet (Dorchies et al. 2006 and
2009) or by subcutaneous injection (Nakae et al. 2008) limits muscle
degeneration. Encouraged by these results and those with EGCG in autoimmune encephalomyelitis (Aktas et al. 2004), a model of multiple sclerosis, we decided to initiate a trial using EGCG administration to
Duchenne muscular dystrophy (DMD) patients. This prospective, multicentric pilot study is aimed to assess safety and tolerability of a
12 months treatment with oral EGCG in doses of up to 10mg/kg body
weight in ambulatory DMD boys aged 5 years or more. The effect of
EGCG on disease-related functional scores will be assessed in this randomized, double blind and placebo-controlled trial. The patients will be
followed by biomarkers including serum creatine kinase (CK) and loco-
551
motor activity will be determined. We expect that similar improvements
as observed in dystrophic mice will be found in DMD patients.
References: Aktas O et al. (2004) Green tea epigallocatechin-3-gallate
mediates T cellular NF-j B inhibition and exerts neuroprotection in autoimmune encephalomyelitis. J Immunol.173, 5794-800.
Dorchies OM et al. (2006) Green tea polyphenols as a potential treatment
for Duchenne muscular dystrophy. Am. J. Physiol. 290, C616-625.
Dorchies OM, et al. (2009). Biofactors 35, 279-294.
Nakae et al. (2008) Subcutaneous injection of epigallocatechin-3-gallate,
a component of green tea, limits the onset of muscular dystrophy in mdx
mice: a quantitative histological, immunohistochemical and electrophysiological study. Histochem Cell Biol.129, 489-501.
Paper No. 2912

FOCUS GROUP: P15 - ENDOTHELIUM IN HEALTH AND
DISEASE
DIFFERENCES IN THE IN VITRO ANTIPLATELET EFFECT
OF DEXIBUPROFEN, IBUPROFEN AND FLURBIPROFEN IN
HUMAN BLOOD
Maria Isabel Ruiz- Moreno(1), JJ Reyes-Vega(1), JA Lopez-Villodres(2),
N Jebrouni(2), JA Gonzalez-Correa(2), JP de la Cruz(2)
(1) University of Malaga, Faculty of Health Sciences, Department of
Pharmacology,Malaga, Spain
(2) University of Malaga, School of Medicine, Department of Pharmacology, Malaga,Spain
Some anesthesiology guides include a possible temporary replacement of
aspirin by a reversible inhibitor of cyclooxygenase. Aim: to compare in
vitro pharmacodynamic prole of dexibuprofen rac-ibuprofen and racurbiprofen in order to determine possible differences in platelet level
action. Methods: The study was carried out in blood samples from
healthy volunteers. Platelet aggregation in whole blood induced with
ADP, collagen and arachidonic acid, platelet thromboxane B2, LPSinduced prostaglandin E2, leukocyte 6-keto-PGF1a and nitric oxide
induced by both constitutive and inducible pathways, were measured
before and after incubation with increasing concentrations of aspirin,
dexibuprofen, rac-ibuprofen and rac-urbiprofen. Concentration that
inhibited or increased 50% of each parameter (IC50 or EC50) were calculated. Results: All the compounds inhibited in a concentration-dependent manner platelet aggregation, thromboxane B2, prostaglandin E2 and
6-keto-PGF1a, and increased calcium-induced nitric oxide production.
Dexibuprofen showed greater antiplatelet potency than rac-ibuprofen and
rac-urbiprofen, as well as a prole similar to that of aspirin. For
instance IC50 values for arachidonic acid-induced platelet aggregation
were 0.85 0.06 lM for dexibuprofen, 14.76 1.22 lM for rac-ibuprofen, 6.39 0.51 lM for rac-urbiprofen and 0.38 0.03 lM for aspirin.
All the compounds inhibited both thromboxane and prostacyclin synthesis, but the ratio IC50 anti-TxB2/IC50 anti-6-keto-PGF1a was
0.21 0.03 for dexibuprofen, 1.05 0.08 for rac-ibuprofen, 0.79 0.11
for rac-urbiprofen and 0.46 0.06 for aspirin. All the compounds
increased calcium-dependent nitric oxide production. Conclusion: The
aryl propionic acid derivative dexibuprofen is more potent antiplatelet
compound and is approaching to the pharmacodynamic prole of aspirin.
Paper No.: 1315

A LABORATORY PACKAGE FOR PERSONALISED
METHADONE THERAPY
Ismail Rusli(1), SC Tan(1), HL Sim(1), M Nurfadhlina(1),
KC Sanjaya(1), MZ Iqbal(1), T Nazila(1), R Fazni(1), n Mohamad(2)
(2) Universitiy Sains Malaysia School of Medical Sciences, Emergency

Department,Kubang Kerian, Kelantan, Malaysia
In 2005 the Malaysian government introduced a comprehensive harm
reduction program that includes the use of methadone substitution therapy for heroin-dependents to control the dual threats of illicit drug use
and HIV. Methadone substitution has repeatedly been shown to reduce
illicit opiate use, crime and health care costs while promoting employment and safer pregnancies. Methadone however has a complex pharmacology. Its effects are inuenced by several drug metabolizing enzymes
that include CYP2B6, CYP2D6, and CYP3A4 and the receptor gene,
OPRM1. Recently we developed a personalized methadone therapy
kit comprising a PCR kit that can detect polymorphisms at CYP2B6 and
OPRM1 gene loci and an ELISA kit that can quantify plasma methadone. The PCR kit comprises primers and mixes that can be used to
detect 6SNPs in CYP2B6 and OPRM1 using a multiplex nested PCR
protocol. The ELISA kit employed uses reagents developed in-house and
is a direct quantitative assay that is specic and sensitive. In a routine
clinical cohort of methadone patients, we found both CYP2B6 and
OPRM1 to be polymorphic. Methadone doses in these patients ranged
10-fold but the resulting plasma methadone varied more that 100-fold
with a large number having concentrations that were probably inadequate
to ensure successful methadone therapy. We conclude that our endogenous kit is a useful adjunct in the management of opiate addiction with
methadone. Used appropriately, it can facilitate methadone dosage adjustments to improve therapeutic outcomes.
Paper No.: 3127

THE ORPHAN GPR50 RECEPTOR PROMOTES
TRANSFORMING GROWTH FACTOR B (TGFB) SIGNALLING
BY INTERACTING WITH THE TGFB TYPE I RECEPTOR
A Saade(1), A Daulat(1), N Ferrand(2), P Delagrange(3), Nathalie
Clement(1), C Prunier(2), R Jockers(1)
(1) Institut Cochin, INSERM U567, CNRS 8104, Universite Paris
Descartes, Paris, France
(2) INSERM UMRS 938, Centre de recherche Saint-Antoine, Paris,
France
(3) Institut de Recherches SERVIER, Suresnes, France
GPCRs (G protein coupled receptors) respond to a variety of stimuli
from ions to proteins. However, despite major efforts of academic and
industrial research more than 100 GPCR remain still orphan. More
recently, ligand-independent functions have been proposed for several
orphan GPCR, where non-orphan GPCRs with dened function are allosterically regulated by orphan GPCRs in heterodimeric complexes (Levoye et al. Embo Rep, 2006, 7:1094-1098). We are now extending this
concept by showing that the orphan GPR50, a member of the melatonin
receptor family, regulates TGFb signalling by directly interacting with
the TGFb type I receptor. This interaction was identied in a tandem
afnity purication assay using GPR50 as bait. Interestingly, expression
of GPR50 potentiates TGFb-induced signalling by increasing the phosphorylation of Smad2/3 and promoting reporter gene activity through
several response elements of the TGFb signalling pathway. Collectively,
our results reveal an unexpected potentiation mechanism of the TGFb
pathway that occurs at the receptor level between the TGFb type I receptor and GRP50. This denes a further ligand-independent function for
the orphan GPR50, which might be relevant for the neuroendocrine functions of TGFb.
(1) Universitiy Sains Malaysia, Institute for Research in Molecular

Medicine (INFORMM), Kubang Kerian, Kelantan, Malaysia
552
Paper No.: 1750
HEALTH AND DISEASE
EFFECT OF CINNAMALDEHYDE (C) ON VASCULAR
FUNCTION IN DIABETIC RATS
Ramzi Sabra, M Khalaf
American University of Beirut, Department of Pharmacology and
Therapeutics, Beirut, Lebanon
Previous studies showed altered responses to vasocative substances in
vascular tissue from diabetic rats, which may involve changes in NO and
prostanoid synthesis. Cinnamaldehyde, the active ingredient in cinnamon, has been shown to reduce fasting blood sugar in diabetes. In this
study we examined whether C (20 mg/kg/day by oral gavage) can prevent vascular dyfunction observed in aortic rings from rats treated with
streptozotocin (STZ, 85 mg/kg i.v., DM rings) compared to those from
rats treated with vehicle (V). Two weeks after STZ, the following was
observed in DM vs. V rings: less contraction by phenylephrine, similar
vasodilation by SNAP (NO donor) and greater vasodilation by acetylecholine. Treatment with C during the second week changed these
responses: greater contraction by phenylephrine, and less vasodilation by
both SNAP and acetylcholine. In rats with longer duration of DM
(4 weeks) in which C was given for the last 2 weeks, aortic rings from
rats not treated with C showed the following in DM vs. V rings: similar
responses to phenylephrine, greater vasodilation by SNAP, and reduced
vasodilation by Ach. Treatment with C again changed this pattern into:
greater contraction by phenylephrine, similar responses to SNAP and
reduced vasodilation by acetylcholine. The differences were annulled by
denuding the endothelium. Blood sugar levels were not inuenced by C.
Conclusion: C alters the vascular abnormalities observed during DM possibly by altering the synthesis of endothelial vasodilators and/or phosphodiesterase activity, and by a mechanism independent of blood sugar
reduction.
Paper No.: 2282

DISEASES
PREVENTIVE EFFECT OF 5-HT1A RECEPTOR AGONIST
BUSPIRONE ON MESENTERIC ISCHEMIA/REPERFUSIONINDUCED INFLAMMATORY INJURIES IN MICE
F Saccani, L Flammini, T Gobbetti, G Vegezzi, P Ghizzardi, S Bertoni,
M Tognolini, V Ballabeni, Elisabetta Barocelli
University of Parma, Department of Pharmacological, Biological Sciences and Applied Chemistries, Parma, Italy
Introduction: 5-HT is recognized as a key element in the control of
enteric functions and large amounts of this amine are detected in the
intestinal lumen and plasma following experimental mesenteric ischemia/
reperfusion (I/R), a clinical problem associated to different pathological
conditions (neonatal necrotizing enterocolitis, mesenteric angina, intestinal infarction). The aim of this study is to evaluate the possible protective effect of putative partial agonists at 5-HT1A (Buspirone) and 5-HT4
(Tegaserod) receptors against mesenteric I/R injuries in mice. Materials:
Mesenteric ischemia was induced by occlusion of the superior mesenteric
artery (45 min) and followed by 5 hrs reperfusion in mice. Sham operated (SO) animals served as controls. Buspirone (10mg/kg i.v.), Tegaserod (0.1mg/kg i.v.) or saline was administered 5 min before ischemia in
I/R or in SO mice. The experiments were performed according to Guiding Principles in the Care and Use of Animals. Results: In I/R mice
oedema formation in intestinal tissues ((wet weight - dry weight)/dry
weight) was reduced only by Buspirone (2.87 vs 4.16 saline, P < 0.01)
whereas the increase of intestinal malondialdehyde levels, index of lipoperoxidation, was prevented by Buspirone (33.83 nmol/g tissue vs saline
92.75 nmol/g tissue P < 0.05) and slightly reduced by Tegaserod
(46.66 nmol/g tissue vs saline 92.75 nmol/g tissue). Intestinal myeloperoxidase activity, index of leukocyte inltration, was reduced only by
Buspirone (5.97 vs saline 22.0 U/g tissue). Conclusion: The ndings
obtained in this preliminary investigation suggest a protective role for 5HT1A receptor activation through reduction of intestinal oxidative stress
and vascular permeability induced by I/R.
Paper No.: 2457

PROTECTIVE EFFECT OF NIGELLA SATIVA EXTRACT AND
THYMOQUINONE ON SERUM/GLUCOSE
DEPRIVATION-INDUCED PC12 CELLS DEATH
Hamid Reza Sadeghnia(1), SH Mousavi(1), Z Tayarani-Najaran(1),
M Asghari(2)
(1) Mashhad University of Medical Sciences (MUMS), School of Medicine, Department of Pharmacology and Pharmacological Research Centre
of Medicinal Plants, Department of Modern Technologies and Sciences,
Mashhad, Iran
(2) Payame Noor University (PNU), Department of Biochemistry, Mashhad, Iran
The serum/glucose deprivation (SGD)-induced cell death in cultured
PC12 cells represents a useful in vitro model for the study of brain ischemia and neurodegenerative disorders. Nigella sativa L. (family Ranunculaceae) and its active component thymoquinone (TQ) have been
known as a source of antioxidants. In the present study, the protective
effects of N. sativa and TQ on cell viability and reactive oxygen species
(ROS) production in cultured PC12 cells were investigated under SGD
conditions. PC12 cells were cultured in DMEM medium containing 10%
(v/v) fetal bovine serum, 100 units/ml penicillin, and 100 lg/ml streptomycin. Cells were seeded overnight and then deprived of serum/glucose
for 6 and 18 h. Cells were pretreated with different concentrations of N.
sativa extract (15.62250 lg/ml) and TQ (1.17150 lM) for 2 h. Cell
viability was quantitated by MTT assay. Intracellular ROS production
was measured by ow cytometry using 2, 7-dichlorouorescin diacetate
(DCF-DA) as a probe. SGD induced signicant cells toxicity after 6, 18,
or 24 h (P < 0.001). Pretreatment with N. sativa (15.62250 lg/ml) and
TQ (1.1737.5 lM) reduced SGD-induced cytotoxicity in PC12 cells
after 6 and 18 h. A signicant increase in intracellular ROS production
was seen following SGD (P < 0.001). N. sativa (250 lg/ml, P < 0.01)
and TQ (2.34, 4.68, 9.37 lM, P < 0.01) pretreatment reversed the
increased ROS production following ischemic insult. The experimental
results suggest that N. sativa extract and TQ protects the PC12 cells
against SGD-induced cytotoxicity via antioxidant mechanisms. Our ndings might raise the possibility of potential therapeutic application of N.
sativa extract and TQ for managing cerebral ischemic and neurodegenerative disorders.
Keywords: PC12, Serum/glucose deprivation, Ischemic insult, Nigella
sativa, Ranunculaceae, Thymoquinone
Paper No.: 3352

EXPERIMENTAL STUDY OF EFFECTS OF TRIPHASIC ORAL
CONTRACEPTIVE PILLS ON FLUCTUATIONS TEAR
Khatereh Safavi Naeini
Islamic Azad University, Arsanjan Branch, Department of Nursing and
Midwifery Arsanjan, Iran
Tear play an important role in protecting and maintaining the function of
the normal ocular surface. Deciency in tear production results in inammation of the conjunctiva and cornea. Tear production was measured
using the standard Schirmer tear test (STT). The STT without use of topi-
553
cal anesthetics is an estimate of basal and reex aqueous production.
Hormonal inuences on tear production may be manifested with the use
of oral contraceptive, when a signicant change from the normal hormonal state occurs. There are a number of reports of dry eye symptomology to contact lens wear by women using oral contraceptive. The
purpose of the present study was to evaluate the effect of triphasic OCPs
on Schimer test in dog as an experimental model for humans. Triphasic
OCPs are a kind of oral combine contraceptive pills that is lowering total
progesterone content in cycle against monophasic OCPs. Because of this
aspect some side effects are lower than monophasic.12 healthy female
mixed breed dogs, with normal ocular examination, ranging in age from
1-2 years were selected for this study. In all dogs STT value were measured before any medications. Six dogs taking oral contraceptive 20 successive daily (experiment group) and six dogs not receive oral
contraceptive (control group).The results showed that no difference in
Schirmer test between experiment group and control group not taking
contraceptive. This study indicates that oral contraceptive have no direct
effect on Schirmer test and tear ow.
Paper No.: 3273

ANTI-INFLAMMATORY EFFECT OF AN EXTRACT OF
MANGIFERA INDICA L. (VIMANG) IN PATIENTS WITH
CHRONIC DERMATITIS
Lorena Safonts(1), M Guevara(1), T Perez(1), C Morales(2)
(1) Biopharmacuetical Laboratories, Havana, Cuba
(2) National Center of Clinical Trials, Havana, Cuba
Introduction: In today context we see an increasing interest to obtain
therapeutics proceeding of natural sources for treatment of chronic dermatitis because of its high incidence and complex treatment. Objective:
To evaluate the anti-Inammatory effect of an extract of Mangifera
indica L. (Vimang cream 1.2%) in patients with chronic dermatitis. Material and Methods: This was a phase II, double-blind controlled trial in 30
outpatient dermatology service with adult chronic dermatitis who were
diagnosed by biopsy, personal history, pattern, evolution, and skin lesions
and who were randomized to apply an extract of Mangifera indica L.
(Vimang cream) or placebo cream twice daily. Clinical evolution, photography, and dermatitis assay Score index (DASI) scoring were done at
baseline and after 6 weeks. Results: Fifteen subjects each received Vimang or placebo. Post intervention, there was a difference of 30% of
improvement of Vimang group, it was signicantly superior (10:1, p =
.0028; 95% CI, 0.01-0.73). 51% of Vimang subject remained positive at
the end of study. Conclusions: The treatment with an extract of Mangifera indica L. (Vimang cream) may be useful in the treatment of
chronic dermatitis, because of the high proportion of patients with satisfactory and partially satisfactory response; besides it was an innocuous
product for topic treatment.
Paper No.: 3234

THE PROTECTIVE EFFECT OF LOW DOSE OF SNP ON
L-DOPA INDUCED APOPTOSIS
Mousa Sahebgharani, Z Sepehrizadeh, M Talebi, Xxxxxxx Saboori,
J Esmaeili, E Jaberi
precipitate progression of the disease. Cellular studies have conrmed

this hypothesis. In the present study, the effects of sodium nitroprusside
(SNP) as NO producer, aminoguanidine (NOS synthase inhibitor) and
COX-2 inhibitor (celecoxib) were studied on the L-DOPA induced apoptosis (programmed cell death) in PC12 cell line (as a neural dopaminergic cell). We used 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay, annexin V-uorescein isothiocyanate test and
quantitative real time RT-polymerase chain reaction for detection of
apoptosis. PC12 cells were exposed to different concentrations of LDOPA (50, 100 and 200 lM/L) for 48 hours. In the other part of
experiments, PC12 cells were exposed to different concentrations of
SNP, aminoguanidine and celecoxib 30 min before treatment with LDOPA. The results of MTT assay and annexin V-uorescein isothiocyanate test indicated that L-DOPA (100 and 200 lM/L) induced apoptosis and low dose of SNP (75 lM/L) but not the other agents,
decreased the apoptotic effect of L-DOPA 100 lM/L. Furthermore,
results of quantitative real-time RT-polymerase chain reaction (RTPCR) revealed that exposure of cells with L-DOPA, increased mRNA
expression of BAX, iNOSA, COX-2 and C-Fos which were reversed by
the pretreatment of cells with low dose of SNP (75 lM/L). Thus it can
be concluded that low dose of SNP shows protective property against
apoptotic effect of L-DOPA.
Paper No.: 2385

HEALTH AND DISEASE
INVOLVEMENT OF SECRETORY PHOSPHOLIPASE A2 IN
STRETCH-INDUCED ABERRANT CONTRACTION IN
PULMONARY ARTERIES ISOLATED FROM PULMONARY
HYPERTENSIVERATS
Maki Saito(1), Y Tanabe(1), A Kamataki(2), T Sawai(2), K Nakayama(1)
(1) Iwate Medical University School of Pharmaceutical Sciences,
Department of Molecular and Cellular Pharmacology, Iwate, Japan
(2) Iwate Medical University School of Medicine, Iwate, Japan
Hemodynamic forces caused by blood pressure and ow play an important role in the regulation of local circulation. We have previously demonstrated excessive stretching evokes contraction in isolated pulmonary
arteries of rabbits, and that the stretch-induced contraction is produced
by endothelium-derived untransformed-prostaglandin (PG) H2 (Saito M
et al, Eur J Pharmacol, 2003;467:151-161). Furthermore, We have found
that pulmonary artery isolated from monocrotaline-induced pulmonary
hypetensive (MCT-PH) rats shows augmented contraction, including
rhythic activity (Kiyoshi A et al, Pugers Arch-Eur JPhysiol,
2003;447:142-149). As it is considered that pulmonary artery in the pulmonary hypertension is chronically exposed to excessive hemodynamic
forces, we speculate that some inammatory pathways such as arachidonate cascade would be activated. The aim of the present study is to clarify whether secretory phospholipase A2 (sPLA2) participates in the
stretch-induced untransformed-PGH2 production and contraction inpulmonary arteries. Indoxam, a selective inhibitor for sPLA2, effectively
abolished the both rhythmic contraction and untransformed-PGH2 production in response to stretching in pulmonary arteries isolated from
MCT-PH rats. Furthermore, we found that the mRNA level of group X
sPLA2 was specically augmented in the pulmonary arteries of MCT-PH
rats as compared with those of controls. These results suggest that
sPLA2-mediated untransformed PGH2 production plays a keyrole in the
enhanced vascular tone of pulmonary arteries caused by acute and/or
chronic exposure to excessive mechanical stress. Thus we herewith propose that the regulation of group X sPLA2 can be a new therapeutic
means against pulmonary hypertension.
Tehran University of Medical Sciences, Department of Pharmacology,

Tehran, Iran
L-DOPA is considered as drug of choice for treatment of Parkinson. But
it has been suggested that chronic treatment with L-DOPA by itself may
554
Paper No.: 1296
EFFECT OF ANCHUSAN, JAPANESE TRADITIONAL HERBAL
MEDICINE, ON MIDAZOLAM METABOLISM IN RATS
Yusuke Saito(1), Y Nishimura(1), N Kurata(2), M Iwase(1), H Yasuhara(1)
(1) Showa University School of Medicine, Department of Pharmacology,
Tokyo, Japan
(2) Showa University, Faculty of Arts and Sciences, Fujiyoshida, Japan
Kampo medicine, Japanese traditional herbal medicine, is widely used in
Japan, however, there are few information about cytochrome
P450(CYP)-mediated interactions. Anchusan is commonly used for the
treatment such as chronic gastritis. It includes several ingredients which
are reported as CYP3A inhibitor or inducer, therefore, it may cause drug
interaction. Thus we investigated the effect of anchusan on CYP3A-mediated metabolism. Anchusan(500mg/kg) was orally administered to rats
once(single) or once a day for one week(multiple). After 2 or
16 hours(single) and 16 hours(multiple) of the nal treatments, midazolam(MDZ) was administered orally and its serum concentrations were
analyzed by HPLC. Hepatic and intestinal CYP3A contents and MDZ 4hydroxylation activity were also examined in the multiple administration
group. The inhibitory effect of anchusan and its seven constituent herbal
extracts on MDZ 4-hydroxylation activity was also investigated using rat
liver microsomes (RLM). The multiple treatment of anchusan resulted in
2.4-fold increase in the AUC and Cmax of MDZ, while hepatic and
intestinal CYP3A contents and the activities were unchanged. In contrast,
no signicant effects were observed by the single treatment of anchusan.
In vitro study showed no remarkable inhibitory effects on CYP3A activity by anchusan and its constituents. However, the preincubation of anchusan, Glycyrrhizae Radix, and Alpiniae Ofcinari Rhizoma with RLM
reduced CYP3A activity in the time and NADPH dependent manner.
These results suggested the possible drug interactions between anchusan
and co-administered drugs metabolized via CYP3A, by the mechanismbased inactivation of this enzyme, or by the accumulation of direct inhibitor generated by intestinal bacteria.
Paper No.: 1330

DISEASES
ANGIOTENSIN II INDUCES HYPERRESPONSIVENESS OF
BRONCHIAL SMOOTH MUSCLE VIA AN ACTIVATION OF
P42/44 ERK IN RATS
kinase (MEK-1/2) inhibitor, inhibited the Ang II-induced phosphorylation of p42/44 ERK in bronchial smooth muscle. Furthermore, the Ang
II-induced BSM hyperresponsiveness to CCh did not appear by pretreatment with U-0126. Interestingly, CCh itself-induced contraction was not
changed by U-0126. In conclusion, although Ang II caused only small
force development in the bronchial smooth muscle, Ang II-induced BSM
hyperresponsiveness through activation of p42/44 ERK is estimated to
play an important role in pathophysiology of bronchial asthma.
Paper No.: 2168

SANOSHASHINTO PREVENTS CARDIAC ISCHEMIA/
REPERFUSION INJURY IN OVARIECTOMIZED RAT VIA
ANTIOXIDANT PROPERTIES
Mayuko Sakanashi(1), T Matsuzaki(1), K Noguchi(1), J Nakasone(1),
M Sakanashi(2), K Chinen(3), M Sakanashi(4), M Tsutsui(1)
(1) University of the Ryukyus School of Medicine, Faculty of Medicine,
Department of Pharmacology, Okinawa, Japan
(2) University of the Ryukyus Hospital, Department of Anesthesiology,Okinawa, Japan
(3) University of the Ryukyus Hospital, Division of Community-Based
Medicine and Primary Care, Okinawa, Japan
(4) University of the Ryukyus, Okinawa, Japan
It is a serious problem that the incidence of acute myocardial infarction
is elevated in post-menopausal women. The present study is designed to
clarify the effects of long-term treatment with Sanoshashinto (SST) on
cardiac ischemia/reperfusion (I/R) injury in post-menopausal rat models.
SST is a Japanese traditional herb medicine for the treatment of menopause-associated symptoms. The clinically relevant concentration of SST
(750 mg/kg/day) was administered into ovariectomized (OVX) female
Wistar rats orally for 4 weeks, from 11 to 15 weeks of age. After the
treatment with SST, the heart was perfused with the Langendorff apparatus, and subjected to 30 minutes of global ischemia and 60 minutes of
reperfusion. Changes in coronary ow, left ventricular (LV) developed
pressure, peak positive and negative LVdP/dt and LV end-diastolic pressure were observed. Myocardial contents of malondialdehyde (MDA), a
marker of lipid peroxidation, and superoxide generation were also measured. The treatment with SST signicantly ameliorated the I/R inducedcardiac dysfunction except for coronary ow. Furthermore, the SST treatment signicantly reduced both myocardial MDA level and superoxide
generation. These results suggest that the cardioprotective effects of SST
against I/R injury in OVX rats would be partly mediated by its antioxidant properties.
Hiroyasu Sakai, Y Chiba, M Hanazaki, M Misawa

Tokyo, Japan
Angiotensin II (Ang II) might be an important mediator in pathogenesis
of bronchial asthma, although mechanisms of airway hyperrsponsiveness
caused by Ang II are not yet clear. Whether p42/44 ERK contributes to
the Ang II-elicited bronchial smooth muscle (BSM) hyperresponsiveness
in rats was examined. The RT-PCR analyses presently revealed that Ang
II AT1a-, AT1b-, AT2-receptors, angiotensinogen, angiotensin-converting
enzyme, but not renin, were expressed in the lungs, trachea and main
bronchi of rats. However the expression of renin mRNA was observed in
kidney (positive control) in rats. The transient contraction was induced
by in vitro application of Ang II. The contraction induced by Ang II was
concentration-dependent, and abolished by losartan, an AT1-receptor
antagonist. The contractions induced by carbachol (CCh), high potassium
ion-depolarization and sodium uoride (NaF; a G protein activator) were
augmented by pretreatment with Ang II. The BSM hyperresponsiveness
induced by Ang II was abolished by losartan. Ang II augmented the
phosphorylation of p42/44 ERK in rat bronchi. U-0126, a p42/44 ERK
Paper No.: 1766

DISEASES
DUAL ROLES OF THE PGE2-EP4 SIGNALLING IN MOUSE
EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS
Daiji Sakata(1), Y Esaki(1), Y Li(1), C Yao(1), E Segi-Nishida(1),
T Matsuoka(1), K Fukuda(2), S Narumiya(1)
(1) Kyoto University Faculty of Medicine, Department of Pharmacology,
Kyoto, Japan
(2) Kyoto University Faculty of Medicine, Department of
Anesthesiology, Kyoto, Japan
Experimental autoimmune encephalomyelitis (EAE) is an animal model
of multiple sclerosis (MS). Although concentrations of prostaglandins
(PGs) are elevated in the cerebrospinal uid of MS patients and in the
spinal cord of mice of EAE, the role of PGs in these diseases remains
555
unknown. We subjected mice decient in each of eight types and subtypes of PG receptors individually to EAE. We also used agonists or
antagonists selective to each of four PGE receptor subtypes, and compared the ndings of these experiments with effects of indomethacin.
Administration of indomethacin during the immunization phase or all
through the experimental period delayed EAE development and suppressed its extent, while that at the onset tended to accelerate the development. Among the mice decient in each PG receptor, only EP4-/- mice
showed signicant suppression of EAE, which was mimicked by administration of an EP4 antagonist, ONO-AE3-208 during the immunization
period.Notably, this inhibitory effect of ONO-AE3-208 was augmented
in EP2-/- mice. The EP4 antagonism during immunization suppressed
generation of Th1 and Th17 subsets and this effect was again augmented
in EP2-/- mice.In contrast, ONO-AE3-208 administered at the onset had
little effect. Instead, administration of an EP4 agonist, ONO-AE1-329, at
the onset delayed and suppressed the disease progression with suppression of increased permeability of the blood brain barrier. Thus, PGE2
exerts dual functions in EAE, facilitating Th1 and Th17 generation
redundantly through EP4 and EP2 in immunization and attenuating invasion of these cells into the brain by enhancing the blood brain barriers
through EP4.
Paper No.: 2338

INFLUENCE OF ALDEHYDE DEHYDROGENASE-2 GENE
POLYMORPHISM ON VASODILATATION INDUCED BY
ORGANIC NITRATES IN HEALTHY HUMAN SUBJECTS
S Sakata(1), K Ichishima(1), H Oniki(1), T Miwa(1), H Arima(1),
K Matsumura(1), T Yoshihara(2), F Takahashi-Yanaga(2), F Shiraishi(2),
Toshiyuki Sasaguri(2)
(1) Kyushu University, Department of Medicine and Clinical Science,
Fukuoka, Japan
(2) Kyushu University, Department of Clinical Pharmacology, Fukuoka,
Japan
Organic nitrates, particularly nitroglycerin (glyceryl trinitrate, GTN), are
used to relieve patients with angina pectoris. However, the response to
GTN is variable among individual patients. Although the reason for this
variability may be complex, it is suggested that mitochondrial aldehyde
dehydrogenase-2 (ALDH-2) is involved in bioactivation of GTN and that
the response to GTN is altered by ALDH-2 gene polymorphism
(Glu487Lys). However, to determine the clinical signicance of this nding, we have to conduct an accurate clinical trial. In particular, we need
to know the effect of GTN on *2/*2 mutant homozygotes whose ALDH2 is inactive, since 3-5% of Japanese population are estimated to have
*2/*2 genotype. The diameter of brachial artery was determined using
ultrasound sonography by a measurer blinded from subjectshf genotypes
before and after administration of GTN (0.3 mg) or isosorbide dinitrate
(ISDN, 1.25 mg) in 60 healthy volunteers (*1/*1, 28; *1/*2, 24; *2/*2,
*8). The maximal increase ratio of the diameter (Dmax-D0)/D0 in
response to GTN was not different among genotypes (total, 18.4 4.51;
*1/*1, 17.8 4.65%; *1/*2, 19.5 4.69%; *2/*2, 19.0 3.27%). However, the time required to attain the maximal dilatation (Tmax) was signicantly longer (P < 0.01) in *2/*2 than *1/*1 (mean, 284 66.5 sec; *1/
*1, 267 58.2 sec; *1/*2, 288 67.1 sec; *2/*2, 332 74.3 sec). In
ISDN, there was no difference in (Dmax-D0)/D0 or Tmax. These results
suggest that ALDH-2 is signicantly involved in bioactivation of GTN,
though other mechanisms may also activate GTN.
Paper No.: 1178

CHANGES IN DRUG-METABOLIZING ENZYME ACTIVITIES
IN THE MULTIPLE HISTAMINE RECEPTOR GENE
KNOCKOUT MICE
Eiichi Sakurai(1), E Sakurai(0), Y Ueda(1), Y Sakurai(1), K Yanai(3)
(1) Tokushima Bunri University, Department of Pharmaceutics, Tokushima, Japan
(2) Iwaki Meisei University, Iwaki, Japan
(3) Tohoku University School of Medicine, Department of Pharmacology, Sendai, Japan
Purpose: Gene knockout mice were used in order to clarify the physiological roles. However, when the effect of drug was observed using gene
knockout mice, the data on disposition of drug are often overlooked. We
studied whether or not the changes in drug-metabolizing enzyme activities occur in the multiple histamine receptor gene knockout mice. Methods: Female H1 receptor gene decient mouse (H1-KO) was mated with
H2 receptor-decient mouse (H2-KO). The male H1/H2 double receptor
knockout (H1/H2 DKO) mice were then mated with H3 receptor-decient mouse (H3-KO; a gift from Dr. Lovenberg to generate the H1/H2/
H3 triple knockout mice (H1/H2/H3 TKO). The metabolism of imipramine (N-demethylation, 2-hydroxylation and N-oxidation) was investigated in the liver microsomes of these knockout mice. In order to
determine the concentrations of imipramine and its metabolites (desipramine, 2-hydroxydesipramine, 2-hydroxyimipramine and N-oxide), an
analytical method using HPLC-PDAD was developed. Results and Discussion: We were able to detect, simultaneously, six PCR products of
each histamine receptor gene for KO and wild type H1, H2 and H3
receptor, respectively using QIAGEN Multiplex PCR kits. The formations to 2-hydroxyimipramine and desipramine of imipramine catalyzed
by CYPs signicantly increased in the liver microsomes of H1-KO, H3KO and H1/H2/H3 TKO mice. Although, there was no signicant difference in N-oxidation activity between wild type and these KO mice, the
formation of N-oxide in H3-KO mice was analysed to be biphasic, indicating gene expression of different type of avin-containing monooxigenase by knockout of H3 receptor gene.
Paper No.: 1225

THE ROLE OF THE HISTAMINE RECEPTORS ON
RESPONSES UNDER RESTRAINT AND PSYCHOLOGICAL
STRESS
Eiko Sakurai(1,2), A Oikawa(2), K Yanai(2)
(1) Iwaki Meisei University, Iwaki, Fukushima, Japan
(2) Tohoku University School of Medicine,
Department of Pharmacology, Sendai, Miyagi, Japan
Stress induces signicant emotional changes and is related to disorders
such as depression and anorexia nervosa. We reported that neuronal histamine is closely associated with acute and chronic stress responses.
However, it is still unclear how the histaminergic neuron system is
involved in attenuating unfavorable stimuli of stress. The histamine
receptor subtypes (H1, H2, H3, and H4) mediate these neuronal actions.
In this study, we investigate the effects of histamine receptors on restraint
stress induced behavioral changes in histamine receptors knockout (KO)
and their wild type mice (WT). Histamine H1, H3 single receptors gene
knockout (H1KO, H3KO), H1/H3 double receptors gene knockout (H1/
H3-DKO), H1/H2/H3 triple receptors gene knockout (TKO) and WT mice
were randomly divided into 3 groups. Mice were restrained in a mouse
holder for 4 h during 5 consecutive days as physical stress group, and
the other mice were set next to the restrained cages (as the psychological
stress group). The control group was kept in a separate room. The body
556
weight was decreased after subjecting to the restraint stress, especially in
H1KO and TKO mice. After subjected to psychological stress, the initial
response time in an open eld was decreased in H1-KO and WT but not
in H3-KO mice. The H1 receptors have an important role of modulating
anxiety and stress. Blocked H3 receptor increases histamine release in the
brain and released histamine binds to H1 receptor. In stressful conditions,
histamine has a pivotal role in modulating stress through the cross talk
between H1 and H3 receptors.
Paper No.: 944

EFFECT OF MOMORDICA CHARANTIA ON BLOOD
GLUCOSE LEVEL IN EXPERIMENTALLY INDUCED
DIABETIC RATS
Mahmed Salahuddin
Aristopharma Ltd, Clinical Research and Medical Services, Dhaka,
Bangladesh
Introduction: Fresh juice of Momordica Charantia (MC) is shown to
reduce blood glucose level in experimentally induced diabetic rats. The
objective was to see the effect of different doses of MC on blood glucose
level in normal and diabetic rats. Methods: 54 adult rats of long Evans
of either sex were taken. In exp-I, 18 rats were distributed equally in
three groups; one group as control and other two groups received two
different doses of MC (1ml and 2ml /100gm of bw daily for 14 days). In
exp-II, 36 rats were made diabetic by single administration of streptozotocin (STZ) i.p. at a dose of 65 mg/kg bw and distributed in four groups,
one group as control, two groups received two different doses of MC
(1ml and 2ml/100gm of bw) and other received glibenclamide at a dose
of 600 lgm/kg bw for same duration. Serum glucose was measured; b
cell number and size of islets were determined. Results: Fresh juice of
MC reduced blood glucose level signicantly (P < 0.05) in diabetic rats
received 1ml MC and Glibenclamide group (P < 0.001) compared to
control (P < 0.001). In histological exam, b cells were increased signicantly in diabetic rats compared to control (P < 0.001). The trans-vertical diameter of Islets in diabetic groups treated by MC and
glibenclamide increased signicantly compared to control (P < 0.001).
Conclusion: MC has signicant hypoglycemic effect in diabetic rats with
increased number of b cells and trans-vertical diameter of islets.
Paper No.: 777

MEDICATION ERRORS IN CRITICAL CARE UNIT
Nicole Salazar, L Escobar, M Jiron
Universidad de Chile, Facultad de Ciencias Qumicas y Farmaceuticas,
Santiago, Chile
Medication errors are considered as the main factor limiting the effectiveness of therapies and patient safety, especially in Intensive Care Units
(ICU). An observational prospective study was carried out in a representative and randomized sample of adult critically ill patients in an ICU of
a Tertiary Hospital in Chile with the aim of determine the frequency and
characteristics of medication errors (ME) in the process of using medications. Data were collected at three different times per day by a trained
and independent observer who recorded the medication use process from
the prescription to the administration. A total of 66 ME were detected in
a total of 194 observed drugs, with a rate of error of 34%. The ME
occurred more frequently during administration and prescription process,
with 25.8% and 9.7% of patients, respectively. ME were detected during
administration in 17.5% (34) of the evaluated drugs mainly by errors in
the rate of administration or wrong time errors. When antimicrobial
drugs were used, a 62.9% of cases presented ME, followed by antivirals

(40%) and anti-hypertensives (35.3%). The frequency ME was higher in
patients receiving 3 or more drugs concomitantly. ME occurs mainly in
the administration stage. To determine the associated risk factors of this
problems are necessary further studies and additional data analyses.
Paper No.: 465

THE EFFECT OF PARAXON ORGANOPHOSPHATE
INSECTICIDE ON ANTIOXIDANT SYSTEM IN RAT BRAIN
TISSUE
Maryam Salehi(1), M Jafari(2), M Saleh Moqadam(1), A Asghari(2),
M Kazemi(3), M Abasnejad(4), M Haggholamali(4)
(1) Razavi Khorasan Payame Noor University, Department of Biochemistry, Tehran, Iran
(2) Baqiyatallah (a.s) University of Medical Sciences, Tehran, Iran
(3) Tehran Azad University, Tehran, Iran
(4) Payame Noor University, Tehran, Iran
Paraxon is a contact organophosphate pesticide and extensively used
both in agriculture. It increases the formation of reactive oxygen species
(ROS) such as superoxide anion, hydrogen peroxide and hydroxyl radical. In physiological condition a balance exits between production and
elimination of free radicals. An imbalance in these processes induces oxidative stress. The aim of this study was to investigate the effects of Paraxon on oxidant-antioxidant system in rat brain tissue. For this propose,
Wistar rats (200-250g) were randomly divided in to ve groups as followed: control, sham (corn oil), and three of groups of Paraxon receiving
different doeses (.3, .7and 1 mg/kg) by intraperitoneal ingection.
24 hours after injection, brain tissue of each group was removed and the
enzyme activities of superoxide dismutase (SOD) and catalase (CAT) as
well as glutathione (GSH) and malondialdehyde (MDA) levels were
determined by biochemical methods. The results show that at concentrations higher than .3 mg/kg paraxon, the activities of brain SOD and CAT
were signicantly increased, comparing with the control. GSH level was
signicantly decreased. There were no signicant changes observed in
brain MDA levels. Our data suggest that an increase in SOD and CAT
activities in brain of rats probably as a function of the increased detoxication capacity. Depletion of tissue GSH is a prime factor, which can
impair the cells defense against the toxic actions of ROS and may lead
to peroxidative cell injury.
Paper No.: 2702

EFFECT OF THREE PLANTS: HYPERICUM HUMIFUSUM,
CRATAEGUS L. AZAROLUS AND MYRTUS COMMUNIS L. ON
ISCHEMIA-REPERFUSION LIVER IN WISTAR MALE RATS
I Salouage, H Ferchichi, T Ayadi, S Bacha, I Ouanes, M Lakhal, Anis
Klouz
National Center of Pharmacovigilance, Laboratory of Clinical
Pharmacology, Tunis, Tunisia
Three species of the genus Crataegus azarolus (Ca), Hypericum humifusum (Hh) and Myrtus communis (Mc) were Tunisian plants rich in antioxidants, particularly in anthocyanin. It was recognized for its antiinammatory, anti-cancer and anti-thrombotic actions. The aim is to evaluate the effect of these three plants on a model of hepatic ischemia-reperfusion in Wistar rat. The model of hepatic ischemia-reperfusion consists
to an induction of hepatic ischemia for 90 minutes in Wistar rats, then
the injection of the plant extract (25 mg/kg) 15 minutes before reperfusion and nally reperfusion for 2 hours. To evaluate the effect of three
plants on ischemia-reperfusion model, we determinate transaminases
557
(AST and ALT) levels and the concentration of MEGX (Monoethylglycinexylidide). In fact, the MEGX test is used to assess the metabolic capacity of the liver by metabolizing lidocaine on his major
metabolite the MEGX. The AST and ALT levels were respectively 4969
and 4281 U/I in no treated groups, 645 and 325UI in group treated by
Ca, 494 and 220 UI in group treated by Hh and 1537 and 1008UI for
group treated by Mc. We noted a signicant increase of MEGX levels in
treated group versus no treated group with any difference between the
three extract plants. Treatment with these extracts permitted to protect
against deleterious effects of ischemia reperfusion in Wistar rat model.
Paper No.: 1475

RENAL SIDE EFFECTS OF SHORT-TERM DEXAMETHASONE
TREATMENT
Ita Samarzija
Zagreb University Faculty of Pharmacy and Biochemistry, Department
of Pharmacology, Zagreb, Croatia
Shortterm glucocorticoid treatment in clinical practice are used in many
cases such as for supportive therapy with setrons (antiemetics) in premedication before providing chemotherapy with high emetogenic drugs,
then, to reduce oedema at high intracranial pressure, by acute interstitial
nephritis and so on. However, the response inside the kidney to the
short-term application of glucocorticoids is not yet well dened. Therefore, the aim of this study was to observe diuretic response to dexamethasone. The experiments were performed on rats. Cumulative urine
volume was collected inside separate metabolic cages during 24 hours,
and electrolyte analysis performed before and after intraperitonealy drug
administration. Results indicated stimulation of diuresis, i.e. urine volume increased in dose-dependent manner: 0.2 mg/kg b.m. by 43%,
0.4 mg/kg by 54% and 0.8 mg/kg by 63% after 24 hours in comparison
to the control values. Time dependent stimulation in diuresis and sodium
excretion was most pronounced within third and eight hours after drug
application. Study results showed that dexamethasone possibly binding
to the high afnity corticoid receptor, which is one of the nuclear receptors and change its transcriptional activity. By changing the activity of
some enzymes (kinase isoforms), its change the activity of ion channels
and Na, K-ATPase, which are involved in the regulation of transport processes and consequentially in the diuretic response. This short-term dexamthasone effect, makes them especially useful for high-dose therapy in
conditions were water retention would be a disadvantage.
Paper No.: 1316

GENETIC POLYMORPHISM AND DRUG INTERACTIONS
MODULATING CYP2D6 AND CYP3A ACTIVITIES HAVE A
MAJOR IMPACT ON OXYCODONE ANALGESIC EFFICACY,
SAFETY AND PHARMACOKINETICS
Caroline Samer(1,2), Y Daali(1,2), M Wagner(3), G Hopfgartner(3),
CB Eap(4,6), MC Rebsamen(5,2), MF Rossier(5,2), D Hochstrasser(5,2),
P Dayer(1,2), JA Desmeules(1,2)
(1) Geneva University Hospitals Faculty of Medicine, Department of
Clinical Pharmacology and Toxicology and Multidisciplinary Pain Centre, Geneva, Switzerland
(2) University of Geneva, Swiss Centre for Applied Human Toxicology
(SCAHT), Geneva, Switzerland
(3) University of Geneva, School of Pharmaceutical Sciences, Life Science Mass Spectrometry, Geneva, Switzerland
(4) Unit of Biochemistry and Clinical Psychopharmacology, Centre of
Psychiatri cNeurosciences, Prilly Lausanne, Switzerlan.

(5) Geneva University Hospital, Service of Laboratory Medicine, Department of Genetics and Laboratory Medicine, Geneva, Switzerland
(6) University of Geneva, School of Pharmaceutical Sciences, Geneva,
Switzerland
Cytochrome P450 (CYP) 2D6 and 3A are the central drug-metabolizing
enzymes responsible for the oxydation of oxycodone. Genetic polymorphisms and/or drug-drug interactions explain their high interindividual
variability, whose impact on the pharmacokinetics and pharmacodynamics of oxycodone remains poorly explored. We conducted a randomized
crossover (5-arm) double-blind placebo-controlled study in 10 healthy
volunteers genotyped for CYP2D6. Oral oxycodone 0,2mg/kg was given
alone or after CYP2D6 and/or CYP3A inhibition (quinidine and/or ketoconazole). Experimental pain (cold pressor, electrical stimulation, thermode), pupil size, psychomotor effects and toxicity were assessed.
Plasma samples of oxycodone and metabolites were collected for 24h.
CYP2D6 (dextromethorphan) and CYP3A (midazolam) phenotypes were
assessed at each session. CYP2D6 activity was correlated with oxymorphone and noroxymorphone AUCs (-0.71 < rhos<-0.92, P < 0.05).
Blocking CYP2D6 reduced oxymorphone and noroxymorphone Cmax
(40%-80%) and increased (70%) noroxycodone AUC. Blocking
CYP3A4 tripled oxymorphone AUC and reduced (80%) noroxycodone
and noroxymorphone AUCs. Shunt to CYP2D6 pathway was observed
after CYP3A4 inhibition. CYP2D6 activity correlated with oxycodone
antinociceptive and pharmacodynamic effects. Cold pressor and pupil
size were unchanged in CYP2D6 poor metabolizer whereas CYP2D6
ultrarapid metabolizers experienced increased effects. CYP2D6 blockade
reduced oxycodone subjective pain threshold SPT (30%). CYP3A4
blockade had a major impact on all pharmacodynamic assessments. Oxymorphone Cmax correlated with pharmacodynamics (rhos= 0.7,
P < 0.05) and was the only independent positive predictor of SPT. Sideeffects were observed after CYP3A4 blockade or in ultrarapid metabolizers. In conclusion, modulations of CYP2D6 and CYP3A activities have
a dramatic impact on oxycodone pharmacokinetics and pharmacodynamics and the effect is dependent on CYP2D6 genetic polymorphism.
Paper No.: 2898

AWARENESS OF THE RISK OF VENOUS THROMBOEMBOLISM AMONG USERS OF ORAL CONTRACEPTIVES
Isidora Samojlik(1), M Panduriv(1), B Bogdanov(2)
(1) University in Novi Sad, Faculty of Medicine, Department of Pharmacology and Toxicology, Novi Sad, Republic of Serbia
(2) The Institute for Pulmonary Diseases of Vojvodina. Novi Sad,
Republic of Serbia
Although oral contraceptives (OC) are the best method of birth control
their usage is associated with certain risk of life threatening adverse reaction (ADR) like venous thromboembolism (VTE). The awareness of
VTE risk is important because in our country OC can be freely purchased in pharmacy without medical prescription. The aim of our study
was to examine the acquaintance of OC users with ADR, contraindications and discontinuation reasons, since, in the last 2 years the number
of VTE associated with OC use was increased form 3.3% to 5.8% in our
region. Data were collected by standardized questionnaire among 50
women who were purchasing OC in pharmacy. We found that they were
mostly well educated, nonsmokers (70%), on average 23 years old and
accustomed to certain type of preparation (Logest - 46%). Most of them
use OC up to 3 years (70%) for birth control (75%) according to medical
advice (56%) or based on friend experience or information from internet/
media (36%). However, 21% of those who were using OC based on
medical advice had neither laboratory nor gynecological examination
before the decision. Although OC users experienced some of ADRs they
knew little about it (only 38%). Conditions in which OC are not to be
used are known for only 12% of questioned. The majority (72%) dont
558
know when to stop with OC and seek medical help. Regarding these
results the need for better education about ADRs of OC together with
the benets of their use should be implemented.
Paper No.: 2452

EFFECT OF PSYCHOTROPIC DRUGS (FLUOXETINE AND
MIRTAZAPINE) ON PHENYLEPHRINE-INDUCED RAT VAS
DEFERENS CONTRACTILITY
Cristina Sampayo(1), I Silva(1), R Pinto(1), B Silva-Lima(1)
University of Lisbon, Faculty of Pharmacy, Laboratory of Pharmacology,
(Pharmacological Sciences Group [iMed.UL (Research Institute for Medicines and Pharmaceutical Sciences)]), Lisbon, Portugal
Effect of psychotropic drugs (Fluoxetine and Mirtazapine) on Phenylephrine-induced rat vas deferens contractility Cristina Mello-Sampayo, Ines
Silva, Rui Pinto, Beatriz Silva-Lima (Pharmacological Sciences Group
[iMed.UL (Research Institute for Medicines and Pharmaceutical Sciences)], Faculty of Pharmacy, University of Lisbon). We described in the
rat vas deferens (rvd) a phenylephrine (Phe) concentration-dependent
increased of the endogenous noradrenaline (NE) release (Pinto et al.,
Pharmacol Toxicol. Oct; 93(4); 191-196. 2003). In order to understand
this mechanism, we studied in Wistar rvd the Phe-induced contractility
and endogenous-NE release in the absence (control) and in presence of
Fluoxetine or Mirtazapine, antidepressants known to cause sexual side
effects. A concentration-dependently decrease of vas deferens contractility was observed with Fluoxetine concentrations equal and above 5lM.
The response was completely abolished at and above 50l M Fluoxetine
concentrations. At lower Fluoxetine concentrations ( 1lM), similar, or
even higher, vas deferens contractile responses were observed. A no
effect was observed with lower concentrations (0.001 to 1.0lM) of Mirtazapine, while higher concentrations (10lM) increased the contractile
response. The endogenous NE released by the rat vas deferens stimulated
with Phe was increased with lower Fluoxetine concentrations while
higher Fluoxetine concentrations reduced it. The NE release was not
modied by Mirtazapines presence. The results suggest that drugs
involved in the modulation of 5-hydroxytryptamine can modulate the vas
deferens contractility through a mechanism where NE also plays an
important role. Future studies are needed to clarify this point, moreover
when drugs involved in the serotonin release (dapoxetin) are used for
treatment of male ejaculation disorders.
Paper No.: 2977

LACRITIN, A NOVEL SECRETAGOGUE, INCREASES TEAR
FLOW IN RABBITS
Sandeep Samudre(1), J Sheppard(2), F Lattanzio(1), G Laurie(3),
R McKown(4), P Williams(1)
(1) Eastern Virginia Medical School, T.R. Lee Center for Ocular Pharmacology, Norfolk, VA, USA
(2) Eastern Virginia Medical School, Department of Ophthalmology,
Norfolk, VA, USA
(3) University of Virginia, Department of Cell Biology, Charlottesville,
VA, USA
(4) James Madison University, Department of Integrated Science and
Technology, Harrisonburg, VA, USA
not well addressed. Lacritin is a newly discovered glycoprotein that

occurs naturally in human tears, produced by lacrimal glands. Human
recombinant lacritin stimulates tear secretion in cultured rat lacrimal acinar cells, leading us to hypothesize that topical application of lacritin will
increase production of tears in vivo. We evaluated numerous lacritin constructs and compared their efcacy to cyclosporin (Restasis), and also
to determine functional domains within the lacritin molecule. Lacritin
(1,10, or 50 lg/ml), N-terminal deletions of lacritin N-24 (50lg/ml)
and N-65 (50 lg/ml), inactive C-25 lacritin (10 lg/ml) or cyclosporin
(0.05%), were administered bilaterally to New Zealand white rabbits
three times daily for 14d (n = 4/group). Lacritin 1,10 or 50 lg/ml
increased tear production by 7 8%, 20 7% and 66 8% over baseline and sustained for 14d (p < 0.01, n = 4). Although cyclosporine initially increased tear ow by 51 4%, it was not sustained after 14 d. N24, C-65 or C-25 did not increase tear production. No local irritant
effects were noted by slit lamp examination. Treatment with lacritin was
better tolerated with cyclosporin. This data suggests that functional
domains that promote tear secretion might reside in both the C and N terminal regions. Glycoproteins, such as lacritin, represent a new, unique
therapeutic approach that may more closely address the pathology of dry
eye.
Paper No.: 1379

HEALTH AND DISEASE
DIFFERENTIAL COX-2-MEDIATED ARACHIDONIC ACID
METABOLISM IN CORONARY AND PENILE ARTERIES
FROM PREDIABETIC OBESE ZUCKER RATS
A Sanchez, C Contreras, N Villalba, P Martnez, A Garca-Sacristan, M
Hernandez, Dolores Prieto
Erectile dysfunction is currently considered an early manifestation of
subclinical vascular disease and a potential marker of coronary artery disease in diabetic patients (Gazarruso et al., Circulation; 110:22-26, 2004).
Since inhibitors of cyclooxygenase-2 (COX-2) have been reported to
increase the risk of myocardial infarction and atherothrombotic events
(Krotz et al., J Vasc Res; 42:312-324, 2005), the purpose of the present
study was to assess the role of arachidonic acid (AA) metabolism via
COX-2 in the regulation of vascular tone of penile and coronary arteries
from obese Zucker rats (OZR). Arteries from OZR and from lean Zucker
rats (LZR) were mounted in microvascular myographs to assess vascular
function and COX expression was determined by both immunohistochemistry and Western blot. Relaxations to both acetylcholine (ACh) and
AA were impaired in penile arteries but not in coronary arteries from
OZR. Selective blockers of COX-1 and COX-2 inhibited both ACh- and
AA-induced relaxations in penile arteries from LZR but not from OZR.
In coronary arteries, the COX-2 inhibitor did not alter either ACh- or
AA-elicited relaxations from LZR, but reduced endothelium-dependent
relaxations to AA in OZR. COX-2 was distributed in the penile endothelium of both LZR and OZR, while it was expressed in the coronary
endothelium predominantly of OZR. Thus, COX-2 is constitutively
expressed and contributes to the endothelium-dependent relaxations in
penile arteries which are impaired in OZR. Endothelial COX-2 is up-regulated in coronary arteries to produce vasodilator prostaglandins in insulin resistant OZR.
Supported by grant SAF2006-09191 from MICINN
Dry eye is an extremely common and debilitating disease with treatment

options being palliative at best and the underlying pathology of dry eye
559
Paper No.: 627
ANTIOXIDANT ACTIVITY OF RHIZOPHORA MANGLE (L.)
BARK AND ITS EFFECT ON REDOX MECHANISMS
INVOLVED IN WOUND HEALING
J Sanchez Calero(1), R Faure Garca(1), G Martnez Sanchez(2), O
Fernandez Limia(1), M Mitjavila Cors(3), E Marrero Faz(1), Yinet Barrese Perez(1)
(1) National Centre for Animal and Plant Health (CENSA), Department
of Chemistry-Pharmacology-Toxicology, San Jose de Las Lajas, Havana,
Cuba
(2) University of Havana, Pharmacy and Food Sciences Institute,
Havana, Cuba
(3) University of Barcelona, Faculty of Biology. Department of Physiology, Barcelona, Spain
Rhizophora mangle (L.) bark aqueous extract and its major component,
high molecular weight polyphenols fraction, were evaluated for antioxidant activity. Different experiments were done: hydroxyl radicals scavenging and iron chelating activities using deoxyribose assay; superoxide
anions tested by xanthine oxidase / xanthine system; superoxide anions
and nitric oxide were evaluated using a cellular line of 264.7 RAW macrophages stimulated with LPS or PMA, detected by Nitrobluetetrazolium
and Griess methods respectively; damage to proteins exposed to Fenton
system was carried out by quantication of SH groups using BSA as
model; lipid peroxidation was tested by MDA measurement and DNA
oxidation was done using Bleomicin/Fe model; different markers representing redox balance were evaluated on a rats model of aseptic wounds:
SOD, CAT, GSH and MDA were measured during the wound healing
process at 0, 3, 7 and 14 days, also the wound area (mm2), with Imagen
Digital Processing System MADIP 3.0 Lab Lapdis, at 7 days after the
wounds were made. Results showed that R. mangle and its fraction have
a remarkable antioxidant activity, achieved by the scavenging ability
against free radicals, while also showed iron chelating properties and
protector effects against the oxidative damage to biomolecules. In the rat
model, in the groups treated with both samples, the wounds area was
reduced (p < 0.05) and the antioxidant biomarkers increased and pro-oxidant indicators decreased (p < 0.05) compared with control group. This
is the rst report showing antioxidant activity of R. mangle and its effect
on redox mechanisms involved in wound healing.
Paper No.: 1688

DMSO-LIPID SOLUBLE CIGARETTE SMOKING PARTICLES
UPREGULATE CONTRACTILE ENDOTHELIN TYPE B AND
THROMBOXANE A2 RECEPTORS OF RAT MIDDLE
CEREBRAL ARTERIES
Hardip Sandhu(1), C-B Xu(0), L Edvinsson(0)
(1) Glostrup Research Institute, Glostrup Uni-ersity Hospital, Department
of Clinical and Experimental Research, Copenhagen, Denmark
(2) University Hospital of Lund, Institute of Clinical Sciences in Lund,
Division of Experimental Vascular Research, Lund, Sweden
Cigarette smoking is a strong risk factor for stroke. We examined if
DMSO lipid-soluble-cigarette-smoking-particles (DSP) enhance the
expression of contractile endothelin type B receptors (ETB) and Thromboxane A2 receptors (TP) in rat middle cerebral arteries (MCAs). Kentucky research cigarettes were smoked with vacuum through a lter and
the smoke particles retrieved on the lter were dissolved in DMSO.
MCAs from adult male Sprague Dawley rats were organ cultured for
24 h in presence of 0.15 ll DSP or DMSO (control). Contractile re-
sponses to ETB receptor agonist sarafotoxin (S6c) or the TP receptor

agonist U46619 were studied by a myograph. Real-time PCR and immunohistochemistry were used to semi-quantify the ETB and TP receptor
mRNA and protein expressions. DSP enhanced S6c-induced contraction
of the MCAs with increased the maximal contrac-tion (Emax) from
60 7% (mean s.e.m. in % of 60 mM K+) to 105 10% (P < 0.05).
The Emax of U46619-induced contraction was also signicantly
increased by DSP, from 112 5% to 148 12% (P < 0.05). Real-time
PCR and immunohistochemistry showed that DSP upregulated both ETB
and TP receptor mRNA and protein expressions in the smooth muscle.
We have demonstrated for the rst time that DSP enhanced the expression of ETB and TP receptors in the MCAs. The enhanced ETB and TP
receptor-mediated contraction by DSP might contribute to cigarette
smoking-associated stroke.
Paper No.: 1235

ANALGESICS
ROLE OF PITUITARY ADENYLATE CYCLASE ACTIVATING
POLYPEPTIDE IN NOCIFENSIVE BEHAVIOURS,
INFLAMMATORY AND NEUROPATHIC HYERALGESIA
Katalin Sandor(1), V Kormos(1), B Botz(1), A Imreh(1), K Bolcskei(2),
B Gaszner(3), D Reglodi(3), J Szolcsanyi(1), N Shintani(4), H Hashimoto(4,5,6), A Baba(4), Z Helyes(1)
(2) University of Pecs, Analgesic Research Laborator and Gedeon-Richter Ltd.,Pecs, Hungary
(3) University of Pecs, Faculty of Medicine, Department of Anatomy,
Pecs, Hungary
Osaka, Japan
(5) Osaka University, Graduate School of Medicine, Center for Child
Mental Development, Osaka, Japan
(6) Osaka University, United Graduate School of Child Development,
Kanazawa University and Hamamatsu University School of Medicine,
Japan
Pituitary adenylate cyclase-activating polypeptide-38 (PACAP-38) and
its receptors have been shown in the spinal dorsal horn, dorsal root ganglia and sensory nerve terminals. Data concerning the role of PACAP in
central pain transmission are controversial, we have recently published
its divergent peripheral effects on nociceptive processes. The aim of this
study was to investigate in PACAP decient (PACAP-/-) mice acute
somatic and visceral nocifensive behaviours, partial sciatic nerve ligation-evoked chronic neuropathic, and resiniferatoxin-induced inammatory thermal/mechanical hyperalgesia to elucidate the role of this
neuropeptide in nociception. Neuronal activation was investigated with
c-Fos immunohistochemistry. In PACAP-/- mice paw lickings in the early
and late phases of the formalin test were markedly reduced and acute visceral
chemonociception was also signicantly attenuated. In these models, markedly smaller c-Fos expression in the periaqueductal grey and the somatosensory cortex was observed in PACAP decient mice. Resiniferatoxin-evoked
mechanical hyperalgesia was decreased, but thermal allodynia increased in
the knockouts. In gene-deleted mice neuropathic mechanical hyperalgesia
did not develop throughout 20 days, albeit the number of c-Fos immunopositive cells was enhanced. These data provide evidence for a role of PACAP in
transmission of acute nociception originating from exteroceptive and
interoceptive regions. The same refers to neuropathic mechanical hyperalgesia. In this model, however, the enhanced number of c-Fos positive
cells may indicate a role for this neuropeptide in the activation of inhibitory neurones. In contrast, it is an inhibitory mediator at the level of the
peripheral sensory nerve endings and decreases their sensitization to heat
with presently unknown mechanisms.
560
Paper No.: 2222
INVOLVEMENT OF H2O2 IN ADENOSINE-MEDIATED
REGULATION OF CORONARY FLOW
Maryam Shari Sanjani(1), B Teng(1), T Krahn(2), S Tilley(3),
C Ledent(4), J Mustafa(1)
(1) West Virginia University, Department of Physiology and
Pharmacology, Morgantown, WV, USA
(2) Bayer Health Care AG, Wuppertal, Germany
(3) University of North Carolina, Chapel Hill, NC, USA
(4) Universite Libre de Bruxelles, Brussels, Belgium
Introduction: Adenosine, released due to increased oxygen demand of the
heart, exerts its physiological effects through its four subtype receptors
(AR): A1, A2a, A2b and A3. A2a AR is known to play a major role in coronary ow (CF) regulation. However, the role of A2b AR needs to be further
elucidated. Therefore, we need to further investigate the role and signalling
pathways of A2a/b ARs in order to better understand the heterogeneity of
CF regulation. Material/Method: We performed Langendorff Heart experiments using two different approaches: 1) pharmacological (using selective
and non-selective AR agonists); 2) genetic (using A2A and A2B knockout
(KO) mice and WT). Results: BAY60-6583 (A2b AR agonist) shows no
effect on CF in A2b KO mice, however, it increases the CF signicantly in
A2a KO (211.0 34.54%) and WT (227.0 79.87%). CGS21680 (A2a
AR agonist) also caused a signicant increase in CF in WT
(203 15.12%) and A2b KO (272 40.38%) mice. These data suggest
the involvement of A2a/2b ARs in CF regulation. Also, exogenous adenosine-induced increase in CF of WT (284 53%), A2a KO (245 46%),
and A2B KO (215 28%) mice were signicantly reduced with catalase
(1,250 u/ml) to 89 12%, 80 20%, and 89 12%, respectively. Furthermore, BAY60-6583-induced increase in CF (232 57%) of WT mice
was signicantly inhibited with glibenclamide (KATP channel blocker) to
114 27%. Conclusion: We propose, for the rst time, that activation of
A2a/b ARs may induce the release of H2O2 which then activates KATP
channels, leading to hyperpolarization and vasodilation.
Supported by NIH grants (HL027339, HL094447, HL071802, and T-32
HL090610).
Paper No.: 1842

B-ARRESTIN 1 AND 2 STABILIZE THE ANGIOTENSIN II
TYPE 1 RECEPTOR IN DISTINCT HIGH-AFFINITY
CONFORMATIONS
Samra Joke Sanni(1), JT Hansen(2), MM Bonde(2), T Speerschneider(2),
GL Christensen(1), S Munk(1), S Gammeltoft(1), JL Hansen(2)
(1) Glostrup Hospital, Departmant of Clinical Biochemistry, Glostrup,
Denmark
(2) The Danish National Research Foundation Centre for Cardiac
Arrhythmia, The Heart Centre, Copenhagen University Hospital, Laboratory for Molecular Cardiology,Copenhagen, Denmark
The Angiotensin II type 1 receptor (AT1R) belongs to family A of seven
transmembrane receptors (7TMR). The receptor has important roles in
the cardiovascular system, and is commonly used as a drug target in cardiovascular diseases. Studies show that 7TMR interaction with G proteins or b-arrestins (b-arrs) often induces higher binding afnity for
agonists. Here, we examined the interaction between AT1R and b-arrs to
analyze for high-afnity phenotypes, and probe for differences between
the AT1R interaction with b-arrestin1 (b-arr1) and b-arrestin2 (b-arr2).
Ligand induced interaction between AT1R and b-arrs was measured by
Bioluminescence Resonance Energy Transfer 2 (BRET2). AT1R- b-arr1
and AT1R-b-arr2 fusion proteins were made and analyzed for differences
using following functional assays: immunocytochemistry, Inositol phospholipid (IP) hydrolysis, and competition radioligand binding. The
BRET2 analysis showed that b-arr1 and -2 are recruited to the AT1R
with similar ligand potencies and efcacies. The AT1R-b-arr fusion proteins showed attenuated G protein signalling and increased agonist binding afnity, while antagonist afnity was unchanged. Importantly, larger
agonist afnity shifts were observed for AT1R-b-arr2 than for AT1R-barr1. Conclusion from our study is that b-arr1 and -2 are recruited to the
AT1R with similar ligand pharmacology and stabilize the AT1R in distinct high-afnity conformations. However, b-arr2 induces a receptor
conformation with a higher agonist-binding afnity than b-arr1. This
demonstrates that b-arrs interact with the AT1R with unique ngerprints,
and suggest that it may be possible to design AT1R biased agonists with
the ability to recruit either of the b-arrs selectively.
Paper No.: 1277
ESTRADIOL INTERACTS WITH GROWTH HORMONE AND
THYROID HORMONES TO REGULATE THE EXPRESSION OF
SUPPRESSORS OF CYTOKINE SIGNALLING (SOCS) IN
LIVER
Ruyman Santana-Farre(1), A Flores-Morales(2), G Norstedt(2),
L Fernandez-Perez(1)
(1) University of Las Palmas de Gran Canaria, Department of Clinical
Sciences, Molecular and Translational Endocrinology Group, Las Palmas
de Gran Canaria, Spain
Stockholm, Sweden
The SOCS proteins negatively regulate cytokine receptor signalling. The
concentration of SOCS proteins is constitutively low but increases rapidly by stimulation with different stimuli. Recently, it has been reported
that 17b-estradiol (E2) induces SOCS expression and in turn negatively
regulates GHR signalling pathway in liver cells. Physiological regulation
of SOCS has not been well characterized. In this work, we investigated
the physiological role of E2, GH and/or T3 on SOCS mRNA expression
in liver. For that purpose, we used adult hypothyroid-castrated (TX-OX)
male rats to minimize the inuence of internal hormones on treatment.
TX-OX rats were treated with E2 benzoate (50 microg/kg; sb; 5 days/
week) for 20 days before hormonal replacement with GH and/or T3 during seven days. Hypothyroidism reduced body weight gain (BWG) and
IGF-I mRNA levels in liver which were recovered by hormonal replacement; in the presence of E2, however, recovery of BWG and IGF-I were
fully abolished. Development of TX-OX increased total cholesterol and
decreased triglyceride in serum. T3 restored serum levels of total cholesterol. E2 did not modify the effects of hypothyroidism or T3 replacement
on serum lipids levels. TX-OX decreased SOCS2 mRNA expression levels and they were restored by GH treatment. In contrast, TX-OX induced
SOCS3 mRNA levels and T3 treatment restored them. E2 treatment
induced SOCS2 mRNA levels. In the presence of E2, however, GH stimulation of SOCS2 was inhibited whereas T3 superinduced SOCS3. Taken
together, these data suggest that interactions of E2 with GH/T3 play a
physiological role in maintaining SOCS levels.
Paper No.: 2374

INFLUENCE OF ESTRADIOL ON GROWTH HORMONEREGULATED LIVER TRANSCRIPTOME IN MALE RATS
Ruyman Santana-Farre(1), A Flores-Morales(2), M Mirecki-Garrido(1),
G Norstedt(2), L Fernandez-Perez(1)
(1) University of Las Palmas of Grand Canaria, Department of Clinical
Sciences -Unit of Pharmacology, Molecular and Translational Endocrinology Group, LasPalmas De Gran Canaria, Spain
561
Stockholm,Sweden & University of Copenhagen, Novo Nordisk Center
for Protein Research,Copenhagen, Denmark
GH is a major regulator of growth and metabolism. Estrogens may modulate GH-regulated endocrine and metabolic functions in liver. To test
this hypothesis, we used adult hypothyroid-castrated (TX-OX) male rats
to minimize the inuence of internal hormones on treatment. TX-OX rats
were treated with E2 benzoate (50 microg/kg; sb; 5 days/week) for
20 days before GH replacement (0.3 mg/kg/day; sb; two daily injections)
during seven days. Hypothyroidism reduced body weight gain, circulating IGF-I, and mRNA levels of IGF-I and male-specic CYP2C11 gene
in liver, which were restored by GH. In contrast, in the presence of E2,
GH was not able to restore the changes induced by hypothyroidism.
CYP2C12, a female differentiated gene, was induced by E2. To obtain
comprehensive information on effects of E2 treatment on GH-regulated
gene expression, we performed microarray analysis of liver transcriptome. In the absence of E2, we identied 218 genes that were up-regulated
by more than 50% by GH treatment, while 139 were down-regulated to
the same extent. In the presence of E2, 172 genes were up-regulated by
GH, while 243 were down-regulated. Administration of E2 to hypothyroid rats, provoked drastic changes (up-regulated genes=382; down-regulated genes=290) in liver transcriptome. A set of 84 genes were
regulated in common by GH and E2. In the presence of E2, the number
of Biological Processes with signicant representation in our set of genes
that were up-regulated by GH treatment was drastically reduced. This
work highlights the inuence of estradiol on male liver which has relevance for several diseases.
Paper No.: 1690

NOVEL PIM KINASE INHIBITOR IMPAIRS PIM-DEPENDENT
STIMULATION OF CANCER CELL MOTILITY
Niina Santio(1,4), R Vahakoski(1,4), E Rainio(1), S Virtanen(3,4),
J Sandholm(1), M Prudhomme(5,6), F Anizon(5,6), P Moreau(5,6),
P Koskinen(1,2)
bo Akademi University, Turku Centre for
(1) University of Turku and A
Biotechnology, Turku, Finland
(2) University of Turku, Department of Biology, Turku, Finland
(3) University of Turku, Institute of Biomedicine and Anatomy, Turku,
Finland
(4) Drug Discovery Graduate School, Finland
(5) Clermont Universite, Universite Blaise Pascal, SEESIB, ClermontFerrand, France
(6) CNRS, UMR6504, Aubie`re, France
Pim family kinases are small constitutively active serine/threonine-specic kinases, elevated levels of which have been detected in human
hematopoietic malignancies as well as in solid tumours. While we and
others have previously shown that the oncogenic Pim kinases stimulate
cell survival, we now report a novel role for them in enhancing cancer
cell motility. We have recently identied 1,10-Dihydropyrrolo[2,3-a]carbazole-3-carbaldehyde (DHPCC-9) as a promising inhibitor for Pim kinases. We now demonstrate that this inhibitor is very effective not only
under in vitro conditions, but also in several cell-based assays. DHPCC9 inhibits phosphorylation of Pim substrates such as NFATc1 and thereby
reduces NFATc-dependent transactivation in lymphoid cells. It also
impairs the anti-apoptotic effects of Pim-1 in cytokine-deprived myeloid
cells. Furthermore, DHPCC-9 slows down migration and invasion of
cancer cells derived from either prostate cancer or squamocellular carcinoma. All these effects are clearly dependent on Pim kinase activity,
since similar results are obtained by silencing Pim expression by RNA
interference. Altogether, our data indicate that DHPCC-9 is an attractive
molecule for cancer drug development to be targeted especially to inhibit
invasiveness of Pim-overexpressing cancer cells.
Paper No.: 1156

DISEASES
DRUG PRESCRIPTION AFTER THIRD MOLAR REMOVAL:
A RETROSPECTIVE STUDY IN THE FACULTY OF DENTAL
MEDICINE OF THE UNIVERSITY OF PORTO/PORTUGAL
Carlos Santos(1), A Calvo(1), T Dionsio(1), JR Lauris(1),
MH Fernandes(2), A Felino(2)
(1) University of Sao Paulo, Bauru School of Dentistry, Department of
Biological Sciences, Baurau, Sao Paulo, Brazil
(2) University of Porto, Faculty of Dental Medicine, Porto, Portugal
This work aimed at performing a retrospective study in the Faculty of
Dental Medicine of the University of Porto regarding drug prescription
after third molar removal since 2003 until 2008 with graduate and postgraduate students. Medical charts from 442 patients who underwent third
molar removal, with or without bone removal, were assessed to obtain
information regarding all drugs prescribed. Anti-inammatory drugs
were prescribed by graduate students when osteotomy was not necessary
in 73.65% of surgeries, less than when ostetomy was necessary (93.85%,
p<.05) and when surgeries were performed by postgraduate students,
with or without ostetomy (95.83% and 98.88%, p<.05). Analgesics were
more prescribed by graduate students in both situations (74.32% without
ostetotomy and 86.15% with ostetotomy) in comparison with postgraduate students (31.25% without ostetotomy and 49.44% with ostetotomy,
p<.05). Corticoids were just prescribed by postgraduate students (25%
without ostetotomy and 65.17% with ostetotomy). Antibiotics were more
prescribed by postgraduate students when ostetomy was necessary
(94.38%) in comparison with surgeries when it was not necessary
(62.50%, p<.05) and in relation to surgeries performed by graduate students (35.81% without ostetotomy and 80% with ostetotomy, p<.05). In
conclusion, graduate and postgraduate students have different patterns of
drug prescription after third molar removal in order to relieve pain and
avoid infection.
Paper No. 3121

FOCUS GROUP: P10 - DRUGS FOR HALF THE WORLD:
PAEDIATRIC CLINICAL PHARMACOLOGY
DISCOVERY OF NOVEL PHARMACOLOGICAL
CHAPERONES FOR PHENYLKETONURIA
S Santos Sierra(1), A Perna(2), J Kirchmayer(2), K Kemter(3),
DD Messing (3), SW Gersting(3), AC Muntau(3), H Glossmann(1),
G Wolber(2), Florian B Lagler(1)
Molecular and Clinical Pharmacology, Innsbruck, Austria
(2) University of Innsbruck, Institue of Pharmacy and Center for
Molecular Biosciences (CMBI), Department of Pharmaceutical
Chemistry, Innsbruck, Austria
(3) Ludwig-Maximilians-University, Department of Molecular Pediatrics,
Dr. von Hauner Childrens Hospital, Munich, Germany
Deciency of phenylalanine hydroxylase (PAH; EC 1.14.16.1) causes
phenylketonuria (PKU) and is the most common inborn error of amino
acid metabolism. Untreated PKU can lead to severe mental retardation.
Disease causing missense mutations lead to a misfolded PAH enzyme
prone to degradation. For many years a very burdensome lifelong phenylalanine restricted diet has been the only therapeutic option. Tetrahydrobiopterin (BH4) the natural cofactor of PAH was recently found to
restore PAH function in a signicant share of PKU patients. BH4 acts as
a pharmacological chaperone (PC) that promotes correct folding of PAH.
In 2008, sapropterin, the synthetic form of BH4 was market approved as
rst PC drug. The costly synthesis of sapropterin, its low stability and
low oral bioavailability (<20%) substantiate the need for alternative molecules. We therefore employed a structure-based molecular modelling to
562
identify novel PC for the treatment of PKU. The examination of the
PAH crystal structure in complex with BH4, the pharmacophore modelling and virtual screening of chemical compound libraries led to the
selection of a set of small molecules with potential PC activity. The binding of PCs to PAH was screened using surface plasmon resonance and
rened by means of tryptophan intrinsic uorescence. PCs that bound
PAH were further in vitro characterized for their ability to stabilise a misfolded PAH mutant and the toxicity dose was assessed in cell culture
experiments. Finally, identied PC candidates were conrmed in vivo
within a murine model of human BH4 responsive PKU.
Paper No.: 1833

PHENOTHIAZINE DERIVATIVES EFFECTS ON
EXPERIMENTAL TACHYARRITHMIAS
Kristina Sarkisyan, M Ivashev
Pyatigorsk State Pharmaceutical Academy, Department of Pharmacology,
Pyatigorsk, Russian Federation
Aim: to investigate the antiarrithmic activity of phenothiazine derivatives
the compound under the laboratory cipher MIKS and ofcinal aethacizine preparation on the models of heart tachyarrithmias (aconitinic and
calcium chloride). Aethacizine is a aetmozine derivative which is applied
in clinical practice under moricizine name in USA. According to the ofcial data aethacizine and aetmozine drugs have been synthesized during
joint investigations carried out at the scientic research pharmacology
institute of the Russian academy of medical sciences and represent aminoacil N-substituted phenothiazine derivatives attributed to the IC class
antiarrithmic drugs. Materials and methods: experiments have been carried out with narcotinized white rats according to the standard techniques. Results: aconitine and calcium chloride infusion caused control
animals death due to ventricular brillation. Animal heart rhythm change
in response to aconitine and calcium chloride administration to all experimental animals was identical to that one of the control group. However,
MIKS and aethacizine combination in all three doses applied in this
experimental series surely increased the animal life duration (time of full
heartbeat stop) and decreased the ventricular brillation expressiveness
in relation to the controls. The new MIKS compound effects in sure to
be higher of the aethacizine one. Conclusion: MIKS compound is certainly a new perspective substance of antiarrithmic activity.
Paper No.: 1377

FOCUSED CONFERENCE GROUP: PW29 - NEW HORIZONS
IN THERAPEUTIC DRUG MANAGEMENT (TDM)
BIODISTRIBUTION OF 18F-LABELED THERMALLY
HYDROCARBONIZED POROUS SILICON NANOPARTICLES
FOR DRUG DELIVERY
Mirkka Sarparanta(1), E Makila(2), T Heikkila(2), J Salonen(2),
V-P Lehto(3), HA Santos(4), J Hirvonen(4), AJ Airaksinen(1)
(1) University of Helsinki, Laboratory of Radiochemistry, Helsinki, Finland
(2) University of Turku, Laboratory of Industrial Physics, Turku, Finland
(3) University of Eastern Finland, Department of Physics, Kuopio, Finland
(4) University of Helsinki, Faculty of Pharmacy, Helsinki, Finland
Porous silicon (PSi) is a lucrative material for controlled drug delivery
applications, as it can be tailored to have dened pore diameter and volume enabling loading with diverse payloads. In addition, the surface can
be modied with variable functional moieties for targeting and imaging.
We have successfully radiolabeled hydrocarbonized porous silicon nanoparticles (THCPSi) with a short-lived positron emitter 18F. The biodistribution of 18F-THCPSi was investigated in male Wistar rats after oral,
subcutaneous and intravenous administration. Control animals received
free uoride as 18F-NaF. The radiolabel was shown to be stable under

physiological conditions in vitro, enabling particle follow-up for up to
six hours in vivo. Radiolabeled nanoparticles passed intact through the
gastrointestinal tract after oral administration and were not absorbed from
the subcutaneous injection site. After intravenous administration they
were rapidly taken up from the circulation by the liver and the spleen.
Only slight detachment of the 18F-label consistent with the uoride biodistribution seen in control animals was observed, illustrating remarkable
label stability in vivo. We conclude that the study of PSi material behavior in a living organism is plausible with 18F-PET, even for slower physiological processes such as passage through the GI tract after oral
administration. Although the investigated material, THCPSi, was shown
to be relatively passive in vivo, it is an interesting platform for the development of drug carriers for oral delivery, and for targeting or circulation
time enhancing modications for intravenous route.
Paper No.: 3287

RECEPTOR LOCALIZATION TO CAVEOLAE REGULATES
DIFFERENTIAL G PROTEIN COUPLING OF MOUSE
BETA3-ADRENOCEPTOR SPLICE VARIANTS
Masaaki Sato, D Hutchinson, B Evans, R Summers
Monash University, Department of Pharmacology, Parkville, VIC,
Australia
Alternative splicing of the mouse b3-adrenoceptor (AR) produces two isoforms that differ at the C-terminus: the b3a-AR couples to Gs and the
b3b-AR couples to both Gs and Gi. To dene residues involved in differential G protein coupling, truncated receptors were expressed in CHO-K1
cells and their properties examined. In cells expressing truncated receptors, maximal cAMP accumulation following stimulation with CL316243
(selective b3-AR agonist) was increased relative to control after pretreatment with pertussis toxin (PTX), suggesting that the b3a-AR is restrained
from coupling to Gi by residues in the C-terminus. Site-directed mutagenesis was used to identify residues in the b3a-AR C-terminus responsible
for inhibition of coupling to Gi. Mutation of a putative caveolin binding
site caused b3a-AR-mediated cAMP accumulation to become PTX-sensitive. Furthermore lipin III, an agent that disrupts lipid rafts as well as
siRNA caveolin-1 also caused the wild-type b3a-AR to become PTX-sensitive. In mouse primary brown adipocytes, (endogenously expressing the
b3-AR) cAMP accumulation in response to CL316243 was signicantly
reduced by pretreatment with lipin III and a marked increase in cAMP
accumulation was observed in cells pretreated with PTX. In addition,
brown adipocytes from caveolin-1 KO mice showed a signicant increase
in cAMP accumulation after pretreatment with PTX. Duolink in situ Proximity Ligation Assays visualized clear interactions between b3a-ARs and
caveolin-1 whereas no interactions were observed in CHO-K1 cells
expressing b3b-ARs. These observations suggest that b3a-ARs are localised to caveolae and that the localization of the receptor plays a specic
role in the determination of G-protein coupling.
Paper No.: 2231

DISEASES
INDUCTION OF DUODENAL ULCERS BY SELECTIVE
CYCLO- OXYGENASE (COX)-1 AND COX-2 INHIBITORS IN
CATS
Hiroshi Satoh, K Amagase, K Takeuchi
Departmentof Pharmacology & Experimental Therapeutics, Kyoto, Japan
563
Background: It has been reported that inhibition of both cyclooxygenase (COX)-1 and COX-2 is required to induce gastric and intestinal damage by nonsteroidal anti-inammatory drugs (NSAIDs). However, the
role of these isoforms in NSAID-induced duodenal ulcers has not been
fully elucidated. Recently we reported that the non-selective COX inhibitor indomethacin (IND) produced lesions in the duodenum and small
intestine in cats. In the present study, we examined the ulcerogenic
effects of a selective COX-1 inhibitor (SC-560) and selective COX-2
inhibitors (celecoxib and meloxicam) on gastrointestinal mucosa in cats.
Methods: Adult cats were used (n = 4-5). NSAIDs were administered
p.o. or s.c. once a day after the morning meal for 3 days. The animals
were sacriced 24 h after the nal NSAID dose and mucosal lesions in
the GI tract were examined. Results: IND (3 mg/kg, p.o.) produced
severe lesions in both the duodenum and small intestine; the mean lesion
area (MLA) was 0.8 0.2 cm2 and 7.7 2.0 cm2, respectively. SC-560
(10 mg/kg, p.o.) caused severe lesions in the duodenum and mild lesions
in the small intestine; MLAs were 1.2 0.1 cm2 and 2.2 2.2 cm2,
respectively. Celecoxib (10 mg/kg, p.o.) and meloxicam (0.6 mg/kg, s.c)
both produced obvious lesions in the duodenum, but there were almost
no lesions in the small intestine; MLAs in the duodenum were
0.6 0.2 cm2 and 0.5 0.3 cm2, respectively. Conclusions: Inhibition
of either COX-1 or COX-2 alone can produce duodenal ulcers in cats,
though inhibition of both isoforms seems to be necessary in induction of
small intestinal ulcers.
Paper No.: 1354

HEALTH AND DISEASE
THYROID HORMONE UP-REGULATES ELASTIN AND LYSYL
OXIDASE GENES IN RAT AORTA
Mitsutoshi Satoh(1), S Yanagino(1,3), T Nishimaki-Mogami(2),
K Suzuki(3), Y Sato(3)
(1) Toho University, Faculty of Pharmaceutical Sciences, Department of
Pharmacology and Toxicology, Chiba, Japan
(2) Toho University Faculty of Pharmaceutical Sciences, Division of
Functional Biochemistry and Genomics, Chiba, Japan
(3) National Institute of Health Sciences, Division of Cellular and Gene
Therap yProducts, Tokyo, Japan
Thyroid hormones (THs) have marked cardiovascular effects. However,
their direct effects on the arteial system have been poorly understood.
We have reported that TH facilitates gene expression of matrix Gla protein in arterial smooth muscle cells via TH nuclear receptors, leading to
prevention of vascular calcication (Circ. Res. 2005; 97, 550-557). To
test a hypothesis that TH also regulates genes associated with arterial
elasticity, we treated rat aortic smooth muscle cells, cultured in the nominally TH-free medium, with 3hf,3,5-triiodo-L-thyronine (T3) for
48 hours, and examined expression of elasticity-associated genes. Quantitative RT-PCRs indicated that a physiological concentration of T3 (2430 pM fT3) facilitated gene expression of elastin and lysyl oxidase
(LOX), which catalyzes cross-linking of elastin, by approximately 2-fold.
These increases were suppressed by RNAi against TRa gene. The protein level of insoluble elastin was also increased by T3, which was inhibited by b-aminopropionitrile, a LOX inhibitor. Consistent with the
ndings in vitro, mRNAs for elastin and LOX were signicantly downregulated by 95% and 91%, respectively, and elastin protein level was
also decreased by 10% in aortic smooth muscle of methimazole-induced
hypothyroid rats (400 mg/L drinking water, 4 weeks), compared to those
of euthyroid animals. Our ndings suggest that TH determines arterial
elasticity at least partly by activation of its nuclear receptors and subsequent up-regulation of elastin and LOX genes in vascular smooth muscle
cells.
Paper No.: 1846

COGITUM EFFECTS AT THE EXPERIMENTAL
GASTROPATHY
Ivan Savenko, A Sergienko
Pjatigorsk State Pharmaceutical Academy (PSPA), Department of Pharmacology, Pjatigorsk, Russian Federation
Aim: Pharmacological validation of the amino acid cogitym gastro protective activity at the experimental steroid-ethanol gastropathy. Cogitum
is a monocomponent synthetic analogue of the asparaginic acid corresponding to it on mechanisms of the body biological action. It is applied
in the complex therapy of the central nervous system disturbances and
asthenic conditions of various genesis. Materials and methods: experiments have bun carried out with wistar white rats, gastropathy was
induced by the intragastral administration of prednizolone in ethanol of
high concentration. In a day gastrectomy was performed, defect quantity
and area, stomach weight, gastric juice volume and acidity have bun
determined. Results: mucous membrane of control was hyperinamed
and almost completely covered with ulcers stomach weights were nearly
twice decreased. Stomach mucous membrane of animals taken cogitum
low doses was partially inamed with separate striped ulcers, the average
stomach weight was three times lower than that of the intact animals.
Cogitum average doses exerted more pronounced gastro protective effect
preserving the mucous membrane in a normal state inducing only separate ulcers. Cogitum high doses exerted the most favourable effect: stomach mucous membrane was slightly pink, practically without in
ammation, with single supercial erosions, stomach mass was preserved. For human body cogitum gastro protective dose is equal to three
ampoules daily. Pharmakotherapeutic impact on the quantity and stomach secretion was proportional to cogitum dose. Conclusions: cogitum is
sure to possess a marked gastro protectiveactivity at the experimental steroid-ethanol gastropathy.
Paper No.: 1718

FOCUSED CONFERENCE GROUP: PW30 - IS ENOS STILL A
VALID THERAPEUTIC TARGET? - THE ERA AFTER THE
DISCOVERY OF ENOS UNCOUPLING
DHEA prevents pulmonary arterial hypertension in a rodent model
of pulmonary hypertension in infants
Jean-Pierre Savineau, T Ducret, J-F Quignard, G Simon, R Marthan,
E Dumas de la Roque
University of Bordeaux, INSERM U 885, Bordeaux, France
Pulmonary arterial hypertension (PAH) is a fatal disease which affects
both adults and newborn infants. We previously showed that dehydroepiandrosterone (DHEA), a steroide hormone, prevents and reverses pulmonary hypertension in an animal model of adult chronically hypoxic rat
(Bonnet et al, PNAS, 2003, 100: 9488-9493). However, no similar study
has been performed in newborn or young animals. We thus investigated
the effect of dehydroepiandrosterone (DHEA) in a model of young rat
suffering from PAH. Two groups (control and DHEA) of 2 weeks old
animals were maintained for 3 weeks in hypobaric chamber to induce
PAH. We observed that DHEA treatment (30 mg/kg every alternate day)
reduces signicantly the mean PA pressure (determined by right cardiac
catheterization), PA reModelling (evaluated by histology) and although
with a less efciency, right cardiac hypertrophy (measured by echography and Fulton index). At the level of the pulmonary artery smooth muscle cell (PASMC), the effect of DHEA appears to involve the following
multiple signalling pathways. DHEA stimulated activity and expression
of the large conductance Ca2 + -activated potassium channel (BKCa) but
inhibited T-type voltage dependant calcium channels as evidence with
the patch clamp technique. It also inhibited serotonin-induced contraction
and had a direct vasorelaxant action on KCl-precontracted vessels.
564
Furthermore, DHEA inhibited PASMC proliferation in vitro. All together
these results indicate that DHEA prevents PAH in newborn rats and suggest that DHEA could be clinically assessed for the management of PAH
in infants.
Paper No.: 3379

CA2 + -DEPENDENT MECHANISM FOR HYDROGEN
PEROXIDE-INDUCED APOPTOSIS IN PANCREATIC B-CELLS
cation between different cell types. Methods: We measured mRNA and

protein expression of Cx36 and Cx43 in rat hippocampus during epileptogenesis in kindling model of epilepsy. The mRNA and protein level
were measured by RT-PCR and western blot, respectively. Results: The
mRNA and protein expression of Cx36 were up-regulated during acquisition of focal seizures and returned to basal levels during development
of generalized seizures, while those of Cx43 did not change during kindling. Conclusion: The results indicate that hippocampal gap junctions
composed of Cx36, has potential role in acquisition of an epileptic focus
during the early kindling stages.
Yurie Sayama, A Noguchi, M Takada, Y Kaneko, T Ishikawa

University of Shizuoka, Graduate School of Pharmaceutical Sciences
Graduate School of Pharmaceutical Sciences, Shizuoka, Japan
Pancreatic b-cell apoptosis induced by reactive oxygen species, including
hydrogen peroxide (H2O2), is known to be one of the causes leading to
functional disorders of b-cells in diabetes. We investigated the mechanism for H2O2-induced b-cell apoptosis, especially focusing on Ca2 + dependent pathways, in the b-cell line INS-1. Apoptosis of INS-1 cells
was induced by the treatment with 100 lM H2O2 for 18 h. The apoptosis
was nearly abolished by BAPTA/AM, a chelator of intracellular Ca2 + ,
by 2-APB, a blocker of inositol 1,4,5-triphosphate (IP3) receptors and
cation channels including store-operated channels (SOCs), and by xestospongin D, a blocker of IP3 receptors, and was partially blocked by
SKF96365, a blocker of SOCs, and by Gd3 + , a non-selective cation
channel blocker. Interestingly, the inhibition of the apoptosis by BAPTA/
AM or 2-APB was markedly reduced when the drug was added 30 min
after the treatment with H2O2. H2O2 induced an elevation of [Ca2 + ]i,
which was composed of two phases, i.e., the initial transient [Ca2 + ]i elevation within 30 min and subsequent slowly developing one. The [Ca2 +
]i elevation, especially the rst phase, was signicantly inhibited by 2APB. On the other hand, H2O2 induced a sustained elevation of mitochondrial Ca2 + concentration, which was signicantly inhibited by 2APB. These results suggest that the initial [Ca2 + ]i transient within
30 min after the addition of 100lM H2O2, which is mediated via IP3
receptors and SOCs, leads to mitochondrial Ca2 + overload, resulting in
b-cell apoptosis.
Paper No.: 854

CHANGES IN HIPPOCAMPAL CONNEXIN 36 MRNA AND
PROTEIN LEVELS DURING ACQUISITION OF SEIZURES IN
THE KINDLING MODEL OF EPILEPTOGENESIS
Paper No.: 2906

CRONO-PHARMACOKINETIC OF IRINOTECAN AFTER
HEPATIC INTRA ARTERIAL PERFUSION IN PATIENTS WITH
METASTATIC COLON CARCINOMA
Francesco Scaglione(1), S Bareggi(1), A Nasisi(2), G Bondiolotti(1),
M Pirovano(2)
(1) University of Milan, Department of Pharmacology, Milan, Italy
(2) S.Carlo Hospital, Milan, Italy
Many studies of biodynamic clearly demonstrates that biophysical and
biochemical processes vary with respect to time of day in a regular and
predictable periodic manner. Some clinical studies reported a safely prole of the nightly administration of irinotecan (CPT-11). Pharmacokinetics and metabolism of CPT-11 are extremely complex. Both CPT-11 and
its metabolite SN-38 are known in an active lactone form and an inactive
carboxylate form. In addition, CPT-11 is subject to extensive metabolic
conversion by both phase I and phase II enzyme systems that may have
circadian variations. The Objective of the study was to evaluate pharmacokinetics of CPT11 and his active metabolite SN38 after hepatica intra
arterial perfusion. serum levels of CPT11 and SN38 were detected,
according to two different time schedules with a 4 hour sinusoidal infusion starting at 10 am or 10 pm in two therapeutic cycles. Blood levels
were collected 1, 2,3,4,5,6,7,8,13,17,28 hours post administration. The
determination of irinotecan (CPT-11) and its active metabolite SN-38 in
human plasma will be performed following the HPLC. Six patients were
enrolled . AUC of CPT11 was 6956 2765 and 5822 734 ng/ml*h
respectively in the day and in the night (p > 0.05), the AUC of SN38
was 2570 1524 and 2026 457 ng/ml*h respectively in the day and
in the night (p > 0.05). No statistical differences were found also in
Cmax or T1/2. The safely prole of the nightly administration of CPT-11
reported in some clinical studies is not probably related to a different PK
prole of the drug.
Mohammad Sayyah(1), S Beheshti(1,2), S Majzoob Zanjani(3),

M Golkar(4), H Sepehri(1), J Babaie(4), B Vaziri(5)
(1) Pasteur Institute of Iran, Department of Physiology and Pharmacology, Tehran,Iran
(2) Tehran University, University College of Science, Department of
Animal Biology, School of Biology, Tehran, Iran
(3) Neda Pharmaceutical Co., Quality Assurance Department, Tehran,
Iran
(4) Pasteur Institute of Iran, Department of Parasitology, Tehran, Iran
(5) Pasteur Institute of Iran, Department of Biothechnology, Tehran, Iran
Introduction: Identication of key molecular changes occurring during
epileptogenesis, provides better understanding of epilepsy and helps to
develop strategies for preventing it. Gap junctional communication is
thought to be involved in epileptogenesis. This communication can be
affected by changes in expression of gap junctional subunits called connexins (Cx). One of the main brain regions involved in epileptogenesis is
the hippocampus in which there is a network of gap junctional communi-
Paper No.: 711

EFFECT OF DICLOFENAC ON COMPOUND ACTION
POTENTIAL
Jelena Scekic, M Drecun, M Bajagic
University of Montenegro Medical School, Department of Physiology,
Podgorica, Montenegro
Introduction: Possible antinociceptive effect of diclofenac is an interesting topic for the research. Study is designed to examine effect of diclofenac on frog sciatic nerve compound action potential (CAP) parameters.
Materials: CAP parameters were measured using extracellular recording
565
techniques, digital storage oscilloscope, Uni-T UT2025C. 24 frog sciatic
nerves were devided into 3 groups, 8 nerves in each group. Nerves were
kept in Ringer solution for 2 hours and control measurements were
taken. Than, nerves were bath for 20 min. in solutions: group I: Ringer
solution-control group, group II: 0.075% diclofenac solution and group
III: 0.25% diclofenac solution. CAP parameters were measured again.
Data were collected and analysed using the statistical computer programe
GrapPad Prism 5.0. Results: After 20 min. incubation in 0.25% diclofenac solution, there was no CAP in group III. In group II diclofenac
decreased amplitude of CAP for 64.82 2.238%, increased latency time
of the onset of the CAP and latency time of the peak of the CAP for
12.71 1.543% and 20.55 2.553%. Data were statistically signicant
in relation to control group for P < 0.01. Depolarisation and half-repolarisation time were increase for 40.46 7.358% and 45.60 10.89%.
Data were statistically signicant in relation to control group for
P < 0.05. There is no statistical signicance between depolarisation and
half-repolarisation time in the group II. Conclusion: Study results showed
that diclofenac statistically signicant decreased amplitude of CAP and
increased nerve conduction time and depolarisation and half-repolarisation time. Results suggest that diclofenac potantially has antinociceptive
effect.
Paper No.: 1917

HEALTH AND DISEASE
NO SYNTHASE AND THROMBOSPONDIN-1 IN THE INITIATION OF FLOW-RELATED REMODELLING OF RAT MESENTERIC RESISTANCE ARTERIES
Paul Schiffers(1), P Lemkens(1), G Boari(2), G Fazzi(1), G Janssen(1), J
Murphy-Ullrich(3), J De Mey(1)
(1) University of Maastricht, Cardiovascular Research Institute, Department of Pharmacology and Toxicology, Maastricht, The Netherlands
(2) University of Brescia, Department of Internal Medicine, Brescia,
Italy
(3) University of Alabama at Birmingham, Division of Molecular and
Cellular Pathology, Department of Pathology, Birmingham, AL, USA
We tested the hypothesis that thrombospondin-1 (TSP1) participates in
the initiation of the reModelling of small muscular arteries in response to
altered blood ow. We measured changes in gene and protein expression
in mesenteric resistance arteries (MA) of 6 week old WKY and SHR rats
exposed for 24 (mRNA), 32 (protein) and 40 hours (protein) to either
increased (HF) or reduced blood ow (LF) in vivo. In both HF and LF
of WKY, the mRNA expression of endothelial nitric oxide synthase
(eNOS), the alpha1 subunit of soluble guanylyl cyclase (sGCalpha1) and
protein kinase G1beta (PKG1beta) were signicantly reduced, whereas
mRNA of TSP1 and tenascin-C (TNC) were markedly increased. In MA
of young SHR, similar results were obtained except that eNOS mRNA
was not reduced in LF arteries. Protein expression of TSP1 was signicantly increased, but only in LF of young WKY and SHR after 32 hours
of ow intervention. Exposure of MA of 12 week old SHR to lmol/L
hep I (peptide fragment of the N-terminal domain of TSP1) in vitro
resulted in a rapid increase in lumen diameter 12 1 + 2% after 3 days)
without alteration in vascular reactivity, media surface area, or cell number. These are the rst observations of reduced gene expression of
eNOS/cGC/PKG and increased expression of TSP1 at the initiation of
arterial remodelling irrespective of its outward or inward outcome. However, protein expression of TSP1 is only upregulated in LF of WKY and
SHR. Furthermore, hep I rapidly and directly reversed pathological
inward arterial remodelling.
Paper No.: 2687

PHARMACOLOGICAL CHARACTERISATION OF AN
ALLOSTERIC SMALL-MOLECULE AGONIST FOR THE
CHEMOKINE RECEPTOR CXCR3
Danny Scholten, M Canals, S de Munnik, M Wijtmans, I de Esch,
M Smit, R Leurs
VU University Amsterdam, Leiden/Amsterdam Center for Drug
Research, Division of Medicinal Chemistry, Amsterdam,
The Netherlands
The chemokine receptor CXCR3 is a G protein-coupled receptor found
predominantly in activated T cells. CXCR3 is activated by three endogenous ligands: CXCL9, CXCL10 and CXCL11. Both CXCR3 and its
chemokines are upregulated in a variety of inammatory diseases such
as rheumatoid arthritis. Moreover, the CXCR3 binding chemokines have
been shown to possess antitumour activity, attributed to the recruitment
of leukocytes. Recently, we have characterized several classes of nonpeptidergic compounds for CXCR3 (e.g. AMG-487, NBI-74330, and
TAK-779) (Verzijl et al, J Pharmacol Exp Ther 2008; 325: 544-55) In
addition, the structure of a small-molecule agonist has been reported in
literature (Stroke et al, Biochem Biophys Res Commun 2006; 349: 2218) and has been synthesized in our lab. Here we provide a detailed characterisation of the pharmacology of this novel small agonist
(VUF10661). This includes several classical G protein-dependent pathway endpoints such as [35S]GTPcS binding, and intracellular cAMP
accumulation using a novel cAMP BRET-based biosensor. In addition,
we have investigated G protein-independent signaling using a b-arrestin
BRET recruitment assay. In summary, we show that VUF10661 activates
both G protein dependent and independent signaling via the CXCR3
receptor. Therefore, VUF10661 represents the rst selective small-molecule agonist for this attractive drug target.
Paper No.: 1097

FOCUSED CONFERENCE GROUP: P19 - GENERAL SESSION HAEMATOLOGY
MODULATED HUMAN MATERNAL AND PREMATURE
NEONATAL ERYTHROCYTE MEMBRANE ENZYME
ACTIVITIES: IN VITRO RESTORATION BY L-CARNITINE
K Schulpis(1), G Vlachos(2), A Antsaklis(2), Charis Liapi(3),
V Stolakis(3,4), A Zarros(3,4), S Tsakiris(4)
(1) Institute of Child Health, Aghia Sophia Childrens Hospital,
Athens, Greece
(2) National & Kapodistrian University of Athens Medical School, 1st
Department of Obstetrics and Gynecology, Athens, Greece
(3) National & Kapodistrian University of Athens Medical School,
Department of Pharmacology, Athens, Greece
Department of Physiology, Athens, Greece
Free radical production and L-carnitine (Carn) are implicated with the
activities of the erythrocyte membrane enzymes acetylcholinesterase
(AChE), (Na+,K+)-ATPase and Mg2 + -ATPase. The aim of this study
was to investigate the effect of the mode of delivery on the above-mentioned maternal and premature neonatal erythrocyte membrane enzyme
activities, as well as to investigate the in vitro effect of Carn (25 lL) on
these activities. Mothers that underwent vaginal delivery (group A,
n = 12), mothers that underwent scheduled caesarean section-assisted
delivery (group B, n = 14) and control age-matched females (group C,
n = 20) were used as erythrocyte donors. All mothers (groups A and B)
gave birth to neonates that were premature, while blood was obtained
pre- and post-delivery as well as from the umbilical cord of their neonates. The acquired erythrocyte membrane enzyme activities were mea-
566
sured spectrophotometrically. Serum total antioxidant status (TAS) was
also determined with a commercial kit. All parameters shared similar levels pre-delivery (in both groups A and B), whereas the activities of
AChE and Na+,K+-ATPase were found increased post-delivery in group
A only. TAS was found decreased post-delivery (in a greater extent in
group A than in group B). Neonates exhibited low but similar (among
the two groups) levels of the above enzyme activities. In vitro incubation
for 1h with Carn restored the above-mentioned modulated activities. In
conclusion, free radical production may modulate the studied maternal
erythrocyte membrane enzyme activities after vaginal delivery of premature neonates, and Carn administration might participate in the restoration
of these activities.
Paper No.: 2973

DISCOVERY
THE ORGANIC SOLUTE TRANSPORTER OSTA-OSTB - A
POTENTIAL DETERMINANT OF DRUG ABSORPTION
Ute Schwarz(1), H Meyer zu Schwabedissen(3), B Urquhart(2),
R Tirona(1,2), R Kim(1,2)
(1) University of Western Ontario, Division of Clinical Pharmacology,
Department of Medicine, London, Ontario, Canada
(3) Ernst Moritz Arndt University of Greifswald, Institute of
Pharmacology,Greifswald, Germany
Introduction: The bidirectional heterodimeric organic solute transporter
OSTa-OSTb has been found to be highly expressed in human ileum and
is recognized for its major role in enterohepatic circulation of bile salts
and sterols, but little is known about its ability to transport xenobiotics.
The objective of this study was to test whether drugs undergo OSTaOSTb transport, to identify potential inhibitors, and to evalulate regional
and interindividudal differences of interstinal OSTa and OSTb expression in humans. Materials/Patients: Transport studies were performed
using transient heterologous expression of OSTa and OSTb in HeLa
cells. Intestinal samples were obtained from healthy subjects undergoing
diagnostic esophagogastro-duodenoscopy or colonoscopy approved by
the Health Sciences Research Ethics Board at the University of Western
Ontario and analyzed by relative quantication using RT PCR and Western blotting. Results: Our ndings demonstrate that the chemotherapeutic docetaxel, the lipid-lowering drugs rosuvastatin and atorvastatin, and
the anti-inammatory agent sulfasalazine are novel substrates of OSTaOSTb, whereas the cholesterol absorption inhibitor ezetimibe, probenecid, as well as sulfasalazine inhibited OSTa-OSTb - mediated taurocholate uptake. We furthermore conmed that OSTa and OSTb are highly
expressed in human ileum and colon, and readily detectable in duodenum with considerable variability among subjects. Conclusion: This
study identied novel drug substrates and inhibitors of OSTa-OSTb, and
suggests OSTa-OSTb as an important intestinal transporter of relevance
to drug dispostion in humans.
Paper No.: 1259

HEALTH AND DISEASE
DISTINCT NADPH-CYTOCHROME P450 REDUCTASE DEPENDENCE OF HEME OXYGENASE-1 AND HEME OXYGENASE-2
Monika Seeanner, JR Kraehling, M Busker, S Behrends
Technical University Braunschweig, Institute of Pharmacology, Toxicology and Clinical Pharmacy, Braunschweig, Germany
P450 reductase (CPR). CPR and heme oxygenases reside in membranes

of the endoplasmicreticulum. A NADPH-mediated interaction between
CPR and HO-1 has been demonstrated by surface plasmon resonance
(Higashimoto et al, JBC 2005; 280:729-737) and uorescence resonance
energy transfer (Wang and de Montellano, JBC2003; 273: 2006920076). To our knowledge no direct interaction has been demonstrated
between CPR and HO-2. We have cloned HO-1, HO-2 and biliverdinreductase (hBVR) from human placenta cDNA and constructed recombinant baculoviruses. A baculovirus encoding CPR was a kind gift by
Dres. Schwarz and Gonzalez. Heme oxygenase activity was determined
in the presence of hBVR by measuring the absorbance of bilirubin.
HO-1 activity was about 10 times higher in the presence of CPR. In
contrast, CPR had no effect on HO-2 activity under the same experimental conditions. HO-1 and HO-2 share high sequence homology in
their core domains but their sequences diverge carboxy terminally
where HO-2contains two conserved heme regulatory motifs. Mutation
of these motifs (C265S/C282S-HO-2 double mutant) did not change
the CPR-independence of HO-2. We conclude that HO-1 and HO-2
differ in their ability to interact with anendogenous CPR-like reducing
system present in Sf9 cells. We hypothesize that similar differences exist
with respect to the interaction of HO-1 and HO-2 with reducing systems
in human cells.
Paper No.: 3179

BLUNTED REWARD SYSTEM ACTIVATION TO EROTIC
STIMULI UNDER PAROXETINE AS COMPARED TO
BUPROPION OR PLACEBO IN HEALTHY SUBJECTS
Angela Seeringer(1), B Abler(2), A Hartmann(2), G Groen(2),
M Walter(3), J Kirchheiner(1)
(1) University of Ulm, Institute of Pharmacology of Natural Products &
Clinical Pharmacology, Ulm, Germany
(2) University of Ulm, Department of Psychiatry, Ulm, Germany
(3) Otto von Guericke-Universitat, Department of Psychiatry,
Magdeburg, Germany
Sexual dysfunction is a common side effect of serotonin reuptake inhibitors like paroxetine when taken for the treatment of depression. As an
alternative treatment Bupropion, a norepinephrine and dopamine reuptake inhibitor, is recommended as no adverse effects concerning sexual
arousal and libido were known. In a functional magnetic resonance imaging experiment we investigated the neural bases of paroxetine-related
sexual dysfunction compared to bupropion and placebo in a double-blind
within-subjects design. We included 18 healthy, heterosexual, male subjects aged 23-34 years without any history of psychiatric disorder or sexual dysfunction. Each subject was scanned three times while watching
video clips of erotic and non-erotic content, once on paroxetine, once on
bupropion and once on placebo. Subjects took 20 mg of paroxetine,
150 mg of bupropion and placebo for 7 days each to reach steady state
conditions. The MRI scans took place on the 7th day, with three weeks
between each two scans in an individual. EPI images were acquired with
a 3 Tesla Siemens Allegra Scanner. Data were analyzed event related
using SPM5. Erotic vs. non-erotic video clips elicited activation in the
hypothalamus and the reward system comprising nucleus accumbens and
ventral tegmental in all three conditions. However, activation of rewardrelated brain areas, particularly the dopaminergic midbrain was signicantly reduced under paroxetine as compared to placebo while activation
under bupropion remained unimpaired. Conclusively, paroxetine reduced
the property of erotic stimulation as a primary reward. Our data may
help understanding the neural mechanisms of SSRI-related sexual
dysfunction.
Unlike nitric oxide synthase which has intrinsic reductase activity, hemeoxygenase (HO) is thought to require the activity of NADPH-cytochrome
567
Paper No.: 530
ANALGESICS
EXPERIMENTAL RESEARCH ON THE INTERACTIONS OF
DIETHYLAMINE WITH OPIOID AND NON-OPIOID
ANALGESICS
ity of Hp extract in rat platelets that express PAR4. Like a PAR4-AP, Hp

extract induced platelet aggregation in platelet rich plasma, an effect
unaffected by NM, whereas Hp extract did not mimic the aggregating
activity of the PAR4-AP in washed platelets. Thus the activities of Hp
extract shown in the present study are considered independent of PAR1,
PAR2 and PAR4.
Ana Segarceanu, I Ghita, O Coman, I Fulga

Carol Davila University of Medicine and Pharmacy, Department of Pharmacology & Pharmacy, Bucharest, Romania
Introduction: The aim of the present study was to investigate the interaction of diethylamine, a possible precursor of anandamide, with other
analgesics. Previous tests have proven the analgesic and sedative properties of diethylamine. Materials and methods. The writhing test and
hot-plate test were conducted on groups of 8 or 10 albino male mice
intraperitoneally injected or orally administered with different doses of
dietyhlamine, ibuprofen, acetylsalicylic acid, morphine or saline solution
and tested at different periods of time. Statistical analysis was performed
using one-way Anova and Dunnetts test. Results: At 1 hour after administration, the writhing test ibuprofen (60 mg/kg) and acetylsalicylic acid
(40 mg/kg) determined an important increasing of the analgesic effect of
diethylamine from 58% to 91% and from 54% to 76% respectively.
Acetylsalicilic acid (25 mg/kg) didnt inuence the analgesic effect of diethylamine. In the hot-plate test, ibuprofen and acetylsalicylic acid in
small doses didnt inuence the analgesic effect of diethylamine but the
same analgesics in higher doses reduced the latency determined by diethylamine with 15.54% and 16.40% respectively. At 2 hours after administration, in the hot-plate test, a subanalgesic dose of morphine increased
the analgesic effect of diethylamine from 1.57% to 30.10%. Conclusion:
Ibuprofen and acetylsalicylic acid increased the intensity of the analgesic
effect of diethylamine in writhing test but not in the hot-plate test. The
association of morphine with diethylamine has a strong analgesic effect
but the mechanisms involved are not clearly established.
Keywords: diethylamine, analgesic, ibuprofen, acetylsalicylic, morphine.
Paper No.: 1137

DISEASES
ANALYSIS OF VARIOUS BIOLOGICAL RESPONSES INDUCED
BY HELICOBACTER PYLORI EXTRACT: INDEPENDENT OF
PROTEINASE-ACTIVATED RECEPTORS?
Fumiko Sekiguchi, Y Maeda, Y Matsumoto, M Matsunami, H Nishikawa,
A Kawabata
Kinki University School of Pharmacy, Division of Pharmacology and
Proteinase-activated receptors (PARs) contribute to inammatory
responses in various tissues and cells. Helicobacter pylori (Hp), a risk
factor for gastritis, gastric ulcer and cancer, contains diverse proteinases
as virulent factors. Thus, we determined whether Hp extract contains proteinases that activate PARs. Cultured Hp was harvested and fractured
with a sonicator in PBS, and the supernatant was ltered through a
0.20 lm lter. The activity of Hp extract was assessed in distinct cell
lines with high expression of PAR1 (normal rat gastric mucosal epithelial
RGM1 cells) and PAR2 (human alveolar epithelial A549 cells and
human colorectal adenocarcinoma HCT-15 cells). A PAR1-activating
peptide (AP), but not Hp extract, increased prostaglandin E2 (PGE2)
release in RGM1 cells. On the other hand, Hp extract increased release
of PGE2 and interleukin-8 (IL-8) in A549 and HCT-15 cells, respectively,
as a PAR2-AP did. Boiling of Hp extract did not affect the PGE2-releasing activity in A549 cells. Nonetheless, the IL-8-releasing activity in
HCT-15 cells was partially suppressed by boiling of the extract and by a
proteinase inhibitor, nafamostat mesilate (NM). Since HCT-15 cells
expressed functional PAR4 in addition to PAR2, we also tested the activ-
Paper No.: 1022

ELUCIDATION OF THE MECHANISM THAT SULFAPHENAZOLE SUPPRESSES CARDIAC ISCHEMIA-REPERFUSION
INJURY. ANALYSIS USING SULFAPHENAZOLE DERIVATIVES
Masaya Sekine(1), A Hamaguchi(1), Y Kobayashi(2), Y Ishihara(1),
M Nakazawa(3), N Shimamoto(1)
(1) Tokushima Bunri University Faculty of Pharmaceutical Sciences,
Laboratory of Pharmacology, Kagawa, Japan
(2) Tokushima Bunri University Faculty of Pharmaceutical Sciences,
Laboratory of Medicinal Chemistry, Kagawa, Japan
(3) Niigata University Faculty of Medicine, Division of Medical Technology, Department of Health Sciences, Niigata, Japan
We have shown that cardiac ischemia-reperfusion injury is suppressed by
treatment with sulfaphenazole (SPZ) which has potent inhibitory action
of cytochrome P450. Because SPZ is reported to have scavenging action
of reactive oxygen species, the mechanism of cardiac protection by SPZ
is not clear. Thus, the aim of this study is to reveal which action is
responsible for an alleviating effect of SPZ on cardiac ischemia-reperfusion injury using SPZ derivatives. We synthesized dichloro-SPZ (2ClSPZ) and acetyl-SPZ (Ac-SPZ) according to the report. While inhibitory
action of 2Cl-SPZ on cardiac P450 is three times potent than that of
SPZ, Ac-SPZ showed almost no inhibitory effect on cardiac P450.
Hydrogen peroxide, superoxide anion and hydroxyl radical were scarcely
scavenged by three compounds. Treatment with 2Cl-SPZ is more effective on cardiac injury induced by ischemia-reperfusion than that with
SPZ. Ac-SPZ had no suppressive effects on reperfusion injury. These
results suggest that suppressive effects of SPZ on cardiac reperfusion
injury are attributed to inhibitory action of cardiac P450.
Paper No.: 759

INTERACTION BETWEEN NITRIC OXIDE AND
DOPAMINERGIC TRANSMISSION IN THE PERIPHERAL
CONTROL OF PENILE ERECTION
Amira Senbel
University of Alexandria Faculty of Pharmacy, Department of Pharmacology,Alexandria, Egypt
Since the peripheral role of dopamine in the mediation of penile erection
was recently identied, its potential role in the modulation of NO action
and whether D1 or D2 receptors are involved in this potential modulation
was investigated. The isolated rabbit corpus cavernosum and measurement of intracavernosal pressure in the anesthetized rat model were
used.The selective D1 antagonist SCH23390 but not the D2 antagonist
sulpiride reduced the inhibitory effect of L-NNA and the potentiatory
effect of sildenal on erectile responses. L-NNA did not change the
inhibitory effect of SCH23390 or the potentiatory effect of apomorphine
but enhanced the effect of high dose fenoldopam on intracavernosal pressure. Similarly, fenoldopam produced 47.30 6.89% potentiation of
relaxation of corpus cavernosum in absence of L-NNA and 80 9.34%
potentiation in its presence at 3 Hz. The effect of sildenal was greatly
568
reduced by pretreatment with SCH23390 or sulpiride and completely
abolished by their combination. This study supports the role played by
D1 receptors peripherally in the control of penile erection. Absence of
NO may potentiate the effect of D1 receptor on erection. Activation of
D1 receptors may be involved in the synthesis of NO in the corpus cavernosum or activate the role of NO in erection.
Paper No.: 760

DISEASES
NOVEL SELECTIVE COX-2 INHIBITOR PYRAZOLE
DERIVATIVE PROVEN EFFECTIVE AS
ANTI-INFLAMMATORY AND ANALGESIC DRUG
Amira Senbel(1), MM Mohy El-Din(1), AA Bistawros(1), A El-Mallah(1),
NA Nour El-Din(1), AA Bekhit(2), H Abdel Razek(2)
(1) University of Alexandria Faculty of Pharmacy, Department of
Pharmacology, Alexandria, Egypt
(2) University of Alexandria Faculty of Pharmacy, Department of
Pharmaceutical Chemistry, Alexandria, Egypt
The introduction of new selective COX-2 inhibitors with high efcacy
and enhanced safety prole would be a great achievement in the development of anti-inammatory drugs. The current study was designed to
screen and assess the anti-inammatory and analgesic activities as well
as some of the expected side effects of some pyrazole derivatives newly
synthesized as potential COX-2 inhibitors in Faculty of Pharmacy, Alexandria University, compared to indomethacin and celecoxib. Twelve
compounds were screened for their anti-inammatory activity using carrageenan-induced paw edema and cotton pellet granuloma tests. Based
on their apparent anti-inammatory activity, 4 drugs with different substitutions were selected for evaluation of their analgesic activity using the
formalin-induced hyperalgesia and hot-plate tests. Compound B2 ((4-(3(4-Methylphenyl)-4-cyano-1H-pyrazol-1-yl)benzenesulfonamide))showed
very promising results. In the single-dose and sub-chronic toxicity studies, compound B2 showed no ulcerogenic effect and produced minimal
effects on renal function. Furthermore, compound B2 was less potent
inhibitor of COX-2 in-vitro than celecoxib, which may indicate lower
potential cardiovascular toxicity. It is concluded that compound B2
seems a promising and safe option for the management of chronic
inammatory conditions. This study recommends more investigation of
the therapeutic effects and toxicity prole of this compound including its
cardiovascular toxicity.
Paper No.: 1958

TRANSIENT NEUROLOGICAL DISTURBANCE IN A PATIENT
WITH LAMOTRIGINE OVERDOSE
of lamotrigine. After a generalised tonic-clonic seizure and a period of

drowsiness, she rapidly became more rousable, but developed hypertonia, clonus and hyperreexia. She also displayed marked dysmetria and
dysdiadochokinesis, more prominent on her left side. All these features
subsequently resolved within 36 hours of admission and she was safely
discharged. Discussion: This case adds to growing evidence that lamotrigine overdose can result in severe neurological impairment, but also
lends support to the notion that such features are often transient. Awareness of this possibility can prevent undue concern, and can reassure clinicians that a period of observation may be a suitable initial approach to
management in such patients.
Paper No.: 866

DISEASE AND THERAPY
EFFECT OF MEBUDIPINE AND DIBUDIPINE TWO NEW
CALCIUM CHANNEL BLOCKERS IN COMPARISON TO
NIFEDIPINE ON VASCULAR FLOW OF ISOLATED KIDNEY
FROM DIABETIC RAT
Lili Sepehr Ara(1), Massoud Mahmoudian(2)
(1) Islamic Azad University, Kazeroon Branch, Department of Biology,
Kazeroon, Iran
(2) Iran University of Medical Sciences, Firoozgar Hospital, Clinical
Research Development Center, Department of Pharmacology, Tehran,
Iran
Calcium channel blockers (CCBs) are used in the treatment of hypertension because of their ability to decrease peripheral vascular resistance.
These drugs also preserve or improve renal function in patients with
essential hypertensive renal disease or diabetic renal disease. Different
classes of CCBs have been introduced. Among these classes dihydropyridine (DHP) derivatives are widely used because of their vasodilating
activity and weak cardiodepressant action. The adverse effect of these
drugs (e. g. negative inotropism, heart blockade, reex sympathetic activation) are agent dependent, not class-related. Thus extensive researches
have been carried out to synthesize new DHPs with improved properties.
Mebudipine and dibudipine are two new 1,4- DHP calcium channel
blockers that recently have been synthesized. In this study we investigated the effect of mebudipine and dibudipine on renal blood ow in the
isolated perfused kidneys from diabetic rats and compared their potencies
to nifedipine. It is concluded that phenylephrine (PE)-induced increases
in perfusion pressure inhibited signicantly in presence of mebudipine
and dibudipine (1, 5, 10 lM). Nifedipine (5, 10 lM) also attenuated
increases in perfusion pressure arising from PE. Calculated EC50s for
inhibition were 1.94 0.43, 2.18 0.47 and 4.38 0.08 for mebudipine, dibudipine and nifedipine respectively. Based on the EC50 values,
mebudipine and dibudipine are signicantly more potent in inhibiting
PE-induced increments in perfusion pressure in isolated kidneys from
diabetic rats as compared to nifedipine.
Keywords: Mebudipine, Dibudipine, Isolated Kidney
Anshuman Sengupta, O Martin, G Lane, P Patel, S Abbasi

Mid Yorkshire Hospitals NHS Trust and Pontefract General Inrmary,
Department of Cardiology, Pontefract, UK
Introduction: Lamotrigine is commonly used in young women with epilepsy; however, this is an age group at particularly high risk of deliberate
self-harm through drug overdose. Awareness of the adverse effects of
lamotrigine toxicity is therefore of importance to doctors involved in
acute medical care. Limited experience of lamotrigine overdose is cited
on Toxbase, the resource utilised by most doctors in the United Kingdom. Cardiovascular sequelae such as PR, QRS and QT prolongation are
well described, as is acute kidney injury secondary to rhabdomyolysis. A
variety of temporary, non-specic neurological complaints are reported,
but convincing neurological features such as pyramidal and cerebellar
signs are rarely described. Patient: A 26 year-old epileptic woman presented to the emergency department after deliberately ingesting one gram
Paper No.: 1332

PLASMA SEROTONIN LEVELS AND INFLUENCE OF
GRANISETRON ON PHARMACOKINETICS AND
PHARMACODYNAMICS OF OMEPRAZOLE IN HEALTHY
SUBJECTS AND PATIENTS WITH PEPTIC ULCER DISEASE
Svetlana Serebrova(1), A Starodubtzev(1), S Kondratenko(2),
G Belyakova(1), N Berdnikova(2)
(1) Institute of Clinical Pharmacology, Analytical Department, Moscow,
Russian Federation
(2) Moscow Medical Academy, Moscow, Russian Federation
569
Introduction: Serotonin plays a role in neurotransmission and possible in
regulation of P-glycoprotein activity. We studied plasma serotonin levels
and inuence of granisetron, a selective antagonist of the 5-hydroxytryptamine3 receptors, on bioavailability and inhibitory activity of omeprazole in healthy subjects and peptic ulcer patients. Materials/Patients: 12
healthy subjects and 12 peptic ulcer patients (2 weeks after treatment)
took part in two pharmacokinetic/pH-monitoring studies of omeprazole
(OM) with two-week interval. One of the days all subjects received granisetron (GR) 1mg 1h before 20mg OM. Laboratory tests were performed by HPLC and EIA. Results: Concentrations of plasma serotonin
were 9.18 8.03ng/ml in volunteers, 137.21ng/ml in patients. Cmax,
Tmax, AUC0-t, Clt, T1/2, MRT, Vd of OM in volunteers were correspondingly 639.8 98.5ng/ml, 1.8 0.1h, 946.6 77.1ngh/ml,
20,8 1,5 l/h, 1.3 0,1h, 2.6 0.2h, 41.4 5.2 l without GR;
913.3 126.7ng/ml, 1.5 0.1h, 1379.9 158.0ngh/ml (P < 0.05),
15.6 1.9 l/h, 1.0 0.1h, 2.3 0,1h, 23.0 5.0 l (P < 0.05),
f = 145.8%, f=142.7% with GR. These parameters in patients were correspondingly 299.0 64.7ng/ml, 2.3 0.3h, 578.5 26.5ngh/ml,
33,0 2,5 l/h, 1.1 0,1h, 2.7 0.8h, 52.4 2.3 l without GR;
171.3 21.8ng/ml (P < 0.05), 1.8 0.2h (P < 0.05), 475.6 64.0ngh/
ml, 44.7 7.5 l/h, 1.3 0,1h, 3.3 0.3h, 87.9 18.9 l (P < 0.05),
f = 82.2%, f = 57.3% with GR. There were signicant differences
between patients and volunteers (without GR) in Tmax, Vd (P < 0.05),
AUC0-, Clt (P < 0.01), Cmax (P < 0.001). f = 61.1%, f = 46.7%.
The time of intragastric pH3 was >24h (with and without GR) in volunteers, 6.2 1.1h without GR, 3.1 1.2h (P < 0.001) with GR in
patients. Conclusion: Bioavailability and inhibitory activity of omeprazole are higher in healthy subjects than in peptic ulcer patients. Granisetron promotes absorption of omeprazole in healthy subjects and reduces
it in patients
Paper No.: 1491

MULTITARGET EFFECTS OF ANXIOLYTIC AFOBAZOL
S Seredenin, Mikhail Voronin, M Yarkova
Zakusov Institute of Pharmacology RAMS, Department of Pharmacogenetics, Moscow, Russian, Federation
Introduction: Afobazol (5-etoxy-2[2-(morpholino)ethylthio]benzimidazole dihydrochloride) was registered in Russia in 2005 as a selective
anxiolytic. In preclinical studies it was revealed the ability of afobazol to
prevent the stress-induced reduction of benzodiazepine binding. Receptor
binding assays in vitro (Cerep, France) showed that afobazol interacts
with MT1 (Ki=1,6*10-5 M), MT3 (Ki=9,7*10-7 M), sigma 1
(Ki=5,9*10-6 M) receptors and MAO-A (Ki=3,6*10-6 M). These results
initiated the searching of the new pharmacological effects of afobazol.
Methods: Western blotting, radiometric assay, experimental models of
global ischemia and intracerebral posttraumatic haemotoma, behavioral
models of depression. Results: In hippocampal neuron cell culture HT-22
afobazol produced protective effect in the models of oxidative stress and
glutamate toxicity at the concentration of 10-8 M. In experimental models of ischemic and hemorrhagic stroke afobazol at the dose range 0.15.0 mg/kg, after course administration starting 6-24h after operation, prevented the animal death, declined the neurological decits score and
improved the recovery of cognitive functions. Afobazol (1 mM) in vitro
inhibited mitochondrial MAO-A activity in rats brain and liver (IC50
0,36 and 0,43 mM correspondingly). In forced swimming and Nomura
tests afobazol (5 mg/kg, i.p.) elicited an antidepressant response similar
to amitriptyline (10 mg/kg, i.p.) effect. Conclusion: Results obtained in
vitro and in vivo studies correspond to receptor activity of afobazol and
provide evidence to extend the indications for use in clinical practice.
Paper No.: 1743

ORAL ANTIBODIES TO S100 PROTEIN IN ATTENTION
DEFICIT AND HYPERACTIVITY DISORDER:
FIRST CLINICAL TRIAL IN CHILDREN
Svetlana Sergeeva, JL Dugina, IA Kheifets, OI Epstein
Materia Medica Holding Company, Department of Research & Development, Moscow, Russian Federation
Tenoten, oral antibodies to S100 protein, were previously found effective
in the treatment of anxiety disorders in adults. Tenoten for children (paediatric formulation) is a promising option for the treatment of attention
decit and hyperactivity disorder (ADHD). In a double-blind placebocontrolled study 50 children (6-12 years) met DSM-IV ADHD criteria
with CGI-ADHD-Severity mild/moderate and ADHDRS-IV-Parent:Inv>22 points (mean baseline in tenoten for children and placebo
groups were 34.2 1.39 and 33.6 1.26 respectively) were enrolled.
The study is a part of currently ongoing multicenter trial. Patients were
randomized to receive either tenoten for children (2 tablets BID, n = 25)
or placebo (n = 25) for 12 weeks. ADHD symptoms were assessed by
ADHDRS-IV-Parent:Inv, CPRS-R:S and CGI-ADHD-Severity. According to both parents and investigators assessment tenoten for children
notably decreased ADHD symptoms: ADHDRS-IV-Parent:Inv total score
reduced by 13.5 points, hyperactivity by 6.7 points and inattention by
6.8 points (5.4, 2.3 and 3.2 in placebo); CPRS-R:S total score reduced
by 12.8 points, hyperactivity by 3.6 points and inattention by 6.1 points
(4.8, 1.8 and 1.7 in placebo). ADHD severity reduced in 9 patients
received TC and 3 patients received placebo. No adverse effects were
reported in both groups, including absence of negative effects on thyroid
gland and cardiovascular system. In summary, the study demonstrated
good clinical efcacy and safety of TC in children with ADHD.
Paper No.: 796

AMPHOTERICIN B MOLECULAR ORGANIZATION AND THE
PARTICLE SIZE AS ESSENTIAL FACTORS TO REDUCE ITS
TOXICITY BY HEMOLYSIS
Dollores R Serrano Lopez, HKR Saldana, JJT Duran, PB Papantonakis,
ST Duran
Complutense University, Department of Pharmacy & Pharmaceutical
Technology, Madrid, Spain
Amphotericin B (AmB) not only possesses broad antimycotic activity,
but also as a highly effective antiparasitic agent. However, its usefulness
is limited due to severe nephrotoxicity and haemolytic adverse effects. In
water, due to low solubility, AmB molecular can adopt several different
aggregation states. In this study it has been compared the toxicity by
haemolysis of several formulations such as Fungizone, dimer (DAmB) and polyaggregate (P-AmB). This last one was subjected to different centrifugation cycles to collect supernatants samples. The AmB
disposition in Fungizone, analized by spectrophotometry, showed the
same spectrum of the experimental D- AmB. However, P-AmB spectrum
was different to the other studied AmB aggregation states. All the supernatants collected had the same spectrum prole of P-AmB and they
differ in their absorption intensity, which is related to their concentration.
In the DLS size determination, decreases in mass have been observed at
each centrifugation cycle; along with a reduction in hydrodynamic mean
size. The haemolysis test, from human erythrocytes, showed D-AmB
was the formulation of highest haemolytic effects; furthermore the haemolysis increases with the number of centrifugation cycles. In conclusion,
self-aggregation of AmB molecules has an important inuence on the
reduction of the toxicity by haemolysis. In the AmB formulations, a
relationship exists between the size of the aggregated AmB and the
570
haemolytic effect, meaning that, the lower the size of the AmB aggregates the higher the toxicity by hemolysis.
Paper No.: 2278

SELECTIVE DETECTION OF CYP3A4 INHIBITION BY
COMPLEMENTARY PRODUCTS
D Sevior(1), J Hokkanen(2), A Tolonen(2), K Abass(3), L Tursas(3),
Olavi Pelkonen(3), J Ahokas(1)
(1) RMIT University, Department of Toxicology, Melbourne, VIC, Australia
(2) Novamass, Oulu, Finland
(3) Oulu University, Oulu, Finland
Inhibition of cytochrome P450 (CYP3A4) is a known cause of adverse
drug interactions. The results obtained of CYP3A4 inhibition studies
vary depending on the selection of probe substrate. In investigating complementary products for their ability to inhibit CYP3A4 we obtained
divergent results depending on the probe used. The probes used were midazolam, testosterone and omeprazole. Analysis of the probes and their
metabolites (1-OH-midazolam, 6b-OH-testosterone, omeprazole sulfone
and 3-OH-omeprazole) was done by a single LC/MSMS run. Complementary products (inhibitors) were all of commercial quality available to
consumers. Methanolic and aqueous extracts were prepared and diluted
to nominal concentrations of 20, 100 and 500 (ug/ml). Eight extracts
caused signicant inhibition of CYP3A4 (IC50 < 100 ug/ml). All but
one of these was detected with omeprazole (detection with either one or
both metabolites). Midazolam hydroxylation was the least sensitive
method with inhibition of CYP3A4 being detected by this reaction with
only one of the investigated extracts. Variability in the IC50s was seen
between the probe substrates; values ranging from 3.6-91 ug/ml. This work
highlights the importance of using more than one substrate when investigating the inhibition of CYP3A4, as results obtained with only one substrate
may not reect the overall inhibition on CYP3A4. CYP3A4 is the most
abundant CYP in the liver and accounts for approximately 50% of all drug
metabolism (Evans and Relling, 1999;286:487-91).Inhibition of CYP3A4
is clinically important and in vitro screening of complementary products
should be mandatory to predict their potential for drug interactions, care
should be taken in selecting probe substrates.
Paper No.: 1185

ROLE OF CARDIAC MITOCHONDRIAL NITRIC OXIDECGMP PATHWAY ON RAT CARDIOMYOCYTE APOPTOSIS
Kazuhiko Seya(1), K Ono(2), S Fujisawa(3), S Motomura(1), K-I Furukawa(1)
(1) Hirosaki University Graduate School of Medicine, Department of
Pharmacology, Hirosaki, Japan
(2) Akita University Graduate School of Medicine, Department of Physiology, Akita, Japan
(3) Akita University Graduate School of Medicine, Department of Biological Informatics and Experimental Therapeutics, Akita, Japan
Recently, we have reported that mitochondrial nitric oxide (NO)-cGMP
pathway exists in rat ventricular myocytes and regulates cytochrome c
release from mitochondria, a major process in apoptosis. The aim of our
study is to conrm the role of this pathway on cardiomyocyte apoptosis.
Left ventricular mitochondria or primary cultured cardiomyocytes enzymatically isolated from adult male Wistar rats were exposed to high
concentration of extramitochondrial Ca2 + , keeping at 1 lM using
Ca-EGTA buffer, to activate mitochondrial NO synthase. Cytochrome c
released from mitochondria was measured by Western blot. Apoptotic

cells were detected by staining with annexin V-FITC. After up to
30 min, 1 lM Ca2 + accelerated cytochrome c release from mitochondria
time-dependently, which was signicantly inhibited by ODQ (1 lM), an
NO-sensitive guanylyl cyclase inhibitor. Apoptosis was induced in cardiomyocytes exposed to high concentration of cytosolic Ca2 + (1 lM) for
100 min, which was signicantly suppressed by ODQ. Furthermore,
1 lM Ca2 + -induced cytochrome c release from mitochondria was signicantly and dose-dependently inhibited by KT5823 (0.3-3 lM), an
inhibitor of cGMP-dependent protein kinase and genistein (30-300 lM),
a tyrosine kinase inhibitor, but not by cyclosporin A (1 lM), a mitochondrial permeability transition pore inhibitor. These results suggest that cardiac mitochondrial NO-cGMP pathway regulates cardiomyocyte
apoptosis induced by cytochrome c release via the undiscovered mitochondrial kinase pathway.
Paper No.: 1273

ENZYMATIC ANALYSIS OF THE VENOM OF HEMISCORPIUS
LEPTURUS, AN IRANIAN SCORPION
Ramin Seyedian(1), K Euikyung(2), A Zare(3)
(1) Bushehr University of Medical Sciences, Department of Pharmacology, Bushehr, Iran
(2) Gyeoungsang National University, Department of Veterinary Medicine, South Korea
(3) Razi Institute of Poisonous Animals, Iran
Hemiscorpius lepturus envenomation exhibits various pathological
changes in the affected tissues, specially blood cells and skin. Envenomed patients are usually treated under close monitoring with intramuscular injection of pepsin derived Iranian antivenin. Materials and
methods: The proteins of H. lepturus venom and Iranian antivenin were
analyzed by SDS-PAGE separately (15 and 12%, respectively). Immunoblotting was carried out by the presence of Razi Institute polyvalent antivenin and anti horse antibody. Zymographic assays were performed on
12% SDS-PAGEs containing respective substrates (gelatin, casein, hyaluronic acid) with and without 1,10 phenantroline, a metalloprotease
inhibitor. The antivenin was used as an antibody for ELISA assay. The
96-well microtitration plates were coated with Iranian IgG F(ab)2, then
incubated with varying concentrations of H. lepturus venom. Results and
Conclusion: The electrophoretic proles of H. lepturus venom showed at
least 10 different protein components, from 3.5 kDa to 260 kDa. Additionally, Iranian antivenin bands were between 10 and 110 kDa. Zymographic assay showed the gelatinolytic, caseinolytic, and hyaluronidase
activities mainly at around 50-60 kDa, 30-40 kDa, and 40-50 kDa,
respectively that disappeared with 1,10 phenantroline Immunoblot analysis showed several antigenic interactions in the range of 30-160 kDa.
This strong antigen/antibody reaction was also demonstrated through
ELISA experiment. Our experiments showed that MMP enzymes like
gelatinase, caseinase and hyaluronidase are present in this venom. In
spite of our results, more extensive studies on the antivenin efcacy will
be necessary for the validation of its use in envenomed patients.
Paper No.: 2888

EVALUATION OF BIOEQUIVALENCY/BIOAVAILABILITY
TRIALS IN OUR CLINIC
Zafer Sezer, A Inal, N Kucuk, O Soydan, A Erenmemisoglu
Erciyes University Faculty of Medicine, Hakan Cetinsaya Good Clinical
Practice Centre, Kayseri, Turkey
571
The concept of bioequivalency has been adopted by the pharmaceutical
industry and national regulatory authorities throughout the world for over
20 years. Bioequivalency/bioavailability trials have been conducted in
our clinic, Erciyes University Medical Faculty Hakan C
etinsaya Good
Clinical Practice Centre,Turkey, since 2000. We want to evaluate the bioequivalency/bioavailability trials which were carried out in our clinic
until the end of the year 2009. The distribution of these 844 trials according to years was as follows: 6 in 2000,10 in 2001, 54 in 2002, 67 in
2003, 107 in 2004, 151 in 2005, 118 in 2006, 123 in 2007, 120 in 2008,
88 in 2009. If we group these trials, we can see that the largest group is
antibiotics (163 trials, %19.31). Furthermore, the group which contains
antihypertensive drugs consists of 151 trials (%17.89), medicines that act
in the central nervous system (antiseizures, antipsychotics, anidepressants)
consists of 79 trials (%9.36), analgesic medicines consists of 76 trials (%9),
peptic ulcus medicines consists of 65 trials (%7.70), antidiabetic medicines
consists of 50 trials (%5.94), antihyperlipidemic medicines consists of 26
trials (%3.08) and the other groups. Both the number of trials and medicine groups which were studied in our clinic change according to years.
For example bioequivalency/bioavailability trials of antibiotics are less
than previous years. Bioequivalency/bioavailability trials are necessary
for control the safety and efcacy of generic medicines.
Paper No.: 604

LEAD POISONING AFTER GUNSHOT INJURIES IN LOWER
PART OF BODY: A CASE REPORT
The Life Line Fluid Store (LLFS) was established in August 1996. To
provide high quality drugs and surgical items to patients at affordable
prices. The LLFS is a pharmacy store that functions within the public
hospital.Provide good standard medicine and surgical items to patients at
the lowest possible rate compared to market prices, but with a marginal
prot for the private contractor who would run the pharmacy store. The
authority controlling LLFS is the Rajasthan Medicare Relief Society
(RMRS). Through competitive bulk purchasing, RMRS obtains medicines at low cost. The LLFS operates on a contractual basis. The LLFS
is a pharmacy store that function within public hospital through an open
tender, RMRS invites bids from suppliers to procure medicines that
LLFS sells to SMS patients at the procurement prices. Due to the tendering process, the prices of drugs and supplies are much cheaper than retail
prices. A committee within the hospital determines the list of items to be
sold and negotiates the sale price of such items. Instead of a tendering
process, lowest-price certicates from the manufacturers are used, and
promotional offers are passed on the customers. The committee also
selects the contractor who would operate the LLFS drug-store on a 24hour basis. The contract for each LLFS is awarded by the MRS to a contractor for a period of 1-2 years.
Paper No.: 792

GATIFLOXACIN INDUCED HYPOGLYCEMIA IN TYPE 2 DM
PATIENT WITH UTI.-A CASE REPORT
K Sharma, Lokendra K Sharma, K Sharma
Lokendra K Sharma, S Kochar, K Sharma, M Mathur, S Dutta

SMS Medical College, Department of Pharmacology, Jaipur, Rajasthan,
India
Objective: Microcytic hypochromic anemia after gunshot injuries in
lower part ofbody: a case report . Design: Case report. No other heavy
metal toxicity hasgained as much public attention as lead due to its
impact on hematopoietic andneuronal system .We report here a rare case
of chronic lead toxicity resultingin microcytic anemia, due to leeching of
lead into blood. A BSF Jawan receivedgunshot injury during a terrorist
attack. Pieces of the projectile had remainedembedded in the left gluteal
region at the back of trunk for a period of about 3years. He was treated
at base hospital and projectiles were not completelyextracted from body
of the patient. He was later referred to the S.M.S. HospitalJaipur. On the
basis of clinical and blood examination the patient wassuffering from
hypochromic microcytic anemia and showed neurological involvementlike tremor and dyspnoea on exertion. Laboratory blood examination
showedhemoglobin = 7.4 g%; blood lead level 39 microg/dl hematocrit
= 23.3%; serumamylase = 103 U/L, ALT = 228 U/L; AST = 183 U/ L,
elevated bilirubin (direct3.83 mg/dl; indirect 1.62 mg/dl); BUN =
23 mg%; serum creatinine = 0.79 mg%. Anabdominal ultrasound examination was normal. In radiological examination,fragment of projectile
were seen. With greater awareness accidental exposure ofchildren and
adult have decreases consequently now not many cases of acute/chronic
lead poisoning are reported.
Paper No.: 790

COST EFFECTIVE DRUG DISTRIBUTION SYSTEM IN
RAJASTHAN (INDIA)
Lokendra K Sharma(1), KK Sharma(1), M Mathur(1), J Chaudhary(2),
R Mandia(1)
SMS Medical College, Department of Pharmacology, Jaipur, Rajasthan,

India
A patient Ramesh 48 years age with Type 2 DM since last 10 years on
OHA attended OPD clinic in private Hospital, Jaipur with presenting complaint of burning Micturiction since last 4-5 days. Patient investigated with
blood & urine examination, at time of visit his Fasting Blood Sugar was
250 mg/dl, HB-14.3, TLC-11,200, Creatinine 1.0 mg/dl. Urine examination showed microalbuminurea, and numerous pus cells under Microscopic
examination. Based on blood & urine test reports patient diagnoses as a
case of urinary tract infection (UTI) & Tab. Gatioxacin 400mg once a day
was prescribed for 5 days. Same day in night patient came to Hospital with
severe sweating is drowsiness. His blood sugar was 38mg/dl at that time,
Inj. Dextrose 100 ml IV give & after 1 hour patient became comfortable
and his blood sugar was 136 mg/dl. Because of hypoglycemia, oral hypoglycemic agent with hold & Gatioxacin given on next day in morning
after breakfast. Again patient developed similar symptoms of sweating &
uneasiness in afternoon & blood sugar level was 42 mg/dl. Based on these
two episodes of severe Hypoglycemia we replaced the tab. Gatioxacin
with tab Cefuroxime & after replacement patient remained comfortable
during next days and his blood sugar level was within range of 160200 mg/
dl. After 3 days oral hypoglycemic agents restarted along with tab. Cefuroxime. Patient remained comfortable during next days. After this patient
has no episode of hypoglycemia.
Paper No.: 3194

EFFECT OF A-LIPOIC ACID ON LIPID PEROXIDATION AND
MICROSCOPIC STRUCTURES OF LIVER, PANCREAS AND
HEART IN STZ DIABETIC RATS
Manju Sharma, KK Pillai, T Anwer, D Ahmad, WA Anjum, MW Siddique
(1) SMS Medical College, Department of Pharmacology, Jaipur,

Rajasthan, India
(2) Government Medical College, Jhalawar, India
Hamdard University, Faculty of Pharmacy, Department of Pharmacology,

New Delhi, India
572
Several recent studies are indicative of a central role of increased oxidative stress in development of late diabetic complications. In addition to
control of blood sugar, control of oxidative stress offers another avenue
for treatment of disease. a-lipoic acid (a-LA) is a naturally occurring
powerful antioxidant. The study assessed the status of lipid peroxidation
and antioxidants in experimental diabetes and evaluated the efcacy of
a-LA supplementation treatment in STZ diabetic rats. Type 2 diabetes
was induced by a single i.p. injection of STZ (100lg/g). a-LA was
administered alone (25 and 50mg/kg, p.o.) and in combination with gliclazide (25mg/kg, p.o.) to STZ diabetic rats. The oxidative stress was
evaluated by measuring reduced glutathione (GSH) content, tissue LPO
levels and catalase activity. Biochemical observations were substantiated
with histological examination of liver, pancreas and heart. The increase
in blood glucose, LPO levels with reduction in GSH content and
decreased catalase activity were the salient features observed in STZ diabetic rats. a-LA had no signicant glucose lowering effects in STZ diabetic rats. Blood glucose concentration did not indicate an interaction
between a- LA and gliclazide. a-LA reverses the reductive imbalance
that occurs in hyperglycaemia. Degenerative changes of liver, pancreatic
a cells and cardiac tissue in STZ-treated rats was minimized to near normal morphology by administration of a-LA as evident by histopathological examination. This improvement is associated with reduction in lipid
peroxidation and effectively preventing a decrease in antioxidant defense
system by a-LA treatment in a dose dependent manner.
Paper No.: 900

FOCUSED CONFERENCE GROUP: PW14 - TRANSLATING
THE HUMAN GENOME: FROM ORPHAN G-PROTEIN
COUPLED RECEPTORS TO NOVEL THERAPEUTIC
THE IUPHAR DATABASE: TOOLS TO NAVIGATE
PHARMACOLOGICAL SPACE
Joanna L Sharman(1), CP Mpamhanga(1), WA Catterall(2), AP
Davenport(3), RR Neubig(4), E Ohlstein(5), JA Peters(6),
M Spedding(7), A Harmar(1), IUPHAR Committee on Receptor
Nomenclature and Drug Classication
(1) University of Edinburgh, Centre for Cardiovascular Science, Edinburgh, UK
(2) University of Washington, Department of Pharmacology, Seattle,
USA
(3) University of Cambridge, Department of Medicine, Cambridge, UK
(4) University of Michigan, Department of Pharmacology, Ann Arbor,
USA
(5) Life Science Development, LLC and Benchmark Consulting Group,
(6) University of Dundee, Centre for Neuroscience, Dundee, UK
(7) Institut de Recherches Internationales Servier (IRIS), Suresnes,
France
The IUPHAR Committee on Receptor Nomenclature and Drug Classication (NC-IUPHAR) maintains an open access database, IUPHAR-DB
(www.iuphar-db.org), providing detailed, expert-driven annotation of the
primary literature on human and rodent receptor systems. Here we report
on recent improvements which increase the scope and utility of the database. Coverage of receptor targets has increased to 616 genes encoding
the full complement of G protein-coupled receptors, voltage-and ligandgated ion channels and nuclear hormone receptors. The coverage and curation of ligand molecules has expanded to include about 3000 distinct
chemical entities (synthetic organic chemicals, natural products and peptides), for which we provide 2D structures, predicted physico-chemical
properties, synonyms, receptor selectivity data and links to co-crystallised 3D structures in the Protein Data Bank (where available). The database search interface now allows navigation of the ligand-chemical
structure space covered by IUPHAR-DB through text, identity, similarity,
substructure and SMARTS-pattern queries. Current work is focused on
assessing the growing data on natural variation at the genomic and proteomic level that can lead to time-, tissue- individual- and disease-specic
differences in receptor function. These activities include assessing the

evidence for in vivo existence of heteromultimeric complexes and deciding the functional relevance of splice variants and polymorphisms. IUPHAR-DB is an established reference resource for pharmacologists; we
believe the new features will extend its usability and facilitate exploratory research. We thank members of NC-IUPHAR and its subcommittees for their valuable contributions.
We are grateful to the British Pharmacological Society, GlaxoSmithKline, Servier, Novartis, UNESCO, Wyeth, Incyte, Millipore and Abbott
for support.
Paper No.: 1038

LATERAL SOMATOSENSORY CORTEX IN SPONTANEOUS
AND PTZ-INDUCED SPIKE-WAVE DISCHARGES OF THE RAT
EVALUATED BY ETHOSUXIMIDE
Fu-Zen Shaw, K-H Yeh
National Cheng Kung University, Department of Psychology, Tainan,
Taiwan
Generalized absence epilepsy is characterized with bilateral spike-wave
discharges (SWDs), particularly in the frontoparietal cortical region. The
lateral somatosensory cortical (LSC) area recently gains attention in the
generation of SWDs from several aspects of evidence in genetically
absence epileptic rats. To further clarify the role of the LSC in the SWD
generation, two models, i.e., Long-Evans rats with spontaneous SWDs
and Wistar rats with low-dose pentylenetetrazol-induced SWDs, were
used with intracortical ethosuximide or saline administration. In both epileptic rat models, ethosuximide in the LSC signicantly reduced SWD
number, shortened SWD duration, and delayed SWD onset compared to
those received saline. In contrast, ethosuximide in the medial somatosensory cortex revealed little effect. Accordingly, the LSC may be essential
for the development of SWDs.
Paper No.: 921

ER STRESS IS INVOLVED IN HYPERPHOSPHORYLATED
TAU-INDUCED CYTOTOXICITY
Yuxian Shen(1), L Feng(0), A Sun(1), Y Shen(1), S Fang(3), J Li(2)
(1) Key Laboratory of Gene Resource Utilization for Genetic Diseases,
Ministry ofEducation and Anhui Medical University, Hefei, PR China
(2) School of Pharmacy, Anhui Medical University, Hefei, PR China
(3) Medical Biotechnology Center, University of Maryland Biotechnology Institute,USA
Introduction: Accumulating evidence indicates that abnormalities in s
phosphorylation play a critical role in the pathogenesis of Alzheimers
disease. Endoplasmic reticulum (ER) stress has been suggested to be
involved in the pathological processes which result in neuronal death in
AD. However, the potential connection of s abnormalities and ER stress
was not explored. Here, we aimed to nd the relationship between hyperphosphorylated s-induced cytotoxicity and ER stress. Materials: Plasmids
encoding GFP and GFP-tagged full length human s; SH-SY5Y and HEK
293T cell lines; phosphatase inhibitor okadaic acid (OA); antibodies to s,
AT-8, s-5, Bip, Chop, GAPDH and the corresponding secondary antibodies; RNA extraction and reverse transcription kit; plasmids extraction kit.
Results and Conclusion: We found that phosphatase inhibitor OA
induced s phosphorylation recognized by AT-8 antibody in SH-SY5Y
cells. s phosphorylation subsequently decreased the viability of SHSY5Y cells markedly. Meanwhile, OA treatment up-regulated the expression of ER stress markers BiP/GRP78 and Gadd153/CHOP in both
mRNA and protein levels. OA exerted less effect on Gadd153/CHOP
573
than BiP/GRP78. Additionally, over-expression of s in HEK 293T cells
resulted in s phosphorylation and cytotoxicity. Over-expression of s also
remarkably up-regulated BiP/GRP78 expression, but the increase of
Gadd153/CHOP expression was not statistically signicant. These results
suggest that s hyperphosphorylation induces ER stress, which may be
involved in the pathogenesis of AD and other s-related neurodegenerative diseases.
Paper No.: 922

UBIQUITIN LIGASE HRD1 FACILITATES DEGRADATION OF
THE Z VARIANT ALPHA-1-ANTITRYPSIN
Yuxian Shen(1), H Wang(0), Q Li(1), A Sun(1), Y Shen(1), F Wang(1),
H Fang(1), L Feng(1), X Zhu(3), X He(2), S Fang(4)
(1) Ministry of Education and Anhui Medical University, Key Laboratory of Gene Resource Utilization for Genetic Diseases, Hefei, PR china
(2) Anhui Medical University, School of Pharmacy, Hefei, PR China
(3) Bengbu Medical College, Department of Pharmacology, PR china
(4) University of Maryland Biotechnology Institute,Medical Biotechnology Center, USA
Introduction: a-1-antitrypsin (AAT) deciency is an autosomal-recessive
disorder that is characterized by the retention of malfolded AAT in the
endoplasmic reticulum (ER) of hepatocytes and by a signicant diminution in the serum levels of AAT, which subsequently causes liver and
lung diseases. AAT Z variant (ATZ) is the most frequent abnormality in
AAT deciency. Previous studies have demonstrated that ubiquitin-proteasome pathway is involved in the degradation of ATZ. However,
detailed mechanisms of ATZ degradation are not fully understood. We
investigated whether ER membrane ubiquitin ligase Hrd1 facilitated the
removal of ATZ through ER-associated degradation. Materials: Plasmids
encoding ATZ, Hrd1 and its mutant form Hrd1C1A; HEK 293T and
HepG2 cell lines; antibodies to AAT, Hrd1, tubulin and the corresponding secondary antibodies; lipofactamine 2000; plasmids extraction kit; In
situ cell death detection kit. Results and Conclusion: We found that Hrd1
decreased intracellular levels of ATZ, especially in the detergent-insoluble fraction, in the model cells transfected with the plasmid encoding
ATZ. The function of Hrd1 in the degradation of ATZ depends on its
intact E3 activity. Moreover, we found that Hrd1 decreased high molecular weight ATZ and increased the solubility of ATZ. Cycloheximide
(CHX) chase and proteasome inhibition experiments showed that the
ubiquitin-proteasome pathway is involved in Hrd1-mediated ATZ degradation. Furthermore, we found that Hrd1 relieved ATZ toxicity and promoted cell survival, implying a potential value for Hrd1 in the treatment
of AAT deciency diseases, since it attenuates intracellular ATZ accumulation and toxicity.
Paper No.: 934

HRD1 EXPRESSION IS ASSOCIATED WITH NEURONAL SURVIVAL AND TAU DEGRADATION IN ALZHEIMERS DISEASE
Yuxian Shen(1), L Feng(1), A Sun(1), Q Li(1), H Hou(2), H Wang(1),
H Fang(1), S Fang(3)
(1) Key Laboratory of Gene Resource Utilization for Genetic Diseases,
Ministry of Education and Anhui Medical University, Hefei, PR China
(2) Hefei National Laboratory for Physical Sciences at Microscale and
Department of Neurobiology and University of Science and Technology
of China, Biophysics, School of Life Science, Hefei, PR China
(3) University of Maryland Biotechnology Institute, Medical Biotechnology Center, USA
Introduction: Neurobrillary tangles (NFTs), which are mainly composed

of highly aggregated s, are the most common pathological protein aggregates found within neurons in the brains of patients with neurodegenerative diseases and have been implicated in mediating neuronal death and
cognitive decits. NFTs in AD are strongly immunoreactive for ubiquitin
suggesting a role of ubiquitin-proteasome pathway in the pathgenisis of
neurodegenerative diseases. However, the underlying mechanism remains
unclear. Here, we investigated the relationship between endoplasmic
reticulum membrane ubiquitin ligase (E3) Hrd1 and s. Materials: Brain
tissues of AD patients and the controls; HEK 293T cell line; plasmids
encoding s, wt Hrd1 and its mutant form Hrd1C1A; antibodies to s,
phosphorylated-s, Hrd1, tubulin and the corresponding secondary antibodies; lipofectamine 2000; RNA extraction and reverse transcription
kit; Plasmids extraction kit. Results and Conclusion: We found an inverse
relationship between Hrd1 expression and NFTs aggregation in the hippocampal neurons of Alzheimers disease and this suggesting that Hrd1
may be a negative regulator of NFTs. This hypothesis was conrmed in
a cell model over-expressing full length human s or knockdown endogenous Hrd1 with siRNA. We demonstrated that Hrd1 signicantly alleviated s cytotoxicity. Hrd1 promoted the degradation of phosphorylated s
as well as total s; the degradation depended on E3 activity of Hrd1.
Hrd1 increased the formation of polyubiquitinated s. These results indicated that Hrd1 may function as an E3 for s and targets s for proteasome
degradation. This is therefore a potential mechanism underlying the
inverse relationship between Hrd1 expression and neuropathology in AD
brain.
Paper No.: 1607

UPREGULATED PANCREATIC NF-J B/INOS EXPRESSION
AND IMPAIRED GLUCOSE STIMULATED INSULIN SECRETION IN MONOSODIUM GLUTAMATE (MSG) INDUCED
OBESE RATS AFTER LONG-TERM FENOFIBRATE
TREATMENT
Zhufang Shen, S Liu
Institute of Materia Medica, Chinese Academy of Medical Sciences &
Peking Union Medical College, Beijing, PR China
Introduction: To investigate the potential effects and mechanisms of
PPARa agonist, fenobrate on b cell function in MSG obese rats.
Methods: We conducted a 12-week study administering fenobrate
(100 mg/kg body weight) in MSG obese rats characterized as obesity,
insulin resistance and dyslipidemia. Hyperglycemic clamp was conducted to determine the two phase insulin secretion. We also evaluated
the changed expression of key genes and protein in pancreatic islet
involved in insulin biosynthesis, glucose sensing and lipids metabolism, inammation and b cell proliferation. Results: We show now
that 8-weeks fenobrate treatment decreased fasting plasma insulin
and impaired two phase insulin secretory during hyperglycemic clamp
compared with control group. Furthermore, fenobrate treatment
reduced activity of Na+-K+-ATPase in pancreatic mitochondrion in
accordance with increased MDA content and signicantly upregulated
iNOS and NF-jB mRNA and protein expression in pancreatic islet. In
addition fenobrate treatment signicantly increased mRNA level of
UCP2 expression but showed no effect on PPAR a expression in pancreas. Conclusions: These ndings demonstrate that abnormally activited NF-jB and iNOS production within islet b cell is a signicant
player in the negative effect of long-term fenobrate treatment in insulin secretion. We presume that the activation of NF-jB may be caused
by a PPARa-independent pathway.
574
Paper No.: 1415
CHEMICAL FORMS OF MERCURIALS DIFFERENTIATE THE
POTENTIAL OF MERCURY FOR TOXICITY FROM THAT
FOR MEDICINAL REMEDY
Paper No.: 1666

ICARIIN ATTENUATES LEARNING AND MEMORY DEFICITS
INDUCED BY D-GALACTOSE IN RATS
Jing-Shan Shi, F Li, Q-H Gong, Q Wu, Y-F Lu
Jing Zhen Shi(1), F Kang(1), Q Wu(2), YF Lu(2), J Liu(2), YJ Kang(3)

(1) Guiyang Traditional Medical College, Department of Pharmacology,
Guiyang, PR China
(2) Zunyi Medical College, Zunyi, PR China
(3) University of Louisville, School of Medicine, Louisville, KY, USA
Background: Mercury (Hg) in traditional Chinese Medicines and Indian
Ayurvedic medicines raises concerns. Hg used in traditional medicines is
in the form of sulde (HgS, cinnabar), which should be toxicologically
inert. Method: HgS and HgS-containing Zhu-Sha-An-Shen Wan (ZSAS)
were compared with mercuric chloride (HgCl2) and methyl mercury
(MeHg) in vitro and in vivo. Results: Cytotoxicity (LC50) towards
human liver HL7702 cells of ZSAS (342 mg Hg/L) and HgS (2079 mg
Hg/L) was much less than HgCl2 (6.8 mg Hg/L) and MeHg (4.1 mg
Hg/L). Acute oral toxicity (LD50) in mice for MeHg was 2 mg/kg, for
HgCl2 was 80 mg/kg, but for HgS or ZSAS, 20 g/kg was not lethal.
Chronic study (60 days) in SD rats showed that both ZSAS (1.4 g/kg,
20-fold of clinical dose) and HgS (0.2 g/kg, equivalent amount to ZSAS)
were much less toxic than HgCl2 (0.02 g/kg) or MeHg (0.001 g/kg), as
evidenced by body weight loss, serology, and histopathology of liver and
kidney. Mercury accumulation in the liver and kidney in ZSAS- or HgStreated groups was slightly but not signicantly higher than controls,
while the mercury contents in HgCl2- and MeHg-treated groups were 315 folds higher. The expression of kidney injury molecule-1 was
increased 60-fold by MeHg, 3-fold by HgCl2, but was unchanged by
ZSAS or HgS. Conclusion: HgS (cinnabar) or HgS-containing ZSAS
was much less toxic than HgCl2 or MeHg, with much lower bioavailability. Thus, the toxic potential of HgS cannot be evaluated by total Hg
content found in traditional medicines.
Paper No.: 1665

FOCUSED CONFERENCE GROUP: P19 - GENERAL SESSION HAEMATOLOGY
EFFECT OF RHYNCHOPHYLLINE ON PLATELET
AGGREGATION AND CYTOPLASMIC FREE CALCIUM
LEVEL IN RABBIT PLATELETS
Jing-Shan Shi, Z-L Xie, M Wu, Q Wu, Z-N Huang, Q-H Gong
Zunyi Medical College, The Key Laboratory of Basic Pharmacology of
Guizhou Province, Zunyi, PR China
Rhynchophylline (Rhy), an alkaloid abstracted from the Chinese traditional herbs (Rhynchophylla), possesses vasorelaxant,antihypertensive
effect. To investigate the effect of Rhynchophylline (Rhy) on rabbit
platelet aggregation and cytoplasmic free calcium level, and search for
the mechanism of action. Rabbit platelet aggregation and cytoplasmic
free calcium level were evaluated by Born method and uorescence
spectrophotometric method respectively. Rhy administered in vitro
inhibited the rabbits platelet aggregation induced by ADP (1.5 105molL-1) and Thr (3Uml-1) in a concentration- dependent manner
(P < 0.05). It was found that Rhy depressed the rise of [Ca2 + ]i in
rabbits resting platelets, and inhibited the rise of [Ca2 + ]i induced by
ADP (1.5 10-5molL-1) and Thr (3Uml-1) (P < 0.05); Rhy markedly inhibited the rabbit platelet aggregation, And the mechanism may
be due to the depression the rise of cytoplasmic free calcium level in
platelets.

Icariin, a avonol isolated from Herba epimedii, was hypothesized to
protect neuron and improve memory function of rats. This study aimed
to investigate whether icariin can attenuate d-galactose (D-gal)-induced
rat learning and memory decits, and explore the potential mechanisms.
We employed the rat model induced by subcutaneous injection of D-gal
(500 mg/kg/d) for four months to evaluate effects of icariin on spatial
learning and memory function by the Morris water maze. Hippocampus
morphological changes were determined by the HE stain. Furthermore,
the levels of brain-derived neurotrophic factor (BDNF) and tyrosine
kinase TrkB mRNA and protein were detected by quantitative real-time
RT-PCR and Western blotting as potential mechanisms. Chronic injection
of D-gal caused rat memory loss; morphologic abnormalities of neurons
in hippocampus region and the reduced expression of BDNF and TrkB.
Icariin (60 mg/kg/d, po) for four months markedly attenuated D-galinduced rats behavioral dysfunction and neurodegeneration, as evidenced
by shortened escape latency and searching distance and rescued morphologic abnormalities, and also elevated the levels of BDNF and TrkB. But
icariin had no signicant inuence on normal rats which werent injected
D-gal. These results clearly demonstrated that D-gal produced learning
and memory decits after chronic administration, and icariin can protect
neurons from D-gal insults and improve the memory loss.
Paper No. 1667

FOCUS GROUP: P14 - ADDICTION AND DOPING:
NEUROBIOLOGICAL AND CLINICAL
INHIBITORY EFFECTS OF DENDROBIUM ALKALOIDS ON
MEMORY IMPAIRMENT INDUCED BY
LIPOPOLYSACCHARIDE IN RATS
Jing-Shan Shi, Y-F Li, F Li, Q-H Gong, Q Wu
Dendrobium alkaloids was hypothesized to attenuate neuroinammation
induced by lipopolysaccharide (LPS). This study investigated the protective effects of Dendrobium Nobile Lindl. Alkaloids (DNLA) on memory
impairment induced by injecting LPS (50 g) into the right lateral ventricle of rats, and explored its potential mechanism. Rats received different
doses of DNLA (40-160 mg/kg/d) by gavage 7 days before LPS injection then for 13 days once daily. The cognitive function of rats was
tested by Morris water maze, the mRNA of tumor necrosis factor receptor 1 (TNFR1) was examined by real-time RT-PCR and TNFR1, nuclear
factor j-B (NF-jB) and phospho-p38 mitogen-activated protein kinases
(p-p38 MAPK) in the hippocampus were detected by Western blotting at
the protein level. Treatments with DNLA could signicantly shorten
escape latency and searching distance compared with LPS-treated group
by approximately 30% (P < 0.05). The expression TNFR 1 was
increased 15 fold by LPS, which was greatly attenuated by 10 fold by
DNLA. Western blotting showed LPS-intensied blots of TNFR 1, NFjB and p-p38 MAPK, and DNLA treatments greatly attenuated immunoblots in a dose-dependent manner. In conclusion, this study clearly demonstrated that DNLA can improve the ability of spatial memory induced
by LPS through reducing the overproduction of TNFR1 and p-p38
MAPK via inhibition of NF-jB signal pathways in rat.
575
Paper No.: 1668
PHARMACOKINETICS OF DENDROBINE, AN ALKALOID
FROM DENDROBIUM NOBILE, IN RATS
Jing-Shan Shi, C Liu, Q Wu, Y-F Lu, Q-H Gong
Paper No.: 1670

RESVERATROL PREVENTS THE INFLAMMATORY PROCESS
AND THE DOPAMINERGIC DEGENERATION INDUCED BY
THE INTRANIGRAL INJECTION OF LIPOPOLYSACCHARIDE
Jing-Shan Shi, F Jin, H Jin, Q Wu, F Zhang, Y-F Lu, Q-H Gong

Pharmacokinetics of nature products represents a relatively under-investigated but interesting area of pharmacodynamics. At present, the dosage
regimens are based either on conventional administration (8~12 h dosage
interval) or on extended-interval dosage (once daily). Obviously, it is
unreasonable and may result in excessive toxicity or suboptimal biological efcacy. These concerns highlight the key role of pharmacokinetic in
course of pre-clinical research for the nature products. We have previously found that Dendrobine, a triterpenoid alkaloid originally isolated
from the stem of Dendrobium Nobile, could decrease inammatory cytokines in microglia and inhibit s phosphorylation (hallmark pathology of
Alzheimers disease). It is, therefore, important to characterize its pharmacokinetic for establishing a safe and effective medical regimen. Evaluate the pharmacokinetic of Dendrobine by intravenously injecting
20 mg/kg dose into rats. This is the rst time that the assay of Dendrobine has been performed using HPLC method with DAD detection. In
conclusion, its pharmacokinetic behaviour in rats is consistent with a
two-compartment open model and characterized by high distribution volumes (Vd = 0.061 L/kg) and short elimination half-lives (t1/2b =
53.4 min). Frequent or excessive dosage hampers it be a promise candidate because of short half-lives. However, it still has a place, and its lifespan could be prolonged if it is better designed by alternative dosing
regimens, and also by rationally combining other drugs based on pharmacodynamic and pharmacokinetic properties.
Paper No.: 1669

REFLECTION OF DENDROBIUM NOBILE LINDL. ALKALOIDS
ON LIPOPOLYSACCHARIDE INDUCED TAU PROTEIN
HYPERPHOSPHORYLATION IN THE HIPPOCAMPUS OF
RATS

Evidence suggests that neuroinammation play signicant roles in Parkinsons disease (PD), where the primary pathology is signicant loss
of the dopaminergic neurons in the substantia nigra. In this study, we
investigated the inuence of resveratrol on the degenerative process of
the dopaminergic neurons of the rat following intranigral injection of
lipopolysaccharide (LPS), a potent inductor of inammation that we
have previously used as an animal model of Parkinsons disease. We
observed dopaminergic neurons and found that resveratrol prevented
the inammatory processes, as the induction of cyclooxygenase-2
COX-2, tumor necrosis factor-a (TNF-a), and inducible nitric oxide
synthase (iNOS) and the consequent dopaminergic degeneration
induced by LPS. Our results suggest that resveratrol could delay the
progression of dopaminergic degeneration in disorders involving
inammatory processes. In summary, intrastriatal LPS served as a
model for inammation-induced dopaminergic neurodegeneration, antiinammatory drugs provided protective properties, and resveratrol may
have therapeutic potential for the treatment of neuroinammation and
PD.
Paper No.: 1671

TETRAMETHYLPYRAZINE INHIBITS RAT LEFT
VENTRICULAR HYPERTROPHY INDUCED
BY ABDOMINAL AORTA COARCTATION:
INVOLVEMENT OF CALCINEURIN
Jing-Shan Shi, J Deng, Q Wu, Y Gao, X-N Huang
Jing-Shan Shi, S Yang, F Li, Q-H Gong, Q Wu, Y-F Lu


Tetramethylpyrazine, one of the active components of Ligusticum chuangxiong Hort, has been reported to inhibit proliferation of vascular
smooth muscle cells induced by angiotensin II (Ang II). This study further investigates whether tetramethylpyrazin has protective effect on Ang
II-induced rat left ventricular hypertrophy and to examine its protective
mechanisms. The rat left ventricular hypertrophy was induced by abdominal aorta coarctation. Tetramethylpyrazine (25, 50 and 100mg.kg-1.d-1,
po) was given the day after surgery for 21 consecutive days. The left
ventricular hypertrophy induced by abdominal aorta coarctation was evidenced by histopathology, and by the increased left ventricular weight,
the cardio-myocyte diameters, and the expression of aterial natriuretic
peptide, calcineurin. Tetramethylpyrazine signicantly ameliorated left
ventricular hypertrophy induced by abdominal aorta coarctation in a
dose-dependent manner. To examine the mechanism of protection, the
expression of calcineurin was determined at the transcript level. Abdominal aorta coarctation induced increases in calcineurin expression, which
was suppressed by tetramethylpyrazine. Tetramethylpyrazine alleviated
left ventricular hypertrophy induced by abdominal aorta coarctation, and
the protection appears to be due, at least in part, to its inhibitory effects
on calcineurin signalling pathways.
Neurobrillary tangles, one of the characteristic pathological features of

Alzheimers disease (AD), are composed of paired helical laments which
are mainly composed of hyperphosphorylated s protein. Relieving the hyperphosphorylation of s protein is one of the effective measures of treating
AD. Here, we injected lipopolysaccharide (LPS, 50 g) into the right lateral
ventricle of rats to induce cell death. The rats were administered different
doses of Dendrobium Nobile Lindl. Alkaloids (DNLA, 3 times in a day by
gavage, and were then injected LPS in the early morning of the second day.
After 2 hours, the right hippocampi of the rats were dissected to observe
the expressions of hyperphosphorylated s protein by western blotting. The
other four rats administered with LPS were continually treated by
DNLA(20 and 80 mg/kg, respectively) thrice daily for one week. The cell
apoptosis in the brain was observed by TUNEL. Compared to the shamtreated rats, LPS injection signicantly increased the expressions of hyperphosphorylated s protein at Ser199-202, Ser396, Ser231, Ser205 and
GSK-3b sites and aggravated the cell apoptosis in the brain, which were
ameliorated with DNLA treatment (P < 0.05). This study suggests that
DNLA could attenuate the hyperphosphorylation of s protein in the hippocampus and relieve the brain cell apoptosis of rats induced by LPS.
576
Paper No.: 1339
EDARAVONE PROMOTES THE PROLIFERATION/SURVIVAL
OF NEURAL PROGENITOR CELLS AFTER GRANULE CELL
LOSS IN THE HIPPOCAMPAL DENTATE GYRUS OF MICE
Paper No.: 2188

EFFECT OF A NOVEL FUNGAL TRIPRENYL PHENOL
METABOLITE (CPD-7) ON ACETIC ACID-INDUCED EMBOLIC
CEREBRAL INFARCTION MODEL IN MICE
Tatsuo Shiba, K Kawada, M Yoneyama, K Ogita
Keita Shibata(1), T Hashimoto(1), K Nobe(1), K Hasumi(2), K Honda(1)
(1) Showa University, Department of Pharmacology, Tokyo, Japan

(2) Tokyo University of Agriculture and Technology, Tokyo, Japan
Our previous study indicated that trimethyltin chloride (TMT) causes

granule cell loss in the hippocampal dentate gyrus selectively 2 days
later, with regeneration of the dentate granule cells. To elucidate mechanisms underlying the modulation of the neurogenesis by cell proliferation
and/or cell survival, in this study, we evaluated the effect of the free radical scavenger edaravone on proliferation/survival of neural progenitor
cells in the dentate gyrus after the damage induced by TMT. From day 2
after being given TMT (2.8 mg/kg, i.p.), edaravone (50 mg/kg, i.p.) was
injected for consecutive 2 days every 12 h. Cells were prepared hippocampal dentate gyrus of mice treated with saline or edaravone after TMT
injection. Edaravone produced a signicantly increase in the number of
nestin-positive cells. The cells then cultured in the DMEM/F12 medium
containing EGF and bFGF for 18 days in vitro. In both saline- and edaravone-treated animals, marked round spheres were formed from cells
adhered to each other and subsequently proliferated from large neurospheres in proportion to the duration of cultivation. In addition, neurospere assay revealed that edaravone produced a signicant increase in the
number of neurospheres at the size of more than 50 microM. These result
suggest that edaravone promotes the proliferation in neural stem/progenitor cells of the hippocampal dentate gyrus after nuronal degeneration in
adult mice.
Paper No.: 1336

CONTRIBUTION OF CYP3A5 ISOZYME TO THE IN VIVO
METABOLISM OF CORTISOL TO 6B-HYDROXYCORTISOL IN
HUMANS
Hiromi Shibasaki-Hirano, S Okamoto, T Furuta
Tokyo University of Pharmacy and Life Sciences, Department of Medicinal Chemistry and Clinical Pharmacy, Tokyo, Japan
Cortisol is metabolized to 6b-hydroxycortisol catalyzed by CYP3A4 and
CYP3A5. We have previously reported evidence for the validity of
endogenous cortisol 6b-hydroxylation clearance as a new index for in
vivo CYP3A phenotyping in humans (Furuta T et al., Drug Metab. Dispos., 2003; 31: 1283-1287). The aim of this study was to investigate the
contribution of CYP3A5 isozyme to the in vivo metabolism of cortisol to
6b-hydroxycortisol by measuring 6b-hydroxylation clearance in subjects
with and without CYP3A5 expressions. Three healthy adult subjects participated in this study. Blood samples were obtained at 10:00, 11:00 and
12:00 every second or third day for 40 days. Urine samples were
obtained for 2 h from 10:00 to 12:00. The 6b-hydroxylation clearances
were calculated as the amount of urinary excreted 6b-hydroxycortisol
during 2-hour urine collection period divided by the corresponding AUC
of cortisol, respectively. The time course data showed that 6b-hydroxylation clearances of cortisol for approximately 40 days were
2.69 0.52 ml/min for subject A (CYP3A5*3/*3), 2.11 0.43 ml/min
for subject B (CYP3A5*1/*3), and 1.90 0.39 ml/min for subject C
(CYP3A5*1/A3), respectively. The mean value of 6b-hydroxylation clearances of cortisol in subject A with no expression of CYP3A5 was higher
than those of heterozygous CYP3A5*1/*3 subjects (subject B and C).
The preliminary experimental results show that the involvement of
human CYP3A5 in the 6b-hydroxylation of cortisol in vivo may be negligible and the CYP3A4 isozyme should be a key determinant of the
pathway in humans.
We previously reported the establishment of novel embolic cerebral

infarction model induced by acetic acid and evaluation of tissue plasminogen activator (t-PA), edaravone argatroban and ticlopidine. The aim of
the present study was to investigate the effect of a novel fungal triprenyl
phenol metabolite (Cpd-7) on acetic acid-induced embolic cerebral
infarction model. Thrombotic occlusion was induced by transfer of acetic
acid-induced thrombus at the right common carotid artery into the brain
of the ddY mice. Infarction area, neurological scores, edema percent and
regional cerebral blood ow were determined as index of efcacy of
Cpd-7. In order to evaluate the mechanism of Cpd-7, plasmin activities
and the expressions of IL-1b, TNF-a and IL-6 mRNA were examined.
Cpd-7 reduced infarction area, neurological score and edema percent in a
dose-dependent manner (0.1, 1, 10 mg/kg) and its therapeutic time window was longer than that of t-PA. Cpd-7 showed plasmin activity in
vivo; additionally, decrease in cerebral blood ow was recovered,
although its plasmin activity was less than that of t-PA. Furthermore, the
expressions of inammatory cytokines mRNA were increased by treatment of t-PA at 3 hr after ischemia. However, these increases were not
induced by treatment of Cpd-7. These results indicate that Cpd-7 shows
the potential of this new therapeutic concept of thrombolysis and antiinammatory effect coincidentally in the cerebral infarction. We theorized that excellent therapeutic time window of Cpd-7 is contributed to
both activities. Therefore, this novel low molecular compound may represent novel approach to the treatment of cerebral infarction.
Paper No.: 3356

EFFECT OF YOKUKAN-SAN, A KAMPO CHINESE
TRADITIONAL MEDICINE ON MGLUR5 KNOCK-OUT
MOUSE, A MODEL MOUSE FOR SCHIZOPHRENIA
Shigenobu Shibata, A Fujimori, T Shimane, Y Nakano, K Horikawa
[Background] As metabolic glutamate receptor mGluR5 knock-out
mouse shows the hyperactivity, decit of Y maze alternative behavior,
and less immobility in behavioral test, we suppose that phenotypes of
this mutant mouse exhibit an animal model of positive symptom of
schizophrenia. Recently, it is clinically evaluated that Yokukan-san, one
of Chinese traditional medicine may be a potential drug for hyperactivity
and positive symptom appeared in dementia and/or schizophrenia. In the
present research we examined whether Yokukan-san can improve the
behavior decits seen in mutant mice in comparison with haloperidol
and risperidone, typical and atypical anti-schizophrenic drugs, respectively. [Result and Discussion] mGluR5 mutant mice showed the metamphetamine-insensitive hyperactivity in open eld test and decit of
Y-maze alternative behavior. Yokukan-san strongly improved the hyperactivity and decit of Y-maze alternative behavior without affecting
normal behavior seen in the control Wild mice. On the other hand, risperidone improved the Y-maze decit with same dose which impaired
the general activity of Wild mice. Haloperidol caused the decit of locomotor activity in both Wild and mGluR5 mutant mice. These results
strongly suggest that Yokukan-sun may be good Chinese traditional herb
medicine for treatment of dementia and/or schizophrenia without carry-
577
ing its side-effect. We are still examining which Shoyaku herb is active
component in Yokukan-san, because 7 Shoyaku herbs are contained in
Yokukan-san.
Paper No.: 1624

DISEASES
INVOLVEMENTS OF RHOA GERANYLGERANYLATION IN
THE PATHOGENESIS OF ALLERGIC BRONCHIAL ASTHMA
Aya Shibuya, S Sato, Y Chiba, M Misawa
Tokyo, Japan
Our previous studies revealed that RhoA-mediated calcium ion sensitization of bronchial smooth muscle contraction is markedly augmented in
mice with antigen-induced airway hyperresponsiveness (AHR), a characteristic feature of allergic bronchial asthma. The RhoA protein is known
to be modulated by posttranslational prenylation, i.e., geranylgeranylation, for its activation. It is therefore possible that the increased expression of RhoA and its activation might enhance the calcium ion
sensitizing signal. In the present study, the effects of geranylgeranyltransferase (GGTase) inhibitor-2133 (GGTI-2133), a direct inhibitor of RhoA
geranylgeranylation, on the augmented bronchial smooth muscle contraction were investigated in the antigen-induced AHR mice. The bronchial
smooth muscle responsiveness to ACh, but not to high potassium ion,
was markedly and signicantly augmented in the repeatedly antigen challenged mice. Western blot analyses revealed that protein levels of RhoA
and GGTase in bronchial smooth muscles of the AHR mice were signicantly increased as compared to those of normal animals. In addition,
marked increases in eosinophil number and interleukin (IL)-4 and IL-13
levels in BALFs were observed in association with the increments of
total and OA-specic IgE in sera in the AHR mice. Both the bronchial
smooth muscle hyperresponsiveness and the airway eosinophilia induced
by antigen exposure were ameliorated by the treatment with GGTI-2133
(5 mg/kg/day, i.p., for 10 days), whereas the treatment had no signicant
effect on the levels of cytokines or IgE. It is thus possible that the inhibition of RhoA geranylgeranylation improves allergic bronchial asthma by
inhibiting both AHR and airway inammation.
Paper No.: 1294

FASTING-INDUCED INCREASE IN GALANIN MRNA
EXPRESSION IN SEVERAL HYPOTHALAMIC NUCLEI
Tomohiro Shiiya, T Niki, H Higuchi
Niigata University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Division of Pharmacology, Niigata, Japan
Fasting changes the gene expression of orexigenic neuropeptides such as
NPY, AgRP, orexin and MCH, and/or anorexigenic ones such as CART
and POMC in hypothalamus. Among various neuropeptides, galanin, an
orexigenic neuropeptide, mRNA was increased remarkably in mouse
hypothalamus by 2 day-fasting, which was detected by RT-PCR. Galanin
peptide is known to stimulate feeding behavior, reduce energy expenditure and regulate release of metabolic hormones. In this study, we investigated fasting-induced change in tissue distribution pattern of galanin
mRNA expression in mouse brain by using in situ hybridization (ISH).
C57BL/6N mice were fasted for 50 hr with free access to water and then
sacriced. The brains were dissected or sliced to perform RT-PCR or
ISH by specic galanin probes. RT-PCR showed that galanin mRNA
level was increased signicantly by 30% by fasting in whole hypothala-
mus, while NPY and AgRP mRNA expression were increased by 100%
and 150%, respectively, in arcuate nucleus (ARC). ISH analysis indicated
that galanin mRNA-containing neurons could be detected in the dorsomedial nucleus (DMN), ARC, lateral hypothalamus (LH) and perifornical nucleus (PeF) in hypothalamus, in the preoptic medial nucleus in
preoptic area and locus coeruleus in pons. Fasting increased the galanin
mRNA level in the DMN, LH and PeF. There was no change in other
area. These ndings suggested that increase in galanin gene expression
in DMN, LH and PeF is important for fasting-induced feeding behavior
and metabolic processes
Paper No.: 3385

THE ZINAXIN CLINICAL PHARMACOLOGICAL
APPLICATION ASPECTS
Evgeniya Shikh, O Shumkova
State Scientic Center, Moscow, Russian Federation
Zinaxin contains 150 mg of Zingiber ofcinale extract, which corresponds to 3000 mg of dry rootstock and also contains 15 mg of Alpinia
galangal extract, which corresponds to 660 mg of dry rootstock. When
producing the preparation, the lipokap technology is implemented,
increased bioavailability. Under bile inuence, a nano granule is dissolved in the intestines, micro capsules penetrate enterocyte and in the
form of liposomes circulate in the blood, which increase bioavailability
by excluding the rst liver passing effect and to supply the preparation to
the ultimate points of appliance. In vitro research (John Hopkins University), showed that Zinaxin suppresses NIO-2 the main enzyme, catalyzing prostaglandin (PGE-2) production. TNF-a suppression and NIO-2
regulation are determined by NF-jkB and Ik-b cascade suppression.
TNF-a expression decrease at m-RNA level in human synoviocytes and
chondrocytes is expressed in dose dependence, which features
expressed chondro protective properties. The Zinaxin clinical
researches among patients with locomotorium desease proved expressed
anti inammatory, analgesic and chondro protective properties: Altman 2001 patients with knee joint osteoarthritis; Wigler patients with
gonarthrosis; E.I. Jakimenko patients with OA. In all researches a good
preparation safety prole is stated. The possibility to application Zinaxin
among patients with locomotorium desease not tolerating NSAIDs is
conrmed by a research including patients with gastro intestinal bleeding in anamnesis; with ulcer perforation in anamnesis; recurrent NSAIDs
induced ulcers in anamnesis; with bronchial obstructionn, induced by
NSAIDs (Kukes, Shikh 2008).
Paper No.: 1965

OVEREXPRESSION OF KININ B1 RECEPTOR INDUCES
INCREASED REACTIVITY OF DBK AND BK IN TRANSGENIC
RAT AORTA
Suma Imura Shimuta, RF da Silva, JB Pesquero, RP Martin,
ES Rodrigues
Federal University of Sao Paulo School of Medicine, Department of
Biophysics, Sao Paulo, Brazil
Bradykinin (BK) mediated by constitutively expressed B2 receptors
(B2R) and Des-Arg9-BK (DBK) by inducible B1 receptors (B1R) are
vasorelaxant, algesic and inammatory agents. B1R is expressed at low
levels in several tissues but highly induced in response to pathological
insults.There is evidence that lack of BK B2R reduces whereas its overexpression increases renal functions. It was found that overexpression of
578
B1R in the endothelial cells increased susceptibility to endotoxic shock
in transgenic rats (TGR(Tie2)). Therefore it was of interest to investigate
the responsiveness of toracic aortic rings to BK and DBK as well as the
receptor gene expression of both B2R and B1R in this rat overexpressing
the constitutive and functional B1R. Concentration-relaxant curves for
BK and DBK were obtained in aorta pre-contracted with nor-epinephrine
(1 lM) and the vascular reactivity was determined. The potency and the
efcacy were markedly potentiated in the relaxing responses to DBK and
an unexpected nding was that BK-induced effect was also potentiated
in TGR(Tie2) compared to the control animal. It was found that the
expression levels of B1R and B2R were also increased in the transgenic
animal. These data explain the increased reactivity of the aorta in
response to BK in addition to DBK. Our results indicate that down regulation of B2R did not occur in rats with endothelial overexpression of
kinin B1R in contrast to that reported in mice overexpressing the B2R. It
is suggested that tissue specic ne tuning between the two subtypes of
kinin receptors may be involved in vascular pathophysiology.
Paper No.: 1421

CINNAMOMI RAMULUS ETHANOL EXTRACT EXERTS
VASORELAXATION THROUGH INHIBITION OF THE
RHO-KINASE SIGNALLING PATHWAY
the proinammatory cytokines interleukin-1b (IL-1b, interleukin 6 (IL6), tumor necrosis factor-a (TNF-a), interferon c (IFN-c) and monocyte
chemoattractant protein-1 (MCP-1), and anti-inammatory cytokine
interleukin-10 (IL-10) in rats with naturally occurring gingivitis (ODUS/
Odu), an animal model of periodontal disease developed and maintained
in our laboratory. These rats are prone to marked plaque formation on
the mandibular incisors, and develop gingivitis and periodontal pockets.
Plasma cytokine levels were measured in ODUS/Odu and control rats
(Res) at 0 (5 weeks of birth, 1, 3, 6, 9, and 12 months after the start of
the experiment using an enzyme-linked immunosorbent assay (ELISA)
kit. The IL-1b, TNF-a, IFN-c and MCP-1 levels were signicantly
greater in ODUS/Odu than in Res throughout the experimental period
(P < 0.001). The IL-6 level was greater in the ODUS/Odu than in the
Res at 6 to 12 months (P < 0.05). However the plasma level of the antiinammatory cytokine IL-10 was signicantly lower in ODUS/Odu than
Res throughout the experimental period (P < 0.001. These ndings were
similar to those in patients with periodontal disease, suggesting that
ODUS/Odu serve as a useful animal model.
Paper No.: 2125
COMMUNICATION OF TOXIC EFFECTS OF DRUGS WITH
POLYMORPHISM GENES GST AND CYP450
Natalya Shipovskaya(1), E Zaklyazminskaya, D Litvinov, L Shelihova
Heung Shin, Y Kang

Dongguk University College of Oriental Medicine, Gyeongju, South
Korea
The Rho-kinase (ROCK) signalling pathway plays an important role in
the pathogenesis of hypertension. We hypothesized that vasodilatory
effect of Cinnamomi ramulus ethanol extract (CRE) is related ROCK
signalling pathway. Rat aortic rings were contracted with phenylephrine
(1 lM) or calpeptin (100 lg/ml) in the presence and absence of endothelium. Treatment with CRE (0.05~1.0 mg/ml) dose-dependently relaxed
the vascular contraction in response to PE or calpeptin in an endothelium-independent manner. To elucidate the fundamental mechanism of
endothelium-independent vasodilative reaction of CRE, ROCK related
signal molecules were measured in endothelium denuded rat aortic rings
in response to calpeptin. CRE treatment for 20 min inhibited the expression of Rho-associated kinase 2 and dephosphorylated myosin phosphatase targeting subunit 1 (MYPT-1), protein kinase C-potentiated
phosphatase inhibitor protein-17 kDa (CPI-17) and myosin light chain
20 kDa (MLC20). Additionally, the phosphorylation of adducin, which
is a downstream target of ROCK2, was inhibited by ECR treatment.
Through XTT assay, we found that ECR (0.05~0.5 mg/ml) did not affect
the viability of human aortic smooth muscle cells (HASMC) and human
umbilical vein endothelial cells (HUVEC). Taken together, these data
indicate that the vasorelaxation effect of ECR occurs via inhibition of
ROCK signal pathway.
Paper No.: 1428

DISEASES
CHANGES OF INFLAMMATORY CYTOKINES IN RATS WITH
NATURALLY OCCURRING GINGIVITIS
Mitsuko Shinohara, A Hidaka, K Iwamoto, K Ohura
(1) Russian National Research Centre of Surgery RAMS, Laboratory of

Medical Genetics, Moscow, Russian Federation
(2) Federal Research Centre of Pediatric Hematology, Immunology and
Oncology, Moscow, Russia
Clinical case: Female patient 3.5 y.o. with neuroblastoma of L5, IV stage
metastasized into a backbone, skeletal bones and bone marrow. Therapy
started under NB-2004 protocol for HRG, with a reduction of Vincristin.
After 3rd block the neurological status impaired up to coma. MRI: Progressive diffusive cortical and subcortical atrophy; bilateral lesion of subcortical nuclei, and brain stem. MR signal increased in subcortical and
cortical frontoparietal zones. Contrast accumulation had been observed in
thalamus, back zone of brain stem, and in frontoparietal area. Severity of
injury was provoked by a toxic encephalopathy, tetraparesis. No data of
infections were found. Because of possible toxic effect of Holoxan, the
methylenic blue as an anti-dot had been used with a positive effect.
Patient required articial pulmonary ventilation about 4 months. We did
perform DNA testing GSTM1, GSTP1 (c.313A>G), and CYP2C9
(c.430C>T, c.1075A>C) gene variants commonly involved in individual
adverse drug effects. The homozygous loss-of-function GSTM1 gene
deletion, and heterozygous low functional variant of GSTP1 were found.
We presume that low functional alleles of phase II detoxication system
can play a role in observed toxic effects of treatment with different level
of severity on children with hemoblastosis and solid tumours, receiving
polychemotherapy. But exact clinical and predictive signicance of this
nding, as well as optimal combination of alleles to be tested in relation
to a given chemiotherapy protocol are to be studied.
Paper No.: 2219
DISCOVERY
TRPC3 MEDIATES THROMBIN-INDUCED ASTROCYTE
ACTIVATION AND UPREGULATES ITS OWN EXPRESSION IN
RAT CORTICAL ASTROCYTES
H Shirakawa, T Nakagawa, Shuji Kaneko
Osaka Dental Hospital, Department of Pharmacology, Osaka, Japan

Periodontal disease is an inammatory condition caused by periodontal
bacteria. Its development and progression are related not only to bacterial
pathogenicity, but also to the host response in the inamed periodontal
tissue. Recent studies have claried the relationship between periodontal
and systemic diseases. In this study, we measured the plasma levels of
Kyoto University Graduate School of Pharmaceutical Sciences,

Department of Moleular Pharmacology, Koyoto, Japn
Reactive astrogliosis is a characteristic response of astrocytes to CNS
injuries including intracerebral hemorrhage, dened by abnormal mor-
579
phology and excessive cell proliferation. Thrombin, a major bloodderived serine protease, leaks into the brain parenchyma upon bloodbrain barrier disruption and can induce brain injury and astrogliosis.
Transient Receptor Potential Canonical (TRPC) channels, Ca2 + -permeable, nonselective cation channels, are expressed in astrocytes and
involved in Ca2 + inux after receptor stimulation, however, their pathophysiological functions in reactive astrocytes remain unknown. Here,
we investigated the pathophysiological roles of TRPC in thrombinactivated cortical astrocytes. Application of thrombin (1 U/ml, 20 h)
upregulated TRPC3 protein, which was associated with increased
Ca2 + inux after thapsigargin treatment. Pharmacological manipulations revealed that the TRPC3 upregulation was mediated by protease-activated receptor 1 (PAR-1), ERK and JNK, and NF-jB
signalling, and required de novo protein synthesis. Ca2 + signalling
blockers BAPTA-AM, cyclopiazonic acid, 2-aminoethoxydiphenyl
borate and a selective TRPC3 inhibitor, Pyrazole-3, attenuated the
TRPC3 upregulation, suggesting that Ca2 + signalling through TRPC3
contributes to its increased expression. Thrombin induced morphological changes at 3 h, upregulated S100B, a marker of reactive astrocytes, at 20 h and increased astrocytic proliferation by 72 h, all of
which were inhibited by Ca2 + signalling blockers and specic knockdown of TRPC3 using siRNA. Intracortical injection of SFLLR-NH2, a
PAR-1 agonist peptide, induced astrocytic proliferation, the majority of
which were TRPC3-immunopositive. These results suggest that thrombin
evokes dynamic TRPC3 upregulation and that TRPC3 contributes to the
pathological activation of astrocytes in part through a feed-forward
upregulation of its own expression.
Paper No.: 1537

HEALTH AND DISEASE
IN VITRO DISSOLUTION KINETICS STUDIES OF
AMLODIPINE TABLETS MARKETED IN RUSSIA UNDER
BIOWAIVER CONDITIONS
IE Shohin(1), GV Ramenskaya(1), GF Vasilenko(1), EA Malashenko(1),
KS Davydova(2), Dmitry A Sychev(2), VG Kukes(2)
(1) Sechenov Moscow Medical Academy, Pharmaceutical Departament,
Moscow, Russian Federation
(2) Sechenov Moscow Medical Academy, Medical Departament,
Moscow, Russian Federation
Introduction. Amlodipine is a dihydropyridine calcium antagonist that
inhibits the transmembrane inux of calcium ions into vascular
smooth muscle and cardiac muscle. According to WHO Guidances,
amlodipine is classied as BCS Class I (high solubility, high permeability). So to establish interchangeability of amlodipine generic drugs
it is possible to perform their in vitro evaluation under biowaiver
conditions. Materials. All dissolution studies were performed using
USP Apparatus 1 (Erweka DT 600, Germany) at 100 rpm. Dissolution media were USP buffer solutions pH 1,2 (hydrochloric acid
solution), pH 4,5 (acetate buffer solution), pH 6,8 (phosphate buffer
solution) at 37 0.5 C, 500 ml. Time points were 10, 15, 20, 30
and 45 min. Drug release was assayed spectrophotometrically using
UV-Visible spectrophotometer (Agilent 8453, USA) at 239 nm. 12
tablets for test and reference preparation were studied to obtain statistically signicant results. Dissolution prole comparison was made
according WHO Guidances using factor f2. Results. Dissolution prole of test and reference amlodipine preparation were equivalent: for
pH 6,8 f2 value was 68, for pH 1,2 and 4,5 proles considered to
be equivalent without statistical treatment (more than 85% released
after 15 min). Conclusion. Dissolution kinetics of test and reference
amlodipine tablets is equivalent under biowaiver conditions that provides evaluation of their interchangeability.
Paper No.: 2176

ROLE OF THE A-2 ADRENOCEPTORS SUBTYPES IN
REGULATION OF GASTRIC MOTOR ACTIVITY IN THE RAT
AND MOUSE
Nashwan Shujaa(1), Z Zadori(1), M Al-khrasani(1), M Rossi(1),
L Hein(2), K Gyires(1)
(1) Semmelweis Universty, Faculty of Medicine, Department of Pharmacology and Pharmacotherapy, Budapest, Hungary
(2) University of Freiburg, Institute of Experimental and Clinical
Pharmacology and Toxicology, Freiburg im Breisgau, Germany
Introduction: Presynaptic a-2-adrenoceptors are classied into three subtypes a-2A-a-2B and a-2C which modulate a number of gastrointestinal
functions including, motility, acid secretion and gastric mucosal protection. Aim: In vitro and in vivo analysis of the role of a-2-adrenoceptors
subtypes in the regulation of gastric motility in rats and mice. Methods:
1.A gastric fundus strips were suspended between two electrodes in
organ bath containing Krebs solution and contractions were induced by
EFS. Drugs were added in a cumulative manner. 2.In vivo the gastric
motility was measured by the balloon method. Gastric motility was stimulated by insulin (5NE i.v.). Results: In Rats 1. Clonidine, oxymetazoline
and ST-91 in a concentration dependent manner inhibited the EFSinduced contractions. 2. Idazoxan, reversed the inhibitory effect of clonidine and ST-91, but not that of oxymetazoline. ARC-239 and BRL
44408 reversed the inhibitory effect of clonidine and ST-91. 3. Intravenously injected clonidine, oxymetazoline and ST-91 inhibited the gastric
motility. 4. The effect of clonidine was antagonized by yohimbine, BRL
44408 and prazosin. In mice 1. Clonidine inhibited the EFS-induced contraction in NMRI, Wild type and a-2B KO and a-2C KO mice, but not in
the a-2A KO mice.2. Its effect was reversed by idazoxane and BRL
44408 but not by ARC-239. Conclusion: 1.Besides a-2A-adrenoceptors
the a-2B-subtype is also likely to be involved in inhibition of gastric
motor activity both in vivo and in vitro in the rat. 2. In the mice the a-2Bsubtype is not involved in the regulation of gastric motility.
Paper No.: 1551

SORTING NEXIN-25 INTERACTS WITH D1 AND D2
DOPAMINE RECEPTORS TO REGULATE RECEPTOR
EXPRESSION AND SIGNALLING
David Sibley, R Free, Y Namkung, L Hazelwood, C Furman,
National Institutes of Health, Molecular Neuropharmacology Section,
Bethesda, MD, USA
Introduction: Our studies employing co-immunoprecipitation assays for
dopamine receptors coupled with mass spectrometry-based sequencing
have identied sorting nexin-25 as a member of the dopamine receptor
signalplex. Mammalian sorting nexins (SNXs) have been suggested to
regulate intracellular trafcking, internalization, and endosomal recycling
or sorting of membrane-bound cargo. The physiological role of SNX25
is unknown. Methods: Using RT-PCR we have found that SNX25 is
expressed in multiple tissues including brain and kidney and that the
endogenous isoform is longer than the one indicated in GenBank.
Results: The full-length SNX25 contains two putative transmembranespanning regions and confocal microscopy shows that it is localized in
distinct clusters at or near the plasma membrane. When SNX25 is overexpressed with dopamine receptors, the receptors show changes in
expression patterns and appear localized to the SNX25 clusters. Overexpression of SNX25 perturbed both endocytosis and recycling of the D2
dopamine receptor. Radioligand binding also show that the expression
580
levels of both D1 and D2 dopamine receptors are increased with SNX25
over-expression. Decreasing the levels of endogenous SNX25 using siRNA causes a subsequent decrease in dopamine receptor expression. Conclusion: These data suggest that SNX25 plays a role in trafcking
through intracellular membrane compartments and regulates both receptor expression and signalling.
Paper No.: 2320

EVALUATION OF CLINICAL RELEVANCE OF DRUG
INTERACTIONS BASED ON METABOLISM: COMPARISON
OF TWO CDK INHIBITORS
Michal Siller(1), P Anzenbacher(1), E Anzenbacherova(2), K Dolezal(3),
M Strnad(3)
(1) Palacky University, Faculty of Medicine and Dentistry, Department
of Pharmacology, Olomouc, Czech Republic
(2) Palacky University, Faculty of Medicine and Dentistry, Department
of Medical Chemistry and Biochemistry, Olomouc, Czech Republic
(3) Palacky University, Faculty of Science, Laboratory of Growth Regulators, Olomouc, Czech Republic
Safety of new drug candidates before its use in clinical practice may be a
critical issue. In vitro study on interaction of drug candidates with main
human drug metabolising enzymes is hence essential. Two experimental
anticancer drugs, olomoucine II and compound 1568, were tested to
evaluate the possible risk of drug drug interactions based on metabolism
by human liver microsomal and intestinal enzymes. Human liver microsomal fraction containing cytochromes P450 (CYP) and UDP-glucuronosyltransferases (UGTs) and supersomes (containing individual
enzymes) were used. Spectroscopic and chromatographic techniques
helped in determination of inhibition effect of drug tested on individual
enzymatic activities; identication of purported metabolites was performed with mass spectrometric detection. Results showed that olomoucine II had considerable potential to interact with CYPs (inhibition of at
least three CYP, substrate of the CYP3A4 enzyme) in vitro. Moreover,
this compound was metabolised by several liver and intestinal UGTs in
vitro. On the other hand, compound 1568 exerted weak interaction with
CYP, however, intestinal UGTs signicantly contributed to metabolism
of this compound. Olomoucin II could possibly affect the metabolism of
other coadministered drugs by both CYP and UGTs if clinically used.
Contrary to olomoucine II, compound 1568 might inuence only the glucuronidation of other drugs in vivo. In conclusion, compound 1568
seems to be more safe in light of drug interactions based on metabolism
in comparison to olomoucin II.
Acknowledgment: The authors thank the MSM6198959216 project and
Grant 303/09/H048 for nancial support.
Paper No.: 2991

ANTIHYPERTENSIVE TREATMENT AND BLOOD PRESSURE
CONTROL IN PRIMARY HEALTH CARE
D Silva(1,3), Isabel Vitorial Figueiredo(1,2), A Figueiras(4),
MT Herdeiro(3), Margarida Caramona(1,2), Jorge Polonia(5,6)
(1) Coimbra University, Faculty of Pharmacy, Laboratory of Pharmacology, Coimbra,Portugal
(2) Centro de Estudos Farmaceuticos (CEF), Coimbra, Portugal
(3) Health Technology Research Center (CITS) - Northern Polytechnic
Health Institute, Famalicao, Portugal
(4) University of Santiago de Compostela, Department of Preventive
Medicine and Public Health, Santiago de Compostela, Spain
(5) University of Porto, Faculty of Medicine, Porto, Portugal

(6) Pedro Hispano General Hospital, Unit of Hypertension, Matosinhos,
Portugal
Introduction: Poor adherence to antihypertensive treatment (AHT) due to
adverse drug reactions is a main cause of uncontrolled blood pressure
(BP). This study relates patients perceived symptoms (S), AHT and BP.
Patients: 124 patients of a Primary Health Care Unit selected by consecutive sampling (age over 18 years old, diagnosis of essential hypertension,
AHT for at least 6 months). Information about clinical data and S was
collected and compared with clinical records. Results: Patients take at
least 2 drugs of different AHT groups (77%). The most used drugs are
diuretics (74%), angiotensin-converting enzyme (ACE) inhibitors (43%)
and angiotensin-II receptor antagonists (ARA) (45%). Therapy of 18%
of patients is different from the clinical records. BP control is independent of treatment adherence, number or type of AHT. The most reported
symptoms were anxiety (74%) and tiredness (63%). Persistent dry cough
(12%) and skin spots (9%) were the less reported. Symptoms are perceived as AR by 24% of the patients and 43% of them associate the
effects to AHT. Logistic regression Modelling shows adjusted signicant
associations between AHT and S. Risk of uncontrolled BP is increased
in men (OR 3.767), in those who feel palpitations (OR 2.737) and in
those who use drugs from the group Others (a2 adrenergic agonists,
nitrates, trimetazidine or codergocrine) (OR 4.364). Patients in secondary
prevention of cardiovascular events have decreased risk (OR 0.207).
Conclusion: Symptoms are signicantly related to AHT. The study provides information about AHT prescription, AR prole and alert doctors
to medical records failures.
Paper No.: 3154

CROSSTALK BETWEEN EXCITATORY A2A, NK1 AND M3
RECEPTORS ON A TRIPARTITE MYENTERIC SYNAPSE
Isabel Silva, D Fernandes, F Ferreirinha, C Vieira, M Duarte-Araujo,
P Correia-de-Sa
Universidade do Porto (UP), UMIB, Instituto de Ciencias Biomedicas
Abel Salazar (ICBAS), Laboratorio de Farmacologia e Neurobiologia,
Porto, Portugal
Gastrointestinal motility relies on the release of acetylcholine (ACh), purines and tachykinins from myenteric motoneurons and subsequent activation of smooth muscle bers. Muscarinic M3 receptors facilitate ACh
release indirectly by increasing adenosine outow acting on A2A receptors on enteric nerve terminals. Interstitial cells of Cajal (ICCs) are
endowed with NK1 and M3 receptors. These cells exhibiting pacemaker
activity are located between enteric nerve terminals and smooth muscle
bers - tripartite synapse. Little is known about the crosstalk between purinergic, tachykinergic and muscarinic receptors in the myenteric plexus.
We used the longitudinal muscle-myenteric plexus of the ileum of
healthy rats (C) and of neonatal capsaicin-treated rats (CAP), which eliminates tachykinergic nerve bres. The muscarinic agonist, oxotremorine
(300lM), and the A2A receptor agonist, CGS21680C (3nM), increased
[3H]ACh release (but not of SP) from stimulated myenteric nerves
respectively by 34 4% (n = 5) and 53 10% (n = 5) in C rats; these
drugs were devoid of effect in CAP animals. Activation of NK1 receptors with s,m-SP (300nM) restored CGS21680C facilitation (46 17%,
n = 6) to control levels in CAP rats. Synergism between A2A and NK1
receptors was conrmed using antagonists of NK1 (L732128, 20nM)
and A2A (ZM241385, 50nM) receptors. In contrast with M3 and NK1
receptors that co-localize on ICCs, the A2A receptors are mainly
expressed on cholinergic neurons. M3 receptor immunoreactivity paralels
the reduction in the number of SP positive nerve bers. Data demonstrate
that NK1 receptors interact synergistically with A2A, but not with M3,
receptors to control ACh release from myenteric nerve terminals.
Supported by FCT.
581
Paper No.: 657
SOLAVETIVONE REDUCES INTRACELLULAR CALCIUM IN
CULTURED MYOCYTES ISOLATED FROM GUINEA-PIG
ILEUM
Joelmir LV Silva(1), F De Andrade Cavalcant(2), VLC Rigoni(1),
TMS Silva(3), TL Nascimento(1), J Aboulaa(1), BA Silva(4),
VLA Nouailhetas(1)
(1) Universidade Federal de Sao Paulo, Brazil
(2) Universidade Federal de Alagoas, Brazil
(3) Universidade Federal Rural de Pernambuco, Brazil
(4) Universidade Federal da Paraba, Brazil
Solavetivone, a sesquiterpene, has been isolated from aerial parts of Solanum jabrense Agra & Nee, a plant only found in collections from the
Pico do Jabre in the State of Paraba, Brazil. We have previously shown
that SV has spasmolytic effect on guinea-pig ileum (GPI) by activating
BKCa thus indirectly blocking L-type VOCCs, therefore decreasing intracellular calcium concentration ([Ca2 + ]i). We investigated the cellular
mechanisms of SV by studying its action on the [Ca2 + ]i and cellular
cycle in cultured GPI myocytes. Experiments were approved by the Ethical Committee in Animal Research from LTF/UFPB. Longitudinal muscle layers isolated from GPI were minced into small pieces and seeded in
culture bottles containing fetal calf serum, glutamine, HEPES, streptomycin and penicillin. Cells were trypsinised, centrifuged, resuspended and
kept in a humidied incubator. Cells were identied by uorescence
microscopy after the reaction with antibody against a-myosin. Confocal
microscopy was used to assess [Ca2 + ]i. Plan time-space uorescent
images were obtained from cells previously loaded with Fluo-3. Cellular
cycle was analyzed by ow cytometry in cells loaded with iodide propidium. Solavetivone (0,1 lM) decreased uorescence in both carbacholand KCl-stimulated cells. Prolonged treatment with solavetivone did not
cause cell arrest, as a similar percentage of cells were observed before
(54 3 and 17 6%) and after solavetivone treatment (57 2 and
14 4%) in Gi/Go and S + G2/M phases, respectively. Collectively
these results corroborate previous results that the spasmolytic effect of
solavetivone on the GPI is due to [Ca2 + ]i reduction, eliminating any
possibility its effect could be to cytotoxicity.
Paper No.: 658

TRACHYLOBANE-360 DECREASES INTRACELLULAR
CALCIUM IN CULTURED MYOCYTES ISOLATED FROM
GUINEA-PIG ILEUM
Joelmir LV Silva(1), RF Santos(2), VLS Rigoni(1), JJF Tavares(2),
MS Silva(2), TL Nascimento(1), J Aboulaa(1), BA Silva(2),
VLA Nouailhetas(1)
(1) Universidade Federal de Sao Paulo, Brazil
(2) Universidade Federal da Paraba, Brazil
The diterpene ent-7a-acetoxytrachyloban-18-oic acid (trachylobane-360),
isolated from the hexanic phase of ethanolic extract of the stem bark of
Xylopia langsdorana. Its spasmolytic effect on guinea-pig ileum (GPI)
showed to be due to blockade of VOCCs and activation of channels-K+.
Furthermore, this trachylobane-360 presents antitumoral effect in leukaemia cells. We now investigated the effect of trachylobane-360 on intracellular calcium concentration ([Ca2 + ]i) and its possible toxicity on the
cellular cycle of cultured GPI myocytes. Experiments were approved by
the Ethical Committee in Animal Research from LTF/UFPB (Protocol
nordm; 0101/08). Longitudinal muscle layers isolated from GPI were
minced into small pieces and seeded in culture bottles containing fetal
calf serum, glutamine, HEPES, streptomycin and penicillin. Cells were
trypsinised, centrifuged, resuspended and kept in a humidied incubator
(CO2 5%). Cells were identied through the reaction with antibody
against a-myosin by uorescence microscopy. Confocal microscopy
(LSM 510 Confocal laser Scanning System) was used to assess [Ca2 + ]i.
Plan time-space uorescent images were obtained from cells loaded with
Fluo-3. Cellular cycle was analyzed by ow cytometry (FACS Calibur)
in cells loaded with 5lg/mL iodide propidium. Trachylobane-360 (105 M) decreased uorescence in carbachol-stimulated cells. Prolonged
treatment with trachylobane-360 did not cause cell arrest, as a similar
percentage of cells were observed before (55 1 and 13 1%) and after
trachylobane-360 treatment (59 2 and 14 2%) in Gi/Go and S+G2/
M phases, respectively. These results strongly corroborate previous
results that the spasmolytic effect of trachylobane-360 on the GPI is due
to [Ca2 + ]i reduction, eliminating any possibility this effect could be due
to cytotoxicity.
Paper No.: 1165

HEALTH AND DISEASE
ROLE OF AT1 AND AT2 RECEPTOR AND NAD(P)H OXIDASE
ON ANGIOTENSIN II-STIMULATED ANTIOXIDANT
ENZYMES ACTIVITY IN THE RAT HYPOTHALAMUS
Jose Silva, M Pastorello, S De Jesus, MG Matos, MR Garrido, A Israel
University Central of Venezuela, Department of Pharmacology, Caracas,
Venezuela
Angiotensin II (AngII) inuences blood pressure via its ability to stimulate NAD(P)H oxidase and produce reactive oxygen species (ROS) such
as O2-, which is sequentially metabolized by the enzymes superoxide
dismutase (SOD), catalase (CAT) and glutathione peroxidise (GPx). ROS
in turn, can activate the mitogen-activated protein kinase (MAPK) pathway which could be involved in events that result in ion channel modulation. The role of oxidative stress in AngII signalling in the
hypothalamus; and the relative involvement of AT1 and AT2 receptors in
these actions are still matter of research. The aim of this study was to
assesses, in vitro, the role of AT1 and AT2 receptors, NAD(P)H oxidase
and protein kinase C in the AngII capacity to stimulate antioxidant
enzymes activity in the rat hypothalamus, a brain structure known to
express a high density of AT1 and AT2 receptors and related with cardiovascular, neuroendocrine and hydromineral control. Male Sprague-Dawley rats (200-240 b.w.), were sacriced by decapitation and
hypothalamus was microdissected under stereomicroscopic control.
SOD, CAT and GPx activity were determined spectrophotometrically and
ERK 1/2 activation by Western blot analysis. In hypothalamus, AngII
increased SOD, CAT and GPx activity. These actions were prevented by
losartan, apocynin and chelerythrine and potentiated by PD 123319.
Likewise, AngII-induced ERK1/2 activation and it was diminished by
apocynin, chelerythrine and losartan, however PD 12319 had no effect.
We demonstrate that in the hypothalamus, AT1 and AT2 receptor/
NAD(P)H oxidase/PKC pathway are involved in AngII-increased antioxidant enzymes activity and ERK1/2 phosphorylation.
Paper No.: 1725

THE MECHANISM OF THE PRESSOR EFFECT OF
EPHEDRINE AND MEPHENTERMINE, THEIR INTERACTION
AT UPTAKE 1 AND THE ROLE OF NITRIC OXIDE
Joseph Simaan
American University of Beirut, Department of Pharmacology and
Therapeutics, Beirut, Lebanon
Ephedrine (E) and mephentermine (M) are a- and b- adrenoceptor agonists that act indirectly by entering across uptake 1 in sympathetic nerve
582
terminals to liberate norepinephrine (NE) from vesicular stores. E also
acts partly directly on receptors. Both exhibit tolerance to their pressor
effect after repeated administration of single doses. Cross tolerance
between E and M, their interaction at the uptake1 site and the role of
nitric oxide (NO) in the tolerance were explored in anesthetized rats in
which mean blood pressure was measured. E produced tolerance from
36+/)1 to 5+/)1 mmHg (-86+/)2%), and M from 23+/)3 to 4+/)1 mm
Hg (-82+/)2%). Blockade of the synthesis of NO with nitro-L-arginine
(15mg/kg) and restoration of the induced rise in pressure to control with
an infusion of nitroglycerin, a NO donor, partly reversed the tolerance to
E by 15+/)3 mmHg (+327+/) 65%) and to M by 7+/)1 mmHg
(+179+/)29%).M after a single dose of E exhibited complete tolerance.
E after M tolerance exhibited tolerance of -18+/)3 mmHg (-46+/)5%),
the residual pressor effect reecting its direct action. It is concluded that
E and M enter across uptake 1 to produce autoblockade of further entry
of their own molecules as well as reciprocal blockade of entry the molecules of each other. NO partly mediates the entry of both E and M across
uptake 1. Supported by funds from the American University of Beirut.
Paper No.: 2419

LYPODYSTROPHY DUE TO ANTIRETROVIRALS. CASE
PRESENTATION FROM RESOURCE-POOR HEALTH CARE
COUNTRY
model for focal epilepsy with secondary generalizations. Materials and

methods: The potential anticonvulsive effect of THIP was investigated in
the murine PTZ kindling model. Male NMRI mice, 4 weeks of age, were
injected with 43 mg/kg PTZ 3 times a week for 4 weeks and scored as
fully kindled if they had clonic convulsions 2 out of 3 times during the 3
last injections. Only fully kindled mice were used for the subsequent anticonvulsive study, where THIP was given s.c. 30 min prior to PTZ
injections in a roman square design. Results: The effect of THIP was
evaluated both by incidence, severity and duration of clonic convulsions
and by latency to clonic convulsions. THIP showed no anticonvulsant
effect in doses up to 4 mg/kg in PTZ kindled mice in any of the parameters used in this study. Conclusion: THIP showed no anticonvulsive
properties in the PTZ kindling model. The effect of THIP on synaptic
transmission and tonic GABAergic inhibition will be investigated using
hippocampal slices from PTZ kindled mice. This may offer an explanation for the apparent lack of anticonvulsant effect.
Paper No.: 617

DESIGN OF NOVEL AND SELECTIVE HDAC8-INHIBITORS
AS ANTICANCER AGENTS
Barij Nayan Sinha, V Jayaprakash,
Abraham Simatupang(1), F Suling(2)
Birla Institute of Technology, Department of Pharmaceutical Sciences,

Ranchi, India
(1) Universitas Kristen Indonesia, Department of Pharmacology, Center

for Study and Service of HIV, Faculty of Medicine, Jakarta, Indonesia
(2) Department of Internal Medicine, Faculty of Medicine, Universitas
Kristen Indonesia, Jakarta Indonesia
Selective inhibitors of Histone deacetylase8 (HDAC8) have gained attention due to signicant correlation of only HDAC8 expresion with
advanced disease and metastatis in neurobalastoma (Oehme I et al, Clin.
Cancer Res. 2009; 15: 91-99). Linker less hydroxamates with aromatic
cap were reported to have selective HDAC8 ihibition (Hrubec KK et al,
Bioorg. Med. Chem. Lett. 2007; 17: 2874-2878). These molecules were
designed by exploiting the large sub-pocket formation when inhibitor
with aromatic linker binds with HDAC8 (Somoza JR et al, Structure
2004; 12: 1325-1334). The movement of residues PHE152 and LYS33
resulted in the formation of large sub-pocket that accomadates the bulkier
aromatic linker portion of the inhibitor. With this background we enumerated a library of linker less pyrazoline based hydroxamates (175 molecules) using Smilib (open source software). 3D-conformers of the
library molecueles were generated using OMEGA (Open eye). Shape
similarity (ROCS, Open eye) and electrostatic similarity (EON, Open
eye) of these library molecules were computed with two potent HDAC8
selective inhibitors (linker less hydroxamates) reported earlier (Hrubec
KK et al, Bioorg. Med. Chem. Lett. 2007; 17: 2874-2878). Top ranked
molecules with a tanimato co-efcient of more than 1.5 were docked on
X-ray crystal structure of HDAC8 (PDB Code: 1T64) keeping PHE152
and LYS33 exible using AutoDock4.0. Selectivity towards HDAC8
was acessed by docking with 1T64 Met274 Leu274 (to mimic tunnel
region of HDAC1-3), an approach successfully employed succesfully by
our group earlier. By this approach we have identied novel and selective pyrazoline based hydroxamate inhibitors of HDAC8.
A 53 years old male patient, came 6 years ago complained chest pain.
Laboratoryndings: Cholesterol total 250 mg/dl, LDL-C 186 mg/dl,
HDL-C 48 mg/dl and TGwas 100 mg/dl. He was diagnosed with angina.
Angiography revealed an 80-90%occlusion of left coronary artery and he
was initiated to have PCI (percutaneouscoronary intervention) but it was
declined by the patient. Since then, patienthas been treated conservatively
with simvastatin, aspilet 80 1 x 1, ramipril 2.5mg, 2 x 0.5 tab, and nitrate
2 x 0.5 tab. Two years afterwards, patient hadsevere diarrhea and body
weight decreased 10 kg. Laboratory ndings (2006): HIV(+), CD4: 55
cells/mm3, VL: 21,500. Antiretrovirals (ARVs) ZDV, 3TC and NVP
areprescribed. Due to economic burden the patient did not have regular
follow-ups.In the last follow-up (2009): patient looked thin, with sunken
cheeksappearance, and skin color is darkened. A lipodystrophy due to
ARVs adverseevents is likely occured to the patient.
Paper No.: 2707

DISEASE AND THERAPY
ANTICONVULSIVE EVALUATION OF THIP IN THE MURINE
PENTYLENETETRAZOLE KINDLING MODEL
Charlotte Simonsen, A Kloppenburg, K Sonderskov, U Kristiansen,
S Hansen,
University of Copenhagen, Faculty of Pharmaceutical Sciences, Department of Pharmacology and Pharmacotherapy,
Introduction: GABAergic neurotransmission is a target for a number of
known antiepileptic drugs, but the potential of extrasynaptic GABAA
receptors as targets for antiepileptic drugs has not been elucidated. Given
the relative selectivity of THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol) for extrasynaptic GABAA receptors we are investigating the
effect of THIP in the murine pentylenetetrazole (PTZ) kindling model, a
Paper No.: 722

DRUG USE PATTERN IN HOSPITAL OUTPATIENTS AND
PRIVATE PRACTICE IN SYLHET CITY OF BANGLADESH
Sharadindu Kanti Sinha, WR Choudhury, MR Choudhury
Jalalabad Ragib-Rabeya Medical College, Department of Pharmacology,
Sylhet, Bangladesh
Prescription audit is a core procedure for evaluation of drug use pattern.
There is a dearth of such studies in Bangladesh. A prospective, cross-sec-
583
tional study was conducted in the Department of Pharmacology of Jalalabab R-R Medical College Hospital (JRRMCH), Sylhet, Bangladesh from
September 2008 to April 2009. Data were recorded in WHO recommended prescribing indicator format from 1200 and 1500 random
samples of prescriptions collected from JRRMCH outpatients and
general practitioners of Sylhet city respectively. Average number of
drugs/prescription, percentage of drugs prescribed in non-proprietary
names, percentage of antimicrobials, injections and from essential
drug list (EDL) was compared byZ test. The average number of
drugs per prescription was almost equal in hospital OPDs (3.28%)
and private practice (3.52%). The use of non-proprietary name was
too low in both the settings. Use of antimicrobials was high inhospital (61.50%) than general practice (35.45%) the result was highly
signicant statistically (p < 0.001). Patients attended in hospital
(20.66%) were treated more with injectables than in general practice
(4.60%) and the results were highly signicant statistically (p < 0.001).
Drugs from EDL were prescribed more in hospital (90.99%) than in general practice (86.23%). Appropriate workable interventions need to
improve the drug use behavior in Bangladesh. A combination of regulatory and non-regulatory interventions directed at producers, providers
and consumers of drugs and qualitative studies should be a part of the
efforts.
Paper No.: 1649

HEALTH AND DISEASE
POTENTIAL BENEFICIAL EFFECT OF ERGOTHIONEINE IN
PROTECTING ENDOTHELIAL DYSFUNCTION
Albert SM Sit(1), EYW Ho(1), RWS Li(1), YW Kwan(2), M Hausman(3),
PM Vanhoutte(1), GPH Leung(1)
(1) The University of Hong Kong, Li Ka Shing Faculty of Medicine,
Department of Pharmacology and Pharmacy, Hong Kong, PR China
(2) The University of Hong Kong, Faculty of Medicine, School of Biomedical Science, Hong Kong, PR China
(3) Total Nutraceutical Solutions, Washington, USA
Endothelial function is often impaired in diabetic patients, leading to
various pathophysiological processes in cardiovascular diseases. A
number of evidence has also demonstrated that reactive oxygen species is one of the major factors for diabetic endothelial dysfunction.
Ergothioneine, a chemical rich in mushroom, has been proposed to
possess potent antioxidant activities, involving the removal of cell
toxic radical species or chelating of metal ions. However, the biological relevance of these ndings is unclear because most of the preceding studies were performed in cell-free systems. In this study, we
aimed to investigate if ergothioneine can enter endothelial cells and
protect endothelial cells against diabetes-induced damage. Human
brain microvascular endothelial cells (HBMECs) were used in this
study. Ergothioneine is a transport substrate of organic cation transporter novel type 1 (OCTN-1) and our result of RT-PCR and Western blotting showed that OCTN-1 was present in HBMECs.
HBMECs were incubated in 5 mM (control) and 25 mM glucose
medium (which mimicked the hyperglycemia in diabetes) in the
absence of presence of ergothioneine. By means of MTT assay, we
found that the viability of HBMECs was signicantly lowered in
25 mM glucose condition but this detrimental effect of high glucose
could be reduced by ergothioneine, at a concentration as low as 10
nM. In conclusion, our result suggests that ergothioneine can be
taken up by endothelial cells and may be a potential protective
agent that can protect endothelial cells, particularly in the case of
diabetes.
Paper No.: 2930

CELL-BASED SCREENING FOR MODULATORS OF
REGULATOR OF G PROTEIN SIGNALLING PROTEIN 9
(RGS9) IMPLICATIONS IN PARKINSONS DISEASE
Benita Sjogren, RR Neubig,
University of Michigan, Department of Pharmacology, Ann Arbour, MI,
USA
Parkinsons disease (PD) is characterized by a loss of dopaminergic neurons leading to reduced dopamine in the striatum and impaired motor
function. Typically PD is treated with L-DOPA to restore dopamine levels a treatment that commonly gives rise to side effects, such as tremor.
Regulators of G protein signalling (RGS) proteins have emerged as
important regulators of signalling via G protein-coupled receptors
(GPCRs). They act as GTPase accelerating proteins at activated Gq and
Gi/o proteins. Compounds that modulate RGS function could selectively
enhance or inhibit GPCR signalling. RGS9 is highly expressed in the
striatum and modulates signalling via dopamine D2 receptors (D2R) and
opioid receptors. RGS9 knockout mice develop worse dyskinesias than
WT mice when treated with L-DOPA. Consequently, RGS9 enhancers
could be useful to improve side effects of L-DOPA treatment in PD.
There is also a great need for pharmacological tools to increase the
understanding of physiological roles of RGS proteins. We have developed a HEK-293 cell line expressing a D2R-Gao fusion protein that is
insensitive to pertussis toxin. These cells also express doxycycline-inducible RGS9 protein and its protein partner Gb5. Quinpirole, a selective
D2R agonist, induces phosporylation of ERK in a time- and concentration-dependent manner which is mediated through a Go pathway. Using
the SureFire AlphaScreen Phospho-ERK assay (Perkin Elmer) we
observe signicant suppression of quinpirole-induced ERK phosphorylation after induction of RGS9 protein expression. This assay is being
developed for high throughput screening for modulators of RGS9 protein
function.
(Supported by NIH-DA023252 and The Swedish research council VR524-2008-6963).
Paper No.: 674

CARDIOPROTECTIVE DRUG MILDRONATE ENHANCES THE
ANTI-DIABETIC ACTIVITY OF METFORMIN IN OBESE
ZUCKER RATS
Elina Skapare(1,2), E Liepinsh(1), B Svalbe(0), M Dambrova(1)
(1) Latvian Institute of Organic Synthesis, Laboratory of Pharmaceutical
Pharmacology, Riga, Latvia
(2) Riga Stradins University, Faculty of Pharmacy, Riga, Latvia
(3) University of Latvia, Faculty of Medicine, Riga, Latvia
Mildronate is a cardioprotective drug whose mechanism of action is
based on regulation of energy metabolism. We have shown earlier that
mildronate lowers blood glucose concentration and protects against diabetes complications in experimental model of type 2 diabetes in GotoKakizaki rats. The present study was carried out to investigate the metabolic effects of mildronate in combination with metformin in an experimental model of obesity and impaired glucose tolerance in obese Zucker
rats. Zucker rats were treated daily with metformin (300 mg/kg), mildronate (200 mg/kg) and combination of both drugs for 4 weeks. The
obtained results show that combination of mildronate and metformin signicantly decreased the rat weight gain for 19%, without affecting the
food intake. Treatment by both drugs separately did not inuence the
weight gain. Both tested drugs and combination decreased to a similar
584
extent fed- and fasted-state blood glucose for 1-2 mmol/l. In addition,
mildronate, metformin and combination signicantly decreased the fed
state plasma insulin concentration for 31%, 29% and 46%, respectively.
Thus, mildronate enhanced the anti-diabetic activity of metformin and
effect was mediated through increased activity of PPAR-a and PPAR-c in
heart and liver, as well as decreased activity in adipose tissue. In conclusion,
these results demonstrate for the rst time that combination of mildronate
and metformin effectively reduces the weight gain, blood glucose and
insulin concentrations in experimental model of obese Zucker rats.
Paper No.: 1752

HEALTH AND DISEASE
STATIN-DEPENDENT EFFECTS ON VASCULAR TONE AND
GENE EXPRESSION IN HUMAN ENDOTHELIAL CELLS
Cristine Skogastierna(1), L Luksha(2), K Kublickiene(2), E Eliasson(1),
A Rane(1), L Ekstrom(1)
(1) Karolinska Institute, Division of Clinical Pharmacology, Department
of Laboratory Medicine, Stockholm, Sweden
(2) Karolinska Institute, Division of Obstetrics & Gynaecology, Department of Clinical Science, Intervention & Technology, Stockholm, Sweden
Aim: HMG-CoA reductase inhibitors (statins) are believed to exert benecial effects against cardiovascular disease beyond correction of dyslipidemia, improving vascular function through modication of vascular
response, decreasing oxidative stress and reducing inammation. There
are however no in vitro studies directly comparing the effects of different
statins on the human vascular endothelium. Methods: Human umbilical
vein endothelial cells were exposed to four commonly used statins (simvastatin, atorvastatin, rosuvastatin and uvastatin). The mRNA expression of genes associated with vascular reactivity and oxidative stress
were analyzed by real-time PCR and promoter activities evaluated in
reporter systems. The formation of two major vasodilators PGI2 and NO
was analyzed by enzyme immunoassay. Moreover, the inuence on the
arterial resistance was assessed ex vivo in isolated human small arteries.
Results: The statins were shown to affect mRNA expression and promoter activity of investigated genes differently. Fluvastatin was the only
compound that signicantly increased the production of both PGI2 and
NO, whereas no induction was detected for the other statins, probably
explained in part by a seven-fold inhibition of the upstream COXenzyme activity. Ex vivo, only uvastatin induced rapid dilatation. Conclusions: Overall, our results indicate that statins could have major
impact on the vascular tone by modication of release of vasoactive factors from the endothelium. Specically, we show that statins may have
very diverse, compound unique effects on vascular response and endothelial health in vitro and that uvastatin seems to have the most favourable effect on vascular function.
Paper No.: 2024

INVESTIGATION OF ANILOCAIN AND LIDOCAIN
ANTIARRHYTHMIC ACTION ON THE BARIUM CHLORIDE
ARRHYTHMIA MODEL
Tatjana A Skorobogatova, VI Pantsurkin, MN Ivashev
Pjatigorsk State Pharmaceutical Academy (PSPA), Department of
Pharmacology, Pjatigorsk, Russian Federation
Aim: to study the anilocain and lidocain antiarrhythmic effect as a comparison drug on the barium chloride arrhythmia. Anilocain is a modern
anesthetic of the amide group of deep and prolonged action that supposes
the presence of its antiarrhythmic activity. Materials and methods: experiments have been carried out with narcotized Wistar rats, which were
implanted polyethylene catheter into the left carotid artery. Heartbeat frequency was registered by means of the computer program Bioshell.
The drugs under study were injected intravenously in low and average
doses two minutes before arrhythmogenic agent injection. The electrocardiogram was registered in the second standard position. Percent decrease
of heartbeat frequency after prophylactic anesthetic administration with
the successive barium chloride injection served as a criterion for antiarrhythmic effect of drugs. Results: animal ventricular extrasystole
occurred fteen-twenty minutes later barium chloride injection. Meanwhile total animal survival was observed. Anilocain and lidocain in low
doses slightly delayed the heartbeat frequency during the rst three minutes. At the same time lidocain average doses was sure to retard the
arrhythmia occurrence for ve minutes and anilocain did it twice longer.
Conclusions: local anesthetic anilocain was found to possess an antiarrhythmic activity (third class antiarrhythmic drugs) under experimental
conditions.
Paper No.: 1306

DISEASES
THE INFLUENCE OF RAMIPRIL ON THE INFLAMMATION
MARKERS AND THE IMMEDIATE PROGNOSIS OF
MYOCARDIAL INFARCTION
Anton Skotnikov, A Vertkin
Moscow State Medical University, Department of Clinical Pharmacology,
Pharmacotherapy & Ambulance, Moscow, Russian Federation
The aim of the research was to nd out the advantages of amprilan (ramipril) in the inuence on the inammation markers and the terminal point
within 1 and 3 months after the myocardial infarction. 60 patients of 4976 years of age with the diagnosis of acute coronary syndrome were
included into the research. The concentration of C-reactive protein, total
cholesterol, triglycerides, cholesterol of high-density lipoproteins and
cholesterol of low-density lipoproteins was determined in all the patients
during the rst 24 hours and 3 months later, with echocardiography
being carried out as well. The amprilan therapy reduced the frequency of
angina pectoris emergence in the post infarction period (65% against
36% in the control group), brought about the deceleration of the hypertrophy intensity growth as well asthe mass of the myocardial left ventricle, and reduction of C-reactive protein concentration in the blood (by
87,6% against 36,4%). In the amprilan group the systolic blood pressure
was reduced by 13,2%, diastolic blood pressure by 9,7%, and 62% of
patients reached the designated blood pressure. In the amprilan group the
level of total cholesterol was reduced by 8,1% against 4,9%, of triglycerides by 10,4% against 7,3% and cholesterol of low-density lipoproteins
by 7,8% against 9,8%, while the cholesterol of high-density lipoproteins
content raised by 8,4% against 2,9% in the control group accordingly.
No recurrent cases of myocardial infarction and, stroke were registered
in the amprilan group (against 4 cases of myocardial infarction e` 1 case
of stroke in the control group).
Paper No.: 2767

RAPID FUNCTIONAL UPREGULATION OF ETB RECEPTORS
IN RAT CORONARY ARTERIES DURING MYOGRAPH
STUDIES
Gry Freja Skovsted(1), AF Pedersen(1), M Sheykhzade(2), L Edvinsson(1)
(1) Glostrup research Institute, Glostrup Hospital, Department of Clinical
Experimetal Research, Glostrup, Denmark
585
Copenhagen, Denmark
In cardiovascular diseases, vasocontractile endothelin ETB receptors are
expressed in the smooth muscle layer of coronary arteries. Interestingly,
a similar receptor upregulation has been observed after organ culture of
arteries. This study aims to examine the time course of the early changes
in ETB receptor function in two different coronary artery segments: the
left anterior descending (LAD) and the septal coronary arteries (SCA).
The functional response to the selective ETB receptor agonist Sarafotoxin
6c was measured during incubation in a wire-myograph. Sarafotoxin 6c
was added cumulatively (1pM - 30 nM) at following time points of incubation: 1, 4, 7, and 24 h. At 1 h of incubation, the ETB receptor-mediated vasoconstriction was almost negligible. However, the contractile
response developed rapidly. In both arteries a 2- and 7-fold increase in
Emax was detected at 4 and 7 h of incubation, respectively. The contractile response reached its maximum at 7 h in LAD, whereas in SCA the
response continued to develop until 24 h of incubation. The transcriptional inhibitor, Actinomycin D (4 lM), or the MEK1/2 inhibitor, U0126
(10lM) attenuated the S6c mediated response signicantly in both arteries. The LAD and SCA displayed the ability to rapidly develop an ETB
receptor-mediated contractile response during incubation in a wire-myograph. This study suggests that the development of the ETB receptor-mediated contractile response depends on a transcriptional upregulation of the
receptor and involves the MEK/ERK type of MAPK.
Paper No.: 2923

SLCO1B1 AND CYP2C9 GENETIC VARIANTS COULD BE
ASSOCIATED WITH FLUVASTATIN INDUCED SIDE EFFECTS
IN SOLID ORGAN ALLOTRANSPLANT RECIPIENTS: CASE
SERIES
N Mirosevic Skvrce(1), L Zibar(2), Nada Bozina(3), VM Sarinic(1),
S Tomic(1)
(2) University Hospital Centre Osijek, Clinic for Internal Medicine, Osijek,
Croatia
(3) University School of Medicine and University Hospital Center, Department of Laboratory Diagnostics, Zagreb, Croatia
Recent studies found out strong associations between variants in the
SLCO1B1 gene, which encodes an organic anion transporter OATP1B1,
and myotoxicity of simvastatin, pravastatin and atorvastatin (The
SEARCH Collaborative Group, N Engl J Med. 2008; 359(8):789-99 and
Voora D et al, J Am Coll Cardiol. 2009; 54(17):1609-16). However, no
such study was done to investigate myotoxicity of uvastatin. According
to the available data, the SLCO1B1 genotypes have no signicant effect
on the pharmacokinetics of uvastatin (Niemi et al, Clin Pharmacol Ther
80: 356366), unlike CYP2C9 polymorphisms (Kirchheiner et al. Clin
Pharmacol Ther 2003; 74(2): 186-94). Using Real-time PCR methods we
genotyped 8 transplant recipients who developed myotoxicity caused by
uvastatin, 80 mg/day, (4 females and 4 males, median age 55 years,
range 44-62; 7 kidney transplant and 1 heart transplant) for
CYP2C9*1,*2,*,3 and SLCO1B1 388 A>G and 521T>C. All patients
were taking cyclosporine, a well known inhibitor of OATP1B1 transporter. Four patients had just CYP2C9 polymorphic variant, one just low
activity SLCO1B genotype and three had both CYP2C9 and SLCO1B
polymorphisms. One patient developed rhabdomyolysis (CYP2C9*3 carrier), one myopathy (SLCO1B1 388A/G and 2C9*2), two had increased
creatine kinase without myalgia (one carrier of 2C9*3, another 2C9*2,
388G/G and 521T/C), three had increased creatine kinase with myalgia
(two are carriers of 2C9*3, one had 388G/G) and one had increased creatine kinase with myalgia and increased transaminases (carrier of 2C9*3,
388A/G, 521T/C). Our preliminary data indicate associations between
myotoxycity of uvastatin with both CYP2C9 and SLCO1B1 polymorphysms.
Paper No.: 1800

EFFECTS OF FULLERENOL ON RAT UTERUS
CONTRACTION AND ANTIOXIDATIVE ENZYMES ACTIVITY
Marija Slavic(1), Z Orescanin-Dusic(1), R Radojicic(2),
S Milovanovic(3), A Djordjevic(4), M Spasic(1), D Blagojevic(1)
(1) University of Belgrade, Institute for Biological Research, Department
of Physiology, Belgrade, Republic of Serbia
(2) University of Belgrade, Faculty of Biology, Belgrade, Republic of
Serbia
(3) University of Eastern Sarajevo, Faculty of Medicine, Foca, Bosnia
and Herzegovina
(4) University of Novi Sad, Department of Chemistry, Faculti of Sciences, Novi Sad, Republic of Serbia
Investigations of biological effects of nanoparticle fullerenol C60(OH)24
have provided evidence for reactive oxygen species (ROS) and/or reactive nitrogen species (RNS) scavenging properties in vitro and in vivo.
The complex set of enzymes called antioxidative defence, balances concentration of ROS toward non-toxic homeostatic levels. Therefore we
evaluated direct pharmacological effect of fullerenol ex vivo and correlation of these effects to changes in endogenous antioxidative defence
activity. Isolated uteri of virgin Wistar rats were treated with fullerenol
dissolved in water or in dimethylsulphoxide (DMSO) and the activity of
antioxidative defence enzymes: manganese superoxide dismutaseMnSOD, copper-zinc superoxide dismutase-CuZnSOD, catalase-CAT,
glutathion peroxidase-GSH-Px and glutathion reductase-GR, was measured at the end of the treatment. Our results provide evidence that fullerenol in concentration of 400lM cumulative decreased DMSO relaxing
effect on spontaneously active uterus. Fullerenol also prevents DMSO
induced decrease in GSHPx activity. Fullerenol doesnt show direct pharmacological effects on isolated rat uterine smooth muscle activity
although has capacity to diminish DMSO induced effects in some extent.
Paper No.: 2297

DISCOVERY
TRANSPORT CHARACTERISTICS OF CAFFEINE IN HUMAN
INTESTINAL CACO-2 CELL MONOLAYER
Libuse Smetanova(1), V Stetinova(1), D Kholova(1), J Kvetina(1),
J Smetana(2), Z Svoboda(1)
(2) University of Defence, Faculty of Military Health Sciences, Hradec
Our study aimed: 1) to characterize the mechanisms of caffeine transport
across the intestinal barrier, 2) to classify caffeine according to Biopharmaceutics Classication System (BCS), 3) to predict drug intestinal
absorption in humans. Caco-2 cell monolayer has been widely accepted
as a potent in vitro model for the rapid screening of the intestinal drug
absorption. The objective of the BCS is to predict in vivo pharmacokinetic performance of drugs from in vitro measurements of permeability
and solubility. Caffeine transport (0.1, 0.3, 1 and 10 mmol/l) was studied
in Caco-2 cell monolayer in apical to basolateral (AP-BL) and basolateral
to apical (BL-AP) direction, under iso-pH 7.4 and pH-gradient (6/7.4)
conditions. The relative contribution of the paracellular route was estimated using Ca2+-free transport medium (opening tight junctions). The
monolayer integrity was checked before the experiment by phenol red
permeability and during the transport studies simultaneously by 14C-mannitol permeability. The caffeine transport seems to be transcellular mediated by passive diffusion (transport linear with time, transport direction
586
and pH independent, displaying non-saturable (rst-order) kinetics, with
high permeability coefcient Papp (AP-BL) = 46.3-53.5 x 10-6 cm/s, Papp
(BL-AP) = 45.6-49.4 x 10-6 cm/s. The paracellular route is only a minor
way of caffeine transport as caffeine Papp was not changed by using
Ca2 + -free medium although Papp of mannitol increased (conrmation of
opening tight junctions). High solubility and high permeability of caffeine rank it among class I of BCS and well absorbed compounds.
The study was supported by the research project GACR 305/08/0535.
Paper No.: 2170

IDENTIFICATION OF G PROTEIN-COUPLED RECEPTOR
(GPCR) TARGETS USING RT-PCR ARRAY: VALIDATION OF
PAR1 AS THE MOST HIGHLY EXPRESSED GPCR IN
REGULATION OF CARDIAC FIBROBLASTS
Aaron Snead, P Insel
(0.25, 0.5 or 1.0 lg/mL) or RPMI 1640 medium (control). In vivo, CdtV
(0.18 mg/kg) or saline (control) was administered subcutaneously to rats
at different time periods: 2 h, 1, 4 or 14 days before or 1 h after administration of carrageenan. In vitro, CdtV reduced signicantly the phagocytic activity of neutrophils (0.25 lg/mL: 38%, 0.5 lg/mL: 36% and
1.0 lg/mL: 34%). The injection of a single dose of CdtV also reduced
the percentage of phagocytosis (2 h: 42%, 1 day: 43%, 4 days: 51%,
14 days: 23% and 1 h later: 39%). These results show that CdtV inhibits
in vitro and in vivo the phagocytic activity of neutrophils, as it has been
described for macrophages. Thus, taking into account that in some
inammatory diseases neutrophils have exacerbated functionality, CdtV
may be an important tool in controlling inammatory diseases.
Paper No.: 2975

MECHANISM OF THE ANTI-HYPERTROPHIC ACTION OF
RGS2 IN CARDIOMYOCYTES
Alina J Sobiesiak, CH Nguyen, P Chidiac
University of California at San Diego, Department of Pharmacology, La

Jolla, CA,USA
Cardiac brosis occurs in advanced age, after injury (e.g. myocardial
infarction) and in numerous disease states (e.g. diabetes mellitus). Fibrosis is characterized by the transformation of broblasts to myobroblasts,
which contribute to the excessive accumulation of collagen-rich extracellular matrix and thereby reduce cardiac function. Previous work has suggested that certain GPCRs may have either pro-or anti-brotic activity. In
order to identify new therapeutic targets for cardiac brosis we used an
unbiased approach (GPCR RT-PCR array) to dene the most abundantly
expressed GPCRs that may function to regulate the broblast/myobroblast transformation in rat cardiac broblasts (rCFs). Pharmacological
analyses were then used on freshly isolated rCFs to validate the most
abundant of these receptors as a functional target. We identied proteaseactivated receptor 1 (PAR1) as the most abundant GPCR in rCFs. Subsequent studies revealed that activation of PAR1 by thrombin elevates
(with distinct kinetics) the expression of a variety of probrotic markers
in rCFs. Thrombin functions via at least two distinct post-receptor mechanisms to regulate broblast/myobroblast transformation, one of which
is dependent on the synthesis of sphingosine-1-phosphate. Our data thus
dene the range of GPCRs expressed by rCFs and together with our subsequent pharmacological results provide a proof-of-principle that such an
approach can identify novel disease-relevant GPCR targets. Supported
by the NIH and the Ellison Medical Foundation.
Paper No.: 694

CROTALUS DURISSUS TERRIFICUS SNAKE VENOM: A
POTENTIAL NATURAL PRODUCT IN CONTROLLING
INFLAMMATORY DISEASES
Tatiane Soares de Lima, S Coccuzzo Sampaio, MC Cirillo
Butantan Institute, Laboratory of Pathophysiology, Sao Paulo, Brazil
Crotalus durissus terricus venom (CdtV) inhibits the spreading and
phagocytic activity of macrophages and also has anti-inammatory properties, inhibiting the paw edema and cell migration induced by carrageenan. Taking into account the role of neutrophils in inammation, the
effect of CdtV on C3b-receptor-mediated phagocytosis by neutrophils
was investigated here. Neutrophils were obtained from the peritoneal
cavity of rats 4 h after intraperitoneal administration of carrageenan
(4.5 mg/kg), and phagocytosis of opsonizaded zymosan was evaluated
after in vitro or in vivo treatment with CdtV. In vitro, neutrophils
(1.2x106 cells/mL) were incubated with different concentrations of CdtV
University of Western Ontario, Department of Physiology and Pharmacology,London, Ontario, Canada

Chronic stimulation of cardiac G protein-coupled receptors, particularly
through Gq/11 and Gs signalling pathways, promotes cardiomyocyte
hypertrophy and development of heart failure. RGS2 potently attenuates
Gq and Gs signals in the heart and has emerged as an important negative
regulator of cardiac hypertrophy. Recent studies suggest that deregulation
of RGS2 contributes to pathogenesis of cardiovascular disease, marking
RGS2 as a potential therapeutic target. We have discovered a novel
RGS2 interaction with the initiation factor eIF2B, which catalyzes a ratelimiting step in mRNA translation. This interaction impedes eIF2B function, leading to an inhibition of de novo protein synthesis. The eIF2Binteracting domain of RGS2 has been localized to a 37-amino acid
sequence within the RGS homology domain. As protein synthesis is
required for cardiac hypertrophy, this interaction may serve as an additional mechanism for the established anti-hypertrophic effect of RGS2.
We examined this possibility using isolated neonatal rat cardiomyocytes
as a model system. Full-length RGS2 and the isolated eIF2B-binding
domain (RGS2-minigene) were expressed in cardiomyocytes using adenoviral gene delivery to test their abilities to block a1- and -adrenergic
receptor-induced hypertrophy. Adrenergic stimulation promoted a hypertrophic response in cardiomyocytes, as determined by increases in cell
size, induction of protein synthesis, and upregulation of a -skeletal actin
and atrial natriuretic peptide mRNA. The hypertrophic phenotype was
eliminated in cardiomyocytes infected with adenovirus encoding either
full-length RGS2 or RGS2-minigene as compared to control adenovirus.
The present results suggest that RGS2-dependent regulation of protein
translation contributes to its ability to attenuate G protein mediated
hypertrophy in cardiomyocytes.
Paper No.: 2075

CLINICAL IMPORTANCE OF THE EVIDENTIARY LEVEL
BASIS FOR DRUG APPROVAL IN ONCOLOGY
Gerald Sokol(1,2,3), L Loftus(2), B Djulbegovic(2), L Cantilena(3)
(1) H. Lee Moftt Cancer Center, Tampa, FL, USA
(2) Florida Cancer Institute/New Hope, Hudson, FL, USA
(3) Uniformed Services University, Bethesda, MD, USA
Introduction: Most nations have a formulated plan for the regulatory
approval of drugs, and international cooperation is ongoing via the ICH.
Cooperative endpoints of drug evaluation for approval attempt to attain a
587
strong evidentiary basis for approval with rigorous criteria for study efcacy, safety, PK, PD, and labeling. Achievement of strong evidentiary
basis is sometimes affected by critical disease need, economics, lack of
alternative treatments as well as public or physician demand for a given
agent. Methods: In this investigation, 23 FDA-approved oncology drugs
from 2004 to 2009 were reviewed to categorize the level of evidentiary
excellence of the approval data based on ICH guidelines. The published
regulatory basis of approval, product labeling, and pertinent literature
were reviewed for these 23 agents to categorize the evidentiary level of
approval by ICH Guidelines. Results: Most agents were found to be
based on Level I evidence but of variable quality. Six of the 23 failed to
contain Level 1 evidence. Discussion and Conclusions: Despite international agreement regarding level of evidentiary basis for drug approval,
dissemination of this information with a newly approved agent is not
routinely performed. Simple regulatory approval alone deprives physicians unschooled in drug evaluation of necessary knowledge for optimum prescribing. Proper dissemination of level of evidence information
should be considered by regulatory agencies.
Paper No.: 1532

DISCOVERY
MDR1 GENE C3435T POLYMORPHISM IS ASSOCIATED WITH
THE RISK OF THREATENED PREGNANCY INTERRUPTION
E Sokova, I Ignatiev, A Prilutsky, Dmitry A Sychev, V Kukes
SCEMAP Roszdravnadzor, Institute of Clinical Pharmacology, Moscow,
Russian Federation
Introduction: Threatened pregnancy interruption (TPI) can occur during
the all period of gestation. The precise cause of TPI is rarely discovered. The placental drug-transporting P-glycoprotein (P-gp) protects the
developing embryo and fetus from toxic substances. A number of SNPs
identied in ABCB1 gene may inuence P-gp expression level and
function. C3435T SNP attracts the most attention because of its ability
to decrease P-gp mRNA stability and so far to reduce P-gp amount in
barrier membranes. Aim of study: to determine whether C3435T polymorphism in MDR1 gene is associated with the risk of threatened pregnancy interruption in Russian women? Materials/Patients: 28 pregnant
Russian women with TPI in the 1st and mid-trimester and 56 without
TPI were genotyped for MDR1 C3435T SNP by PCR-RFLP method.
Results: The allele/genotype frequencies of MDR1 C3435T polymorphism were: 0% CC, 71,43% CT, 28,57% TT in group with TPI and
17,86% CC, 67,86% CT, 14,28% TT in group without TPI. There were
a signicant difference in genotype (chi-square criteria, p = 0,030) frequencies between groups with/without TPI. Conclusion: MDR1 gene
C3435T polymorphism is associated with threatened pregnancy interruption in Russian women; TT genotype is the risk factor this disorder
occurrence.
Paper No.: 1709

REVERSAL OF SUPPRESSIVE ACTION OF BETA-AMYLOID
ON LONG TERM POTENTIATION IN RAT HIPPOCAMPAL
SLICES BY DONEPEZIL
Elena Solntseva, N Kapai
Russian Academy of Medical Sciences, Center of Neurology, Department of Brain Research, Moscow, Russian Federation
gets for donepezil, N-methyl-D-aspartate receptors (NMDARs) and

sigma1 receptors, are described in literature. It is generally believed that
b-amyloid peptides (Ab) contribute to the pathogenesis of AD. Low concentrations of Ab were shown to impair long-term potentiation in hippocampus (LTP), a cellular model for learning and memory. The purpose
of our study was to determine whether Ab1-42 effect on LTP can be
reversed by therapeutically-relevant concentration of donepezil (0.5 lM).
Possible involvement of NMDARs into mechanisms of donepezil action
was also studied. LTP was investigated on rat hippocampal slices. Population spikes (PS) were recorded from the pyramidal layer of the CA1
region. LTP was induced by one train of high frequency stimulation
(100 Hz, 1 s). NMDA-currents were investigated on acutely isolated
CA1 pyramidal neurons by the patch-clamp technique in the whole-cell
conguration. In the control group, the amplitude of PS 30 min post-tetanus reached 153 14% (n = 11). Ab (200 nM) markedly suppressed the
LTP induction or even caused the depression of PS: the amplitude of PS
30 min post-tetanus was 82 15% (n = 7). This suppression of LTP
could be prevented when donepezil was co-administered with Ab: the
amplitude of PS 30 min post-tetanus was 161 24% (n = 7). We also
demonstrated that NMDA-current was affected by neither Ab nor donepezil. We have concluded that donepezil antagonizes the suppressive
action of 200 nM Ab1-42 on LTP in rat hippocampal slices in
NMDARs-independent manner.
Paper No.: 860

DISCOVERY
RELAXATORY EFFECT OF MAGNESIUM ON MESENTERIC
VASCULAR BEDS DIFFERS FROM NORMAL AND
STREPTOZOTOCIN INDUCED DIABETIC RATS
Nepton Soltani(1), F Kharazmi(1), M Keshavarz(2)
(1) Hormozgan University of Medical Science, Department of Physiology, Bandar Abbas, Hormozgan, Iran
(2) Tehran University of Medical Science, Faculty of Medecine, Department of Physiology, Tehran, Iran
Magnesium deciency has recently been proposed as a novel factor
implicated in the pathogenesis of diabetes complications. The vessel
magnesium mechanism is not very well known. The present study was
designed to determine the mechanism which is involved in magnesium
sulfate vasorelaxation in diabetic rat vessels. Diabetes was induced by a
single tail injection of streptozotocin. Eight weeks later, superior mesenteric arteries of control and diabetic animals were isolated and perfused
according to the McGregor method. Prepared vascular beds were constricted with phenylephrine to induce 70-75% of maximal constriction.
Magnesium sulfate at concentrations of 0.001 M to 0.1 M was added
into the medium and perfusion pressure was then recorded. Mesenteric
bed baseline perfusion pressure in intact and denuded endothelium of
diabetic groups was higher than controls. In all groups, relaxant
response to magnesium in mesenteric bed was attenuated after endothelium removal, but a relaxatory effect appears at high concentration. In
the presence of N (omega)-nitro-L-arginine methyl ester (L-NAME),
magnesium-induced relaxation was signicantly suppressed in intact
mesenteric bed of control animals but in diabetics, the relaxant response
was slightly inhibited. Gutamic acid could not suppress magnesium
relaxation in intact mesenteric bed of diabetic animals, but in presence
of Theopheline magnesium-induced relaxation was signicantly suppressed in intact mesenteric bed of diabetic. From the results of this
study, it can be concluded that magnesium-induced endothelium dependent and endothelium independent vasorelaxation are mediated by nitric
oxide in control rats while in diabetic animals adenosine receptor may
be involved.
Donepezil is a potent acetylcholinesterase inhibitor used for the treatment

of Alzheimers disease (AD). Two additional therapeutically relevant tar-
588
Paper No.: 2755
EFFECTS OF POLYMORPHISM OF MDR1, SLCO1B1 AND
CYP3A4 ON THE PHARMACOKINETICS OF SIMVASTATIN IN
HEALTHY KOREANS
parison with recently X-ray adrenoceptors obtained structures and previous D2DR models were carried out. Then, ex-ligand and residues
docking methodology were used for studying the capacity to use these
models in ligand recognition and afnity estimation studies. Docking
results suggest optimal prediction of binding mode and approximation in
binding afnities for ligand tested.
Hankil Son(1,2), SB Jang(1,2), YJ Lee(1,2), LA Lim(1,2), D Lee(1,2),

S Cho(1,2), J Chung(1,2), K Park(1,2)
(1) Yonsei University College of Medicine, Department of Pharmacology, Seoul, Korea
(2) Brain Korea 21 Project for Medical Science, Seoul, Korea
The purpose of this study was to investigate the effects of the polymorphism of CYP3A4*4, MDR1 and SLCO1B1 on simvastatin pharmacokinetics in healthy Koreans. The subjects received a 40 mg of simvastatin
orally once (Study I; n = 11) or daily from day 1 to 8 (Study II; n = 13).
Blood samples were collected up to 48 hours after dosing for studyn
and day 8 of study II, and for up to 24 hours for day 1 of studyo`.
Plasma concentrations of simvastatin and simvastatin acid, the active
metabolite, were analyzed by HPLCCMS/MS method. AUC, Cmax, tmax,
and t were determined by a non-compartment method using WinNolin.
The genotypes of CYP3A4*4 and selected SNPs of MDR1 (c.1236C>T,
c.2677G>A/T, c.3435C>T) and SLCO1B1 (c.388A>G, c.521T>C, g.11187G>A) were investigated. Haplotype analyses of MDR1 and
SLCO1B1 genotypes assembly were performed using Haploview 3.2.
Pharmacokinetic differences between the genotype or haplotype groups
were evaluated using the Kolmogorov-Smirnov test or the Kruskal-Wallis
test with S-PLUS 7.0. P-values less than 0.05 were considered statistically signicant. The respective mean of AUC48h, AUC and Cmax of
simvastatin acid were 67.2% (p = 0.0152), 66.2% (p = 0.0152) and
61.8% (p = 0.0676) higher in subjects with MDR1 c.1236TT genotype
than in those with the MDR1 c.1236CC or c.1236CT genotype. Other
genotypes did not have a signicant effect on the pharmacokinetics of
simvastatin acid. This study showed that the MDR1 polymorphism
markedly affected the pharmacokinetics of simvastatin acid, indicating
that genetic variation in P-glycoprotein function may have clinically
important inuences on the risks and benets of simvastatin treatment.
Paper No.: 546

7TM-HOMOLOGY MODELLING OF LONG FORM OF HUMAN
AND RAT D2 DOPAMINE RECEPTOR AND VALIDATION FOR
EVALUATING LIGANDS RECOGNITION
Paper No.: 2082

ACTIVATION OF GABA B RECEPTORS ABOLISHES
DOPAMINE RELEASE INDUCED BY SYSTEMIC
AMPHETAMINE IN THE RAT LATERAL SEPTUM
Ramon Sotomayor-Zarate, K Araya, P Pereira, P Carreno, K Gysling,
G Quiroz, E Blanco
(1) Millennium Institute of Stress and Addiction, Laboratory of
Biochemistry Pharmacy, Department of Cell and Molecular Biology,
Santiago, Chile
The Lateral Septum (LS) is a brain nucleus involved in various mental
disorders such as anxiety and drug addiction. In 2005, we demonstrated
that intravenous morphine administration produces a dose-dependent
increase of dopamine (DA) release in LS (Sotomayor R et al, J Neurosci
Res 81: 132-139). This morphine effect is produced through an inhibition
of GABAergic tone in Ventral Tegmental Area (VTA). In the present
study, we investigated whether systemic amphetamine, known to release
DA in the Nucleus Accumbens (Nacc), will also release DA in the LS.
Microdialysis probes were implanted in the LS and Nacc of anesthetized
Sprague-Dawley rats, and DA extracellular levels were determined by
HPLC coupled to electrochemical detection. Our results show that systemic amphetamine administration (2 mg/Kg i.p.) induced a signicant
increase in DA extracellular levels in Nacc but not in LS. Interestingly,
intra LS perfusion of amphetamine induced a signicant increase in DA
extracellular levels in LS. We next tested if the GABAergic neurotransmission in LS is responsible for the lack of response of LS DA to systemic amphetamine administration. A signicant increase in LS DA was
induced by systemic amphetamine administration when CGP-52432
(50 lM), a GABA-B selective antagonist, was perfused through the microdialysis probe in LS. Our results suggest that the systemic administration of amphetamine induces an increase in LS GABA release and the
consequent activation of GABA B receptors counteracting the direct
effect of amphetamine on LS DA release.
Funded by Grants MSI P06/008-F and FONDECYT 3095007 (RSZ) and
1070340 (KG).
Marvin A. Soriano-Ursua(1), JO Ocampo-Lopez(2), JG Trujillo-Ferrara(3), J Correa-Basurto(4)

(1) Escuela Superior de Medicina-Instituto Politecnico Nacional,
Departamento de Fisiologa y Farmacologa, Mexico City, Mexico
(2) Laboratorio Medico Qumico Biologico, Cerro San Antonio, Mexico
(3) Escuela Superior de Medicina-Instituto Politecnico Nacional,
Departamento de Bioqumica, Mexico City, Mexico
(4) SEPI-Escuela Superior deMedicina-Instituto Politecnico Nacional,
Laboratorio de Modelado Molecular y Bioinformatica, Mexico City,
Mexico
The D2 dopaminergic receptor (D2DR) is considered target for treatment
of Parkinson Disease, as well as for other disorders in the Central Nervous System (CNS) like schizophrenia. For obtaining insights about
ligand recognition and drug design, 3-D models for D2DR have been
built based in rst-principles theoretical and computational techniques
and/or the knowledge about the 3-D structure of some rhodopsine molecules. Recently, 3-D structures of closer related receptors like adrenoceptors have been elucidated. Here, we have built 3-D models of the long
form of human and rat D2DRs. 3-D models were obtained for homology
Modelling and minimized on CHARM27 with NAMD2 program. Com-
Paper No.: 2097

SESSION - ONCOLOGY
STUDY IN VITRO OF THE MECHANISM OF ACTION BY
WHICH COUMARIN AND 7-HYDROXYCOUMARIN
PROMOTES APOPTOSIS IN TWO HUMAN LUNG
ADENOCARCINOMA CELL LINES
Maribel Soto-Nunez, P Cuautle-Rodrguez, JA Molina-Guarneros
UNAM Faculty of Medicine, Department of Pharmacology, Mexico DF,
Mexico
Introduction: 7-hydroxycoumarin is the main biotransformation product
of coumarin in human. Coumarin and 7-hydroxycoumarin, at concentrations below 150 ug/ml, have an antiproliferative effect in cells of human
lung carcinoma, mediated by a decreased expression of cyclin D1(Jimenez-Orozco FA et al, Lung Cancer 2001; 34:185-194). At concentrations
greater than 150 &ug/ml, produces morphological changes characteristic
of apoptosis only on adenocarcinomas (Lopez-Gonzalez JS et al, Lung
589
Cancer 2004; 43:275-283). The aim of this study was identify the mechanism of action by which coumarin and 7-hydroxycoumarin promotes
apoptosis in lung adenocarcinoma cell lines A-549 and A-427. Materials:
Coumarin and 7-hydroxycoumarin, human adenocarcinoma lung cell
lines A-549 and A-427 (ATCC). Results: Coumarin and 7-hydroxycoumarin treatment (2mM, 24h) induce morphological changes characteristics
of apoptosis in A-549 and A-427 cells. The treatment with 7-hydroxycoumarin induce an increase of 40% of caspase 3 enzimatic activity in
A-549 cells and 12% in A-427 cells. The treatment with 7-hydroxycoumarin also induce an increase in cleavage of PARP and in the expression
of Bax with a concomitant decrease of Bcl-2. Conclusions: 7-hydroxycoumarin induces cell apoptosis, associated with the regulation of the
Bcl-2 family proteins, enzymatic activation of caspase 3 and cleavage of
PARP in both lung adenocarcinoma cell lines. These results indicate that
coumarin and 7-hydroxycoumarin might be a prospect drug for the treatment of lung adenocarcinomas. CONACYT98729
Paper No.: 3398

THE RAAS HYPERTENSION PHYSIOLAB PLATFORM:
A SYSTEMS MODELLING APPROACH TO HYPERTENSION
Antoine Soubret(1), A Sarkar(2), M Hallow(2), B Bieth(2), A Georgieva(2), A Lo(3), J Beh(3), S Ermakov(3), S Ramanujan(3), M Rodrigo(3),
D Keefe(2), A Charney(2)
of the signals transduced by the AT1 receptor. Among the pathophysiological actions, it is well known that Ang II exerts pro-inammatory
effects by inducing the production of adhesion molecules and cytokines,
including in the vascular inammation events associated with hypertension and atherosclerosis. Since most of the described actions of Ang(1-7)
counteract those elicited by AngII, in this work we investigated the role
of the Ang(1-7)-Mas receptor activation in the inammatory response
induced by LPS in mouse peritoneal macrophages. Transcriptional
expression patterns of the Mas receptor and the pro-inammatory TNF-a
and IL-6 cytokines were determined by real-time PCR. Our results
showed that Mas receptor transcripts were up-regulated by 8-fold in
LPS-induced macrophages as compared with the control group. Furthermore, the observed increased expression of both TNF-a and IL-6 cytokines in LPS-induced macrophages was signicantly attenuated by
Ang(1-7) treatment; a phenomenon abolished by the Ang(1-7)-Mas
receptor antagonist A779. Taken together our ndings demonstrate a possible anti-inammatory activity of the Ang(1-7)-Mas receptor axis in
LPS-induced macrophages.
Paper No.: 2023

HEALTH AND DISEASE
ADDING EZETIMIBE TO STATIN HALTS PROGRESSION OF
CAROTID PLAQUE
J David Spence
(1) Novartis Pharma AG, Basel, Switzerland

(2) Novartis Pharmaceuticals, NJ, USA
(3) Entelos Inc., Foster City, CA, USA
The RAAS Hypertension model presented here captures the pathophysiological mechanisms of hypertension and its impact on end-organ status
using a deterministic Modelling approach. The resulting RAAS hypertension model features a modular design with four key modules, namely,
systemic, renal, cardiovascular and nervous systems. Here, we present
the features of the rst generation RAAS hypertension platform which
includes a representation of the kidney as well as the RAAS pathway
and its interactions with sodium and water regulation. The methodology
and the different Modelling tools used for model parameterization and
simulation will be described. The application of the platform to simulate
systemic and renal biomarkers in selected patient phenotypes following
treatment with angiotensin-converting-enzyme inhibitors (ACEI), angiotensin-receptor blockers (ARB) and calcium channel blockers (CCB) is
reported. These simulations demonstrate that the platform is a valuable
tool to elucidate multi-faceted disease progression patterns in various
hypertensive patient phenotypes and to characterize the effect of various
pharmacological options to treat hypertension.
Paper No.: 3312

EVIDENCE FOR AN ANTI-INFLAMMATORY ROLE OF THE
ANG(1-7)-MAS RECEPTOR AXIS IN LPS-INDUCED
MACROPHAGES
Laura Souza, C Costa-Neto
University of Sao Paulo, Department of Biochemistry and Immunology,
Ribeirao Preto, Brazil
The renin angiotensin system (RAS) is classically involved in maintaining cardiovascular function by regulating blood pressure and electrolyte
homeostasis. The classical signalling pathway of this system is represented by angiotensin II (AngII) binding to either AT1 or AT2 receptors,
followed by G-protein signalling cascade activation. More recently, alternative pathways within the RAS have been described, such as the
Ang(1-7)-Mas receptor axis, which it has been shown to counteract some
University of Western Ontario, Robarts Research Institute, Stroke Prevention & Atherosclerosis Research Centre, London, Ontario, Canada
In recent years failure to show benet of ezetimibe on carotid intimamedia thickness (IMT) despite substantial lowering of LDL has raised
controversy regarding possible harmful effects of ezetimibe. We studied
carotid progression/regression in patients being followed with measurement of carotid total plaque area (TPA), a phenotype that is more closely
related to coronary risk factors and which more strongly predicts coronary risk than IMT. Included in the study were all 150 patients (43%
female) who had carotid plaque measurment performed annually for two
years before and two years after initiation of ezetimibe. Usually ezetimibe was added because the patient could not tolerate high doses of statins. The mean (SD) of baseline characteristics of the patients were: age
65.3 10.4 years,
BP
Systolic
141 19.6,
BP
Diastolic
78 12 mmHg; TChol 4.17 0.94, Trig 1.30 0.64, HDL
1.40 0.48. LDL declined from 2.53 0.85 to 2.04 0.98 mmol/L
(p < 0.0001). 10.5% were taking no statins because of myopathy; 37%
were on low-dose statin, 14.3% on medium dose and 2.1% on high-dose
statins (eg. <20mg, 20-40mg or 80mg of atorvastatin, or equivalent doses
of other statins). 16.5% were taking brates, and 5.9% were taking niacin. Before ezetimibe TPA progressed by 5.3mm2 in 2 years; after ezetimibe TPA declined by -3.8mm2 in two years (p2 = 0.12). Ezetimibe
has the expected effect on carotid atherosclerosis when atherosclerosis is
measured. It is better to measure plaque than IMT when assessing antiatherosclerotic effects of therapies.
Paper No.: 1333

THE EFFECT OF ANTI-PARKINSONIAN DRUGS ON OXIDATIVE STRESS INDUCED PATHOLOGICAL [3H]DOPAMINE
EFFLUX AFTER IN VITRO ROTENONE EXPOSURE IN RAT
STRIATAL SLICES
Beata Sperlagh(1), E Milusheva(2), M Baranyi(1), Z Hracsko(1),
S Vizi(1)
(1) Hungarian Academy of Sciences, Institute of Experimental Medicine,
Department of Pharmacology, Budapest, Hungary
590
(2) Bulgarian Academy of Sciences, Institute of Neurobiology, Soa,
Bulgaria
Introduction: Recent studies pointed to the role of environmental toxins
causing mitochondrial dysfunction and the pathological interaction of
mitochondrial dysfunction and oxidative stress is believed to play a
determinant role in the development of the neurodegeneration. Methods:
An in vitro model of mitochondrial dysfunction with subsequent oxidative stress was elaborated and utilized to study the effect of drugs, currently used for the treatment of Parkinsons disease, on pathological
H2O2-evoked [3H]dopamine efux and the formation of toxic dopamine
metabolites in rat striatal slices. Results: 60 min rotenone (0.1-10
microM) pretreatment decreased dopamine content and [3H]dopamine
uptake, as well as ATP level and energy charge of the slices. In addition,
a robust potentiation of H2O2-evoked [3H]dopamine efux and the formation of dopamine quinone in the efuent was detected. L-DOPA (200
microM) markedly elevated resting but not 100 microM H2O2-evoked
and electrically-induced [3H]dopamine efux. Furthermore, L-DOPA
promoted the formation of dopamine quinone. Ropinirole (100 nM) did
not affect resting and H2O2-evoked [3H]dopamine efux and inhibited
the electrically-evoked release only in untreated slices. L-deprenyl, at
concentration of 0.01 microM potentiated, whilst between 1-50 microM
diminished H2O2-evoked [3H]dopamine efux. Rasagiline (0.01-50
microM) slightly inhibited H2O2-evoked [3H]dopamine efux, and it
was able to prevent the generation of dopamine quinone. Conclusions: A
future concept of antiparkinsonian drug design could be the inhibition of
mitochondrial dysfunction, oxidative stress and pathological dopamine
release with simultaneous augmentation of electrically-evoked [3H]dopamine release, which may have disease-modifying potential in addition to
symptomatic improvement.
Paper No.: 1265

HEALTH AND DISEASE
INVOLVEMENT OF ENDOTHELIAL SPHINGOLIPID METABOLISM IN RELEASE AND ACTION OF ENDOTHELIUMDERIVED CONTRACTING FACTORS
Leon Spijkers, R van der Akker, A Alewijnse, S Peters
Academic Medical Center, Department of Pharmacology, Amsterdam,
The Netherlands
Hypertension is associated with endothelial dysfunction favoring vascular
contraction. This is partly mediated by elevated endothelium-derived
contractile factor release. We and others have previously shown that
sphingolipids, a family of bioactive membrane lipids, can modulate
endothelium-derived relaxing factor signalling. Accordingly, we hypothesized that sphingolipids also play a regulatory role in the release and/or
action of endothelium-derived contracting factors and that this is especially relevant in hypertension. Lipidomics analysis by mass spectrometry revealed signicantly elevated levels of several sphingolipid species
in arterial tissue and blood plasma of spontaneously hypertensive rats
(SHR) compared to normotensive Wistar Kyoto rats (WKY). Pharmacological inhibition of sphingosine kinase in isolated carotid arteries from
SHR induced marked transient and endothelium-dependent contractions,
mediated by the release of TXA2. In line with aforementioned, sphingosine kinase inhibition augmented endothelin-1-induced contractions, in
isolated aorta of SHR, likely by augmenting endothelin-1-induced thromboxane A2 production. This augmentation of endothelin-1 contraction
was endothelium-dependent. In summary, we show that hypertension is
associated with marked alterations in sphingolipid biology, correlating
with augmented endothelin-1 and thromboxane A2 signalling. This study
was performed within the framework of Top Institute Pharma project T2108.
Paper No.: 2029

COMBINED TREATMENT WITH SERTINDOLE AND
CLOZAPINE - REPORTS OF CASES OF PATIENTS WITH
DRUG-RESISTANT SCHIZOPHRENIA
Bozena Spila(1), A Grzywa-Celinska(2), A Urbanska(1)
(1) Lublin Medical University, Department of Psychiatry, Lublin, Poland
(2) Lublin Medical University, Department of Internal Medicine, Lublin,
Poland
Introduction: Drug-resistance in schizophrenia is dened as the failure in
achieving the appropriate therapeutic effect by the only use of adequately
high dose of clozapine of (600-800 mg/d) over at least 6 weeks. Materials/Patients: We report the cases of 3 patients (2 men and 1 woman) with
the medical history suggesting the drug-resistant schizophrenia, in whom
all neuroleptics available in Poland were used without any effect. Patients
were treated in the Department of Psychiatry of Medical University of
Lublin between 2008 and 2010. The applied combined therapy consisted
of sertindole of 16 - 20 mg/d and clozapine of 100-600 mg/d doses.
Results: The signicant improvement of the psychical state was achieved
during the rst two months of gradually introduced treatment. The scores
in CGI scale felt down from 5 to 3 points. According to the GAF scale
the evaluation of the functioning improved from 30 to 70 points. With
regard to positive symptoms measured by the use of BPRS-4 the signicant lowering of productive symptoms from 18 to 9 points was observed.
The intensity of negative symptoms measured using the BNS scale lowered meanly from 20 to 15 points. The heart function was monitored
strictly according to the recommendations of the physician. Neither the
QT segment nor the QTc was remarkably prolonged. No arrhythmias
were observed. Conclusion: The used combined treatment of sertindole
and clozapine was found to be effective and safe for patients with drugresistant schizophrenia.
Paper No.: 1461

PHARMACOKINETIC INTERACTION BETWEEN DULOXETINE AND SECOND-GENERATION ANTIPSYCHOTICS IN
PATIENTS WITH PSYCHOTIC DISORDERS
Edoardo Spina(1), C DArrigo(1), V Santoro(1), U Mico`(2),
MR Muscatello(2), G Pandolfo(2)
Medicine & Pharmacology, Messina, Italy
(2) University of Messina, Department of Neurosciences, Psychiatric and
Anesthesiological Sciences, Messina, Italy
Introduction. Antidepressants are often used in combination with novel
antipsychotics and this may result in pharmacokinetic drug interactions.
Aim of the present study was to evaluate the effect of duloxetine on the
steady-state plasma concentrations of the second-generation antipsychotics clozapine, olanzapine, risperidone in patients with schizophrenia or
schizoaffective disorder. Patients and Methods. Twenty-two outpatients
stabilized on clozapine (250-500 mg/day, n = 7), risperidone (2-4 mg/
day, n = 7) or olanzapine (10-20 mg/day, n = 8) received adjunctive duloxetine, 60 mg/day, over 6 weeks. Plasma concentrations of clozapine,
olanzapine, risperidone and their major metabolites were measured by
HPLC at baseline and after 2, 4 and 6 weeks of duloxetine coadministration. Results. Plasma concentrations of clozapine and olanzapine were
not signicantly modied during concomitant administration of duloxetine. On the other hand, plasma concentrations of risperidone active fraction (sum of the concentrations of risperidone and its metabolite 9hydroxy-risperidone) increased signicantly from 46 ,,b 12 ng/mL at
baseline to 58 ,,b 14 ng/ml at nal evaluation (P < 0.01). Adjunctive
duloxetine therapy was generally well tolerated except in one patient in
591
the risperidone group who developed mild extrapyramidal symptoms
during the second week of duloxetine augmentation. Conclusion. These
ndings indicate that duloxetine, at therapeutically recommended doses,
does not affect the plasma levels of the new antipsychotics clozapine and
olanzapine, while it may cause a clinically signicant elevation of those
of risperidone. This effect is presumably related to an inhibitory effect of
duloxetine on CYP2D6-mediated 9-hydroxylation of risperidone.
Paper No.: 2301

DISEASES
SUSTAINED IMPROVEMENT OF GLYCEMIC CONTROL
REDUCES URINARY LEUKOTRIENE E4 EXCRETION IN
PATIENTS WITH TYPE 1 DIABETES
Francoise Stanke(1), R Boizel(2), G Bruttmann(2), PY Benhamou(2),
S Halimi(2)
(1) Grenoble University Hospital, INSERM ERI17, Laboratory of
Pharmacologie, Grenoble, France
(2) Grenoble University Hospital, Department of Endocrinology-Diabetology-Nutrition, Grenoble, France
Objective: Leukotrienes are pro-inammatory mediators suspected to be
involved in the onset of cardiovascular diseases. The urinary excretion of
LTE4 (LTE4-UE) is a marker of in vivo cysteinyl leukotriene levels. It is
increased in type 1 diabetes (T1). We investigated the effect of an
improved glycemic control on LTE4-UE in patients with diabetes. Materials and methods: We measured LTE4-UE and other inammatory mediators at baseline and after a 3-month intensive insulin therapy in 20 T1
and 19 type 2 (T2) patients. Results: Glycated haemoglobin (HbA1c)
and 11-dehydro-thromboxane B2 signicantly decreased after insulin
therapy whereas hsCRP and sICAM levels remained unchanged in T1
and T2. LTE4-UE decreased by 32% (P = 0.0079 vs baseline) in T1, not
in T2; this change was correlated to baseline HbA1c (r = 0.695,
P = 0.0042). Conclusions: Hyperglycemia induced a reversible activation
of the 5-Lipoxygenase pathway in T1. Despite a similar HbA1c improvement, it was not reversed in T2, suggesting a multifactorial activation.
Paper No.: 3225

HEALTH AND DISEASE
SMALL AND INTERMEDIATE CALCIUM-ACTIVATED K
CHANNELS ARE INVOLVED IN RELEASE OF NO AND EDHF
IN SMALL ARTERIES
Edgaras Stankevicius(1,3), C Krjgaard(1), T Dalsgaard(1), L Beck(1),
AD Hughes(2), U Simonsen(1)
(1) University of Aarhus, Department of Pharmacology, Aarhus,
Denamark
(2) Imperial College, Department of Clinical Pharmacology, London, UK
(3) Kaunas University of Medicine, Department of Physiology, Kaunas,
Lithuania
The present study addressed whether activation of calcium-activated K
(KCa) channels leads to release of NO and EDHF-type relaxation in
small arteries. Quantitative PCR and patch clamp studies were performed
in HUVEC. Vasorelaxation and release of NO studied in rat mesenteric
and retinal arteries. Quantitative PCR showed expression of small (SKCa)
and intermediate (IKCa) conductance KCa, while expression of large-conductance (BKCa) KCa was below detection limit in primary human umbilical vein endothelial cells. In whole cell patch clamp studies, NS309 (11000 nM) induced calcium-dependent increases in current which were
inhibited in the presence of blockers of SKCa, apamin, of IKCa, charybdotoxin and TRAM-34, while a blocker of BKCa, iberiotoxin, and a
blocker of KATP channels, glibenclamide, did not change NS309-evoked

increases in current. In pressurized rat mesenteric small arteries contracted with U46619, NS309 evoked endothelium-dependent vasodilatation was reduced in the presence of ADMA and inhibited by apamin,
charybdotoxin, and TARM-34, while iberiotoxin did not alter vasodilatation. A combination of ADMA, apamin, and charybdotoxin abolished
NS309 vasodilatation. Microsensor detection of NO revealed NS309
increased NO concentration small mesenteric arteries. In retinal arteries
bradyknin induced release of NO and relaxation. The present ndings
support that openening of SKCa and IKCa channels leads to endotheliumdependent vasorelaxation associated with release of NO and EDHF-type
vasodilatation in small arteries.
Paper No. 1169

FOCUS GROUP: PW34 - STATE-OF-THE-ART OF BASIC AND
CLINICAL PHARMACOLOGY IN SERBIA
INFLUENCE OF TICLOPIDINE ALONE AND IN COMBINATION WITH ASPIRIN ON LIVER FUNCTION TEST IN RATS
Zorica Stanojevic(1), R Mitic(1), S Stevic(1), M Dejanovic(2), Z Bukumiric(3)
(1) Medical Faculty Pristina, Institute of Pharmacology and Toxicology,
Kosovska Mitrovica, Republic of Serbia
(2) Medical Faculty Pristina, Institute of Physiology, Kosovska Mitrovica, Republic of Serbia
(3) Medical Faculty Pristina, Institute of Medical Statistics and Informatics, Kosovska Mitrovica, Republic of Serbia
A combination of aspirin and ticlopidine has been proven to reduce the
frequency of haemorrhagic and vascular complications after coronary
artery stenting. Ticlopidine is often associated with abnormal liver function test values. The purpose of this study was to evaluate the inuence
of ticlopidine, administered alone and in combination with aspirin, on
liver function parameters. The experiment was conducted on white laboratory rats, type Wistar. Twenty four rats were divided in three groups
and they received one of the following treatments for three days: group I
- control; group II - ticlopidine (125 mg/kg/day i.p.) and group III - ticlopidine+aspirin combination (125 mg/kg/day+50 mg/kg/day i.p.). After
the treatment the animals were anaesthetised with ether and blood for
further analyses was taken by cardiopunction. Total cholesterol serum
level was signicantly increased in group treated with ticlopidine compared to control (P < 0.01). In group treated with ticlopidine+aspirin
combination total cholesterol serum level was increased, but not signicantly. Also, the total bilirubin concentration and serum activity of alkaline phosphatase were signicantly elevated only in ticlopidine treated
group compared to control (P < 0.001). Serum AST and ALT activities
were not signicantly elevated in all treating groups. Based on obtained
results it can be observed that the values of liver function parameters are
higher in group treated with ticlopidine than in group treated with ticlopidine+aspirin combination.
Paper No.: 1531

CHARACTERIZATION OF BIMODAL MULTIVALENT
INTERACTIONS FOR THE BIFUNCTIONAL MUSCARINIC
RECEPTOR (MACHR) ANTAGONIST AND
B2-ADRENOCEPTOR AGONIST (MABA), THRX 198321
Tod Steinfeld, AD Hughes, U Klein, JAM Smith, M Mammen,
(1) Theravance, Inc., Department of Molecular and Cellular Biology, San
Francisco,CA, USA
Using a multivalent design strategy, we have identied a class of compounds referred to as MABAs that may offer clinical benet for patients
592
with respiratory disease. THRX-198321 is a bimodal bifunctional multivalent ligand functioning as a mAChR antagonist (M3 IP1 accumulation
pKI=10.1 0.2) and b2AR agonist (cAMP accumulation pEC50 = 9.3
0.0). mAChR agonist (oxotremorine) activity was competitively inhibited by THRX-198321 (M3 pKB=10.3 0.9). Furthermore, b2AR agonist activity of THRX-198321 was competitively inhibited by the b2AR
antagonist, propranolol. These data are consistent with THRX-198321
binding interactions at the orthosteric sites of each receptor. In kinetic
studies designed to detect allosteric modulators, THRX-198321 retarded
the dissociation of radiolabeled orthosteric ligands from mAChR
(pEC50,diss=6.7 0.1) and b2AR (pEC50,diss=3.8 0.2) in a manner
consistent with an allosteric interaction. Similar studies conducted with
molecular fragments of THRX-198321 suggest that different regions of
the ligand bind to these orthosteric and allosteric sites. Inhibition binding
data for an analog of THRX-198321 further support the hypothesis that
the ligand binds to a site outside of the b2AR orthosteric pocket. These
data suggest the existence of a novel allosteric site on b2AR. Free energies were calculated from afnities determined in radioligand binding
studies. That the free energy values for binding of THRX-198321 are
similar to the sums of free energies for pairs of fragments, strongly suggests that THRX-198321 binds in a multivalent manner at mAChR and
b2AR. In summary, THRX-198321 is a potent bimodal bifunctional multivalent ligand at mAChR and b2AR that binds to orthosteric and allosteric sites.
Paper No.: 1933

THE HLA CLASS I B*1801 ALLELE INFLUENCES HEPATOCELLULAR EXPRESSION OF AMOXICILLIN-CLAVULANATE
LIVER DAMAGE AND OUTCOME IN SPANISH PATIENTS
C Stephens(1,10), MA Lopez-Nevot(2), RJ Andrade(3,10), F Ruiz-Cabello(2), MC Fernandez(4), C Guarner(5,10), G Soriano(5,10), M RomeroGomez(6,10), JM Navarro(7), JL Sols-Herruzo(8), J Costa(9), Maribel
Isabel Lucena(1,10)
(1) Servicio de Farmacologa Clnica, Hospital Universitario Virgen de la
Victoria,Malaga, Spain
(2) Servicio de Analisis Clnicos, Hosptial Virgen de las Nieves, Granada, Spain
(3) Unidad de Hepatologa, Hospital Universtario Virgen de la Victoria,
Facultad Medicina, Malaga, Spain
(4) Servicio de Farmacologa Clnica, Hospital Torrecardenas, Almeria,
Spain
(5) Servicio de Aparato Digestivo, Hospital San Pau, Barcelona, Spain
(6) Unidad de Hepatologa, Hospital Universitario de Valme, Sevilla,
Spain
(7) Servicio de Aparato Digestivo, Hospital Costa del Sol, Malaga, Spain
(8) Servicio de Aparato Digestivo, Hospital 12 de Octubre, Madrid,
Spain
(9) Servicio de Farmacologa Clnica, Hospital Germans Trias i Pujol,
Barcelona, Spain
(10) Centro de Investigacion Biomedica en Red: Enfermedades Hepaticas y Digestivas(CIBERehd), Barcelona, Spain
Introduction: Amoxicillin-clavulanate (AC)-DILI is mostly expressed as
cholestatic/mixed (Chol/Mix) damage in North-European populations.
However, up to 30% of Spanish subjects have hepatocellular (HC) ACDILI, which may bear a genetic basis. Methods: To investigate possible
associations between HLA alleles and the susceptibility to developing
AC-DILI and ascertain if such putative associations are phenotype specic, high resolution genotyping of HLA class I (A, B and C) and class
II (DRB1, DQB1) loci was performed by PCR with sequence specic
oligonucleotide probes in 57 Spanish patients diagnosed with AC-DILI
and 400 healthy Caucasian controls. Results: The HLA class I B*1801
allele was found to be signicantly more frequent in HC type of DILI
(10/17,59%; P = 0.00005) and related to cases of younger age (55 vs
66 years), requiring hospitalization (76% vs 46%) and having worse
DILI outcome (1 death, 2 liver transplants vs 0) as compared to those

with HLA class II alleles DRB1*1501-DQB1*0602, where Chol/Mix
type of injury predominated (12/13, 92%; P = 0.009). The A*1010 allele
and haplotype RB1*0701-DQB1*0202 play a protective role in AC-DILI
development (23.5% vs 7%; P = 0.006 and 30% vs 15%; P = 0.03,
respectively). Conclusions: Hence, the HLA class I allele B*1801 predisposes to AC-DILI of HC type with a worse outcome, while the class II
haplotype DRB1*1501-DQB1*0602 play a role in Chol/Mix damage.
Ethnic differences in B*1801 allele frequencies could explain potential
variations in hepatocellular AC-DILI manifestation between different
populations.
Funding Spanish Medicine Agency. CIBERehd is funded by ISC III
Paper No.: 1738

DISEASES
NOVEL PAN-JAK INHIBITOR SHOWS EFFICACY IN BOTH
STEROID SENSITIVE AND STEROID INSENSITIVE
INFLAMMATORY PATHWAY
Laurie Stevens(1), P Fenwick(2), W Liu(1), I Kilty(1), M Yeadon(1),
L Donnelly(2)
(1) Pzer, Department of Allergy and Respiratory, Sandwich, UK
(2) Imperial College London, National Heart and Lung Institute, London,
UK
Corticosteroids are a widely used therapeutic strategy for the modulation
of inammation observed in COPD. However, whilst steroids may
reduce exacerbation rate the pulmonary inammation is largely corticosteroid insensitive. This may be due to dysfunction of the glucocorticoid
response pathways, or enhanced stimulation of pathways not modulated
by corticosteroids such as JAK/STAT mediated signal transduction. The
aim of this study was to determine the effect of a pan-JAK inhibitor on
IL-2 stimulated IFNc from peripheral T lymphocytes and the subsequent
IFNc induced release of CXCR3 chemokines from lung epithelial cells
in comparison to dexamethsone. Human peripheral blood mononuclear
cells (PBMCs) were pre-treated with pan JAK inhibitor (PF-956980) or
dexamethsone and stimulated with anti-CD3/IL-2. In parallel, human primary bronchial epithelial cells were pre-treated with PF-956980 or dexamethsone prior to stimulation with IFNc for 24h. Release of IFNc from
PBMC and CXCR3 chemokines from epithelial cells were measured by
ELISA. PF-956980 inhibited IFNc release from PBMCs (EC50
41 7nM n = 5). Dexamethsone was 10-fold more potent in this assay.
By contrast, dexamethsone had no effect on the release of CXCL9,
CXCL10 and CXCL11 from epithelial cells. However, all three chemokines were inhibited by PF-956980 (EC50 0.4 0.1 lM, 1.0 0.3lM
0.5 0.2lM n = 4, respectively). Based on this model system, we can
conclude that PF-956980 inhibits both release of IFNc and the downstream induction of CXCR3 chemokines, in contrast to dexamethasone,
which was ineffective at inhibiting the latter. Thus, a pan-JAK inhibitor
may have additional benets to steroids for the treatment of inammatory conditions such as COPD.
Paper No.: 2448

ON FUTURE PHYTOPHARMACOLOGY
Jakob Stiglmayr(1,2), J Milbradt(1,2), E Neu(1,2,3), SK Gupta(4),
J Foltinova(5), M Schratz(6), W Seidenbusch(1,3), G Weber(7), N
Senn(1,6), R Neu(1,6), U Welscher(1,2,3), M Michailov(1,2,3)
Muenchen, Germany
593
(2) University of Erlangen-Nuernberg, Institute of Botany, Erlangen, Germany
Austria
(4) All Indian Institute of Medical Sciences, Institute of Pharmacology,
New Delhi, India
(6) University of Innsbruck, Faculty of Education/Dean, Innsbruck, Austria
Austria
effect in comparison to the control and group treated with silymarin.

Observing the values of the parameters, which indicate the intensity of
lipid peroxidation, we saw that treatment with silymarin in comparison
with doxorubicin treatment leads to a signicant decrease of prooxidative
parameters activity (lipid peroxidation, 2.25:0.28 P < 0.05) and an
increase of the parameters, which are in negative correlation with the oxidative stress (catalase, 5.98:0.40, P < 0.05). On the basis of the results it
can be concluded that silymarin prevents toxic effects of doxorubicin on
myocardial and liver function.
Introduction: Herbal-drugs are origin of modern pharmaco-therapy and

till today actual. Bio-active plant-products are omnipresent: Angio-cardiac, gastro-intestinal, renal, neuro-&psychotropic effects of coffee, tea,
cacao (methylxanthines - caffeine, theophylline, theobromine), garlic
(allium sativum contains also phytoncides), pepper (capsaicin), etc. are
well known. Necessity of systematic actual scientic evaluation of medical plants is evident: Traditional African, American, Asian (Chinese,
Indian/ayurveda, Japanese, etc.), Australian, European herbal-therapy
could be better applied in modern therapy. Conception: IUPHAR-aims
about global education/ research, section on natural products are coincident with ICSD/IAS-projects on new models for humanization, higher
effectiveness, internationalization of science, medicine, ecology via foundation of international universities (IU: B. Russells proposal, British
*Nobel Laureate) (Michailov et al, Br.J.Urol. 2004;94:302; Neu, Stiglmayr et al, Acta-Pharm.Sinica 2006;27/1:411). Creation of 1st international
institute for phytopharmacology (IIPP) via network of national departments for botany (incl. botanical gardens), pharmacology, physiology,
biophysics/-chemistry, etc. from important countries could be basis for
global agency for phytopharmacological research/education, incl. on
local, national/German, European level, publications in int. journals, similar to Palmenblatt: editors Stiglmayr, Wel, also documentation centre
for herbal-drugs. Conclusion: Proposed conception incl. IIPP could open
a new dimension in herbal-medicine and its application in context of
combined therapy, supporting UNO-Agenda21 for better health, ecology,
etc. in developing and industrial countries.
Dedicated to moral support (1989-2010) of Hon. ICSD-members: Jean
Dausset* (medicine)/France, Donat-P. Hader, Walter Wel (botany),
Dieter G. Weiss (biology)/Germany, Yuan T. Lee* (chemistry)/China,
Rita Levy-Montalcini* (biol./pharmacol.: 100 years)/Italy, Lord George
Porter* (chemistry)/UK, Andrej Saccharov* (peace)/Russia, Karan Singh
(past minister for health)/India
Paper No.: 1856

EXPRESSION OF NMDA RECEPTOR SUBUNITS NR1 AND
NR2A AND METABOTROPIC MGLUR1 IMMUNOREACTIVITY
IN THE INTERTRIGEMINAL REGION OF THE LATERAL
PONS IN RATS
Paper No.: 595

INTERACTION OF SILYMARIN AND DOXORUBICIN IN RAT
Nebojsa Stilinovic, R Sederino, A Raskovic, M Mikov, V Jakovljevic,
S Vukmirovic
Medical University of Novi Sad, Department of Pharmacology, Toxicology & Clinical Pharmacology, Novi Sad, Republic of Serbia
Silymarin, presents a complex of avonolignans with a broad spectrum
of action. The main reason of the cardio and hepatotoxicity is the production of free radicals, lipid peroxidation and DNA damage of these tissues. The aim of investigation was to examine effects of interaction of
silymarin and doxorubicin on heart function during continuous infusion
of lidocaine and verapamile, also activity of indicators of oxidative stress
from liver homogenate. The animals were anaesthetised with urethane
and the prepared jugular vein was connected to the infusion pumps with
verapamil and lidocaine. Then animals were sacriced and livers were
taken for biochemical examination. The results showed that treatment
with silymarin and doxorubicin in combination causes statistically significant increase of verapamil dose necessary to produce rst change, continuous reaction and toxic effect in comparison to the groups treated with
silymarin or doxorubicin alone. Treatment with doxorubicin alone causes
statistically signicant increase of lidocaine necessary to produce toxic
Milan Stoiljkovic(1), S Pesic(1), N Stojiljkovic(2), V Nikolic(1)

(1) University of Nis School of Medicine, Department of Pharmacology,
Nis, Republic of Serbia
(2) University of Nis School of Medicine, Department of Physiology,
Chemostimulation of intertrigeminal region (ITR) neurons by glutamate
evoked immediate apnea proposing the ITR as a regulatory site for
apneic reexes. Previous studies (Radulovacki et al, 2007;156:40-6; Stoiljkovic et al, 2009;165:137-42) demonstrated presence of functional glutamate receptors in the ITR, and postulated their role in both central and
reexive apnea, but specifying only putative signalling pathways. The
aim of the present study was to examine the expression of glutamate
receptors within the ITR by detecting their subtype-specic immunoreactivity. Fifteen adult male Sprague-Dawley rats were transcardially perfused and brains sections were processed immunohistochemically using
polyclonal antibodies for NMDA-NR1, NMDA-NR2A and mGluR1a
receptors. Labeled neurons in the ITR were analyzed using light microscope and computerized image analysis system for quantication of relative receptors levels as the mean of integrated optical density (IOD).
Specicity of labeling was veried by the omission of primary antibodies
in adjacent slices from each series. Light microscopic analyses demonstrate that NMDA-NR1-immunoreactivity was intense and mainly localized in the neuronal somata with sparse distribution on primary dendrites
or extracellular matrix, NMDA-NR2A-immunoreactivity was generally
weak and only localize on somata, while mGluR1a-immunoreactivity
was primarily neuronal/dendritic since moderate staining intensity was
observed in both neuronal cytoplasm and extracellular matrix. Quantitative analyses of IOD showed very strong expression of NMDA-NR1
(5.8 0.41), moderate expression of NMDA-NR2A (3.1 0.12) and
strong-to-moderate expression of mGluR1a (4.6 0.23). Our data conrm the presence of specic glutamate receptor subtypes in the ITR
which are apparently involved in signalling pathways by which this
region modulates apnea expression.
Paper No.: 2819

HUMAN CEREBROSPINAL FLUID CONTAINS EXOSOMES
THAT REPRESENT A NOVEL RESERVOIR FOR
THERAPEUTIC BIOMARKER DISCOVERY
Jonathan M Street(1), RTA Chalmers(2), TS Walsh(2), DJ Webb(1), JW
Dear(1)
(1) University of Edinburgh, Clinical Pharmacology Unit, Edinburgh, UK
(2) Royal Inrmiary of Edinburgh, Edinburgh, UK
594
Neurodegenerative diseases such as transmissible spongiform encephalopathies and Alzheimers dementia involve the accumulation of abnormal proteins within brain tissue. Previously, these proteins have been
shown to be released into the extra-cellular space in association with exosomes. Formed and released as part of the endosomal pathway, exosomes
are small lipid membrane bound vesicles approximately 60-100 nm
diameter with a cup-shaped morphology (under electron microscopy)
and several known protein markers including otillin-1 and TSG-101.
Exosomes have not been previously demonstrated in human cerebrospinal uid (CSF) but have been identied in CSF from sheep. We hypothesise that human CSF will also contain exosomes and these will be a
potential source of protein biomarkers. CSF was collected from patients
undergoing thoraco-abdominal aortic aneurysm repair (with full ethical
approval). This group was chosen as they have a CSF drain inserted
peri-operatively as part of routine clinical management. The CSF was ultracentrifuged to purify exosomes. The exosomal markers TSG101 and
otillin-1 were found to be enriched in the ultracentrifugation pellet over
the unfractionated CSF. Using sucrose gradient centrifugation, the density of the microvesicles was established as 1.14 - 1.20 g.cm-3, consistent with the presence of exosomes. Immunoelectron microscopy
revealed otillin-1 positive particles consistent in size and shape with
previous reports for exosomes. Alpha-2-macroglobulin, which is
involved in the clearance of A-beta amyloid, was identied in the exosomes by tandem mass spectrometry. We conclude that human CSF contains exosomes and we are currently performing mass spectrometry to
further dene their proteome.
Paper No.: 2875

DISEASES
DEVELOPMENT OF AN IN VITRO MODEL TO INVESTIGATE
CHANGES IN KIDNEY-DERIVED EXOSOMES WITH DISEASE
Jonathan M Street(1), M Bailey(1), F Frame(2), TS Walsh(2),
DJ Webb(1), JW Dear(1)
(1) University of Edinburgh, Clinical Pharmacology Unit, Edinburgh,
UK
(2) Royal Inrmary of Edinburgh, Department of Critical Care, Edinburgh, UK
Human urine contains exosomes, small vesicles (approximately 60100 nm diameter) with a distinct cup-shaped morphology under electron
microscopy and several characteristic protein markers such as otillin-1.
We have developed a new in vitro model, utilising a murine cortical collecting duct (mCCD) cell line, to investigate changes in kidney-derived
exosomes with disease. We collected tissue culture medium from the
mCCD cells following incubation in exosome-depleted media. We also
collected urine from human subjects with severe sepsis and healthy volunteers (with full ethical approval). Exosomes were isolated by ultracentrifugation. Western blot analysis of the cell media demonstrated
enrichment of otillin-1 in the ultracentrifuge pellet. Sucrose density centrifugation showed that otillin-1 localised to a density of 1.12
1.16 g.cm-3, consistent with previous reports for exosomes. Stimulation
of the cells with vasopressin caused an enrichment of aquaporin 2 in the
exosomes and stimulation with the cytokine TNF-a caused enrichment
of the transcription factor c-Jun in the exosomes (without altering the
amount of otillin-1 in either case). No change in cellular apoptosis or
necrosis was observed as measured by annexin V / propidium iodide
FACS analysis. Western blot analysis demonstrated a signicant increase
in c-Jun in exosomes isolated from patients with severe sepsis over those
isolated from healthy controls. We conclude that changes in the exosomal
proteome reects changes in the physiological and pathological state of
kidney cells. Exosomal c-Jun may be a useful biomarker of kidney cell
inammation and injury.
Paper No.: 1469

CARDIOVASCULAR EFFECTS OF DEXMEDETOMIDINE IN
AWAKE A2A-ADRENOCEPTOR KNOCK-OUT MICE
Tomi Streng, V Ranta-Panula, S Tikka, M Scheinin
University of Turku, Department of Pharmacology, Drug Development
and Therapeutic, Turku, Finland
Activation of a2-adrenoceptors (a2-AR) by agonists (e.g. dexmedetomidine) is known to induce hypotension and bradycardia in mice and other
mammalians. The contributions of the three a2-AR subtypes to the cardiovascular effects of dexmedetomidine are not known in detail. In the
current study, dexmedetomidine was administered s.c., i.p. and i.v.
(30 lg/kg) to awake C57/Bl (WT) and a 2A-KO (KO) mice. a2A-KO
mice (from BK Kobilka; Stanford University; see Altman et al., Mol
Pharmacol. 1999;56:154-61) were used to dene the role of this receptor
subtype in dexmedetomidines cardiovascular effects. Telemetry transmitters (from DSI) were implanted in the left carotid artery. In WT mice, as
expected, dexmedetomidine very markedly depressed heart rate and
blood pressure, with similar maximal effects independent of the route of
administration. The effect duration was somewhat shorter after i.p. than
after i.v. or s.c. administration. In KO mice, both effects were signicantly attenuated compared to the WT mice, but not abolished. Signicant depression of heart rate and blood pressure was seen in a2A-KO
mice, too, which indicates that the a2A-AR is not the only a2-AR subtype that mediates bradycardia and hypotension in mice. Interestingly, no
blood pressure increases were seen in a2A-KO mice after dexmedetomidine administration, at least when measured in 10-min epochs. The
awake mouse may not be the ideal model organism to investigate blood
vessel constriction responses mediated by a2-AR activation previously
observed in humans.
(Snapir et al., Br J Anaesth. 2009;102:38-46; Snapir et al., Anesthesiology 2006;105:902-10).
Paper No.: 3417

ANTIMICROBIAL CONSUMPTION AND POLYPHARMACY IN
PATIENTS ON IMMUNOSUPPRESSIVE THERAPY
Jan Strojil, P Langerova, K Urbanek
Palacky University, Department of Pharmacology, Olomouc, Czech
Republic
Patients on chronic immunosuppression often require complex therapeutic regimes involving multiple drugs, their antibiotic consumption is
higher, with higher incidence of resistant strains. As a pilot study for a
planned prospective study of antibiotic consumption and antimicrobial
resistance in immunosuppressed patients, we analyzed 1,796,761 prescriptions issued the University hospital in Olomouc between January
2005 and December 2009. Of these, 30,953 (1.7%) were for immunosuppressive agents but a total of 131,404 (13.7%) of prescriptions were
issued to patients on immunosuppressive therapy (2,858 of 167,537
patients). Antimicrobial agents are the fourth most common drug group
other than immunosuppressants (after corticoids, mineral supplements,
anti-ulcer drugs), with 21.4 antimicrobial prescriptions per 100 patient
visits. Antibiotic consumption in this patient group is signicantly different from the general population, the top three antibiotics were sulfamethoxazole and trimethoprim, amoxicillin with an inhibitor, and ooxacin,
representing 42%, 21%, and 7% of all antibiotic prescription to immunosuppressed patients, respectively. Itraconazole and uconazole represented 56% and 41% of antimycotics drugs, and aciclovir and
valaciclovir represent 61% and 23% of antivirals. Other frequently prescribed drugs included mineral supplements, corticosteroids, anti-ulcer
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drugs and lipid-lowering agents. We identied patients with multiple prescriptions, non-rational combinations and uncoordinated prescription
from multiple clinics, some tens of different drugs. The results from this
retrospective analysis will be used for a prospective study of antimicrobial consumption and resistance prevalence in patients on immunosuppressants.
Paper No.: 2334

GENTAMICIN PHARMACOKINETICS ESTIMATED USING
RAT MODEL OF EARLY SEPSIS WITH CAPILLARY LEAK
SYNDROME
adherence to dose timing on the efcacy of levooxacin in communityacquired pneumonia (CAP) using pharmacokinetic/ pharmacodynamic
(PK/PD) methods. Methods: Free AUC0-24h values after levooxacin
dosage (500 mg once daily) were derived from a previously published
PK model of levooxacin in CAP. MIC distribution data for S Pneumoniae were obtained from EUCAST data base and the index AUC0-24h/
MIC90, a target PD predictor of bacterial eradication (target> 33.8
against S. pneumoniae in patients with CAP) was calculated. Simulated
scenaria of levooxacin PK/PD for different extended intervals between
doses were studied. Results: The simulations showed that delays beyond
the scheduled time for dose intake not always allow adequate AUC024h/MIC90 > 33.8 index values to be maintained. Particularly, delays
above 6 hours beyond the scheduled time do not guarantee bacterial
eradication. Conclusion: This type of analysis could be used to quantify
the probability of success of antimicrobial therapy in non complying
patients and also to explore the drugs capacity for forgiveness.
Sarka Studena(1), J Martinkova(1), J Chladek(1), D Slizova(2),

O Krs(2), J Zahora(3)
(1) Charles University Faculty of Medicine, Department of Pharmacology, HradecKralove, Czech Republic
(2) Charles University Faculty of Medicine, Department of Anatomy,
Hradec Kralove,Czech Republic
(3) Charles University Faculty of Medicine, Department of Biophysics,
HradecKralove, Czech Republic
A model of early sepsis with capillary leak syndrome (CLS) was developed using lipopolysaccharide (LPS) and interleukin-2 (IL-2). The goal
was to explore the impact of CLS on pharmacokinetics (PK) of gentamicin (GE). Male Wistar rats (260420 g) were divided to groups 1) intact
rats (IR) given saline 30 min before GE (0.8 or 1.6 mg/kg by 20-minutes
i.v. infusion); 2) LPSIL-2 rats given 1 mg/kg LPS + 15 lg/kg IL-2 prior
to GE infusion. Biochemical, hematological data, respiratory and cardiac
functions were analysed during 360 minutes. The PK of GE was estimated by Kinetica v4.4. In acid-base status hypocapnic alkalosis was
diagnosed. Serum lactate was increased by 40% (p = 0.001) and plasma
albumin decreased by 14% (p = 0.001). Serum creatinine increased by
34% (P < 0.001), while creatinine clearance differentiated insignicantly
(3.6 vs. 2.4 mL/min. in IR vs. LPSIL-2, respectively; p = 0,31). Using
the model of early sepsis, LPSIL-2 rats compared to IR examined by the
light microscopy revealed: pulmonary interstitium and alveoli inltrated
with macrophages and inammatory exudate. Disposal of Cardiogreen,
which is linked to plasma proteins, manifested marked CLS in the lungs.
The spleen was overlled with erythrocytes. No changes were found in
other organs. Pharmacokinetics of GE: Volume of distribution: 0.068 vs.
0.092 L/kg; p = 0.055. GE clearance: 1.1 vs. 2.6 mL/min/kg; p = 0.02.
GE elimination half-life: 70 vs. 51 min.; p = 0.041 in intact rats vs.
LPSIL-2 rats, respectively. A model of early sepsis with CLS showed
signicant differences in gentamicin pharmacokinetics that might be
important for clinical practice.
Paper No.: 2369

INFLUENCE OF NON-COMPLIANCE ON ANTIMICROBIAL
THERAPY WITH LEVOFLOXACIN BASED ON
PHARMACOKINETIC/ PHARMACODYNAMIC SIMULATION
Maria Elena Suarez Gonzalez(1), N Carral(1), R Calvo Duo(1),
J Lukas(2)
(1) University of Basque Country, Faculty of Medicine, Department of
Pharmacology, Leioa, Vizcaya, Spain
(2) University of Basque Country and Dynakin SL, Derio, Vizcaya,
Spain
Introduction: Non-compliance with antibiotic therapy can impact treatment efcacy via failure to achieve optimum drug concentrations. The
aim of this study was to asses the consequences of irregular patient
Paper No.: 496

DEVELOPMENT AND VALIDATION OF BIOANALYTICAL
METHOD OF AMLODIPINE BESYLATE IN HUMAN PLASMA
Ganesh P Subedi, GM Khan
Kathmandu University, Pharmacy Department, Dhulikhel, Kavre, Nepal
Amlodipine Besylate is a long acting dihydropyridine calcium antagonist
developed for the treatment of angina and hypertension. In this study, a
new reversed-phase high performance liquid chromatography method is
developed for the bioanalytical assay of amlodipine besylate in human
plasma. The instrumentation consisted of RP-HPLC of Shimadzu, solvent delivery system LC 20 AD, autosampler 20 AC, UV visible detector
and the analytical column Luna 5 lmC18 (2)100A of size 250X4.6 mm.
Two plasma extraction procedures were experimented, using dichloromethane without using nitrogen drying and methanol by using nitrogen drying method. Methanol with nitrogen drying method was optimized for
analysis of large number of samples as it was easy and less time consuming. After the development of bioanalytical method the nal mobile
phase was Acetonitrile: Buffer (0.01M, pH 3.5), in the ratio of 35:65, the
pH of the mobile phase was 3.5 adjusted with 10% v/v Orthophosphoric
acid. The equipment settings were ow rate 2.0 ml/min, oven temperature 35oC and wavelength 239 nm. Under these conditions the retention
time of propanolol hydrochloride (IS) 10 ng/ml and amlodipine besylate
was 2.933 and 5.825 minutes respectively. The method was validated for
accuracy, recovery, precision, linearity within day and between day analysis and calibration curve. The LOQ was 4 ng/ml and the %RSD and
accuracy deviation were 6.75% and 9.23% at this concentration. The
LOD was 2.5 ng/ml. The calibration curve was found to be linear at concentrations 4-20 ng/ml with coefcient of regression (r) to be 0.99 and
mean %RSD of 13.01.
Paper No.: 765

THERAPEUTIC TARGETS
UPREGULATION OF ENDOGENOUS ANTIOXIDANT AND
PHASE 2 ENZYMES BY DIETARY SUPPLEMENTATION OF
SCOPOLETIN, A PLANT DERIVED COUMARIN, IN RAT
LIVER: POSSIBLE ROLE IN PROTECTION AGAINST
CHRONIC DISEASES WITH AN UNDERLYING OXIDATIVE
STRESS
Sudhakar Raja Subramaniam, EM Ellis
University of Strathclyde, Strathclyde Institute of Pharmacy and BiomedicalSciences, Glasgow, Scotland, UK
596
Dietary antioxidants, including polyphenolic compounds, are considered
to be benecial in the prevention of chronic diseases associated with oxidative stress including cancer, neurodegenerative diseases, atherosclerosis, and rheumatoid arthritis. Among the possible mechanism of
protection against these diseases include the antioxidant dependent induction of detoxifying enzymes and the regulation of intracellular redox status. Coumarins are phenolic compounds widely distributed in legumes
and fruits, and are reported to possess antioxidant, antiinammatory, neuroprotective and antitumor properties. Therefore, in this study we investigated the effect of dietary supplementation of 7-hydroxy-6methoxycoumarin, scopoletin, on the expression and activities of antioxidant and phase 2 detoxifying enzymes by immunoblotting and enzyme
assays in male albino Wistar strain rats. In addition, intracellular glutathione levels were also measured to establish the redox state of the hepatocytes. Dietary supplementation of scopoletin (0.5% w/w) to the rats for
7 days signicantly increased the expression of NADPH: quinone oxidoreductase 1 (NQO1) and heme oxygenase 1 (HO1) to 2-fold and 4-fold
respectively in liver. Parallel to these changes, scopoletin feeding to the
rats also resulted in a signicant enhancement of specic activities of
enzymes viz. glutathione S-transferase (3-fold), glutathione reductase
(15-fold) and aldoketo reductase (7-fold) in liver as compared with vehicle diet fed control (P < 0.05). Scopoletin consumption by rats also
showed a marked increase in total intracellular glutathione levels by 2fold in liver. In conclusion, the diet driven induction of antioxidant and
phase 2 detoxifying enzymes by scopoletin suggest the potential value of
this plant derived coumarin against oxidative stress induced liver diseases.
Paper No.: 3206

IMPORTANCE OF ACID INHIBITION FOR GASTROESOPHAGEAL REFLUX DISEASE WITH LOW-DOSE ASPIRIN
Mitsushige Sugimoto(1), M Nishino(2), C Kodaira(2), M Yamade(2), T
Uotani(2), M Ikuma(2), K Umemura(3), H Watanabe(4), T Furuta(1)
(1) Hamamatsu University School of Medicine, Center for Clinical
Research, Hamamatsu, Japan
(2) Hamamatsu University School of Medicine, First Department of
Medicine, Hamamatsu, Japan
(3) Hamamatsu University School of Medicine, Department of Pharmacology, Hamamatsu, Japan
(4) Hamamatsu University School of Medicine, Department of Clinical
Pharmacology, Hamamatsu, Japan
Background and Aim: Aspirin used widely as an antithrombotic drug
increases a risk for gastrointestinal mucosal injury, especially in stomach,
intestine and colon. However, how gastric acid should be inhibited for
the prevention of aspirin-related esophageal mucosal injury is unclear.
We intended to determine an intragastric pH value necessary for preventing aspirin-induced esophageal mucosal injury. Methods: Fifteen Japanese volunteers were dosed for 7 days at 3-way randomly crossover trial:
aspirin 100 mg, aspirin 100 mg plus rabeprazole 10 mg or lansoprazole
15 mg. All subjects underwent gastroduoenal endoscopy and 24-hour intragastric pH monitoring on day 7. Results: Seven individuals (46.7%)
were developed esophageal mucosal injury with aspirin 100 mg alone.
Rabeprazole signicantly inhibited acid secretion irrespective to
CYP2C19 genotypes and decreased the incidence of aspirin-related
esophageal injury and symptoms according to increasing pH value. The
median 24-hour pH in grade A GERD was signicantly lower [1.5
(1.1 1.9)] than that in non-GERD [4.5 (0.8 6.4), P = 0.04]. When
individuals achieved more than 5 of 24-hour mean intragasric pH and
less than 40% of percentage time of pH <4.0 irrespective to kinds of
PPIs, nobody were developed the esophageal mucosal injury by dosage
of aspirin. The development of GERD by aspirin intake related with
occurrence of gastric erosions (p < 0.001). Discussion: Development
of aspirin-induced esophageal and gastric mucosal injury depended on
intragastric pH value. Concomitant PPI therapy with aspirin may prevent

advanced effects for gastrointestinal mucosal tract by low-dose aspirin.
Paper No.: 2181

ANALYSIS OF PROARRHYTHMIC EFFECTS OF TYPICAL
IKR BLOCKERS USING HUMAN ES/IPS CELL-DERIVED
CARDIOMYOCYTES
Atsushi Sugiyama(1), T Kitamura(2), F Nomura(1), T Kaneko(1),
P Sartipy(3), K Fukuda(4), K Yasuda(1)
(1) Tokyo Medical and Dental University, Tokyo, Japan
(2) Mitsubishi Chemical Medience Corporation, Japan
(3) Cellartis AB, Japan
(4) Keio University School of Medicine, Japan
Proarrhythmic effects of IKr blockers were assessed using human embryonic stem (ES) cell-derived cardiomyocytes (CMs). Field potential (FP)
waveforms of ES-CMs clusters were recorded using on-chip multi electrode arrays (MEAs) system. FP duration at 90% repolarization level
(FPD90) of consecutive beats under stable automaticity was measured.
Poincare plots with FPD90(n) versus FPD90(n + 1) were prepared. The
mean orthogonal distance from the diagonal to the points of the Poincare
plot was determined as beat-to-beat variability of repolarization (BVR).
E-4031 (0.1 nM-1 uM) as well as dl-sotalol (0.1 uM-1 mM) signicantly
prolonged FPD90 in a concentration-related manner together with an
increase of BVR in ES-CMs clusters (n = 4 for each drug). Next, proarrhythmic effects of E-4031 (0.03, 0.1 and 0.3 mg, iv /kg/10 min) and dlsotalol (1 and 3 mg, iv /kg/10 min) were conrmed using the chronic
atrioventricular block dogs (n = 4 for each drug). Similar extents of
changes to those observed in ES-CMs clusters were conrmed for the
QT interval and its BVR, respectively, at corresponding drug concentrations. Torsades de pointes was induced in 0, 2 and 4 out of 4 animals by
the low, middle and high dose of E-4031, whereas it was observed in 0
and 4 out of 4 animals by the low and high dose of dl-sotalol, respectively. These results indicate that analysis of FPD90 of ES-CMs clusters
may become a promising marker for predicting potential of drug-induced
TdP. In this meeting, the results of human induced pluripotent stem (iPS)
cell-derived CMs will be also shown and discussed.
Paper No.: 3397

ROUTINE USE OF PHARMACOGENETIC TESTS FOR
DETECTION OF CYP2C19 GENETIC POLYMORPHISMS IN
CLOPIDOGREL NON-RESPONDERS IN RESOURCE LIMITED
SETTING
Chonlaphat Sukasem(1), R Tunthongb(2), V Pairoj(1),
C Srichunrasamia(1), P Vathesatogkit(2), P Sritarab(2),
W Chantratita(1)
(1) Ramathibodi Hospital, Center for Pharmacogenomics and Theranostics, Deapartment of Pathology, Bangkok, Thailand
(2) Ramathibodi Hospital, Department of Medicine, Bangkok, Thailand
Genetic variation in the CYP2C19 gene have gradually more been documented as determinants of the conversion of clopidogrel and the variability in antiplatelet effect of clopidogrel. We believe that, from a genetic
background, we may be able to select dosages of clopidogrel more
appropriately and individually, and these genetic polymorphisms can also
predict a response to adjusted-dose of clopidogrel. The aim of this study
was to determine the frequencies of the CYP2C19 allele in Thai Clopidogrel non-responders population. A total of 103 patients with cardiovascular disease treated with clopidogrel were recruited in this study. The
597
genotypes of CYP 2C19 gene (*1/*2*3) were determined by multiplex
allele-specic polymerase chain reaction and AmpliChip CYP450 test.
The genotyping analysis of participants demonstrated that 44 patients
(42.72%) were homozygous wild-type (CYP2C19*1/*1), 27 patients
(26.21%) were heterozygous CYP2C19*1/*2, 6 patients (5.82%) were
heterozygous CYP2C19*1/*3, 19 patients (18.44%) were homozygous
CYP2C19*2/*2, 6 patients (5.83%) were heterozygous CYP2C19*2/*3
and homozygous CYP *3/*3 was found in 1 patient (0.97%). Therefore,
42% was predicted as extensive metabolizer (EM), 32% as intermediate
metabolizer (IM) and 26% as poor metabolizer (PM). It can conclude
that clopidogrel non-responders were strongly associated with mutant
CYP2C19 genotype, particularly in patients who carried at least one
mutant allele; CYP2C19*2. Thus pharmacogenetic testing of CYP2C19
prior using these drugs will help physician to select the suitable dosage
for individual patient resulting in improve the efcacy and safety of the
drug therapy.
Paper No.: 2204

EFFECTS OF REDUCTION OF SYMPATHETIC NERVOUS
TONE ON VENTRICAL MYOCYTESCONTRACTION AND THE
RESPONSE OF B1 AND B2 ADRENOCEPTOR TO
ISOPRENALINE INTHE AGED RATS
(3) Duquesne University, Departments of Chemistry and Biochemistry,

Pittsburgh, PA,USA
Recognition of the psychostimulants cocaine and amphetamine by the
dopamine transporter (DAT) protein is principally responsible for the
euphoria and addiction associated with these drugs. The crystal structure
of a distantly related bacterial leucine transporter served as template to
create a 3-D DAT computer molecular model. Ligand docking to the
model revealed potential substrate and inhibitor pockets, conrmed by in
vitro pharmacology. An inhibitor pocket dened by the DAT model to be
within the extracellular vestibule, above the external gate of the primary substrate pocket, was used for virtual screening of a structural
library of compounds. High-throughput docking and application of pharmacophore constraints within this vestibular pocket identied a compound structurally dissimilar to the classic monoamine transporter
inhibitors. The compound displaced binding of cocaine analogs at all
three monoamine transporters, usually with high nanomolar Ki values
and within two fold of cocaines afnity at the norepinephrine transporter. Although a weak DAT inhibitor, this compound reduced by three
fold the potency of cocaine in inhibiting DAT-mediated cellular uptake
of dopamine. To our knowledge, the present ndings are the rst to successfully employ receptor-based Modelling to identify high afnity
monoamine transporter ligands. Screening using monoamine transporter
models provides a rapid, low cost discovery process that should accelerate identication of ligand scaffolds and provide lead compounds in
combating psychostimulant addiction and in treating other monoaminerelated CNS diseases.
Hong Sun(1), Z Zhao, L Gao, Q Wu, Y Hao, C Xu, Y Sun, C Yan

(1) Xuzhou Medical College, Xuzhou, PR China
The present study was to investigate the effects of centrally reducing
sympathetic nervous tone on cardiomyocytes contraction and to analyze
the contribution of b-adrenoreceptors (b-ARs) subtypes-b1 and b2-AR,
to contraction in isolated ventricular myocytes of the aged rats. SpragueDawley rats were randomly divided into an adult group, an aged group
and an aged with clonidine group. After pretreated by CGP20712A (the
selective b1-ARantagonist), or ICI118,551 (the selective b2-AR antagonist) or no pretreatment, the contractile responses of cardiomyocytes of
each group were observed respectively under the perfusion of KrebsHenseleit solution containing isoprenaline. The results showed that the
contractile response to isoprenaline was restored by clonidine in the aged
rats. CGP20712A depressed the cardiomyocytes contractile response in
adult group more evidently than that in the aged group and in the aged
with clonidine group. The contractile amplitude of myocytes incubated
with ICI118,551 dropped in the aged group, especially in the aged with
clonidine group. But ICI118,551 had no effect on the contractile amplitude of adult cardiomyoctyes. The contents of b1-adrenoceptor and b2adrenoceptor in cardiomyoctes had no differences among each group.
These results suggested that reducing the central sympathetic tone
improved the ventricular myocytes contractile, and the b2-adrenoceptors
played an important role in the aged rat heart.
Paper No.: 522

DISCOVERY
DISCOVERY OF A MONOAMINE TRANSPORTER INHIBITOR
VIA HIGH THROUGHPUT MOLECULAR MODEL DOCKING
AND PHARMACOPHORE MODELING
Christopher Surratt(1), M Indarte(2), Y Liu(1), J Madura(3)
Paper No.: 2486

MOLECULAR MECHANISMS UNDERLYING THE LACK OF
D-OPIOIDS-INDUCED REWARDING EFFECTS ASSOCIATED
WITH THE DOWNREGULATION OF CENTRAL
DOPAMINERGIC TRANSMISSION UNDER THE
NEUROPATHIC PAIN-LIKE STATE
Tsutomu Suzuki, S Imai, M Narita, K Niikura, N Kuzumaki, M Narita
Tokyo School of Pharmacy and Pharmaceutical Sciences, Department of
Toxiclogy, Tokyo, Japan
Neuropathic pain is most difcult to manage the pain, and loses the motivation with decreasing the quality of life. Dopaminergic neurons in the
ventral tegmental area (VTA) mainly innervate to the nucleus accumbens
(N.Acc) and medial prefrontal cortex (mPFC). It has been reported that
the ascending dopamine projection from the VTA to the N.Acc is mostly
related to motivational functions and the reinforcing effects of opioids.
The present study was then undertaken to investigate the alteration of
central dopaminergic system under the neuropathic pain induced by sciatic nerve ligaion in mice. In the present study, we demonstrated that
phosphorylated-tyrosine hydroxylase (p-TH) immunoreactivity in the
VTA was dramatically attenuated by sciatic nerve ligation. Furthermore,
the sciatic nerve ligation dramatically attenuated the d-opioid receptormediated G-protein activation without changing d-opioid receptor expression in the VTA. This phenomenon resulted in the decrease in dopamine
release induced by the d-opioid receptor agonist in the N.Acc. Under
these conditions, the sciatic nerve ligation suppressed the rewarding
effects induced by microinjection of d-opioids into the VTA of mice.
These ndings suggest that alterations of dopaminergic function could be
responsible for the suppression of opioid dependence under the neuropathic pain-like state by sciatic nerve ligation.
(1) Duquesne University Mylan School of Pharmacy, Division of Pharmaceutical Sciences, Pittsburgh, PA, USA
(2) University of Texas Health Science Center at Houston, School of
Health Information Sciences, Houston, TX, USA
598
Paper No.: 1932
DISEASE AND THERAPY
FUNCTION OF BK-VDCC COMPLEX IN VASCULAR SMOOTH
MUSCLE CELLS
Yoshiaki Suzuki, H Yamamura, S Ohya, Y Imaizumi
Nagoya City University Graduate School of Pharmacy, Department of
Molecular and Cellular Pharmacology, Nagoya, Japan
Large-conductance Ca2 + -activated K+ (BK) channel contributes to
diverse biological functions ranging from modulation of neurotransmitter
release to regulation of smooth muscle tone. In some tissues, it has been
reported that BK channel activation requires Ca2 + delivery through voltage dependent Ca2 + channel (VDCC), and BK channel forms molecular
complex with VDCC. Here we examined single-molecule imaging of
BK-VDCC complex in plasma membrane using total internal reection
uorescence (TIRF) microscopy. We also examined functional coupling
between BK channel and VDCC in mouse aortic smooth muscle cells
(ASMCs) using patch clamp technique. When co-expressing BKa-YFP
with VDCCa1C-CFP in HEK293 cells or primary cultured ASMCs, most
of BK channels co-localized with VDCCs, and these channels demonstrated uorescence resonance energy transfer (FRET) interaction.
Recently, it has been reported that some signaling proteins accumulate in
caveolae and organize functional microdomain. To examine the possible
involvement of BK-VDCC complex in caveolae, we used caveolin-1
knockout (Cav1-KO) mice, genetically lacking caveolae. When coexpressing BKa-YFP with VDCCa1C-CFP in ASMCs isolated from
Cav1-KO mice, the molecular interaction of these two channels was signicantly smaller than that in ASMCs from wildtype mice. In patch
clamp analysis, BK current coupling with VDCC in ASMCs from Cav1KO mice was signicantly smaller than that in ASMCs from wildtype
mice. These results suggest that a substantial part of BK channels in
ASMCs compose molecular complex with VDCCs and accumulate in
caveolae, where functional Ca2 + microdomain is supposed to be formed.
Paper No.: 1699

THE ANTIHYPNOTIC AND ANTIHYPOXIC EFFECTS OF
MILDRONATE
Baiba Svalbe(1,2), R Muceniece(2), E Vavers(1), E Liepinsh(1),
L Zvejniece(1)
(2) University of Latvia, Riga, Latvia
Mildronate (3-(2,2,2-trimethylhydrazinium)propionate), a structural analogue of L-carnitine, is used in clinics as an anti-ischemic drug for neurological applications. The effects of mildronate on the nervous system
have primarily been evaluated after long-term treatment, which signicantly decreases the tissue concentrations of L-carnitine. However, the
effects of acute administration of mildronate have not been systematically
examined before. Therefore, this study was undertaken to investigate the
acute effect of mildronate on alcohol-induced impairments by testing
effects on loss of righting reex (LORR) and decreased cerebral blood
ow. We also studied the effect of mildronate on changes in blood ow
and oxygen saturation in rat brain after short-term occlusion of a. carotis
communis (CCA). Mildronate administration exerted a dose-dependent
antihypnotic effect: at dose of 200 mg/kg mildronate signicantly lowered the effect of ethanol-induced LORR. The anti-hypnotic activity of
mildronate was partially blocked after pre-treatment with L-NAME.
Mildronate at doses of 100 and 200 mg/kg decreased the magnitude of
the drop in oxygen saturation; this effect was statistically signicant at a
dose of 200 mg/kg. Moreover, mildronate partially inhibited the ethanolinduced reduction in cortical cerebral blood ow. In conclusion, the acute
administration of mildronate protects against ethanol-induced narcosis, as

well as disturbed cortical blood ow and oxygen saturation after shortterm CCA occlusion. These ndings indicate that acute administration of
mildronate could be benecial for the treatment of alcohol intoxication.
Paper No.: 3419

ACTIVATION OF LIVER X RECEPTORS (A AND B) BY HIV
PROTEASE INHIBITORS ATAZANAVIR AND RITONAVIR AND
INHIBITION BY NON-NUCLEOSIDE REVERSE
TRANSCRIPTASE INHIBITOR EFAVIRENZ
Jenny Svard(1), JP Spiers(1), F Mulcahy(2), M Hennessy(1)
(1) Trinity College, Department of Pharmacology & Therapeutics,
Dublin, Ireland
(2) St Jamess Hospital, Department of Genitourinary Medicine and
Infectious Diseases, Dublin, Ireland
Liver X receptors are nuclear receptors associated with regulation of cholesterol metabolism and lipid biosynthesis. Although LXR isoforms
appear to have common activators, they differ in their tissue distribution;
LXRa is highly expressed in liver, intestine, adipose tissue and macrophages whereas LXRb is ubiquitously expressed. While some HIV protease inhibitors (PIs) activate pregnane X receptor (PXR), it remains to be
established if they are ligands of LXRs. Recent data indicates that LXR
and PXR may share activators.The present study examined the ability of
several PIs (atazanavir (ATV), fosamprenavir (FOS), lopinavir (LPV)
and ritonavir (RTV)) and one non-nucleoside reverse transcriptase inhibitor (efavirenz (EFV)) to activate LXRa and LXRb using a luciferase
reporter assay in HepG2 cells. The synthetic LXR agonist T0901317
was used as a positive control. Data were normalised to internal standard
(pRL-TK), analysed by one-way ANOVA with Dunnetts post hoc analysis and presented as meanSEM. ATV and RTV activated (10uM;
P < 0.05) LXRa (fold increase ATV 1.74 0.31, RTV 2.56 0.76) and
LXRb (fold increase ATV 1.53 0.40 and RTV 1.73 0.65) compared
to vehicle control (DMSO). FOS and LPV had no effect on LXRa/b
activation at 10uM. Interestingly, EFV (10uM) decreased basal activation
of LXRa and LXRb by 72% and 93% respectively. In conclusion, we
show that antiretroviral agents differentially activate both LXR isoforms
in HepG2 cells. As LXR is intrinsically coupled to the regulation of
ATP-binding cassette transporters, glucose transporters, lipoproteins and
transcription factors, this expands our understanding of the metabolic
abnormalities associated with antiretroviral treatment.
Paper No.: 2940

DETERMINATION OF OLANZAPINE IN HUMAN PLASMA BY
A VALIDATED LC-MS/MS METHOD
Dobrin Svinarov, L Kasabova
Medical University of Soa, TDM & Department of Clinical Pharmacology, Soa, Bulgaria
Development and validation of a LC-MS/MS determination of olanzapine (OLZ) was performed with the aim to be applied for pharmacokinetic analysis and therapeutic drug monitoring. OLZ and olanzapine-D3
(OLZD3, internal standard) were extracted from 50 lL of human plasma
with 1-chlorobutane. Chromatographic separation was performed on a
C18 analytical column with isocratic elution, utilizing a mobile phase
consisting of methanol and ammonium acetate-formic acid buffer. Positive-ion electrospray ionization and selected reaction monitoring were
used to follow the predominant transitions: collision energy (CE)=23 at
m/z 313?256 for OLZ, and CE=23 at m/z 316?256 for OLZD3. Selectiv-
599
ity was assessed with 6 different individual sources of human plasma,
and conrmed with matrix effect (ME) averaging 110-111% for OLZ,
109-113% for OLZD3, and relative ME of 98-102% for OLZ. Accuracy
and precision (within-run and between-runs) were all within 10%.
Extraction recoveries averaged 83-91% (OLZ) and 83-95% (OLZD3);
linearity range was 0.03232.96 lg/L, R2 > 0.99. Freeze-thaw stability
was determined for three cycles each lasting 24 h, post-preparative stability was documented for 48 h at 8oC, short-term stability at ambient temperature was proven for 4 h in the dark and for 2 h at daylight; stock
solution stability and long term stability in plasma - for 60 days at 20oC. With run time of less than 2.5 min, a throughput of over 150 samples per working day can be achieved. The method was validated according to FDA requirements and allows the accurate and precise
determination of OLZ in human plasma.
Paper No.: 513

REDUCED ACTIVITY OF CYP2C9 IN THE APPLICATION OF
A COMPLEX PHARMACOTHERAPY AT RUSSIAN PATIENTS
WITH DECOMPENSATION OF CHRONIC HEART FAILURE
Dmitry A Sychev(1), A Levanov(2), A Savchenko(1), G Ramenckaya(1),
V Dmitriev(1), S Bogoslovscaya(2), V Kukes(1)
this GAD DSM-IV-TR criteria combine both presentation of anxious tension and low energy (being easily fatigued). Developed in Russia novel
drugs Afobazol (benzimidazole derivate) and Ladasten (2-aminoadamantane derivate) present in their spectrum of psychotropic activity not only
anxiolytic but psychostimulating properties as well. Afobazol and Ladasten 2-week double-blind placebo-controlled efcacy and safety studies
were conducted in patients aged 18-50 years old diagnosed with GAD
according DSM-IV-TR criteria. Trials endpoints included evaluation of
Symptomatic scale, HAM-A, CGI, Spielberger state-anxiety scale and
self-being-activity-mood scale score changes from baseline at each postrandomization visit. These studies showed that such a combination of
both anxiolytic and psychostimulating action provided better outcome
than seen during benzodiazepine treatment. This improved efcacy can
be explained by combined simultaneous reduction of both emotional tension and low energy symptoms. It was shown that Afobazol demonstrated more anxiolytic than psychostimulating properties and Ladasten
more psychostimulating than anxiolytic properties. Such psychotropic
effects ratios provide opportunity for differentiated treatment of patients
depending on domination of anxiety or low energy symptoms in the
GAD course. Moreover Afobazol and Ladasten treatment was associated
with favorable changes of psychophysiological parameters and cognitive
functioning and was characterized by good safety prole. Afobazol and
Ladasten discontinuation didnt produce any withdrawal symptoms.
Thus, drugs with both anxiolytic and psychostimulating action provide
new opportunities for anxiety disorders treatment.
(1) Sechenovs Moscow Medical Academy, Department of Clinical Pharmacology, Moscow,Russian Federation
(2) Saratov State Medical University, St Petersburg, Russian Federation
CYP2C9 is involved in the biotransformation of a number of widely
used drugs: NSAIDs, oral anticoagulants, oral hypoglycemic drugs, losartan, etc. It is known that decompensation of chronic heart failure
(CHF) is reduced activity of microsomal liver enzymes. Aim: to study
the dynamics of the activity of CYP2C9on the application of a complex
pharmacotherapy of patients with decompensation of CHF. Materials and
methods. The study included 10 patients with decompensation of
CHF(IV NYHA), mean age was 67 years (55-78). All patients suffer
from coronary heart disease. Patients carried standard complex pharmacotherapy (parenteral diuretics, ACE inhibitors, digoxin). CYP2C9 activity was evaluated against the concentration of losartan to its metabolite
E-3174 (L/M) in daily urine after administration of losartan at a dose of
50 mg. CYP2C9 activity were studied on admission and before discharge of patients from hospital. The concentration of losartan and its
metabolite E-3174 in urine was determined by high performance liquid
chromatography (HPLC). Results: After application of a complex pharmacotherapy (diuretics, ACE inhibitors, digoxin, spironolactone) at
patients with decompensation of CHF, we observed decreasing ratio L/M
from 0,74(0,08-2,70) to 0,43 (0-1,06), p = 0,043. This was accompanied
by increased tolerance to physical load from 75 meters (40-100) to 250
meters (150-320) according to the 6-minute test with walking, p = 0,032.
Therefore, we can assume that patients with decompensation of CHF
should be applied drug-substrates of CYP2C9 in minimal doses, increasing the dose to the therapeutic medium only in compensated patients.
Paper No.: 1627

DRUGS WITH BOTH ANXIOLYTIC AND
PSYCHOSTIMULATING ACTION: NEW OPPORTUNITIES
FOR ANXIETY DISORDERS TREATMENT
Sergey Syunyakov, D Chumakov, E Teleshova, G Neznamov
Russian Academy of Medical Sciences, Zakusovs Institute of
Pharmacology, Moscow, Russian Federation
Paper No.: 2758

IMPAIRED NEUROGENIC RELAXATION OF THE SPHINCTER
OF ODDI OF RABBITS WITH INSULIN RESISTANCE
Zoltan Szilvassy, J Nemeth, R Sari, B Peitl
University of Debrecen, Department of Pharmacology and Pharmacotherapy and Pharmapolis Debrecen Ltd, Debrecen, Hungary
Aim: To study if non adrenergic, non cholinergic (NANC) relaxation of
the rabbit sphincter of Oddi (SO) was inuenced by insulin resistance
(IR) associated with either hypercholesterolaemia or chronic partial hepatic denervation (CHSD). Methods: The SO muscle rings were prepared
from normal rabbits and from those made IR either by cholesterolenriched diet or by cut of the anterior hepatic plexus (CHSD). The hypercholesterolaemia and CHSD decreased insulin sensitivity determined
by hyperinsulinaemic euglycaemic glucose clamping. Changes in isometric tension were measured in SO rings subjected eld stimulation before
and after successive incubations in NANC solution (guanethidine, atropine, L-NAME and L arginine). Concentrations of neuropeptides (VIP,
CGRP and somatostatin) were determined from the incubation medium,
whereas NO was measured from SO tissue at maximum contractile
responses. For control to these experiments served SO rings from normal
rabbits. Results: Rings from normal rabbits responded to FS with contractions followed by relaxation with an increase in neuropeptide and
NO concentrations. In NANC solution, FS produced similar increases in
neuropeptide release and NO synthesis with monophasic relaxation. L
NAME reversed NANC relaxation; the L-arginine reversed the effect of
L-NAME. In rings from hypercholesterolaemic IR rabbits, the NANC
relaxation was not seen with no signicant increase in release of NO or
sensory neuropeptides. In CHSD sphincters, there were no signicant
increases in NO with an attenuated increase in neuropeptide release.
Conclusion: NANC relaxation is impaired in SO from IR rabbits. This is
possibly underpinned by a decrease in neuropeptide release triggered by
NO.
Drugs with anxiolytic action in spectrum of psychotropic activity are traditionally used for generalized anxiety disorder (GAD) treatment. Along
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Paper No.: 1814
DISEASES
INVESTIGATION OF THE ROLE OF TRPV1 CAPSAICIN
RECEPTORS AND TACHYKININS IN DEXTRANE-SULPHATE
INDUCED MOUSE COLITIS MODEL
Istvan Szitter(1), E Pinter(1), G Pozsgai(1), K Sandor(1), A Kemeny(1),
A Perkecz(1), K Elekes(1), Z Helyes(1), J Szolcsanyi(1), J Quinn(2)
(1) University of Pecs Faculty of Medicine, Department of Pharmacology
and Pharmacotherapy, Pecs, Hungary
(2) University of Liverpool, Department of Molecular Biology, Liverpool, UK
Data concerning the role of Transient Receptor Potential Vanilloid 1
(TRPV1) cation channels and neurogenic inammation in colitis are contradictory, the mechanisms are not clearly understood. Tachykinins
encoded by the preprotachykinin A (TAC1) gene such as substance P
(SP) and neurokinin A (NKA) induce oedema and activate inammatory
cells via predominantly tachykinin NK1 and NK2 receptors, respectively.
The present study investigated the role of TRPV1 receptors and the
tachykinin system in dextrane-sulfate (DSS)-induced colitis in wildtype,
TRPV1-/-, TAC1-/-, NK1-/- and double knockout (TAC1-/-/NK1-/-) mice.
Colitis was induced by oral DSS solution administration for 7 days, control animals received tap water. Disease activity index was assessed on
the basis of weight loss, stool consistency and blood content. Histological analysis and semiquantitative scoring were performed. IL-1b and
myeloperoxidase activity referring to accumulated granulocytes and macrophages were measured from bowel homogenates with ELISA and
spectrophotometry, respectively. The symptoms were signicantly less
severe in all gene-decient groups after the fth day of the experiment.
Histopathological changes and myeloperoxidase activity in the distal
colon were signicantly reduced in TRPV1-/- and TAC1-/-/NK1-/- mice,
but IL-1b production was not altered in any groups.It concluded that
TRPV1 cation channel activation increases the clinical severity, inammatory histopathological changes and granulocyte-macrophage accumulation in DSS-induced colitis but are not important in inammatory
cytokine production. The role of tachykinins in this model is more complex, TAC4 gene products (hemokinins) of non-neural origin also seem
to be involved.
Paper No.: 2493

CREATING TRPV1 KNOCK DOWN MICE BY TENTIVIRAL
TRANSGENESIS
in TRG neurons by microuorimetry. We observed that siRNA could

inhibit the TRPV1 expression more than 80% in various eukaryotic cell
lines and almost 100% in TRG neurons. We also tried the shRNA
approach. Here the DNA encoded interfering RNA sequence is cloned
behind a RNS polymerase III promoter (e.g. U6) in a DNA expression
plasmid. Three effective shRNA constructs could inhibit the TRPV1
expression more than >80%. The best shRNA construct was cloned into
a lentiviral system. The designed lentiviral vectors carrying shRNA were
used to create transgenic mice by microinjection. We tested the offsprings (F1 generation) of the founder transgenic mice and F2 generation
by capsaicin instillation into eyes and induction of neurogenic inammation. Our results indicate that shRNA can act in transgenic mice and inhibit the expression of TRPV1.
Paper No.: 2715

INVESTIGATION OF BIOAVAILABILITY OF NANOSIZED
TELMISARTAN IN RAT
Anita Sztojkov-Ivanov(1), G Filipcsei(2), G Heltovics(2), F Darvas(2),
I Zupko(1)
(1) University of Szeged, Department of Pharmacodynamics and Biopharmacy, Szeged, Hungary
(2) Nangenex Inc., Budapest, Hungary
Pharmacokinetic prole is a crucial feature of drug development which is
responsible for a 40% failure rate in clinical trials. The selection of drug
candidates in preclinical stage for early clinical phase is largely based on
their pharmacokinetic behavior. On the other hand, improvement of oral
bioavailability currently used agents is an outstanding possibility for
researchers involved in generic development. The aim of the work was
the characterization of the action of NanActive technology on the
serum concentrations of orally administered telmisartan in rat. Nanosized
and reference telmisartan (30 mg/kg) was administered in physiological
saline to both fasted and fed male rats. A currently available telmisartan
tablet was additionally administered to a group of rats. Blood samples
were collected after 15, 30 and 45 min, 1, 2 and 6 hours. Telmisartan
was extracted from serum samples and analyzed by a HPLC system with
gradient elution (methanol - phosphate buffer) and UV detection at
310 nm. Our results demonstrate that nanosized telmisartan prepared by
NanActive technology ensures signicantly more efcient absorption
than reference item evidenced by earlier maximal serum concentration as
well as substantially (roughly 6-fold) higher area under the curve (AUC)
value. Our results can be considered as a proof of technology which is
presumably applicable for many other established drugs with limited oral
availability.
Eva Szoke(1), DM Toth(2), K Kvell(1), B Bender(3), Z Bosze(3),

Z Sandor(2), J Szolcsanyi(1)
(1) University of Pecs, Department of Pharmacology and Pharmacotherapy, Pecs, Hungary
(2) University of Pecs, Analgesic Research Laboratory, Pecs and Gedeon
Richter Plc, Budapest, Hungary
(3) Agricultural Biotechnology Center, Godollo, Hungary
A novel method has appeared to investigate the function of genes by discovery of RNA interference (RNSi). RNSi is a post-transcriptional
silencing method and in the course of it we can destroy the mRNA of a
target gene with a sequence-specic double stranded RNA (dsRNA).
Our aim was to knock down the TRPV1 capsaicin receptor which is a
promising target for development new analgesic and anti-inammatory
drugs. In our rst experiments we used chemically synthesized siRNA
which is specic for TRPV1 mRNS. It was co-transfected with a reporter
plasmid carrying the ratTRPV1eGFP fusion gene into various eukaryotic
cell lines. The expression of the green uorescent protein was measured
by ow cytometry. Furthermore, we examined the efciency of siRNA
Paper No.: 2852

POTENTIALLY INAPPROPRIATE MEDICATION LEADING TO
ADVERSE DRUG REACTIONS AND HOSPITALISATION
Jacek Szymanski(1), S Schmiedl(1), S Holt(1), K Farker(2),
B Drewelow(3), M Hippius(4), K May(5), PA Thurmann(1)
(1) University Witten/Herdecke, Philipp Klee-Institute of Clinical
Pharmacology, HELIOS Clinic Wuppertal, Department of Clinical
Pharmacology, Wuppertal, Germany
(2) University of Jena, Institute of Pharmacology and Toxicology,
Department of Clinical Pharmacology, Regional Pharmacovigilance
Center Weimar, Germany
(3) University of Rostock, Institute of Clinical Pharmacology, Regional
Pharmacovigilance Center Rostock, Germany
601
(4) University of Jena, Institute of Pharmacology and Toxicology,
Department of Clinical Pharmacology, Regional Pharmacovigilance Center Jena, Germany
(5) University of Greifswald, Regional Pharmacovigilance Center Greifswald, Department of Clinical Pharmacology, Germany
A German list of Potentially inappropriate medication (PIM) was
recently introduced for elderly patients. We aimed to analyse PIM-related
adverse drug reactions (ADR) in a cohort of patients with serious ADR
leading to hospitalisation, the German Network of Regional Pharmacovigilance Centres (NRPC). The German PIM list was developed using a
delphi survey on drugs suspected for an increased ADR risk in elderly
patients (Holt S et al. BJCP 2009; 68 Suppl.1: 19). All patients admitted
between 2000 and 2007 to the NRPC (Schneeweiss S et al. BJCP 2001;
52: 196-200) were analysed using documented medical history, medication and causality assessment. 1122 (18%) of 6181 patients (age
70 16 years, 60% females) received PIM. The most commonly
observed PIM were sotalol (138), pentoxifylline (99), doxazosin (93) and
amitriptyline (91). Psychotropics were the most frequently concerned
class of drugs. In 407 patients (7% of all patients with ADR and 36% of
PIM-users) PIM-drugs were assessed as possibly related to cause an
ADR. Sotalol (66), indometacin (50) and pentoxifylline (47) were most
frequently involved resulting in cardiac arrhythmia, gastrointestinal
bleeding and syncope. A case-based ADR database allows to evaluate
PIM-related risks. Our ndings have to be discussed in context with prescribing frequencies, different age groups and polypharmacy in our
cohort. The strength and usefulness of a PIM-list has to be evaluated in
nursing homes, geriatric in- and outpatients and with special regard to
age-specic ADR such as decline of cognitive function and falls.
Supported by: BMBF Fo 01ET0721, BfArM Fo 2.1-68502-201
Paper No.: 2985

EFFECT OF COMBINED PSYCHIATRIC AND CLINICAL
PHARMACOLOGICAL AUDIT
Eva Sdder(1), B Brock(3), B Klindt Poulsen(3), U Bartels(2)
(2) Risskov Hospital, Department of Psychiatric, Aarhus, Denmark
(3) Aarhus Hospital, Department of Clinical Pharmacology, Aarhus,
Denmark
Introduction: Interactions are often overlooked. Examples are clozapine
and smoking, which induce metabolism by 50%. Sct. Johns Wort
decreases AUC and Cmax of amitriptyline/nortriptyline by 40/20%.
SSRI increase the risk of bleeding by reducing the effect of thrombocytes. Tramadol and Sct. Johns Wort increase serotonin and, therefore,
concomitant treatment with e.g. SSRI, SNRI, and MAOI increase the risk
of serotonin toxicity. In a medical audit we focused on these drugs and
registration of smoking and use of herbal medicine. Materials/patients:
Eight psychiatry wards were included. At each ward 20 medical records
were chosen at random. Medication at admission and smoking/herbal
medicine were obtained from the medical records. Results: 158 patients
were included. Smoking was registered in 41 cases (25%). Herbal medicine in 11 cases (7%). Clozapine treatment: 7 patients (4%); smoking
was only registered in 2 cases. Nortriptyline/amitriptyline: 6 patients
(4%); herbal medication was not registered in any case. Concomitant
treatment with SSRI and NSAID: thirteen patients (8%); none were in
concomitant treatment with PPI. Tramadol + >1 other drug that potentially increase risk of serotonin toxicity: ve patients (3%). Conclusion:
This audit demonstrated a high incidence of lack of clinical relevant registrations and lack of relevant prescription of PPI. The potential of an
audit conducted by a team consisting of a psychiatrist and a clinical pharmacologist was demonstrated. There is an unmet need for increased
attention on medication at psychiatric wards.
Paper No.: 3350

THE STUDY OF EFFECT OF IOHEXOL ON SCHIRMER TEAR
TEST VALUES IN CLINICALLY NORMAL CATTLE
Aboutorab Tabatabaei Naeini, J Ceverie
Shiraz University School of Veterinary Medicine, Department of Clinical
Sciences, Shiraz, Iran
Knowledge of normal tear production parameters is important for the differential diagnosis of the most eye diseases. Tear play an important role
in maintaining the health and normal function of the conjunctiva and cornea. Dacryocystography has been widely used in the assessment of the
nasolacrimal duct system.The water-soluble agent iohexol is currently
widely used in nervous and vascular lesions in diagnostic radiology and
also employed in dacryocystography. The aim of this study was to investigate the effects of iohexol on tear production and normal uctuations of
Schirmer tear test (STT) values in cattle. Tear production was measurd in
10 Holstein Frisian cattle with the same range of age. All animals were
healthy as determined by clinical and paraclinical examination. Tear production was measured using the standard Schirmer tear test with commercial tear ow test strip.The STT of ten normal cattle were
determined, which were done pre-and post-treatment of iohexol during
dacryocystography. Schirmer tear test was performed at 0 h, 2 h and
weeks pre-and post-dacryocystography with iohexol. Cattle were housed
outdoors throughout the study. There were no signicant differences in
tear production before and after administration of iohexol. The slight
uctuations in STT may be as a result of individual and/or environmental
changes. Iohexol did not change STT values of the cattle in this study.
No signs of conjunctivitis or corneal inammation were detected in any
of the eye examined.
Paper No.: 2190

ATRIAL ENLARGEMENT AND DECREASED HEART RATE IN
SHRSP.Z-LEPRFA/IZMDMCR RATS, A MODEL OF
METABOLIC SYNDROME
Yukari Tada, S Kagota, N Nejime, H Wakuda, K Nakamura,
M Kunitomo, K Shinozuka
Mukogawa Womens University, Department of Pharmacology,
Nishinomiya, Hyogo, Japan
Metabolic syndrome has been markedly increasing in occurrence and
associated with the onset of cardiovascular diseases, such as ischemic
heart disease. Metabolic syndrome is a risk factor for atrial hypertrophy
and brillation, however, the effect on atrial pacing is still uncertain. We
therefore investigated changes in atrial size and heart rate in SHRSP.ZLeprfa/IzmDmcr rats (SHRSPZF) with metabolic syndrome. We compared responses in male 16-week-old SHRSPZF to lean littermates
(SHRSP) and normotensive Wistar-Kyoto rats (WKY). Heart rate in
vivo, beating of isolated atrial strips, and protein expression of endothelial nitric oxide synthase (eNOS) in atrium were determined by tail-cuff,
myograph, and western blot methods, respectively. The heart rate was
signicantly faster in SHRSP than in WKY, but signicantly slower in
SHRSPZF than in SHRSP. Atrial weight was greater in SHRSPZF than
in SHRSP and WKY. The atrial rate decreased in SHRSPZF and SHRSP
compared with that in WKY. Atrial weights were correlated inversely to
heart rate or atrial rate in SHRSPZF. Protein expression of eNOS in
atrium was signicantly increased in SHRSPZF compared with that in
WKY and SHRSP. Chronotropic responses in isolated atria from WKY
were depressed by nitroprusside and nitroglycerin in concentrationdependent manners. Tertiapin, an inhibitor of inwardly rectifying potassium channels (Kir), signicantly inhibited the negative chronotropic
response induced by nitroprusside whereas 4-aminopyridine, apamin and
602
glibenclamide had no effect. These results suggest that metabolic syndrome in this model was associated with atrial enlargement and decrease
in heart rate, possibly the latter via activating Kir by NO.
Paper No.: 2203

HEALTH AND DISEASE
ELECTROPHARMACOLOGICAL ANALYSIS OF TRIGGERED
ACTIVITY IN THE PULMONARY VEIN MYOCARDIUM OF
THE GUINEA PIG
Akira Takahara, T Sugimoto, K Takeda, I Namekata, H Tanaka,
Toho University of Pharmaceutical Sciences, Department of Pharmacology, Funabashi, Japan
Introduction: The pulmonary vein is known to be an important source of
ectopic beats, initiating frequent paroxysms of atrial brillation. In this
study, we pharmacologically analyzed triggered activity elicited in the
isolated pulmonary vein from the guinea pig. Methods and Results:
Immediately after the termination of burst pacing (pacing cycle length of
100-200 ms, n = 100), spontaneous activities accompanied with phase-4
depolarization were detected in 46 out of 48 pulmonary vein preparations. Such triggered activities were not observed in 8 isolated left atria.
The resting membrane potential of the pulmonary vein myocardium was
more positive than that of the left atrium. The incidence of triggered
activities increased in a frequency-dependent manner. Verapamil (1
microM), ryanodine (0.1 microM) and pilsicainide (10 microM) suppressed the occurrence of triggered activities. Carbachol (0.3 microM)
hyperpolized the resting membrane potential of the pulmonary vein myocardium, and completely inhibited the occurrence of triggered activities.
Conclusion: These results suggest that the pulmonary veins have more
arrhythmogenic features than the left atrium through lower resting membrane potential. Intracellular Ca2 + overload may be associated with
spontaneous activity of the pulmonary vein.
Paper No.: 852

DISEASES
PROSTAGLANDIN E2 INHIBITS ADVANCED GLYCATION END
PRODUCTS-INDUCED ACTIVATION OF HUMAN
MONOCYTES
Hideo Takahashi(1), K Liu(1), H Wake(1), S Mori(2), M Nishibori(1)
(1) Okayama University Graduate School of Medicine, Dentistry and
Pharmaceutical Sciences, Department of Pharmacology, Okayama, Japan
(2) Shujitsu University, Okayama, Japan
Introduction: Advanced glycation end products (AGEs) subtypes, proteins or lipids that become glycated after exposure to sugars, induce complications in diabetes. Among the various AGE subtypes, AGE-2 and
AGE-3 have been indicated to play roles in inammation in diabetic
patients. The engagement of AGEs and receptor for AGEs (RAGE) activates monocytes. Since the engagement of intercellular adhesion molecule-1 (ICAM-1), B7.1, B7.2 and CD40 on monocytes with their ligands
on T-cells plays roles in cytokine production, we investigated the effects
of AGE-2 and AGE-3 on the xpressions of ICAM-1, B7.1, B7.2 and
CD40 on monocytes, the production of IFN-c and TNF-a and the lymphocyte proliferation in human peripheral blood mononuclear cells
(PBMC) and their modulation by PGE2. Methods: Flow cytometry was
used to examine the expression of ICAM-1, B7.1, B7.2 and CD40. Cytokine production and cAMP was determined by ELISA. Lymphocyte proliferation was determined by [3H]-thymidine uptake Results and
Conclusion: AGE-2 and AGE-3 induced the expressions of adhesion
molecule, the cytokine production and the lymphocyte proliferation.
PGE2 concentration-dependently inhibited the actions of AGE-2 and
AGE-3. The effects of PGE2 were mimicked by an EP2-receptor agonist,

ONO-AE1-259-01, and an EP4-receptor agonist, ONO-AE1-329. An
EP2-receptor antagonist, AH6809 and an EP4-receptor antagonist,
AH23848, inhibited the actions of PGE2. These results as a whole indicated that PGE2 inhibited the actions of AGE-2 and AGE-3 via EP2-/
EP4-receptors. PGE2 increases cAMP levels. The effects of PGE2 were
reversed by a PKA inhibitor, H89, and mimicked by dbcAMP and an
adenylate cyclase activator, forskolin.
Paper No.: 1457

IMPACT OF CYP3A5 AND ABCB1 GENE POLYMORPHISMS
ON PLASMA DISPOSITION OF FENTANYL AND ITS
CLINICAL RESPONSES IN CANCER PATIENTS WITH OPIOID
SWITCHING TO FENTANYL TRANSDERMAL SYSTEM
Yoshiaki Takashina(1), T Naito(1), Y Mino(1), K Ohnishi(2),
J Kawakami(1)
(1) Hamamatsu University School of Medicine, Department of Hospital
Pharmacy, Hamamatsu, Japan
(2) Hamamatsu University School of Medicine, Oncology Center,
Hamamatsu, Japan
Introduction: Fentanyl is metabolized by CYP3A, and its passage across
the brain-brain barrier is limited by p-glycoprotein. Inuence of their
gene polymorphisms on clinical response to fentanyl has not been fully
claried. The aim of this study was to evaluate the plasma disposition of
fentanyl and incidence of rescue medication and adverse effects based on
CYP3A5 and ABCB1 gene polymorphisms in cancer patients with opioid switching to fentanyl transdermal system. Patients: Sixty Japanese
cancer patients treated with fentanyl transdermal reservoir system according to the guideline in Japan were enrolled. Plasma fentanyl concentration was determined at 192 hours after opioid switching. Rescue
medication and adverse effects caused by opioid switching were evaluated. Results: Plasma fentanyl concentration normalized with measured
absorption rate was signicantly higher in CYP3A5*3/*3 than *1/
*1 + *1/*3 group. There were no signicant differences in the measured
absorption rate normalized plasma fentanyl concentration between
ABCB1 gene polymorphisms. The incidence of central adverse effects
was signicantly higher in CYP3A5*3/*3 than *1/*1 + *1/*3 group.
With respect to rescue medication, ABCB1 1236TT group was signicant less than 1236CC+CT group. No signicant differences were
observed in the incidence of rescue medication and adverse effects
between ABCB1 G2677A/T and C3435T. Conclusion: CYP3A5*3 affects
the plasma disposition of fentanyl and the incidence of central adverse
effects. ABCB1 C1236T decreases the incidence of rescue medication
after opioid switching. CYP3A5*3 and ABCB1 C1236T would contribute to interindividual difference in clinical response to fentanyl in cancer
patients with opioid switching to fentanyl transdermal system.
Paper No.: 1676

HEALTH AND DISEASE
PROTON MEDIATES PERIVASCULAR ADRENERGIC AND
CGRPERICG NEUROTRANSMISSION IN THE RAT
MESENTERIC ARTERY
Shingo Takatori, K Hirai, H Fujiwara, X Jin, S Egutchi, Y Kitamura,
H Kawasaki
Okayama University, Department of Clinical Pharmaceutical Science,
Okayama, Japan
We reported that nicotine stimulates presynaptic nicotinic ACh receptors
in adrenergic nerves and activates TRPV1 located on CGRP-containing
vasodilator nerves (CGRPergic nerves) resulting in vasodilation. The aim
603
of this study is to investigate whether proton acts as an axo-axonal transmitter in perivascular nerves. Rat perfused mesenteric vascular beds
without endothelium were contracted by perfusion with Krebs solution
containing methoxamine and the pH levels of perfusate were measured
with a pH meter. Perfusion of nicotine (1-100 lM) or capsaicin for
1 min and periarterial nerve stimulation (PNS) lowered pH levels of the
perfusate concomitant with g vasodilation. Cold-storage denervation
(4C for 72 h) of the preparation abolished pH lowering induced by nicotine and PNS. Guanethidine inhibited PNS- and nicotine-induced lowering of pH levels. Mecamylamine blunted nicotine-induced pH lowering
and vasodilation, but TRPV1 antagonist capsazepine and ruthenium red
inhibited only nicotine-induced vasodilation. The study using a uorescent pH indicator demonstrated perivascular pH decrease after nicotine
applied outside small mesenteric artery. Immunohitochemical study
showed dense innervation of adrenergic tyrosine hydroxylase (TH)-like
immunoreactivity (LI) and CGRP-LI-containing nerves and both
appeared in the same neuron. CGRP-LI and TRPV1-LI-containing
nerves also appeared in the same neurons. Application of HCl in
denuded preparations induced pH lowering and vasodilation, which was
inhibited by denervation, capsazepine, capsaicin pretreatment and
CGRP8-37 without affecting pH lowering. These results suggest that
excitement of adrenergic nerves releases protons to activate TRPV1 in
CGRPergic nerves and thereby induce vasodilation. It is also suggested
that CGRPergic nerves release proton associated with exocytosis.
Paper No.: 1642

DISEASES
IL-16 ACCELERATES CELL PROLIFERATION MEDIATED BY
ERK- CYCLIN D1 PATHWAY IN HEPATOCELLULAR
CARCINOMA
Yuko Takeba, N Matsumoto, T Kumai, M Watanabe, Y Kinoshita,
S Kobayashi
St. Marianna University School of Medicine, Department of Pharmacology, Kawasaki, Kanagawa, Japan
Several cytokines are known to play an important role in immune
response, angiogenesis, cell growth or differentiation in hepatocellular
carcinoma (HCC). To identify tumor-specic cytokines in HCC, human
HCC tissues and adjacent non-tumor tissues were analyzed by proteomic
approach using antibody-based protein microarray technique. The results
were compared with those from hepatic hemangioma, liver metastasis of
colon cancer from Japanese and normal liver tissues from Caucasian
transplantation donors. Protein levels and localization of identied cytokines were analyzed using Western blotting and immunohistochemistry.
Among the several cytokines, the expressions of the IL-1 receptor antagonist, macrophage migration inhibitory factor (MIF), plasminogen activator inhibitory-1(PAI-1) and IL-16 were increased in the HCC and in the
paired adjacent non-tumor tissue as compared with the tissue from normal livers. IL-16 level in the HCC was higher than the hepatic hemangioma or the liver metastasis of colon cancer. Furthermore, we found that
IL-16 signicantly increased cell proliferation in HCC cell line (HepG2
and Huh 7) in vitro. The proliferative response in HCC is mediated by
the ERK- cyclin D1 pathway. These results suggest that IL-16 may be
involved in cell growth mechanism in HCC.
Epilepsy is characterized by recurrent seizures caused by an excessive

discharge of cerebral neurons. In clinical practice, patients with epilepsy
are frequently associated with depression and cognitive impairment.
However, whether epilepsy is associated with psychic function remains
unclear. The present study was undertaken to clarify the effect of kindling with epileptic seizure on psychic function using a behavioral pharmacology test. Pentylenetetrazol kindled mice showed no signicant
difference in the locomotor activity and muscle relaxation in comparison
to the naive mice. On an emotional test, pentylenetetrazol kindled mice
showed no signicant difference of immobility time in a forced swimming test in comparison to the naive mice. In addition, imipramine signicantly decreased the duration of immobility in both naive and kindled
mice. Kindled mice showed an anxiolytic effect on an elevated-plus
maze test. We also investigated the social approach of kindled mice using
the three chamber social test. Naive mice showed a signicant preference
for social novelty in this test, while the kindled mice showed no interests.
These results suggested that in regard to their psychic functions, pentylenetetrazol kindled mice have specic characteristics such as anxiolytic
actions and social approaches.
Paper No.: 1127

INCREASE IN HIPPOCAMPAL SYNAPTOSOMALASSOCIATED PROTEIN 25 IN MICE ADAPTED TO CHRONIC
VARIABLE STRESS
Hiroshi Takeda, K Miyagawa, T Takeuchi, M Tsuji
International University of Health and Welfare, School of Pharmacy,
Division of Pharmacology, Tochigi, Japan
The ability to adapt to stress is an important defensive function of a living body, and impairment of this ability may contribute to some stressrelated disorders. Thus, the examination of brain mechanisms involved
in stress adaptation could help to pave the way for new therapeutic strategies for stress-related psychiatric disorders. The present study was tried
to examine the behavioral characteristics of mice subjected to chronic
variable stress (CVS), and also characterize changes in protein expression
in the hippocampus of this stress model. The daily schedule of CVS was
as follows: Day 1, restraint stress; Day 2, foot shock stress; Day 3, water
deprivation; Day 4, cold swimming stress; Day 5, psychological stress;
Day 6, food deprivation; Day 7, tail-pinch stress. The exploratory behaviors of mice were measured by hole-board test 24 h after the nal CVS
exposure, and then brain samples were collected. Exposure to CVS for 1
and 2 weeks produced the increase in head-dipping behavior and the
decrease in locomotor activity, respectively. These results suggest that
CVS may induce different types of emotional abnormalities depend on
the duration of stress exposure. On the other hand, exposure to CVS for
4 weeks did not induce any behavioral changes, indicating the development of stress adaptation. Proteome analysis revealed a signicant
increase in synaptosomal-associated protein 25 in the hippocampus of
mice adapted to CVS for 4 weeks. The present ndings suggest that
plasticity of transmitter exocytosis in the brain might be involved in the
development of stress adaptation.
Paper No.: 896

EMOTIONAL BEHAVIOR IN PENTYLENETETRAZOL
KINDLED MICE
Paper No.: 1687

HEALTH AND DISEASE
X6-POLYUNSATULATED FATTY ACIDS, BUT NOT OTHER
FREE FATTY ACIDS, EVOKE ENDOTHELIAL DYSFUNCTION
BY INHIBITING STORE-OPERATED CA2 + ENTRIES
Kenshi Takechi, K Suemaru, H Araki
Kazuhiko Takeuchi, J Wei, N Inui, H Watanabe
Ehime University Graduate School of Medicine, Division of Clinical

Pharmacology & Pharmacy, Ehime, Toon, Japan
Hamamatsu University School of Medicine, Department of Clinical

Pharmacology and Therapeutics, Hamamatsu, Japan
604
Introduction: Free fatty acid (FFA) levels are higher in patients with metabolic syndrome and diabetes mellitus susceptible to cardiovascular diseases, and circadian rhythm of FFA levels has similar pattern to that of
cardiovascular events, vasospastic angina, myocardial infarction and
ischemic stroke; high in the morning and low in the evening. However,
mechanical pathways through which FFAs contribute to vascular disorders are not completely understood. Thus, the present study was
designed to elucidate effects of major FFAs in human plasma on endothelium-dependent vascular relaxation (EDR) and endothelial Ca2 + signalling. Methods: Primary porcine aortic endothelial cells (PAECs) were
used for measurements of intracellular Ca2 + /Mn2 + concentrations and
NO production with fura-2/AM and DAF-FM/DA uoroscopy, and of
prostacyclin production by assessing 6-keto prostaglandin F1a with
enzyme immunoassay. Results: In PAECs, two omega6-polyunsatulated
fatty acids (x6PUFAs), arachidonic (AA, 2lM) and linoleic (LA, 2lM)
acids suppressed bradykinin (BK, 10nM)-induced prostacyclin and NO
productions by 5% and 7% at 0.1lM, by 41% and 59% at 5lM respectively, and abolished prostacyclin and NO productions at 10lM. Moreover, the x6PUFAs suppressed BK-induced endothelial Ca2 + responses
and thapsigargin-stimulated non-selective cation (Mn2 + ) channels in
dose-dependent manner. However, other FFAs (10lM), palmitic, stearic
and oleic acids, did not affect BK-induced prostacyclin and NO productions, or Ca2 + responses in PAECs. Conclusion: Among major FFAs,
only x6PUFAs, AA and LA, affect store-operated Ca2 + entry followed
by suppressing endothelial functions; NO and prostacyclin productions,
thus which may trigger cardiovascular events.
Paper No.: 2287

MUSCARINIC ACETYLCHOLINE RECEPTOR SUBTYPES
INVOLVED IN CARBACHOL-INDUCED HCO3- SECRETION IN
MOUSE DUODENUM IN VITRO
Koji Takeuchi, M Koyama, K Takahashi, E Aihara, N Kurata, S Hayashi
Japan
HCO32+
secretion in the
Cholinergic agents are known to stimulate the
duodenum through an increase of intracellular Ca via the activation of
muscarinic acetylcholine receptors (mAChRs). We investigated the roles
of mAChRs subtypes in the cholinergic regulation of HCO3- secretion
using mAChR M1~M5 knockout (KO) mice and demonstrated the
importance of M4-receptor in the response to carbachol (CCh). The duodenal mucosa of mice, stripped of the muscular layers, was mounted on
an Ussing chamber and HCO3- secretion was measured. CCh stimulated
duodenal HCO3- secretion in a dose-dependent manner, and this action
was totally inhibited by atropine given serosally 30 min before CCh. The
HCO3- response to CCh was similarly observed in M2- and M5-KO
mice, while signicantly decreased in M1-, M3- and M4-KO mice. The
decreased response in M4-KO mice, but not M1- or M3-KO mice, was
reversed by the co-addition of CYN154806 the SST2 receptor antagonist.
Somatostatin decreased the HCO3- response to CCh, in a CYN154806inhibitable manner. Immunohistochemial study revealed the localization
of M4 receptors in the duodenal D cells. The medium somatostatin level
in the WT mouse duodenum was decreased in response to CCh. The
duodenal HCO3- response to CCh is mainly mediated by the activation
of M1 and M3 receptors and modied by M4 receptors but does not
involve other mAChR subtypes. It is assumed that the activation of M4
receptors inhibits the release of somatostatin from D cells and results in
enhancement of the HCO3- response by minimizing the negative inuence of somatostatin.
Paper No.: 2288

DISEASES
ROLES OF PROSTAGLANDINS, NITRIC OXIDE AND
ENTEROBACTERIA IN PATHOGENESIS OF ISCHEMIC
ENTERITIS IN RATS
Koji Takeuchi, S Kojima, A Kuki, Y Nakamori, Y Komatsu, T Kotani
Departmen tof Pharmacology & Experimental Therapeutics, Kyoto,
Japan
We recently established an animal model of ischemic enteritis, yet the
pathogenic mechanism remains fully unexplored. We investigated the
roles of prostaglandins (PGs), nitric oxide (NO) and enterobacteria in the
development of ischemic enteritis in rats. Under ether anesthesia, the
superior mesenteric artery (SMA) of Male SD rats was exposed, and a
calibrated stenosis was produced by placing a needle on a blood vessel,
ligating both the vessel and needle, and then removing the needle from
the ligature. After the operation, the animals were fed normally and
killed various days later up to 3 days. Various agents were given for
2 days after the operation. The stenosis of SMA caused the mucosal
ischemia in the small intestine within 3 days. This model of enteritis was
accompanied by enterobacterial invasion in the mucosa. These lesions
was prevented by treatment with ampicillin (antibiotic), aztreonam
(gram-negative bacterium specic antibiotic), L-NAME (nonselective
NOS inhibitor) or aminoguanidine (selective iNOS inhibitor), while
markedly aggravated by indomethacin (nonselective COX inhibitor) and
rofecoxib (selective COX-2 inhibitor). The deleterious effect of indomethacin was abrogated by the co-administration of PGE2 or AE1-329
(EP4 agonist). The expressions of iNOS and COX-2 were both up-regulated in the small intestine in a time-dependent manner following the
onset of ischemia, together with the increase of mucosal NO and PGE2
contents. These results suggest that enterobacteria, especially gram-negative bacteria play a major pathogenic role in this model of ischemic
enteritis. And iNOS/NO is deleterious, while COX-2/PGE2 prevent the
ischemic enteritis through EP4 receptors.
Paper No.: 1319

THE MECHANISM OF GINGIVAL OVERGROWTH AS A SIDE
EFFECT OF NIFEDIPINE
Reiri Takeuchi(1), H Matsumoto(1), Y Akimoto(2), A Fujii(3,4)
(1) Nihon University School of Dentistry, Department of Oral Molecular
Pharmacology, Matsudo, Chiba, Japan
(2) Nihon University School of Dentistry, Department of Oral Surgery,
Matsudo, Chiba, Japan
(3) Nihon University School of Dentistry, Department of Clinical
Pharmacology, Matsudo, Chiba, Japan
(4) Nihon University, University Research Center, Matsudo, Chiba,
Japan
Introduction: We have been investigating the cause of nifedipine (NIF)induced gingival overgrowth. Several studies revealed that the growth of
gingival broblast contributed to this disease. In the present study, we
report that the cause of this disease is higher proliferative and lower
apoptotic activities in gingival broblast obtained from NIF reactive
patients (NIFr) than those in broblast obtained from NIF non-reactive
patients (NIFn). Materials: NIFn and NIFr cells were cultured to semiconuent and stimulated by bFGF and IGF-I, and then cell proliferation,
cell cycle distribution, and expression of cell cycle regulating proteins
were analyzed. Cells were also stimulated by LPS, and apoptotic cell,
cell cycle distribution, and expressions of apoptosis control genes and
proteins were analyzed. Results and Conclusion: In the presence of bFGF
605
and IGF-I, the proliferation and the progression to S and G2/M phases
from G0/G1 phase in NIFr were signicantly increased than those in
NIFn. Expressions of cell cycle regulating proteins (cyclin A, cyclin E,
cdk2, phospho-thr160-cdk2, and phospho-ser807/811-Rb induced by
bFGF, and phospho-ser780-Rb and E2F-1 by IGF-I) in NIFr were greater
than those in NIFn. In the presence of LPS, the apoptosis and the G1/S
transition in NIFr were signicantly decreased and increased, respectively, than those in NIFn. The c-myc gene and protein in NIFr showed
signicant expressions than those in NIFn, and expressions of p53 and
bcl-2 gene and protein were similar in both cells. Thus, NIF-induced gingival overgrowth may be induced by greater proliferation and lesser
apoptosis of NIFr compared to those of NIFn.
Paper No.: 1424

CEVIMELINE RAISES SALIVA VOLUME AND LEVEL OF
SUBSTANCE P IN SALIVA
Masaharu Takeyama, K Amada, S Tsuda, Y Sato, H Itoh
Ca2 + exchanger (NCX) (Asano S et al., J. Neurochem. 1995; 64:235238). Further, we have recently demonstrated that SNP induces caspaseindependent apoptosis in cultured astrocytes by stimulating mitogen-activated protein kinases (MAPKs) such as extracellular signal-regulating
kinase (ERK), c-jun N-terminal protein kinase (JNK) and p38 MAPK
(Kawasaki T et al., Glia 2007; 55: 1325-1333). In this paper, we examined whether NCX is involved in nitric oxide-mediated astrocyte apoptosis. Astrocytes were isolated from cerebral cortices of 1-day-old rats as
previously reported, and subjected to pharmacological experiments. The
cell injury was determined by MTT assay and Hoechst 33342 staining
method. The specic NCX inhibitor SEA0400 blocked SNP-induced
phosphorylation of ERK, JNK and p38 MAPK, and protected cells from
apoptosis. SNP-induced phosphorylation of ERK, JNK and p38 MAPK
depended on external Ca2 + , and SNP treatment caused an increase in
45
Ca2 + inux through the reverse mode of NCX. In addition, SNPinduced 45Ca2 + inux and cell injury were reduced in NCX1 siRNAtransfected cells. Inhibitors of intracellular Ca2 + -dependent enzymes
such as calpain (ALLN) and calmodulin (triuoperazine) blocked SNPinduced ERK phosphorylation and decrease in cell viability. Furthermore, the guanylate cyclase inhibitor LY83583 and the cGMP-dependent
protein kinase inhibitor KT5823 inhibited SNP-mediated cell injury.
These ndings suggest that NCX is involved in SNP-mediated astrocyte
apoptosis by a cGMP-dependent pathway.
Oita University Hospital, Department of Clinical Pharmacy, Oita, Japan

Introduction: Cevimeline is a muscarinic agonist available orally for use
in treatment xerostomia. Cevimeline has been shown to improve the
symptoms of dry mouth in patients with Sjogrens Syndrome. The salivary glands are supplied with nerve ver containing neuropeptides, such
as substance P (SP) and vasoactive intestinal peptide (VIP). These peptides are important modulators of salivation. We examined the effects of
cevimeline on neuropeptide levels in saliva and saliva volume in healthy
subjects. Methods: Five healthy male volunteers participated in this
study. Each subjects received information about the studys scientic purpose, which was approved ethics Committee of Oita University, and gave
informed consent. Cevimeline (30mg, Saligren Cap. Nippon Kayaku,
Tokyo) or placebo was orally administered with water. Saliva was sampled before and after administratins. Results: Cevimeline signicantly
increased saliva volume (at 60, 90min) compared to placebo. Cevimeline
signicantly increased SP (at 90, 120 min) in saliva compared to placebo. Correlation (r = 0.61) was found between saliva volume and saliva
SP levels. Cevimeline did not change VIP in saliva signicantly. Conclution: Cevimeline increases SP in saliva and their increase correlate with
enhanced saliva volume. These results might suggest that cevimeline
improves dry mouth by stimulating SP neuron locally.
Paper No.: 1419

DISCOVERY
NA+/CA2 + EXCHANGER IS INVOLVED IN NITRIC OXIDEMEDIATED APOPTOSIS IN CULTURED ASTROCYTES
Kazuhiro Takuma(1), T Kitao(1), T Kawasaki(1), Y Inoue(1),
A Ikehara(1), T Nashida(1), Y Ago(1), T Matsuda(1,2)
(1) Osaka University Graduate School of Pharmaceutical Sciences, Laboratory of Medicinal Pharmacology, Osaka, Japan
(2) Osaka University Graduate School of Medicine, The Osaka-Hamamatsu Joint Research Center for Child Mental Development, Department of Experimental Disease Model,Osaka, Japan
Nitric oxide (NO) acts as a key signal-modifying molecule in many pathophysiological conditions. We have previously found that sodium nitroprusside (SNP), an NO donor, stimulates the activity of the Na+/
Paper No.: 2011

ETHANOL AND THE CANNABINOID SYSTEM INTERACTION
AT GABAERGIC SYNAPSES WITHIN THE BASOLATERAL
AMYGDALA
Giuseppe Talani(1,2), DM Lovinger(2)
(1) University of Cagliari, Department of Experimental Biology, Section
of Neuroscience, and Center of Excellence for the Neurobiology of
Dependence,Cagliari, Italy
(2) National Institute on Alcohol Abuse and Alcoholism/National Institutes of Health, Section on Synaptic Pharmacology, Laboratory for Integrative Neuroscience, Bethesda, MD, USA
Several studies showed that basolateral amygdala (BLA) plays a crucial
role in ethanol (EtOH) reinforcing actions. Activation of CB1 cannabinoid receptors in BLA leads to a presynaptic inhibition of neurotransmitter release at GABAergic synapses, while EtOH potentiates this release.
Strong activation of BLA pyramidal neurons leads to postsynaptic production of endocannabinoids (eCBs) that transiently suppress GABA
release with a phenomenon called DSI. Thus, EtOH and the eCB system
may interact in modulating GABA release in BLA. Using rat BLA coronal slices we performed whole cell patch clamp recordings of spontaneous IPSCs from pyramidal neurons. Application of 50 mM EtOH
increased both amplitude and frequency of sIPSCs. DSI protocol elicited
a decrease in sIPSC frequency without any signicant change in amplitude. In the presence of EtOH (50 mM) DSI was eliminated. While the
CB1 agonist WIN55212-2 (5microM) reduced sIPSC frequency with no
signicant change in amplitude, and prevented the increase in both frequency and amplitude in the presence of EtOH, the antagonist
SR141716A (1microM) also blocked the EtOH effect on sIPSCs. In
addiction, the effect of EtOH on sIPSC was reduced in CB1 KO and heterozygote mice compared to WT genotype. Finally, the adenylate cyclase
inhibitor DDA decreases the EtOH effect on sIPSC amplitude and frequency. These ndings suggest that EtOH alters eCBs signaling in BLA.
This interaction between EtOH and eCBs at BLA GABAergic synapses
could be relevant for the behavioral effects induced by the association of
these two drugs of abuse.
606
Paper No.: 2012
LITHIUM POTENTIATES THE EFFECTS OF
THIOCOLCHICOSIDE ON NEURONAL EXCITABILITY IN
RAT DENTATE GYRUS
Giuseppe Talani(1), N Obili(1), E Fadda(1), R Uras(1), G Cordeddu(1),
C Fois(1), S Prinzis(1), A Frau(1), V Piras(1), L Siuni(1), P Niola(1),
PL De Riu(2), GP Sechi(2), G Biggio(1), E Sanna (1),
(1) University of Cagliari, Department of Experimental Biology, Section
of Neuroscience, Cagliari, Italy
(2) University of Sassari, Department of Neurosciences, Sassari, Italy
Thiocolchicoside (TCC) is used clinically for its muscle relaxant, antiinammatory, and analgesic properties. Our previous electrophysiological
studies have indicated that TCC is a competitive antagonist of GABAARs, an action that results in a proconvulsant and convulsant activity in
rats and humans. Recent preliminary evidence has suggested that pretreatment of rats with lithium, at concentrations found in humans treated
with lithium under clinical conditions, markedly enhances the convulsant
effects of TCC. We thus aimed at evaluating the effects of the interaction
between lithium and TCC on neuronal excitability in the rat dentate
gyrus (DG). Hippocampal slices were incubated in the absence or presence of 1 mM LiCl for 6 h, and fEPSP were then recorded. LiCl incubation induced a signicant increase of the I/O responses of fEPSPs
compared to control conditions. Patch-clamp analysis in DG granule cells
revealed that LiCl incubation enhanced the frequency of mEPSCs and
reduced paired-pulse facilitation ratio suggesting that lithium increases
the probability of glutamate release. Application of 1 uM TCC potentiated fEPSPs with a greater extent in slices incubated with LiCl compared
with those with vehicle. These results indicate that treatment of slices
with LiCl increases the excitatory postsynaptic responses in DG granule
cells, and markedly potentiates the effects of TCC. The strong increase
of DG neuronal excitability induced by the association of lithium and
TCC is consistent with the pharmacological observation of an enhanced
convulsant activity in rats and suggests that some caution should be
taken when such drug combination is used in clinical practice.
Paper No.: 2891

INDUCTION OF KININ B1 RECEPTOR BY STIMULATION OF
TRPV1
S Talbot, J Pena Dias, Rejean Couture
University of Montreal, Department of Physiology, Montreal, Quebec,
Canada
Kinins are vasoactive peptides involved in pain and inammation; they
act through the activation of two G-protein coupled receptors (R),
namely B2 (constitutive) and B1 (inducible). B1R is induced by proinammatory cytokines and oxydative stress. The capsaicin receptor,
TRPV1, is known to increase the oxidative stress and to be modulated
by pro-inammatory mediators. A link between B1R and TRPV1 is suggested as both are involved in pain processes. This study aims at investigating the role of TRPV1 in the expression of B1R and its implication in
B1R-induced hyperalgesia in the tail-ick test. The induction of B1R was
studied in Wistar rats, 24 h after s.c. treatment with 1-50 mg/kg capsaicin. In capsaicin-treated rats, intrathecal des-Arg9-BK (B1R agonist)
evoked a rapid and transient hyperalgesia at 1 min post-injection . The
B1R agonist had no effect in control rats. Capsazepin (TRPV1 antagonist) or antioxydant therapy with N-acetyl-L-cysteine prevented the
induction of B1R (mRNA and binding sites) in the spinal cord and the
B1R-induced hyperalgesia. The latter response was also prevented by a
pretreatment with antagonists/inhibitors of NMDAR, NK-1R and nitric
oxide synthase. Confocal microscopy revealed the presence of B1R on

microglia, astrocytes and C-bres. This highlights a new target and
mechanism for B1R induction and establishes a link between pro-inammatory mediators (substance P, NO and glutamate), TRPV1, oxidative
stress and thermal nociception
Paper No.: 2892

DISEASES
NEW ROLES FOR KININ B1 RECEPTOR IN THE CONTROL
OF CORE TEMPERATURE
Sebastien Talbot, H de Brito Gariepy, R Couture
University of Montreal, Department of Physiology, Montreal, Quebec,
Canada
Kinins are vasoactive peptides involved in inammation; they act
through the activation of two G-protein coupled receptors (R), namely
B2 (constitutive) and B1 (inducible). The B1R is induced by bacterial
lipopolysaccharides (LPS) and diabetes. LPS-induced fever is inhibited
by B1R antagonists and is absent in B1R knockout mice. The role of
B1R in thermoregulation is, however, unknown. This study aims to
investigate the role of peripheral B1R in control of core temperature
using a model oftype I diabetes in which B1R is upregulated. Diabetes
was induced with streptozotocine (65 mg/kg; i.p.) in Sprague-Dawley
rats. Four days later, rectal temperature was measured in awaked rats
after i.p. injection of increasing doses ofdes-Arg9-BKand Sar-D-Phe8des-Arg9-BK (B1R agonists). BK (B2R agonist) was also injected for
comparison. Results showed a transient (5-30 min) and dose-dependent
increase of rectal temperature (+1.0oC) with either B1R agonist but no
response with BK in STZ rats. B1R agonists had no effect in control rats.
B1R agonist-induced hyperthermia was blocked by antagonists/inhibitors
of B1R (SSR240612), Cox-2 (niumic acid) and nitric oxyde synthase
(L-NNA), and was absent in vagotomized rats. B1R mRNA was signicantly increased in TRPV1-positive neurons of the vagus nerve in STZ
rats. This study provides the rst evidence that B1R can affect core temperature through a vagal sensory mechanism, involving prostaglandins
and NO.
Paper No.: 1801

DISEASES
THE STUDY OF EFFECTS OF PSYCHOTROPIC DRUG
LADASTEN ON LPS-INDUCED CHANGES CYTOKINES
PROFILE AND BEHAVIOR IN MICE C57BL/6
Anna Tallerova, L Kovalenko, E Shipaeva, M Yarkova, A Durnev,
S Seredenin
SI Zakusov State Institute of Pharmacology RAMS, Department of Drug
Toxicology, Moscow, Russian Federation
The present study was to examine the effects of psychotropic drug Ladasten (N-(2-adamantyl)-N-(para-bromophenyl)-amine) on lipopolysaccharide (LPS)-induced elevated levels of proinammatory cytokines and
depressive-like behavior in mice C57Bl/6. Male C57BL/6 mice were
used in experiments. Ladasten (30, 50 mg/kg, i.p.) administrated 5 days,
on a 5 day after 1h last injection of drug LPS was given (100mkg/kg
i.p.). To determine the concentration of cytokines in serum (after 2h LPS
injection) by ow cytometry we used FlowCytimix mouse Th1/Th2
10plex set as provided by BenderMedSystems. The behavioral changes
were studied after 2h LPS injection in an actometer and elevated plusmaze (EPM). For statistic analyses we used Anova; post-hoc: Fisher
LSD test and Mann-Whitney U test.After LPS administration concentra-
607
tion of cytokines TNF-a and IL-6 in serum mice increased 3,8 and 332,1
times respectively. 5 days administration of ladasten before endotoxin
injection, induced reduction of level pro-inammatory cytokines TNF-a
and IL-6 in serum in 3,2 and 1,7 times respectively. Administration of
LPS decreased spontaneous motor activity of mice in 1,8 times and in
EPM signicantly decreased number ofclose-, open-arms entries and
time spent in the open-arms 59%, 82% and 81% respectively. Ladasten
signicantly increased spontaneous locomotor activity on 26,9% after
endotoxin injection, time spent in the open-arms and number ofopenarms entries 68% and 69% respectively. Psychotropic drug Ladasten suppress relies of proinammatory cytokines TNF-a and IL-6 and limit the
depressive-like effects of LPS in mice C57Bl/6, nding suggests that Ladasten is a possible drug-candidate for depressive disorders.
Paper No.: 1615

ISOPHTHALIC ACID DERIVATIVES TARGETED TO THE C1
DOMAIN OF PKC INDUCE CELL CYCLE ARREST IN HELA
CELLS
Virpi Talman(1), E Ekokoski(1), A Olli(2), G Boije af Gennas(3),
J Yli-Kauhaluoma(3), RK Tuominen(1)
(1) University of Helsinki, Faculty of Pharmacy, Division of Pharmacology and Toxicology, Helsinki, Finland
(2) University of Helsinki, Institute of Biotechnology, Helsinki, Finland
(3) University of Helsinki, Faculty of Pharmacy, Division of Pharmaceutical Chemistry, Helsinki, Finland
Protein kinase C (PKC) is an important regulator of cell proliferation,
differentiation and apoptosis. It has been linked with numerous severe
diseases, such as cancer, and it is therefore an important drug target. Our
research group has discovered derivatives of isophthalic acid as novel
PKC ligands that target the C1 domain within the regulatory part of the
enzyme. The compounds bind to PKC at submicromolar concentrations
and modify PKC activation in living HeLa cells (Boije af Gennas et al,
J. Med. Chem. 2009; 52: 3969-3981). This poster describes the effects of
these isophthalates on HeLa cell proliferation. HeLa cells were treated
with different concentrations of the compounds and imaged real-time
using a continuous cell culturing platform with integrated optics and an
automated phase-contrast camera (Cell-IQ, Chipmantech, Tampere,
Finland). Visual observation suggested that the active isophthalates
induced cell cycle arrest and a distinct change in cell morphology
(including cell elongation). The Cell-IQ Analyser software was taught
to recognize dead, dividing, normal and elongated HeLa cells. Analysis
conrmed the results of visual observation. Cell cycle markers were analyzed from lysates of Hela cells that had been treated with one of the
active compounds, and the amounts of cyclin E and Rb protein were
found to be downregulated. Although the mechanism behind the effect is
currently unknown, the results point out the potential of these C1 domain
ligands as lead compounds for drug development.
Paper No. 3126

PRELIMINARY EXPERIMENTAL DATA REGARDING THE
BIOAVAILABILITY AND BIODISTRIBUTION OF
SYSTEMICALLY ADMINISTERED 99MTC-COUPLED AA124
SILICA NANOPARTICLE
Bogdan I Tamba(1), C Stefanescu(2), I Jaba(1), O Mungiu(1),
A Wirth(3), U Pieles(3)
(3) Institute of Chemistry and Bioanalytics, University of Applied Sciences Northwestern Switzerland (FHNW), Muttenz, Switzerland
Aim of investigation. The organ distribution and availability of Technetium coupled silica nanoparticles (SNP) in guinea pigs was studied
in order to determine the possible uses as a drug carrier. Methods.
Groups of 4 animals were treated with 1mCi/kg/animal 99mTc-coupled
AA124 SNP (100 nm in size and carrying OH groups on the surface),
administered intravenously. Control groups received 1mCi/kg animal
99mTc. A dual-head Siemens gamma camera with parallel collimators
was used. The following whole body image acquisitions were made:
dynamic image acquisition for 60 seconds (1 image/sec) (radioisotopic
angiography to point out vascularization); dynamic image acquisition
for 4 minutes (1 image/sec); static planar images (FA/FP), every 15,
duration 2 h and then every 30 for 4 hours. Results. Our preliminary
data for the SNP tested showed the bioavailability and biodistribution
for the rst 4 hours. The specic results support the idea of using the
AA124 as container for modular drug delivery system with usage in
pain control drugs. Conclusion. Inovative painkillers for tomorrow
require intelligent carriers for improved analgesia and patient compliance. AA124 SNP is a possible candidate as a delivery system for
such drugs.
Keywords: Nanoparticles, technetium, body distribution, analgesia.
Acknowledgements: Work partially supported by the Romanian Ministry
of Education and Science, grant CNCSIS IDEI 1734/ 2009.
Paper No.: 1362

DISCOVERY
EVIDENCE FOR A KEY CONTRIBUTION OF TMEM16A
PROTEIN TO THE CALCIUM-ACTIVATED CHLORIDE
CURRENT IN RAT PULMONARY ARTERY SMOOTH MUSCLE
CELLS
Paolo Tammaro, B Manoury, A Tamuleviciute
(University of Manchester, Faculty of Life Sciences, Manchester, UK
Calcium-activated chloride channels (CaCCs) are of key importance in
many physiological processes. In vascular smooth muscle, these ion
channels are activated in response to agonist-induced Ca2 + -release and
their opening results in membrane depolarisation, increased Ca2 + -entry
through L-type Ca2 + channels and vasoconstriction. The molecular identity of vascular CaCCs is not fully understood. Here we present evidence
that TMEM16A (or Anoctamin1), a member of the transmembrane 16
(TMEM16) protein family is a major component of the calcium-activated
chloride current (ICaCC) in pulmonary artery smooth muscle cells (PASMCs). Patch-clamp analysis in acutely isolated PASMCs revealed an
ICaCC with biophysical characteristics, including properties of outward
rectication, activation and deactivation kinetics and single-channel conductance, very similar to those of heterologous TMEM16A currents
(Caputo et al. (2008) Science 322, 590-594; Yang et al. (2008) Nature
455, 1210-1215; Schroeder et al. (2008) Cell 134, 1019-1029). High levels of TMEM16A mRNA were identied in both rat and human pulmonary arteries and various other vascular smooth muscle cell types, while
the closely related TMEM16B/Anoctamin2 was about 50 times less
abundant. Downregulation of TMEM16A gene expression with small
interfering RNA in primary cultured PASMCs was accompanied by
almost total loss of whole-cell CaCC currents. Our data suggest that
TMEM16A is a major component of ICaCC in rat PASMCs and may help
the development of CaCC modulators for treating vascular disorders
including hyper- and hypo-tension.
(1) Centre for the Study and Therapy of Pain, Gr. T. Popa University
of Medicine and Pharmacy, Iasi, Romania
(2) Department of Nuclear Medicine, Gr. T. Popa University of
Medicine and Pharmacy, Iasi, Romania
608
Paper No.: 1654
MULTIPLE EFFECTS OF L-ASCORBIC ACID ON
CONJUGATION REACTIONS IN COLON CARCINOMA CELLS
Hiroomi Tamura, H Tsuruta, M Shimizu
Paper No.: 955

HPLC METHOD DEVELOPMENT & VALIDATION FOR THE
DETERMINATION OF IMATINIB MESYLATE IN HUMAN
SERUM
Ka Liong Tan, R Hassan, SH Gan
Keio University Faculty of Pharmacy, Tokyo, Japan

Vitamin C has been known to modulate conjugation reactions in the gastrointestinal tract for more than 30 years although its precise molecular
mechanism has not yet been elucidated. To elucidate this effect of vitamin C, we investigated the effects of L-ascorbic acid (AA) on the conjugation of 1-naphthol (1-NA) within Caco-2 cells. Methods: Conjugation
reactions of 1-NA in Caco-2 cells were monitored by HPLC analyses.
The microsomal UGT activity and cytosolic SULT activity were measured using 1-NA as a substrate. Gene expression was measured by realtime PCR analyses. Efux activity of MRPs in Caco-2 cells was measured using calcein as a probe. Results: Following AA treatment of
Caco-2 cells for 24 h, the accumulation of both glucuronyl- and sulfoconjugates of 1-NA was decreased in a dose-dependent manner with
IC50 values of 28 mM and 42 mM, respectively. AA did not inhibit the
in vitro enzymatic activity of phenol SULT, whereas it moderately inhibited UGT activity in vitro. AA induced the expression of UGT1A family
genes but it reduced the expression of SULT1A family genes, resulting
in changes in the corresponding enzymatic activities in AA-treated Caco2 cells. Furthermore, AA reduced the gene expression of efux transporters in Caco-2 cells. The efux activity of MRP was also reduced by AA
treatment, corresponding to the gene expression. Conclusion: Our ndings indicate that intake of AA may reduce conjugation reactions in the
gastrointestinal tract via modulation of the activities of conjugation
enzymes and transporters.
Paper No.: 1391

ADP RECEPTOR P2Y13 INDUCE APOPTOSIS IN PANCREATIC
B-CELLS

Imatinib mesylate is highly effective in the treatment and management of
Chronic Myeloid Leukaemia. In this study, a new RRLC coupled with
UV detector method for the determination of imatinibs plasma concentrations is described. The effects of 1) using different buffer types 2) different buffer pHs 3) adding organic modiers such as triethylamine, 1octanesulfonic acid sodium salt and acetic acid 4) changing column temperature 5) changing mobile phase composition 5) varying the injection
volume and 6) changing UV detector wavelength were investigated.
Chromatographic separation of imatinib was successfully achieved using
an isocratic ow rate of 0.5 ml/min (30:70 Acetonitrile: Potassium Dihydrogen Phosphate + Triethylamine 0.10%) on an Agilent ZORBAX
Eclipse C18 reversed phase column (75 X 4.6 mm I.D., 3.5 lm) at
25oC. The total run time was only 5 minutes. The pH of the buffer-triethylamine combination was adjusted to 2.5 with 95% orthophosphoric
acid. Detection was performed at 267 nm. Pyrilamine Maleate was used
as an internal standard. The precision as well as accuracy meets the
guideline laid by the FDA. The developed HPLC method was found to
be rapid and suitable for the determination of plasma imatinib concentrations for a clinical study to evaluate patients adherence to daily oral therapy, to investigate drugdrug interactions and pharmacokinetic/
pharmacodynamic relationships.
Paper No.: 1133

GENE EXPRESSION PROFILING OF HUMAN
MESENCHYMAL STEM CELLS FOR IDENTIFICATION OF
SURROGATE MARKERS FOR IN VITRO CULTURE STAGE
Chanyuan Tan(1), A Salehi(2), S Svensson(1), B Olde(1), D Erlinge(1)

Shihori Tanabe, Y Sato, T Suzuki, T Yamaguchi, K Suzuki
(1) Lund University, Department of Cardiology, Lund, Sweden
(2) Lund University, Department of Clinical Sciences, Malmo, Sweden
Pancreatic b-cell loss caused by apoptosis represents a key factor in the
pathogenesis of both Type 1 and Type 2 diabetes (Mathis D et al, Nature
2001; 414: 792-798; Butler AE et al, Diabetes 2003; 52: 102-110). Since
the inuence of purinergic signalling in b-cell apoptosis has not been
much investigated, we examined the role of the ADP receptor P2Y13
using the pancreatic insulinoma-cell line MIN6c4 as a model system.
Real time-PCR revealed high expression of the ADP receptors P2Y1 and
P2Y13. Adding the ADP analogue, 2MeSADP, to MIN6c4 cells induced
calcium inux/mobilization and inhibition of cAMP production by activation of P2Y1 and P2Y13, respectively. These effects were completely
blocked by specic antagonists. 2MeSADP reduced cell proliferation as
well as increased Caspase-3 activity; both these effects could be fully
reversed by the P2Y13 receptor antagonist MRS2211. We further discovered that blocking the P2Y13 receptor results in enhanced ERK1/2, Akt/
PKB and CREB phosphorylation suggesting that these mechanisms may
be involved in b-cell survival. Taken together, these results indicate that
P2Y13 is a proapoptotic receptor in b-cells as the P2Y13 receptor antagonist MRS2211 is able to protect the cells from ADP induced apoptosis.
Stimulation of b-cell survival through inhibition of functional P2Y13
receptors could serve as a novel strategy for diabetes therapy and b-cell
transplantation.
National Institute of Health Sciences, Division of Cellular and Gene

Therapy Products, Tokyo, Japan
Human bone marrow-derived mesenchymal stem cells (hMSCs) are
multi-potent and expected for application as cellular therapeutics. However, the phenotypic features of hMSCs are changed after a long-term in
vitro culture, and their multilineage differentiation potential is decreased
by repeated passagings. To determine factors that indicate culture stage
of hMSCs for quality assessment, we obtained gene expression proles
of six batches of hMSCs in early (passage #4-5), middle (#7-9), and late
(#22-28) stages, using Affymetrix HG-U133 Plus 2.0 Arrays. Gene
ontology analysis and network analysis suggested that expression of gene
sets related to cell-cell interaction, cellular movement, cell cycle,
or cancer showed signicant changes by the passagings. Quantitative
RT-PCR analysis conrmed the passage-dependent alterations in the
expression of at least 4 genes; nephroblastoma overexpressed gene
(NOV), necdin homolog (mouse) (NDN), EPH receptor A5 (EPHA5),
runt-related transcription factor 2 (RUNX2), which are associated with
cell proliferation, cell proliferation or neuronal development, protein phosphorylation, and osteoblast differentiation, respectively. Our
ndings suggest that these genes can be surrogate markers for culture
stage, which are useful for quality assessment of cell-therapy products or
tissue-engineered products containing hMSCs.
609
Paper No.: 3182
MECHANISM OF ACTION OF COMPOUNDS ISOLATED
FROM CECROPIA GLAZIOUI SNETH, A PLANT USED TO
TREAT HYPERTENSION IN BRAZILIAN FOLK MEDICINE
Mirtes Tanae(1), MTR Lima-Landman(1), TMA Lima(2), KKY Isla(2),
C Souccar(1), AJ Lapa(1)
(1) Federal University of Sao Paulo, Department of Pharmacology, Sao
Paulo, Brazil
(2) Centro de Biotecnologia da Amazonia - CBA, Manaus, AM, Brazil
Cecropia species are used in Brazilian folk medicine to treat asthma and
hypertension. The aqueous extract (EA, 1 g/kg/day p.o.) of C. glazioui
leaves decreased the systolic blood pressure of rats. Partition with n-butanol yielded a fraction (FBu) 5 times more active than EA. FBu inhibited
calcium inux in smooth muscle (Lima-Landman et al. Phytomedicine
2007;14:309-313). This work aimed to identify the hypotensive compound(s) and to study the mechanisms of action. FBu purication by
HPLC yielded avan-3-ols (catechin and epicatechin), procyanidins (B2,
B3, B5 and C1) and avonoids (orientin, isoorientin and isovitexin)
(Tanae et al., Phytomedicine 2007; 14:309-313). The compounds were
tested in anesthetized rats, in rat or guinea-pig aorta adrenergic tonus and
in cultured rat cardiomyocites loaded with fura-2. The i.v. injection of
procyanidins and avan-3-ols (30300 lg/kg) induced hypotension, but
the avonoids did not. The hypotensive compounds (100 lM) did not
alter atria and papillary muscles responses in vitro. In rat and g-p aorta,
epicatechin, orientin and procyanidin C1 (30 lM) relaxed the adrenergic
tonus; procyanidin was the most active. The relaxation was inhibited
after endothelial removal and by L-NAME (10-5l M); on endothelium
denuded aorta, 4-aminopyridin (10-4 M) further inhibited relaxation
induced by procyanidin C1 (30 lM). In rat cardiomiocytes, procyanidin
C1 (30 lM) reduced the calcium inux measured by the MnCl2 (104 M) quenching. The results indicate that avan-3-ols, procyanidins and
orientin may induce NO synthesis in endothelial cells. In addition, blockade of L-type calcium channels may contribute to vascular relaxation
leading to hypotension.
Paper No.: 1349

ACTIVATION OF JNK SIGNAL BY NO IN COCHLEAR SPIRAL
LIGAMENT FIBROCYTES FOLLOWING INTENSE NOISE
EXPOSURE
these proteins. Taken together, our data y suggest that intense noiseinduced hearing loss is, at least in part, due to NO generation and the following activation of JNK signal in the spiral ligament brocytes of lateral wall.
Paper No.: 1733

FOCUSED CONFERENCE GROUP: P19 - GENERAL SESSION CELL BIOLOGY
ANALYSIS OF GENE EXPRESSION IN THE SPONTANEOUSLY
CARTILAGE CALCIFICATION INSUFFICIENT (CCI) RAT
Masami Tanaka(1,2), I Yokomi(2,5), M Watanabe(2), K Sudo(3),
H Satoh(4), T Igarashi(5), A Seki(6), S Kobayashi(2)
(1) St. Marianna University Graduate School of Medicine, Institute for
Animal Experimentation, Kawasaki, Japan
(2) St. Marianna University School of Medicine, Department of Pharmacology, Kawasaki, Japan
(3) Tokyo Medical University, Animal Reseach Center, Tokyo, Japan
(4) University of Tokyo Graduate School of Frontier Sciences, Laboratory of Tumor Cell Biology, Tokyo, Japan
(5) Animal Care Co., Ltd., Japan
(6) Hamri Co., Ltd, Japan
Rats with spontaneous cartilage calcication insufciency (CCI rat,
mutant of the Sprague-Dawley strain) discovered at our institute develop
dwarsm accompanied by the shortness of all 4 limbs and the overall
body length as a systemic change. CCI rats are characterized by systemic
ossication deciency of cartilage, and it was revealed that decient
endochondral ossication is present. The bone mineral density in the
tibia based on CT images was similar between CCI and normal rats, and
growth cartilage calcication was considered to be delayed in CCI rats.
We compared mRNA expression in the growth plate of the hind legs
between 4-week-old CCI and normal control rats. In the growth plate of
the CCI rats, the expression level of FGF18 mRNA was markedly high,
and that of Indian hedgehog (Ihh) mRNA and bone type alkaline phosphatase (BAP) mRNA was extremely low. These results suggested that
Ihh expression is inhibited due to enhanced FGF18 expression in the
growth plate of CCI rats, and resulting decreases in the expression of
BAP inhibit cartilage calcication. In addition, the expression level of
type X collagen, matrix metalloproteinase 9 and dentin matrix protein 1
mRNA was markedly low while that of c-type natriuretic peptide mRNA
was extremely high. In the future, we intend to clarify the mechanism of
cartilage calcication insufciency in CCI rats, and use this animal to
elucidate the mechanism of cartilage calcication.
Hajime Tanaka, R Nagashima, T Yamaguchi, A Matsushita, K Ogita

Sensorineural hearing loss can be caused by a variety of insults including
acoustic trauma, exposure to ototoxic drugs, infections, and aging. There
is compelling evidence that nitrite oxide synthetase (NOS) is increaced
the expression and c-Jun N-terminal kinase (JNK) signals are activated
in the cochlea during acoustic overstimulation. The purpose of this study
was to evaluate the effects of L-nitroarginine methyl ester (L-NAME:
nitric oxide synthase inhibitor) and the mechanism underlying JNK signal activation by NO following intense noise. Adult male Std-ddY mice
were exposed to 8 kHz octave band noise of 110 dB SPL for 1 h. LNAME (1 mg/kg, i.p.) was administered 30 min before the onset of
intense noise exposure. The threshold shift of the auditory brainstem
response (ABR) was determined on 0-7 days after exposure. The expression of phospho-JNK (p-JNK) and phospho-c-Jun (p-c-Jun) were examined in the spiral ligament brocytes of lateral wall. Noise exposure
produced the threshold shift at frequencies of 4, 12, and 20 kHz. Immunostaining revealed that noise was effective in increasing the expression
of p-JNK and p-c-Jun in the spiral ligament brocytes. L-NAME had the
ability to prevent the threshold shift and the increase in expression of
Paper No.: 1443

HEALTH AND DISEASE
DIFFERENT CONTRIBUTIONS OF RHO-KINASE TO THE
VASOCONSTRICTIONS CAUSED BY 5-HT AND
NORADRENALIN IN THE ISOLATED HUMAN INTERNAL
THORACIC ARTERY AND SAPHENOUS VEIN
Naoko Tanaka(1), T Kanai(1), Y Matsuo(1), M Kuwabara(2), E Nakamura(3), K Nakamura(3), Y Asada(4), H Hisa(5), R Yamamoto(1)
(1) Kyushu University, School of Pharmaceutical Sciences, First Department of Pharmacology, Nobeoka, Japan
(2) Kuwabara Clinic, Japan
(3) Nobeoka Hospital, Department of Cardiovascular Surgery, Nobeoka,
Japan
(4) Miyazaki Medical College, First Department of Pathology, Nobeoka,
Japan
(5) Kyushu University, School of Pharmaceutical Sciences, Second
Department of Pharmacology, Nobeoka, Japan
610
Our aim was to clarify the relative contributions of Rho-kinase to the vasoconstrictions caused by 5-hydroxytryptamine (5-HT) and noradrenalin
(NA) in the isolated human internal thoracic artery (ITA) and saphenous
vein (SV). We investigated the effects of fasudil, a Rho-kinase inhibitor,
on the 5-HT- and NA-induced vasoconstrictions of isolated these human
vessels. The motor activities of ITA and SV obtained from patients
undergoing coronary bypass surgery were measured isometrically without endothelium. Fasudil signicantly inhibited 5-HT- and NA-induced
vasoconstrictions in both ITA and SV. In SV, the inhibitory effect of
fasudil on NA-induced vasoconstriction was greater than that on the 5HT-induced one. In ITA, on the other hand, there was no signicant difference between the inhibitory effect of fasudil on NA-induced vasoconstriction and that on the 5-HT-induced one. These observations indicate
that Rho-kinase plays an important role in 5-HT- and NA-induced vasoconstrictions in human blood vessels. However, the contributions of
Rho-kinase to the constrictions of ITA and SV were different. Furthermore, the contributions of Rho-kinase to the 5-HT- and NA-induced vasoconstrictions were also different. These results suggest that effect of a
Rho-kinase inhibitor differs in the various pathophysiological conditions
when ITA and SV are used as a graft for bypass surgery.
Paper No.: 701

ACUTE TOXICITY STUDIES AND PHYTOCHEMICAL
SCREENING OF AN ERECTILE DYSFUNCTION HERBAL
TREATMENT; KOSTELETZSKYA BEGONIIFOLIA (ULBR)
(2) Mahidol University Faculty of Dentistry, Department of Hospital

Dentistry, Bangkok, Thailand
(3) Mahidol University Faculty of Dentistry, Department of Oral and
Maxillofacial Surgery, Bangkok, Thailand
Introduction: High mobility group box 1 (HMGB1) is a conserved
nuclear protein that acts in the extracellular environment as a primary
proinammatory signal. HMGB1 binding results in the receptor for
advanced glycation end products (RAGE)-dependent sustained NF-jB
activation which perpetuates the cellular inammatory response. The
existence of RAGE and HMGB1 in dental pulp lesions has not yet been
elucidated. Patients: Human dental pulp samples were obtained from
teeth having a clinical diagnosis of various disease states of reversible
and irreversible pulpitis and of healthy teeth. Results: Immunostaining of
specimens from inamed pulp tissues showed that strong RAGE was
over-expressed and conned to the capillary endothelial cells, cell periphery of odontoblast, broblast. Healthy pulp tissues showed only faint
RAGE expression in the capillary endothelial cells and broblast-like
cells but not in the odontoblast cell layer. Immunoblot analysis demonstrated that the levels of RAGE and HMGB1 protein were both signicantly higher in the inammatory pulp tissues in comparison with those
in the healthy tissues. The level of nuclear-to-cytosolic translocation of
HMGB1 was quantitated by an ELISA. The acute-phase of pulpitis
response to inammation correlated well with changes in the corresponding cytosolic HMGB1 protein levels. Conclusion: A discovery of RAGE
and HMGB1 in excessive inammatory pulp may help to clarify the
acceptably novel mechanism in the pulp disease. Continue expression
of RAGE and HMGB1 following disease states may act as an important
amplication signal for progressive tooth destruction. RAGE and
HMGB1 Antagonist might have a valuable therapeutic role for the management of pulpitis.
Julius Kihdzee Tanayen(1,2), J Oloro(1), AA Ganafa(2)

(1) Kampala International University-Western Campus, Department of
Pharmacology and Toxicology, Kampala, Uganda
(2) Mbarara University of Science and Technology, Department
of Pharmacology andTherapeutics, Mbarara, Uganda
Kosteletzkya begoniifolia is an indigenous Ugandan plant used for long
in northern Ugandan folk medicine to treat erectile dysfunction. In this
study, a scientic screening of the medicinal plant was initiated. Acute
toxicity studies and phytochemical screening were done on the ethanolic
extract of the root bark. Using the Lorkes 1983 method, acute toxicity
of the plant was evaluated. Swiss albino mice were used. The ethanolic
extract of Kosteletzkya begoniifolia root bark did not cause any mortality
at the doses studied, 6000, 8000 and 10,000g/kg body weight when
administered orally. The animals did not show any clinical sign of toxicity while observed for two weeks. The phytochemical screening was
done following the procedures used by Sofowora et al, 1983 as recorded
in Trease and Evans, 2002. Among phytochemicals tested, those found
present were saponins, terpenoids, free amino acids and arginine in particular. This study led to the nding that the plant is appreciably safe and
indicated the presence of phytochemicals that have been associated with
pharmacological activity. The presence of arginine could bring about the
claimed activity in erectile dysfunction. The recommendation is for subacute and chronic toxicity studies to be done. Proper models should be
used to study its erectile effects.
Paper No.: 1767

EXPRESSION OF THE RECEPTOR FOR ADVANCED
GLYCATION END PRODUCTS (RAGE) AND HIGH MOBILITY
GROUP BOX 1 (HMGB1) IS RELATED TO HUMAN DENTAL
PULPITIS PROGRESSION
Paper No.: 3217

GAS CHROMATOGRAPHY MASS SPECTROMETRY (GC/MS)
METHOD OPTIMIZATION & VALIDATION FOR DETECTION
OF PARAQUAT
Suk Peng Tang(1), SA Sulaiman(1), SH Gan(2)
(1) University Sains Malaysia, School of Medical Sciences, Department
of Pharmacology, Kubang Kerian, Kelantan, Malaysia
(2) University Sains Malaysia, Human Genome Center, Kubang Kerian,
Kelantan, Malaysia
Paraquat is an extremely toxic herbicide and is still extensively used in
third world countries. To date, there are no clinically-available proven
antidotes for paraquat. In this study, a new validated gas chromatography
mass spectrometry (GC/MS) method for quantifying paraquat is
described. The method involved an initial chemical reduction step for
both paraquat and ethyl paraquat (used as internal standard) using
sodium borohydride-nickel choride to their respective perhydrogenated
products, followed by a GC/MS injection. For optimization of GC/MS
parameters, the effects of varying injector temperature and oven temperature programmes were investigated. Chromatographic separations of
paraquat and ethyl paraquat was successfully achieved on a Varian VF5ms capillary column (30 m x 0.25 mm I.D x 0.25 lm lm thickness)
with a total run time of less than 15 minutes. The method meets the
requirements by the FDA for validation and is applicable to a routine
sample analysis.
Salunya Tancharoen(1), T Tengrungsun(2), T Suddhasthira(3)

(1) Mahidol University Faculty of Dentistry, Department of Pharmacology, Bangkok, Thailand
611
Paper No.: 3360
EFFECTS OF A STANDARDIZED EXTRACT OF CENTELLA
ASIATICA ECA 233 ON SECOND DEGREE BURN WOUND IN
RATS
Boonyong Tantisira, M Tantisira, K Wannarat
Chulalongkorn University Faculty of Pharmaceutical Sciences,
Department of Pharmacology and Physiology, Bangkok, Thailand
Effects of standardized extract of Centella asiatica ECa 233 on second
degree burn wound induced by 90c hot plate was conducted in six
groups of male Wistar rats with six animals in each. No burn and burn
without treatment (untreated) as reference groups, another four groups
were treated with gel base, 0.05, 0.1 and 0.5% ECa 233 gel. Topical
application of 100 mg of the gel was made once daily and evaluation
was made on day 3, 7 and 14 post burning. Wound treated with ECa 233
appeared to have less exudates and mild degree of inammation, smaller
wound size and more epithelialization. Hair formation was clearly visualized in 0.05%ECa 233 treated group on day 14. Rate of wound healing
in all ECa 233 treated group were statistically higher than untreated and
gel base treated groups on day 7 whereas cutaneous blood ow was signicantly different only from untreated group. Burn created oxidative
stress which was reduced by the treatment of gel base and ECa 233,
however, signicant suppression of malondialdehyde (MDA) was
observed only in all ECa treated groups on day 7. Histologically, fewer
sign of inammation, higher density of hair follicles, angiogenesis and
fully developed epithelialization and keratinization were observed in all
ECa 233 treated groups. The present study demonstrates signicant
wound healing effects of ECa 233, in which 0.05% seems to be an optimal concentration, on burn wound in normal rats. Increase of cutaneous
blood ow and suppression of oxidative stress may partly explain the
results observed.
Paper No.: 3361

A RANDOMIZED, SINGLE BLIND, PLACEBO CONTROLLED
TRIAL ON THE EFFECTS OF A STANDARDIZED EXTRACT
OF CENTELLA ASIATICA ECA 233 ON MINOR APHTHOUS
ULCERS
Mayuree Tantisira(1), C Ruengprasertkit(1), B Tantisira(1),
N Hongprasong(2)
(1) Chulalongkorn University Faculty of Pharmaceutical Sciences,
Department of Pharmacology and Physiology, Bangkok, Thailand
(2) Chulalongkorn University, Faculty of Dentistry, Bangkok, Thailand
A randomized, single-blind, placebo controlled trial was conducted on
24 eligible subjects with minor recurrent aphthous ulceration (MiRAU).
They were randomly divided into four groups of treatments : 0.05, 0.10
and 0.20% of a standardized extract of Centella asiatica ECa 233 oral
paste and a placebo oral paste. Subjects were instructed to apply the oral
paste three times per day (after breakfast and lunch and at bedtime) for
10 days or until the ulcer was completely healed. Numbers of ulcer, size
and pain score were daily evaluated and recorded in a log diary booklet
by subjects themselves. In comparison to the ulcer size in day 0, signicant reduction in ulcer size was initially noted at day 9 in a placebo
group whereas they were at day 2, 6 and 4 in 0.05, 0.10 and 0.20% ECa
233 treated groups, respectively. In accordance with observation on ulcer
size, signicant reduction of pain score was initially observed at day 2, 4
and 3 in 0.05, 0.10 and 0.20% ECa 233 treated groups, respectively
whereas their respective value in placebo group was found at day 6. In
parallel with its efcacy, no unwanted effect of ECa 233 was reported or
noted. In conclusion, the present study demonstrated for the rst time
that the oral paste containing ECa 233 was safe and effective in reducing
pain and ulcer size of MiRAU suggesting a potential of a remedy from

natural source for the management of oral minor aphthous ulcers.
Paper No.: 1056

TRANSFECTION OF A MUTANT MU-OPIOID RECEPTOR
INTO MICE PERIAQUEDUCTAL GRAY RESULTED IN
NALOXONE-MEDIATED ANTINOCICEPTION WITHOUT
TOLERANCE AND DEPENDENCE
Pao-Luh Tao(1), SH Chou(1), HI Ma(2), PY Law(3), HH Loh(3)
(1) National Defence Medical Centre, Department of Pharmacology,
Taipei, Taiwan
(2) Tri-Service General Hospital, National Defence Medical Centre,
Department of Neurological Surgery, Taipei, Taiwan
(3) University of Minnesota, Department of Pharmacology, Minneapolis,
USA
Naloxone, an opioid antagonist, was shown to have full agonistic property in a mutated mu-opioid receptor (MORS196ACSTA). Previously,
we found that naloxone could elicit antinociceptive effect determined by
tail-ick test, but not by hot plate test in mice locally transfected with
MORS196ACSTA into spinal cord (S2-S3). Since the midbrain periaqueductal gray (PAG) is another major site for opioid analgesic action, we
have injected dsAAV-MORS196ACSTA-EGFP into ventral lateral area
of PAG (A-3.28 mm, L 0.3 mm, V-3.0 mm) in ICR mice in the present study. After 2 weeks of virus injection, signicant MORS196ACSTA-EGFP was expressed at PAG and naloxone elicited signicant
antinociceptive effects determined by both tail-ick test and hot plate
test. On the other hand, there was no tolerance or signicant dependence
was shown after sub-chronic treatment of naloxone (10 mg/kg, s.c., bid
for 6 days) . The acute antinociceptive effect of naloxone (10 mg/kg,
s.c.) could be blocked by CTOP (mu-opioid receptor antagonist, 50 ng,
i.c.v.), further demonstrate that such naloxone effect was the direct consequence of activation of the mutated mu-opioid receptors. (Supported by
NSC 97-2320-B-016-005-MY3; Taiwan, ROC and NIH/NIDA (1 RO1DA023905), USA)
Paper No.: 1024

DISCOVERY
GRAPEFRUIT JUICE MARKEDLY REDUCES THE PLASMA
CONCENTRATIONS OF THE OATP2B1 SUBSTRATE
ALISKIREN
Tuija Tapaninen, PJ Neuvonen, M Niemi
University of Helsinki, Department of Clinical Pharmacology, Helsinki,
Finland
The aim of this study was to investigate the effect of grapefruit juice, an
inhibitor of cytochrome P450 3A4 (CYP3A4) and some organic anion
transporting polypeptides (OATP), on the pharmacokinetics of the antihypertensive drug aliskiren, a substrate of OATP2B1, P-glycoprotein, and
CYP3A4. In a randomized crossover study with two phases and a washout period of two weeks, eleven healthy volunteers ingested either
200 ml normal strength grapefruit juice or water three times daily for ve
days. On day three, they ingested a single 150-mg dose of aliskiren with
grapefruit juice or water. Plasma aliskiren concentrations were measured
up to 72 h and its excretion into urine up to 12 h by liquid chromatography-tandem mass spectrometry. Grapefruit juice reduced the mean peak
plasma aliskiren concentration (Cmax) by 77% (range, 42-91%,
P < 0.001) and the mean area under the plasma aliskiren concentrationtime curve by 62% (range, 15-72%, P < 0.001). The Cmax of aliskiren
612
occurred later during the grapefruit juice phase (median, 1 h) than during
the water phase (0.5 h) (P = 0.016). The elimination half-life of aliskiren
was shortened from 26.1 to 23.6 h by grapefruit juice (P = 0.020).
Grapefruit juice reduced the mean amount of aliskiren excreted
unchanged into urine by 66% (range, 6-81%, P < 0.001), but had no signicant effect on its renal clearance. In conclusion, grapefruit juice markedly reduces the plasma concentrations of aliskiren, probably by
inhibiting the OATP2B1-mediated inux of aliskiren in the small intestine.
Paper No.: 2469

ENVIRONMENTAL RISK FACTORS AND OCCURRENCE OF
GASTRITIS AND COLITIS IN A MESTIZO POPULATION OF
XICO
CHABEKLUMIL, STATE OF CHIAPAS, ME
metered-dose inhalers (Ventolin, Salamol, Respigen). Each inhaler was

administered according to manufacturers recommendations via a volumatic spacer (adults 2 puffs, children 1 puff). Participants perception in
terms of strength of taste, appeal of taste, and willingness to use
the inhaler regularly were recorded using a 100mm visual analogue scale.
We recruited 118 participants (67 adults, 51 children (5-16 years)) from
the Wellington region. There were no signicant differences between
adults and childrens perception of taste. Ventolin had a signicantly
milder strength of taste than either Respigen or Salamol (P < 0.001).
While all 3 brands of inhalers had similar mean scores for appeal of
taste, signicantly more participants gave low scores (30/100) to Salamol and Respigen (p = 0.01). Despite all three inhalers being similar in
their appeal of taste, people were signicantly more willing to use
Ventolin, with 48% of participants ranking Ventolin as the best tasting
inhaler (P < 0.01). Despite both containing alcohol as excipient, there
was only a weak correlation between Salamol and Respigen in appeal
of taste (r2 = 0.14, P < 0.001). This has widespread implications for
treatment adherence, among lower-income families in countries like New
Zealand where only Salamol and Respigen are fully subsidised.
Diana C Tapia-Pancardo(1), R Jesus-Sandoval(2), L Aburto-Cortes(3),

M Angeles-Gamino(3), XE Nolasco-Velazquez(3), MM Valera-Mota(4),
R Villalobos-Molina(1)
(1) Fes-Iztacala, U.N.A.M., Division de Investigacion y Posgrado, Tlalnepantla, Mexico
(2) Carrera de Medicina, Fes-Iztacala, U.N.A.M., Tlalnepantla, Mexico
(3) Carrera de Enfermeria, Fes-Iztacala, U.N.A.M., Tlalnepantla, Mexico
(4) Carrera de Optometria, Fes-Iztacala, U.N.A.M., Tlalnepantla, Mexico
Extreme poverty occurs in rural communities in Mexico, like Chabeklumil, Chiapas where economic situation is precarious, leading to poor
health among its inhabitants. People in Chabeklumil eat a lot of irritant
food like coffee and hot pepper, which damage their gastrointestinal
tract; furthermore, men consume drugs since early ages. These individuals live with diseases that are preventable. We aimed to identify most frequent environmental risk factors for gastritis and colitis occurrence,
according to population life styles, age and sex in Chabeklumil, Chiapas.
The study was observational, descriptive, transversal and prospective;
319 questionnaires were applied to individuals of both genres from
11 years of age, low income and socio-economic status, after they signed
the informed consent. An individual le was opened by the nurses in
Chabeklumil. The predominant genre was female (56%), with 37%
among adulthood (19-59 years of age); among males (44%), 29%
included adults. We identied the following environmental risk factors to
develop gastritis and colitis: irritant food (32%), stress (20%), long periods of daily fasting (15%) in females. Males reported long periods of
daily fasting (57%), irritant food (25%), alcohol and tobacco consumption (21%), and stress (9%). We concluded that females are most affected
by environmental risk factors for gastritis, which occurs mainly due to
lifestyle, since women are precluded of a better life standard, they work
hard in the eld, and start early in the day without eating a meal which
they eat late in the day, once arriving home.
Supported by PAPCA and SEDESOL.
Paper No.: 3300

TASTE PERCEPTIONS OF SALBUTAMOL-CONTAINING
INHALERS: A RANDOMISED CROSSOVER TRIAL
Jolyn Tay, TR Ingham, B Jones, N Pierse, C Burgess
University of Otago, Department of Medicine, Wellington, New Zealand
There are many factors that can affect patients adherence to their asthma
management plan, but taste has been shown to be the most important
medication characteristic, even above cost. We conducted a randomised,
double-blind, crossover trial to assess adults and childrens perception of
the taste of three different brands of salbutamol containing CFC free
Paper No.: 2185

INVESTIGATION OF MORPHINE PREFERENCE IN SWISS
AND C57BL/6J MICE
David Taylor, B Bernard, D Malone
Monash Institute of Pharmaceutical Sciences, Department of Medicinal
Chemistry and Drug Action, Parkville, Melbourne, VIC, Australia
Voluntary oral morphine provides a useful method to investigate opioid
drug addiction. Whilst rats will not voluntarily consume morphine solution when given a choice, after a period of involuntary morphine consumption, approximately 25-30% of rats will voluntarily consume
morphine. Mice on the other hand will consume morphine without the
no choice exposure. The present study investigates whether a correlation
exists between the innate morphine preference and that following no
choice morphine in inbred (C57BL/6J) and outbred (Swiss) mice. Mice
were housed individually and provided with a choice between 0.4 mg/ml
morphine in 0.2% w/v saccharin and 0.2% w/v saccharin solution for
3 weeks. This was followed by 1 week of water alone, then 3 weeks of
no choice morphine. After another week of water alone a second 3 week
choice period commenced. During the rst choice period all mice displayed an innate morphine intake. Although they displayed the same
preference C57BL/6J mice consumed more morphine than Swiss
(22.2 mg/kg compared with 15.8 mg/kg). During the no choice period
C57BL/6J mice continued to consume more morphine than Swiss mice.
In the second choice period, morphine preference increased signicantly
in C57BL/6J mice who displayed a narrower range of preference values
than Swiss. In Swiss but not C57BL/6J mice there was a correlation
between the morphine intake in the second choice phase and the innate
and no choice morphine intake and the rst choice preference. From
these results it is suggested that comparisons between outbred or inbred
strains need to be considered cautiously.
Paper No.: 2226

DISCOVERY
DRUG TRANSPORTERS IN HUMAN MAMMARY EPITHELIAL
CELLS
Lisa Bee-Gek Tee(1), TF Sim(1), D Williams(1), A Crowe(1), K Lett(2)
(1) Curtin University, School of Pharmacy, Perth, WA, Australia
(2) University of Western Australia, Perth, WA, Australia
613
Despite the benets, breastfeeding may increase the risk of drug exposure to nursing infants via breast milk if lactating mothers are receiving
drug treatment. To ensure safety of breastfed infants while allowing
mothers to receive pharmacotherapy, it is vital that we identify, characterise and understand the mechanism of the transport system in lactating
mammary glands. P-glycoprotein and multi-drug resistance-associated
proteins efux transporters may play an important role in protecting nursing infants from drug accumulation in breastmilk. Objective: The aim of
this study is to identify and characterize the MDR1, MDR3, and MRP1
active efux drug transporters in the lactating human mammary epithelial
cells (HMECs). Hypothesis: We proposed that if these active efux transporters are present in the lactating HMEC forming the blood-milk barrier,
they may play a role in preventing drug entry into the breast milk. Methods: Primary HMEC cultures were grown from human breast milk samples. Transporter prole in the HMECs was compared to four
immortalized cell lines; CaCo-2, BT-549, MCF-7 and MCF-10A. Protein
expressions of these transporters were examined by western blotting.
Localization and activity were analysed using immunouorescence technique and active efux assays, respectively. Results: Our study demonstrates the presence of MDR1, MDR3, and MRP1 in the HMECs. Active
efux activity in the HMEC and CaCo-2 was inhibited by Vinblastine.
Conclusion: The identication of MDR1, MDR3 and MRP1 efux transporters and the demonstration of their functional activities in the HMECs
suggest that they may play a role in preventing the accumulation of drug
in the breast milk.
Paper No.: 2974

DISCOVERY
PHARMACOKINETIC ANALYSIS OF IRINOTECAN IN
OATP1B2 KNOCKOUT MICE
Wendy Teft(1,2), S Mansell(1,2), S Welch(1,3), R Kim(1,2,4)
(1) University of Western Ontario, Department of Medicine, London,
Ontario, Canada
(2) University of Western Ontario, Department of Medicine, Division of
Clinical Pharmacology, London, Ontario, Canada
(3) University of Western Ontario, Department of Oncology, London,
Ontario, Canada
(4) University of Western Ontario, Department of Pharmacology and
Physiology,London, Ontario, Canada
Irinotecan is commonly used in the treatment of metastatic colorectal
cancer. The metabolic pathway of irinotecan is complex with the enzyme
UGT1A1 playing a role in its metabolism. Recently, human hepatic drug
uptake transporters OATP1B1 and OATP1B3 have been shown to affect
the disposition of irinotecan and its active metabolite SN-38. Therefore,
expression and function of hepatic OATP transporters may play an
important role a patients response to irinotecan therapy. To more fully
clarify the role of hepatic OATP1B transporters to irinotecan disposition
we utlized newly created Oatp1b2 (murine homolog of human OATP1B1
and 1B3) knockout mice to test the hypothesis that absence of this transporter would result in a lower liver-to-plasma ratio of irinotecan and
SN38. Male wildtype and Oatp1b2 null mice (8-12 wks, n = 8/group)
were injected with irinotecan (10mg/kg, i.v.) and a pharmacokinetic
study was preformed by sampling blood via saphenous vein puncture at
15, 30, 60 and 90 min post-injection (p.i.). At 120 min p.i. mice were
euthanized and blood and liver was collected. Drug levels were determined in plasma and liver homogentates by LC-MS/MS. The plasma
and liver levels of irinotecan and SN-38 in the wildtype and and knockout groups were not signicantly different. This suggests that Oatp1b2
does not play a major role in the hepatic uptake of irinotecan in mice.
Currently, we are also assessing plasma levels of irinotecan and SN-38 in
patients treated with this agent to determine the effect of SNPs in
OATP1B1 and 1B3 to irinotecan disposition.
Paper No.: 2366

HEALTH AND DISEASE
IMPAIRED ARTERIAL SMOOTH MUSCLE CALCIUM
SIGNALLING AND ULTRASTRUCTURAL DETERIORATION
APPEAR LINKED IN THE ELDERLY
Teresa Tejerina(1), J Navarro-Dorado(1), M Alonso(2), M Ramajo(1),
S Redondo(1), C van Breemen(3), N Fameli(3)
(1) University Complutense of Madrid School of Medicine. Department
(2) University Complutense of Madrid School of Medicine, Hospital Clnico San Carlos, Service of Surgery II, Madrid, Spain
(3) University of British Columbia, Department of Anesthesiology, Pharmacology and Therapeutics, Vancouver, BC, Canada
Introduction: Adrenergic activation of systemic arterial smooth muscle
(ASM) is characterized by asynchronous Ca2 + waves, which are due to
regenerative release of sarcoplasmic reticulum (SR) Ca2 + (Iino M et al.,
EMBO J. 1994;13:50265031). To maintain repetitive Ca2 + waves the
SR must be relled from the extra-cellular space via plasma membrane
(PM)-SR junctions (20-nm-wide PM-SR appositions). We have recently
reported that human mesenteric ASM derived from older patients does
not exhibit asynchronous Ca2 + oscillations and that this signalling malfunction appears to be associated with a low density of PM-SR junctions
(Dai J et al., Eur. J. Pharmacol. 2009 Dec 11). We have also computationally simulated the process required for matching the rate of relling
to the rate of SR Ca2 + release during the maintained Ca2 + oscillations
(Fameli N et al, Cell Calcium 2007;42:565575). Methods: Human mesenteric artery rings from abdominal surgery were processed for transmission electron microscopy and smooth muscle cell images were obtained
from 80-nm sample sections. Results: Our ultrastructural observations on
human ASM cells and previous model results allow us to calculate that
the density of PM-SR junctions in smooth muscle of elderly patients (SR
apposing less than 2% of the PM) falls below the threshold for maintaining an SR relling rate required for the maintenance of asynchronous
Ca2 + waves (PM-SR apposition about 12% of the PM). Conclusion: We
conclude that degradation of arterial Ca2 + signalling during human aging
appears to be associated with a loss of PM-SR junctions.
Paper No.: 3272

CORONARY ARTERY BYPASS GRAFTING SURGERY
WORSENS OXIDATIVE STRESS AMONG PATIENTS WITH
CORONARY DISEASE
Teresa Tejerina(1), MC M. Carnero(2), J Munoz-Marin(3),
JA Gonzalez-Correa(3), E Gallardo(3), J Navarro-Dorado(1),
M Ramajo(1), S Redondo(1)
(1) University Complutense of Madrid School of Medicine. Department
Spain
(3) Universidad de Malaga, School of Medicine, Department of Pharmacology, Malaga, Spain
Introduction: Oxireduction enzymatic mechanisms in human beings can
be physiologically altered due to inammatory or infectious stimuli to
the organism. Inammatory response to heart surgery has been widely
studied, but, up to date, little is known about its possible inuence over
oxidative stress. We studied the variations of multiple oxidative stress
related products and enzymes in a cohort of patients who underwent a
coronary artery bypass grafting surgery (CABG). Materials: We measured 2 hours before and 24 after CABG the concentration of malondialdehidic acid (MDA), nitrates, and peroxynitrites (all of them
614
considered to be prooxidative markers); and reducted glutation and mithocondrial superoxide dismutase (SOD-Mn) (antioxidative markers) in a
cohort of patients with coronary disease in a single center. Results: 119
patients were included in the present study. Statistically signicant differences were detected in the mean plasmatic MDA concentration before
(0.148 mmoL/L (SD 0.12)) and after (0.283 mmoL/L /SD 0.16) surgery
(P < 0.001). Higher concentrations of peroxynitrates (p = 0.443) and
nitrates (p = 0.078) were also detected, though differences did not reach
statistical signicance. On the other hand, lower levels of reducted glutation and SOD-Mn were detected after surgery (p = 0.94 and p = 0.070),
though differences were not signicant. Conclusions: CABG surgery
worsens the oxidative stress in patients with coronary disease higher oxidation activity and greater concentrations of its products and a lower
antioxidant activity.
Paper No.: 1906

METHOTREXATE INFLUENCES NEITHER PHARMACOKINETICS NOR CONCENTRATION-EFFECT RELATIONSHIP OF
INFLIXIMAB IN ANKYLOSING SPONDYLITIS
David Ternant(1), F Lauferon(2), D Mulleman(1), D Wendling(2),
E Ducourau(2), J-P Valat(2), P Goupille(2), G Paintaud(1)
(1) CNRS UMR 6239 GICC, Tours, France
(2) Tours University Hospital, Department of Rheumatology, Tours,
France
Methotrexate (MTX) was shown to inuence the pharmacokinetics of
iniximab (IFX), an anti-TNF-a monoclonal antibody, in rheumatoid
arthritis. The objective of our study was to analyse the inuence of MTX
on IFX pharmacokinetics and pharmacokinetic-pharmacodynamics (PKPD) in axial ankylosing spondylitis (AAS). Twenty-six AAS patients
were included in this prospective, bicentric and comparative study which
lasted 24 weeks. Patients were randomly assigned to IFX alone (MTXarm) or to combined MTX-IFX (MTX+ arm), and were treated by 5 mg/
kg IFX infusions at weeks 0, 2, 6, 12 and 18. Iniximab concentrations
were measured before and after each infusion, and weekly. The recommanded clinical endpoint in AAS, the BASDAI score, was measured at
each visit. The pharmacokinetics of IFX were described using a twocompartment model with rst order distribution and elimination constants. The relationship between IFX concentration and BASDAI score
was described using an indirect response model with inhibition of BASDAI input. A population approach was used. The inuence of MTX
was tested on each pharmacokinetic and PK-PD parameter. Pharmacokinetic and BASDAI data were satisfactorily described by our models. Infliximab elimination half-life was 14 days. A high body surface area, the
previous use of an anti-TNF-a biopharmaceutical and the presence of
antibodies toward IFX signicantly modied IFX pharmacokinetics.
Methotrexate inuenced neither pharmacokinetics nor PK-PD of IFX.
Because MTX inuences neither pharmacokinetics nor PK-PD of IFX in
AAS, its combination with IFX in this rheumatic disease cannot be recommanded.
Paper No.: 3106

HEALTH AND DISEASE
RENOVASCULAR HYPERTENSION INCREASES THE
HYPOTHALAMIC CONTENT OF SEROTONIN IN RATS
David Ternant(1), F Lauferon(2), D Mulleman(1), D Wendling(2),
E Ducourau(2), J-P Valat(2), P Goupille(2), G Paintaud(1)
(1) CNRS UMR 6239 GICC, Tours, France
(2) Tours University Hospital, Department of Rheumatology, Tours, France
Methotrexate (MTX) was shown to inuence the pharmacokinetics of

iniximab (IFX), an anti-TNF-a monoclonal antibody, in rheumatoid
arthritis. The objective of our study was to analyse the inuence of MTX
on IFX pharmacokinetics and pharmacokinetic-pharmacodynamics (PKPD) in axial ankylosing spondylitis (AAS). Twenty-six AAS patients
were included in this prospective, bicentric and comparative study which
lasted 24 weeks. Patients were randomly assigned to IFX alone (MTXarm) or to combined MTX-IFX (MTX+ arm), and were treated by 5 mg/
kg IFX infusions at weeks 0, 2, 6, 12 and 18. Iniximab concentrations
were measured before and after each infusion, and weekly. The recommanded clinical endpoint in AAS, the BASDAI score, was measured at
each visit. The pharmacokinetics of IFX were described using a twocompartment model with rst order distribution and elimination constants. The relationship between IFX concentration and BASDAI score
was described using an indirect response model with inhibition of BASDAI input. A population approach was used. The inuence of MTX
was tested on each pharmacokinetic and PK-PD parameter. Pharmacokinetic and BASDAI data were satisfactorily described by our models. Infliximab elimination half-life was 14 days. A high body surface area, the
previous use of an anti-TNF-a biopharmaceutical and the presence of
antibodies toward IFX signicantly modied IFX pharmacokinetics.
Methotrexate inuenced neither pharmacokinetics nor PK-PD of IFX.
Because MTX inuences neither pharmacokinetics nor PK-PD of IFX in
AAS, its combination with IFX in this rheumatic disease cannot be recommanded.
Paper No.: 1957

DELAY OF MONITORING MAY ADD TO PRE-EXISTING
TOXICITY RISK IN ELDERLY PATIENTS TREATED WITH
POTENTIALLY NEPHROTOXIC ANTIBIOTICS: CASE SERIES
STUDY
Hundie Tesfaye(1), R Prusa(1), B Jedlickova(1), J Lukaskova(2),
M Palivoda(3), V Pavelkova(4)
(1) Faculty Hospital Motol, Division of Clinical Pharmacology, Department of Clinical Biochemistry and Pathobiochemistry, Prague, Czech
Republic
(2) KlinLab, Prague, Czech Republic
(3) Mediterra-Hospital, Intensive Care Unit, Sedlcany, Czech Republic
(4) Masaryk Hospital, Division of Infectious Diseases, Usti nad Labem,
Czech Republic
Introduction: Elderly patients are prone to drug toxicity due to existing
physiologic organ function deterioration and polymorbidity. Thus, drug
overdose may be fatal particularly in this population. Aminoglycoside
and glycopeptide antibiotics are known nephrotoxins.We describe clinical
case series of elderly patients on amikain and vancomycin therapy, who
developed renal function impairment in association with toxic drug levels
revealed after delayed monitoring and its consequences. Patients and
methods:Data of seven patients (66-80 years old), one male and two
females treated with amikacin and four females on vancomycin therapy
were included in the study. Serum drugs levels determined by uorescent polarization immunoassay (FPIA) were used to guide dose adjustment. Results: The patients in the case series demonstrated evident renal
function impairment manifested by 2.5-10 fold serum creatinine elevation. Trough levels of the drugs were 2 -15 fold above the recommended
range. In one case of amikacin overdose, haemodialysis was required.
Underlying ischemic heart disease and sepsis were characteristic for these
patients with abnormal drug levels. In conclusion, these cases illustrate
that elderly patients on aminoglycoside or glycopeptide antibiotics doses
are at increased risk of suddenly renal function deterioration, unless
timely monitored regardless of initially normal serum creatinine. In cases,
where aminoglycoside or glycopetide antibiotics are indicated in elderly
patients, obtaining serum levels immediately before the second dose and
accordingly dosing regimen adjustment is warranted as the therapeutic
615
drug monitoring provided without delay may ensure safer and more
effectively cost-saving therapy leading to favourable outcomes.
.
Paper No.: 3161

STRUCTURAL ANALYSIS OF TWO SELECTIVE INHIBITORS
OF G PROTEIN-COUPLED RECEPTOR KINASE 2
expression of pro-inammatory mediators in lipopolysaccharide (LPS)stimulated HAPI microglial cells. Compound 092 is a pure compound
fractionated from the methanol extract of Curcuma comosa, an indigenous plant of Thailand traditionally and widely used as an anti-inammatory agent for the treatment of uterine inammation. Our results showed
that compound 092 at the dose of 0.1, 0.5 and 1 micromolar signicantly
inhibited the release of NO and PGE2 in a dose-dependent manner. Furthermore, the expression of inducible nitric oxide synthase (iNOS) and
cyclooxygenase (COX)-2 was also signicantly attenuated by compound
092 in both mRNA and protein levels. These results suggest that compound 092 exerts anti-inammatory effects and may possess therapeutic
potential in treating inammation-related neurodegenerative diseases.
Keywords: Microglia: Curcuma comosa; NO; PGE2; iNOS; COX-2
David Thal(1,2), R Yeow(2), J Tesmer(0)

(1) University of Michigan, Chemical Biology Doctoral Program, Ann
Arbor, MI, USA
(2) University of Michigan, Life Sciences Institute, Ann Arbour, MI,
USA
(3) University of Michigan, Department of Pharmacology, Ann
Arbour, MI, USA
G protein-coupled receptors (GPCRs) are key regulators of cell physiology, controlling processes that range from glucose homeostasis to contractility of the heart. Phosphorylation of these receptors by GPCR
kinases (GRKs) is a major regulatory mechanism for the desensitization
of activated receptors. Since, the discovery of a link between the overexpression of GRK2 and heart failure, GRK2 has been considered a pharmaceutical target for the treatment of cardiovascular disease. Several lead
compounds have now been discovered that show both exquisite selectivity for GRK2 and therapeutic potential for the treatment of heart failure.
To understand how these drugs achieve their selectivity, we have determined the crystal structure of the bovine GRK2-Gc complex in the
presence and absence of two GRK2 selective drugs. Our results indicate
that the drugs bind in the active site of GRK2 in manner that is very similar to the AGC kinase inhibitor balanol, and induce similar changes in
the structure of the enzyme. Given that these compounds are much less
potent against other GRKs, we have made mutations converting residues
in GRK2 in the P-loop and aB-aC helices to the analogous residues in
GRK1. Our discussion will focus on potential mechanisms by which
these inhibitors achieve selectivity and their kinetics of dissociation compared to those of other more non-selective GRK2 inhibitors. Supported
by grants from the NIH and AHA.
Paper No.: 523

FOCUSED CONFERENCE GROUP: P09 - NFLAMMATION
AND IMMUNOPHARMACOLOGY: NEW TOOLS FOR OLD
DISEASES
INHIBITORY EFFECTS OF (3S)-7-(3,4-DIHYDROXYPHENYL)1-PHENYL-(1E)-1-HEPTEN-3-OL, A NOVEL COMPOUND ISOLATED FROM CURCUMA COMOSA ON NITRIC OXIDE AND
PROSTAGLANDIN E2 PRODUCTION IN LIPOPOLYSACCHARIDE-INDUCED MICROGLIAL ACTIVATION
Paper No.: 2901

DEVELOPMENT AND APPLICATION OF A QUANTITATIVE
LC-MS/MS METHOD FOR ENDOCANNABINOIDS
Brian Thomas(1), S Warren(1), Q Zhan(1), J Sasay(2), D Russell(2),
A Howlett(2)
(1) RTI International, Department of Analytical Chemistry and
Pharmaceutics, Research Triangle Par, NC, USA
(2) Wake Forest University, Winston-Salem, NC, USA
Endocannabinoids, including the lipid-derived signaling molecules
anandamide and 2-arachidonoyl glycerol, are produced in response to
specic stimuli, interact with cannabinoid receptors (CB1R and CB2R),
and are degraded via enzymatic means and hydrolysis. The endocannabinoid system acts both centrally and peripherally to modulate a variety of
physiological processes including cognition, memory, nociception, appetitive behaviors, immune response, energy balance, lipoprotein metabolism, insulin sensitivity, and glucose homeostasis. Pharmacological
manipulation of the endocannabinoid signaling system is therefore of signicant interest as a means of therapeutic treatment for a variety of physiological diseases and disorders, including obesity and metabolic
syndrome. In order to understand how the modulation of endocannabinoid levels relates tochanges in physiological processes, or can be
affected by pharmacological treatments, we have developed a sensitive
LC-MS/MS method using a Waters ultra-performance liquid chromatograph coupled to an Applied Biosystems API4000 Qtrap. Over 15 endocannabinoids can be quantitated using synthetic substances as reference
standards, with a limit of quantitation of approximately 1 ng/mL or g tissue. Other endocannabinoids and lipid molecules can be characterized on
a relative basis or as compared to control treatments. The sensitivity and
relevance of this assay has been demonstrated in cell cultures and brain
tissues after pharmacological manipulation. These studies indicate that
LC-MS/MS can provide a valuable quantitative tool to investigate endocannabinoid function and the role of endocannabinoids in pysiological
disorders and disease.
Anusorn Thampithak(1), Y Jaisin(1), B Meesarapee(1), N Poopyruchpong(1), Y Sanvarinda(1,2)

(1) Mahidol University, Faculty of Science, Department of Pharmacology, Bangkok, Thailand
(2) Mahidol University, Faculty of Science, Center for Neuroscience,
Bangkok, Thailand
Microglia are the resident immune cells in the central nervous system
(CNS). Excessive production of pro-inammatory mediators such as
nitric oxide (NO) and prostaglandin E2 (PGE2) from activated microglia
play an important role in the pathogenesis of several neurodegenerative
diseases including Parkinsons disease, Alzheimers disease, and AIDS
dementia. In the present study, we investigated the effects of (3S)-7-(3,4dihydroxyphenyl)-1-phenyl-(1E)-1-hepten-3-ol, or compound 092 on the
Paper No.: 3418

DISCOVERY
MOLECULAR DETERMINANTS FOR SELECTIVE
RECOGNITION OF ANTIDEPRESSANTS IN THE HUMAN
SEROTONIN AND NOREPINEPHRINE TRANSPORTERS
Mette Thomsen, L Srensen, J Andersen, K Strmgaard, AK Kristensen
University of Copenhagen, Department of Medicinal Chemistry,
Copenhagen, Denmark
616
The monoamine neurotransmitters serotonin and norepinephrine are
involved in control of human mood and behaviour. The serotonin transporter (SERT) and the norepinephrine transporter (NET) belong to the
family of Na+/Cl- coupled neurotransmitter transporters and mediates the
uptake of synaptically released serotonin and norepinephrine in the brain.
Competitive inhibitors of SERT and NET regulate the extracellular levels
of serotonin and norepinephrine and are used as therapeutics for wide
range of affective disorders, including depression. The majority of antidepressant drugs are SERT and/or NET inhibitors in the form of the
selective reuptake inhibitors (SSRIs), the selective norepinephrine reuptake inhibitors (NRIs), or the dual-activity serotonin-norepinephrine reuptake inhibitors (SNRIs). The selectivity prole of antidepressants at
SERT and NET is important for therapeutic efcacy. At present, little is
known about the molecular mechanism of action of many antidepressants; including the structure of the drug binding pocket, drug orientation
in the binding pocket and the specic residues that determine drug selectivity. The present study examines the role of amino acid residues within
the antidepressant binding pocket in human SERT and NET for antidepressant potency and selectivity. Previous mutational analyses of residues
within these binding pockets have identied several mutations that control antidepressant potency. Here, we determine the effect of 6 of these
mutations on the inhibitory potency of 12 clinically used antidepressants;
including prototypic SSRIs, NRIs, and SNRIs. The resulting mutational
dataset allows construction of models for the structural basis for antidepressant selectivity at monoamine transporters.
Paper No.: 2894
PATIENTS PREFERENCES FOR WRITTEN INFORMATION
ABOUT EFFECTS AND UNDESIRABLE SIDE EFFECTS OF
DRUGS
Petra A Thurmann(1,2), V Mulders(1), D Simic(3), O Herber(3,4),
D Schwappach(5), S Wilm(3)
(1) University of Witten/Herdecke, Department of Clinical Pharmacology, Wuppertal, Germany
(2) HELIOS Klinikum Wuppertal, Philipp Klee-Institute for Clinical
Pharmacology,Wuppertal, Germany
(3) University of Witten/Herdecke, Institute of General Practice and FamilyMedicine, Wuppertal, Germany
(4) University of Dundee, School of Nursing & Midwifery, Dundee, UK
(5) Swiss Patient Safety Foundation, Zurich, Switzerland
Package information leaets (PILs) are often perceived as too long,
deterrent and difcult to understand by patients. We thus investigated
which information patients desire about their drugs and how this information should be presented. We conducted six focus groups with n = 57 patients with diabetes, arterial hypertension and hypercholesterolemia
to elicit their wishes for written drug information. Out of the interview
data attributes (e.g. prevalence of side effects) and their corresponding
levels (e.g. graphical versus numerical description of prevalence) were
derived by content analysis in a multidisciplinary team including
patients. These ndings were used in a stated-preference technique in
1000 people aged 50 years and above in order to identify the relative
importance of each attribute. Focus groups revealed that PILs cause emotional reactions such as anxiety, uncertainty or dissatisfaction in patients
resulting in a broad spectrum of reactions, ranging from seeking for help
to non-compliance. Patients demand readable, attractive and short PILs
written in simple language with an accentuation of important information
by colour or typeface. Out of this information, 16 scenarios for the preference choice testing were developed. Modern media such as CDs were
requested as well. Most existing PILs do not satisfy patients needs for
useful and understandable information about their drugs. Discrete-choice
analysis will reveal the most important features for patients to be tested
in a subsequent randomised controlled trial evaluating the impact of a
specically designed drug information brochure on patients knowledge,
satisfaction and self-reported compliance.
Funded by the Federal Ministry of Education and Research (01GX0751)
Paper No.: 431

A2-ADRENOCEPTOR MEDIATED INCREASES
PHOSPHORYLATION OF MAP-2C IN RAT
PHEOCHROMOCYTOMA CELLS (PC12 CELLS) VIA A
PROTEIN KINASE C- AND MITOGEN ACTIVATED PROTEIN
KINASE-DEPENDENT MECHANISM
Lu Tie, X-J L
School of Basic Medical Sciences, Health Science Center, Peking
University, Department of Pharmacology, Beijing, PR China
Adrenoceptors mediate effects of endogenous catecholamines and have
been shown to affect the neuronal development. Microtubule-associated
protein-2 (MAP-2) is an important cytoskeleton protein whose phosphorylation in response to extracellular signal is involved in the regulation of
neurite outgrowth and neuronal plasticity. The present study was
designed to determine the effect of activation of adrenoceptor by epinephrine on MAP-2 phosphorylation in differentiation PC12 cells and, if
so, to explore the mediating mechanism. We found that epinephrine
could signicantly increase the phosphorylation of MAP-2c at ser136 in
a dose- and time- dependent manner in differentiated PC12 cells as well
as microtubule arrays. Differentiated PC12 cells express a2A-adrenoceptor, whose antagonists could block these effects of epinephrine mentioned, and clonidine which is the agonist of a2-adrenoceptor could
mimic the effect of epinephrine. Epinephrine could partially protect
against NGF withdrawal induced neurite retraction. Moreover phosphorylation of ERK and PKC was induced by epinephrine, and ERK and
PKC specic inhibitors concentration-dependently prevented epinephrine-induced phosphorylation of MAP-2c at ser136. In addition, pretreatment of PC12 cells with epinephrine partly inhibited 30 lM nocodazole
induced neurites retraction. These ndings suggest that epinephrine
induces phosphorylation of MAP-2c at ser136 through a a2-adrenoceptor
mediated, ERK/PKC-dependent signalling pathway, which may contribute to the stabilization of neurites.
Paper No.: 1252

INCREASED VULNERABILITY OF PERINATALLY
DRUG-EXPOSED RATS TOWARDS SUBSEQUENT
DRUG ABUSE
Julia Timar(1), M Sobor(1,2), S Gyarmati(1), P Riba(1),
M Al-Khrasani(1), S Furst(1,3)
(1) Semmelweis University, Department of Pharmacology and Pharmacotherapy, Budapest, Hungary
(2) National Institute of Pharmacy, Budapest, Hungary
(3) Hungarian Academy of Sciences, Neuropsychopharmacology
Research Group, Budapest, Hungary
The sensitivity towards subsequent drug challenge was checked in rats
exposed to morphine (MO) or methylenedioxymethamphetamine
(MDMA) in utero and afterwards via the maternal milk. The dams were
treated from the day of mating until weaning with MO (10 mg/kg/day)
or MDMA (3 mg/kg/day). Offspring of saline (S)-treated dams served as
controls. Naloxone (NX)-induced withdrawal was checked in MOexposed offspring on postnatal day 21. The conditioned place preference
(CPP) induced by MO or MDMA in MO-exposed offspring and that of
MDMA in MDMA-exposed offspring was checked in adulthood. The
NX-induced conditioned place aversion (CPA) was also measured in
MO-exposed adult offspring. NX failed to precipitate signicant physical
or psychic withdrawal symptoms. In CPP test the reinforcing capacity of
617
both MO and MDMA was signicantly more marked in MO-exposed
animals than in the S-exposed ones. Similar results were observed for
MDMA in MDMA-exposed rats. The CPA-inducing effect of NX was
signicantly higher in MO-exposed rats. The enhanced MO effect in
MO-exposed offspring was observed also after weaning and late adolescent. The results indicate that perinatal drug-exposure may enhance the
vulnerability towards subsequent drug abuse albeit no characteristic withdrawal symptoms were demonstrated after ending the exposure.
This study was supported by Hungarian grants OTKA K-60999 and
ETT-441/2006.
Paper No.: 840

THE FEASIBILITY OF MONITORING PAEDIATRIC ADVERSE
DRUG REACTIONS VIA SPECIALIST CLINICS
Mansour Tobaiqy(1), D Stewart(2), PJ Helms(1), CM Bond(1),
AJ Lee(1), JHG Williams(1), JS McLay(1)
(1) Division of Applied Health Sciences, University of Aberdeen, UK
(2) School of Pharmacy and Life Sciences, Robert Gordon University,
UK
The UK Yellow Card Scheme for reporting ADRs is limited by reliance on opportunistic reporting of ADRs, lack of a denominator and
lower than expected reporting rates. This is particularly true for children,
who are frequently prescribed off- label medicines, which are associated
with an increased incidence of ADRs. Research focusing on a targeted
population at increased risk of ADRs may improve the effectiveness of
ADR monitoring. The aim of the proposed research was to develop a targeted, systematic ADR monitoring system to detect ADRs associated
with attention decit hyperactivity disorder (ADHD) medicines in children. Five specialist paediatric and psychiatric clinics within Grampian,
UK, for the management of ADHD were approached and agreed to participate. Children under 16 years of age (n = 278) on ADHD pharmacological treatments were identied from participating clinic electronic
record databases. Parents received an invitation letter from their childs
specialist medical consultant together with a participant information leaflet and ADR questionnaire to be returned directly to the researchers, followed by reminder letters after one month. Parents had the option of
completing and returning the questionnaire by post or telephoning the
researchers to describe their responses. Within 2 months 107 questionnaires were returned (38.5%), 101 of which were completed (98 by post,
3 by phone). Eighty two questionnaires (81%) reported 93 potential
ADRs and 229 uncomfortable symptoms; the majority of which were
related to methylphenidate. This study demonstrated the feasibility of targeting high risk populations through specialist clinics, highlighting ongoing concerns about the safety of psychotropic medications.
binant CYP enzymes. Methods: Recombinant CYPs including CYP2C8,

CYP2C9, or CYP2J2 were incubated with losartan and AA, and the
reactions were terminated by adding ice-cold acetonitrile to the samples.
Remaining AA was detected by newly developed LC/MS/MS method
using deuterium-labeled AA as internal standard. Results and Conclusion: Linear calibration curves were obtained to determine AA up to 200
uM with r > 0.999. The degree of substrate (AA) elimination was
decreased by losartan in incubation medium containing either of the
recombinant CYP2C8, CYP2C9, and CYP2J2 enzymes. The magnitude
of inhibition by losartan was almost the same for CYP2C8 and CYP2C9,
but slightly weaker for CYP2J2. Losartan is an inhibitor of metabolism
of AA with a potential effect on the formation of vasoactive agents
derived from this pathway.
Paper No.: 1553

CLINICAL PHARMACOLOGY FOR UNDERGRADUATE
MEDICAL STUDENTS IN SERBIA
Zoran Todorovic, M Prostran
University of Belgrade School of Medicine, Department of Pharmacology, Clinical Pharmacology & Toxicology, Belgrade, Republic of Serbia
Clinical Pharmacology (CP) has been adopted as a core subject in undergraduate medical education at the School of Medicine, University of Belgrade (SMUB) since 2004. Current reform of undergraduate curriculum
and syllabus at SMUB was inevitable due to several reasons (e.g. curriculum adjustment to the Bologna process, low student success rates,
implementation of the integrated curriculum etc.). In particular, Pharmacology and Toxicology course has been moved back to the 3rd year of
the undergraduate medical curriculum. Consequently, it was necessary to
introduce a separate course dedicated to better prescribing and rational
drug use for students more advanced in clinical subjects. Currently, CP
course is taught during 11th semester as the 40-hour program that
involves general CP topics (e.g. individualization of pharmacotherapy),
rational drug prescribing (e.g. P-drug formulary) and methods of drug
use assessment (compliance, quality of life etc.). Both drug- and disease
(patient)-oriented approach is used in teaching. The CP course deals with
basic issues of pharmacovigilance (main concept, system and methods of
collecting data), and students are expecting to develop skills, attitudes
and knowledge necessary for independent assessment of drug safety.
Rapid feedback assessment data (surrogate markers - nal examination
success and evaluation of the course) would help us to compare traditional versus new curriculum. However, only long-term feedback or primary endpoints (e.g. prescription follow-up and adverse drug events
reporting improvement) would test whether the current reforms are the
right answer to the shortcomings of traditional pharmacology training.
Paper No.: 2216

EFFECT OF LOSARTAN ON METABOLISM OF
ARACHIDONIC ACID USING RECOMBINANT CYTOCHROME
P450 (CYP) ENZYMES
Paper No.: 949

DEVELOPING A MODEL CHECKLIST FOR THE
EVALUATION OF THE DISPENSING SCORES IN THE
PHARMACY
Takaki Toda(1), N Inotsume(1), K Sakamoto(1), S Takeuchi(1),

T Hayakawa(1), B Ask(2), E Eliasson(2), A Rane(2)
HZ Toklu(1), GA Dulger(1), Bulent Gumusel(2), E Yaris(3),

NI Klayoncu(3), A Akici(4)
(1) Hokkaido Pharmaceutical University School of Pharmacy, Department ofPharmaceutics, Otaru, Japan
(2) Karolinska Institute, Department of Clinical Pharmacology, Stockholm, Sweden
(1) Marmara University School of Pharmacy, Department of Pharmacology, Istanbul,Turkey

(2) Hacettepe University School of Pharmacy, Ankara, Turkey
(3) Karadeniz Technical University School of Medicine, Trabzon, Turkey
(4) Marmara University School of Medicine, Istanbul, Turkey
Introduction: Losartan inhibits paclitaxel and uvastatin metabolism

using recombinant CYP2C8, CYP2C9, and CYP3A4. We have studied
the effect of losartan on arachidonic acid (AA) metabolism using recom-
Rational use of drugs (RUD) is a growing need throughout the world.

Some medical schools have developed teaching methods (e.g. Groningen
618
model) for the prescribers. On the other hand, only the medical doctors
and dentists have the right to prescribe in Turkey. To develop teaching
methods for pharmacy students, we have prepared a model checklist for
evaluating the dispensing behaviors of the pharmacist. The checklist consists of three main parts, the rst section evaluates checking the properties of the patient and the prescription. The second part is mainly for
evaluating the information supplied to the patient about his/her medical
condition and the medication. The third and the last part is based on the
evaluation of the communication skills of the pharmacist. The students
were subjected to a simulated case scenario and their dispensing behaviors were scored in accordance to this form. The checklist used in the
medical schools was called as OSCE (objective structured clinical
examination). We may suggest that our unique checklist to be called as
OSPE; (objective structured practical examination) that is friendly useful for evaluation of under/post graduate level pharmacists practices. In
conclusion, drug centered pharmacotherapy is often thought by the lectures given by the pharmacologists in the medical and pharmacy schools.
Since new teaching methods based on the implementation of the theoretical pharmacotherapy knowledge to practice are developed, a practical
examination needs a model checklist for the evaluation of the dispensing
score. However, our checklist may be improved or revised in regard to
the specic needs.
Paper No.: 1334

DOSE RESPONSE FOR SIMVASTATIN IN A RAT MODEL FOR
THE EVALUATION OF MYOPATHY
(2) Chinese University of Hong Kong, School of Pharmacy, Shatin,

Hong Kong, PR China
The pharmacokinetics of labetalol show wide inter-subject variability, the
genetic causes for which are largely undetermined. We retrospectively
examined the effects of some common polymorphisms in candidate
enzymes and transporters in relation to labetalol pharmacokinetics in 39
healthy male Chinese subjects taking part in bioequivalence studies with
labetalol 200mg. The CYP3A5*3, CYP2C19*2 and *3, CYP2D6*10,
SLCO1B1 521T>C, 388A>G, and ABCB1 1236C>T, 2677G>T and
3435C>T polymorphisms were examined for associations with labetalol
pharmacokinetic parameters. There was no signicant effect of
CYP3A5*3 or CYP2D6*10 polymorphisms, but subjects with the
CYP2C19*2/*2 genopyte (n = 5) had higher Cmax (255 80 vs.
156 66 ng/mL) and AUC0-24 (1249 383 vs. 460 160 h. ng/mL)
compared to subjects with the *1/*1 genotype (n = 22) and *1/*2 or *1/
*3 subjects (n = 10) had intermediate values. Subjects with the ABCB1
2677T/A or T/T (n = 13) or ABCB1 3435T/T (n = 8) genotypes also
had higher AUC0-24 (826 411 vs. 456 102 h.ng/mL for 2677G>T;
850 401 vs. 478 139 h.ng/mL for 3435C>T), and lower clearance
(266 116 vs. 422 111 L/h for 2677G>T; 245 89 vs. 416 117 L/
h for 3435C>T) compared to the wild types, but it was difcult to
separate the effect from the two ABCB1 polymorphisms. There was no
signicant effect of the other polymorphisms. The pharmacokinetics
of labetalol were signicantly affected by CYP2C19 and probably
ABCB1 polymorphisms but not by the common CYP2D6 and CYP3A5
variants.
Brian Tomlinson(1), VWL Mak(1), TWC Lo(2), DK Rowlands(2),

M Hu(1), B Chadwick(3), AE James(2)
(1) Chinese University of Hong Kong, Department of Medicine and
Therapeutics, Hong Kong, PR China
(2) Chinese University of Hong Kong, Laboratory Animal Service
Centre Research Unit, Hong Kong, PR China
(3) Vet Path Asia, Hong Kong, PR China
Simvastatin is widely used in clinical practice to lower LDL-cholesterol
and reduce cardiovascular events, but higher doses are associated with an
increased risk of myopathy. Supplementation with certain currently available products, such as coenzyme Q10, might ameliorate this muscle toxicity and it would be useful to have an animal model of simvastatininduced myopathy to test the effect of such treatments. To develop a rat
model of statin-induced myopathy, male Sprague-Dawley rats aged
4 weeks were treated with oral doses of simvastatin (100, 300 and
1000 mg/kg/day) dissolved in carboxymethyl cellulose (CMC) or CMC
vehicle for 20 days. Rats receiving the highest dose of simvastatin
(1000 mg/kg/day) showed evidence of myopathy but failed to thrive and
died or were sacriced on day 6. Rats receiving the low and medium
doses had only mild and inconsistent evidence of muscle damage and
there were no signicant changes in plasma creatine kinase in any group.
With the 300 mg/kg/day dose, the relative weight of adrenals, liver and
spleen tended to increase, suggesting some degree of organ damage. A
longer period of treatment with this dose or a higher dose than 300 mg/
kg/day may provide a better model to test coenzyme Q10 or other substances which might reduce muscle toxicity.
Paper No.: 2229

PHARMACOGENETICS OF LABETALOL ARE INFLUENCED
BY CYP2C19 RATHER THAN CYP2D6
Paper No.: 1617

THE BIDIRECTIONAL REGULATION OF VITAMIN D3 ON
CALCIFICATION IN HUMAN JOINTS AND LIGAMENTS
Xinglong Tong, Y Xu, Y Xu
Xinglong Medical and Pharmaceutical Institute of Hebei, Department of
Basic Research, Shijiazhuang, PR China
Vitamin D3 (VD3) is a key regulator for calcium metabolism. It is generally accepted that application of VD3 for a long time in human can
induce calcication of joints and ligaments. However, based on a clinical
observation in 42120 patients with calcication of joints and ligaments
during almost 20 years, we found that VD3 produced bidirectional
effects on the calcium metabolism in joints and ligaments. In patients
with hyperosteogeny, such as osteoarthritis, rheumatoid arthritis, ankylosing spondylitis, spinal stenosis syndrome, calcication in longitudinal
posters ligamenta, administration of VD3 (i.m. 300 ku/week for 8 weeks)
dramatically absorbed abnormal calcication in joints and ligaments if
the patients had no history of fracture, bone surgery and tuberculosis of
bones and joints. But once patients had any one of above disease history,
VD3 exhibited an opposite effect, i.e. promoted calcication and so thus
hyperosteogeny was deteriorated. The active form of VD3, 1, 25(OH)2
VD3 was signicantly increased after the administration of VD3 and the
levels of Ca and P in serum were also enhanced. The result suggested
that 1, 25(OH)2 VD3 was the effector responsible for the bidirectional
regulation of VD3. Although the mechanism underlying the bidirectional
regulation of VD3 on calcication need to be further investigated, our
observation has provided evidence disagreed with a general result that
VD3 can only promotes calcication of joints and ligaments. The study
suggests a novel use for VD3 to treat calcication of human joints and
ligaments.
Brian Tomlinson(1), BSP Fok(1), OQP Yin(2), SSW Ko(1), CWY Tam(1),
TTW Chu(1), M Hu(1), VHL Lee(2)
(1) Chinese University of Hong Kong, Department of Medicine &
Therapeutic, Shatin, Hong Kong, PR china
619
Paper No.: 2817
MANGANESE ENHANCES AGONIST BINDING TO 5-HT1A
RECEPTORS BY INHIBITING GUANOSINE NUCLEOTIDE
BINDING TO RECEPTOR-COUPLED G-PROTEINS
Lauri Tontson, S Parkel, A Rinken
University of Tartu, Institute of Chemistry, Tartu, Estonia
Manganese is an essential trace nutrient for all forms of life, while
chronic overexposure causes toxicity (manganism) that manifests with
various Parkinsons disease-like symptoms in humans. We used 5-HT1A
selective
agonists
and
antagonist
([3H]8-OH-DPAT
and
[3H]WAY100635, respectively) and [35S]GTPcS in radioligand binding
studies to investigate how Mn2 + affects serotonergic signal trasmission
in rat brain membrane preparations and preparations of Sf9 cell membranes expressing human 5-HT1A receptors alongside heterotrimeric Gproteins. We found that millimolar Mn2 + (compared to Mg2 + ) caused
an increase in high-afnity agonist binding to rat hippocampal membranes, but not in rat cortical membranes. In Sf9 cell membranes coexpression of Gi proteins with 5-HT1A receptors was required to achieve
high afnity agonist binding and its moderate modulation by Mn2 + ions.
The potencies of guanosine nucleotides for inhibition of high-afnity
agonist binding to 5-HT1A receptors were highest in hippocampal membranes, and Mn2 + in comparison with Mg2 + decrased these potencies in
all studied preparations. This means that by inhibiting guanosine nucleotide binding, Mn2 + acts as an enhancer for agonist binding to 5-HT1A
receptors. Differeces in bivalent cation sensitivity between hippocampal
and cortical membranes lead us to speculate that 5-HT1A receptors in
these tissues are coupled to different signal transduction systems (subtypes of G proteins and/or other regulatory proteins) but further studies
are necessary to nd the most sensitive targets of Mn2 + modulation.
Paper No.: 682

HEALTH AND DISEASE
FP AND TP RECEPTORS ARE RESPONSIBLE FOR
INCREASED VASOCONSTRICTION INDUCED BY
PROSTAGLANDIN F2ALPHA IN PREECLAMPTIC HUMAN
UMBILICAL VEIN
Gokce Topal Tanyilmaz(1), SB Uydes Dogan(1), M Kucur(2),
A Gezer(3), R Madazli(3), O Ozdemir(1), X Norel(4)
(1) Istanbul University, Faculty of Pharmacy, Department of Pharmacology, Istanbul, Turkey
(2) Istanbul University, Cerrahpasa Medical Faculty, Fikret Biyal
Research Laboratory, Istanbul, Turkey
(3) stanbul University, Cerrahpasa Medical Faculty, Department of
Obstetrics and Gynecology, Istanbul, Turkey
(4) INSERM U698: Haemostasis, Bio-engineering and Cardiovascular
Remodelling, CHU X. Bichat, Paris, France
Preeclampsia is dened as an elevation in blood pressure and proteinuria
developing after 20 weeks of gestation. Although, its etiology is not fully
understood, one of its classical diagnostic criteria, hypertension secondary to increased vasoconstriction is one of the major underlying pathophysiological event in this syndrome. In this study, we examined the role
of PGF2a in preeclamptic and normal human umbilical vein (HUV).
These veins were dissected out of cords collected after caesarean deliveries, cut as rings and then set up in organ bath. The concentration-effect
curves induced by PGF2a (endogenous agonist of FP receptor) and uprostenol (FP receptor selective agonist) were determined in the absence or
presence of BAY u3405 (TP receptor selective antagonist). The major
metabolite of PGF2a, 13,14-dihydro-15-keto- PGF2a concentration was
measured in maternal and umbilical cord serum by ELISA. PGF2a and
uprostenol produced concentration-dependent contraction of isolated

HUV. These contractions were signicantly greater in HUV preparations
derived from preeclamptic women. In addition, BAY u3405 didnt modify the concentration-effect curves induced by PGF2a in preparations
obtained from normotensive women. However, in preeclampsia BAY
u3405 reduced the contractions induced by PGF2a. Furthermore, there
was no signicant difference between preeclamptic and normotensive
groups concerning 13,14-dihydro-15-keto-PGF2a concentration in maternal serum. Nevertheless, this concentration was signicantly increased in
preeclamptic group when measured in the umbilical serum. In conclusion, our results suggest that FP and TP receptors activation by PGF2a
could be involved in umbilical vasospasm observed in preeclampsia.
This work was supported by Research Fund of Istanbul University. Project number: 490/05052006
Paper No.: 508

STUDY OF THE EFFECTS OF NATURAL ZEOLITE MODIFIED
WITH ZINC (ZZ) AND ITS CELLULAR SIGNALLING
MECHANISMS IN DIABETES
Amarilys Torres Domnguez(1), O Leon(1), G Rodrguez(2),
LC de Menorval(3), M Casanova(1), L Rodriguez(1)
(1) University of Havana, Department of Basic Chemistry, Havana, Cuba
(2) University of Havana, Faculty of Physics, Zeolites Engineering
Laboratory, Institute of Materials and Reagents (IMRE), Havana, Cuba
(3) Universite Montpellier II, UMR 5072 -CNRS, Laboratoire des
Agregats Moleculaires et Materiaux Inorganiques, Montpellier, France
Diabetes is a serious problem of health at world for its high prevalence
and its complications. Recently several studies in animal models and
clinical trials have been reported a relationship between the zinc functions and the oxidative stress associated to Diabetes. This has been associated among other factors to changes in the zinc metabolism that
modify the insulin answer and metallothioneins antioxidative properties.
Zeolites, in particular the clinoptilolite, have emerged as promissory
materials for medical applications for its properties. In Cuba have been
designed and developed several modied zeolites as active principle for
drugs formulation for their demonstrated pharmacological properties.
One of them is the active zeolitic product activated with zinc ions, was
named ZZ. Taking into account we studied in vitro and in vivo the kinetics of Zinc release from ZZ, the kinetics of carbohydrates adsorption in
ZZ, and antihyperglyceamia effect and its effects on oxidative stress
markers levels on streptozotocin-induced diabetes model in rats. The Zn
release study demonstrated that ZZ act like the Zn control releaser in gastric and intestinal uid. ZZ has glucose adsortive capacity, as the same as
other carbohydrates. The ZZ treatment reduced the plasma glucosa and
prevented oxidative stress. The results of this study show that repeated
administration of ZZ might play a role in the control of diabetes and its
complications.
Paper No.: 806

NON-ETHANOL FRACTIONS ISOLATED FROM RED WINE
HAVE POTENT VASODILATION ACTIVITY IN ISOLATED
RAT AORTA
Milena Trandalovic(1,2), M Janzek(2), L Ziberna(2), M Lunder(2),
S Kreft(3), D Janes(3), G Drevensek(2)
(1) University of Nis, Faculty of Medicine, Department of Pharmacolgy,
(2) University of Ljubljana, Faculty of Medicine, Institute of Pharmacology and Experimental Toxicology, Ljubljana, Slovenia
(3) University of Ljubljana, Faculty of Pharmacy, Ljubljana, Slovenia
620
The protective effect of red wine on the cardiovascular system is considered to be solely due to the polyphenolic component. Indeed, experimental evidence indicates that red wine polyphenols can induce pronounced
endothelium-dependent relaxations of isolated arteries. However, there
might be other compounds with similar action. Therefore, our aim was
to study the vasorelaxation activity of red wine fractions prepared by
using different organic solvents. Thoracic aortic rings obtained from male
Wistar rats were mounted in standard organ baths lled with KrebsHenseleit solution, maintained at 37 C with a 95% O2 - 5% CO2 mixture. Rings were divided into three groups: control group (intact aortic
rings), endothelium denuded rings and rings with inhibited nitric oxide
synthase (NOS) activity by application of L-NNA. After inducing submaximal contraction in all studied groups, chemically characterized isolated fractions of petroleum ether, diethylacetate, butanol, diethylether
and water were applied in increasing concentrations, ranging from
10000- to 10-times dilutions. Vascular tone was measured isometrically
through mechano-electrical transducer. All tested fractions relaxed precontracted rat aortic rings in a concentration dependent manner in the
control group, but neither in endothelium-denuded aortic rings nor in
rings with inhibited NOS. The maximum relaxation was observed in
water- (13.90 3.26%) and diethylacetate-fraction (38.96 10.56%). In
contrast, high concentration of butanol-fraction (-31.91 14.66%)
caused contraction. Further chemical analysis has led us to characterization of individual substances held responsible for observed activity. Our
results show that non-ethanol fractions have strong endothelium-dependent vasodilation activity, dependent on the NOS activity.
Paper No.: 2654

NEGATIVE IMPACT OF METHAMPHETAMINE ON
GLUTAMATERGIC NEURON-ASTROCYTE INTERACTION
Ana Soa Travassos, T Morgadinho, J Goncalves, AP Silva, CF Ribeiro,
FC Pereira
University of Coimbra Faculty of Medicine, Institute of Pharmacology
and Experimental Therapeutics/IBIL, Coimbra, Portugal
Methamphetamine use may cause addiction, schizophrenia-like symptoms, attention and memory decits, learning problems and compromised
decision making. We recently demonstrated that the glutamate receptor
levels are altered following METH exposure. The glutamatergic system
is intimately related to the glutamate-glutamine cycle between neurons
and astrocytes. The effect of METH on this neuron-astrocyte cross-talk
is not understood. Our aim was to assess the impact of METH on astrocyte-related glutamatergic function in rodents by evaluating the following
markers: i) the glutamine/glutamate ratio, ii) glial brillary acidic protein
(GFAP); ii) glutamine synthetase (GS), an astrocytic enzyme which recycles synaptic glutamate. Adult C57BL/6 mice (3-4 month-old) were sacriced 4, 24 and 72h following a single dose of METH (30 mgkg-1 i.p.).
Caudate-putamen, frontal-cortex and hippocampus were dissected for the
analysis of glutamine/glutamate ratio, GFAP and GS. Aminoacids were
evaluated by HPLC-ED whereas protein expression levels were assessed
by Western blot. METH treatment evoked an increase in striatal glutamine/glutamate ratio at 72 h and a two-fold increase of both GFAP and
GS expression at this time-point. Frontal-cortex exhibited decrease in
glutamine/glutamate ratio at 4h when compared to 24 h and a 3-fold
increase in GS expression at 72h comparing to control. Frontal-cortex
and hippocampus showed a 50-60% increase in GFAP expression at
24 h. Although hippocampal glutamine/glutamate ratio at 4 h was different from control and 24 h, GS expression remained unchanged throughout the 3 time-points. Our results suggest that this METH regimen
produces astrogliosis and that astrocytes play a role on glutamatergic
neurotransmission disorder.
Paper No.: 2717

PRO-APOPTOTIC EFFECT OF OUABAIN IN JURKAT AND
A549 CELLS
Annalisa Trenti, F Cusinato, S Luciani, L Trevisi
University of Padova, Department of Pharmacology and Anaesthesiology, Padova,Italy
Recently cardiac glycosides derivatives have been studied as potential
anticancer agents. Ouabain binds to Na/K-ATPase and activates signalling cascades resulting in changes of cell proliferation, differentiation or
apoptosis in a cell-specic manner (Schoner W and Scheiner-Bobis G,
Am J Physiol Cell Physiol 2007, 293: C509-36). Apoptotic effect of ouabain was studied in two cancer cell lines: Jurkat and A549. Cell viability
was determined by methyl thiazolyl tetrazolium (MTT) assay after 24 h
incubation with ouabain (1-100 nM). Apoptosis was ascertained by analysis of annexin V/propidium iodide binding by ow cytometry, analysis
of caspase-3 activation and DNA laddering. Levels of antiapoptotic Bcl2 protein and phosphorylated ERKs were determined by western blotting. Ouabain induced apoptosis in both cell lines in a concentrationdependent manner. Decrease of Bcl-2 protein expression was an early
event. In Jurkat ouabain-induced apoptosis was preceded by a decrease
of mitochondrial potential and an increase of caspase-3 activity. DNA
laddering of ouabain-treated Jurkat was characteristic of caspase-dependent apoptosis. In A549 cells no changes in caspase-3 activity and mitochondrial potential were found, and a smear pattern of DNA
fragmentation was observed. Ouabain activated ERKs in both cell lines,
however inhibition of MEK/ERK pathway with U0126 or PD98059 did
not affect the apoptotic action of ouabain. These ndings indicate that
ouabain, at the same concentrations that induce antiapoptotic and proliferative effects in normal cells, has a citotoxic action in cancer cells.
Apoptotic pathways activated by ouabain are different depending on the
cell line. The mitochondrial-dependent intrinsic apoptotic pathway predominates in Jurkat cells.
Paper No.: 2435

THE SAFETY OF ARISTOLOCHIA TAGALA CHAM. AND
HOMNAWAKOD RECIPE IN WISTAR RATS: RENAL
FUNCTION STUDY
Pinpat Tripatara(1), S Booranasubkajorn(2), S Soukasem(1),
W Onlamul(2), S Huabprasert(1,2), A Lumlerdkj(2)
(1) Mahidol University, Faculty of Medicine, Siriraj Hospital,
Department of Pharmacology, Bangkok, Thailand
(2) Mahidol University, Faculty of Medicine, Siriraj Hospital, Bangkok,
Center of Applied Thai Traditional Medicine, Thailand
A dried root of Aristolochia tagala Cham. (ATC) is often used in Thai
traditional medicine as antipyretics, anti-inammatory agent, muscle
relaxant, appetite-enhancing agent, and analeptics (Sathornviriyapong S
et al, 2007; 41: 420-32). However, some Aristolochia species have been
reported to cause nephrotoxicity due to aristolochic acid and its derivatives (Tran HTB et al, 2008; 6). Here, we have investigated whether
ATC in Homnawakod herbal recipes causes nephrotoxicity in vivo. Male
Wistar rats (200-210g) were randomly devided into 7 groups in which
the rats were intragastrically administered water (i, N = 6); 10 mg/kg (ii,
N = 6), 30 mg/kg (iii, N = 5), and 100 mg/kg ATC (iv, N = 3); Homnawakod containing 10 mg/kg (v, N = 5) and 30 mg/kg ATC (vi,
N = 5); and compound same dose as in group vi but 30 mg/kg ATC
was subtracted from the recipe (vii, N = 5). The minimum dose (10mg/
kg) of ATC was equivalent to that in highest dose of Homnawakod herbal recipe currently used in human. Serum creatinine and urea were measured before and 7, 14 and 21 days after the treatment. There was no
signicant difference in serum creatinine and urea between groups either
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at baseline, 7, 14 or 21 days after the treatment. In addition, the values
serially measured up to 21 days were not signicant changed from baseline. These results suggest that (i) Aristolochia tagala Cham. and Homnawakod, at the dose equivalent to that used in human, do not cause
acute nephrotoxicity in rats, and (ii) the continuous exposure in this
study did not demonstrate the nephrotoxicity.
Paper No.: 861

COMPOUNDING OF EXTEMPORANEOUS ITEMS IN
PHARMACIES IN SOUTH AFRICA
Ilse Truter(1), M Lubbe(2), N Butler(3), D Nazer(4), A Gous(5),
D Bayever(6), M Naidoo(7), P Naidoo(8), V Tlala(9), S Putter(10)
(1) Nelson Mandela Metropolitan University, Drug Utilization Research
Unit (DURU), Department of Pharmacy, Port Elizabeth, South Africa
(2) North-West University, Potchefstroom, South Africa
(3) University of the Western Cape, Bellville, South Africa
(4) Tshwane University of Technology, Pretoria, South Africa
(5) University of Limpopo, Medunsa Campus, South Africa
(6) University of the Witwatersrand, Johannesburg, South Africa
(7) Rhodes University, Grahamstown, South Africa
(8) University of KwaZulu-Natal, Durban, South Africa
(9) South African Pharmacy Council, Arcadia, South Africa
(10) Management Sciences for Health, Pretoria, South Africa
Compounding of an extemporaneous item for a specic patient is one of
the services delivered in pharmacies. The primary aim was to determine
the extent of provision of extemporaneous compounding services in
pharmacies in South Africa, and to determine the time it takes. A
national research project was undertaken during 2008 by the South African Pharmacy Council on the services for which a pharmacist may levy
a fee. A total of 597 pharmacies were surveyed by eldworkers. The
focus of this study was on one component of the larger study, namely
extemporaneous compounding. One hundred and fty-eight pharmacies
provided extemporaneous compounding services during the study period
(79.75% were community pharmacies). A total of 201 extemporaneous
items were prepared by these pharmacies (79.10% by community pharmacies). Most prescriptions (80.10%) were rst-time acute prescriptions,
followed by repeat prescriptions (14.43%). Many products were dermatological applications. Compounding was divided into three phases,
namely interpretation and evaluation of the prescription (Phase I), preparation and labelling of the prescribed medicine (Phase II), and provision
of information and instructions to the patient (Phase III). The weighted
mean time (all types of pharmacies) for Phase I was 92.84 (SE = 11.00)
seconds, for Phase II was 521.85 (SE = 39.49) seconds, and for Phase III
was 82.76 (SE = 12.17) seconds. In total, it took a weighted mean time
of 669.25 (SE = 39.44) seconds to compound an extemporaneous item
(all three phases), that means it took approximately 11 minutes for the
extemporaneous compounding of an item.
Paper No.: 2032

ANTIPSYCHOTIC PRESCRIBING TO ADULTS:
A COMMUNITY PHARMACY DATABASE ANALYSIS
Ilse Truter
Africa for 2008. The database consisted of 1 289 922 central nervous
system (CNS) medication records. Antipsychotics only constituted
3.25% of CNS medicine. A total of 7 658 patients 18 years and older
received 29 883 antipsychotic products at a sales value of R12 720
308.72. The average cost per antipsychotic product was R425.67. The
average age of patients was 47.06 (SD = 18.17) years. Most patients
(64.08%) were female. Over 40% of the antipsychotics were prescribed
to patients between 30 and 50 years of age. The Lorenz curve was used
to illustrate skewness in prescribing. Ten percent of patients received
35.37% of antipsychotics. A high percentage of atypical antipsychotics
(86.09%) were prescribed. Quetiapine was the most frequently prescribed
antipsychotic (41.11%), followed by risperidone (24.44%) and olanzapine (10.87%). The Dened Daily Dose (DDD) for quetiapine is 400 mg
(oral). The Prescribed Daily Dose (PDD) for quetiapine in this study was
only 121.75 (SD = 148.15) mg. The dosages of antipsychotics should be
investigated in relation to the diagnoses for which they are prescribed. It
is therefore recommended that diagnoses be included in databases and
also that qualitative studies be conducted to determine possible side
effects experienced by patients.
Paper No.: 2033

PRESCRIBING FOR PARKINSONS DISEASE IN THE PRIVATE
SECTOR IN SOUTH AFRICA
Ilse Truter
Nelson Mandela Metropolitan University, Drug Utilization Research Unit
(DURU),Department of Pharmacy, Port Elizabeth, South Africa
Parkinsons disease (PD) is a motor system disorder, which is the result
of the loss of dopamine-producing brain cells. PD usually affects people
over the age of 50 years. The primary aim of the study was to determine
the prescribing patterns of antiparkinsonian drugs in a private health care
setting in South Africa. A retrospective exposure-cohort drug utilisation
study was conducted on prescription data of a pharmacy group in South
Africa for 2008. The database consisted of 1 289 922 central nervous
system (CNS) medication records. Antiparkinsonian medicine only constituted 1.10% of CNS medicine. A total of 2 341 patients were prescribed one or more products for PD (ATC group N04). The average age
of patients was 64.03 (SD = 16.16) years, with 58.48% female patients.
Two-thirds of patients (66.29%) were 60 years or older. A total of 11
738 products for PD were prescribed. The average cost per product was
R326.38 (SD = R296.85). Patients received on average 5.01 (SD = 6.46)
antiparkinsonian items over the year. A quarter (26.50%) of prescriptions
was rst time prescriptions. Dopaminergic agents accounted for 80.19%
of prescribing frequency and 88.97% of prescribing cost. Levodopa
accounted for 61.00% of prescriptions for dopaminergic drugs, followed
by pramipexole (25.35%). Most products were available as tablets
(97.22%). The Lorenz curve was used to illustrate skewness in prescribing. Ten percent of patients received 39.90% of products for PD. Studies
on the disease duration, severity and cost-effectiveness of PD treatment
in South Africa are needed.
Paper No.: 1278

COLLOIDS IMPROVE MORPHINE EXCRETION IN ELDERLY
PATIENTS AFTER SINGLE INTRAVENOUS DOSE
Nelson Mandela Metropolitan University, Drug Utilization Research Unit

(DURU),Department of Pharmacy, Port Elizabeth, South Africa
Stergios Tsartsalis(1), M Vasileiadis(2), E Flioni(2),

M Myronidou-Tzouveleki(1)
Conventional antipsychotics are increasingly being replaced in clinical

practice by atypical antipsychotics. The primary aim of the study was to
determine antipsychotic prescribing patterns to adults in a primary care
setting in South Africa. A retrospective exposure-cohort drug utilisation
study was conducted on prescription data of a pharmacy group in South
(1) Aristotle University of Thessaloniki Medical School, Department of

Pharmacology,Thessaloniki, Greece
(2) Theagenio Anticancer Hospital of Thessaloniki, Intensive Care
Unit,Thessaloniki, Greece
622
Introduction: Morphine as part of postoperative analgesic regimen often
induces problems and adverse effects in elderly patients. Material/
Patients: Fifty-eight surgical patients, age 68-83 y, received postoperative
intravenous morphine 6mg/kg. They were randomised in two groups. In
Group Col (n = 28), 1L of colloid solution (hydroxyethyl-starch) and in
group Cry (n = 30) 1L of crystalloid solution (Ringers Lactated) was
infused after morphine administration. After ten hours, urine specimen
was measured for morphine-3b-glucuronide (M3bG) (TDx 11.2 Abbott
Lab.). Exclusion criteria were renal or hepatic failure, severe anaphylactic
reactions to morphine or altered mental state. All patients were within
normal limits of creatinine clearance. M3bG levels were compared with
unpaired t-test, Welch corrected (GraphPad Instat 3.06) and a = 0.01 was
considered statistically signicant. Results: In group Col, M3bG levels
were signicantly higher (3516.5 2286 ng/ml) than in group Cry
(631 427.5 ng/ml), resulting in p < 0.0001. Condence Intervals in
group Col and Cry were 2379 4653 and 424 837, respectively. Conclusion: From our perspective, higher levels of M3bG in group Col
means faster excretion of morphine metabolites than in group Cry. In
result, in order to avoid any adverse effects in elderly patients, colloid
infusion helps better and with lesser volumes than with appropriate volume of crystalloids.
Paper No.: 2389

HETEROMERIZATION OF HCMV ENCODED CHEMOKINE
RECEPTORS
Pia Tschische(1), K Tadagaki(2), M Kamal(2), R Jockers(2),
M Waldhoer(1)
(1) Medical University of Graz, Institute of Experimental and Clinical
(2) Universite Paris Descartes, Institut Cochin, INSERM U567, Paris,
France
Human cytomegalovirus (HCMV) is a widespread pathogen that
encodes four G protein-coupled receptors (GPCRs) with high homology to endogenous chemokine receptors, namely US28, US27, UL33
and UL78. It is suggested that HCMV uses chemokine mimicry to
hitchhike the host organism for immune evasion, cellular reprogramming, tissue targeting and cell entry. Based on the observation that
cellular chemokine receptors form homo- and hetero-oligomers and
that virally encoded GPCRs (vGPCRs) mimic the function of cellular
receptors, we set out to investigate whether vGPCRs also homo- and
hetero-oligomerize. To elucidate whether vGPCRs form hetero-oligomers we conducted BRET (bioluminescence resonance energy transfer) experiments. Furthermore, we studied the functional
consequences of receptor heteromerization on receptor trafcking by
conducting immunouorescence assays. The signaling of heteromerizing vGPCRs was assessed by performing inositol phosphate accumulation assays and luciferase reporter gene assays to check the
activation of the transcription factor NF-jB. We nd that vGPCRs
are indeed able to form homo-and hetero-oligomers. Furthermore, this
heteromerization has functional consequences on the signaling proles
of the HCMV encoded receptors. By identifying the potential of
vGPCRs to form homo-and hetero-oligomeric complexes similarly
to endogenous chemokine receptors we may be one step closer to
gaining a better understanding of the cellular mechanisms implied by
HCMV to evade the immune system.
Paper No.: 1441

NEURONAL INSULIN RESISTANCE IN THE NUCLEUS
TRACTUS SOLITARII INDUCES HYPERTENSION IN THE
FRUCTOSE FED RATS
Ching-Jiunn Tseng(1), B-R Chen(1), W-Y Ho(2), W-H Cheng(3),
P-W Cheng(1)
(1) Kaohsiung Veterans General Hospital, Department of Medical
Education and Research, Kaohsiung, Taiwan
(2) Zuoying Armed Forces General Hospital, Institute of Clinical
Medicine, Taiwan
(3) National Yang-Ming University, Institute of Clinical Medicine,
Taiwan
Insulin resistance was thought as the major etiology of hypertension of
the metabolic syndrome. Our previous study demonstrated that insulin
plays a cardiovascular regulatory role in the nucleus tractus solitarii
(NTS). Recently, many studies reported that IRS1S307 phosphorylation
inhibited normal insulin signal transduction and increased oxidative
stress, which raise the risk of diabetes mellitus and cardiovascular diseases. The aims of this study are to investigate whether the neuronal cells
in the NTS will develop insulin resistance and further induce hypertension through activate p38MAPK phosphorylation by ROS in the metabolic syndrome rats. In our study, Wistar-Kyoto (WKY) rats were fed
with 10% fructose water as their drinking water for second weeks.
Another group of fructose-fed WKY rats were fed with insulin sensitizer,
rosiglitazone, and antioxidant, Tempol, since the 2th week. These results
indicated that the neuronal insulin resistance in the NTS can induce
hypertension and the underline pathogenesis of insulin resistance in the
NTS was increased ROS in the NTS, which activate p38MAPK and then
phosphorylate IRS1S307. In conclusion, the neuronal cells in the NTS
may develop insulin resistance in fructose-fed rats, and the neuronal
insulin resistance in the NTS contributes to the hypertension of metabolic
syndrome. The pathogenesis of IRS1S307 phosphorylation is activated
p38MAPK which in turn is activated by ROS in the NTS.
Paper No.: 1685

POST-TREATMENT WITH AN ACTIVE COMPONENT OF
SCUTELLARIAE RADIX (TCHI-2) REDUCES LPS-INDUCED
ACUTE LUNG INJURIES AND FATALITY
Tzu-Ling Tseng(1), M-F Chen(2), Y-H Hsu(3), TJF Lee(4,5)
(1) Tzu Chi University, Institute of Medical Sciences, Hualien, Taiwan
(2) Tzu Chi General Hospital, Neuro-Medical Scientic Center, Hualien,
Taiwan
(3) Tzu Chi General Hospital, Department of Pathology, Hualien, Taiwan
(4) Tzu Chi University, Institutes of Life Science, and Pharmacology and
Toxicology, Hualien, Taiwan,
(5) Southern Illinois University School of Medicine, Institute of Pharmacology, Springeld, IL, USA
Acute respiratory distress syndrome (ARDS) is a devastating clinical
problem. It is caused by excessive secretion of proinammatory and
inammatory mediators, resulting in diffuse alveolar damage, disruption
of alveolar epithelium, and capillary injury. The aim of this study was to
assess possible role of a puried plant extract (TChi-2) in the treatment
of lipopolysaccharide (LPS)-induced acute lung injury in male SpragueDawley rats and lethality in mice. 24 hrs after its application, LPS
(10 mg/kg, either iv or ip) signicantly elevated plasma tumor necrosis
factor-a (TNF-a) and nitric oxide (NO), increased pulmonary edema, and
thickened interalveolar septa in lung tissues. These changes were prevented by posttreatment of TChi-2 (15 mg/kg, iv or 30 mg/kg, ip),
623
administered one or six hrs after LPS-challenge. These treatments also
signicantly attenuated LPS-induced release of late cytokine high mobility group box 1 (HMGB1), and activation of NF-jB in lung tissues.
Treatment with TChi-2 also signicantly increased survival rate in LPStreated mice. These results suggest a promising role of TChi-2 in treating
LPS-induced acute lung injury.
(Supported by National Science Council, Tzu Chi University, and Tzu
Chi Foundation).
Paper No.: 1125

SA13353, A TRANSIENT RECEPTOR POTENTIAL VANILLOID
1 AGONIST, ATTENUATES LEUKOCYTE INFILTRATION IN
LPS-INDUCED ACUTE LUNG INJURY AND OVALBUMININDUCED ALLERGIC AIRWAY INFLAMMATION
Fumio Tsuji, M Murai, K Oki, H Inoue, M Sasano, H Aono
Santen Pharmaceutical Co., Ltd., Research and Development Center,
Nara, Japan
We recently demonstrated that SA13353, a transient receptor potential
vanilloid 1 (TRPV1) agonist, reduced the severity of the symptoms of
kidney injury (Ueda K et al, J Pharmacol Exp Ther 2009; 329: 202-209),
arthritis (Murai M et al, Eur J Pharmacol 2008; 588: 309-315) and
encephalomyelitis (Tsuji F et al, Eur J Pharmacol 2010; 627: 332-339) in
disease models. Here, we investigated the effects of orally administered
SA13353 on leukocyte inltration in LPS-induced acute lung injury and
ovalbumin-induced allergic airway inammation. For LPS-induced acute
lung injury, rats were exposed to the inhalation of aerosolized LPS. Four
hour after the end of the LPS inhalation, the bronchoalveolar lavage uid
(BALF) was collected for the analysis of leukocyte inltration and cytokine. For ovalbumin-induced allergic airway inammation, mice were
immunized ovalbumin by intraperitoneal injection, and exposed to the
inhalation of aerosolized ovalbumin repeatedly. One day after the last
ovalbumin inhalation, BALF was collected for the analysis of leukocyte
inltration and cytokine. Drugs were ollary administered 30 min before
LPS or ovalbumin inhalation. In LPS-induced lung injury, SA13353
attenuated neutrophil inltration, and the increase of TNF-a and CINC-1
levels. In allergic airway inammation, SA13353 tended to inhibit leukocyte inltration, and attenuated the increase of IL-4 and IL-12p40. These
results suggest that somatosensory TRPV1 may play an anti-inammatory role in lung inammation.
Paper No.: 1694

ANALGESICS
SA14867 ((+)-3-ACETYL-6-CHLORO-2-[2-(3-(N-(2-ETHOXYETHYL)-N-ISOPROPYLAMINO)PROPOXY)-5-ME THOXYPHENYL]BENZOTHIAZOLINE O,OHF-DIACETYL-L-TARTARATE),
A NEWLY SYNTHESIZED J-OPIOID AGONIST, SHOWS
ANTI-NOCICEPTIVE AND ANTI-PRURITIC EFFECTS
Fumio Tsuji, Y Tsukahara, M Niwa, M Nakamura, M Sasano, H Aono
Santen Pharmaceutical Co., Ltd., Research and Development Center,
Nara, Japan
We synthesized SA14867 ((+)-3-Acetyl-6-chloro-2-[2-(3-(N-(2-ethoxyethyl)-N-isopropylamino)propoxy)-5-methoxyphenyl]benzothiazoline
O,O-diacetyl-L-tartarate), a selective ja opioid agonist, and investigated
its anti-nociceptive and anti-pruritic effects. In functional binding assay,
SA14867 showed about >31000 and 2200 times higher afnity for the j
opioid receptor than for the l and d opioid receptors, respectively. For
the evaluation of anti-nociceptive effects, we used acetic acid-induced
writhing, formalin test and silver nitrate-induced arthritis model in rats.
SA14867 inhibited acetic acid-induced writhing and formalin test by oral

administration. The ED50 values of SA14867 for acetic acid-induced
writhing, formalin test rst phase and second phase were 1.9, 9.4 and
6.4 mg/kg, respectively. These values were smaller than those of asimadoline, U-50488H, and tramadol. SA14867 also showed anti-nociceptive
effects in silver nitrate-induced arthritis as strong as U-50488H, tramadol
and morphine, and stronger than asimadoline. The ED50 value of
SA14867 for hyperalgesia of the arthritis was around 10 mg/kg. For the
evaluation of anti-pruritic effects, we used 5-hydroperoxyeicosatetraenoic
acid (HPETE)- and substance P-induced pruritic models in mice, and
morphine-induced pruritic model in rhesus monkeys. SA14867 showed
anti-pruritic effects on 5-HPETE- and substance P-induced pruritic models at 1 to 3 mg/kg. SA14867 also attenuated the scratching reactions in
morphine-induced pruritic model in monkeys. These results suggest that
SA14867 may be expected for the anti-nociceptive and anti-pruritic drug.
Paper No.: 1152

THE 5-HT7 RECEPTOR MEDIATES FEAR CONDITIONING
THROUGH THE AMYGDALOID EXTRACELLULAR SIGNALREGULATED KINASE SIGNALLING IN MICE
Minoru Tsuji, K Miyagawa, T Takeuchi, H Takeda
International University of Health and Welfare, School of Pharmacy,
Division of Pharmacology, Tochigi, Japan
Molecular cloning studies have revealed the existence of 14 different
genes, each encoding a distinct serotonin (5-HT) receptor subtype. The
5-HT7 receptor is the most recently identied member of the family of
G-protein-coupled 5-HT receptor subtypes. Recent studies have suggested that 5-HT7 receptors may play a role in the regulation of emotional as well as cognitive functions. Fear conditioning is an associative
learning paradigm for studying the neurobiological mechanisms of aversive learning memory and also for understanding the root of fear-related
disorders. The present study examined whether 5-HT7 receptors are
involved in the mechanism of fear conditioning using the selective 5HT7 receptor antagonist DR4004. Male ddY mice weighing 30-40g were
used. Conditioning was performed in a third trial in which a tone was
followed by an electrical foot-shock. Context- and tone-dependent fear
was examined in tests conducted 24 and 48 hr after conditioning, respectively. DR4004, when administered intraperitoneally either 30 min before
or immediately after conditioning, caused a signicant decrease in context- and tone-dependent fear. In contrast, neither of the doses of
DR4004 when administered 30 min before the tests had signicant
effects. Biochemical analysis revealed that pretreatment with DR4004
before conditioning inhibits the activation of extracellular signal-regulated kinase (ERK) in the amygdala induced by context-dependent fear.
Additionally, DR4004 did not modify the sensitivity toward painful electrical stimuli. These results suggest that 5-HT7 receptors may play an
important role in the acquisition and/or retention of fear conditioning
accompanied by, at least in part, the activation of ERK signalling.
Paper No.: 1301

BAG1 REGULATES CHAPERONE-MEDIATED TRIAGE
DECISIONS ON DEGRADATION OF CHRONIC
MYELOGENOUS LEUKEMIA ONCOPRTEIN, BCR-ABL
Fujiko Tsukahara, Y Maru
Tokyo Womens Medical University, Department of Pharmacology,
Tokyo, Japan
Degradation of BCR-ABL oncoproteins by heat shock protein 90
(Hsp90) inhibitors in chronic myelogenous leukemia is expected to over-
624
come resistance to ABL tyrosine kinase inhibitors. However, the precise
mechanisms still remain to be uncovered. We found that while c-Cbl E3
ligase induced ubiquitin-dependent degradation of mature and phosphorylated BCR-ABL proteins, another E3 ligase CHIP [carboxyl terminus
of the heat shock cognate protein 70 (Hsc70)-interacting protein]
degraded immature BCR-ABL proteins. The ability of CHIP to suppress
BCR-ABL-dependent cell growth was more prominent than that of cCbl. To investigate how immature BCR-ABL proteins were recognized
by CHIP-induced protein degradation pathway, a series of molecular
chaperones were screened for binding to BCR-ABL protein. Interestingly, Bag1, a nucleotide exchange factor for Hsc70, directly bound
BCR-ABL with a high afnity, which was enhanced by CHIP and
Hsp90 inhibitors, inhibited by imatinib and competed with Hsc70. Induction of Bag1 alone resulted in BCR-ABL degradation, which was
enhanced by CHIP. When endogenous Bag1 was knocked down by antiBag1 siRNA, Hsp90 inhibitor geldanamycin (GA)-induced degradation
of BCR-ABL was signicantly attenuated in K562 cells. In contrast,
Hsc70 inhibited CHIP, but not c-Cbl, -induced BCR-ABL degradation.
When Hsc70 was knocked down, Bag1 stimulated the CHIP-induced
growth inhibition. CHIP appears to sort newly synthesized Hsp90unchaperoned BCR-ABL to the proteasome not only by inhibiting
Hsc70 and thereby promoting Bag1 to bind BCR-ABL but also by ubiquitinating BCR-ABL. Bag1 may direct CHIP/Hsc70-regulated protein triage decisions on BCR-ABL immediately after translation to the
degradation pathway.
Paper No.: 1310

BODY TEMPERATURE REGULATION AFTER DIETARY
INTERVENTION OF THREE KINDS OF HIGH-FAT DIETS
Hiromi Tsushima(1), M Mori(2), K Yamada(3), H Okuyama(3),
Y Hashimoto(4), T Ohkubo(5), H Hibino(5)
(1) Kinjo Gakuin University, College of Pharmacy, Nagoya, Japan
(2) Nagoya City University Madical School, Nagoya, Japan
(3) ORC Kinjo Gakuin University, Nagoya, Japan
(4) Aichi-Gakuin University School of Dentistry, Nagoya, Japan
(5) NOF Corporation, Japan
High-fat diet is found to inuence not only the metabolisms but also
neuronal functions in the central nervous system (CNS). In this study,
body temperature (BT) regulation was investigated using rats given
20 W/W%-sh oil (HF)-, soybean oil (HS)- or lard (HL)-containing
diet. BT was measured with telemetry units implanted intraabdominally.
After dietary intervention for 20 weeks, basal BT of the HS- and the
HL-diet groups was signicantly low all day, compared with the normal
diet group. And, the weight of the HL-diet group was the heaviest
among the other three groups. The intracerebroventricular injections of
IL-1bn(50 ng/5 microl) increased BT in all groups. However, the
increases of the HS-diet group were remarkable from 2 hr after administration. On the other hand, bombesin (50 ng/5 microl) injected into
the ventricle did not change BT of the HS-diet group, although BT of
the other three groups was strongly decreased after the administration.
The n-6/n-3 fatty acid ratio in the hypothalamus of the HF-diet group
became to be low, compared with the HS-diet group. These ndings
suggested that the high fat feeding inuences neuronal activity which is
related to BT regulation.
Paper No.: 2065

IN VITRO RELEASE AND PERMEATION CHARACTERISTICS
OF THREE NIMESULIDE GEL PRODUCTS (SULIDIN, NIMES
AND NIMELID GELS) IN TURKEY
F Cankat Tulunay(1), L Kaytanlioglu(2), K Embil(2), A Okyar(3),
G Baktir(3)
(1) Ankara University Medical School, Department of Pharmacoloy and
ClinicalPharmacology, Ankara, Turkey
(2) Embil Pharmaceutical Co. Ltd, Turkey
(3) Istanbul University Faculty of Pharmacy, Istanbul, Turkey
The in vitro release of nimesulide (1%) from Sulidin LPS through
synthetic membrane and dermatomed porcine skin was investigated and
its release characteristics was compared with commercially available gel
products (Nimes and Nimelid Gel). In vitro release studies were performed using Franz-type diffusion cells (diffusion area 1.77cm2). The
receptor compartment (12mL) was maintained at 32 0.5C. The receptor solution was a mixture of PEG400 and aqueous Brij58 solution
(0.5%, w/v) (40:60, v/v). 200 mg of gel was applied on the membrane.
At predetermined intervals, receptor uid samples were collected and the
amount of nimesulide was determined by a validated HPLC method. The
results show that the cumulative amounts of nimesulide released from
Sulidin 1% LPS Gel formulation through synthetic membranes were
signicantly higher than those of other two commercial gels. The superior diffusion characteristics of the patented Sulidin 1% LPS Gel
could be explained by its lamellar penetration properties, suggesting a
high absorption prole with a faster onset of action. This superior release
characteristics of Sulidin 1% LPS Gel is supported with ongoing in
vitro permeation studies of nimesulide from gel products across excised
porcine skin.
Paper No.: 2099

DISCOVERY
EFFECT OF GARLIC, GINKGO AND ST. JOHNS WORT ON
THE PHARMACOKINETICS OF FEXOFENADINE IN RATS
Jasmina Turkanovic, MB Ward, RW Milne
University of South Australia, Sansom Institute, Adelaide, SA, Australia
Induction of drug transporters is an important mechanism for herb-drug
interactions. This study investigated the effects of garlic and ginkgo on
the pharmacokinetics of fexofenadine, a P-glycoprotein (P-gp)/ Organic
anion transporting polypeptide (Oatp) substrate, in the isolated perfused
rat liver (IPRL) and the in vivo rat. Male rats were orally dosed with garlic (120 mg/kg), ginkgo (860 mg/kg), St. Johns wort (SJW; 1000 mg/
kg; positive control) or milli Q water (control) for 14 days. On day 15,
either the livers of rats were perfused with fexofenadine for 1 h, or the
animals were dosed with fexofenadine (orally or intravenously) and
blood collected for 8 h. In the IPRL, garlic, ginkgo and SJW signicantly (p < 0.05) increased the biliary clearance of fexofenadine with
respect to perfusate by 71%, 121% and 234%, respectively. SJW
increased the biliary clearance relative to the liver concentration by 64%.
The ratio of liver to perfusate concentrations signicantly increased in all
treated groups. In vivo, SJW signicantly decreased AUC (22%) and
increased the clearance (28%) of intravenous fexofenadine. Garlic signicantly increased the AUC (47%) of oral fexofenadine. There were no signicant changes following pretreatment with ginkgo. Results suggest that
garlic and ginkgo induce the activity of Oatp within the liver, whereas
SJW induces both P-gp and Oatp. Garlic increases the oral absorption of
fexofenadine, possibly via induction of intestinal Oatp. SJW and ginkgo
do not signicantly affect the intestinal absorption of fexofenadine, however a dual effect on Oatp and P-gp transport cannot be excluded.
625
Paper No.: 1860
DISEASES
MKP-1 NEGATIVELY REGULATES THE EXPRESSION OF
IL-8, IL-6 AND COX-2 IN HUMAN A549 EPITHELIAL CELLS
Tuija Turpeinen, R Nieminen, E Moilanen, R Korhonen
The Immunopharmacology Research Group, Medical School, University
of Tampere and Tampere University Hospital, Tampere, Finland
Introduction: Mitogen-activated protein kinases are major signalling
pathways in inammation and negatively regulated by MAP kinase phosphatases (MKP). In the present study, the effect of MKP-1 on IL-6, IL-8
and COX-2 expression in A549 human lung epithelial cells was investigated. Materials: A549 cells were stimulated with cytokines (TNF, IL-1b
and IFNa). The expression of IL-6, IL-8, COX-2 and MKP-1 was determined by quantitative RT-PCR and Western blot or ELISA. p38 and
JNK phosphorylation was detected by Western blot. MKP-1 expression
was silenced by siRNA. Result: Cytokine induced p38 and JNK phosphorylation, and IL-6, IL-8 and COX-2 expression in A549 cells. p38
inhibitors SB202190 and BIRB 796 inhibited MK2 phosphorylation (a
p38 substrate) and IL-6, IL-8 and COX-2 expression in a dose-dependent
manner. An aminopyridine-based compound JNK inhibitor VIII dosedependently inhibited c-Jun phosphorylation (a JNK substrate), but did
not alter IL-6, IL-8 or COX-2 expression. Down-regulation of MKP-1
with siRNA enhanced p38 phosphorylation and increased IL-6, IL-8 and
COX-2 expression in A549 cells. Conclusions: MKP-1 limited the
expression of proinammatory factors IL-6, IL-8 and COX-2 in human
epithelial cells by reducing the phosphorylation of p38 MAP kinase. The
results suggest that MKP-1 negatively regulates inammatory gene
expression in human cells and is a potential anti-inammatory target.
Paper No.: 2315

CLINICAL EFFICACY AND ADVERSE EFFECTS OF THE
PARTIAL DOPAMINERGIC AGONIST ARIPIPRAZOLE
COMPARED WITH OTHER ANTIPSYCHOTICS
Ingunn Fride Tvete(1), M Klemp(2), J Gaasemyr(3), B Natvig(3),
T Skomedal(2), I Aursnes(2)
(1) Norwegian Computing Center, Oslo, Norway
(2) University of Oslo, Department of Pharmacology, Oslo, Norway
(3) University of Oslo, Department of Mathematics, Oslo, Norway
Introduction: We wanted to examine the efcacy and degree of adverse
effects connected with atypical antipsychotic drugs by using a previously
described Bayesian statistical method that included both direct and indirect comparisons simultaneously. Patients: We used the results of 30 double blind, randomized studies including comparisons of four atypical
antipsychotics and haloperidol, head-to-head or against placebo. We calculated the success ratios for drugs against placebo and thereafter the relative success ratios for one drug against another. With uniform priors, we
calculated and ranked posterior estimates of success ratios with the use
of the BUGS statistical program and 10 000 iterations. Correspondingly
we calculated the risk ratios for weight gain and for occurrence of extrapyramidal symptoms. Results: Aripiprazole was the least effective among
the new antipsychotics with a success ratio of 1.44 (95% posterior credibility interval (CI) 1.24-1.66) against placebo. Aripiprazole had the lowest weight gain among the new antipsychotics with a weight gain ratio of
0.94 (CI 0.66-1.28) against placebo. Aripiprazole showed a risk ratio of
1.20 (CI 0.96-1.48) compared with placebo for occurrance of extrapyramidal symptoms. It ranked in the middle compared with the others, with
haloperidole the worst and olanzapine the best. Conclusion: We found
clozapine, olanzapine and risperidone to have signicantly better clinical
efcacy than aripiprazole, which corresponds with its effect being a par-
tial dopaminergic agonist as well as an antagonist. Among these aripiprazole gives the lowest weight gain. As to risk of extrapyramidal
symptoms, aripiprazole stands in the middle among the atypical antipsychotics and haloperidol.
Paper No.: 2303

IN-VIVO PHENOTYPING FOR CYP3A4 BY DETERMINATION
OF A SINGLE-POINT PLASMA CONCENTRATION AND
URINARY EXCRETION OF MIDAZOLAM AND ITS
METABOLITES
Shinya Uchida(1), M Miura(1), S Misaka(1), Y Katoh(1), S Yamada(1),
K Ohashi(2), H Watanabe(3), N Namiki(1)
(1) University of Shizuoka, School of Pharmaceutical Sciences,
Department of Pharmacy Practice & Science, Shizuoka, Japan
(2) Oita University, School of Medicine, Japan
(3) Hamamatsu University School of Medicine, Japan
Cytochrome P450 (CYP) 3A4 has a large inter- and intra-indivisual variation of the activity. Midazolam (MDZ) is predominantly metabolized to
1- and 4-hydroxymidazolam (1OHMDZ and 4OHMDZ) by CYP3A4,
and is considered as a probe of CYP3A4. The determination of MDZ
clearance (CL) for CYP3A4 phenotyping requires collecting multiple
plasma samples of MDZ. The aim of this study was to evaluate whether
a single plasma concentraion and urinary excretion of MDZ and its
metabolites predict CL of MDZ in healthy volunteers. Nineteen volunteers received the i.v. (5, 15, 30 lg/kg) and p.o (15, 50, 100 lg/kg)
MDZ on day 1 and day 2, respectively. Plasma concentrations of MDZ,
1OHMDZ and 4OHMDZ were determined until 10 hr after administrations in both trial days. Urine samples were collected until 24 hr after i.v.
and p.o. administrations of MDZ, and were pretreated with b-glucuronidase. Concentrations in plasma or urine were determined by LC/MS/MS.
There were signicant correlations between area under curves of MDZ
and plasma concentrations at all sampling points after the i.v. and p.o.
administration. Plasma concentration ratios (1OHMDZ/MDZ) at 6 hr
after i.v. and p.o. administrations signicantly correlated with CL of
MDZ. Although metabolic ratios in urine were not signicantly correlated with CL of MDZ, there was a weak correlation between CL and
the urinary excretion of metabolites corrected by MDZ dose after the
p.o. administration. These results suggest that plasma concentration ratios
and the urinary excretion of metabolites corrected by the dose could predict CL of MDZ.
Paper No.: 1920

WHAT IS THE FUNCTIONAL ROLE OF THE HYDROPHOBIC
DOMAIN IN THE N-TERMINAL TAIL OF THE
ALPHA2C-ADRENOCEPTOR?
Staffan Uhlen, JA Jahnsen
University of Bergen, Department of Medicine/Centre for Pharmacy,
Bergen, Norway
Pharmacology of adrenoceptors: eighth satellite meeting. Introduction:
The N-terminal tail of the a2C-adrenoceptor is 49 amino acids long. The
outermost 22 amino acid sequence is predicted to be a signal peptide.
The
sequence
is
markedly
hydrophobic,
i.e.
MASPALAAALAVAAAAGPNASG. Materials: 14 N-terminally modied
a2C-adrenoceptors were constructed. The modications included addition
of the inuenza virus hemagglutinin signal peptide, addition of a FLAG
epitope, various truncations of the N-terminal tail, and combinations of
these changes. Receptor constructs, transiently expressed in COS-7 cells,
626
were quantied by radioligand binding, and FLAG-epitoped receptor
constructs were immunostained by antibodies M1 and M2. Results and
Conclusion: The results indicate that the predicted signal peptide in the
N-terminal tail of the a2C-adrenoceptor is not a functional cleavable signal peptide. On the other hand, this stretch is important for receptor
expression in the cell membranes, since truncated receptors showed
much lower expression levels compared to the wild type receptor. A general nding is that the N-tail has a great impact on receptor translocation
to the plasma membrane. The translocation is not an all-or-nothing process. Instead, the different receptor constructs showed a wide range of
translocation efciences. For a well-translocated receptor construct, a
large fraction of newly synthesized receptors seems to be transported
from the ribosomes to the plasma membrane. For a less well-translocated
receptor construct, a smaller fraction is translocated. This indicates there
is a statistical chance for an individual receptor molecule to get transported to the membrane, or alternatively to be degraded intracellularly.
Paper No.: 877

HEALTH AND DISEASE
ENDOTHELIAL CELLS DURING A PARABOLIC FLIGHT
WHICH CHANGES APPEAR?
Claudia Ulbrich(1), J Pietsch(1), J Bauer(2), D Grimm(3)
(1) Charite Universitatsmedizin Berlin, Berlin Germany
(2) Max-Planck Institute for Biochemistry, Martinsried, Germany
(3) Aarhus University, Department of Pharmacology, Denmark
Since several years our group investigates the behaviour, the biochemistry and the physiology of cells during their exposition towards simulated
microgravity. In this context human endothelial cells have been characterized under the conditions of simulated microgravity using a Random
Positioning Machine. One of the most interesting results during these
investigations was the cognizance that short-term effects as well as longterm changes could be found in human cells. In 2008 and 2009 our
group participated in the DLR parabolic ight campaigns (PFC). At the
end of the PFC we were able to show that all cells had stayed vital during the parabolic ight, there had been no signicant enhancement of
apoptosis or necrosis. With the help of Gene Arrays we could show that
the gene activation process is already initiated after 22s in real weightlessness. More than 5700 genes have been investigated. By means of
these data we developed a list of genes which have just been further
investigated via TaqMan PCR. We followed the question if such a short
time period of real weightlessness would also have an inuence on the
structure of the cytoskeleton using immunouorescence and Western blot
analysis. Here we found amongst others changes of cytokeratin, vimentin, a-tubulin and F-Actin. So far our results are promising. The next step
is to investigate the transcription behaviour of the genes during apoptosis, signal transduction and cell cycle.
Paper No.: 2691

PRESCRIPTION AUDIT AS AN IMPORTANT TOOL IN THE
ANALYSIS OF THE PRESCRIPTIONS FOR THE CHILDREN
Canan Uluoglu(1), HZ Guney(1), T Ercanly(2)
(1) Gazi University Medical School Department of Pharmacology,
Ankara, Turkey
(2) Altynova Pharmacy, Altindag, Ankara
Introduction: Presription mistakes constitute an important problem for
the appropiate usage of drugs. Prescription audit is an important tool for
the determination of the quality in the prescriptions. To understand the
present situation and implement the principles of the audit, the prescrip-
tions in the eld must be examined. Methods: The prescriptions of the

adult patients obtained from a pharmacy in Ankara were audited. A total
number of 100 prescriptions that were written for adult patients were
included in the study. The prescriptions were audited according to the
criteria about the patient, physician and the drug. The best score was
100. The drug cost for each prescription was calculated. The most common mistakes in the prescriptions were also recorded. Results: The most
common three mistakes in the prescriptions are: 1. The total amount of
the drug prescribed, is not sufcient enough to supply the frequency of
usage that has been mentioned in the prescription. 2. The amount is not
mentioned in the suspensions. 3. Dosage is not mentioned. The prescription audit result was 73,5 points on average. Usually the gender, age and
the adress of the patients are missing. The average cost per prescription
was 13 TL (6.2 Euros). Conclusion: The quality of the prescriptions need
to be improved and the physicians need to be aware of the evaluation.
Prescription audit might serve an important tool for the training of the
physicians for the appropriate prescriptions.
Paper No.: 939

THE IMPACT OF A GRONINGEN MODEL OF
PHARMACOTHERAPY TRAINING FOR GENERAL
PRACTITIONERS IN KAYSERI, TURKEY
Hasan Basri Ulusoy, T Sumak, S Sahin, H Gultekin
Erciyes University Faculty of Medicine, Department of Pharmacology,
Kayseri, Turkey
Groningen Model of rational pharmacotherapy teaching is a kind of
problem based learning method developed by World Health Organisation. The impact of a rational pharmacotherapy training given by this
model to twenty four general practitioners (GPs) for two days in Kayseri,
Turkey was investigated. The Objective Structured Clinical Examinations
(OSCEs) were made using simulated patients, before and after the training. In this study, the OSCE scores were evaluated and prescriptions
written in these examinations were analysed. The average score of GPs
in second (after the training) OSCE was signicantly higher than the
average score obtained in rst (before the training) OSCE (92.38 1.42
vs 37.21 4.11). The average score of prescriptions in the second OSCE
was signicantly higher than the average score in the rst OSCE
(75.88 2.46 vs 51.42 2.22). There are inappropriate drugs in 62.5%
of prescriptions in the rst OSCE while there is no inappropriate drug in
prescriptions in the second OSCE. The 37.5% of prescriptions in the rst
OSCE were hardly readable, this ratio was increased to 42% in prescriptions in the second OSCE. The results of this study revealed that the GPs
do not apply rational pharmacotherapy rules in making their treatment
plans and consequently write inappropriate drugs to their patients. Fortunately, the quality of the pharmacotherapy given by GPs were improved
tremendously after the training. According to the results, this pharmacotherapy training was very successful. These good results indicate that
Groningen Model of rational pharmacotherapy training is a very effective method.
Paper No.: 1001

EVALUATION OF THE RATIONAL PHARMACOTHERAPY
CLERKSHIP AT ERCIYES UNIVERSITY FACULTY OF
MEDICINE, KAYSERI, TURKEY
Hasan Basri Ulusoy, T Sumak, E Eroglu, H Gultekin
Erciyes University Faculty of Medicine, Department of Pharmacology,
Kayseri,Turkey
627
The rational pharmacotherapy clerkship is done using World Health
Organisation / Groningen Model in the 5th year of Erciyes University
Faculty of Medicine. At the beginning and the end of the clerkship,
The Objective Structured Clinical Examinations (OSCEs) are made
using simulated patients. In this study, the OSCE scores of total 256 students in 2008-2009 and 2009-2010 academic year were evaluated and
prescriptions written in these examinations were analysed. The average
score of students in the second (at the end of the clerksip) OSCE was
signicantly higher than the average score obtained in the rst (at the
beginning of the clerksip) OSCE (90.5 4.4 vs 24.4 1.2; P < 0.001).
The average score of prescriptions in the second OSCE was signicantly
higher than the average score in the rst OSCE (4.46 0.05 vs
1.63 0.16; P < 0.001). There are inappropriate drugs in 87.5% of prescriptions in the rst OSCE and in 7.2% of prescriptions in the second
OSCE. The results of this study revealed that the students do not apply
rational pharmacotherapy rules in making their treatment plans at the
beginning of the clerkship and consequently write inappropriate drugs to
simulated patients; on the other hand the clerkship improves quality of
the pharmacotherapy given by the students signicantly.
Paper No.: 699

EVALUATION OF HAEMATINIC ACTIVITY OF NAGA
BHASMA(NB) IN PHENYLHYDRAZINE INDUCED
HAEMOLYTIC ANAEMIA IN RATS
Vaishali R Undale(1), AV Yadav(1), PN Jagtap(1), CD Upasani(2),
AV Bhosale(1)
(1) PDEAs S.G.R.S. College of Pharmacy, Saswad, Pune, India
(2) SNJBs Sureshdada Jain College of Pharmacy, Chandawad, India
Anaemia constitutes a serious health problem in many countries. In anemia, there is decreased level of circulating hemoglobin, less than 13g/dl
in males and 12g/dl in females and decrease in the oxygen carrying
capacity of RBCs. The empirical use of incinerated preparations in the
treatment of anaemia dates from ancient times. These incinerated preparations are Lauha Bhasma, Naga bhasma, Abhrak Bhasma etc. Naga
Bhasma, the incinerated lead is reported to be used in treatment of various disorders. The present study was undertaken to assess the haematinic
activity of Naga Bhasma. The study was carried out on Wistar rats of
either sex weighing about150-200g. Anaemia was induced by an oral
administration of phenylhydrazine 40mg/kg for a period of 2 days. Haemoglobin was measured on 3rd day. Rats with Hb less than 11g/dl were
selected and dosed with Naga Bhasma 250,500,1000 mg/kg day for
15 days and Hb was checked on 0, 3rd,7th,15th day .Oral administration
of Phenylhydrazine decreased Hb from 14g/dl at day 0 to 11g/dl at day
3. Naga Bhasma induced a signicant increase in Hb concentration to 13
at the dose of 250mg/kg p.o. after 48 hrs. Naga bhasma increased Hb
values in anaemic rats. Thus, it can be concluded that Naga Bhasma
exhibits signicant haematinic activity in rats.
Keywords: Anaemia, Phenylhydrazine, Haemoglobin, Naga bhasma
Paper No.: 1258

INCIDENCE, SEVERITY AND AETIOLOGY OF
DRUG-INDUCED ACUTE PANCREATITIS
severity of drug-induced pancreatitis during a two-year period in a tertiary hospital. The study was conducted as a retrospective analysis of all
cases of AP in the University Hospital in Olomouc (1432 beds) in 20062007. All recorded cases of AP were re-evaluated and those meeting the
contemporary criteria of acute pancreatitis were included in the study
cohort. The inclusion criteria were met by 170 medical les. Druginduced disease was diagnosed in 8 patients; in one there were two consecutive cases caused by the same drug. The proportion is 5.3% of the
total number of AP cases (or 11.2% of non-biliary cases) and adverse
drug reaction is the third most frequent cause of the AP. Of the eight
patients in this study, six were women, three suffered from Crohns disease and two from a haematological malignancy. Azathioprine was the
most frequent causative factor (three cases in two patients) of druginduced AP in our cohort; all the other causative drugs were documented
only in single cases: mesalazine, dexamethasone, ramipril, mycophenolate mofetil, cytarabine and valproate. The diagnosis seems to be underestimated because of the difculties in determining the causative agent
and the need for a retrospective re-evaluation of the suspected causative
factors. Drug-induced AP is more probable in younger persons, women
and patients suffering from Crohns disease.
Supported by grant MSM 6198959216
Paper No.: 2319

EFFECTS OF BERGAMOT ESSENTIAL OIL AND ITS
EXTRACTIVE FRACTIONS ON SH-SY5Y HUMAN
NEUROBLASTOMA CELL GROWTH
MR Ursino(1), E Trapasso(1), G Calapai(2), P Bramanti(3), Michele
Navarra(1)
(1) University of Messina, Pharmaco-Biological Department, Messina,
Italy
Medicine and Pharmacology, Messina, Italy
A number of studies show that about 70% of anticancer drugs are natural
or semisynthetic products (Newman and Cragg, J. Nat. Prod., 2007) and
that molecules present in essential oils may have antitumor activity (Edris, Phytother. Res., 2007). Bergamot essential oil (BEO), obtained from
peels of Citrus bergamia fruits, is a basic component of fragrances. It
comprises a volatile fraction (93-96% of total) containing monoterpene
and sesquiterpene hydrocarbons and a nonvolatile fraction (4-7% of
total) consisting essentially of coumarins and psoralens (Costa et al.,
Flav. Fragr. J., 2010). Our study provides novel insights into the eld of
the cytotoxic effects of BEO, which could be exploited in the discovery
of new molecules with antiproliferative activity. Treatment of SH-SY5Y
cells with increased dilutions of BEO and its extractive fractions (ranging
from 0.1 to 0.5 microL/ml for 6, 24, 48 and 72 hours), although with different efcacy, reduces the cell proliferation in a time and concentrationdependent manner (MTT test and cell count). The reduction of cell
growth rate seems to be correlated to the cytotoxic action of BEOs, as
assessed by the trypan blue assay. Furthermore, BEO and its extractive
fractions modify the progression of cells trough the cell cycle (ow
cytometry analysis). In particular there is an increase of cell population
in the S phase and a reduction of both G1 and G2 phases of the cell
cycles for all the BEOs tested.
Karel Urbanek, I Vinklerova, D Hajdu, V Prochazka

Palacky University Faculty of Medicine, Department of Pharmacology,
Olomouc, Czech Republic
Drug-induced acute pancreatitis (AP) is considered to be a rare diagnosis.
The aim of this study was to determine the incidence, aetiology and
628
Paper No.: 1438
HEALTH AND DISEASE
PROTECTIVE EFFECT OF AMLODIPINE AGAINST THE
DECREASE IN BONE DENSITY IN RATS
Kentarou Ushijima, Y Liu, E Ishikawa, T Maekawa, Y Motosugi,
H Ando, A Fujimura
Paper No.: 2905

HEALTH AND DISEASE
SENSITIVITY TO RHO KINASE INHIBITOR Y-27632 IS
INCREASED IN THE MESENTERIC ARTERIES OF L-NAME
HYPERTENSIVE RATS
Sonmez Uydes-Dogan, F Gezerler, S Takir, FI Alp, O Ozdemir
Jichi Medical University, Department of Clinical Pharmacology, Shimotsuke, Tochigi, Japan
Istanbul University, Faculty of Pharmacy, Department of Pharmacology,

Istanbul, Turkey
Introduction: Hypertensive patients have an increased risk of osteoporosis. A recent study has demonstrated that anti-hypertensive therapy
reduced a risk of fracture in these patients. In this study, we investigated
whether amlodipine protects against the reduction in bone density in
stroke-prone spontaneously hypertensive rats (SHR-sp). Materials: Oral
dosing of amlodipine was started when SHR-sp were 3-months old, and
continued for 3 months. At the end of the experiment, bone density of
femur and serum concentrations of calcium, parathyroid hormone (PTH)
and C-telopeptide of type I collagen (CTx), reecting osteoclast activity,
were measured. Results: Bone density of femur was signicantly lower
in SHR-sp than in WKY rats. The bone density dose-dependently
increased by the treatment with amlodipine. In addition, amlodipine
reduced serum concentrations of calcium, PTH and CTx. Conclusion:
This study showed that amlodipine prevents the reduction in bone density during the repeated dosing in SHR-sp. Amlodipine might exert its
effect through a direct inhibition of osteoclast function, and a suppression
of PTH secretion and subsequent inhibition of osteoclast activity.
Activation of Rho-Rho kinase pathway is suggested to be involved in

the pathophysiology of hypertension. Herein, we investigated vascular
effects of a Rho kinase inhibitor Y-27632 in a hypertension model
induced by chronic NO synthesis inhibition. NO synthase inhibitor, LNAME (60mg/kg) was given daily in drinking water to male Wistar rats
(200-250g) for 5-6 weeks. Thereafter, isolated carotid and mesenteric
arteries of hypertensive and control rats were studied parallely in a wiremyograph system. Following the determination of endotelial and smooth
muscle relaxant capacities, the effects of Rho kinase inhibitor Y-27632
(10-10- 10-4M) was assessed at increasing concentrations in hypertensive and control arteries precontracted with prostaglandin F2a. Endothelium-dependent relaxations were signicantly reduced whereas
endothelium-independent relaxations remain unchanged in the arteries of
hypertensive rats compared to controls. Y-27632 elicited comparable
maximal relaxations in carotid and mesenteric arteries of L-NAME
hypertensive and control rats. Whereas, augmented relaxations to lower
concentrations of Y-27632 were observed in mesenteric arteries of LNAME hypertensive rats. Our results showed that responsiveness to Rho
kinase inhibitor Y-27632 is maintained in the arteries of hypertensive rats
where endothelial reactivity is impaired. Additionally, an increased sensitivity to Y-27632 was observed in the mesenteric arteries of hypertensive
rats which suggests that Rho kinase activity is importantly enhanced.
The diverse vasorelaxant prole of Y-27632 in carotid and mesenteric
arteries of L-NAME hypertensive rats may be related to differences in
the relative contribution of Rho kinase in these arteries.
The present work was supported by the Research Fund of Istanbul University. Project No. T-3136.
Paper No.: 2018

EFFECT OF 2-IMINOBIOTIN AND N-OMEGA-PROPYL-LARGININE ON LEARNING AND MEMORY IN RATS
T Utkan(1), SS Gocmez(2), IK Celikyurt(1), Oguz Mutlu(1), N Gacar(1),
F Aricioglu(3)
Kocaeli, Turkey
(3) Marmara University Pharmacy Faculty, Istanbul, Turkey
Nitric oxide (NO) is a messenger molecule that is produced in brain and
it has physiological role in the basis of memory formation. On the other
hand NO participates in many neuropathological states. Previous studies
with nonselective NO synthase (NOS) inhibitors have produced contradictory results in learning experiments. The aim of this work was to
observe the effect of two NOS inhibitors (2-iminobiotin (2-IB) neuronal
NOS (nNOS) and inducible NOS (iNOS) inhibitor, N-omega-propyl-Larginine (NPLA) nNOS and endothelial NOS (eNOS) inhibitor) on learning and memory in comparison with scopolamine in rats. 2-IB (30 mg/
kg) and NPLA (20 mg/kg) were administered intraperitoneally (i.p.) and
30 min. prior to a trial. Scopolamine (1mg/kg, i.p.) was administrated
20 min prior to a trial and considered as a reference drug for learning
impairment. In three-panel runway (TPR) task, the number of times an
animal pushed an incorrect panel gate (errors) and the time required for
the animal to obtain food pellets (latency) were recorded. In step-through
type passive avoidance (PA) task, the latency was carried out 24h after
training and cut-off time was set to be 300 sec. Scopolamine signicantly
impaired learning and memory in TPR and PA task compared to control
group. Administration of 2-IB impaired learning in both tests. Pretreatment with NPLA also impaired learning in TPR but not in PA test. The
results indicate that inhibition of nNOS and eNOS are more involved in
learning than iNOS. Therefore, agents inhibiting NO formation might
have a negative effect on learning and memory function.
Paper No.: 902

EXPRESSION OF PHOSPHODIESTERASE (PDE) ISOENZYMES
PDE4, PDE5 AND PDE11 IN THE HUMAN SEMINAL VESICLES
- AN IMMUNOHISTOCHEMICAL STUDY
Stefan Uckert(1), J Sonnenberg(2), V Steffenhagen(2), M Kuczyk(1),
P Hedlund(3)
(1) Hannover Medical School, Department of Urology & Urological
Oncology, Hannover, Germany
(2) Institute for Biochemical Research & Analysis, Sexual Function
Research Unit, Hannover, Germany
(3) Lund University Hospital, Department of Clinical & Experimental
Pharmacology, Lund, Sweden
Introduction: Phosphodiesterase isoenzymes PDE4 (cyclic AMP PDE),
PDE5 (cyclic GMP PDE) and PDE11 (Dual Substrate PDE) have been
shown to play a role in the control of human genitourinary tract tissues.
There a hints from clinical studies that PDE5 inhibitors can retard the
male ejaculatory response. In order to elucidate further the signicance
of PDE isoenzymes in the regulation of the ejaculatory system, we have
investigated the expression and distribution of PDE4, PDE5A and
PDE11A in the human seminal vesicles (SV). Material/Patients: Cytosolic supernatants prepared from isolated human SV tissue were subjected to Dot-blot analysis using specic anti-PDE antibodies. Using
immunohistochemistry (laser uorescence microscopy), the occurrence
629
of PDE4A, PDE4B, PDE5A, PDE11A, the vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide (CGRP) and protein gene
product 9.5 (PGP 9.5) was examined in sections of SV tissue. Results:
Dot-blot analysis revealed the expression of PDE4A, PDE4B, PDE5A
and PDE11A in the SV tissue. The expression of the PDE isoenzymes
was mainly limited to the glandular epithelium and slender nerve bers
transversing the sections. In nerve bers and nerve endings, PDE4A and
PDE4B were found co-localized with VIP, while PDE5A- and PDE11Apositive nerve bers/endings also presented immunosignals specic for
CGRP and PGP 9.5, respectively. Conclusion: The results indicate that
cAMP- and cGMP-PDE isoenzymes are involved in the control of secretory activity and efferent neurotransmission in the SV. These ndings
might be of importance with regard to the identication of new therapeutic avenues to treat premature ejaculation.
Paper No.: 903

IS THERE A SIGNIFICANCE OF THE CYCLIC AMP
SPECIFIC PHOSPHODIESTERASE 4 (PDE4) IN THE CONTROL
OF PROSTATE SMOOTH MUSCLE? A FUNCTIONAL AND
BIOCHEMICAL APPROACH
Introduction: Rho-associated kinase (ROK) causes calcium-independent

contraction of smooth muscle and has been shown in male penile erectile
tissue. It is not known if ROK or related proteins are involved in the control of vaginal blood ow. Thus, we evaluated by means of functional
experiments and immunohistochemistry (IHC) the ROK pathway in
human vaginal vessels. Materials/Patients: The effects of the Rho-kinase
inhbitor Y 27632 on the concentration-response curve to phenylephrine
(PE) of isolated human subepithelial vaginal arteries (diameter < 100
lm) were investigated. The distribution of ROK-1, ROK-2, RhoA and
RhoGDI in sections of full wall specimens of the vagina was elucidated
by means of immunohistochemistry. Results: The contractile response to
PE of the vaginal arterial segments was signicantly reduced in the presence of 0.1 lM and 1 lM of Y 27632. In PE-contracted preparations,
the addition of Y 27632 (10 nM - 10 lM) produced relaxant responses
with a mean -log IC50 of 6.4 and a relaxation of 91% at a concentration
of 10 lM. IHC revealed a mesh-work of a-actin immunoreactive arterioles in the subepithelium of the vaginal specimens. Immunoreactions for
ROK-1, ROK-2, RhoA and RhoGDI were observed in the smooth musculature of these vessels. Conclusion: a-adrenoceptor activation contracts
subepithelial human vaginal arteries via ROK-sensitive mechanisms.
Based on our ndings, a role for this signalling pathway in the control of
vaginal blood ow might be considered.
Stefan Uckert, D Schonfeld, G Kedia, M Kuczyk

Hannover Medical School, Department of Urology & Urological
Introduction: The use of inhibitors of the cyclic GMP-phosphodiesterase
type 5(PDE5) to treat lower urinary tract symptomatology (LUTS) suggestive of benignprostatic hyperplasia has been suggested. In contrast,
only a few studies haveaddressed the signicance of the cyclic AMP signalling and related proteins(for example, PDE4) in the control of the
human prostate. Materials/Patients:The effects of the PDE4 inhibitors rolipram, Ro 20-1724 and ZK 803616 on thetension induced by norepinephrine of isolated human prostate tissue wereinvestigated in
comparison to the PDE5 inhibitors sildenal, DASPP and MSPP,and
BAY 13-1197, a NO-independent activator of the soluble guanylyl
cyclase (1nM - 10 lM each). PDE activity was isolated from the microsomal fractionprepared from specimens taken from the transition zone
and subjected tobiochemical analysis. Results: Rolipram, Ro 20-1724
and ZK 803616dose-dependently reversed the tension induced by NE of
the isolated prostatetissue by 32%, 59%, and 53%, respectively. The
PDE4 inhibitors were as effectiveas MSPP (54%) and more effective
than BAY 13-1197 (18%), sildenal (17%), andDASPP (14%). Only a
single peak of PDE activity, likely to represent PDE4, wasisolated from
the microsomal fraction. The activity was sensitive to papaverine(nonspecic PDE inhibitor), and the PDE4 inhibitors rolipram and Ro 201724(0.01 lM - 100 lM). Conclusion: Our results present evidence for a
signicanceof the cyclic AMP signalling in controlling prostate smooth
muscle function.These ndings might be of importance for the identication of new drug optionsto treat LUTS.
Paper No.: 904

RHO-KINASE RELATED PROTEINS IN HUMAN VAGINAL
ARTERIES - FUNCTIONAL AND IMMUNOHISTOCHEMICAL
STUDIES
Paper No.: 928

IN VITRO ANTIMICROBIAL ACTIVITY OF SOME HERBAL
EXTRACTS AGAINST ORAL PATHOGENS
Surena Vahabi, E Naja
Shaheed Beheshti Medical University (SBMU), Department of
Periodontics, Tehran,Iran
Introduction: Dental plaque, plays an important role in the development of caries and periodontal disease. Improved understanding of
infectious nature of dental disease has dramatically increased an interest in chemical methods of plaque control. Since some chemical
materials including Chlorhexidine has some side effects, use of natural agents can be a useful alteration. Materials: Hydro alcoholic
(50:50) extracts of 6 plants were taken using succilate method, then
4 dilutions of extracts (20, 40, 80, And 100%w/v) were put on
blood agar media and cultured with one of the bacteria using 4mm
paper discs. Antimicrobial activities of these extracts were examined
by disc diffusion method and mean of diameters of inhibition zone
of each bacterium in different dilutions was reported. An a error
level was set equal to 0.05. Results: Hydro alcoholic extracts of Punica granatum and Trigonella foenum-graceum had strong antibacterial activity respectively and their antibacterial activities were
signicantly less than Chlorhexidine and more than Irsha and Miswak. The hydro alcoholic extracts of Scrophularia striata and Fumaria parviora showed less antibacterial activity in comparison with
the rst 2 ones and it was signicantly less than Chlorhexidine and
Miswak and more than Irsha. Carthamus tinctorius had the weakest
antibacterial activity and it was signicantly less than Chlorhexidine
and Irsha mouthwashes and Miswak extract. Conclusion: High antibacterial effects of some of these herbal extracts recommend more
studies to demonstrate practical approaches of using natural materials
on the oral biolms.
Stefan Uckert(1), E Waldkirch(1), M Kuczyk(1), P Hedlund(2)

(1) Hannover Medical School, Department of Urology & Urological
(2) Lund University Hospital, Department of Clinical & Experimental
Pharmacology, Lund, Sweden
630
Paper No.: 1444
TRPC2 CHANNELS MODULATE BOTH CALCIUM- AND
CAMP-MEDIATED SIGNALLING IN RAT THYROID FRTL-5
CELLS
Minna Vainio(1), P Sukumaran(2), C Lof(2,3), K Tornquist(2,4)
(1) University of Turku, Department of Biology, Laboratory of Animal
Physiology, Turku, Finland
bo Akademi University, Department of Biology, Turku, Finland
(2) A
(3) Turku Graduate School of Biomedical Sciences, Turku, Finland
(4) The Minerva Foundation Institute for Medical Research, Helsinki,
Finland
TRPC (canonical transient receptor potential) channels are putative
receptor- and store-operated cation channels that play a fundamental role
in the regulation of cellular calcium homeostasis. RT-PCR screening of
the rat thyroid FRTL-5 cells revealed the presence of TRPC2 channels
which are previously known to have a physiological function in the acrosome reaction in mature sperm and in pheromone sensing in the vomeronasal sensory organ. The TRPC2 channels were knocked down using
shRNA and the effect was conrmed by TRPC2 Western blot analysis.
The physiological effect of channel knock-down was studied by measuring ATP-stimulated calcium responses and activation of adenylyl cyclases by forskolin or thyrotropin (TSH). Single cell uorometry showed
that the ATP -evoked calcium response was decrease in the TRPC2
knock down cells, compared with wild type cells. Intracellular cAMP
content was measured using a commercial cAMP kit (AssayDesigns Inc.,
Ann Arbor, MI, USA). Both the forskolin- and TSH-evoked increase in
cellular cAMP concentrations was enhanced in TRPC2 knock down
cells, compared with wild type cells. No change in basal cAMP- or calcium levels was detected in TRPC2 knock-down cells, compared with
wild type cells. The expression of TSH receptor protein was up-regulated
in TRPC2 knock-down cells. Our results suggest that TRPC2 channels
are involved in receptor-mediated calcium and cAMP signalling and
modulate the function of adenylyl cylases in rat thyroid FRTL-5 cells.
Paper No.: 2744

HIGH-THROUGHPUT SCREENING FOR NOVEL PROSTATE
CANCER DRUG TARGETS
Paula Vainio(1), K Ketola(1), J-P Mpindi(3), S Kilpinen(3), P Kohonen(1), V Fey(2), S Gupta(1), M Perala(2), O Kallioniemi(1,2,3),
K Iljin(1,2)
(1) University of Turku, Turku Centre for Biotechnology, Turku, Finland
(2) VTT Medical Biotechnology, Turku, Finland
(3) Institute for University of Helsinki, Molecular Medicine Finland
(FIMM), Helsinki, Finland
There is an urgent need for more efcient and more targeted methods of
prostate cancer treatment. Here, we combined information from gene
expression studies of prostate cancer tissues in vivo and RNA interference studies of prostate cancer cell lines to nominate potential targets
and pathways for therapeutic exploration. We selected 300 prostate and
prostate cancer-specic genes from our body- and disease-wide gene
expression database (in silico Transcriptomics, www.genesapiens.org).
To identify therapeutically relevant genes and pathways, we then performed high-throughput screening (HTS) with an siRNA library targeting
300 genes (4 siRNAs per gene, a total of 1200 individual siRNAs) in
several prostate cancer cell lines (VCaPs, MDA-Pca-2bs, LNCaPs and
LNCaPs cultured in androgen deprivation). Cell viability and apoptosis
were studied as end-points. The expression of targets in clinical tumors,
as well as results from the siRNA screening, highlighted a signicant
role for enzymes associated with the production of bioactive lipids and
signalling molecules, especially the arachidonic acid pathway, in prostate

cancer cell proliferation. As expected, known regulators of prostate cancer growth in the arachidonic pathway were found to impact cell proliferation. However, there were also four novel cell growth regulatory genes
along this pathway, suggesting strong dependency of tumor cells on arachidonic acid metabolism. Other genes and pathways identied in the
HTS analysis included several AR and ERG-related as well as actin cytoskeleton associated genes. These results illustrate the power of highthroughput RNAi coupled with systems biological data analysis in the
exploration of potential new target genes for prostate cancer.
Paper No.: 2208

SIMILAR ADENOSINE A1 RECEPTOR ALLOSTERIC
AGONISTS REQUIRE DIFFERENT ORTHOSTERIC SITE
CONFORMATIONS FOR STIMULUS TRANSMISSION
Celine Valant(1), L Aurelio(2), P Scammells(2), P Sexton(1),
A Christopoulos(1)
(1) Monash Institute of Pharmaceutical Sciences, Drug Discovery Biology Laboratory, Melbourne, VIC, Australia
(2) Monash Institute of Pharmaceutical Sciences, Department of Medicinal Chemistry and Drug Action, Melbourne, VIC, Australia
The adenosine A1 receptor (A1R) is the rst GPCR for which positive
allosteric modulators of agonist function were identied. The current
study investigated the pharmacology of two novel aminobenzoylthiophene derivatives, VCP678 and VCP783, as allosteric modulators and allosteric agonists of the A1R; these two compounds only vary from one
another by the absence (VCP678) or presence (VCP783) of a chlorine
group. Both allosteric ligands caused a concentration-dependent increase
in the potency of the orthosteric agonist, (-)-N6-(2-phenylisopropyl)adenosine (R-PIA), as well as an activation of the A1R in their own right in
ERK 1/2 phosphorylation assay, with VCP678 exhibiting over a 10-fold
higher afnity for the allosteric site than VCP783. The agonistic effects
of each allosteric ligand were inhibited by the orthosteric antagonist 8cyclopentyl-1,3-dipropylxanthine (DPCPX) to different extents, consistent with an allosteric interaction characterized by different degrees of
negative cooperativity between each agonist-antagonist pair. Intriguingly,
divergent effects were noted in A1R-mediated ERK 1/2phosphorylation
after irreversible alkylation of the orthosteric binding site using the
chemoreactive orthosteric ligand, 8-cyclopentyl-3-[3-[[4-(uorosulphonyl)benzoyl]oxy]propyl]-1-propylxanthine (FSCPX). Specically, the
agonist effect of VCP678 remained unaltered after orthosteric site alkylation, suggesting that this allosteric agonist can maintain a robust receptor activation irrespective of orthosteric site occlusion, whereas the
agonist effect of VCP783 was abolished, suggesting that the conformational switch triggered by the latter allosteric agonist requires a different
orthosteric site conformation for stimulus transmission. These studies
highlight that even small structural variations in allosteric A1R ligands
can have profound effects on activating receptor conformations via a
common allosteric site.
Paper No.: 1363

EFFECTS OF MEXIDOL AND HEMANTANE IN ANIMAL
MODELS OF PARKINSONISM
Elena Valdman, L Nerobkova, I Kapitsa, T Voronina, O Popova,
N Zolotov
Zakusov Institute of Pharmacology RAMS, Department of Psychopharmacology, Moscow, Russian Federation
631
Novel antiparkinsonian drug hemantane (N-2-adamantyl hexamethylenimine hydrochloride) was shown to reduce motor disturbances in animal
models of parkinsonism. Complex mechanism of action, including antioxidant activity, was determined. Efcacy and safety of monotherapy by
hemantane 25 mg daily was proved in open-label clinical study in
patients with early stages of Parkinson disease. Mexidol (2-ethyl-6methyl-3-oxypyridine succinate) is an antioxidant widely used in neurological disturbances treatment. The aim of the study was to assess the
effects of mexidol and its combination with hemantane in animal models
of parkinsonism. Experiments were held in male albino rats and C57BL/
6 mice. Tremor was induced by arecoline, catalepsy by haloperidol, parkinsonian syndrome by MPTP. Mexidol reduced rigidity, oligokinesis
and tremor, but it effect was lower than of hemantane. It was shown that
the effects of combination hemantane (10 mg/kg) and mexidole (200 mg/
kg) was higher compared to that of hemantane alone. In mice with
MPTP induced parkinsonism the signicant increase in the levels of malone dialdehyde (MDA) and dienic conjugates in brain were determined.
Both drugs administered before MPTP reduced the increase of MDA and
dienic conjugates. The combination of hemantane and mexidol was more
active for reducing MDA levels. The results obtained proved the antioxidant action of hemantane and the rationale for using of hemantane with
mexidol for the treatment of Parkinson disease.
Paper No.: 1909

DEVELOPMENT OF NEW CATECHOL-O-METHYLTRANSFERASE INHIBITORS FOR TREATMENT OF PARKINSONS
DISEASE
(2) University of Helsinki, Faculty of Pharmacy, Division of Pharmaceutical Technology, Helsinki, Finland
(3) University of Turku, Laboratory of Industrial Physics, Department of
Physics, Turku, Finland
Tuberculosis (TB) is a major health problem worldwide, with approximately 1.7 million people dying annually from the disease. The long
current drug regimen, the emergence of drug resistant strains and
HIV co-infection have resulted in a resurgence in research efforts to
address the urgent need for new anti-tuberculosis drugs (Rivers et al,
Drug Discov. Today, 2008, 13: 1090-1098). With respect to multidrug-resistant TB, ethionamide (ETA) is a structural analog of isoniazid that is typically used in case of loss activity of the front-line
drugs. However, the activation of ETA has remained obscure and
some reports suggest that its metabolites are toxic. Porous silicon
(Psi) materials can be used as carriers of drug molecules and have
several advantages over the existing materials for drug delivery (Salonen et al, J. Pharm. Sci., 2008, 97: 632-653), overcoming most of
the problems related to the oral delivery of poorly soluble drug molecules (Kaukonenet et al, Eur. J. Pharm. Biopharm. 2007, 66: 348356), such as poor dissolution/solubility and/or pharmacokinetic properties in the intestinal lumen, poor permeation properties in the GI
tract, as well as high intestinal or hepatic rst pass metabolism. With
this work, we pretend decrease toxicity associated to ETA and
improve biological prole, leading this drug to rst line agents.
For that, we investigated a novel oral formulation technology of Psi
microparticles loaded with ETA. The dissolution proles and the in
vitro toxicity and permeability tests in Caco-2 cell lines of the drug
formulation are evaluated.
Nuno Vale(1), N Micaelo(2)

(1) University of Porto Faculty of Sciences, Department of Chemistry
and Biochemistry, Porto, Portugal
(2) AlcantaraBio, Tecmaia, Portugal
AlcantaraBio is a protein- and drug-design start-up company, specialized
in the discovery and improvement of new drugs using computational
structure-based design methodologies. We are located in Tecmaia Science
Park near Oporto, Portugal. Our mission is to discover new drugs and
support the early stages of drug development of academic and industrial
partners using structure-based computational tools. We create highly
focused, tailor-made libraries of drug candidates towards specic biological targets, and provide a deep structural understanding of the molecular
mechanism of drug action. Our recent work has been focused on the
development of a new catechol-O-methyl transferase (COMT) inhibitor
for Parkinsons disease. A new library of high afnity COMT inhibitors
was designed based on the nitrocatechol group. The drug-COMT structural complexes and binding afnities were evaluated with an improved
proprietary docking and scoring algorithm. We are proposing a new series of COMT inhibitor candidates with predicted high binding afnity
and logP<5. In-silico studies have demonstrated that our new drug candidates have higher afnity relative to well-known COMT inhibitors, such
as: Entacapone, Nebicapone and Tolcapone. These new drug candidates
are going to be synthesized and screened for biological activity.
Paper No.: 1910

NEW TIMES, NEW TRENDS FOR ETHIONAMIDE
Nuno Vale(1), HA Santos(2), E Makila(3), J Salonen(3), J Hirvonen(2),
P Gomes(1)
Paper No.: 1911

SREENING OF PORTUGUESE PROPOLIS AND ITS PLANT
SOURCES FOR INHIBITORY ACTIVITY AGAINST
PATHOGENIC PROTOZOA
Nuno Vale(1), S Falcao(1,2), V Batista(2), C Freire(1), L Maes(3),
M Villas-Boas(2)
(2) CIMO Escola Superior Agraria, Instituto Politecnico de Braganca,
Campus d eSta. Apolonia, Braganca, Portugal
(3) University of Antwerp,Laboratory of Microbiology, Parasitology and
Hygiene Antwerp, Belgium
Propolis is a beehive product with a very complex chemical composition,
widely used in folk medicine because of its several therapeutic activities.
Its biological properties and chemical composition may vary according
to the geograc location and to the different plant sources. Recently, we
reported the mechanism for extration and propolis sample preparation
(Falcao et al, Anal. Bioanal. Chem., 2010, 396: 887-897). After that, we
developed a new study for Portuguese propolis and plant samples found
around the hive, and we present in this study the activity of these samples against pathogenic protozoa. Two different raw propolis samples
from the northeast and centre of Portugal, both acquired from local beekeepers, were analysed in the present work. For the plant samples we
collect the buds exudates and surface material present on the leaves,
stems of Populus nigra male and female specimens and Cistus lanadifer.
These samples were screened for in vitro activity against the pathogenic
protozoa Plasmodium falciparum, Leishmania infantum, Trypanosoma
cruzi and Trypanosoma bruci. To assess selectivity of action, cytodoxicity against MRC-5 broblast cells was also evaluated. We present in this
communication interesting results, according to criteria set up by
the WHO Special Programme for Research & Training in Tropical
Diseases.
632
Paper No.: 605
ETHIC AND PSYCHIATRY CLINICAL TRIALS DESIGN
R Valle Cabrera, Y Mendoza Rodrguez, Yinet Barrese Perez
Paper No.: 2808

EVALUATION OF A GLUCAGON CHALLENGE TEST AS A
TOOL IN DIABETES RESEARCH
National Coordinating Centre for Clinical Trials, Havana, Cuba
Marloes GJ Van Dongen(1), BF Geerts(1), S Bhanot(3), ML de Kam(1),

AF Cohen(1), JA Romijn(2), J Burggraaf(1)
This paper constitutes a review of the main ethical concerns about

research in psychiatry. The most frequent designs, their advantages and
disadvantages, as well as the scales more frequently used in this type of
studies, were analyzed. International standards such as the Declaration of
Helsinki and Good Clinical Practice Guidelines were taking into account,
considering also regulations from the regulatory agencies of United
States and Europe. Once analyzed all the available information, we conclude that informed consent and methodology, where the use of placebo
have a primordial place, are the most important ethical aspects in the
investigation in psychiatry. Also, the design of the study depends on the
illness, the medication to evaluate, the objectives, the hypothesis and the
duration of the study. On the other hand, the scales of evaluation used in
research are valid tools to measure what is sought, but it is vital to establish inter-rater reliability in order to increase the precision of the results
of the study.
Paper No.: 606

THE IMPACT OF PSYCHIATRY CLINICAL TRIALS
OVER THE CLINICAL PRACTICE. THE CUBAN
EXPERIENCE
R Valle Cabrera, Y Mendoza Rodrguez, Yinet Barrese Perez
National Coordinating Centre for Clinical Trials, Havana, Cuba
Five of the ten leading worldwide disability causes (major depression, schizophrenia, bipolar disorders, alcoholism and obsessive compulsive disorders) are mental problems. They are relevant as in poor
as in rich countries, and all predictions indicate there will be a dramatic increase in the coming years The National Coordinating Centre
for Clinical Trials (CENCEC) is a Clinical Research Organization.
Three clinical trials in psychiatry had been performed, since CENCECs creation up to now. The present paper had the objective of
evaluating the impact of these psychiatry clinical trials over the clinical practice in Cuba. With that purpose, quality and efciency indicators (structure, process and results) of the hospitality services were
analysed. The conduction of clinical trials in psychiatry had a positive impact over the clinical practice in Cuba. Those trials permitted
the institutions to achieve the acquisition of material resources to better the infrastructure of the medical services needed to treat psychiatric patients. Besides that, vitally important was the training of a
huge number of specialists throughout the Island, which permitted
the update of the national and international art of mental illness, the
acquisition of skills and competence in the use of psychotherapy,
how to handle placebo response and suicide risk, the acquisition of
specic and updated bibliography, usually not available in the country, the introduction of diagnostic and assessment tools to clinical
practice and the possibility of treatment for some patients with the
rst line of medications.

(2) LUMC Leiden University Medical Center, Leiden, The Netherlands
(3) ISIS Pharmaceuticals, Carlsbad, USA
Relative glucagon excess in type 2 diabetes mellitus contributes to
increased hepatic glucose production and postprandial hyperglycemia.
Hence, tests assessing the contribution of glucagon in glucose homeostasis, such as the hyperglucagonemic challenge described by Petersen et al.
(2001; 44(11): 2018-24), may be a tool in diabetes research. However,
the relationship between glucagon levels and (the increase in) glucose
levels induced by this challenge are largely unknown. Therefore, we
investigated glucagon-induced hyperglycaemia in 12 overnight fasted
adult volunteers. We assessed the repeatability and inter- and intra-individual variability of a glucagon challenge consisting of a 6 hrs [6,6-2H2]
glucose infusion and 3 hrs simultaneous infusion of 3 ngkg-1.min-1glucagon, 0.1 ugkg-1min-1somatostatin and 4 mUm-2min-1insulin.
Each subject had two challenges 6 weeks apart. Blood samples for
(labeled) glucose (measured by GC-MS), insulin and glucagon levels
were taken at regular intervals. After initiation of the glucagon infusion,
serum glucose increased rapidly from a basal level of ~5 mmol/L to a
steady state level of ~11 mmol/L, reaching a maximum at 90 min. The
AUC of glucose had an inter-individual variability of 12.5% and an
intra-individual variability of 4.0%. For the hepatic glucose production
(calculated using Steeles equations) inter-individual variability was
13.4% and intra-individual variability was 10.7%. The CV was 21%/
11%, resp for insulin and 21%/18% for glucagon. In conclusion, the
standardized glucagon challenge has low intra- and inter-individual variability and is a promising tool to further explore the contribution of glucagon in normal and altered glucose homeostasis.
Paper No.: 1229

THE EFFECT OF 5 ARTIFICIAL SWEETENERS ON CACO-2
CELLS
Amorel Van Eyk
University of the Witwatersrand, Department of Pharmacology, Johannesburg, South Africa
Introduction: Some articial sweeteners (AS) have been associated with
the development of tumours in animals. In this study, the effects of 5 AS
on Caco-2 cells were studied. Materials and Methods: Caco-2 cells were
cultured at 37 degrees Celsius and 5% CO2 in DMEM medium (5%
FCS, streptomycin and penicillin). MTT assays were performed (24-72h)
with 5 AS (0-100mM: sodium saccharin (SS), sodium cyclamate (SC),
acesulfame-K (A-K) and sucralose (Suc) and 0-35mM: aspartame (Ast)).
Cell morphology changes were noted. Possible free radical scavenging
activity and effect on ascorbic acid antioxidant activity of AS (10mM)
were measured using the DPPH assay. DNA damage was detected using
the alkali single cell gel electrophoresis assay (10mM AS, 24h). Results:
Cells became bigger, atter, granular and less well dened with increasing cell necrosis at concentrations >50mM for SS, SC, Suc and A-K and
>20mM for Ast. IC50 values of AS were found to be 24h>48h>72h.
IC50 values at 24h: A-K>SC>Suc>Ast>SS; at 48h: SC>SS>SucGAK>Ast and at 72h: SC>SS>A-K>SucGAst. Aspartame indicated possible
free radical scavenging activity as well as slight alteration of ascorbic
acid antioxidant activity. Sucralose and sodium saccharin indicated possi-
633
ble DNA damage. Conclusions: AS cause changes in cell morphology at
higher concentrations. Longer exposure of Caco-2 cells to AS decreases
IC50 values and possible DNA damage was indicated by alkaline comet
assays (sucralose and sodium saccharin). Further research needs to be
done to clarify the DNA damage indicated by AS.
Paper No.: 1776

THE ANTICANCER AND METAL CHELATING PROPERTIES
OF 8-HYDROXYQUINOLINE DERIVATIVES
Robyn L Van Zyl, A Hibbins, M McLaverty
Iron and copper are essential metals required for numerous biochemical
processes and chelation of these metals result in cell death. The 8-hydroxyquinoline (8-OH) class of compounds are effective monoprotic bidentate chelating agents that have a wide spectrum of pharmacological
properties. These include in vitro inhibitory effects on bacteria, fungi,
mycobacteria, amoeba and cancer. The chemotherapeutic properties of 8OH and 14 derivatives were evaluated in vitro against erythroleukaemia
(K562), T-cell leukaemia (Jurkat) and immortilized keratinocytes (HaCaT) using the tetrazolium cellular viability assay. The metal chelating
and free radical scavenging properties were also investigated. Of the 19
compounds tested, 8-OH was the most active against all three cell lines
(IC50 value less than 15lM) with 5,7-dimethyl-8-OH displaying the next
best inhibitory activity and the best safety index (K562: 18.2; Jurkat:
8.5). 2-Amino-8-OH inhibited cell growth (IC50 range: 9.93 18.41lM)
and along with 5-amino-8-OH and 5,7-dimethyl-8-OH scavenged the
DPPH free radical. All compounds chelated iron with 2-methyl-8-OH
being 20x more potent than 8-OH and 6x more potent than EDTA. Structure-activity analysis of these results has indicated the validity for the further synthesis of 8-OH derivatives.
Paper No.: 1777

DISEASES
THE ANTIMICROBIAL PROPERTIES OF 8-HYDROXYQUINOLINE DERIVATIVES AND INTERACTION WITH VITAMIN B12
Robyn L van Zyl, M McLaverty, SF van Vuuren
The emergence of multi-drug resistant strains has compromised the successful treatment of infections. To circumvent this, derivatives of compounds with known activity need to be investigated for antimicrobial
activities. 8-Hydroxyquinoline (8-OH) is an effective metal chelating
agent that has a wide spectrum of pharmacological properties, including
antibacterial, antifungal, anti-amoebic and pesticidal activity. Thus, 8-OH
and 12 derivatives were tested in vitro on Staphylococcus aureus, S. epidermidis, Pseudomonas aeruginosa, Candida albicans and Cryptococcus
neoformans using the microdilution minimum inhibitory concentration
(MIC) method. 8-OH and 2-amino-8-OH were active against S. epidermidis (MIC: 10.75 and 22.48lM, respectively), with 2-amino-8-OH
being twice as effective as 8-OH against P. aeruginosa. The most potent
of the compounds tested was 5-chloro-7-iodo-8-OH against C. albicans
and C. neoformans (MIC: 0.77 and 0.51lM, respectively), which was
more similar in activity to amphotericin B (MIC: 1.35 and 0.34lM,
respectively). 5-Chloro-7-iodo-8-OH was ve times more potent than
amphotericin B against C. albicans and twice as active against C. neoformans. However, 5-chloro-7-iodo-8-OH was withdrawn for oral use due
subacute myelo-optico-neuropathy, as a result of 5-chloro-7-iodo-8-OH
decreasing vitamin B12 bioavailability. Concurrent supplementation with
vitamin B12 may prevent this syndrome, but would it interfere with the
antimicrobial effects of 5-chloro-7-iodo-8-OH? A combination of these
two compounds resulted in a synergistic interaction when tested against
C. neoformans. Thus, further investigation into the activity of the active
compounds is warranted.
Paper No.: 3349

DISEASES
GLPG0259, A SMALL MOLECULE KINASE INHIBITOR BEING
DEVELOPED FOR RHEUMATOID ARTHRITIS, SHOWS GOOD
SAFETY, TOLERABILITY AND PHARMACOKINETICS IN
HEALTHY VOLUNTEERS
Frederic Vanhoutte(1), J Beetens(1), L Gheyle(2), E Vets(2), W Haazen(2), F Namour(3), N Vandeghinste(1), S Blockhuys(1), P Wigerinck(1)
(1) Galapagos NV, Mechelen, Belgium
(2) SGS - Clinical Pharmacology Unit, Antwerp, Belgium
(3) Galapagos SASU, Romainville, France
GLPG0259, an inhibitor of the protein kinase MAPKAPK5, blocks the
release of mediators of inammation and cartilage erosion in human cells
in vitro. The compound also demonstrates bone protection and reduced
inammation in the mouse collagen-induced arthritis (CIA) model.
Therefore, GLPG0259 may offer a promising new therapeutic approach
for the treatment of rheumatoid arthritis (RA). A Phase I program was
initiated to assess the safety and tolerability as well as the pharmacokinetic (PK) prole of single and multiple ascending doses of the compound, including exploration of interaction with methotrexate.
GLPG0259 was administered orally in a dose range of 1.5 to 150 mg as
single doses and in a dose range of 25 to 75 mg q.d. for 14 days as multiple doses. Lastly, a dose of 50 mg as an oral solution was compared to
a capsule formulation in fasted and fed state. GLPG0259 was generally
safe and well tolerated with no adverse effects on ECG, vital signs or
laboratory parameters in healthy volunteers. GLPG0259 showed a dose
proportional PK prole over the dose range tested, supporting a oncedaily oral dosing regimen. After q.d. dosing, steady-state was reached
within one week. Methotrexate, the current standard treatment for RA,
and GLPG0259 had no inuence on the PK proles of each other,
thereby conrming the feasibility of combining GLPG0259 with methotrexate in future trials. A similar safety and PK prole was observed for
the capsule formulation of GLPG0259, which will be used in the Phase
II efcacy trial in RA patients.
Paper No.: 2268

HEALTH AND DISEASE
GENOMIC CHANGES CAUSED BY CHRONIC EXPOSURE TO
HIGH CHOLESTEROL OR FISH OIL IN NATIVE AND REGENERATED PORCINE CORONARY ENDOTHELIAL CELLS
Paul M Vanhoutte(1), Mary YK Lee(1), Y Wang(1), HF Tse(2)
(1) The University of Hong Kong, Department of Pharmacology & Pharmacy, Hong Kong,PR China
(2) The University of Hong Kong, Department of Medicine, Jong Kong,
PR China
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The genomic changes associated with endothelial lesions caused by balloon angioplasty and hypercholesterolemia or those affected by dietary
sh oil in porcine coronary arteries were analyzed using large scale
microarray gene expression proling. Pigs were fed with high cholesterol
(CHL) diet or sh oil diet rich in omega-3 unsaturated fatty acids. Arterial endothelial denudation was performed by percutaneous transluminal
coronary angioplasty of the left anterior descending artery (LAD). Cells
from left circumex (native cells) and LAD (regenerated cells) were harvested for primary culture and subsequent genomic microarray experiments four weeks after the surgery. The plasma levels of LDL-C,
triglycerides and arachidonic acid were elevated while those of HDL-C
were reduced in the CHL group. Fish oil treatment reduced the plasma
levels of arachidonic acid, LDL-C, HDL-C, and the LDL-C/HDL-C
ratio. Only 27 genes showed differential regulations, whereas 385 genes
were altered similarly by hypercholesterolemia and regeneration. In addition, hypercholesterolemia induced 396 gene changes that were independent of endothelial regeneration. Fish oil signicantly reduced the
number of gene changes associated with regeneration (176 genes).
Regeneration-induced changes of 72 genes were either completely or
partially blocked by sh oil. Thus, the present study identies endothelial
genomic changes associated with both hypercholesterolemia-induced
prelesional responses and postlesional damage caused by regeneration.
The genes responsible for the potential protective effects of sh oil were
determined.
Paper No.: 2927

EFFECTS OF SINGLE IN VIVO DOSES OF GABOXADOL, A
HIGH-EFFICACY AGONIST OF EXTRASYNAPTIC GABA-A
RECEPTORS, ON GLUTAMATERGIC TRANSMISSION IN VTA
DA NEURONS
Paper No.: 532

QUNIDINE AND ASPIRIN KINETICS IN BLOOD AND
CENTRAL NERVOUS SYSTEM OF RATS AFTER
INTRAARTERIAL APPLICATION
Velibor Vasovic, S Vukmirovic, V Jakovljevic, M Mikov, B Milijasevic,
I Mikov
Medical Faculty of Novi Sad, Department of Pharmacology, Toxicology
& Clinical Pharmacology, Novi Sad, Republic of Serbia
We have tested the kinetics of quinidine and aspirin in blood and CNS
of rats after the intra-arterial application. The experiments were done on
the anesthetized white rats (n = 60) which were given quinidine and aspirin by retrograde intra-arterial bolus injection (15 seconds) into the right
axillary artery. By this way of application, quinidine and aspirin were
directed towards truncus brachiocephalicus and then, through blood circulation, to the head and CNS respectively, avoiding the inuence of the
peripheral organs, that could have changed their absorption, distribution,
metabolism and excretion. Rats (at least 6) were sacriced by decapitation 30, 60, 90, 120 and 240 seconds after quinidine and aspirin administration. Blood samples were taken from the left jugular vein, and brain
was washed out and divided to brainstem, cerebellum and hemispheres
of cerebrum in order to enable determination of the concentration of
quinidine and aspirin by standard spectrophotouorometric methods.
Quinidine and aspirin signicantly differ regarding their respective kinetics in blood and CNS. Maximum concentration of quinidine in the CNS
is reached with delay, in relation to its maximum concentrations in blood,
and the concentrations are usually higher in CNS than in blood. On the
contrary, concentrations of aspirin are considerably (about 30 times)
lower in CNS than in blood. Central kinetics of quinidine, more than that
of aspirin, indicates the existence of two compartments in CNS; one,
which consists of brainstem and cerebellum where concentrations are
higher and the other, which consists of hemispheres of cerebrum.
Elena V Vashchinkina, AE Panhelainen, OY Vekovischeva, ER Korpi

University of Helsinki, Department of Pharmacology, Helsinki,Finland
Midbrain ventral tegmental area (VTA), representing a critical brain
regions for drug-dependent behavioral modulation, contains functional
extrasynaptic GABA-A receptors (Xiao et al, J Physiol. 2007; 580:73143). Their involvement in the regulation of VTA dopamine (DA) neurons
remains poorly known. Therefore, effects of gaboxadol, a high-efcacy
agonist of extrasynaptic GABA-A receptors, on glutamatergic transmission in VTA DA neurons were studied. Tyrosine hydroxylase-EGFP
transgenic mouse line was used to aid identication of VTA DA neurons
in midbrain slices in vitro. Pharmacologically isolated peak height ratios
between GluA (AMPA) and GluN (NMDA) receptor-mediated evoked
excitatory currents using whole-cell patch-clamp conguration were
determined. A single injection of 6 mg/kg gaboxadol, i.p., signicantly
(P < 0.01) increased the GluA/GluN ratio in VTA DA neurons in slices
obtained 24 h after the injection (control 0.42 0.09, n = 7 mice, vs. gaboxadol 0.75 0.25, n = 7 mice). In previous experiments, the ratio
increased after a single injection of ethanol, morphine, diazepam and zolpidem (Heikkinen et al, Neuropsychopharmacology 2008, 34: 290298).
The effects of gaboxadol (6 mg/kg) on locomotor activity were followed
for 12h by Ethovision video tracking method to show behavioral
responses. Gaboxadol-treated mice were less active (shorter walking distance and lower mobility) than their controls, especially during rst 4 h
after the injection. Our results suggest that the enhancement of the extrasynaptic GABAergic transmission may modify the glutamate neurotransmission in the VTA DA neurons and might regulate activity of drug
reward system.
The study was supported by the Finnish Foundation for Alcohol Studies.
Paper No.: 853

A TWO-STEP COMPETITION BINDING APPROACH TO
DETERMINE UNLABELLED LIGAND-RECEPTOR COMPLEX
DISSOCIATION RATES
Georges Vauquelin(1), M Wennerberg(2), A Balendran(2), A Packeu(1)
(1) Free University Brussels - VUB, Brussels, Belgium
(2) AstraZeneca R&D, Molndal, Sweden
The dissociation rate of unlabelled ligand-receptor complexes is often
estimated indirectly from their ability to slow-down the association of
subsequently added radioligand molecules. The presently explored twostep competition binding approach is particularly suited for intact plated
cell binding studies. It consists of preincubating the receptor-preparation
with a wide range of ligand concentrations, washing off free ligand molecules, adding radioligand and monitoring its receptor binding after a
xed time. Based on the rationale that binding of both ligands is mutually exclusive and that they bind according to the law of mass-action to a
single class of sites, the unlabelled ligands disociation rate can be estimated from the upward shift that the competition curve experiences after
washing. The relevance of this approach is illustrated by simulations and
by monitoring the dissociation behaviour of unlabelled D2 dopamineand CB1 cannabinoid receptor antagonists in intact cell binding studies.
For the D2 receptor, binding of spiperone, haloperidol and (+)-butaclamol was very tight while raclopride, sulpiride and clozapine dissociated
swiftly. Yet, the high partitioning of clozapine in the cell membrane
could contribute to its prolonged effect. For the CB1 receptor: rimonabant dissociated much faster than taranabant. Compared to previous paradigms, the two-step competition binding approach only needs
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measuring radioligand binding at a single time point and the unlabelled
ligands dissociation rate is easily estimated. Above all, it also spots the
capability of unlabelled ligands to partition in cells and to be subsequently released therefrom.
Paper No.: 3210

HEALTH AND DISEASE
EFFECT OF SULODEXIDE ON AORTIC VASODILATION
CAPACITY AND ASSOCIATED MORPHOLOGICAL CHANGES
IN RATS WITH STREPTOZOTOCIN-INDUCED DIABETES
Jose Vazquez(1), Y Mathison(1), E Romero(1), C Suarez(2)
(1) Universidad Central de Venezuela, Faculty of Medicine, School Jose
Mara Vargas, Caracas Venezuela
(2) Universidad Central de Venezuela, Faculty of Medicine, AnatomyPathologic Institute, Caracas Venezuela
Endothelial dysfunction (ED) is observed in patients with hypercholesterolemia, arterial hypertension, obesity and diabetes mellitus. Recent evidences suggest the involvement of glycosaminoglycans(GSG) in ED. We
evaluated the effect of sulodexide (SLD), a natural GSG used in albuminuria and ischemic diabetes treatment, on arterial relaxation and vascular morphological changes in a diabetic type I model. Diabetes was
induced, in Sprague-Dawley rats by streptozotocine (STZ) administration, 60 mg, iv. Rats were divided into four groups; I: control, II: diabetics, III: control + SLD, IV: diabetics treated with SLD (15 mg/day).
After three months period, phenylephrine precontracted aortic rings were
used to evaluate acetylcholine (ACh) and sodium nitroprusside (NPS)
relaxation capacity. Light microscopy of aorta was done with several
staining procedures. In vitro, SLD did not change smooth muscle tone in
resting or phenylephrine precontracted aortic rings. In diabetic rats, ACh
relaxation was 28.8 35.1% lower than in control rats. Diabetic rats treated with SLD showed aortic ACh relaxation similar to control rats. No
signicative statistical difference was found in endothelium-independent
NPS relaxation, between the different groups. Light microscopy histological studies revealed important morphological alterations, particularly in
intima and adventitia layers of aortic artery; those changes were dramatically reversed in SLD treated rats. Our experiments support the conclusion that SLD is a potential drug for improving endothelial dysfunction
in diabetes.
Paper No.: 1539

GENDER DIFFERENCES IN PHARMACOKINETICS OF
TACROLIMUS IN RENAL TRANSPLANT RECIPIENTS
Radmila Velickovic-Radovanovic(1,3), M Mikov(2),
A Catic-Djordjevic(1), G Paunovic(3), V Djordjevic(1,3)
(1) Nis University Faculty of Medicine, Department of Pharmacy, Nis,
Republic of Serbia
(2) Novi Sad University Faculty of Medicine, Novi Sad, Republic of
Serbia
(3) Nis University Faculty of Medicine, Clinic of Nephrology, Nis,
Republic of Serbia
The possible inuence of gender on tacrolimus disposition and response
in renal transplant patients is an issue of medical importance. Monitoring
of tacrolimus blood concentration is of utmost importance due to its narrow therapeutic index and highly variable pharmacokinetics. The aim of
this study was to detect interpatient pharmacokinetic variability of tacrolimus due to patients gender and to assess the predictability of individual
tacrolimus concentrations using abbreviated AUC measurements. The
purpose is to nd the best sampling time to predict the exposure of tacrolimus in renal transplant recipients. First oral dose tacrolimus pharma-
cokinetics studies have been conducted in 20 Republic of Serbian renal

transplant recipients (10 man/10 women) -on triple immunosuppressive
therapy. The rst tacrolimus oral dose (0.05 mg/kg) was given at day 5
posttransplant. Blood concentrations were measured by microparticle
enzyme immunoassay method. Associations between the each sampling
time point of concentrations and AUC12 were evaluated by Pearson correlation coefcients. Abbreviated sampling equations were derived by
multiple, stepwise regression analyses. The variance in the strength of
association between predicted AUCp and AUC12 was reected by linear
regression coefcients. AUC12 showed remarkable interindividual variations after the rst tacrolimus oral dose. Our results show signicant difference between men and women. C2 seems to be indicator of total body
exposure to tacrolimus in women. Alternatively, the concentrations at 1,4
and 12 hours as an abbreviated AUC were as good predictor as a full
pharmacokinetic study for men.
Paper No.: 1980

PROTEINASE ACTIVATED RECEPTOR 2 (PAR-2) AND TOLL
LIKE RECEPTOR 4 (TLR4)RECEPTORS CROSS-TALK IN RAT
VASCULAR REACTIVITY
Valentina Vellecco, M Bucci, V Brancaleone, F Roviezzo, R Meli,
G Mattace Raso, G Cirino
University of Naples Federico II, Department of Experimental Pharmacology,Naples, Italy
Introduction: PAR2 is a G protein-coupled receptor activated by proteases
or by synthetic peptides called PAR2-APs. PAR2 is widely distributed, in
particular in endothelial cells. A role for PAR2 has been proposed in vascular homeostasis and innate immune response. TLR4 is the LPS- specic receptor involved in immune response. Recent evidence suggest a
possible cross-talk between PAR2 and TLR4 signalling. Aim: To investigate the possible cross-talk between PAR2 and TLR4 in vascular district
in LPS-induced septic shock. Methods: After 4 and 8h from LPS injection, rat aorta was used for in vitro studies in isolated aortic rings model;
in vivo studies were performed by measuring blood pressure in anaesthetised rats. All experiments were performed with TLR4 inhibitors: curcumine or resveratrol. In aorta homogenates immunoprecipitation of TLR4
and immunoblot for TLR4 and PAR2 were performed. Results: PAR-2,
but not TLR4, expression was enhanced in endotoxemic rat aortas.
PAR2-AP-induced vasorelaxation was increased in LPS-treated rats aortas. Curcumine and resveratrol signicantly reduced PAR2-induced vasorelaxation and PAR2-induced hypotension in both nave and
endotoxemic rats. Imunoprecipitates revealed PAR2/TLR4 association in
both nave and endotoxemic rats. Western blot of supernatants of immunoprecitates showed the presence of PAR-2 exclusively in endotoxemic
rat aortas. Conclusions: In vasculature PAR2 and TLR4 reside and cooperate in molecular signalling in both physiological and pathological conditions. Following LPS priming there is an increase in PAR-2 expression
that is not engaged in the interaction with TLR4. The unengaged quote
of PAR2 mediates the hypotensive effect related to this receptor in shock.
Paper No.: 2350

MUTATION OF THE S1P1 RECEPTOR DIFFERENTIALLY
AFFECTS SIGNALLING BY AGONISTS
Dennis Verzijl(1), P Van Loenen(1), C De Graaf(2), R Didier(3),
S Peters(1), A Alewijnse(1)
(1) Academic Medical Center, Department Pharmacology and Pharmacotherapy, Amsterdam, The Netherlands
636
(2) Vrije Universiteit, Department Pharmacochemistry, Amsterdam, The
Netherlands
(3) Universite de Strasbourg, Structural Chemogenomics Group, UMR
7200 CNRS, Illkirch, France
The sphingosine-1-phosphate type 1 receptor (S1P1) belongs to the family of G protein-coupled S1P receptors, a class of receptors which play
important roles in the cardiovascular system and in immune regulation.
FTY720 is a novel immunomodulatory drug which is in Phase III clinical trials for the treatment of multiple sclerosis. The phosphorylated form
of this compound, FTY720-P, has afnity for all S1P receptor subtypes
except the S1P2 receptor. The S1P1 receptor seems to be to most important target for the immunomodulatory effects observed for FTY720. In
this study we determine the role of amino acids located in the transmembrane domains of the S1P1 receptor in activation by S1P and synthetic
agonists, including FTY720-P. For example, mutation of tyrosine Y2.57
to phenylalanine (Y2.57F) did not signicantly affect the potency of any
of the tested agonists, whereas mutation to alanine (Y2.57A) resulted in
a 100-fold lower potency for S1P and a 10-fold reduction in potency for
most synthetic agonists. Remarkably, the potency of FTY720-P did not
change, indicating a different mode of activation of the S1P1 receptor by
S1P and FTY720-P. Indeed, computational modeling studies suggest a
different binding mode for FTY720-P compared to S1P. Although a
direct interaction of Y2.57 with S1P or FTY720-P seems unlikely, Y2.57
appears to be involved in stabilizing the binding pocket for S1P but not
for FTY720-P.
Paper No.: 1243

DISEASES
MINOCYCLINE REVERSES BEHAVIOR AND
NEUROCHEMICAL CHANGES IN THE MODEL OF GLOBAL
BRAIN ISCHEMIA IN RATS
Glauce SB Viana(1,2), AGG de Carvalho(1), AVB Peixoto(1),
CCB Bezerra(1)
Paper No.: 1244

DISEASES
MINOCYCLINE REVERTS NEUROCHEMICAL AND
BEHAVIOR ALTERATIONS IN THE PD MODEL OF
6-OHDA-INDUCED STRIATAL LESIONS IN RATS
Glauce Viana(1,2), PLF Tavares(1), IPP Xavier(1), IR Holanda(1), GM
de Andrade(2)
(2) Faculty of Medicine of Juazeiro do Norte, Ceara, Brazil
Minocycline (M), a second generation tetracycline, has attracted interest
for its potential therapeutic uses in neurodegenerative diseases. Previously, we showed that minocycline exhibits potent anti-inammatory and
antioxidant effects. We evaluated the effects of Minocycline in an experimental model of PD, by injecting 6-OHDA into the right striatum of
male Wistar rats. The stereotaxic surgery was conducted in sham-operated (SO), 6-OHDA-lesioned and 6-OHDA+M (10, 25 and 50 mg/kg,
i.p., daily, 7 days) groups. The 1st injection of M was given 1 h before
surgery. After 15 days, the rotational behavior test, induced by the apomorphine injection was performed, followed by the open eld test which
evaluates the locomotor activity. At the next day, the animals were sacriced, and the striatum dissected for neurochemical measurements (determinations of dopamine - DA and their metabolites concentrations) by
HPLC. Results showed that while the 6-OHDA-lesioned animals presented an increased number of rotations, as compared to the SO group,
this effect was signicantly reduced by the minocycline treatment. The
reduced locomotor activity shown in 6-OHDA-lesioned animals was also
reverted by M. On the contrary, while the 6-OHDA-induced lesions led
to signicant decreases in rearing and grooming behaviors, these effects
were even intensied in the M groups as compared to the SO group. The
6-OHDA-lesioned rats presented signicant reductions in the levels of
DA, DOPAC and HVA, and these changes were signicantly and dosedependently reversed by the minocycline treatment. We conclude that
minocycline is a potential candidate and an alternative for the treatment
of neurodegenerative diseases, such as PD.
(2) Faculty of Medicine of Juazeiro do Norte, Ceara, Brazil
Minocycline is a second generation tetracycline which in addition to its
antimicrobial properties has been reported to exert neuroprotective
effects. Previously, we showed that minocycline exhibits potent antiinammatory and antioxidant effects. We studied the minocycline (M)
treatment in adult male Wistar rats submitted to global ischemia in the
following groups: sham-operated (SO), ischemic (ISC), and the combination of ISC+M. Animals were anesthetized and had their common carotid
arteries clamped for 30 min, when clips were removed for reperfusion.
The SO had their carotid arteries exposed without clamping. After the
surgery, the tests were performed the following week. M (25 and 50 mg/
kg, p.o., daily for 1 week) treatment signicantly recovered the locomotor activity, and rearing and grooming behaviors affected by ischemia.
Although no change was seen after ischemia in either, memory acquisition or retention, the animals presented a better performance as far as
memory retention is concerned after minocycline. Ischemia signicantly
increased the time to nd the platform in the water maze test, indicating
an impairment of spatial memory and hypocampal dysfunction. The minocycline treatment completely reversed this effect, and values were
close to or even lower than those of the SO group. Furthermore, signicant increases in striatal DA, DOPAC and HVA concentrations were
observed in the ISC+M50 group, as compared to the ISC group. We conclude that minocycline acts as a dopaminergic agonist, and this effect
together with the drug anti-inammatory and antioxidant activities are
probably responsible for its neuroprotective action.
Paper No.: 1908

BIOTRANSFORMATION OF A NEW ANXIOLYTIC
APHOBAZOLE IN RATS
Anastassia Viglinskaya(1), S Seredenin(2), V Zherdev(1),
G Kolyvanov(1), A Litvin(1), T Mozhaeva(3), D Bastrigin(1)
(1) Russian Academy of Medical Sciences, SI Zakusov of Pharmacology,
Department of Pharmacokinetics, Moscow, Russian Federation
Department of Pharmacogenetics, Moscow, Russian Federation
Department of Chemistry, Moscow, Russian Federation
Selective anxiolytic aphobazole [2-(2-morpholinoethylthio)-5-ethoxybenzylimidazole dihydrochloride] was developed in Zakusov Institute of
Pharmacology Russian Academy of Medical Sciences. The aim of the
study was identication of aphobazole metabolites in blood plasma and
urine of rats. Aphobazole was administered intraperitoneally and intragastrally using probe in dose 25 mg/kg in male Wistar rats (200h}20 g).
Mass-spectra of aphobazole metabolites were registered on the liquid
chromatograph with mass-selective and diode array detectors (Agilent
Technologies, Model 1100, USA). The aphobazole metabolism study
permitted to detect 17 products of biotransformation of parent drug. The
metabolites were numbered in accordance with increasing of the retention times of elution. According to the results of comparative analysis of
637
chromatographic and mass-spectral properties of compounds, M-3
(hydroxylated at benzimidazole cycle), M-6 (oxidated at heteroatom of
sulfur), M-7 (hydroxylated at aliphatic radical), M-11 (oxidated at mopholine fragment), M-14 (cyclized at nitrogen atom), and synthesized standards their complete identity was found. Other metabolites were
characterized by values of the molecular ions. Metabolite M-11 was
identied as main metabolite. In this way the basic directions of aphobazole biotransformation in rats were consisted in the formation of
metabolites which hydroxylated at aromatic ring of benzimidazole cycle
and oxidated at mopholine ring. In urine of rats as major metabolites of
aphobazole were detected M-3 and its glucuronic acid conjugate as well
as M-6, M-7, M-11 and an additional biotransformation product which in
blood plasma was not registered - sulfone of aphobazole.
Paper No.: 2021

LSP4-2022 IS A NEW SELECTIVE AND POTENT MGLU4
AGONIST WITH ANTIHYPERALGESIC EFFECT
CYP2B6 metabolises various clinically important drugs which include

the nonnucleoside reverse transcriptase inhibitor, efavirenz. The allele
frequencies of CYP2B6-516G>T have been investigated and were found
to vary signicantly between different populations. A single nucleotide
change, 516G>T, inuences efavirenz plasma concentration. The homozygous allele (T/T) is associated with reduced CYP2B6 activity leading
to higher efavirenz plasma concentrations and consequently more drug
related side-effects. We examined the CYP2B6-516G>T polymorphism
in 60 HIV-infected children on efavirenz based therapy. The CYP2B6516G>T genotypes, G/G; G/T and T/T were distributed as follows: 42%,
35% and 23%, respectively. The CYP2B6-516T allele frequency was
41% and within the Hardy-Weinberg equilibrium (P = 0.10). Efavirenz
plasma levels were taken at steady state, one month post HAART initiation. The mean (SD) plasma efavirenz concentrations were signicantly
different and correlated with CYP2B6 genotypes as follows: 1.70 (0.98),
3.84 (3.94) and 5.32 (2.91) `g/ml for the G/G, G/T and T/T genotypes,
respectively (P = 0.0003). This is the rst study to evaluate efavirenz
plasma concentrations in association with the CYP2B6-516G>T polymorphism in South African HIV-infected children. We can conclude that
there is a role for considering CYP2B6 genotypes in deciding the appropriate dosage for efavirenz in this paediatric population.
Bruno Vilar(1), J Busserolles(2), I Brabet(1), B Ling(2), F Acher(3),

J-P Pin(1), A Eschalier(2), C Goudet(1)
(1) Institut de Genomique Fonctionnelle, Montpellier, France
(2) Laboratoire de Pharmacologie Moleculaire, Clermont-Ferrand, France
(3) Laboratoire de Chimie et de Biochimie Pharmacologiques et Toxicologiques, Paris, France
Chronic pain is often associated with an overactive glutamate neurotransmission. Finding pharmacological tools capable to counteract this overactivity represents an interesting challenge with potential therapeutic
application for the treatment of chronic pain. Metabotropic glutamate
receptors (mGluRs) are G-protein coupled receptors which modulates
glutamatergic neurotransmission. They are divided in three groups,
mGlu4, 6, 7 and 8 belong to group III and are mainly presynaptic receptors. Group III mGluRs are especially abundant all along the pain neuraxis and particularly in the dorsal horn of the spinal cord, a key
localization for the regulation of ascending informations. To date, there
is a crucial need for developing new tools to better understand the role
of these particular mGluRs in chronic pain. This study describes the
molecular and behavioural pharmacological characterization in animal
model of pain of LSP4-2022, a new ligand that has been developed on
the basis of a virtuals screening approach. Taken together, our results
reveal that LSP4-2022 is a potent and selective agonist of mGlu4 with
antihyperalgesic effect, and conrms that mGlu4 may be considered as a
potential therapeutic target in chronic pain.
Paper No.: 706

PREVALENCE OF CYP2B6-516G>T POLYMORPHISM IN
BLACK SOUTH AFRICAN HIV-INFECTED CHILDREN ON
EFAVIRENZ BASED ANTIRETROVIRAL THERAPY
Michelle Viljoen(1), C Dandara(2), M Rheeders(1), KR Conradie(3), HS
Kruger(3), H Gous(4), TM Meyers(4)
(1) North-West University, Department of Pharmacology, Potchefstroom,
South Africa
(2) University of Cape Town, Faculty of Health Sciences, Division of
Human Genetics, Cape Town, South Africa
(3) North-West University, TReNDS-Centre of Excellence for Nutrition,
Potchefstroom, South Africa
(4) Chris Hani Baragwanath Hospital, Harriet Shezi Childrens Clinic,
ECHO, Soweto, South Africa
Paper No.: 1381

IMPAIRED CA2 + HANDLING IN PENILE ARTERIES FROM
PREDIABETIC ZUCKER RATS: INVOLVEMENT OF
RHO-KINASE
N Villalba, A Garca-Sacristan, M Hernandez, Dolores Prieto
Metabolic syndrome is a cluster of metabolic and cardiovascular abnormalities including obesity, insulin resistance, dyslipidemia and hypertension, which usually precede type 2 diabetes. Both endothelial
dysfunction and augmented vasoconstriction are present in penile arteries
(PAs) from prediabetic obese Zucker rats (OZR) (Villalba et al., Am J
Physiol 297: 696, 2009). However, whether this abnormal arterial reactivity represents an alteration in vascular smooth muscle Ca2 + handling
is unknown. The aim of the present study was to investigate the role of
myolament Ca2 + sensitivity in the impaired penile vasoconstriction of
OZR. PAs from OZRs and lean Zucker rats (LZRs) were mounted on
microvascular myographs for simultaneous measurements of intracellular
Ca2 + concentration ([Ca2+]i) and tension. The relationships between
[Ca2+]i and contraction for the a1-adrenergic vasoconstrictor phenylephrine (Phe) were left shifted and steeper in OZR than in LZR,
although the magnitude of the contraction was similar in both groups.
The Rho-kinase inhibitor Y-27632 attenuated the vasoconstriction
induced by Phe to a greater extent in OZR than LZR. In LZR, Y27632 inhibited and augmented Ca2 + entry elicited by submaximal
and maximal concentrations of Phe, respectively, but the inhibitor did
not alter the reduced Phe-induced Ca2 + entry in OZR. Addition of Y27632 on the steady-state increases in [Ca2+]i and force induced by a
high K+ solution reduced tension to a greater degree in arteries from
OZR compared to LZR without changing [Ca2+]i. RhoK-II expression
was augmented in arteries from OZR. These results suggest a Rhokinase-mediated enhanced myolament Ca2 + sensitivity in penile
arteries during metabolic syndrome.
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Paper No.: 2472
CB1 CANNABINOID RECEPTOR EXPRESSION IS
REGULATED BY GLUCOSE AND FEEDING STATE IN
MURINE PANCREATIC ISLETS
Paper No.: 3136

THE PHARMACOKINETICS AND PHARMACODYNAMICS OF
BIOMARKERS IN PATIENTS WITH LEFT VENTRICULAR
SYSTOLIC DYSFUNCTION (LVSD) AND CHF TREATED WITH
EPLERENONE
Rafael Villalobos-Molina(1), A Vilches-Flores(1), NL Delgado-Buenrostro(1), G Navarrete-Vazquez(2)
John Vincent(1), J Cumps(2), F Zannad(3), J Ketelslegers(2)
(1) Fes-Iztacala, U.N.A.M., Unidad de Biomedicina, Tlalnepantla, Mexico

(2) Facultad de Farmacia, U.A. Morelos, Cuernavaca, Morelos, Mexico
Endocannabinoids are closely involved in food intake and energy balance from carbohydrates and lipids. Beside hypothalamic neurons, pancreatic islet cells express CB1 cannabinoid receptor, however little is
known about its physiological role and regulation in these cells. Gene
expression of many islet proteins is dependent on glucose concentration,
we studied CB1 receptor expression in response to fasting and feeding
states. Pancreas or islets were isolated from 16-h fasted adult Wistar rats,
with or without 1.5 g/kg glucose oral-intake. CB1, insulin and glucagon
were analyzed by immunouorescence. Islets from feed animals were
cultured with different glucose concentrations, and 1lM anandamide, or
rimonabant analog [1-(4-chlorophenyl)-2-(2,4-dichlorophenyl)-N-(1-piperidinyl)-1H-benzimidazole-5- carboxamide]. CB1, insulin, glucagon, glucokinase, and PDX-1 expression were determined by real-time RT-PCR.
CB1 receptor expression in pancreatic islets is up-regulated during fasting, and decreases in response to glucose intake or feeding. In cultured
islets, 16 mM glucose and analog reduced CB1 mRNA, as well as
anandamide in low-glucose media. Peptides expression increased in islets
+ anandamide at 5 mM glucose. Analog augmented PDX-1 and glucokinase mRNA at 16 mM glucose. Data suggest that endocannabinoids
have an important role, along with feeding state, in gene expression of
pancreatic islets.
Paper No.: 2473

METABOLIC SYNDROME PREVALENCE IN UNIVERSITY
XICO CITY
STUDENTS OF ME
Rafael Villalobos-Molina(1), JR Jimenez-Flores(1), AR Mendez-Cruz(1),
NY Rodrguez-Soriano(1), MI Mendoza-Ramos(1), M Murguia-Romero(1), SC Sigrist-Flores(1), M Rodriguez-Salazar(2), L Anguiano(2), E
Gonzalez-Dalhaus(2)
(1) Fes-Iztacala, U.N.A.M., Unidad de Biomedicina, Tlalnepantla, Mexico
(2) Coordinacion de Servicios Estudiantiles, U.A.C.M., Mexico
Metabolic syndrome is a pathological condition that clusters abdominal
waist index, overweight, dyslipidemia, hyperglucemia, insulin resistance,
hypertension, hyperuricemia, and is considered a previous step of type 2
diabetes development. We aimed to identify biomarkers for MS in
young, rst year students of Mexico city university. 592 students (68%
women, 32% men, mean 22 years of age) were evaluated for anthropometry, vital signs and lab blood measurements (conducted by CARPERMOR, an international certied clinical lab). They signed an informed
consent letter. Results shown that 55% of the students have at least 1
metabolic alteration, 15% showed insulin resistance and 15% MS. Other
dysfunctions were observed. In a medical interview each student was
informed of the outcome. We conclude that MS seems to start at early
ages in Mexican population. It will be interesting to work with younger
people in order to determine if MS associated pathologies are already
present.
Supported in part by grant PICDS08-69 from ICyT-GDF, Mexico.
(1) Pzer Inc, Department of Clinical Sciences, New York, NY, USA
(2) Universite Catholique de Louvain, Cliniques Universitaires St-Luc,
Brussels, Belgium
(3) Inserm, Centre dInvestigations Cliniques CIC Inserm CHU, Nancy
CIC-P 9501, Dommartin-les-Toul, France
Eplerenone is a selective inhibitor of the mineralcorticoid MR receptor.
In the EPHESUS trial in 6600 patients it signicantly improved all-cause
death, sudden cardiac death, and heart failure hospitalization in patients
with LVSD and CHF post AMI. In a subset of 476 patients, neuro-hormones (aldosterone, BNP, Nt-proBNP, Big-ET-1), inammatory markers
(Il-6, osteopontin, hs-CRP, cortisol) and collagen remodeling biomarkers
(PINP, PIIINP, ICTP, TIMP-1) were measured at baseline, 1, 3, 6 and 9
months, except BNP which was measured at baseline and 1 month.
Neuro-hormones and inammatory markers were analyzed using a
Mixed Model (SAS 9.1) with treatment (eplerenone vs placebo), biomarker values at baseline and each visit, age, gender and renal function as
xed effects: collagen biomarker changes from baseline were analyzed
using a mixed-effect model with change from baseline (dependent variable), treatment (xed effect) and patient (random effect) The magnitude
of baseline neuro-hormones was related to mortality and morbidity outcomes. Treatment with eplerenone resulted in a signicant (p < 0.01)
increase in serum aldosterone, and a signicant decrease in neuro-hormones (BNP, Nt-proBNP, Big-ET-1), inammatory markers (osteopontin,
IL-6), collagen synthesis (PINP, PIIINP) and degradation (ICTP) biomarkers while decreases in TNF-a, hs-CRP, cortisol and TIMP-1 were not
signicant. The magnitude of the changes in neuro-hormones was related
to the baseline values. In addition, reductions in neuro-hormones were
predictive of outcomes. Eplerenone signicantly reduced neuro-hormones, some inammatory biomarkers and collagen remodeling biomarkers. The reductions in neuro-hormones were predictive of mortality and
morbidity and may underlie some benecial actions of eplerenone.
Paper No.: 3123

PERIPHERAL BLOOD MONONUCLEAR CELL GENE
EXPRESSION PROFILES IDENTIFY SUBJECTS WITH MAJOR
DEPRESSIVE DISORDER (MDD) AMONG HEALTHY
CONTROLS
J Vistisen(2), J Tamm(1), R Artymyshyn(1), I Antonijevic(1), M Das(1),
A Abdourahman(1), Birgitte Sgaard(2)
(1) Lundbeck Research USA, Translational Research, Department of
Biological Research, Paramus, USA
(2) H. Lundbeck A/S Clinical Pharmacology & Translational Medicine,
Copenhagen, Denmark
Patients diagnosed with major depressive disorder (MDD) represent a
heterogeneous population with respect to underlying disease biology.
Segmentation based on readily accessible biomarkers may aid the development of more targeted and effective treatments. To dene disease-relevant biomarkers, we evaluated gene transcription patterns in peripheral
blood samples from depressed patients (enrolled in clinical trials for
depression) vs. healthy controls. A hypothesis-driven approach was used
to assess transcription of a limited number of genes associated with
diverse physiological processes, e.g. monoamine neurotransmission,
receptor signalling, inammation, transcription factors, and others. Blood
639
samples were collected in PaxGene tubes from 307 severely depressed,
174 mild/moderately depressed patients and 198 normal controls. RNA
was isolated and reverse transcribed, and transcription levels were
assessed using quantitative PCR. All data were normalized to 7 reference
(housekeeping) genes. Gene expression patterns for depressed vs. control
samples were then analyzed using several algorithms (Support Vector
Machine, Random Forest, and Relevance Vector Machines combined
with multi objective genetic optimization algorithms). No single gene
expression change was sufciently predictive; however there were multiple two-gene pairs whose expression patterns could correctly distinguish
a severely depressed patient from control with > 90% accuracy. An 11gene signature was identied that could distinguish moderately
depressed patients from control with > 90% accuracy. Our data show that
changes in blood transcription patterns of a small hypothesis driven gene
set can be diagnostic of depression. These ndings could be used to
guide treatment strategy and also to identify novel biological networks /
targets for disease modication.
Paper No.: 3215

ONCOLOGY CLINICAL TRIALS IN CROATIA
limited efcacy and severe side effect limitations. Interestingly, the glial
cell line derived neurotrophic factors GDNF and Artemin (ARTN) seem
to normalize abnormal pain behaviour. On the other hand, others neurotrophins, like NGF, acts as pain mediator and its administration results in
pronounced hyperalgesia. Our results demonstrated that NGF was statistically increased by CCI in the ipsilateral dorsal root ganglia (DRG)
(140%), in the spinal cord (350%) and in the periaqueductal grey matter
(PAG) (180%). Conversely, ARTN was decreased bilaterally in DRG
(50% ipsi, 30% controlateral), spinal cord (40%) and PAG (30%). GDNF
levels decreased in ipsilateral DRG (30%), whereas the nervous damage
did not modify its protein expression in the CNS. Chronic treatment with
the antihyperalgesic compound acetyl-L-carnitine (ALCAR; 100 mgkg-1
i.p. twice daily for 14 days) was able to prevent the increase of NGF levels. In conditions of pain relief, ALCAR normalized peripheral and central alterations of GDNF and ARTN levels. Characteristically, sham
animals that underwent the same ALCAR treatment, showed increased
levels of ARTN both in the DRG (160%) and in the spinal cord (240%).
These data underline the involvement of neurotrophins on painful condition, moreover this research point out ARTN like a possible player in the
mechanism of the antihyperalgesic effect of ALCAR. Taken together, the
results suggest that neurotrophic modulation might be a new pharmacological tool to soothe chronic pain.
Dinko Vitezic(1,2,3), M Lovrek(4), S omic(4)

(1) University of Rijeka School of Medicine, Rijeka, Croatia,
(2) University Hospital Centre Rijeka, Rijeka, Croatia
(3) Central Ethics Committee, Croatia
The Croatian system of the ethical and scientic review of clinical trials
is carried out through a centralized procedure (Central Ethics Committee,
CEC). The aim of this study is to present data concerning clinical trials
in the eld of oncology. The analysis was performed using CEC data in
relation to clinical trials assessment from January 2007 to the end of
2009. During the investigated period CEC reviewed 291 clinical and
non-interventional trials, out of which 57 in the eld of oncology. Clinical trial applications were retrospectively analyzed according to the type
of trial-site, research area, and the type of trial. The highest number of
approved oncology trial sites was at the university hospital centres. More
than half of oncology trials (n = 35) assessed were phase III trials, followed by phase II trials (n = 12). Most trials were conducted in the eld
of breast cancer (n = 16) followed by malignant neoplasms of lymphoid
and haematopoietic tissue. In most of phase III trials, investigational
medicinal product was investigated as add-on therapy to reference regimen. The proportion of clinical trials in the eld of oncology proves to
be the most challenging eld of medicine today. Further, a centralized
system of clinical trials review is very efcient and offers an identical
approach to all clinical trials during and after the assessment process.
Paper No.: 1915

ANALGESICS
ACETYL-L-CARNITINE INCREASES ARTEMIN LEVEL AND
PREVENTS NEUROTROPHIC FACTOR ALTERATIONS IN
NEUROPATHIC PAIN STATE
Elisa Vivoli, L Di Cesare Mannelli, A Salvicchi, A Bartolini, C Ghelardini,
Paper No.: 1196

ANTI-PARKINSONIAN EFFECTS OF MDMA IN THE RAT:
CONTRIBUTIONS OF 5-HT1A-RECEPTORS AND SEROTONIN
TRANSPORTERS AND ROLE OF CHIRALITY
Andreas von Ameln-Mayerhofer(1), M Hupkes(0), C Riegel(1), S SeegerArmbruster(1), NY Lettfuss(1)
(1) University Tubingen, Department of Neuropharmacology, Tubingen,
Germany
(2) University Tubingen, Department of Animal Physiology, Tubingen,
Germany
In the present study, we investigated the antiparkinsonian effect of
MDMA in both haloperidol induced catalepsy (HIC) and unilateral 6OHDA lesion models of PD. In the HIC experiment, we investigated the
role of 5-HT1A-receptors by means of the selective antagonist WAY
100635 (WAY) and/or the role of serotonin transporters (SERT) by
means of the selective serotonin reuptake inhibitor citalopram (CIT).
MDMA did very potently antagonize the HIC. Both WAY and CIT did
reduce the therapeutic effect of MDMA dose dependently, indicating an
important role of two pharmacological targets of MDMA: 5-HT1A-receptors and SERT. The aim of the 6-OHDA-experiment using lesioned animals was the investigation of MDMA enantiomers. In these animals, SMDMA dose dependently reduced motor impairment of the contralateral
forepaw, indicating a potent antiparkinsonian effect of MDMA also in an
animal model with higher face validity. Moreover, in all treated animals,
MDMA did not lead to dopaminergic or serotonergic neurotoxicity over
at least 21 days of consecutive treatment with the therapeutic dose. In
conclusion, MDMA exerts its anticataleptic effects by binding at both
the 5-HT1A-receptors and SERT. S-MDMA also shows potent antiparkinsonian effect in unilaterally DA-depleted animals. Thus, MDMA does
represent a valuable tool for the development of novel antiparkinsonian
agents this strategy should apparently aim at combined targeting of
both 5-HT1A-receptors and SERT.
University of Florence, Department of Pharmacology, Florence, Italy

Neuropathic pain is a personally devastating and costly condition affecting 3-8% of the population. Current pharmacological treatments have
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Paper No.: 1150
DETERMINATION OF PSEUDOCHOLINESTERASE SERUM
ACTIVITY AMONG AGRINION PESTICIDE APPLICATORS
PRE- AND POST-EXPOSED TO ORGANOPHOSPHATES (FENTHION AND DIMETHOATE)
G Vrioni(1), H Souki(2), K Kasiotis(3), M Katramadou(2), Haris Carageorgiou(2)
(1) National & Kapodistrian University of Athens, Medical School,
Department of Microbiology, Cology, Athens, Greece
(2) National & Kapodistrian University of Athens, Medical School,
Department of Pharmacology, Cology, Athens, Greece
(3) Benaki Phytopathological Institute, Laboratory of Pesticides Toxicology, Athens
Organophosphate pesticides are used in agriculture in order to raise crop
production. The aim of this study was to investigate the biological effects
of two organophosphates, fenthion and dimethoate, among 270 farm
workers in the area of Agrinion (Western Greece) by measuring the
serum pseudocholinesterase activity during the years 2004-2006. The
pseudocholinesterase activity was photometrically analyzed before and
after exposing to organophosphates. According to our results: a) the median pseudocholinesterase activity was reduced in the spraying period in
comparison with the non spraying period (7604 U/L and 9557 U/L consequently, P < 0.001), b) a statistically signicant difference in the median pseudocholinesterase activity was also observed between the two
organophosphates (fenthion applicators -26.87%, dimethoate applicators
-15.95%, P < 0.001), c) the duration of organophosphates spraying inuenced the mean pseudocholinesterase activity and it was -9.83% after 1
day, -23.81% after 2-3 days and -25.26% after 4-7 days of spraying, d)
in occupational applicators reduced levels of the mean value of pseudocholinesterase activity was observed in exposure periods (5446 U/L) and
in non exposure periods (6950 U/L), as well, e) the extend of using protective equipment inuenced the mean levels of pseudocholinesterase
activity: -23.9% without any protection, -15% with partial protection and
-7.4% in those applicators with full protective equipment. In conclusion,
the mean value of serum pseudocholinesterase activity was reduced after
the exposure in the two organophosphates and this reduction of the pesticides applicators depends on the particular organophosphate, the duration
of the exposure, and the use or not of personal protective equipment.
Paper No.: 1329

ANALGESICS
STRUCTURE-ACTIVITY RELATIONSHIP STUDY OF NOVEL
FENTANYL ANALOGUES
S Vuckovic(1), Z Nesic(1), K Savic Vujovic(1), M Milovanovic(2), Milica
Prostran-Veselinovic(1)
(1) University of Belgrade School of Medicine, Department of Pharmacology, Clinical Pharmacology & Toxicology, Belgrade, Republic of Serbia
(2) University of Belgrade School of Veterinary Medicine, Belgrade,
Republic of Serbia
Fentanyl is the prototype of the 4-anilidopiperidine class of synthetic
opioidanalgesics. From the aspect of structure-activity relationship (SAR)
studies,it was interesting to introduce methyl, ethyl, propyl, iso-propyl,
butyl,benzyl, phenethyl, as well as carbomethoxy group in the position 3
of thepiperidine ring and test the newly synthetized fentanyl analogues
(all in theracemic form) for the antinociceptive activity by using tailimmersion test inrats. It has been revealed that the presence of an alkyl
group substituent inposition 3 of the piperidine ring generally decreases
or completely inhibits theanalgesic activity compared to fentanyl. The
exceptions are the (+/))cis-3-Mefentanyl (about 8 x fentanyl) and (+/

))cis-3-Et fentanyl (about 1.5 x fentanyl). With increasing voluminosity
of the alkyl group, the potencydecreases rapidly. The relative cis/trans
stereochemistry is important since thecis isomers are 1.5 - V6 times more
active than the trans isomers. Also, it wasfound that the introduction of a
carbomethoxy group in position 3 of thepiperidine ring of fentanyl
decreased antinociceptive activity by a factor of 2and 10 in (+/))cis and
(+/))trans 3-carbomethoxy fentanyl, respectively.(+/))cis 3-carbomethoxy fentanyl seems to be equipotent to the (+/))cis 3-propylfentanyl.
The observed similarity in antinociceptive potency could result fromthe
similar voluminosity of the 3-carbomethoxy and 3-propyl group. Inconclusion, it seems that a steric factor (voluminosity of the group and thecis/trans isomerism) has a predominant role in the antinociceptive
potency of3-substituted fentanyl analogues, while the nature of the substituent isprobably irrelevant.
Paper No.: 602

ANALGESICS
INFLUENCE OF BILE ACID DERIVATES ON MORPHINE
ANALGESIC EFFECT IN MICE
Sasa Vukmirovic, V Vasovic, D Lendak, M Mikov, M Vukmirovic Papuga,
N Stilinovic
Novi Sad University Medical Faculty, Department of Pharmacology,
Toxicology & Clinical Pharmacology, Novi Sad, Republic of Serbia
As well as other surfactants bile acids and their salts can be used for
improvement of xenobiotics absorption. There is data on the potential of
bile acids to aid nasal, intestinal, buccal, transdermal, ocular, rectal and
pulmonary absorption. Their salts also improve drug permeability through
blood-brain barrier. Inuence of two newly synthesized bile acids derivates, namely sodium salt of monoketocholic acid MKH-Na and methyl
ester of monoketocholic acid MKH-Me on morphine (2mg/kg intramuscular administration) analgesic effect was examined in this research. Analgesic effect was measured by antinociceptive hot plate method.
Interaction was estimated by detection of changes in analgesic effect of
morphine combined with bile acids (bile acids were subcutaneously
administrated in a dose of 4 mg/kg 20 minutes before morphine administration) compared to analgesic effect of the same dose of morphine given
alone. Analgesic effect was measured on 10 minute intervals for 90 minutes from morphine administration. White male mice (bw 20-30g) were
used in this study. Liposoluble methyl ester of monoketocholic acid did
not show interaction with morphine, what was in contrary to our expectations since morphine is a liposoluble drug. However hidrosoluble sodium
salt of monoketocholic acid increased the analgesic effect of morphine in
rst 20 minutes from morphine administration. According to the time
point when interaction reached statistically signicant difference it can be
presumed that after intramuscular administration of morphine, sodium salt
of monoketocholic acid increases morphine absorption and transport to
brain and in that way increasing morphine analgesic effect.
Paper No.: 1528

INFLUENCE OF CLONIDINE WITHIN ANALGESIC
COMBINATIONS ADMINISTERED EPIDURALY ON
POSTOPERATIVE ANALGESIA QUALITY
Jelena Vukovic(1), L Grbovic(2), Z Milan(3), V Dragojevic-Simic(1),
D Jandric(1), A Kovacevic(1)
(1) Military Medical Academy, Center for Clinical Pharmacology,
(2) University of Belgrade, Faculty of Medicine, Belgrade, Republic of Serbia
(3) St Jamess University Hospital, Leeds, UK
641
The elimination of pain is an intermission of undesirable mechanisms
which affect patients recovery. Clonidine administered epiduraly in combination with local anesthetic or opioid analgesic augment and prolong
their effect, ameliorating quality of analgesia. Study determineted effectiveness of clonidine within analgesic combinations for epidural analgesia in the immediate postoperative period of large vascular operations.
Sixty patients were divided at random into three groups, I group were
receiving epidural analgesic combination bupivacaine 0.125% and morphine 0.1mg/ml, II group bupivacaine 0.125% and clonidine 5`g/ml and
III group morphine 0.1mg/ml and clonidine 5`g/ml. When pain intensity
in motionless state reached 3rd grade, assessed by 010 grade scale,
patients received 5ml bolus, followed by continuous infusion of initial
rate 5ml/h, adjusted during the 24 hours period according to each
patients needs. Additional analgesic was morphine 2mg i.v. The quality
of epidural analgesia was satisfactory for most of the patients of all
groups all the time, and requirement for additional analgesic was similar.
Analgesic effect appared rst in II group (10.50 2.76min), with statisticaly signicant difference related to III group (12.35 3.73min). At the
time of maximal effect, statistically signicant difference wasnt present.
At the end of 24 hours period, pain was the lowest in III group (0.30
0.66 grade), but less than 1 grade in all groups. Duration of analgesic
effect was the longest in III group (12,90 3,08h). Clonidine showed
sinergistic effect with bupivacaine and morphine, analgesia level was
adequate and comparable with those achieved with standard combination bupivacaine-morphine. Continuous monitoring of patients was obligate.
Paper No.: 2697

DISEASES
PROINFLAMMATORY AND CATABOLIC ROLE OF
ADIPOKINES LEPTIN AND ADIPONECTIN IN ARTHRITIS
Paper No.: 1786

CA2 + -SIGNALLING OF A2B-ADRENOCEPTORS IN A7R5
VASCULAR SMOOTH MUSCLE CELLS
Anne Vuorenpaa, A Huhtinen, M Scheinin
University of Turku, Department of Pharmacology, Drug Development
and Therapeutics, Turku, Finland
Vascular a2-adrenoceptors (a2-ARs) play important roles in vascular
physiology and pathophysiology. Based on studies on KO mice lacking
subtypes of a2-ARs (a2A, a2B, a2C), it has been demonstrated that a2BARs on vascular smooth muscle (VSM) cells are mainly responsible for
the initial hypertensive phase of the biphasic blood pressure response
elicited by intravascularly administered a2-AR agonists, whereas central
a2A-ARs mainly mediate the hypotensive phase. Several studies performed with mammalian blood vessels suggest that a2-ARs constrict
VSM cells by increasing intracellular calcium levels [Ca2+]i. In order to
investigate the effects of a2B-AR activation on [Ca2+]i in VSM cells we
generated and characterized a VSM cell line stably expressing the human
a2B-AR at a density of ~1,5 pmol receptor/mg membrane protein (Huhtinen & Scheinin, Eur J Pharmacol. 2008;587:48-56). Fluorescence
microscopy was used to monitor [Ca2+]i responses in A7r5-a2B-cells.
Activation of a2B-ARs evoked robust [Ca2+]i increases: DF340/F380
(mean SEM): with 10 nM dexmedetomidine 0,462 0,064; with 100
nM oxymetazoline 0,303 0,03; with 100 nM brimonidine 0,22 0,11.
Agonist-induced calcium responses were absent in wild-type A7r5 cells.
Removal of extracellular Ca2 + (calcium-free buffer), inhibition on L-type
Ca2 + -channels by nimodipine (1 lM) and depletion of intracellular Ca2
+
stores by thapsigargine (1 lM) abolished the [Ca2+]i responses to dexmedetomidine. The results indicated that a2B-AR activation leads to
mobilization of intracellular Ca2 + stores and Ca2 + entry through L-type
Ca2 + channels in A7r5-a2B VSM cells.
Katriina Vuolteenaho(1), A Koskinen(1), S Juslin(1), R Nieminen(1),

T Moilanen(2), E Moilanen(1)
(1) University of Tampere Medical School and Tampere University Hospital, Immunopharmacology Research Group, Tampere, Finland
(2) Coxa, Hospital for Joint Replacement, Tampere, Finland
Adipokines leptin and adiponectin regulate energy metabolism and apetite. Interestingly, they have recently been related also to the regulation
of inammatory responses. In arthritis, proteolytic degradation of cartilage is mediated by matrix metalloproteinases (MMPs) and IL-6 contributes to inammation and joint damage. In the present study, the
association of leptin and adiponectin to MMPs and IL-6 in synovial uid
from osteoarthritis (OA) patients was assessed, and further, the effects of
these adipokines on human OA cartilage were investigated. Synovial
uid samples were obtained from 100 OA patients (62 females, BMI
30.8 0.6kg/m2, age 70.0 1.0 years; meanSEM). Cartilage samples
for tissue culture were collected from OA patients undergoing total knee
replacement surgery. Synovial uid leptin correlated positively with
MMP-1 (r = 0.41, p < 0.001) and MMP-3 (r = 0.51, p < 0.001) as well
as with IL-6 (r = 0.33, p < 0.002). There was also an association
between adiponectin and IL-6 (r = 0.39, p < 0.001), MMP-1 (r = 0.31, p
< 0.004) and MMP-3 (r = 0.27, p < 0.001). In cartilage culture, leptin
and adiponectin enhanced production of MMPs. Leptin-induced MMP-1,
-3 and -13 production were dependent on activation of PKC, JNK and
NF-jB pathways. The p38 pathway was involved in the adiponectin
effect on MMP-3 production. Both adiponectin and leptin induced IL-6
production. In addition, both adiponectin and leptin enhanced also
expression / production of inammatory factors, inducible nitric oxide
synthase, prostaglandin H synthase-2 and IL-6, by a MAP kinase depedent manner. The ndings support the idea of leptin and adiponectin as
catabolic factors in the pathogenesis of OA, and as possible link between
obesity and osteoarthritis.
Paper No.: 1064

PRESCRIPTIONS ANALYSIS OF ANALGESIC MEDICINES BY
PRIMARY CARE PHYSICIAN
Nidaa Wadi, M Bhupathyraaj, KAI Harthy
Oman Medical College, Department of Pharmacy, Muscat, Oman
Introduction: The need for achieving quality care system is not because
of nancial responses but appropriate use of medicines. The main objective of this study is to analysis the quality of prescriptions in term of adequacy and clarity of information continued. Method: The methodology
used was adopted from the World Health Organization study protocol.
The study was conducted in Oman, a cross-section survey of all prescriptions received over a month in January 2009. A 206 photocopied prescriptions dispensed in two centres were collected. Result: The tables/
capsules dosage form of analgesic are widely prescribed by prescriber as
the mean 64% of both centres in comparison with other dosage form. As
prescribing information written the prescriptions also evaluated, for the
dose, dosage form, duration shows a satisfactory result which their mean
are 99.5%,98.7% & 85.6% respectively, but half of the prescriptions
mean 47% lack of information about analgesic frequency. Conclusion:
The overall layout of all prescriptions medicines in the health centres
was quite acceptable. There is a need to regulate the layout of prescription and include all the important information. The effective intervention
need a collaborative work to new physicians to improve prescription
behaviour and skill. Implement a rational use guidelines will reducing
misuse, overuse, and underuse of medicines. A political will form decision makers so that these interventions are likely to be implemented.
642
References:YounisMZ.et.alPharmacoecon.OutcomesRes.2009;(3):24350
(PubMed);Sarkar PK.A.IndianJMedEthics2004;1:112;Lindnet.al.JClinPsychopharmaco,1999;19:13240(PubMed); WHO1994.
Paper No.: 1002

HEALTH AND DISEASE
PEROXYNITRITE STIMULATES PULMONARY ARTERY
ENDOTHELIAL CELL PROLIFERATION AND MAY BE
GENERATED IN PULMONARY HYPERTENSION
Roger Wadsworth(1), EO Agbani(1), P Milliken(1), AT Demiryurek(2),
P Coats(1), A Mills(1), A McPhaden(3), T Hoehn(4)
(1) University of Strathclyde, Institute of Pharmacy and Biomedical Sciences, Glasgow, UK
(2) Glasgow Royal Inrmary, Glasgow, UK
(3) Heinrich-Heine-University, Dusseldorf, Germany
Pulmonary hypertension is associated with oxidative stress, endotheliumdysfunction, proliferation of endothelial cells and remodelling of pulmonaryarteries with lumen narrowing. A history of peroxynitrite
exposure was revealedby detection of nitrotyrosine in endothelium and
smooth muscle of pulmonaryarteries of infants with pulmonary hypertension. Nitrotyrosine was also found inthe endothelium and smooth muscle
of pulmonary arteries of rats withexperimental pulmonary hypertension,
correlating with development of rightventricular hypertrophy. Lung homogenates from rats with pulmonary hypertensionhad enhanced generation of superoxide, and antioxidant therapy reduced pulmonaryartery
pressure. In cultured bovine pulmonary artery endothelial cells,peroxynitrite 0.2lM stimulated cell proliferation which was inhibited by theperoxynitrite scavenger ebselen 5lM. Endothelial cell proliferation was
alsostimulated by the peroxynitrite donor SIN-1 2lM. Peroxynitrite
stronglyincreased ERK phosphorylation peaking at 30 min. Phosphoprylation of ERK andcell proliferation were prevented by the MEK inhibitor
U0126 5lM, Raf-1 kinaseinhibitor 10lM and by the ras inhibitor FPTIII
25lM. Peroxynitrite-induced cellproliferation and ERK phosphorylation
was prevented by the PDGF and EGF receptorblockers AG-1296, AG1478 and imatinib. In pulmonary artery endothelial cells, lowering oxygen concentration to 5% stimulated cell proliferation and increasednitrotyrosine in several target proteins. It is concluded that
pulmonaryhypertension is associated with peroxynitrite formation, and
that peroxynitriteis capable of stimulating proliferation of pulmonary
artery endothelial cellsvia Ras, Raf, MEK and ERK. Peroxynitrite may
act via growth factor receptors.
Paper No.: 880

HEALTH AND DISEASE
EDHF-MEDIATED RESPONSES IN THE RENAL
CIRCULATION OF DOUBLE TRANSGENIC HYPERTENSIVE
MICE DEVELOPING RENAL FAILURE
Ludovic Waeckel(1), F Bertin(1), N Clavreul(1), R Kohler(2),
P Sansilvestri-Morel(1), S Simonet(1), C Vayssettes-Courchay(1),
H Wulff(3), TJ VerbeurenN(1), M Feletou(1)
(1) Institut de Recherches Servier, Suresnes, France
(2) University of South Danemark, Institute Medical Biology, Physiology
and Pharmacology, Odense, Denmark
(3) University of California, Department of Pharmacology, Davis, CA,
USA
(L-NAME: 100 mg/kg/d for 4 weeks; ARSL) they develop malignant

hypertension and renal failure. The purpose of the present work was to
assess renal endothelial function by measuring changes in perfusion pressure in isolated perfused kidneys of female AR, ARSL, and the corresponding wild type mice under control (C57BL6) or the same salt and
L-NAME regimen (C57BLSL). The salt diet and L-NAME treatment
produced a small but signicant increase in blood pressure in C57BLSL
and a massive increase in ARSL. Only the latter developed renal failure
evidenced by increased microalbuminuria, glomerulosclerosis, tubulopathy and arteriolar wall hypertrophy associated with a marked increase in
brosis marker gene expression. In isolated perfused kidneys, methacholine induced similar vasodilatations in the four groups of mice that were
maintained in the presence of indomethacin and L-nitro-arginine (L-NA).
NS309 and SKA-31, two activators of endothelial calcium-activated
potassium channels of small (KCa2.3 or SKCa) and intermediate conductance and (KCa3.1 or IKCa), induced long-lasting vasodilatations that
were signicantly more sustained in AR and ARSL kidneys than in the
other groups. The combination of apamin plus TRAM-34, blockers of
KCa2.3 and KCa3.1, respectively, abolished the L-NA- and indomethacinresistant vasodilatations to methacholine, NS309 and SKA-31. These
results indicate that EDHF-mediated responses play a preponderant role
in the murine kidneys and that these responses are maintained and even
enhanced in mice with an exacerbated renin-angiotensin system.
Paper No.: 881

HEALTH AND DISEASE
ENDOTHELIAL DYSFUNCTION IN THE ISOLATED AORTA
OF DOUBLE TRANSGENIC HYPERTENSIVE MICE
DEVELOPING AORTIC ANEURYSM
Ludovic Waeckel, N Clavreul, P Sansilvestri-Morel, S Simonet,
C Vayssettes-Courchay, TJ Verbeuren, M Feletou
Institut de Recherches Servier, Suresnes, France
Double transgenic mice expressing both the human angiotensinogen and
renin genes (AR) are moderately hypertensive. However, when submitted
to a high-salt diet (8%) plus a N-w-nitro-L-arginine-methyl-ester treatment (L-NAME: 100 mg/kg/d for 4 weeks; ARSL) they develop malignant hypertension. The purpose of this work was to assess aortic
morphological changes and the endothelial function by recording
changes in isometric tension in isolated aortic rings of female AR,
ARSL, and the corresponding wild type mice under control (C57BL6) or
the same salt and L-NAME regimen (C57BLSL). The salt diet and LNAME treatment produced a small but signicant increase in blood pressure in C57BLSL and a large increase in ARSL. The latter exhibited
ventricular hypertrophy and enhanced thoracic aortic weight. Half of
ARSL developed suprarenal abdominal aortic aneurysms, which were
associated to a higher systolic blood pressure. A fth of these were grade
IV aneurysm (Daugherty scale, Br J Pharmacol, 2001), characterized by
extracellular matrix accumulation, elastin laminae disruption, aortic dissection and inammatory cell inltration. When compared to other
groups, receptor-independent contractions to KCL were signicantly
smaller in ARSL aortic rings with aneurysm. Contractile responses to
norepinephrine and U 46619 were similar in the four groups of mice.
Acetylcholine- and sodium nitroprusside-induced relaxations were significantly impaired in ARSL aorta. Additionally, acetylcholine-induced
endothelium-dependent contractions were enhanced in ARSL aorta.
These results indicate that in aorta of ARSL both endothelial (nitric oxide
and EDCF) and smooth muscle dysfunctions are observed. These dysfunctions precede the formation of aortic aneurysms.
Double transgenic mice expressing both the human angiotensinogen and

renin genes (AR) are moderately hypertensive. However, when receiving
a high-salt diet (8%) plus a N-w-nitro-L-arginine-methyl-ester treatment
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Paper No.: 1305
HEALTH AND DISEASE
HISTIDINE-RICH GLYCOPROTEIN INHIBITS THE
ANGIOGENIC RESPONSE INDUCED BY THE COMBINATION
OF HIGH MOBILITY GROUP BOX 1 AND HEPARIN IN
MATRIGEL PLUG ASSAY
dopaminergic neurite outgrowth. Conditioned medium from striatal cells

alone had no effect on the dopaminergic neurite outgrowth, suggesting
that diffusible factor had little effect on the neurite outgrowth in our system. RGDS, an ihibitor of integrin, suppressed dopaminergic projection
to the striatal cell region. These results suggest that striatal neurons promote dopaminergic neurite outgrowth through cell adhesion molecules
such as integrin.
Hidenori Wake(1), S Mori(2), HK Takahashi(1), K Liu(1),

M Nishibori(1)
(1) Okayama University Graduate School of Medicine, Dentistry & Pharmaceutical Sciences, Department of Pharmacology, Okayama, Japan
(2) Shujitsu University School of Pharmacy, Japan
Angiogenesis consists of complex processes mediated by several factors
and is associated with inammation and tumor growth. High mobility
group box 1 (HMGB1) is released from necrotic cells as well as macrophages and exerts proinammatory roles. In inammatory site, extravasation of plasma protein due to vascular hyperpermeability occurs, so that
proinammatory cytokine like HMGB1 may interact with plasma factors
like histidine-rich glycoprotein (HRG). Recently, we reported that
HMGB1 in complex with heparin induces angiogenesis in matrigel plug
assay. In the present study, we examined the effect of HRG, an antiangiogenic plasma protein with constant high levels, on heparin-dependent HMGB1-induced angiogenesis. HRG completely inhibited the
angiogenic response induced by the combination of HMGB1 and heparin, with concomitant decrease in tumor necrosis factor-a and vascular
endothelial growth factor-a 120 expression. HRG inhibited the diffusion
of a complex of HMGB1 with heparin from matrigel into surrounding
tissue. HRG also competed with HMGB1 for heparin binding in vitro.
Taken together, these results suggested that HRG in plasma might control
the angiogenic activity of HMGB1 by regulating the interaction with
heparin or heparan sulfate. HRG may be a potential drug for the treatment of angiogenesis-related disease conditions including tumor growth.
Paper No.: 1320

RECONSTRUCTION OF THE NIGROSTRIATAL
PROJECTIONS IN DISSOCIATED PRIMARY CULTURES: A
ROLE OF STRIATAL CELLS IN DOPAMINERGIC NEURITE
OUTGROWTH
Seiko Wakita(1), Y Izumi(1), T Kume(1), H Sawada(2), A Akaike(1)
Department of Pharmacology, Kyoto, Japan
(2) Utano National Hospital, Department of Neurology and Clinical
Research Center,Center for Neurological Diseases, Kyoto, Japan
The main pathological feature of Parkinson disease is a selective degeneration of the nigrostriatal pathway. Regeneration of the neuroprojection
is a potential therapeutic approach, but the mechanisms underlying the
dopaminergic projections are not well understood due to the absence of a
suitable system for studying neural projections. Our aim is to reconstruct
the nigrostriatal projections of dopaminergic neurons using primary dissociated neruons and to investigate the effects of striatal cells on dopaminergic neurite outgrowth. Mecencephalic cells, striatal cells and spinal
cells were dissected from rat embryos, then we formed a mecencephalic
cell region next to a striatal or spinal cell region on a plastic coverslips.
After paired-culturing for 11 days, dopaminergic neurons in the mesencephalic cell region extended their neurites to the strital cell region, but not
to the spinal cell region. The dopaminergic projecions were increased in
a striatal cell density-dependent manner. Immunouorescent microscopy
showed that dopaminergic axons elongated along striatal neurons. In
addition, astrocytes derived from the striatum were cultured next to the
mesencephalic region, but striatal astrocytes did not facilitate dopaminergic neurite outgrowth. We next investigated the roles of striatal cells on
Paper No.: 2197

DISCOVERY
HIGHLY SENSITIVE AND RAPID ASSESSMENT OF
P-GLYCOPROTEIN ACTIVITY IN CACO-2 CELLS
Hirkazu Wakuda(1), Y Tada(1), N Nejime(1), S Kagota(1),
K Nakamura(1), K Umegaki(2), S Yamada(3), K Shinozuka(1)
(1) Mukogawa Womens University, Department of Pharmacology,
Nishinomiya, Hyogo, Japan
(2) Center of Information, National Institute of Health and Nutrition,
Japan
(3) University of Shizuoka School of Pharmaceutical Sciences, Department of Pharmacokinetics and Pharmacodynamics and Global Center of
Excellence (COE), Japan
Purpose: The aim of the present study is to establish a highly sensitive
and rapid method for evaluating P-glycoprotein activity in Caco-2 cells.
Methods: Using a time-lapse confocal laser-scanning microscopy, we
measured the change in uorescence of residual rhodamine 123 in Caco2 cells. Horizontal uorescence images of rhodamine123 in both the apical and central sites of these cells were captured for 90 min. Results:
Continuous and signicant quenching of the uorescent intensity of rhodamine 123 in both the apical and central sites of the Caco-2 cells
occurred over time during the measurement. However, the mean rate of
rhodamine 123 quenching in the apical sites of Caco-2 cells was signicantly larger (P < 0.05) than that of the central sites, 3.4 +/- 1.1 and -0.7
+/- 1.1%/min, respectively. The decreases in rhodamine 123 intensity in
both sites were abolished in the presence of 0.01 mM digoxin. Conclusions: The residual rhodamine 123 assay in the apical site of Caco-2 cells
as imaged by confocal laser scanning microscopy is a valuable screening
tool for studying both inhibition and induction of P-glycoprotein activity
and expression. This method may be useful for predicting P-glycoprotein
mediated alterations in the intestinal absorption of drugs.
Paper No.: 2489

DEVELOPMENT OF AN IN VITRO ASSAY TO EVALUATE
SUNSCREEN EFFICACY
Nicole Walter, C Surber, G Kunze
Spirig Pharma Ltd., Department of Research & Development, Egerkingen, Switzerland
There has been a recent increase in consumer awareness for protection
against sunlight. Exposure to sun and UV-irradiation initiates a cascade
of cellular pathways leading to cell damage. Current legislation and
guidelines restrict the use of in vivo assays and have driven the need for
a reliable, reproducible and predictive in vitro test system to quantify the
protection efcacy of sunscreens. The goal of our research is to develop
a test system with a sufcient sensitivity to detect the inuence of a formulation on different cellular pathways involved in UV-damage. We
have used human normal keratinocytes and HaCat cells as test models.
They were irradiated with different doses of UV and released cytokines
(e.g. IL-1a) were quantied using an ELISA assay. The chemical ben-
644
zalkoniumchloride served as positive control. There was a dose dependent release, evident in both cell types tested. Interleukin 1a levels were
approximately four times lower (80pg/ml) than those after benzalkoniumchloride treatment (400pg/ml). In conclusion, we have shown that cytokine release from both keratinocyte models is UV-triggered in a dose
dependent manner. Although we could demonstrate the release of cytokines, the level was lower than for the positive control. In order to
increase the sensitivity of the assay so that the effect of the sunscreen formulations can be more accurately assessed, future research will involve
other markers of cell injury and assays (e.g. cyclobutane pyrimidine
dimers).
Paper No.: 665

ASSOCIATION BETWEEN THE NEUREGULIN 1 GENE
POLYMORPHISMS WITH SCHIZOPHRENIA IN THREE
ETHNIC GROUPS IN MALAYSIAN POPULATION
Ching Lee Wan, LH Lian, NZ Zainal, Z Mohamed
Neuregulin 1 (NRG1) has been identied as a possible susceptible gene
that can lead to schizophrenia. A number of studies have replicated an
association in various populations, i.e Scottish (Stefansson et al, 2003;
72: 83-87), Japanese (Fukui et al, 2006; 396: 117-120) and Chinese
(Zhao et al, 2004; 41: 31-34). Thus, the aim of this study was to correlate
four single nucleotide polymorphisms (SNPs) of SNP8NRG241930,
SNP8NRG243177, SNP8NRG221533 and rs1081062 in NRG1 with
schizophrenia in the three ethnic groups of in the Malaysian population.
A total of 79 Malay, 154 Chinese, and 84 Indian schizophrenic patients,
as well as 160 Malay, 164 Chinese, 93 Indian healthy controls were
genotyped in this study. In our results, we were unable to demonstrate
any
signicant
association
between
SNP8NRG241930,
SNP8NRG243177 and rs1081062 with schizophrenia in all three ethnic
groups for genotypic frequencies p > 0.05. However, we found strong
evidence for the association of SNP8NRG221533 with schizophrenia in
the Chinese and Indians, where p < 0.0001. Haplotype analysis based on
these four SNPs have shown an signicant association with schizophrenia in the Chinese and Indian ethnic groups (p < 0.0001). The preliminary results suggest that the single markers of SNP8NRG221533 could be
a susceptibility SNP to schizophrenia disease in Malays, Chinese and
Indians, wheares SNP8NRG241930 and SNP8NRG243177 of the NRG1
gene, are not likely to be so. As a conclusion, data supports the possibility of NRG1 gene as a susceptibility gene for schizophrenia in the
Malaysian population.
Paper No.: 592

A VALIDATED STEREOSELECTIVE ASSAY FOR
3,4-METHYLENEDIOXYMETHAMPHETAMINE IN HUMAN
URINE SAMPLES
Wan Raihana Wan Aasim(1), SH Gan(1), SC Tan(2)
(1) University Sains Malaysia School of Medical Sciences, Department
of Pharmacology, Kubang Kerian, Kelantan, Malaysia
(2) University Sains Malaysia, Veterinary Forensic Laboratory, USAINS
Holding Sdn. Bhd., Pulau Pinang, Malaysia
3,4-methylenedioxymethamphetamine (MDMA) is a commonly abused
chiral amine drug. As MDMA is stereoselectively metabolised in vivo by
cytochrome P450 enzymes, a stereospecic assay was developed and
validated to enable quantitative analysis of MDMA and its metabolites
3,4-hydroxymethamphetamine
(HHMA),
3,4-hydroxyamphetamine
(HHA), 4-hydroxy-3-methoxymethamphetamine (HMMA), 4-hydroxy-3methoxyamphetamine (HMA), 3,4-methylenedioxyamphetamine (MDA),
amphetamine (AM) and methamphetamine (MA) in human urine samples. In the developed method, sample preparation is simplied by carrying out simultaneous extraction and derivatization using a solution
containing 3 mL hexane/ethyl acetate (1:2 v/v) + 0.15% triethylamine as
the extraction solvent and a chiral derivatization reagent (R)-a-methoxya-(triuoromethyl)phenylacetyl chloride (R-MTP) which is added to 2
mL urine samples. The sample is mixed, centrifuged and the organic
layer is removed and dried. The residue is subjected to a second derivatization with N-methyl-N-trimethylsilyltriuoroacetamide (MSTFA) to
enable analysis of metabolites possessing hydroxyl groups. Analysis was
by gas chromatography-mass spectrometry. The assay was validated
using a 4-day protocol. Under the conditions used, the assay exhibited
excellent linearity (R2 > =0.999) from 10 - 500 ug/L per enantiomer for
MDMA, HHMA, HMMA, AM and MA and 2 - 100 ug/L per enantiomer for MDA, HHA and HMA. Recoveries for all analytes were > 70%.
At three concentrations tested, precision and accuracy were demonstrated
to be within acceptable limits with coefcients of variations < 15% and
accuracy 10%. The developed method is sensitive and the simplied
sample preparation approach enables the rapid analysis of samples making it well suited for future studies on the stereoselective disposition of
MDMA and metabolites in humans.
Paper No.: 936

THERAPEUTIC TARGETS
PROTECTIVE EFFECT OF L-CARNITINE AGAINST
H2O2-INDUCED NEUROTOXICITY IN NEUROBLASTOMA
(SH-SY5Y) CELLS
Chunbo Wang(1), Z Han(2), L Zhu(2), J Yu(3), J Ye(4), J Tong(1)
(1) Department of Pharmacology, Medical College, Qingdao University,
Qingdao, Shandong Province, PR China
(2) The Afliated Hospital of Medical College, Qingdao, Shandong
Province, PR China
(3) Department of Pharmacology, Laiyang medical school, Shandong
Province, PR China
(4) Department of Pathophysiology, Medical College, Qingdao University, Qingdao, Shandong Province, PR China
L-carnitine is an endogenous mitochondrial membrane compound and
some studies have reported that L-carnitine could effectively protect various cells against oxidative injury both in vitro and in vivo. In the present
study we used the human neuroblastoma SH-SY5Y cell line as an in vitro model and assessed the effect of L-carnitine on hydrogen peroxide
(H2O2)-mediated oxidative stress and neurotoxicity. Cells in culture were
treated with different concentrations of H2O2 alone or pretreated with Lcarnitine. 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide
(MTT) assays, transmission electron microscopy, agarose gel electrophoresis, biochemical methods, and Western blotting were employed in the
present study. Our results showed that pretreatment with L-carnitine 3
hours inhibited H2O2-induced cell viability loss, morphological changes,
intracellular ROS generation and LPO in a concentration-dependent manner. Endogenous antioxidant defense components including total antioxidative capacity (T-AOC), glutathione peroxidase, catalase and superoxide dismutase were also promoted by L-carnitine. Meanwhile, H2O2induced down-regulation of Bcl-2, up-regulation of Bax, DNA damage
and apoptosis were also inhibited in the presence of L-carnitine. Taken
together, these results suggest that L-carnitine may function as an antioxidant to inhibit H2O2 -induced oxidative stress as well as regulation of
Bcl-2 family and prevent the apoptotic death of neuronal cells, which
might be benecial for the treatment of oxidative stress in neurodegenerative diseases.
Keywords: Apoptosis; L-carnitine; Oxidative stress; SH-SY5Y cells
645
Paper No.: 2160
EFFECT OF CTOL ON THE PROLIFERATION OF AF BY
BRDU
Chunxia Wang, L Hou
Southern Medical University, Nanfang Hospital, Department of
Pharmacy, Guangzhou, PR China
Objective: To explore the effect of CTOL (Changtong Oral Liquid) on
proliferation of cultured broblasts from adhesive peritoneum, and prepare the laboratorial foundation for further study on its molecular therapeutic mechanism. Methods: (1) Preparation of medication serum:
Twenty SD male rats were randomly divided into 4 groups: normal
serum group, 3 CTOL groups (low, medium and high dose). After
administration of medication at the different dosages for 7 days, serums
were obtained from the abdominal arteries of all rats. (2) In vitro culture
of broblast: In our study, the passage 3~8 cells were used in order to
maintain comparability. (4)Proliferation assay of broblasts: Fibroblasts
in desired condition were seeded on 24-well plates. The media were treated by adding different treatments, such as CTOL medium, normal rat
serum, PAI-1, tPA, respectively. Untreated cells were used as control.
after 24h, 10lM bromodeoxyuridine (BrdU) was applied to the culture
media for 4h to label the nuclei of cells that had differentiated, and then
immunocytochemistry was performed to detect BrdU positive cells after
xation with 4% PFA. Results: When CTOL medium, PAI-1 and tPA
were added to the cultures, the fraction of BrdU positive cells was significantly changed (P < 0.001) (CTOL medium). However, positive cells of
normal rats serum had no signicant change. Conclusions: CTOL as
well as tPA could evidently restrain the proliferation of broblasts. However, PAI-1 accelerated the growth of AF, which may have implications
for clinical utilization of CTOL to prevent peritoneal adhesions.
Paper No.: 1692

DISCOVERY
ACTIVATION OF M3 MUSCARINIC ACETYLCHOLINE
RECEPTORS AND ALPHA1-ADRENOCEPTORS INDUCES
AQP5 TRAFFICKING IN RAT PAROTID ACINAR CELLS
Di Wang(1), G Cho(1), M Shono(2), Y Ishikawa(1)
(1) University of Tokushima Graduate School, Institute of Health Sciences, Department of Pharmacology, Tokushima, Japan
(2) University of Tokushima Graduate School, Support Center for
Advanced Medical Sciences, Tokushima, Japan
The activation of M3 muscarinic acetylcholine receptor (mAChR) and
a1-adrenoceptor increases salivary uid secretion. Large volume of saliva
uid is secreted from acinar cells. Aquaporin-5 (AQP5) is a membrane
water channel protein that is highly expressed in salivary glands. We
visualized the translocation of AQP5 in rat parotid acinar cells in
response to M3-mAChR and a1-adrenoceptor activation. Immunohistochemical studies using anti-AQP5, anti-otillin-2, and anti-GM1 antibodies were performed to investigate subcellular distribution of AQP5 and
lipid rafts. Confocal microscopy revealed that most of AQP5, otillin-2
and GM1 were diffusely distributed throughout apical cytoplasm under
control conditions. Cevimeline or phenylephrine was intravenously
injected into rat tail vein. After 6 to 10 min of injection, AQP5 was predominantly associated with the apical plasma membrane (APM). Then
AQP5 internalized to the cytoplasm from the APM with giving rise to
buds together with otillin-2. Atropine and phentolamine inhibited cevimeline- and phenylephrine-induced translocation of AQP5, respectively.
AQP5 and GM1 were recognized in detergent-insoluble fraction under
control conditions, but their amounts decreased in this faction after 10
min of cevimeline injection. Most of salivary AQP5 was detected in the
detergent-soluble fraction, whereas salivary GM1 was detected in the

detergent-insoluble fraction, indicating that salivary AQP5 is not associated with lipid rafts. Thus, AQP5 are located in recycling rafts under
control conditions. Upon M3-mAChR and a1-adrenoceptor activation,
AQP5 is trafcking to the APM and then dissociated from lipid rafts at
the APM. Although the dissociated AQP5 was released into saliva,
undissociated AQP5 was internalized to the cytoplasm.
Paper No.: 1693

DISEASE AND THERAPY
ASSESSMENT OF SALIVARY AQP5 AS A BIOMARKER FOR
XEROSTOMIA
Di Wang(1), F Iwata(2), Y Pan(3), M Muraguchi(2), K Ooga(2),
Y Ohmoto(2), M Takai(2), X-J Li(3), T Mori(2), Y Ishikawa(1)
Sciences, Department of Pharmacology, Tokushima, Japan
(2) Otsuka Pharmaceutical Co. Ltd, Research Institute of Pharmacological & Therapeutical Development, Japan
(3) Peking University Health Science Center, Beijing, PR China
Aquaporins (AQPs) are a family of membrane proteins that function as
water channels. AQP5 is highly expressed in salivary glands. In response
to the activation of M3 muscarinic acetylcholine receptor (mAChR),
AQP5 is translocated to the apical plasma membrane (APM) together
with lipid rafts. Some parts of AQP5 are then released into saliva. We
investigated whether salivary AQP5 was useful tool for a diagnosis of
xerostomia. Participants including healthy adults and patients gently
chewed the Salivette swab. After centrifugation, the saliva was subjected
to ELISA system. To develop ELISA system, we generated anti-AQP5
antisera against recombinant human AQP5 protein. The amount of
AQP5 in unstimulated saliva showed a diurnal variation with high levels
during waking hours and an age-related decrease in salivary AQP5 was
coincident with the amount of saliva. Cevimeline, a mAChR agonist,
induced to release AQP5 to saliva. Changes in salivary AQP5 levels after
cevimeline-administration occurred simultaneously with changes in saliva ow rates. Confocal microscopy revealed that AQP5 showed a diffuse pattern in acinar and duct cells of rat parotid glands under
unstimulated conditions. At 6 min after cevimeline-injection, AQP5 was
predominantly associated with the APM and was localized in the lumen.
In patients with diabetes mellitus or Sjogrens syndrome, the decrease of
salivary secretion was concomitant with low salivary AQP5 levels. In
Alzheimer patients, donepezil-administration increased salivary secretion
and salivary AQP5 levels. Thus, salivary AQP5 levels correlated with
salivary secretion in both health and disease, suggesting that salivary
AQP5 was useful in assessing xerostomia.
Paper No.: 1901

EFFECTS OF APPLE POLYPHENOLS ON OXIDATIVE DNA
DAMAGE INDUCED BY ETHANOL
Fang Wang, J Yang, J Du, C Wu
Shenyang Pharmaceutical University, Department of Pharmacology,
Shenyang, PR China
Introduction: Increasing evidence supports that the generation of excess
amounts of reactive oxygen species (ROS) during the metabolism of ethanol leads to oxidative DNA damage in the organism, which may contribute to the development of various potential pathologies. It is well
known that apple polyphenols (AP) from apple extract show various biological activities and pharmacological functions as a natural and effective
646
antioxidant. However, the protective effect of AP on oxidative DNA
damage indued by ethanol has not been examined previously. Materials:
Mice and rat cerebellar granular neurons were used to investigate the
protective effect of AP on ethanol induced oxidative DNA damage.
Results:AP administration for 1d and 3d inhibited signicantly ethanolinduced oxidative DNA damage in the cerebellum and hippocampus of
mice. AP also prevented ethanol-induced oxidative DNA damage in primary cultures of rat cerebellar granule neurons by using single cell gel
electrophoresis. Further study revealed that AP inhibited ethanol-induced
increase of urinary 8-OhdG, a reliable marker of ROS-induced DNA
base modication by using HPLC-ECD. Moreover, AP have the tendency of further increase the expression of DNA repair enzyme8-oxoguanine DNA glycosylase (OGG1) induced by ethanol in cerebellum
and hippocampus of mice. Conclusion: AP exert a powerful protective
effect against oxidative DNA damage induced by ethanol and the protective effect is associated with the increase in oxidative DNA damage
repair capacity..
Paper No.: 2150

THE EFFECTIVENESS AND SAFETY OF ARSENIC TRIOXIDE
WITH AND WITHOUT ALL-TRANS RETINOIC ACID IN THE
TREATMENT OF ACUTE PROMYELOCYTIC LEUKAEMIA:
A META-ANALYSIS
Hao Wang, Z-X Rong, H Qi, Y Zhang, B-S Wang, H-Z Chen
Shanghai Jiaotong University School of Medicine Institute of Medical
Sciences, Department of Pharmacology, Shanghai, PR China
2-arachidonyl glycerol is a substrate for cyclo-oxygenase-2, resulting in

the biosynthesis of PG-glyceryl esters. PGE2-glyceryl ester has received
particular attention and a pharmacologically distinct receptor that preferentially recognizes PGE2-glyceryl ester has been suggested. To assist in
further characterizing PGE2-glyceryl ester pharmacology, the following
were employed (a) a cell model (monocyte-derived human osteoclasts)
that elicited a Ca2 + signal response to PGE2-glyceryl ester but not PGE2
(b) a molecular that selectively blocked PGE2-glyceryl ester effects
(AGN 217673). AGN 217673 contains the same oxabicycloheptane scaffold that was used to design potent PGF2 s ethanolamide antagonists
(AGN 211334-6). The key structural determinant for PGE2-glycerylester
antagonism was replacement of the monalkylamide moiety of AGN
211334-6 with a serinolamide. Thus, AGN 217673 did not block the
effects of PGF2 s -ethanolamide in the feline iris sphincter preparation.
AGN 217673 exhibited no meaningful antagonism at human recombinant EP1-4, DP, FP, or IP receptors: it was, however, a TP antagonist.
Nevertheless, AGN 217673 is, to the best of the authors knowledge, the
rst compound shown to selectively block the effects of PGE2-glyceryl
ester vs. PGE2 and may be useful pharmacological tool.
Paper No.: 501

PHYTOESTROGENIC EFFECTS OF CATALPOL IN T47D AND
MDA-MB231 CELLS IN CULTURE
Jifeng Wang, J Niu, Q Hao, P Zhao
Beijing University of Chinese Medicine, Department of Biochemistry,
Beijing, PRChina
Arsenic, a natural substance, has been used as a traditional Chinese medicine for more than 1000 years. Since 1970s, arsenic trioxide (ATO) has
been successfully applied in China for the treatment of acute promyelocytic leukemia (APL). This meta-analysis assessed the effectiveness and
safety of ATO combined with all-trans retinoic acid (ATRA) in APL.
Medical databases (MEDLINE, Cochrane Library, and Chinese Scientic
Journal Database) were searched up to December 2009. Eligible studies
included prospective randomized trials comparing ATO/ATRA with ATO
alone in both newly diagnosed and relapsed APL subjects. Eight studies
involving 457 patients were included. Compared with ATO alone, induction therapy with ATO/ATRA signicantly increased the complete remission (CR) rate (RR: 1.09, 95% CI: 1.001.19, P = 0.04), shortened the
time to achieve CR (WMD: -6.51, 95% CI: -11.32-1.70, P = 0.008),
and improved the molecular remission rate after consolidation therapy
(RR: 1.74, 95% CI: 1.142.66, P = 0.01) and the 1-year disease-free survival rate (RR: 1.22, 95% CI: 1.001.50, P = 0.05). There were no statistically signicant differences between two treatments in terms of early
death (RR: 0.63, 95% CI: 0.341.17). The incidences of the main
adverse events, such as liver dysfunction, gastrointestinal reactions, and
cardiac dysfunction were similar in patients receiving ATO/ATRA or
ATO alone. These results suggested that ATO/ATRA could synergistically improve the overall outcome of newly diagnosed and relapsed APL
patients through an increase in the molecular remission and long-term
survival rates, supporting the use of ATO/ATRA as an effective treatment
for all APL patients previously untreated with ATO.
To demonstrate the phytoestrogenic effects of catalpol, the proliferation

and cell cycle and the levels of ERa and and ERb protein and gene pS2
mRNA expression were measured on breast cancer cellsestrogen receptor positive T47D cells and estrogen receptor negative MDA-MB231cells. Result:(1) The cells proliferation was enhanced signicantly by
treatment with catalpol in the range of10-7M to 10-6M for T47D cells
indicated by MTT assay. The proliferation effects were inhibited by adding antiestrogen ICI182 780(10-8 M). (2) There was no signicant difference on the proliferation of MDA-MB-231 cells by treatment with tested
concentration of catalpol as well as by co-treatment with antiestrogen
ICI182 780(10-8 M) compared with solvent control group. (3) Catalpol
up-regulated pS2 mRNA expression of T47D cells at tested concentration 10-7M or 10-6M. and they increased the level in T47D cells, ERa
protein expression as well. 10-6M catalpol increased the level of ERa
protein expression T47D cells. However, 10-7M catalpol did not show
remarkable effects to the level of ERb protein expression in T47D cells.
Conclusion: Catalpol possessed phytoestrogenic effects by up-regulating
pS2 gene expression and the receptor subtype of ERa and ERb expression. The phytoestrogenic effects were mediated by regulating ER. There
was a positive correlation between ER expression and pS2 gene expression.
This work was supoorted by the 111 project B70007,PR China and by
the International Science and Technology Cooperation Program of China,
2008DFA31970 to Dr. Niu Jianzhao
Paper No.: 3110

DISEASES
IDENTIFICATION OF A SELECTIVE ANTAGONIST FOR
PROSTAGLANDIN (PG) E2-GLYCERYL ESTER: AGN-217673
Paper No.: 1230

INVESTIGATIONS ON THE ANTITUMOR EFFECTS AND
MECHANISMS OF 13 COMPOUNDS
J Wang(1), David Woodward(1), L Ford(2), R Ross(2), M Rogers(2)

(1) Allergan Inc., Department of Biological Sciences, Irvine, USA
(2) University of Aberdeen, Scotland, UK
Juan Wang, XH Chen

Shanghai Institute of Pharmaceutical Industry, Department of Pharmacology, Shanghai, PR China
647
Introduction: The study was conducted to nd out active compounds
with potent tumor inhibition ability from 13 compounds screening in vitro and in vivo, also showed their mechanisms. Materials: (1) The antiproliferative effect of compounds against 7 human carcinoma cell lines
in vitro was observed by MTT assay. (2) The antitumor effect in vivo
was evaluated with the model of nude mice bearing A549 xenografts.(3)
The effects on cell cycle distribution and inducement of apoptosis and
the active caspase-3 of A549 were assessed by ow cytometry analysis.
(4) The effects on the expression of apoptosis-related gene were assessed
by RT-PCR. Results: (1) 3810, 3813, 3818 showed signicant growth
inhibitive effects on most of the cell lines. (2) There were signicant differences in the tumor weight between 3810, 3818 and negative control
group (P < 0.01). (3) They all had the ability to induce apoptosis of
A549, showed time-dependent manner. 3810, 3818 block cells in G0-G1
phase, while 3813 blocked cells in G2-M phase signicantly. They
induced apoptosis of A549 by activated caspase-3 with a time-dependent
manner . (4) 3810 up-regulated the p21 mRNA . Conclusion: 3810,
3813, 3818 exhibited potent antitumor activity in vitro and in vivo. The
mechanisms of them were associated with inducement of tumor cells
apoptosis, inducement of G0-G1 or G2-M phase arrest, activation of caspase-3 and up regulation of p21 mRNA, these three active compounds
have the extensive development perspective.
Paper No.: 1212

DISCOVERY
INTERACTION BETWEEN TROXERUTIN AND BOVINE
SERUM ALBUMIN
L Wang, X Li, Y Li, Y Chen, Y Fan, Ming Xue
The characteristics of the binding reaction of troxetutin with the bovine
serum albumin (BSA) and the quenching mechanism of the uorescence
of BSA by troxerutin by the uorescence and ultra violet-visible absorption spectra were investigated. The dynamic quenching constants and static binding constants the Stern-Volmer equation and the double
reciprocal Lineweaver-Burk equation were determined. The number of
binding site was calculated with double logarithmic equation and the
main binding force was discussed by thermodynamic equations. The
binding distance and the energy transfer efciency between the donor
(BSA) and the acceptor (troxerutin) were obtained based on Forsters
nonradiative energy transfer method. Troxerutin effectively quenched the
uorescence of BSA via static quenching processes. The binding constant KA was calculated to be in the order of 106. The number of binding site was approximately equal to 1, the binding distance was 1.97nm,
the energy transfer efciency was 0.529, and the binding force was
mainly hydrophobic force. The data indicated there was a strong interaction between troxerutin and BSA. Troxerutin effectively quenched the
intrinsic uorescence of BSA via static quenching mechanism, and the
binding mode was mainly driven by the hydrophobic interaction.
Keywords: troxerutin, bovine serum albumin, uorescence spectrometry,
interaction
Paper No.: 1080

POPULATION PHARMACOKINETICS OF CARBAMAZEPINE
AND ITS METABOLITE IN CHINESE CHILDREN WITH
EPILEPSY
Aim: Few drugs pharmacokinetic parameters of children are available

since traditional study (frequent sampling) is impossible in children. Population pharmacokinetics (PPK) opens a new way to get individual PK
parameters. To individual therapy of carbamazepine (CBZ) in children
with epilepsy. PPK of CBZ and the most important metabolite CBZE
was investigated in Children. Methods: HPLC method was developed
for monitoring CBZ and CBZE simultaneously. Demography from pediatric patients was collected in detail. PPK model was set up using NONMEM software. Basic model: xed factors were added one by one
(DOFV>3.84), full model: backward elimination step (DOFV>10.8)then
nal model and stability validation. ME, MSE, SME, WRES and 95%
CI were induced to evaluate the accuracy and precision. Individual
parameters were calculated using 1-2 concentrations by Bayesian estimation. Levels were predicted to conduct individualized therapy. Results:
229 concentrations were collected at different times after dosing during
steady-state. Data from 168 patients with normal renal/hepatic functions
were analyzed. One-compartment model with rst-order absorption and
elimination best described the data. CBZ-PPK: Cl/F(l/kg/h)= 0.103
(0.676^(Wt/28.62))EXP(ETAC1), V/F(l/kg)=2.68 (1.8^(Dose/
16.66))EXP(ETAV). CBZE-PPK: Cl/F(l/kg/h)= 0.369 (1-0.281
VPA)(0.855^(Wt/28.62))EXP(ETACl),
V/F(l/kg)=
11.7
(1.46^(Dose/16.66))EXP(ETAV). Prediction of CBZ and CBZE in new

patients were satisfactory for clinical application. Conclusion: PPK models of CBZ and CBZE for Chinese children were successfully established. Individual PK parameters of CBZ and CBZE obtained are pivotal
to optimize CBZ therapy for children with epilepsy.
Paper No.: 2093

ATTENUATION OF ISCHEMIA-INDUCED CA2 + OVERLOAD
AND LV MECHANICAL DYSFUNCTION BY INHIBITORS OF
LATE INA
Lianguo Wang(1), B Vacher(2), B Le Grand(2), A Clanachan(1)
(1) University of Alberta, Department of Pharmacology, Edmonton,
ALB, Canada
(2) Centre de Recherche Pierre Fabre, Castres, France
Myocardial ischemia-reperfusion (IR) injury causes LV mechanical dysfunction that is associated with excessive accumulation of intracellular
[Ca2+] (Ca2 + overload). To examine directly if a persistent Na+ inux
(late INa) underlies Na+-dependent increases in [Ca ] during IR, we utilized isolated working rat hearts to measure simultaneously LV work and
[Ca2+] (indo-1 uorescence). In untreated hearts (n = 5), diastolic [Ca2+]
was increased after 20 min of global ischemia by 56 5%. Lidocaine
(10 lM, n = 5), an inhibitor of both peak and late INa, inhibited the
increase in [Ca2+] to 25 3%. Increases in [Ca2+] were also inhibited by
the late INa inhibitors, ranolazine (10 lM, n = 4, to 30 2%) and F
15845 (3 lM, n = 5, to 28 3%; 10 lM, n = 4, to 24 5%). During
reperfusion (20 min) of untreated hearts, diastolic [Ca2+] remained elevated by 15 3%. Greater recovery was observed with lidocaine (10
lM, to 2 1%), ranolazine (10 lM, to 4 1%) or F 15845 (3 lM, to 5
2%; 10 lM, to 3 3%). Recovery of LV work (30 9% of pre-ischemic values in untreated hearts) was enhanced by lidocaine (10 lM, to 69
3%), ranolazine (10 lM, to 78 3%) or F 15845 (3 lM, to 72 6%;
10 lM, to 95 3%). Thus, attenuation of Na+-induced Ca2 + overload
by late INa inhibitors may be a useful approach to enhance post-ischemic
LV mechanical function during IR. Moreover, as Ca2 + overload triggers
additional deleterious processes, these data suggest that inhibitors of late
INa may elicit a range of cardioprotective actions.
Li Wang, Y Liu
Peking University First Hospital, Department of Pediatric Neurology,
Beijing, PRChina
648
Paper No.: 1182
HEALTH AND DISEASE
AGES BREAKERS REVERSE ENDOTHELIAL CELL
DYSFUNCTION AND ENHANCE THE ANTIHYPERTENSIVE
EFFECT OF NIFEDIPINE IN DIABETIC-HYPERTENSIVE RATS
Paper No.: 1689

STUDY ON ABSORPTION KINETICS OF BAICALIN IN
EXTRACTED SCUTELLARIA AFFECTED BY RUBARB
ANTHRAQUINONES
Lili Wang, B Zhang, G Chen, S Li
Ping Wang, X Meng, Y Zhang, H Liu
Beijing Institute of Toxicology and Pharmacology, Beijing, PR China

Chengdu, PR China
Combining drugs with complementary mechanisms of action has been

believed as a well-advised means for Aging hypertension control. AGEs
breaker, a new class of candidate drug, target a fundamental pathological
process in aging cardiovascular dysfunction could be a better choice.
This study evaluated the effects of two AGEs breakers ALT-711 and C36
on endothelial cell protection and the efcacy of Nifedipine. Diabetic
and diabetic-hypertensive rats were STZ-induced and treated with 1%
NaCl drinking water for continuous 12 wks. After treated with ALT-711
and C36, respectively for another 4 wks, the isolated aorta vessels function and the effect of Nifedipine were observed. In addition, plasma NO,
ET-1 and Ang- II contents and their associated gene expression in aorta
and kidney were assayed. Nifedipine had more potent anti-hypertensive
effect on diabetic-hypertensive rats pre-treated with AGEs breakers compared with vehicle treated rats. Meanwhile, AGEs breakers treated rats
exhibited signicantly increased plasma NO content, decreased gene
expressive level of preproendothelin and Angiotensinogen in aorta and
renal tissues. Moreover, the endothelial-dependent vasodilation induced
by ACh in AGEs breakers treated diabetic rats signicantly augment.
Those results indicated that AGEs breakers could efciently enhance the
anti-hypertensive effect of Nifedipine. The underling mechanism is
mainly related to the improvement of vascular endothelial cell dysfunction, and may also be benet of the regulation of AGEs breaker on local
renal RAS system. As candidates of adjuvant drug for treating diabetic
and aging cardiovascular complications, AGEs breakers have a great
potential.
In Chinese traditional medicine, Scutellaria are usually used with the

Rhubarb Anthraquinones. And baicalin is one of the most important
components of Scutellaria, and has many effects to some diseases. To
observe the effect of Rhubarb Anthraquinones to absorption kinetics of
baicalin, rats were given orally extracted scutellaria only and extracted
scutellaria compatibility with Rhubarb Anthraquinones respectively, and
its portal vein blood was collected b yregularly time (15-250min, 11
times); HPLC was used to determine baicalin concentration in biological
samples and the two-site absorption model ts to the plasma concentration-time data. The HPLC method is simple, sensitive, accurate, precise
and stable, and suitable for the study of baicalin in vivo; the baicalin concentration in portal vein blood was improved signicantly by Rhubarb
Anthraquinones (Cmax, AUC and MRT in group, P < 0.01). The compatibility of extracted Scutellaria and Rhubarb Anthraquinones may
enhance the absorption degree of baicalin, prolong the absorption time,
which will be absorption evidence to the compatibility of two herbs.
Paper No.: 2182

ANALYSIS ON QUALITY CONTROL OF DRUG CLINICAL
TRIALS
Shaohua Wang
Paper No.: 1216

DISCOVERY
STRUCTURAL ELUCIDATION OF THE METABOLITES OF
TANSHINONE I AND ITS ANALOGUE DIHYDROTANSHINONE
I IN RATS BY HPLC-ESI-MSN
M Wang, H Dai, X Li, Y Li, L Wang, Ming Xue
Tanshinone I and its analogue dihydrotanshinone I are major active components isolated from Salvia miltiorrhiza Bunge and Salvia Przewalskii
Maxim. These compounds have been found to possess signicant antibacterial, antidermatophytic, antioxidant, anti-inammatory and anticancer activities. Fifteen phase I metabolites and two phase II metabolites of
tanshinone I and dihydrotanshinone I in rats were elucidated and identied by a sensitive HPLCCESICMSn method in detail. The molecular
structures of the metabolites are presented on the basis of the characteristics of their precursor ions, product ions and chromatographic retention
times. The results indicate that the phase I metabolites are biotransformed
through four main pathways: dehydrogenation, hydroxylation, furan ring
cleavage and oxidation metabolism. Phase II metabolites were identied
as the sulfated conjugates which showed a characteristic neutral loss of
80 Da. The biotransformed pathways of tanshinone I and its analogue dihydrotanshinone I are presented on the basis of the investigation. This
investigation provides the valuable information on tanshinone metabolism which is essential for understanding the safety and efcacy of these
drugs as well as for the development of novel drug.
Key words: Structural elucidation; tanshinone I; dihydrotanshinone I;
metabolites; LCCESICMSn
Qingdao Municipal Hospital, National Medical Institution Conducting

Clinical Trials, Qingdao, PR China
The quality of clinical trials is the key to test the design of clinical trial,
the author with many years of management experience in clinical
research puts forward some suggestions and measures about Quality
Control of Drug clinical trials, including the establishment of a sound
organization and management system; establishment of regulations and
standardized operating procedures; implementment of GCP and the current laws and regulations; following the pilot programs and SOP strictly;
the building of the research team; establishment access system of monitor; establishment three level quality control system; carrying out electronic data management and practicing clinical research coordinator
(CRC)in clinical trials.
Paper No.: 528

MICROENVIRONMENT IMPROVING EFFECT OF OCTACOSANOL IN 6-HYDROXYDOPAMINE-INDUCED PARKINSONIAN
MODELS IN RATS
Tao Wang(1), P Zuo(2), P Chan(1)
(1) Xuanwu Hospital of Capital Medical University, Beijing Institute of
Geriatrics and Department of Neurobiology and Neurology, Beijing, PR
China
College, Institute of Basic Medical Sciences, Department of Pharmacology, Beijing, PR China
649
Background: Increasing evidence from human and animal studies suggested that oxidative stress is an important pathogenesis mediator in Parkinsons disease (PD) pathogenesis. In the nigrostriatal systems,
oxidative stress impairs the balance between neuronal cell death and cell
survival determined by the ratio of proNGF and NGF, and probably initiates the death cascade mediated by proNGF-p75NTR-sortilin complex.
Meanwhile, the anti-apoptotic effects mediated by NGF through TrkA
receptors were inhibited to augment the apoptosis of dopaminergic neurons. Methods: behavioral tests, biochemical, immunohistochemical, and
molecular biological methods were used to investigate the protective
effects of Octacosanol in a unilateral 6-OHDA-treated rat model of PD.
Results: The present results indicated that oral administration of Octacosanol signicantly improved the behavioral impairments in rats induced
by 6-OHDA and dose-dependently preserved the free radical scavenging
capability of the striatum. Octacosanol treatment also effectively ameliorated morphological appearances of nigrostriatal neurons and decreased
the apoptotic cells induced by 6-OHDA. In addition, Octacosanol strikingly blocked the 6-OHDA-induced increased expression of proNGFp75NTR-sortilin death signalling complex and its downstream effector
proteins in striatum. Meantime, Octacosanol reversed the decreased levels of NGF, its receptors TrkA and p-Akt which together mediated the
cell survival pathway. Conclusions: In summary, these ndings implicated that the anti-parkinsonism effects afforded by Octacosanol might
be mediated by its neuro-microenvironment improving potency through
retrieving the ratios of proNGF: NGF and their respective receptors
p75NTR: TrkA in vivo. Taking its excellent tolerability and non-toxicity,
Octacosanol may be a promising agent for PD treatment.
Paper No.: 509

NEUROPROTECTIVE EFFECTS OF MORRONISIDE AGAINST
TRANSIENT FOREBRAIN ISCHEMIA IN THE RAT CORTEX
Wen Wang, L Li, J Xiang, H Ai, L Zhang, P Wang, L Li
Xuanwu Hospital of Capital Medical University, Key Laboratory for
Neurodegenerative Diseases, Beijing, PR China
Morroniside, an iridorid glycoside isolated from Cornus ofcinalis Sieb.
et Zucc. and known as shang-zhu-yu in Chinese, and C. ofcinalis have
been used to treat cerebrovascular disease in Traditional Chinese Medicine for a long time. In the present study, the neuroprotective potential of
morroniside was investigated in middle cerebral artery occlusion-induced
focal cerebral ischemia/reperfusion injury in the rat cortex. Nissl staining
showed that neuronal injury was signicantly lessened after treatment
with morroniside (30, 90, and 270 mg/kg). Morroniside (270 mg/kg)
treatment signicantly decreased the level of malondialdehyde, increased
the content of glutathione, and increased the activity of superoxide
dismutase in ischemic brain tissues (P < 0.05). Morroniside (270 mg/kg)
also signicantly decreased the expression of caspase-3 protein in these
tissues. These ndings demonstrated that morroniside could signicantly
protect the brain from damage induced by transient forebrain cerebral
ischemia. The antioxidant and anti-apoptotic properties of morroniside
may contribute to the neuroprotective potential of morroniside in cerebral
ischemic damage.
Paper No.: 1637

DEVELOPMENT OF 2-(1-HYDROXYPENTYL)-BENZOATE, A
PRO-DRUG FOR TREATMENT OF CEREBRAL ISCHEMIA
AND DEMENTIA
Stroke in the aging individual, could result in cerebral dysfunction and

induce vascular dementia and AD like pathology. The drugs for treatment of stroke and prevention of dementia are needed. 2-(1-hydroxypentyl)-benzoates (DL-PHPB) is the pro-drug of dl-NBP (3-nbutylphthalide) which was approved by SFDA in 2002 for treatment of
ischemic stroke. In the rat model of transient focal cerebral ischemia
(MCAo), dl-PHPB signicantly increased cerebral blood ow, reduced
the infarct volume and cerebral edema. In addition, dl-PHPB inhibited
the platelet aggregation thrombus formation. Meanwhile, dl-PHPB
improved mitochondria dysfunction by regulating energy metabolism
and inhibiting apoptotic pathway in mitochondria. In the vascular dementia and AD animal models, dl-PHPB markedly improved the learning
and memory decits and neuronal loss. Especially it can reduce the phosphorylation of s protein in brain. Toxicological researches showed that
the LD50 of oral and intravenous administration of dl-PHPB was 4.11g/
kg and 421 mg/kg, respectively. And no obvious toxic effects were
observed during the long-term toxicological studies. Pharmacokinetic
researches show that dl-PHPB converted to dl-NBP quickly and completely in vivo. The relative bioavailability of converted dl-NBP from
dl-PHPB was 50% higher than the dl-NBP given directly. Therefore,
it is promising to develop dl-PHPB to a new anti-cerebral ischemia
and anti-dementia drug candidate. DL-PHPB was approved for Phase I
clinic trial by SFDA recently. It obtained global IP protection by PCT
patent.
Paper No.: 837

LIQUORICE, PHARMACOLOGICAL AND CLINICAL
EXPERIMENTS FROM THE TRADITIONAL CHINESE
MEDICINE TO THE MODERN RESEARCH
Xiaoying Wang, H Zhang, X Gao
Institute of TCM Research, Tianjin University of Traditional Chinese
Medicine, Tianjin, China
Liquorice is a widely used herbal medicine native to southern Europe
and parts of Asia, and has benecial applications both in the medicinal
and the confectionery sectors. However, there is different evidence of
liquorice efcacy between traditional Chinese medicine (TCM), modern
pharmacological experiments and clinical trials. Unlike the usage in Europe, liquorice in TCM always been used with other herbs in a single prescription. Liquorice is applied in as many as half of all Chinese herbal
formulas as a unique guide drug to enhance the effectiveness of other
ingredients and reduce toxicity, as well as improve avor. Modern pharmacological experiments have proved that liquorice possesses multiple
constituents (glycyrrhizin, glycyrrhizic acid, liquiritin, etc.) and actions
(effects on digestive system, adrenocortical hormone function, anti
inammation, anti allergy, etc.). According to some researches, the
guide effect of liquorice was in part due to the induction of hepatic
microsomal cytochrome P450. Side effects of long-term use of liquorice
such as hypertension and edema were indicated as well. The molecular
mechanism of liquorice was also been reported such as the modulation
of intestinal P-glycoprotein. Formulas with liquorice were proved to be
effective by the evidence over the centuries and a number of randomized
controlled trials in recent years. Because of the decient quality of most
clinical trials on liquorice, reliable clinical data in human is still lacking.
The overview and comparison between traditional uses of liquorice with
the important recent scientic studies provide a useful way of developing
further indications for this ancient drug.
Xiaoliang Wang, J Li, S Xu, N Feng, L Wang, Y Hu, Y Peng

China
650
Paper No.: 1839
DISEASES
TUMOR NECROSIS FACTOR-RELATED APOPTOSIS
INDUCING LIGAND PROMOTES EARLY ATHEROGENESIS IN
APOLIPOPROTEIN(E)-DEFICIENT MICE
attenuated MI-induced mesenteric artery dilatation dysfunction, elevated

the activation levels of PI3K, Akt and eNOS (P < 0.05). Inhibition with
either LY294002 or L-NAME both markedly inhibit exercise restored vasorelaxation. Present study demonstrate that the reversal of dilatation
function of mesenteric artery by exercise training is most likely associated with attenuated endothelial cells injury, increased the activation of
PI3K/Akt mediated eNOS cascaded.
Yan Wang, F Jiang, Y Zhang, C Zhang, Y Zhao

Shandong University, Qilu Hospital, Key Laboratory of Cardiovascular
Remodelling and Function Research, Jinan, PR China
Tumour necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL)
is a type II membrane protein of the TNF superfamily primarily involved
in regulating cell apoptosis. Both vascular endothelial cells and smooth
muscle cells express TRAIL receptors. Previous studies have demonstrated that TRAIL suppresses the development of advanced atherosclerotic lesions in apolipoprotein(E)-decient mice. However, the effects of
TRAIL on the early stages of atherogenesis are not clear. To test this we
systematically overexpressed TRAIL by adenoviral vector administration
in ApoE-decient mice before the onset of atherosclerosis, followed by a
high-fat diet for 8 weeks. En face oil-red O staining showed that TRAIL
expression markedly increased the early atherogenesis, and this was further conrmed with a model of periarterial cuff-induced atherogenesis in
the carotid artery. Immunohistochemistry and ow cytometry analyses
demonstrated that TRAIL was mainly expressed in circulating and inltrating leukocytes. TUNEL labeling and RT-PCR results indicate that
vascular cell apoptosis and inammation are unlikely to have a major
role in the TRAIL effects. Interestingly, we found that TRAIL increased
the content of intra-plaque smooth muscle cells, and TRAIL-induced
lesions generally exhibited a stable phenotype. In conclusion, TRAIL
may have differential roles in modifying the progression of early versus
advanced atherosclerotic lesions.
Paper No.: 2153

HEALTH AND DISEASE
EXERCISE IMPROVES THE DILATATION FUNCTION OF
MESENTERIC ARTERY IN POST-INFARCTION RATS:
INVOLVED THE MECHANISM OF PI3K/AKT MEDIATED
ENOS CASCADED
Y-H Wang(1), S-P Wang(1), H-K Jiang(1), S-F Chen(1), M Zhao(1),
J Yang(1), J Zhang(1), Y Cai(1), S-J Fan(1), Y-L Tian(1), X-J Yu(1),
Wei-Jin Zang(1)
Xian Jiaotong University, Department of Pharmacology, Xian,
PR China
Considerable evidence demonstrates that myocardial infarction (MI)
induces endothelial dysfunction of peripheral resistance arteries. This
study was designed to examine the effects of MI-related mesenteric
artery dilatation function and its underlying mechanisms, with specic
focus on the role of exercise training in reversing dilatation function of
mesenteric artery in post-MI rats. Adult male Sprague-Dawley rats were
divided into three groups: sham operation, MI, and MI+exercise training.
The protocol of MI by ligation of the left anterior descending coronary
artery, MI+exercise training subjected to a 8 weeks trained on a rodent
treadmill (60 min/day, 5 days/week). Mesenteric artery endothelial cells
ultrastructure, vascular relaxation function, the expression and activation
of phosphatidylinositol-3 kinase (PI3K), kinases Akt/protein kinase B
(Akt) and endothelial nitric oxide synthase (eNOS) were determined. MI
caused mesenteric artery endothelial cells injury evidenced by rough and
uneven cell membrane surface and digitationes in cell nucleus, caused
signicant endothelial dysfunction evidenced by signicantly attenuated
ACh-induced vasorelaxation in mesenteric artery (P < 0.01), and
decreased the expression and activation of PI3K, Akt and eNOS (P <
0.05). Exercise training restored the morphosis of endothelial cells and
Paper No.: 847

DISCOVERY AND CHARACTERISTICS OF INDUCIBLE
SPINAL D-AMINO ACID OXIDASE AS A NOVEL TARGET FOR
THE TREATMENT OF CHRONIC PAIN
Yong Xiang Wang, N Gong, Z-Y Gao, J-L Huang
Shanghai Jiao Tong University School of Pharmacy, Shanghai, PR China
Human D-amino acid oxidase (DAO) is an enzyme catalyzing deamination of certain D-amino acids and expressed in the kidneys, liver, and
central nervous system including the spinal cord. We have recently
explored the nociceptive role of spinal DAO in chronic pain by using
techniques of DAO mutation, enzyme inhibition, and direct spinal injection. Formalin-induced persistent hyperalgesia but not nociceptive pain
was reduced by 60% in ddY/DAO-/- mice compared with ddY/DAO+/+
mice. Systemic injection of DAO inhibitors blocked spinal nerve ligation-induced mechanical allodynia and formalin-induced hyperalgesia in
animals but not acute pain such as in the hotplate and tail ick tests or
formalin nociception. Systemic administration of DAO inhibitors also
inhibited basic and formalin-upregulated DAO activity from the spinal
cord. Correlated to their inhibitory effects on spinal cord derived DAO
activity, direct intrathecal injection of a series of structure-unrelated
DAO inhibitors blocked formalin-induced hyperalgesia (but not carrageenan-induced heat hyperalgesia, a model for peripheral sensitization)
with calculated maximum inhibition of 60%, higher than that (50%) of
intrathecal MK-801, an NMDA receptor antagonist. Additionally, spinal
nerve ligation and peripheral formalin challenge increased spinal DAO
gene expression and enzymatic activity. We for the rst time demonstrated that spinal DAO is a novel target for the treatment of chronic pain
including neuropathic pain with following characteristics: 1) selective tissue distribution; 2) specic central sensitization-mediated pain transduction; 3) inducible up-regulation by nerve damage or stimulation; 4) and
high pain transmission efcacy, at least compatible to that of NMDA
receptors, a well-demonstrated human target of chronic pain.
Paper No.: 2127

THE ACTION MECHANISM AND METABOLISM CHARACTERISTICS OF THE EFFECTIVE COMPONENTS GROUPS OF
XIAOXUMING DECOCTION FOR ANTI-ISCHEMIA
Yuehua Wang(1), N Zhou(1), X He(1), J Hu(1), H Zhang(1), Y Ding(2),
H Qin(1), J Zhang(1), Z Li(3), G Du(1)
(1) Chinese Academy of Medical Sciences, Institute of Materia Medica,
National Pharmaceutical Screening Centre, Beijing, PR China
(2) Tsinghua University, PR China
(3) Jiangsu Institute for Food and Drug Control, PR China
Xiaoxuming decoction (XXM) is one of the Chinese materia medica prescription which has been used for treatment of stroke and the sequela of
stroke. In this study, one new research method was established using
modern scientic theories and techniques. First of all, the neuroprotective
effects of different components of XXM on multiple targets related to
cerebral ischemia were studied through high throughput screening
651
method, and by comprehensive analysis the effective components groups
(ECG) was obtained. Then, the effects of ECG were evaluated in both
acute and chronic ischemic rat models. The results also showed that the
ECG could alleviate impairments induced by ischemic. Subsequently, the
action mechanism and metabolism characteristic of ECG were studied,
and the statistical analysis suggested that the action mechanism of Chinese materia medica prescription is considered to be the interaction of
two complicated systems, that is, the complex material system composed
of active drug ingredients and the complex biological system composed
of drug targets under pathological conditions. Actually, The function of
Chinese materia medica prescription may be described with the effective
components groups of Chinese materia medica prescription. Modern science technology will push the development of Chinese medical prescription greatly.
Key word: Chinese materia medica prescription, effective components
groups, cerebral ischemic
Acknowledgements: The study was supported by the National Natural
Science Foundation (No. 30630073) and the Ministry of Health Community Project (No. 200902008).
Paper No.: 1582

DISEASE AND THERAPY
ANGIOTENSIN II INHIBITES IKR /HERG CHANNELS
THROUGH PROTEIN KINASE C-E
Yuhong Wang, J Xu, X Chen, Y Xu
(2) Shandong Center for Disease Control and Prevention,Shandong

Jinan, PR China
Introduction: To study the protection effect of erythropoietin(EPO) on
newborn rats with hypoxic-ischemic brain damage(HIBD) and its possible molecular mechanism. Materials: This experiment have prepared neonatal HIBD rats model Seven-day-old rats were randomly divide into
three groups: Sham-operated group,HIBD group and rhEPO therapy
group(n = 8 in each subgroup) based on different time points at
6h,12h,24h,48h and 72h after HIBD.In sham-operated group, rats were
performed operation by taking median neck incision and separated the
left common carotid artery, and hypoxic-ischemic was not conducted. In
HIBD group, the left common carotid artery of rats were separated and
ligated. Then they were put under hypoxic environment for 2h.In rhEPO
therapy group, rats were instantly given single dose of rhEPO(5000IU/
Kg) by intraperitoneal injection following the hypoxic-ischemic brain
damage. All rats were decapitated at different time points and brain tissues were collected. Then general morphological change of the brain
was observed. The pathological changes of brain tissue were observed
under light microscope by HE stain. And the expressions of bcl-2,bax in
cerebral cortex were detected. Results: After HIBD, general morphological changes of brain showed abnormal at different degrees, the brain tissue signicant edema was apparent HIBD at 24-48h and above
mentioned changes weakened after accepting rhEPO therapy. The expression of bcl-2 at different time points in rhEPO therapy group were
increased, and the expression of bax were declined in rhEPO compared
with that of HIBD group(P < 0.01). Conclusion:rhEPO may inhibit neuronal apoptosis and provide a protective effect for hypoxia-ischemiainduced neuronal injury of neonatal rats.
Hebei Medical University, Department of Pharmacology, Shijiazhuang,

PR China
Human ether-a-go-go-related gene (hERG) encodes the pore forming
subunit of the channel underlying the rapidly activating delayed rectier
K+ current (IKr), which is crucial for the repolarization of cardiac action
potentials. We previously reported that angiotensin II (Ang II) inhibited
IKr /hERG through PKC pathway (Wang YH et al, Br J Pharmacol.2008;154:429-39.). The present study was designed to investigate
which PKC isoenzyme was functionally involved in mediating the inhibitory action of Ang II on IKr /hERG. The whole-cell patch-clamp technique was used to record IKr /hERG in freshly isolated guinea pig
ventricular cardiocytes or HEK 293 cells co-transfected with hERG and
human Ang II type 1 (AT1) receptor gene. Activation of AT1-receptors with
Ang II reduced IKr /hERG current amplitudes. The inhibitory effects of Ang
II were signicantly inhibited by general PKC inhibitor bisindolylmaleimide I but were little affected by the conventional PKC inhibitor Go-6976
and Go-6983. However, inclusion of a PKC-e translocation inhibitor peptide epsilonV1-2 in the intracellular solution signicantly reduced the inhibition of Ang II on Ikr/hERG. Western blot analysis showed that activation
of AT1 receptor by Ang II selectively promoted the translocation of PKC-e
but not PKC-a, from the cytosol to membrance in ventricular myocytes.
Moreover, the study revealved that the regulation of AngII on hERG was
ablated in a mutant hERGDPKC channel lacking 17 among 18 putative
PKC-dependent phosphorylation sites. Our results demonstrated that by
specically activation of PKC-e, Ang II elicited inhibitory effect on IKr /
hERG through channel phosphorylation.
Paper No.: 2444

STUDY ON PROTECTION EFFECT OF ERYTHROPOIETIN ON
NEWBORN RATS WITH HYPOXIC-ISCHEMIC BRAIN
DAMAGE
Yunxiao Wang(1), W Liu(2), Y Cao(1), Y Mengn(1)
(1) The Afliated Hospital of Medical College, Qingdao University,
Department of Pharmaceutical preparation section, Qingdao, PR China
Paper No.: 464

DISEASES
R-FLURBIPROFEN REVERSES MULTIDRUG RESISTANCE,
PROLIFERATION AND METASTASIS IN GASTRIC CANCER
CELLS BY P75NTR INDUCTION
Zhipeng Wang, H Jin, Q Mei, D Fan
Fourth Military Medical University, State Key Laboratory of Cancer
Biology & Xijing Hospital of Digestive Diseases, Department of
Pharmacology, Xian, PR China
To study the effects and possible mechanisms of nonsteroidal anti-inammatory drugs (NSAIDs) on multidrug resistance (MDR), proliferation
and metastasis in human gastric cancer cells. Methods Gastric cancer
cells SGC7901 and MKN45, and MDR-resistant lines were treated with
ibuprofen, R-urbiprofen (R-ur), indomethacin and aspirin. MTT, plate
clonogenic, and soft agar assays detected the effects of R-ur on proliferation and MDR. Western blot measured p75NTR, P-gp, Bcl-2, CyclinD1,
CDK4, Caspase3, MMP9 and phosphorylated p38, among others. MTT
assays evaluated drug sensitivity of MDR cells, and ow cytometry analyzed cell cycle, apoptosis and uptake of adriamycin. R-ur effects were
validated in vivo in nude mice. Immunohistochemistry detected tumor
p75 and antiogenesis. Assays for in vitro wound healing, migration, invasion, and in vivo metastasis measured effects of R-ur on metastasis. A
dominant negative p75NTR and siRNA determined p75-dependence.
Results NSAID treatment prevented proliferation and induced expression
of p75NTR. R-ur was the most efcient inducer. Caspase 3 and activated p38 increased, and P-gp, Bcl-2, metalloproteases and cell cycle
factors were reduced. Anti-proliferative, anti-MDR and anti-migratory
effects, and pro-apoptotic effects of R-ur were validated in vivo. R-ur
action required induction of p75 via the p38 signalling pathway. Conclusions R-ur reversed MDR and inhibited proliferation and metastasis of
gastric cancer cells by upregulation of p75NTR via p38. R-ur does not
inhibit cyclooxygenase (Cox) enzymes, therefore, expression of MDR,
apoptotic, metastatic and cell cycle factors were altered, independently of
652
Cox. NSAIDs are possible therapeutics for gastric carcinoma, and
p75NTR a potential target for reversing malignancy.
Paper No.: 1631

VKORC1 AND CYP2C9 GENOTYPING USING A RAPID
SINGLE NUCLEOTIDE POLYMORPHISM (SNP) DETECTION
SYSTEM NAMED SMART AMPLIFICATION PROCESS
(SMARTAMP) METHOD
Minoru Watanabe(1), N Matsumoto(1), T Kumai(1), M Tanaka(2),
Y Mitani(3), Y Hayashizaki(3), S Kobayashi(1)
(1) St. Marianna University School of Medicine, Department of Pharmacology, Kawasaki, Kanagawa, Japan
(2) St. Marianna University School of Medicine, Institute of Animal
Experiments, Kawasaki, Kanagawa, Japan
(3) RIKEN Yokohama Institute, Yokohama, Japan
Warfarin has established role in the prevention of thromboembolic
events, but the maintenance dose varies between individuals. The polymorphism of two genes (CYP2C9 and VKORC1) those being involved
in the PK and PD of warfarin is considered as one of the cause for individual difference of warfarin maintenance dose. Although single nucleotide polymorphism (SNP) analysis was performed by the various
methods until now, long time and considerable effort were required to
get these SNPs results. Recently, a rapid SNP detection system named
smart amplication process (SmartAmp) method was developed. Two
hundred and sixty-three patients with anticoagulation therapy were genotyped for VKORC1 (-1639G>A) and CYP2C9*3 (1075A>C) using this
SmartAmp method. Each SNP analysis by the SmartAmp method could
be performed at one step, and analysis was possible within about 1 hour.
In CYP2C9*3 (1075A>C), the number of *1/*1 (AA), 1/*3 (AC), and
*3/*3 (CC) were 257, 6, and 0 patients, respectively. In VKORC1 (1639G>A), the number of GG, GA, and AA were 2, 68, and 193
patients, respectively. These results were almost identical to those have
been reported formerly for Japanese population. This SmartAmp method
was impressively easy to use, and the results were reliable. This new
technique will enable us to start taking care of the patients with warfarin
on the same day that visited a hospital for the rst time.
Paper No.: 1015

DISCOVERY
ELECTROPHYSIOLOGICAL PROPERTIES OF YM-244769, A
NOVEL AND POTENT NA+/CA2 + EXCHANGE INHIBITOR, IN
CARDIAC VENTRICULAR MYOCYTES OF GUINEA PIG
ELECTROPHYSIOLOGICAL PROPERTIES OF YM-244769, A
NOVEL AND POTENT NA+/CA2+EXCHANGE INHIBITOR, IN
CARDIAC VENTRICULAR MYOCYTES OF GUINEA PIG
Yasuhide Watanabe(1), J Kimura(2), K Yamashita(1), T Yamakawa(1),
T Iwamoto(3)
(1) Hamamatsu University School of Medicine, Deparment of Health
Science, Hamamatsu, Japan
(2) Fukushima Medical University, School of Medicine, Fukushima,
Japan
(3) Fukuoka University, Faculty of Medicine, Fukuoka, Japan
+
2 +
exchange (NCX) inhibitor would be

A potent and selective Na /Ca
very useful not only for studying the physiological roles of NCX but also
for potentially offering a new drug therapy for cardiovascular and neuronal diseases. In the present study, we examined electrophysiological
properties
of
YM-244769(N-(3-aminobenzyl)-6-{4-[(3-uoroben-
zyl)oxy]phenoxy} nicotinamide), a novel benzyloxyphenyl NCX inhibitor, in mammalian heart. Inward and outward NCX currents (INCX)
were measured in single cardiac ventricular myocytes of guineapig by
using the whole-cell voltage-clamp technique. YM-244769 suppressed
the bi-directional INCX in a concentration-dependent manner (0.01
3lM). The IC50 values of YM-244769 for the bi-directional outward
and inward INCX were both about 0.1lM. YM-244769 suppressed the
uni-directional outward INCX (Ca2 + entry mode) with the IC50 value of
0.05 lM. In contrast, the blocking effect on the unidirectional inward
INCX (Ca2 + exit mode) was less potent, because 10 lM YM-244769
inhibited it only by about 50%. At 5 mM intracellular Na+ concentration,
YM-244769 suppressed INCX more potentially than it did at 0
mM[Na+]i. Intracellular application of trypsin via the pipette solution did
not change the blocking effect of YM-244769. YM-244769 inhibits the
Ca2 + entry mode of NCX more potently than the Ca2 + exit mode in cardiac myocytes. The inhibition of YM-244769 is [Na+]i dependent and
trypsin-insensitive. These characteristics are comparable to those of KBR7943, but its efcacy is much higher, suggesting that YM-244769 is a
representative NCX inhibitor in existing benzyloxyphenyl derivatives.
Paper No.: 2434

NICOTINIC ACETYLCHOLINE RECEPTOR STIMULATION
ENHANCES THE DNA SYNTHESIS AND PROLIFERATION OF
MOUSE INDUCED PLURIPOTENT STEM CELLS
Yasuhiro Watanabe(1), T Ishizuka(1), T Hiramoto(1)
National Defense Medical College, Department of Pharmacology,
Tokorozawa, Japan
Previous studies reported that nicotine stimulates the proliferation of
endothelial progenitor cells through nicotinic acetylcholine receptors
(nAChR).However, the effects of nAChR on the proliferation of induced
pluripotent stem (iPS) cells remains unknown. In the present study, we
examined whether nAChR stimulation regulates the proliferation or pluripotency of mouse iPS cells. Mouse iPS cells were cultured under feederfree conditions in the presence of leukemia inhibitory factor. Twenty-four
hours after re-plating, the cells were treated by nicotine, hexamethonium
(a3b4 nAChR antagonist), or mecamylamine (a4b2 nAChR antagonist)
for 8-24 h. MTT assay and BrdU incorporation assay revealed nicotine
(100-300 nM) signicantly enhanced the DNA synthesis and proliferation of the cells. Pretreatment with mecamylamine (1lM) signicantly
reduced the DNA synthesis and proliferation enhanced by nicotine, but
pretreatment with hexamethonium did not affect them. Alkaline phosphatase staining revealed that the cells were under undifferentiated state
independent of nAChR stimulation. These results suggest that a4b2
nAChR stimulation may enhance the DNA synthesis and proliferation of
mouse iPS cells without affecting the pluripotency.
Paper No.: 2724

DEVELPOMENT OF A NOVEL THERAPEUTIC SYSTEM OF
ADENOVIRUS GENE THERAPY IN COMBINATION WITH
MACROMOLECULAR DRUG THERAPY
Yoshiteru Watanabe(1), N Koizumi(1), Y Yamagishi(1), E Hagiwara(1),
H Mizuguchi(2), M Fujii(1)
(1) Showa Pharmaceutical University, Department of Pharmaceutics &
Biopharmaceutics, Tokyo, Japan
Osaka, Japan
Adenovirus (Ad) vectors induce the clathrin-dependent endocytosis and
macropinocytosis in infected cells. The adenoviral particles are taken up
653
by the clathrin-dependent endocytosis into cells. Furthermore, macropinocytosis have a role to release adenoviral particles from endosome to
cytosol. Thus clathrin-mediated uptake of adenovirus together with signal-stimulated macropinocytosis leads to viral effective infection. We
focused the macropinocytosis induced by Ad vector for drug delivery,
because macropinocytosis can be used delivery of macromolecule and
nanoparticle in Ad vector infected cells. We have made a hypothesis that
combination of Ad gene expression and delivery of therapeutic macromolecule in the target cells are used more effective therapeutic effect
compared to each therapeutic method. The aim of the present study was
to develop a novel therapeutic system of Ad gene therapy in combination
with macromolecular drug therapy. We observed that Ad-induced macropinocytosis in the infected HepG2 cells. Also, we found that macropinocytosis induced by Ad vector can be delivered a model of
macromolecular drug, FITC-dextran (M.W. 40,000), into the cells. Furthermore, we investigated the uptake of FITC-dextran into the cell by
each adenoviral receptor, which are coxsackievirus and Ad receptor
(CAR), integrins and heparan sulfate glycosaminoglycans (HSG), binding- ablated mutant Ad vector. The HSG binding-ablated mutant Ad vector induced macropinocytosis leader than conventional Ad vectors. These
results suggested that the HSG binding-ablated Ad vector is a useful tool
for the therapeutic system of Ad gene therapy in combination with macromolecular drug therapy.
Paper No.: 1628

DISEASES
POSSIBLE INVOLVEMENT OF SRC FAMILY KINASES IN THE
RAT BRONCHIAL SMOOTH MUSCLE CONTRACTION
Yu Watanabe, H Sakai, Y Chiba, M Misawa
Tokyo, Japan
The protein serine/threonine kinases such as myosin light chain kinase
and protein kinase C are believed to play important roles in the contraction of smooth muscles. In addition, an involvement of tyrosine kinases
in the contraction has also been suggested in vascular smooth muscles.
Src family kinases belong to the non-receptor protein tyrosine kinases,
but their functional importance in bronchial smooth muscle is unclear. In
the present study, the roles of Src family kinases in the contractions
induced by carbachol (CCh) and high potassium ion- depolarization were
determined in rat bronchial smooth muscles. Both the CCh- and high
potassium ion-induced contractions were signicantly inhibited by nonselective protein tyrosine kinase inhibitors, genistein and ST638. Interestingly, the contraction induced by high potassium ion-depolarization was
more sensitive to genistein and ST638 than that induced by CCh. On the
other hand, the contraction induced by high potassium ion, but not by
CCh, was inhibited by SU6656 (a selective Src family kinase inhibitor).
In addition, orthovanadate, a potent protein tyrosine phosphatase inhibitor, caused force development in bronchial smooth muscle, concentration-dependently. The contraction induced by orthovanadate was
signicantly inhibited by nicardipine, a voltage dependent calcium ion
channel inhibitor. In conclusion, Src family kinases might be involved in
the activation of voltage-dependent calcium ion channels in rat bronchial
smooth muscles.
Paper No.: 2679

OLIGOMERIZATION OF CHEMOKINE RECEPTORS CXCR3
AND CXCR4
Anne Watts(1), M van Lipzig(1), S Jahnichen(1), R Alimahomed(1),
G Zaman(2), M van der Lee(2), N Siderius(1), H Vischer(1), M Smit(1),
R Leurs(1)
(1) VU University Amsterdam, Leiden/Amsterdam Center for Drug
Research, Division of Medicinal Chemistry, Amsterdam, The Netherlands
(2) Schering Plough Research Institute, Molecular Pharmacology Unit,
Oss, TheNetherlands
Chemokine receptors (CKRs) form a large family of GPCRs that are
involved in immune cell chemotaxis. A number of CKRs were shown to
dimerize, which appears to be of importance for functionality of the
receptors and of drugs targeting these CKRs. The CKRs CXCR3 and
CXCR4 are coexpressed on activated T cells and certain tumor cells and
are consequently potential drug targets in autoimmune disease, HIV
infection and cancer. In this study, we show oligomerization between
CXCR3 and CXCR4 using resonance energy transfer and coimmunoprecipitation. Oligomerization between CXCR3 and CXCR4 modulates
radioligand binding kinetics and results in negative binding cooperativity
between the endogenous ligands of the two receptors. Taken together,
these data indicate that CXCR3 and CXCR4 form oligomers with consequences for receptor function.
Paper No.: 2439

THE EVALUATION OF QUALITY OF PHARMACOLOGICAL
TREATMENT IN ELDERLY PATIENTS
Martin Wawruch(1), L Kostkova(1), A Macugova(1), K Strateny(1),
M Kuzelova(2), D Jezova(1,3), A Dukat(4), V Kristova(1)
(1) Comenius University, Faculty of Medicine, Department of
Pharmacology and Clinical Pharmacology, Bratislava, Slovakia
(2) Comenius University, Faculty of Pharmacy, Department of
Pharmacology and Toxicology, Bratislava, Slovakia
(3) Slovak Academy of Sciences, Institute of Experimental
Endocrinology, Bratislava, Slovakia
(4) Comenius University, Faculty of Medicine, Bratislava,
2nd Department of Internal Medicine, Slovakia
Introduction: Elderly patients represent a vulnerable group of the population regarding the pharmacotherapy. There are some medications, the
administration of which is associated with increased risk of adverse
effects in older patients. Patients: The sample of our study was selected
from 1045 patients aged 65 or more hospitalized in a general hospital.
Three quality indicators were used to analyze the pharmacotherapy: use
of potentially inappropriate medications, adverse drug reactions (ADRs)
leading to hospital admission and polypharmacy. Results: Almost two
thirds of patients were taking 6 drugs or more. One fth of patients were
treated with potentially inappropriate medications, which included mainly
cardiovascular and psychotropic drugs. Heart failure and depression
appeared to be predictors of potentially inappropriate medication use.
Falls represented the most common ADR leading to hospital admission.
Conclusion: Our study conrmed that the prevalence of potentially inappropriate medication use in hospitalized older patients is high. The specic criteria of potentially inappropriate medication that reect regional
differences in prescription habits should be created. The results of our
study indicate the necessity to pay more attention to the specic aspects
of pharmacotherapy in elderly patients. This study was supported by
grants VEGA 1/0135/09 and 1/0314/08.
654
Paper No.: 402
DISEASES
B LYMPHOCYTE STIMULATOR RECEPTORS AND AUTOIMMUNE DISEASES
Wei Wei
Anhui Medical University, Institute of Clinical Pharmacology, Hefei,
PR China
B lymphocyte stimulator (BLyS, also known as TALL-1, BAFF,
THANK And zTNF4) plays a key role in B lymphocyte differentiation,
survival and activation. The elevated levels of BLyS and APRIL homotrimers, circulating heterotrimeric complexes of BLyS and APRIL have
also been shown to be elevated in serum from patients with systemic
immune-based rheumatic diseases (including SLE, RA, Reiters syndrome, psoriatic arthritis, polymyositis, and ankylosing spondylitis), and
have been shown to induce B cell proliferation in vitro. B cell membrane
receptors evolve and change throughout the B cell life span. TACI,
BCMA and BAFF-R are present on both immature B cells and mature B
cells. TACI is also expressed on activated T cells. TACI-Ig is a recombinant fusion protein built with the extracellular ligand binding portion of
TACI. It blocks activation of TACI by April and BLyS. Decreasing the
levels of BLyS and APRIL in vivo is expected to result in a decrease in
circulating mature B cells and Ig-secreting cells, and a consequent drop
in Ig levels. TACI-Ig inhibited collagen-induced arthritis in DBA-1 mice
and B lymphocytes and antibodies in MRL/lpr mice.
Paper No.: 2449

ON HUMAN RELEVANCY OF PHARMCO-TOXICOLOGICAL
EXPERIMENTS ON EXAMPLE OF GENITO-UROLOGY
Ursula Welscher(1,2,3), J Stiglmayr(2), J Milbradt(2), E Neu(1,2,3), HW
Bauer(4,5), R Wilkowski(5), N Moro(1), MC Michailov(1,2,3),
R Schatzle(1), L Zebuhr(1), W Seidenbusch(3.1), J Foltinova(6)
Muenchen, Germany
Germany
Austria
(4) FU Berlin, Medical Faculty, Berlin, Germany
(5) University of Muenchen, Medical Faculty, Muenchen, Germany
Introduction: Immense numbers of publications about pharmacological
animal experiments in genito-urology are not evident for human relevancy. About differences in motor reactions of animal&human preparations (surgical-tissue) was reported (Michailov et al, Urol. int.1983;
38:234-242; Neu, Stiglmayr et al, Acta-Pharm.Sinica 2006;27/1:411;
Ber. Bayer.Ges.Gyn./Alete-Wiss. 1989; 243-250; Welscher et al, ActaPhysiol., EPHAR-2007 Bratislava; 191/658:52; J.Physiol.Sci.; IUPS2009 Kyoto; 59/1:241). Method: Spontaneous contractions (SC) & to
electrical neurogenic (CENS: 10/100Hz, 0.3ms, 3s)/myogenic (CEMS:
10Hz, 40ms, 3s) stimulation of vesical detrusor (D), pyelon (P), ureter
(U), myometrium (M), vas deferens (VD) are recorded. Preparations:
human (H), guinea-pig (GP), rat (R). Results (recent&earlier): 1. Ions. HSC-amplitudes increase 2-times stronger after CaCl2 (2.1mM) than GP.
KCl-inuence (5.6mM) on SC-frequency is reciprocal. 2. Hormones.
Prostaglandin A1 (10-100nM) has stimulatory effect on H-U-SC, but
inhibitory on GP-U (a). Prostaglandin F2a inhibits CENS of GP-VD, but
potentiates H-VD (b). CEMS of GP-VD is potentiated, of H inhibited
(c). H/GP/R-Sprague-Dawley-D reacts to adrenaline (0.1-10lM) with

relaxation, R-Wistar-D with contraction (d). 3. Drugs. -sympathomimetics buphenin (1-100lg/ml) has inhibitory effect of GP-SC, but stimulatory of H (a). Combined herbal extract (UrolR (rad.rubiae, rad. taraxaci,
etc.: 0.1-10lg/ml) augments H-, but inhibits GP-SC (b). 4. Toxicants.
High sensitivity of H-D to mercury (HgCl2: 0.1-100nM), GP-D (110lM), also 5. to X-irradiation: H-D (1-30Gy/min, threshold doses 15Gy), GP-D (50Gy). 6. Varia. Differences in reactivity (1-5), other factors (thermo-induced contractions: 37-5C, etc.), are observed also in H/
GP-M. Conclusion: According to medical/bio-ethics IUPHAR could recommend large application of isolated H-preparations in pharmaco-physiological research supporting H-relevancy of results & reducing animal
experiments.
Paper No.: 1383

HEALTH AND DISEASE
20-HETE ACTIVATION OF TRPV1 IN SMALL DIAMETER
PRIMARY SENSORY NEURONS
Hairuo Wen(1,2), J Ostman(1), C Panayiotou(2), J Priestley(1),
M Baker(1), A Ahluwalia(2)
(1) Queen Mary University of London, Blizard Institute of Cell and MolecularScience, London, UK
(2) Queen Mary University of London, William Harvey Research Institute, London, UK
An interaction between blood vessels and their primary afferent innervation has been suggested to underlie intraluminal pressure-induced constriction (Scotland, RS. et al, Circ Res. 2004; 95: 1027-34), linking the
release of the arachidonic acid metabolite 20-hydroxyeicosatetraenoic
acid (20-HETE) to the activation of the vanilloid receptor, TRPV1, on
unmyelinated sensory nerve endings in the vessel wall. We have now
demonstrated an action of 20-HETE on TRPV1 in vitro consistent with
this proposal. Whole-cell patch-clamp experiments were performed on
primary sensory neurons from adult C57/BL6 (WT) and TRPV1 knockout (KO) mouse dorsal root ganglia and on HEK293 cells expressing
human TRPV1 (Caterina, MJ. et al, Nature. 1997; 389:816-24). Heterologously expressed hTRPV1 was activated by 20-HETE (1-100 lM) with
a maximum response at 100 lM (EC50 = 12.48 1.08, n = 7), while it
had no effect in untransfected control cells (n = 5). 20-HETE (10 lM)
also evoked currents in DRG neurons (I =23.89 pA/pF, n = 39), that were
reduced by ~90% (I =2.47 pA/pF, P < 0.01, n = 31) in neurons from
TRPV1 KO mice. Calcium imaging conrmed the activity of 20-HETE (30
lM) with a rise of [Ca2+] i (F/F0 = 1.26 0.05, n = 25, P < 0.001) in WT
neurons but not in KO (F/F0 = 1.04 0.02, n = 10). We conclude that 20HETE is a novel activator of TRPV1, consistent with its proposed role as a
local excitatory mediator in an interaction between the endothelium and
perivascular C-bres. Funded by The Wellcome Trust.
Paper No. 2896

FOCUS GROUP: P09 - NFLAMMATION AND IMMUNOPHARMACOLOGY: NEW TOOLS FOR OLD DISEASES
EXPRESSION AND FUNCTIONALITY OF LEPTIN RECEPTOR
(OB-R) IN THE HUMAN NEAR-TERMMYOMETRIUM
Maeva Wendremaire(1,2), F Lirussi(0), M Sediki(0), F Goirand(2),
C Peyronel(3), S Lionnais-Couvreur(3), M Dumas(2), P Sagot(3),
M Bardou(1,2),
(1) CHU Dijon Faculty of Medicine, INSERM CIC-P 803, Dijon, France
(2) University of Dijon Faculty of Medicine, LPPCE (EA 2979), Dijon,
France
(3) CHU Dijon, Department of Gynaecology and Obstetrics, Dijon,
France
655
Leptin is produced by adipose tissue and involved in the regulation of
foodintake, energy homeostasis and reproduction. As leptin is secreted
by theplacenta, circulating levels of leptin increase during pregnancy.
Maternalobesity is associated with a wide spectrum of delivery disorders,
such asdelayed and difcult labor. It has been suggested that leptin is a
potentinhibitor of myometrial contractility in vitro. However, leptin receptorsexpression and functionality have never been demonstrated, as of
today in humanmyometrium. This study was aimed to assess leptin
receptor (Ob-R) expression,and intracellular signalling pathways induced
by leptin, in human pregnantmyometrium. Myometrial biopsies were
obtained from 10 women with uncomplicatedpregnancy who delivered
by caesarean section at term. Leptin receptors weredetected by immunohistochemistry, RT-PCR and western blotting experiments. Ob-Rintracellular signalling pathways, JAK/STAT, ERK1/ERK2, p38 MAPK and
PI3K/AKTwere investigated by western blotting experiments performed
in tissueschallenged with leptin (10-10 to 10-7 M). RT-PCR and western
blottingexperiments (n = 5) showed that both long and short isoforms are
expressed inhuman pregnant myometrium. Immunochemistry staining
conrmed the localizationof the receptors in the smooth muscle cells (n
= 3). Leptin induced activation ofextracellular signal-regulated kinase
(ERK1/2) after a 60-minute stimulation, ina concentration dependent
manner. Our results demonstrate for the rst timethat leptin receptors are
expressed and functional in the myometrium of pregnantwomen. Stimulation of these receptors activates ERK1/2 pathway. Our worksupports
the need for further studies on the role of leptin in human pregnancy.
Paper No. 2897

ORAL PRESENTATION
FOCUS GROUP: P09 - NFLAMMATION AND IMMUNOPHARMACOLOGY: NEW TOOLS FOR OLD DISEASES
LEPTIN PREVENTS LPS-INDUCED APOPTOSIS IN AN
IN VITRO HUMAN MODEL OF MYOMETRIAL
INFLAMMATION
Maeva Wendremaire(1,2), F Lirussi(0), M Sediki(0), F Goirand(2),
S Lionnais-Couvreur(3), C Peyronel(3), M Dumas(2), P Sagot(3),
M Bardou(1,2)
(1) INSERM CIC-P 803, CHU Dijon (France)
(2) LPPCE (EA 2979), Faculte de Medecine, Dijon (France)
(3) Service de Gynecologie-Obstetrique, CHU Dijon (France)
Maternal obesity is associated with many delivery disorders, such as
delayed orpost-term delivery. Obesity increases plasma leptin levels. We
have evidencedthe presence of leptin receptors in human pregnant myometrium. A role for leptinin delivery is suggested since leptin inhibits,
in vitro, myometrialcontractility, suggesting a role in obesity-related
delivery disorders. Deliveryis characterized by a down-regulation of proliferation-related genes, alongwith the up-regulation of inammatory,
contraction and apoptosis associatedgenes. This study assessed the ability
of leptin to interfere with LPS-inducedapoptosis, in a validated model of
human myometrial inammation. Myometrialbiopsies, obtained from 14
women with uncomplicated pregnancy, delivered bycaesarean section at
term, were dissected and allowed to stabilize at 37C for48h. They were
stimulated with LPS (10 lg/ml) for 48h without or with leptin(10-10 to
10-7 M). Apoptosis was assessed by chromatin condensation andexpression of pro- and anti-apoptotic markers. DAPI staining showed thatLPSinduced increase in apoptotic bodies and chromatin condensation wasprevented by leptin treatment. LPS challenge was associated with an increasedexpression of pro-apoptotic protein BAX and cleaved caspase-3
and a decreasedexpression of anti-apoptotic protein BCL2 that were prevented by leptin in aconcentration dependent manner. Anti-apoptotic
effects of leptin were abolishedafter pretreatment with a selective Ob-R
antagonist. These results show thatleptin prevents LPS-induced apoptosis
in human myometrium and provide newinsight into the physiology
of delivery disorders in obese women. They suggestOb-R as a
potential therapeutic target for the pharmacological management
ofpreterm labor.
Paper No.: 1322

HEALTH AND DISEASE
REDUCED ACTION OF SILDENAFIL IN PULMONARY
ARTERIES FROM MICE LACKING THE PKGI OR THE BKCA
CHANNEL
Matthias Werner(1), S Lia(1), M Nelson(1,2), A Gurney(3)
(1) University of Manchester, Faculty of Medical and Human Sciences,
Division of Cardiovascular and Endocrine Science, Manchester, UK
(2) University of Vermont, Department of Pharmacology, Burlington,
Vermont, USA
(3) University of Manchester, Faculty of Life Sciences, Manchester, UK
Pulmonary arterial hypertension (PAH) is a severe disease with impaired
exercise tolerance, heart failure and short life expectancy. Sildenal, one
of the newest drugs to treat PAH, elevates the concentration of cGMP in
pulmonary artery smooth muscle (PASM) by inhibiting its degradation.
The cGMP-dependent protein kinase I (PKGI) and the large-conductance, Ca2 + -activated potassium (BKCa) channel are considered to be
important mediators of sildenal-induced pulmonary vasodilation. To
address the roles of these proteins directly, myography experiments using
PA rings from wild type mice and from mice that lack either the PKGI
(PKGI-/-) or the BKCa channel (BKCa-/-) were performed. PA rings were
constricted with 50mM KCl, washed then pre-constricted with the aadrenoceptor agonist phenylephrine (1lM) and sildenal applied at
increasing concentrations. Constriction induced by depolarising PASM
with high [K+] did not differ between wildtype and PKGI-/- or BKCa-/-,
suggesting no change of voltage-dependent Ca2 + currents in the knockout models. Similarly, no difference in contractile force in response to
phenylephrine was detected between the genotypes. Sildenal dosedependently relaxed pre-constricted wildtype PA rings. This relaxing
effect was almost entirely abolished in PA rings from PKGI-/- mice. In
BKCa-/- PA rings, the sildenal effect was not abolished, however the
dose-response-curve was shifted signicantly towards higher concentrations (wildtype: pEC50 = 7.5 0.5, n = 6; BKCa-/-: pEC50 = 4.0 0.5, n
= 9, P < 0.001). These results demonstrate an absolute requirement for
PKGI and cross-activation of the cAMP signalling pathway seems not to
be involved in the action of sildenal. Likewise, the BKCa channel
appears to be an important effector of sildenal relaxation in pulmonary
arteries.
Paper No.: 3266

A MULTIPLEXED PRIMER EXTENSION DENATURING HIGH
PERFORMANCE LIQUID CHROMATOGRAPHY METHOD
FOR THE IDENTIFICATION OF 3 ABCC2 (MRP2) GENETIC
POLYMORPHISMS
Ian Westley(1,2), J Coller(3), M Ward(2), A Evans(2), R Morris(1,3),
B Sallustio(1,3)
(1) The Bazil Hetzel Institute, The Queen Elizabeth Hospital. Department
of Clinical Pharmacology, Adelaide, SA, Australia
(2) University of South Australia., School of Pharmacy and Medical
Sciences, Adelaide, SA, Australia
Australia
Mutations in the genes coding for transporters cause or contribute to
many human genetic disorders. The aim of this study was to develop a
multiplexed primer extension denaturing high performance liquid chromatography (PE-dHPLC) method to determine three MRP2 genetic polymorphisms in the promoter region (-24C>T), exon 10 (1249G>A) and
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exon 28 (3972C>T) in a single extension reaction. Genomic DNA was
extracted from whole blood (EDTA-anticoagulant) samples. PCR reactions contained 100ng DNA, primers (0.2lM), PCR buffer, dNTP
(200lM) and 1.0U Taq DNA polymerase. Puried PCR products were
analysed by sequencing and PE-dHPLC. For sequencing, puried PCR
products were treated with isopropranol precipitation, Big dye sequencing mix, and either forward primer (0.2lM, -24C>T and 3972C>T SNPs)
or reverse primer (0.2lM, 1249G>A SNP). The PE reaction contained
puried PCR product, primers (5ng/lL), detergent-free Thermopol buffer, dNTP/ddGA (2.5mM) and 1.0U Therminator. PCR PE product
(10lL) was injected into Transgenomic Wave Nucleic Acid Fragment Analysis System (Transgenomic Incorporated, USA). Mobile
phase consisted of Transgenomic Buffer A (0.1M TEAA) and a linear gradient (18-39%) of Transgenomic Buffer B (0.1M TEAA:
25% Acetonitrile) at 1.2mL/min (rt = 9min). Samples were separated using a Transgenomic DNASep cartridge with UV detection
(260nM) and analysed using standard Transgenomic Wave oligonucleotide purication parameters for oligonucleotides of length 15-50
without uorescent labels. Within each extension reaction, primers were
incubated with water only and the master mix was incubated with no
PCR template to detect non-specic dimer formation. A 100% identity
match was shown with the PE-dHPLC genotype and sequenced genotype
samples (n = 15).
Paper No.: 1571

CHARACTERISATION OF THE MUSCARINIC RECEPTOR
SUBTYPE MEDIATING CONTRACTION IN THE MOUSE
PROSTATE GLAND
Paper No.: 2174

HEALTH AND DISEASE
ALBUMIN-FACILITATED EXTRAVASATION OF SIRNA
ACROSS VASCULAR ENDOTHELIUM IN VIVO
Paul White, S Lau, N Cao, B Graham, C Pouton, B Boyd
Monash University, Department of Pharmaceutical Biology, Parkville,
VIC, Australia
A major bottleneck of siRNA therapeutics is delivery across the vascular
endothelium to desired cells. This study aimed to bypass the endothelial
barrier by harnessing the transcytosis mechanism of the serum protein
albumin to reach targeted cardiomyocytes. In situ albumin-siRNA conjugates were produced by reacting 5-amine-modied siRNA with succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate to yield thiolreactive siRNA that irreversibly links to cysteine residues within albumin. An IGF-I-receptor siRNA sequence which produced 90% knockdown in vitro was used. In situ conjugation of activated amine-modied
siRNA to albumin in mouse serum occurred within minutes of addition
and was found to be nuclease stable using SDS-PAGE. Intravenous
injection of this activated siRNA (1 mg/kg) in mice signicantly reduced
left ventricle IGF-IR mRNA to 64.12 4.14% (meanSEM), whilst a
control siRNA (unconjugated) had no effect (n = 4, P > 0.05). In a second strategy, albumin and siRNA were formulated into nanoparticles
using electrostatic attraction via a cationic lipopeptide to facilitate escape
from the endosome. Imaging of myocardium from mice treated with
nanoparticles containing Alexa-488-siRNA showed clear evidence of
extravasation and cellular uptake after administration of siRNA-albuminlipopeptide particles but not siRNA alone or siRNA mixed with albumin.
siRNA-albumin constructs are therefore capable of extravasation to the
myocardium resulting in silencing in this otherwise silencing-resistant
organ.
Carl White, J Short, S Ventura

Monash University, Monash Institute of Pharmaceutical Sciences, Parkville, VIC, Australia
Benign prostatic hyperplasia (BPH) is a disorder caused by the ageing
prostate gland physically impeding the smooth ow of urine through the
urethra. Lower urinary tract symptoms due to BPH arise from an enlargement of the prostate and an increase in prostatic smooth muscle tone. In
humans and mice, the majority of prostatic contraction is attributed to
activation of a1-adrenoceptors by noradrenaline release from sympathetic
nerves. Electrical eld stimulation of the isolated mouse prostate in the
presence of prazosin or guanethidine elicits a residual non-adrenergic
contraction that is mediated by acetylcholine acting at muscarinic receptors. Using isolated organ bath studies we investigated the muscarinic
receptor subtype responsible for the cholinergic component of contraction. Exogenous application of the muscarinic receptor agonist carbachol
elicits reproducible, and concentration dependent contractions of the isolated mouse prostate that are sensitive to hexamethonium (10 lM) and
prazosin (0.3 lM) but not physostigmine (10 lM). Furthermore, concentration-response curves to carbachol were shifted rightwards in a concentration dependent manner in the presence of muscarinic receptor
antagonists. The rank order of antagonist afnities (pKB -values) to the
carbachol-elicited response was: atropine (9.06) > 4-DAMP (9.03) > hexahydrosiladifenidol (7.85) para-uoro-hexahydrosiladifenidol (7.47) >
himbacine (7.35) pirenzepine (7.32) > methoctramine (6.1). Such an
antagonist prole is indicative of the M3 muscarinic receptor subtype.
Therefore the non-noradrenergic, cholinergic component of smooth muscle contraction in the mouse prostate appears to be mediated by the M3
muscarinic receptor subtype.
Paper No.: 2841

EVALUATION OF A LIMITED SAMPLING STRATEGY FOR
DETERMINATION OF THE MYCOPHENOLIC ACID-AUC IN
DIFFERENT RENAL PEDIATRIC PATIENTS
Martin Wiesen(1), M Feldkotter(2), H Stutzer(3), C Muller(1)
(1) University Hospital of Cologne, Department of Pharmacology,
Cologne, Germany
(2) University Hospital of Cologne, Division of Pediatric Nephrology,
Department of Pediatrics, Cologne, Germany
(3) University Hospital of Cologne, Institute of Medical Statistics, Informatics and Epidemiology, Cologne, Germany
In a retrospective investigation, a limited sample strategy (LSS) for determination of AUC0-12h from mycophenolic acid (MPA)-trough levels
(C0) and MPA-levels after 30 (C0.5) and 120 min was evaluated in a
renal pediatric patient collective. Fifty-eight patients aged 1.6-20.6 years
(4 patients over 18 years) were included between 2004 and 2010: 40
patients after renal transplantation (RT, m:f = 25:15, nAUC=59), 14
patients with nephrotic syndrome (NS, m:f = 5:9, nAUC=26), and 4
patients with Henoch-Schonlein purpura nephritis (HSPN, m:f = 3:1,
nAUC=5). C0, C0.5, and C2 serum samples were collected after oral
intake of a steady-state maintenance dosage of 120-1000mg Cellcept PO
BID. MPA-concentrations were determined by an evaluated HPLCmethod with an internal standardization. Based on a two-compartment
model, MPA-AUCs0-12 were calculated from individualized PK-parameters using the Bayesian tting method. Correlations between MPAAUC0-12
and the sum of P
corresponding C0, C0.5, and C2
P
( C0,C0.5,C2) and C0 and C2 ( C0,C2) levels were characterized by
linear regression analysis respectively. Mean MPA-AUC in all patients
was 37.2 21.4 (range 8.6-144.5) mg*h/L. For RT, NS, and HSPN
657
patients, mean MPA-AUC was 35.7 17.4, 41.2 29.7, and 33.9 14.3
mg*h/L. Correlation between MPA-AUC and RC0,C0.5,C2 was r2 =
0.806 (p < 0.0001) and between MPA-AUC0-12 and RC0,C2 r2 = 0.851
(p < 0.0001). Assuming a target MPA-AUC of 20-60mg*h/L, the derived
formula AUC=6.102 + RC0,C0.5,C2*1.855 falsely predicted 13 MPAAUCs within or off this range. In contrast, the derived formula
AUC=5.567 + RC0,C2*5.598 falsely predicted 10 MPA-AUCs, thus
appearing preferable for estimation of MPA-AUC0-12h. A LSS using C0
and C2 levels may sufciently predict MPA-AUCs0-12h in pediatric
patients. However, further clinical investigations are needed to evaluate
this approach.
Paper No.: 1902

CELL MEMBRANE CROSSING OLIGOMERS - A NOVEL
ANTISENSE THERAPY FOR REDUCTION OF
APOLIPOPROTEIN B-CONTAINING LIPOPROTEINS
Georg Wietzorrek(1), Y Nakada(1), B Werner(2), H Bock(2),
T Lindhorst(2), H Dieplinger(1)
nib alaninate (400 mg) was administered concomitantly with ketoconazole (400 mg). Plasma samples for PK analysis of brivanib were collected
up to 48 hrs on Day 1 and Day 8 and analyzed using a validated liquid
chromatography-tandem mass spectrometry method. The PK properties of
brivanib were investigated using non compartmental analysis. Results: Peak
concentrations for brivanib were rapidly absorbed alone and with ketoconazole. T-Half estimates were consistent across treatments (~12 h). Cmax
was similar; AUC (INF) was 16% higher in the presence of ketoconazole;
however, the 90% CI were within 80-125% of the reference value. Conclusion: Concomitant administration of brivanib alaninate with ketoconazole
was associated with no clinically signicant increase in brivanib exposure.
Brivanib alaninate may be safely co-administered with ketoconazole and
other CYP3A4 inhibitors with no dose adjustments.
Paper No.: 3240

SUPPORTED CELL-MEMBRANE SHEETS AS A MODEL
SYSTEM TO ANALYZE PATHWAYS OFINTRACELLULAR
GPCR SIGNALLING
Volker F Wirth, TC Mller, KL Martinez

Molecular & Clinical Pharmacology, Innsbruck, Austria
(2) Ugichem GmbH, Innsbruck, Austria
Elevated plasma concentrations of LDL and other apolipoprotein B
(apoB)-containing lipoproteins represent well-recognised risk factors for
developing cardiovascular diseases. Current pharmacological interventions show limited effects, particularly in patients with genetic hyperlipemia and call for a novel therapeutic approach. Here we report results
from an approach to lower plasma LDL concentrations by reducing apoB
production in liver cell culture and mice using a novel antisense
approach: cell-membrane crossing oligomers (CMCOs), whose unique
structure allows penetration of cell membranes without needing an additional carrier. In an in-vitro-approach in mouse primary cell culture, a
reduction of 75% apoB production could be observed. 3-day i.v. treatment of adult c57bl mice resulted in reduction of total serum cholesterol
by 10%, non-HDL cholesterol by 30%, triglycerides by 20% and apoB
by 50%. ApoB protein was reduced in liver homogenates by 30%
whereas apoB mRNA was not altered. These results proof the efcacy
and safety of this novel antisense strategy to lower atherogenic lipoproteins in mice by means of cell-membrane-crossing oligomers.
Paper No.: 1098

LACK OF DRUG-DRUG INTERACTION BETWEEN
KETOCONAZOLE AND BRIVANIB
Daphne Williams, S Syed, D Lathers, G Kollia, A Gupta, J Pursley, Y-Q
Xia, E Masson
Bristol-Myers Squibb, Department of Discovery Medicine & Clinical
Pharmacology, Princeton, NJ, USA
Purpose: Brivanib alaninate is the ester prodrug of the active moiety brivanib, an oral selective inhibitor of the vascular endothelial growth factor
(VEGF) and broblast growth factor (FGF) signalling pathway which is
in phase III for the treatment of advanced colorectal cancer and hepatocellular carcinoma. Brivanib is primarily metabolized by the liver, including CYP3A4 and other metabolic pathways. To evaluate the effect of
ketoconazole on the pharmacokinetics (PK) of brivanib, an open-label
study was conducted in 16 healthy subjects. Methods: On Day 1, brivanib alaninate (400 mg) was administered as a single dose. On Days 4 to
7, ketoconazole (400 mg) was administered once daily. On Day 8, briva-
University of Copenhagen, Bio-Nanotechnology Laboratory, Department

of Neuroscience and Pharmacology & Nano-Science Center, Copenhagen, Denmark
G-protein coupled receptors (GPCRs) are key players in the regulation of
a broad variety of cellular processes. Thus, they represent a main group
of target proteins for commercially available drugs and in drug discovery.
One common challenge for the in-vitro analysis of GPCR activity is to
reconstitute these transmembrane proteins in a suitable environment
while conserving both structure and function of the receptor proteins. We
present a method to transfer fragments of native cell membranes to solid
substrates suitable for uorescence microscopy down to single molecule
tracking. The supporting surface is coated with positively charged polylysine and electrostatically attached to the negatively charged cell membrane. The attached parts of the membranes can be ripped off by
mechanical stress from the intact cells and remain as patches on the surface (Perez et al., Advanced Functional Materials 2006, 16:306-12).
These supported cell-membrane sheets (SCMS) allow easy control of
both the geometry and the orientation of the probes: The lipid bilayer is
aligned along the supporting surface while the intracellular side of the
membrane is exposed to the surrounding solution. Both lipid and protein
composition of the membrane patches is conserved; the intracellular
domains of transmembrane receptors can be directly manipulated. In
addition to this, the absence of background uorescence from the cytoplasm and other cellular components qualies this system for highly sensitive measurements like single molecule spectroscopy or single particle
tracking (Perez et al., J. Molecular Biology 2006, 363:918-30).
Paper No.: 3390

THE STRUCTURAL AND FUNCTIONAL LANDSCAPE OF THE
ADP RECEPTOR P2Y12
Doreen Wittkopf(1), M Coster(1), A Kreuchwig(2), K-U Simon(1),
G Kleinau(2), D Thor(1), G Krause(2), T Schoneberg(1)
(1) University of Leipzig, Institute of Biochemistry, Leipzig, Germany
(2) Leibniz Institute of Molecular Pharmacology, Berlin, Germany
P2Y12-like receptors, among them the clopidogrel-sensitive ADP receptor P2Y12, belong to the family of rhodopsin-like G protein-coupled
658
receptors (GPCR). Despite considerable sequence similarity, members of
the P2Y12-like receptor group display high specicity towards distinct
nucleotides. The structural basis of ligand specicity is still unknown
although recent crystal structures of GPCR give a rst insight in the
molecular landscape of these receptors. However, these homology models have only a limited predictive value and need additional renement
based on structure-function studies. Here, we combine the information
from naturally occurring P2Y12 variants (orthologs) together with highthroughput saturation mutagenesis, in vitro pharmacological testing and
bioinformatic analysis to generate a dynamic GPCR model. We focused
on the ADP receptor P2Y12 as a model system because it is the best
characterized prototype of P2Y12-like receptors. Saturating mutagenesis
of a receptor portion spanning transmembrane helix 6 to 7, in which each
amino acid position in the human P2Y12 was replaced with all other
possible amino acids (~900 mutants) and functional in vitro-testing provided a detailed picture of the functional effects of every position. Analyses revealed that ~13% of the mutants present wild-type function and
~83% showed partial or complete loss-of-function. The structural conservation found by comparing P2Y12 orthologs strongly correlated with the
functional importance of the respective positions in the in vitro tests. Our
saturating mutagenesis data set together with the ortholog data set were used
to compile a dynamic molecular model of the P2Y12 in which individual positions are depicted spatially and by their biophysical properties.
Paper No.: 1791
STEREOTYPY IN THE DEER MOUSE, CORRELATION WITH
CORTICO-STRIATAL OXIDATIVE STATUS AND DOPAMINE
TURNOVER, AND RESPONSE TO COMBINED FLUOXETINE/
RISPERIDONE TREATMENT
De Wet Wolmarans(1), M Guldenpfennig(1), B Harvey(1), D Stein(2),
J du Preez(1)
(1) North-West University, Department of Pharmacology, Potchefstroom,
South Africa
(2) University of Cape Town, South Africa
The deer mouse presents with stereotypical movements, that are variable
within a population, and which closely resemble behaviours seen in
OCD. OCD is linked to altered redox status. Since dopamine can promote oxidative stress and also increase stereotypies, we postulated that
deer mouse stereotypy may be correlated with abnormalities in this
regard. Deer mice were separated into different stereotypical cohorts.
Frontal DOPAC and HVA, as well as oxidized GSSG and reduced GSH
glutathione were determined by tandem LC-MS. SOD activity was determined using a SOD kit. Saline, uoxetine (5mg/kg/day), risperidone
(2mg/kg/day), and a combination of these, were administered to high stereotypical mice for 3 weeks. Behavioural analyses were performed, animals sacriced, and the above mentioned neurochemical parameters
determined. While chronic treatment with uoxetine separated from control in the 3rd week, with risperidone being completely ineffective. The
combination though was effective in attenuating stereotypy. This provides evidence for the predictive validation of the model. Cortical GSH
and GSSG levels were lower in HSB mice, compared to NS mice, and
showed a signicant correlation with the severity of stereotypy, suggesting a decient glutathione system in this stereotypy.
Paper No.: 1412

PROTECTION OF CATHELICIDIN-ENCODING LACTOCOCCUS LACTIS IN MURINE ULCERATIVE COLITIS
Intrarectal administration of mouse cathelin-related antimicrobial peptide

(mCRAMP) has been shown to effectively reduce intestinal inammation
in mice with acute colitis. With the aid of bioengineering, Lactococcus
lactis could be used as a delivery tool and production machinery for
mCRAMP in order to provide a more convenient way for oral administration and reduce the production cost. Lactococcus lactis NZ3900 was
transformed with mCRAMP gene, and the addition of the inducer
nisin followed by mCRAMP production. Ulcerative colitis (UC) in
mice was induced by 3% dextran sulfate sodium (DSS) dissolved in
drinking water for 7 days. Eight log colony forming unit (cfu) or 10
log cfu of L. lactis NZ3900 or the transformed strains with or without
mCRAMP induction were given as a parallel treatment. Sulfasalazine
(SASP) was used as a reference drug for UC. Body weight, fecal microora populations, clinical symptoms and histological examinations
of colonic tissues were measured. Myeloperoxidase (MPO) activity
and malondialdehyde (MDA) level were also evaluated to reect the
degree of inammation. SASP, L. lactis and its transformants could
improve the clinical symptoms and revert the MDA level after DSS treatment. In difference from SASP, L. lactis encoded with mCRAMP also
preserved the crypt integrity and mucous thickness. The number of apoptotic cells and MPO activity were decreased. The increases of aerobes
and anaerobes in UC animals were also signicantly prevented. L. lactis
encoded with mCRAMP is a better therapeutic agent when compared to
SASP in the treatment of UC.
Paper No.: 2194

HEALTH AND DISEASE
BOTH D- AND L-ARGININE INCREASE CONTRACTIONS TO
DEXMEDETOMIDINE IN THE PRESENCE OF L-NAME IN
THE RAT AORTA WITH ENDOTHELIUM
Emily SW Wong(1), RYK Man(1), PM Vanhoutte(1), KFJ Ng(2)
(1) The University of Hong Kong, Department of Pharmacology and
Pharmacy, Hong Kong,PR China
(2) The University of Hong Kong, Department of Anaesthesiology, Hong
Kong, PR China
Dexmedetomidine is an anesthetic agent which can cause relaxation (due
to the release of NO) and contraction (due to activation of a1 and a2 adreneoceptors) of isolated arteries. The present study investigated whether
or not L-arginine or D-arginine affected the balance between relaxation
and contraction in response to dexmedetomidine. Thoracic aortae with
endothelium were isolated from male Sprague Dawley rats (10 week
old), and suspended in organ chambers for isometric tension recording.
Cumulative concentrations of dexmedetomidine were added to quiescent
aortic rings, incubated with L-NAME (NOS inhibitor) and/or L-arginine
or D-arginine for 40 minutes. Dexmedetomidine caused concentrationdependent contractions in the presence of L-NAME (EMax (% of 60
mM KCl): 48.20 5.49, logEC50: -6.61 0.18) which were potentiated signicantly by both L-arginine (EMax: 81.76 4.42, logEC50: 6.68 0.09, P < 0.05) or D-arginine (EMax: 127.4 3.55, logEC50: 6.90 0.05, P < 0.05). These potentiations were reversed by (S)-(2boronoethyl)-L-cysteine (BEC, arginase inhibitor). No such potentiation
was observed in preparations without endothelium or during contractions to a1-adrenergic agonists. To conclude, both L-arginine and Darginine potentiate a2-mediated contractions by dexmedetomidine in
endothelium intact rat aortae. This potentiation likely involves a product
of arginase.
Clover Ching Man Wong, CH Cho

Shatin, NT, Hong Kong, PR China
659
Paper No.: 1288
HEALTH AND DISEASE
CHRONIC TREATMENT OF VITAMIN D DERIVATIVES
REDUCE ENDOTHELIUM-DEPENDENT CONTRACTIONS IN
THE AORTA OF THE SPONTANEOUSLY HYPERTENSIVE
RAT
Michael Sze Ka Wong, RYK Man, PM Vanhoutte
The University of Hong Kong, Department of Pharmacology and Pharmacy, Hong Kong, PR China
The available evidences suggest that vitamin D has cardiovascular effects
besides regulating calcium homeostasis. Previous studies demonstrated
that 1,25-dihydroxyvitamin D3, the major metabolite of vitamin D,
acutely reduce endothelium-dependent contractions induced by acetylcholine. To examine the chronic effect of 1,25-dihydroxyvitamin D3, rats
were treated with the vitamin D derivative for 6 weeks. The serum 1,25dihydroxyvitamin D3 level was signicantly higher than the control
while the mean arterial blood pressure was signicantly lower. Aortic
rings with or without endothelium were used for organ bath experiments.
The release of prostacyclin and thromboxnane A2 after acetylcholine or
A23187 stimulation were measured. The cytosolic-free calcium concentration was measured by confocal microscopy with the uorescent dyes
Fluo-4. Real time PCR was used to compare the mRNA level of COX-1,
prostacyclinsynthase, thromboxane synthase and eNOS between the control and treated groups. Both acetylcholine- and A23187-induced endothelium-dependent contractions were reduced signicantly in the treated
group. The acetylcholine- induced release of prostacyclin and the
A23187-induced thromboxname A2 was reduced in the treated group.
There was no signicant difference in cytosolic free calcium concentration caused by acetylcholine or A23187 between control and treated
groups. COX-1 mRNA level was signicantly inhibited in the treated
SHR. These results demonstrate that chronic treatment of 1,25-dihydroxyvitamin D3 modulates vascular tone by inhibiting the expression
level of COX-1 mRNA which is a completely different mechanism as in
the acute treatment. This chronic effect to EDCF may account for one of
the factors that reducing the mean arterial blood pressure in the SHR
rats.
Paper No.: 2970

RAPID CHARACTERIZATION OF BIMATOPROST
PHARMACOLOGY IN HUMAN OCULAR CELLS BY USING
CELLULAR DIELECTRIC SPECROSCOPY
David Woodward(1), J Wang(1), D Piwnica(2), R Carling(3),
C Cornell(3), J Martos(3), D Stamer(4)
(1) Allergan, Inc., Department of Biological Sciences, Irvine, CA, USA
(2) Cerep SA, USA
(3) Selcia Ltd, USA
(4) University of Arizona, USA
Bimatoprost is widely used as anti-glaucoma therapy but, to date, the cellular localization and characterization of its effects in individual ocular
cell types has not been achieved. To accomplish this, primary cultures of
human endothelial cells of Schlemms canal (ECSC), trabecular meshwork (TM), and ciliary muscle (CM) cells were prepared from donor
eyes. Cellular dielectric spectroscopy permits rapid pharmacological and
second messenger characterization in primary cells in a 96 well format.
It is a real time, non-invasive, label-free assay, which measures impedance. Bimatoprost produced a concentration-dependent change in cell
monolayer impedance in ECSC, TM, and CM cells, with EC50 (nM)
values of 1.6, 4.3, and 1.4, respectively. These effects were susceptible to
the prostamide antagonist AGN 211334. Bimatoprost effects were insen-
sitive to cholera toxin and pertussis toxin but were abolished by phorbol
12-myristate, 13-acetate. AGN 211334 exerted properties consistent with
inverse agonism in TM cells but not ECSC cells . The pharmacology of
bimatoprost and AGN 211334 in TM cells identied by cell dielectric
spectroscopy was conrmed in its entirety by studying hydraulic conductivity in a TM cell monolayer. In conclusion a bimatoprost-sensitive prostamide receptor was identied and rapidly characterized using cellular
dielectric spectroscopy. Bimatoprost potently stimulates ECSC, TM, and
CM cells and the receptor is Gq coupled. Cellular dielectric spectroscopy
represents a useful technique for rapid characterization of pharmacology
and second messenger signaling in human primary cells.
Paper No.: 977

DISEASE AND THERAPY
PROTEIN KINASES MODULATE STORE-OPERATED
CHANNELS IN PULMONARY ARTERY MYOCYTES
Bin-Nan Wu(1), I-S Chen(1), D Welsh(2), Z-K Dai(3), I-J Chen(1)
(1) Kaohsiung Medical University, Department of Pharmacology, Kaohsiung, Taiwan
(2) University of Calgary, Smooth Muscle Research Group and Department of Physiology and Pharmacology, Calgary, Alberta, Canada
(3) Kaohsiung Medical University Hospital, Division of Pediatric Pulmonology and Cardiology, Department of Pediatrics, Kaohsiung, Taiwan
Background and purpose: This study is the rst to investigate whether
protein kinase G (PKG), protein kinase A (PKA) and protein kinase C
(PKC) are involved in the regulatory mechanisms of store-operated channel (SOC) activation in pulmonary arteries. Experimental approach: Pulmonary artery smooth muscle cells (PASMCs) were enzymatically
dissociated from rat intralobar pulmonary arteries. Conventional whole
cell, cell-attached and inside-out patch-clamp electrophysiology were
used to monitor SOCs in isolated smooth muscle cells. Key results: Initial experiments noted that the Ca2 + -ATPase inhibitor cyclopiazonic
acid (CPA, 10 lM) initiated a whole cell current that was reduced by the
selective SOC blocker SKF-96365 (50 lM). Subsequent work using both
cell-attached and whole cell congurations revealed that the PKG and
PKA inhibitors, KT5823 (3 lM) and H-89 (10 lM), also stimulated
SOC activity; this augmentation was attenuated by the potent SOC
blocker Ni2 + (1 mM). Finally using the inside-out conguration, the
PKC activator phorbol 12-myristate 13-acetate (PMA, 10 lM) was conrmed to modestly stimulate SOC activity although this augmentation
appeared to be more substantial following the bath application of 10 lM
inositol 1,4,5-triphosphate (Ins(1,4,5)P3). Conclusion and Implications:
SOC activity in PASMCs was stimulated by the inhibition of PKG and
PKA and the activation of PKC. Physiologically, SOC activation would
result in the increase of intracellular calcium ([Ca2+]i), which leads to
pulmonary arteriopathy.
Paper No.: 1455

HEALTH AND DISEASE
ROLE OF HYDROGEN SULFIDE (H2S) IN CONDUIT AND
RESISTANCE ARTERIES FROM SEPTIC RATS
Chin-Chen Wu(1), S-J Chen(2), C-T Chiu(1), M-H Liao(1)
(1) National Defence Medical Centre, department of Pharmacology,
Taipei, Taiwan
(2) Kang-Ning Junior College of Medical Care and Management, Taipei,
Taiwan
Severe hypotension, an important characterization of circulatory failure
in late sepsis, causes tissue hypoperfusion and leads to multiple organ
660
failure. It can largely be accounted for NO overproduction. Recently,
H2S has been reported that associated with inammation and it may
cause vasodilatation. Thus, we hypothesized that H2S decreased conduit
and resistance vessel tone and contributed to hypotension in sepsis. In
this study, we examined effects of sodium hydrosulde (NaHS, a donor
of H2S) on thoracic aortas (TA) and mesenteric arteries (2nd branch,
MA)) from rats with sepsis, and evaluated whether H2S could modulate
effect of NO. The vascular reactivity to norepinephrine (NE) was
examined in vivo and ex vivo, whereas the response to NaHS of TA
and MA were measured ex vivo. In addition, the changes of hemodynamics, blood glucose, hepatic and renal functions were also monitored. Our preliminary results showed that (i) rats with sepsis
manifested hypotension, hypoglycemia, and organ dysfunction, (ii) the
NaHS-induced contraction (at low concentrations) in aortic rings from
septic rat was enhanced, (iii) the relaxation elicited by NaHS (at high
concentrations) was not signicantly different from control rats, (iv)
PAG (an inhibitor of H2S formation) attenuated the NaHS-induced
relaxation in TA and MA from both groups, whereas L-NAME (an
inhibitor of NO formation) potentiated the NaHS-induced relaxation in
TA from both groups, and (v) L-NAME attenuated NaHS-induced relaxation in MA from the SOP group only. In conclusion, our results suggest
that H2S plays a minor role in vascular hyporeactivity to NE in endotoxin-induced sepsis.
Paper No.: 2100

APOPTOSIS OF U937 CELLS INDUCED BY BRUCEA
JAVA NICA OIL EMULSION
Chunfu Wu(1), H Zhang(1), J Yang(1), L Wang(1), F Zhou(2),
W Zhang(1)
(1) Shenyang Pharmaceutical University, Department of Pharmacology,
Shenyang, PR China
(2) Shenyang Military General Hospital, Department of Hematology,
Shenyang, PR China
Introduction: Brucea Javanica oil emulsion (BJOE) has been used
clinically in China for years to treat lung cancer, prostate cancer and
gastrointestinal cancer, but whether it can be used to treat acute myeloid leukemia (AML) has not been reported. In this study, we investigated the anti-cancer effects of BJOE in vitro, and elucidated its
underlying molecular mechanisms.Materials/Patients: Human AML cell
lines U937 and patients leukemia cells were used to test the antileukemia activity of BJOE. Lymphocytes from fourteen cases of newly
diagnosed AML patients and four healthy volunteers were isolated to
test the therapeutic relevance of BJOE. Results: BJOE concentrationdependently decreased the viability of U937 cells in MTT assay. Cell
cycle examination, cleaved poly (ADP-ribose) polymerase (PARP) and
DNA fragmentation indicated that BJOE signicantly induced apoptosis in U937 cells. Apoptosis of U937 induced by BJOE were associated with the activation of Caspase-3, Caspase-8, Caspase-9, and
PARP. The protein levels of Bcl-2, Bax and Mcl-1 were modulated
by BJOE. BJOE induced Bid cleavage and decreased c-FLIP, but the
levels of DR4, DR5 and Fas were not affected. Furthermore, BJOE
induced apoptosis in patients leukemia cells and showed low cytotoxicity against peripheral blood lymphocytes from healthy volunteers.Conclusion: Our study gives strong evidence for the rst time
that BJOE can induce apoptosis in both cultured and primary AML
cells with lower cytotoxicity on peripheral blood lymphocytes. The
present study indicated that BJOE may have therapeutic relevance in
the treatment of human leukemia.
Paper No.: 2101

OXIDATIVE DNA DAMAGE INDUCED BY ETHANOL IS
ASSOCIATED WITH ITS ADH1B/ALDH2 METABOLIC
PATHWAY IN HUMAN PERIPHERAL LYMPHOCYTES
Chunfu Wu, Y Yan, J Yang, Y Mou, L Wang,
Shenyang Pharmaceutical University, Department of Pharmacology,
Shenyang, PR China
Introduction:Numerous studies have indicated that ethanol induces genotoxicity in human organs. The purpose of present study is to investigate
oxidative DNA damage induced by ethanol in human peripheral lymphocytes and its possible mechanism. Materials: Human peripheral lymphocytes were used to investigate the oxidative DNA damage induced by
ethanol in vitro. Results :By comet assay and intracellular 8-OHdG
detection, which was detected by owcytometry, we proved that ethanol
induced oxidative DNA damage could be self-repaired by lymphocytes.
Furthermore, it was revealed that ethanol was metabolized through
ADH1B/ALDH2 pathway pathway by using inhibitors of CYP2E1,
ADH1B and ALDH2 and RT-PCR assay. In addition, base excision
repair (BER) system was found to be activated in DNA repair process.
Conclusion: we offered direct evidence that ethanol could induce oxidative DNA damage in lymphocytes in vitro and its mechanism was possibly associated with the ethanol metabolism. Moreover, ethanol-induced
DNA damage could be repaired by lymphocytes and BER system was
involved in this DNA repair process.
Paper No.: 2199

NEUROPROTECTION OF GINSENOSIDE RG1 AGAINST
AB25-35-INDUCED NEUROTOXICITY INPRIMARY
CULTURED RAT CORTICAL NEURONS VIA NUCLEAR
STEROID RECEPTORS ACTIVATION
Jiang Wu, Z Wang, X Zhang, W Zhu, Y Shen, R Cui, J Lin, Y Su, Y Lou
Zhejiang University, Institute of Pharmacology & Toxicology and
Biochemical Pharmaceutics, Hangzhou, PR China
Present study aimed to investigate the neuroprotective effects of ginsenoside-Rg1 (Rg1), one of the ginsenosides with steroid structure, against bamyloid peptide25-35 (Ab25-35)-induced insults in primary cultured ratcortical neurons. In addition, activation of nuclear steroid receptors, such
as the glucocorticoid receptor (GR) and the progesterone receptor (PR)
may be involved in the neuroprotective effects of Rg1. Our results
revealed that Rg1 conspicuously increased cell viability and attenuated
neuronal apoptosis after Ab25-35 exposure. Rg1 maintained the expression of choline acetyltransferase (ChAT) of neurons and protected neurons from mitochondrial membrane potential (um) loss. Alteration of
ERK1/2 phosphorylation and the Bcl-2 family expression were observed
in the presence of Rg1. Meanwhile, Ab25-35 was less effective in nitrotyrosine when co-incubated with Rg1, compared with that treated
alone. Interestingly, the neuroprotective effects of Rg1 were nearly abolished by RU486, an antagonist for both GR and PR. Furthermore, pretreatment with Rg1 markedly triggered nuclear translocation of GR and
PR. The computational receptor-ligand docking data also supported Rg1
had certain afnities to GR and PR. Finally, Rg1 showed an anti-inammation-like effect similar to glucocorticoid and progesterone to inhibit
Ab25-35-induced NF-jB activation. In summary, these results provide
novel insights into the underlying mechanisms of Rg1 anti-apoptotic
function induced by Ab25-35 treatment, suggesting that GR and PR
could be important regulatory factors targeted by Rg1.
This work was supported by the National Natural Sciences Foundation
of China[Grants 30672564, 30472112, 30171121].
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Paper No.: 2198
CHARACTERISTICS OF JUNCTOPHILIN 3 AND 4
EXPRESSIONS IN NEURONS DERIVED FROM MOUSE
EMBRYONIC STEM CELLS
Jiaying Wu, Y Gao, H Wang, X Zhang, W Zhu, J Qian, Y Shen, H Yu,
Q Wang, Y Lou,
(1) Zhejiang University, Institute of Pharmacology & Toxicology and
Biochemical Pharmaceutics, Hangzhou, PR China
Junctophilins (JPs) are essential proteins which contribute to formation
of junctional membrane complexes. JP-3 and JP-4 are subtypes of JPs
predominantly distributed in the brain and play important roles in keeping intracellular calcium homeostasis and subsequent neuronal functions.
However, their expressions are not documented in the process of development, we established an in vitro model of neural differentiation of
mouse embryonic stem cell line D3 (ESc) induced by retinoic acid (RA)
to mimic embryonic neural development. The differentiated neurons
were characterized by neuron marker btubulin III. JP-3 and JP-4 expression was examined by western blot and ow cytometry. The results
showed that (i) differentiated neurons expressed both JP-3,4 in mRNA
and protein levels. (ii) interestingly, the expression of JP-3,4 in the three
substyle of neurons was distincted, the most in glutamatergic neurons,
the second in cholinergic neurons, and the least in GABAergic neurons.
(iii) the calcium induced calcium release (CICR) process could be
detected with workstation for alive cell imaging in neurons derived from
mouse ESc, indicating the JPs were functionally matured. Taken
together, our results demonstrated the JP3, 4 expression pattern in the
process of RA-induced neurogenesis. Furthermore, small moleculer such
as RA may bias ESc differentiation to generate functional neural derivatives, which shed light on the potential utility in regenerative medicine.
This work was supported by the National Natural Sciences Foundation
of China [Grant No. 30973600, 90813026]
potentials, but did not appear to be the sole factor affecting their cytotoxicity. chemical forms of metals determine metal toxicity, and both toxicokinetic and toxicodynamic factors are involved in toxicity potentials of
metals with different chemical forms
Paper No.: 1659

PROTECTIVE EFFECT OF RUTAECARPINE AGAINST
MYOCARDIAL HYPERTROPHY IN RATS
Qin Wu, L Deng, X-N Huang, J-S Shi
Rutaecarpine, one of the indole-quinoline alkaloid of Medicinal Evodia
Fruit, has been reported to cut down blood pressure, inhibited proliferation of vascular smooth musle cells. This study aims to investigate
whether rutaecarpine (Rut) has protective effect on rat cardiocyte hypertrophy in vitro, and to probe its protective mechanisms. The rat cardiocyte hypertrophy in vitro was induced by angiotensin(1 +/-10-7mol L-1),
and Rut (1 +/-10-5, 1 +/-10-6 and 1 +/-10-7 mol.L-1) was given at the
same time. The cardiocyte hypertrophy in vitro induced by angiotensino`was evidenced by cardio-myocyte diameter, cardio-myocyte protein
level and the expression of aterial natriuretic peptide. Rut signicantly
ameliorated rat cardiocyte hypertrophy in vitro induced by angiotensin in
a dose-dependent manner. To examine the mechanism of protection, the
content of nitric oxide (NO) and the activity of nitric oxide synthase
(NOS) was determined. Rut signicantly increased the content of NO
and the activity of NOS in the rat cardiocyte hypertrophy in vitro was
induced by angiotensin. These results demonstrate that Rut alleviates the
rat cardiocyte hypertrophy in vitro was induced by angiotensino`, and
the protection appears to be due, at least in part, to its increasing effects
on NO- release.
Paper No.: 1658

CHEMICAL FORM OF METALS UNDERLYING POTENTIAL
CYTOXICITY OF METAL-CONTAINING TRADITIONAL
MEDICINES IN VARIOUS CELL LINES
Paper No.: 1567

LUTEIN, A NATURAL CAROTENOID, INHIBITS PDGF-BB
AND OXIDATIVE STRESS-INDUCED SIGNALLING IN
VASCULAR SMOOTH MUSCLE CELLS
Qin Wu, Y-F Lu, J-Z Shi, J Liu
Wen-Bin Wu(1), Y-C Lin(2)

(1) Fu-Jen Catholic University, School of Medicine, Taipei, Taiwan

(2) TaoYuan General Hospital, Department of Health, Department of
Occupational Medicine, TaoYuan, Taiwan
Mercury and arsenic are frequently found in traditional medicines, justiably raising public concerns. However, the forms of metals used in traditional medicines are mainly sulde forms, such as mineral arsenicals
(realgar, As4S4) and mineral mercurials (cinnabar, HgS). The addition of
these mineral realgar or cinnabar has been claimed for their efcacy to
improve health. However, arsenic is known for its carcinogenic and toxic
effects and mercury for its toxicity. To address their potential toxicity, a
Hg- and As-containing traditional medicine An-Gong-Niu-Huang Wan
(AGNH) were compared to common mercurials (cinnabar, HgS, HgCl2
and MeHg) and common arsenicals (realgar As4S4, As2S2, As2O3, NaAsO2, and Na2HAsO4) for their cytotoxicity in rat liver TRL1215 cells
using the MTS assays. MeHg is the most potent toxicant with LC50 of 4
nM, followed by NaAsO2 (25 nM), HgCl2 (50 nM), Na2HAsO4 (60
nM), As2O3 (30 nM), and As2S2 (50 nM). In comparison, the LC50 of
realgar (As4S4, 250 nM), and cinnabar and HgS is over 50,000 nM. The
toxicity of As- and Hg-containing AGNH is in the range of 5,0000 nM.
Approximately 10,000-fold difference exists between MeHg and HgS,
and 40-fold difference exists between NaAsO2 and As4S4. Cellular
accumulation of As and Hg accumulation correlated with their toxic
Atherosclerosis, a cardiovascular disease (CVD) of the large arteries, is

the primary cause of heart disease and stroke. In westernized societies, it
is the underlying cause of about 50% of all deaths. Vascular smooth muscle cells (VSMCs) play an important role in promoting the progression
of atherosclerosis. Carotenoids, including carotene, lycopene, lutein, and
etc., are present in many foods, particularly in (Chinese) herbal medicine,
fruits, vegetables, and sh. Numerous epidemiological studies have demonstrated that intake of carotenoids can prevent cardiovascular diseases.
In this study, we investigated lutein effects on PDGF-BB-induced signalling pathway and functions in VSMCs. We found that lutein inhibits
PDGF-BB-induced signalling pathway, including PDGF receptor-b
(PDGFR-b), PLCc, and MAPKs phosphorylation (activation), in a concentration- and time-dependent manner in VSMCs. Moreover, lutein
inhibited oxidative stress (H2O2)-induced ERK1/2 and p38 MAPK activation in VSMCs. In a functional study, lutein inhibited PDGF-BBinduced SMC migration at concentrations of 5 and 10 lM. Taken
together, our results provided evidence showing that lutein inhibits
PDGF-BB- and H2O2-induced signalling and migration in aortic SMCs,
662
suggesting the possible role of lutein in preventing atherosclerosis. The
results presented here may help us to better understand the benecial
effects of carotenoids in CVD prevention.
Paper No.: 1331

T-TYPE CALCIUM CHANNELS MEDIATE NICOTINEINDUCED CEREBRAL NEUROGENIC NITRERGIC
VASODILATION
Yin-Chieh Wu(1), HC Lee(2), PY Chen(1), MH Nam(3), MF Chen(4),
TJF Lee(3,5)
(1) Tzu Chi Universuty, Institute of Pharmacology & Toxicology,
Hualien, Taiwan
(2) Tzu Chi Universuty, Institute of Medical Research, Hualien, Taiwan
(3) Tzu Chi Universuty, College of Life Science, Hualien, Taiwan
(4) Tzu Chi Hospital, Department of Research, Hualien, Taiwan
(5) Southern Illinois University School of Medicine, Department of Pharmacology, Springeld, IL, USA
Nicotine has been shown to stimulate a3b2-nicotinic acetylcholine receptors (nAChRs) located on cerebral perivascular sympathetic nerves, causing release of norepinephrine (NE). The released NE binds b2adrenoceptors located on the neighboring parasympathetic nitrergic
nerves, leading to NO release and vasodilation. The exact mechanism
involving calcium in this axo-axonal interaction in regulating cerebral
vascular tone is not established. The possible involvement of T-type calcium channel in mediating NE-elicited NO release, therefore, was examined in porcine cerebral arteries. Results from in vitro tissue bath studies
indicated that the neurogenic nitrergic dilation of isolated porcine basilar
arteries induced by nicotine (100 microM) was inhibited by T-type calcium channel blockers, mibefradil and NNC55-0396 (3 microM), which
did not affect that induced by transmural nerve stimulation (TNS). The
nicotine-induced vasodilation was not affected by a selective Cav2.1 P/Q
type calcium channel blocker (omega-agatoxin TK), a selective P type
calcium channel blocker (omega-agatoxin IVA), or an N-type calcium
channel blocker (omega -conotoxin GVIA). Furthermore, nicotineinduced NE release from sympathetic nerves in isolated circle of Willis
was not inhibited by mibefradil or NNC55-0396. These T-type calcium
channel blockers did not affect nicotine-induced inward currents in
a3b2-nAChR-expressing oocytes either. These results suggest that inhibition of nicotine-induced vasodilation by mibefradil and NNC55-0396
was not due to inhibition of nAChRs or NE release. It is concluded that
T-type calcium channels located on nitrergic nerve terminals are involved
in mediating nicotine-induced neurogenic NO release and dilation in porcine basilar arteries.
(Supported by NCS, Tzu Chi University, and Tzu Chi Foundation).
Paper No.: 2644

DISEASE AND THERAPY
LOCALIZATION OF BKCA CHANNELS AND EFFECT OF BKCA
CHANNELS ON CGRP RELEASE IN THE RAT TRIGEMINAL
NUCLEUS CAUDALIS
Helle Wulf-Johansson(1), DV Amrutka(1), A Hay-Schmidt(2), A Poulsen(3), D Klaerke(3), J Olesen(1), I Jansen-Olesen(1)
(1) Glostrup Hospital, University of Copenhagen, Faculty of Health Sciences, Department of Neurology & Danish Headache Centre, Glostrup,
Denmark
(2) University of Copenhagen, Faculty of Health Sciences, Department
of Neuroscience and Pharmacology, Copenhagen, Denmark

(3) University of Copenhagen, Faculty of Life Sciences, IBHV, Department of Physiology and Biochemistry, Frederiksberg, Denmark
Large conductance calcium-activated potassium (BKCa) channels are
membrane proteins contribut-ing to electrical propagation through neurons. BKCa channels are located pre-synaptic and limit the duration of
Ca2 + entry through voltage-gated Ca2 + channels and thus, regulate the
duration of transmitter release. Therefore, opening of pre-synaptic BKCa
channels may serve as protection or emergency brakes on hyperactive
neurons in reducing the release of neurotransmitters. Calcitonin generelated peptide (CGRP) is a neuropeptide released from the trigeminal
nerve. CGRP plays an important role in migraine by enhancing transmission of migraine pain through the trigeminal nucleus caudalis (TNC). We
investigated the expression of BKCa channels in the rat TNC. Furthermore, the effect of the BKCa channel blocker iberiotoxin and the BKCa
channel opener NS11021 on CGRP release was investigated using isolated TNC tissues. The BKCa channel protein was found in the perikarya
of small oval neurons in the substantia gelati-nosa layer of TNC wherease a rich supply of CGRP nerve bres was found in the marginal layer
using immunouorescence. Isolated TNC tissues were used and CGRP
release was measured using an enzyme-linked immunoassay (EIA) kit.
Iberiotoxin (0.1 nM to 1 nM) caused a concentra-tion-dependent increase
in CGRP release from theTNC. The increase in CGRP release induced
by iberiotoxin was signicantly attenuated by pre-treatment with 0.1 mM
NS11021. This suggests that BKCa channel openers might be potential
candidates for migraine treatment due to its inhibition of CGRP release
and prevention of neuronal hyperexcitibility in the TNC.
Paper No.: 1524

PHARMACOLOGICAL CHARACTERIZATION OF THE FIRST
POTENT AND SELECTIVE CYSTEINYL LEUKOTRIENE 2
RECEPTOR ANTAGONIST
Frank Wunder(1), H Tinel(1), R Kast(1), A Geerts(2), E-M Becker(1),
P Kolkhof(1), J Hutter(1), J Erguden(3), M Harter(3),
(1) Bayer Schering Pharma AG, Cardiovascular Research, Wuppertal,
Germany
(2) Bayer Schering Pharma AG, Target Research, Wuppertal, Germany
(3) Bayer Schering Pharma AG, Medicinal Chemistry, Wuppertal,
Germany
Cysteinyl leukotrienes (CysLTs) have been implicated in the pathophysiology of inammatory and cardiovascular disorders. Their actions are
mediated by two different CysLT receptors, designated CysLT1 and CysLT2, respectively. Here, we report the discovery of 3-({[(1S,3S)-3-carboxycyclohexyl]amino}carbonyl)-4-(3-{4-[4-(cyclo-hexyloxy)buto
xy]phenyl}propoxy) benzoic acid (HAMI3379), the rst potent and
selective CysLT2 receptor antagonist. In a CysLT2 receptor reporter cell
line HAMI3379 antagonized leukotriene C4 (LTC4-) and D4- (LTD4-)
induced intracellular calcium mobilization with IC50 values of 3.8 and
4.4 nM, respectively. In contrast, HAMI3379 exhibited very low potency
on a recombinant CysLT1 receptor cell line (IC50 > 10,000 nM). In addition, HAMI3379 did not exhibit any agonistic activity on both CysLT
receptor cell lines. In binding studies using membranes from the CysLT2
and CysLT1 receptor cell lines, HAMI3379 inhibited [3H]-LTD4 binding
with IC50 values of 38 nM and > 10,000 nM, respectively. The effects
were compared with those of zarlukast, a specic CysLT1 receptor
antagonist, and BAY u9773, a dual CysLT1/CysLT2 receptor antagonist
and partial CysLT2 receptor agonist. In isolated Langendorff-perfused
guinea pig hearts HAMI3379 concentration-dependently inhibited and
reversed the LTC4-induced perfusion pressure increase and contractility
decrease. In contrast, zarlukast was found to be inactive in this experimental setting. In conclusion, HAMI3379 was identied as a potent and
selective CysLT2 receptor antagonist. Using this compound we could
663
show that cardiac CysLT effects are predominantly mediated by the CysLT2 receptor.
Paper No.: 2167

DISEASES
CARNOSOL MODULATES NF-JB ACTIVITY BY DUAL
MECHANISMS IN ENDOTHELIAL CELLS
selective mGluR7 allosteric antagonist. In vivo microdialysis demonstrated that cocaine priming signicantly increased extracellular DA in
the NAc, VP and DS, while increased extracellular glutamate only in the
NAc. Pretreatment with AMN082 failed to alter basal or cocaineenhanced extracellular DA in the NAc, VP or DS, but dose-dependently
inhibited cocaine-induced increases in glutamate in the NAc. This effect
was also blocked by intra-NAc local perfusion of MMPIP. These data
suggest that mGluR7 activation inhibits cocaine-triggered reinstatement
by a glutamate-, but not DA-, dependent mechanism in the NAc. The
present ndings support the potential use of AMN082 in the treatment of
cocaine relapse.
Supported by NIDA IRP.
Being-Sun Wung
National Chiayi University, Department of Microbiology and Immunology, Chiayi, Taiwan
Under inammatory conditions, there is increased expression of specic
cell adhesion molecules on activated vascular endothelial cells, which
increases monocyte adhesion. In our current study, we demonstrate a
putative mechanism for the anti-inammatory effects of carnosol, a diterpene derived from the herb rosemary. Our results show that both carnosol and rosemary essential oils inhibit the adhesion of TNFa-induced
monocytes to endothelial cells and suppress the expression of ICAM-1 at
the transcriptional level. Moreover, carnosol was found to exert its inhibitory effects by blocking the degradation of the inhibitory protein IjBa in
short term pretreatments but not in 12 hour pretreatments. Our data show
that carnosol reduces IKK-b phosphorylation in pretreatments of less
than 3 hours. In TNFa-treated ECs, NF-jB nuclear translocation and
transcriptional activity was abolished by up to 12 hours of carnosol pretreatment and this was blocked by Nrf-2 siRNA. The long-term inhibitory effects of carnosol thus appear to be mediated through its induction
of Nrf-2-related genes.The inhibition of ICAM-1 expression and p65
translocation is reversed by HO-1 siRNA. Carnosol also upregulates the
Nrf-2-related glutathione synthase gene and thereby increases the GSH
levels after nine hours of exposure. Treating ECs with a GSH synthesis
inhibitor, BSO, blocks the inhibitory effects of carnosol.In addition, carnosol increases p65 glutathionylation. Hence, our present ndings indicate that carnosol suppresses TNFa-induced singling pathways through
the inhibition of IKK-b activity or the upregulation of HO-1 expression.
The resulting GSH levels are dependent, however, on the length of the
carnosol pretreatment period.
Paper No.: 2036

THE SELECTIVE MGLUR7 AGONIST AMN082 INHIBITS
COCAINE-TRIGGERED RELAPSE BY A GLUTAMATE
MECHANISM IN THE NUCLEUS ACCUMBENS IN RATS
Paper No.: 2741

THE ROLE FOR L-CARNITINE IN STZ-INDUCED DIABETIC
MICE PERIPHERAL NERVE INJURY
Yunqiu Xia, W Zhong, S Tao, C Wang, J Dong
Qingdao University Medical College, Department of Pharmacology,
Qingdao, PR China
Diabetic neuropathy is a common complication even the glucose level
was controled well in diabetic patients. We hypothesized that metabolic
disorders especially fatty acid was involed in diabetic peripheral hyperalgesia due to absolutely or relativly lack of insulin. L-carnitine is an
amino-acid that transport of fatty acids through the membrane of mitochondria, it may play a role in the peripheral never metabolism.The
experiment was carried out on Kunming 9 weeks age male mice. Control
group (one group fed with normal diet, another fed with high-calorie
diet); STZ-induced diabetes high-calorie diet (18% fat, 20% sugar, 3%
cholesterol) mice; STZ-induced diabetes normal diet mice. The mice
body weight were signicent increased both in high-calorie diet groups.
STZ-induced diabetes high-calorie diet mice are obesity (type 2 diabetes). Tail-ick latency was measured in 49 constant temperature water
bath. Hyperalgesia was observed in type 2 diabetes mice from 1-6 weeks
after injection STZ. However, in STZ-induced nomal diet diabetic mice,
the tail-ick latency signicant shortening was appeared from 6-12
weeks treatment with STZ. For therapeutic study, L-carnitine was administered once daily (Ig 250mg/kg) from 1-6 weeks when tail-ick latency
shortening appeared in type 2 diabetes mice. Compare to the group without L-carnitine treatment type 2 diabetic mice, the tail-ick latency siginicant prolonged after administration L-carnitine 3-week, but did not
normalized. Our results indicated that peripheral nevers are more susceptible to injury in high-calorie diet diabetes mice, and the L-carnitine
could improve this damage.
Zheng-Xiong Xi, X Li, J Li, E Gardner

National Institute on Drug Abuse, Intramural Research Program,
Baltimore, MD,USA
We have recently reported that activation of the metabotropic glutamate
receptor 7 (mGluR7) by AMN082 inhibits intravenous cocaine selfadministration and cocaine-enhanced brain-stimulation reward by a
GABA-dependent mechanism in the ventral pallidum (Li et al., Neuropsychopharmacology, 2009, 34:1783-96). In the present study, we further
investigated the role of mGluR7s in cocaine-induced reinstatement of
drug-seeking behavior. Systemic administration of AMN082 (1-20 mg/
kg, i.p., 30 min prior to cocaine) produced a dose-dependent inhibition
of cocaine-triggered reinstatement. Microinjections of AMN082 into the
nucleus accumbens (NAc), ventral pallidum (VP), or dorsal striatum
(DS) also inhibited cocaine-triggered reinstatement in a dose-dependent
manner. This effect was blocked by the co-administration of MMPIP, a
Paper No.: 2497

HEALTH AND DISEASE
EFFECTS OF CYP7A1 POLYMORPHISMS ON THE FASTING
PLASMA CONCENTRATIONS OF BILE ACIDS, BILE ACID
SYNTHESIS MARKER, AND TOTAL CHOLESTEROL IN
HUMANS
Xiaoqiang Xiang, PJ Neuvonen, M Niemi
University of Helsinki, Department Clinical Pharmacology, Helsinki,
Finland
Cholesterol 7a-hydroxylase (CYP7A1) is the rate-limiting enzyme of bile
acid production in humans. Our aim was to investigate the effects of
664
CYP7A1 polymorphisms on the fasting plasma concentrations of bile
acids, bile acid synthesis marker, and total cholesterol in humans. Fasting
blood samples were drawn from 109 healthy volunteers. The following 7
haplotype-tagging single nucleotide polymorphisms (SNP) of CYP7A1
were determined using TaqMan allelic discrimination: rs3808607,
rs1125226, rs10957057, rs11786580, rs10504255, rs8192879, and
rs1023652. Plasma concentrations of 16 endogenous bile acids, their
synthesis marker (7a-hydroxy-4-cholesten-3-one), and cholesterol were
measured by liquid chromatography-tandem mass spectrometry. Phenotype-genotype association analysis showed some, but not many,
signicant differences. Total plasma bile acid concentration was only
affected by rs11786580. The concentrations of tauroursodeoxycholic
acid, taurolithocholic acid, taurocholic acid, glycocholic acid, ursodeoxycholic acid, chenodeoxycholic acid and hyodeoxycholic acid were
inuenced by several SNPs of CYP7A1. CYP7A1 polymorphism had
no effect on bile acid synthesis marker. Only the CYP7A1
rs8192879 polymorphism affected total plasma cholesterol concentration.
For this SNP, total plasma cholesterol concentrations in subjects with CT
variant genotype were 11.7% higher than in those homozygous for the C
allele (P < 0.05). In general, common CYP7A1 polymorphisms are unlikely to considerably affect cholesterol metabolism and bile acid homeostasis
Paper No.: 992

SESSION - ONCOLOGY
AST1306, A NOVEL IRREVERSIBLE INHIBITOR OF THE
EPIDERMAL GROWTH FACTOR RECEPTOR 1 AND 2,
EXHIBITS ANTITUMOR ACTIVITY BOTH IN VITRO AND
IN VIVO
Hua Xie(1), L Lin(1), Y Jiang(2), J Guo(2), J Ding(1)
(1) Shanghai Institute of Materia Medica, Department of Anti-tumor
Pharmacology, Shanghai, PR China
(2) Shanghai Allist Pharmaceuticals, Shanghai, PR China
Despite the initial response to the reversible, ATP-competitive quinazoline inhibitors that target ErbB-family, such a subset of cancer patients
almost invariably develop resistance. Recent compelling evidence demonstrated that irreversible ErbB inhibitors were signicantly active to
override this resistance. Several irreversible inhibitors are now under
preclinical or clinical studies. Here, we found that AST1306, a novel
anilino-quinazoline compound, inhibited enzymatic activities of wild
type EGFR and ErbB2 as well as EGFR T790M/L858R resistant
mutant both in cell-free and cell-based levels. Importantly, AST1306
functions as an irreversible inhibitor, most likely by covalent interaction with Cys797 and Cys805 in the catalytic domains of EGFR and
ErbB2, respectively. Further studies showed that AST1306 inactivated
their downstream pathways and thereby inhibited the proliferation of a
panel of cancer cell lines. Detailed insights manifested that ErbB2
expression experienced with a consistently higher sensitivity to
AST1306 across various cell lines. And in parallel, ErbB2, but not
EGFR, silencing decrease the sensitivity of SK-OV-3 cells to
AST1306. As a consequence, AST1306 caused potent tumor growth
suppression in ErbB2-overexpressed xenograft models, but exhibited
weak suppression in EGFR-overexpressed tumor xenografts. Moreover,
tumor growth inhibition in SK-OV-3 xenograft models was correlated
with both EGFR and ErbB2 inhibition, and notably, 24 h targets inhibition were observed after single dose AST1306 treatment. The above
ndings, combined with a signicant restriction of tumor growth challenged by AST1306 in FVB-2/Nneu transgenic breast cancer mouse
model, dene AST1306 as a selective irreversible ErbB2 and EGFR
inhibitor with ErbB2 being more sensitive.
Paper No.: 742

ELLUCIDATION OF CROTALUS DURISSUS CASCAVELLA
NATRIURETIC PEPTIDE MODE OF ACTION IN ISOLATED
RAT KIDNEY: EXPRESSION OF GC-A AND GC-C RECEPTORS
Rafael Matos Ximenes(1,2), JSAM Evangelista(3), RS Alves(4), ARC Jorge(1), PCP Sousa(1), AMC Martins(4), MEA Moraes(1), MM Rabello(5),
MZ Hernandes(5), MH Toyama(6), A Havt(1), HSA Monteiro(1)
(1) Federal University of Ceara, Department of Physiology and Pharmacology, Fortaleza, Ceara, Brazil
(2) Federal University of Pernambuco, Academic Center of Vitoria,
Vitoria de SantoAntao, Pernambuco, Brazil
(3) State University of Ceara, College of Veterinary, Fortaleza, Ceara,
Brazil
(4) Federal University of Ceara, Department of Clinical and Toxicological Analysis, Fortaleza, Ceara, Brazil
(5) Federal University of Pernambuco, Department of Pharmaceutical
Sciences, Recife, Pernambuco, Brazil
(6) Paulista State University, Sao Vicente Unit, Campus of Litoral Paulista, SaoPaulo, Brazil
Natriuretic peptides are a family of endogenous hormone peptides that
participates in homeostasis of volume, osmosis and pressure regulation
of circulatory system. Recently, several analogues were isolated from
Viperidae snake venom. Renal and vascular effects of Crotalus durissus
cascavella natriuretic peptide (NPCdc) were reported by our group,
including natriuresis, diuresis and vasoactive action. In order to investigate which receptor is responsible to the action of NPCdc into isolated
perfused rat kidneys, we tested, by real time PCR, the expression of the
mRNA of guanylate cyclase A (GC-A) and guanylate cyclase C (GC-C)
receptors. The NPCdc showed up regulation of GC-C, presenting a signicant 4.01 fold increase (p = 0.003) when kidneys received 0.1lg/mL.
However, after been tested with a higher dosage (0.3lg/mL) we found a
1.03 fold increase, which was not signicant different from the control
values. In addition, the expression of GC-A was signicantly down regulated with a 37.73 (p = 0,0024) and 32.90 (p = 0,0024) fold reduction
when kidneys were treated with 0.1 and 0.3lg/mL, respectively. With
these results we noticed a possible intracellular connection between the
expression of GC-C and GC-A receptors in kidney after NPCdc treatment. This result was also seen when rats were submitted to high-salt
ingestion for 10 days (Fonteles MC et al, Regulatory Peptides 2009;
158: 6-13). In silico studies, using macromolecular docking after homology Modelling of NPCdc showed its afnity for GC-A (NPR-A) receptor. Other kidneys guanylate cyclase receptors may also be involved in
this effect and will be investigated at a near future.
Paper No.: 897

INHIBITORY EFFECTS OF SCHISANDRIN A AND
SCHISANDRIN B ON CYP3A ACTIVITY
Hua-Wen Xin, W-L Li, M-W Su
Wuhan General Hospital, Department of Clinical Pharmacology, Wuhan,
Hubei, PRChina
Aim: To investigate the inhibitory effects of schisandrin A (sch A) ands
chisandrin B (sch B) on CYP3A activity in rat liver microsomes and the
mechanism of this interaction in vitro and in vivo. Methods: Simultaneous determination of midazolam and its metabolism, 1-hydroxy-midazolam, by HPLC. Results: In vitro, sch A and sch B inhibited CYP3A
activity with IC50 values of 6.60 and 5.51 lM and Ki values of 5.83
and 4.24 lM, respectively. The inactivation of CYP3A activity by sch A
and sch B was time- and concentration-dependent manner (sch A:
KI=4.51 lM, Kinact=0.134 min-1; schn B: KI=3.01 lM, Kinact=0.112
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min-1). In vivo study, we estimated that CYP3A activity in the liver
according to the amount of 1-hydroxy-midazolam formed per mg of the
rat liver microsomes. The results showed the liver CYP3A activities were
reduced 15% after repeated oral administration of inhibitors (sch A
(16mg/kg), t-test, p = 0.047, or sch B (16mg/kg) t-test, p = 0.041). Conclusions: These results in vitro and in vivo conrmed that effects of sch
A and sch B would have potential inhibitory effects on CYP3A activity
in rat liver microsomes. In vitro, the dilution assay plots and double-reciprocal plots showed the inhibitory effects were reversible and sch A and
sch B mostly belonged to mixed non-competitive inhibition in complete
inhibition.
Paper No.: 1899

OESTROGEN CONFERS CARDIOPROTECTION BY
UP-REGULATING B2-AR
C Xu, A Liu, Hong Sun
PCR) and protein (Western blot) levels. The involvement of intracellular

MAPK pathways was studied by using Western blot and specic inhibitors for extracellular signal-regulated kinases (ERK1/2) and p38 MAPK.
Our results show that LDL increased the ETB2 receptor-mediated vasoconstriction and mRNA expression for VSMC ETB2 receptors in concentration- and time-dependent manners. LDL induced activation of
ERK1/2 and p38 MAPK in the VSMC. Inhibitors for ERK1/2 (PD98059
and U0126) and p38 (SB203580 and SB239063) MAPK abolished the
LDL effects. In conclusion, we have demonstrated for the rst time that
LDL increases VSMC ETB2 receptor expression through activation of
ERK1/2 and p38 MAPK pathways. Understanding the intracellular
MAPK signal mechanisms may provide new strategies for treatment of
cardiovascular disease.
Paper No.: 997

PROTECTIVE EFFECT OF ROLIPRAM ON MEMORY IMPAIRMENTS IN RATS INJECTED WITH AMYLOID-(25C35)
Jiangping Xu(1), C Wang(1), Y Cheng(1), H Zhang(2)
XuZhou Medical College, Department of Physiology, XuZhou, PR

China
Oestrogen plays a cardioprotective role in female rat hearts subjected to
ischemiareperfusion injury, and the effects of oestrogen are associated
with decreased cardiomyocyte contraction and expression of b1-AR, and
increased expression of b2-AR. We hypothesized that oestrogen may also
protect the heart by increasing the physiological role of b2-AR, that
improves contraction, decreases myocardial injury and apoptosis. Female
Sprague-Dawley rats were subjected to bilateral ovariectomy (OVX) or
sham operation (Sham). A subgroup of OVX rats received oestrogen
(E2) replacement (40 lg kg kg-1 day-1) for 4 weeks. Cardiomyocyte
shortening was evaluated in the presence of isoprenaline (ISO).The presence of lactate dehydrogenase (LDH) and creatine kinase (CK) activity
in the culture medium was determined. We also determined the percentage apoptosis in myocytes from rats in each group. b2-AR antagonist,
ICI118,551 signicantly increased cardiac contractility, the number of
apoptotic cells, myocardial injury in sham group during ischemia, while
there was no difference in OVX group, and all of which were reversed
by treatment with E2. Oestrogen confers cardioprotection at least partly
by up-regulating the physiological role of b2-AR during ischemia,
decreasing cardiomyocyte contraction, ischemic insult, and apoptosis.
Paper No.: 2063

LDL INCREASES VASCULAR ETB2 RECEPTOR EXPRESSION
VIA ACTIVATION OF ERK1/2 AND P38 MAPK PATHWAYS
(1) Southern Medical University, Department of Pharmacology, Guangzhou, PR China

(2) West Virginia University Health Sciences Center, Morgantown, WV,
USA
Phosphodiesterase-4 (PDE4) inhibitor blocks the hydrolysis of cAMP via
inhibition of PDE4 and is attractive candidate for novel anti-AD cognitive disorder drugs.We examined whether treatment with Rolipram, a
cyclic nucleotide phosphodiesterase type 4 inhibitor (PDE4), would
reverse the learning impairment induced by Ab(2535). Our aim was to
study the effects of CREB and pCREB levels in the hippocampus and
cortex of the rat after injection of the Ab(2535). We wanted to determine whether Rolipram improves the memory correlated with CREB
and pCREB. In addition we examined the working spatial memory in a
Morris water maze and passive avoidance task. The animals received a
16-day treatment of Rolipram after the Ab(2535) injection. Our results
showed a signicant increase of pCREB in the hippocampus and cortex
of Rolipram-treated rats. There were impairments in the spatial memory
evaluated in the Morris water maze and passive avoidance task. Our
results suggest that Rolipram may protect memory against Ab(2535)
caused impairments by inhibiting the effects of PDE4 in the hippocampus and cortex and suggests a possibility of PDE4 inhibitor rolipram as a
potential therapeutic agent against memory impairments in rats induced
by Ab(2535).The PDE4 inhibitor may serve as a cognitive enhancer for
therapeutic treatment of age-related cognitive decline and Alzheimers
dementia (AD).
Keywords: Phosphodiesterase 4 inhibitors; CREB; pCREB; Rolipram
Acknowledgements: This work was supported by grants from National
Natural Science Foundation (No. 30672453 and 30973518).
Cang-Bao Xu, J-P Zheng, Y Zhang, W Zhang, L Edvinsson

Lund University, Institute of Clinical Sciences, Division of Experimental
Vascular Research, Lund, Sweden
In cardiovascular disease, there is increased expression of endothelin type
B2 (ETB2) receptors on the vascular smooth muscle cells (VSMC) and
enhanced ETB2 receptor-mediated VSMC contraction, which contribute
to the pathogenesis of cardiovascular disease. The present study was
designed to investigate if the cardiovascular risk factor low density lipoprotein (LDL) increases ETB2 receptor expression and if intracellular
mitogen-activated protein kinases (MAPK) is involved. Mesenteric arteries were isolated from rats and cut into ring segments. The segments
were incubated with different concentrations of LDL (50, 100 and
200mg/L) for up to 48 hrs. Effect of LDL on ETB2 receptor expression
was examined at functional contraction (myograph), mRNA (real-time
Paper No.: 1327

EFFECT OF THREE PURINE ALKALOIDS IN TEA ON
LEARNING AND MEMORY FUNCTIONS OF RATSS EXPOSED
TO RESTRAINT STRESS
Jie-Kun Xu(1), W-K Zhang(2), Q-G Wang(1), K Hiroshi(3), X-S Yao(3)
(1) Beijing University of Chinese Medicine, School of Preclinical Medicine, Beijing, PR China
(2) National Center for Pharmaceutical Screening, Institute of Materia
Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, PRChina
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(3) Jinan University, Institute of Traditional Medicine & Natural Products, Colleg eof Pharmacy, Jinan, PR China
It is well known that tea is a popular drink which can make head clear.
Although numerous reports had described purine alkaloids in tea such as
caffeine increase alertness and cognitive performance, the differences
among the effects of different purine alkaloids on rats exposed to
restraint stress have not been investigated so far. The present work aims
at studying the effects of caffeine, theobromine and theacrine on learning
and memory functions of rats exposed to acute and chronic restraint
stress. The step-through test was used to determine the effects of caffeine, theobromine and theacrine (at 10 and 30 mg/kg) on non-spatial
memory functions of rats exposed to acute restraint stress. The Morris
water maze was used to assess the effects of three purine alkaloids on
spatial learning and memory functions of rats exposed to chronic restraint
stress. The results indicated that the chronic stress exposure was associated with impaired performance in learning and memory tasks, and acute
stress exposure had the adverse effects. However, treatment of caffeine,
theobromine and theacrine all showed signicant improvement in learning and memory abilities of rats either acute or chronic stress exposure.
In addition, both theobromine and theacrine have more signicant effects
than that of caffeine. These ndings indicated that purine alkaloids present in tea have a potential to increase the learning and memory functions,
however their action mechanisms are different from those of restraint
stress.
Paper No.: 1049

WNK IMPROVES LEARNING AND MEMORY BY
MODULATING THE NEUROTRANSMITTER LEVELS IN RATS
AND MICE WITH VASCULAR DEMANTIA
Li Xu, J-X Liu, W-H Cong
Chinese Academy of Medical Sciences, Xiyuan Hospital, Research &
Development Centre, Beijing, PR China
Introduction: WNK, one of traditional Chinese medicines, is effective to
manage the vascular dementia (VaD). The present research is aimed to
explore its effects on the neurotransmitter levels, learning and memory
ability in rats and mice with VaD. Methods and materials: Three kinds of
animal models were employed in this experiment to evaluate the efcacy
of WNK comprehensively. (1) Rat model of two artery occlusion (2VO)
for 3 months; (2) Rat model of multi-infarct dementia (MID); (3) Mouse
model of acquired memory impairment. Results: In Morris water maze,
the latent time to nd the platformfor 2VO rats treated with WNK at dosage 8.25,16.5,33.0 mg/kg were signicantly cut down respectively. Acetylcholine level in brain tissue was increased meanwhile the
acetylcholine esterase activity was inhibited by WNK. The pyramidal
neurons in hippocampal region were in an orderly compact structure with
plump nuclear membrane, indicating the injury was signicantly alleviated. In addition, the hippocampus ratio against body weight, 5-HT levels in cortex and hippocampus were all signicantly increased by WNK.
For MID rats, the latent time were also signicantly shortened by WNK
at dosage of 16.5 and 33.0 mg/kg respectively. And the acquired memory
impairment in mice induced by scopolamine and reserpine were
improved, the error numbers and latencies in step-down test signicantly
decreased by WNK at dosage 22 and 44 mg/kg respectively. Conclusion:
WNK improves the learning ability and restores the memory both in rats
and mice by modulating the neurotransmitter levels.
Paper No.: 2110

DISEASES
PHOSPHODIESTERASE 4 INHIBITION ATTENUATES
NICOTINE-INDUCED AIRWAY HYPERRESPONSIVENESS TO
KININ RECEPTOR AGONISTS
Yuan Xu, Y Zhang, L-O Cardell
Karolinska Institute CLINTEC, Division of Ear, Nose and Throat
Diseases, Stockholm, Sweden
Cigarette smoke causes airway inammation and hyperreactivity (AHR).
It also increases the risk for and severity of asthma. The present study
examines if nicotine, one of the most important components of cigarette
smoke, can induce AHR to kinin receptor agonists, and if phosphodiesterase (PDE) 4 might have a role in such a process. Murine tracheal segments were cultured for 1, 2 or 4 days in serum-free DMEM medium in
presence of nicotine (1 or 10 microM) or vehicle (DMSO). The contractile responses induced by kinin B1 receptor agonist des-Arg9-bradykinin,
and B2 receptor agonist bradykinin, were analyzed with myographs. The
kinin receptor mRNA expressions were quantied using real-time PCR
and the corresponding protein expression assessed with confocal-microscopy-based immunohistochemistry. Four days of organ culture with nicotine concentration-dependently increased B1 and B2 receptor-mediated
airway contractions. This was accompanied by elevated mRNA and protein expressions for both receptors, which suggests involvement of transcriptional mechanisms. However, no such effects were seen during the
rst two days. The nicotine effects were abolished by nicotinic receptor
antagonists MG624 and hexamethonium. Further, both non-specic PDE
inhibitor theophylline (10 and 100 microM) and specic PDE4 inhibitor
YM976 (10 nanoM and 1 microM) concentration-dependently attenuated
the nicotine-induced increase in contractile responses, without affecting
the control segments. In analogy, the elevated kinin receptor mRNA
expressions were also suppressed by YM976. Altogether, this suggests a
role for PDE4 activation in nicotine-induced AHR to kinin receptor agonists. Thus, PDE4 might be a therapeutic target in novel attempts to
treat cigarette-smoke-associated airway diseases.
Paper No.: 1721

IS PHYTOTHERAPY EFFECTIVE FOR THE URINARY
DYSFUNCTION IN PATIENTS WITH BENIGN PROSTATIC
HYPERTROPHY?
Shizuo Yamada, M Abe, Y Ito, S Onoue
University of Shizuoka School of Pharmaceutical Sciences, Department
of Pharmacokinetics & Pharmacodynamics, Shizuoka, Japan
Plant extracts are widely used for the treatment of benign prostatic hyperplasia (BPH) and related lower urinary tract symptoms. In fact, phytotherapeutic agents, including Saw palmetto extract (SPE), are very
popular in many European countries as herbal remedies represent up to
80% of all drugs prescribed for these disorders. Numerous pharmacodynamic mechanisms of SPE have been proposed, including inhibition of
5s-reductase. Our previous studies have shown that SPE may improve
lower urinary tract symptoms by acting on pharmacologically relevant
receptors in the prostate and bladder of rats and human (J. Urol., 173:
1395, 2005; Urology, 69: 1216, 2007). To identify active constituents in
SPE, n-hexane and diethyl ether extracts of SPE were fractioned by silicagel column chromatography followed by reversed-phase HPLC. The
fractions containing lauric acid and oleic acid, which are major constituents in SPE, displayed signicant binding activity of muscarinic, s1adrenergic and 1,4-dihydropyridine receptors, suggesting that these fatty
acids are major principles for pharmacodynamic effects of SPE. Com-
667
mercially available agents of lauric acid and oleic acid also exerted similar binding activities of these receptors as the fatty acids fractioned from
SPE. The plasma concentrations of both fatty acids reached maximum
levels at 3 hr after single oral administration of SPE at 600 mg/kg. Further, clinical study has shown that repeated administration of SPE in
patients with BPH receiving long-term therapy with s1-blockers
improved signicantly residual urine. Thus, the present study suggests
that SPE is effective for the urinary dysfunction in patients with BPH.
Paper No.: 1722

NONINVASIVE EVALUATION OF BRAIN MUSCARINIC
RECEPTOR OCCUPANCY OF OXYBUTYNIN, DARIFENACIN
AND IMIDAFENACIN IN RATS BY POSITRON EMISSION
TOMOGRAPHY
as possible. We have developed an apparatus named Yubi-MR (Japan

System Planning) to reduce oxidative stress in blood by radiation of electromagnetic waves to a nger. Yubi-MR mimicked a NMR-Pipetector
which is widely used as a dehydrator of Fe(III) ions by reduction through
inducing negatively charged water. Blood samples from healthy 9 volunteers before/after irradiation by Yubi-MR subjected to analyze a reactive
oxygen metabolites (d-ROMs) and a biological antioxidant potential
(BAP) by FRAS4 (Wismerll). D-ROM value was 249.6 16.7
(meanSE) before the irradiation. After the irradiation with electromagnetic waves for 10 min at 1.0 x 10-3 dBlV/m x 3 intensity, d-ROM signicantly dropped to 230.4 14.8 (meanSE) (P < 0.01). In contrast,
BAP was not changed at all (before/after = 2401.1 37.5 vs 2401.2
37.0, meanSE). In vitro radiation to blood by Yubi-MR showed the
similar reductive effect in d-ROM value. Since the radiation by Yubi-MR
or Pipetector is believed to form negatively charged water that provides
free-electrons for reduction of Fe(III) ions, Yubi-MR might work at a
similar way in the blood, resulting in reduction of oxidative stress
(dROM). This Yubi-MR is the rst oxidative stress-reducer, possibly
applicable to clinical therapeutic use for reducing oxidative stress in
patients with oxidative stress-involved diseases.
Shizuo Yamada(1), A Yoshida(1), Y Ito(1), H Tsukada(2)

(1) University of Shizuoka School of Pharmaceutical Sciences, Department of Pharmacokinetics & Pharmacodynamics, Shizuoka, Japan
(2) Hamamatsu Photonics K. K., Central Research Laboratory, Japan
An overactive bladder (OAB) with symptoms of frequency, urgency and
urge incontinence is very common in the geriatric population. Antimuscarinic agents are widely used for the treatment of OAB, but they are
also associated with anticholinergic side effects. Notably, agents that can
cross the blood brain barrier (BBB) and bind to muscarinic receptors in
the brain have risk of causing central nervous system (CNS) dysfunction
including cognitive impairment. Such side effects are of great concern in
elderly patients due to the increase in the BBB permeability with age.
The current study was undertaken to characterize noninvasively muscarinic receptor occupancy in the rat brain after i.v. injection of oxybutynin,
darifenacin and imidafenacin using small animal PET. In PET study, i.v.
injection of oxybutynin but not darifenacin or imidafenacin at pharmacological doses decreased signicantly binding potential of [11C](+)3-MPB
in the rat cerebral cortex and corpus striatum in a dose-dependent manner. Similarly, in in vivo autoradiographic experiment, oxybutynin dosedependently reduced binding of [11C](+)3-MPB in the brain, whereas
darifenacin and imidafenacin did not. Following i.v. injection of each
agent, signicant binding of muscarinic receptors in the brain of rats was
observed after the injection of oxybutynin but not darifenacin or imidafenacin. In conlusion, oxybutynin but not darifenacin or imidafenacin
has potential side effects on the CNS in patients with OAB. The results
reveal noninvasive characterization of brain receptor occupancy by PET
to be a powerful tool for precise evaluation of adverse CNS effects of antimuscarinic agents in pre-clinical and clinical evaluations.
Paper No.: 542

HEALTH AND DISEASE
RADIATION OF ELECTROMAGNETIC WAVES TO A FINGER
USING YUBI-MR REDUCES OXIDATIVE STRESS IN BLOOD
BY ELECTRON-TRANSFER VIA NEGATIVELY CHARGED
WATER FORMATION
Naomasa Yamamoto(1), Y Koike(2), K Kumano(3), N Yonehara(2)
(1) Ohu University School of Pharmaceutical Sciences, Department of
Biochemistry, Koriyama, Fukushima, Japan
(2) Ohu University School of Pharmaceutical Sciences, Department of
Pharmacology, Koriyama, Fukushima, Japan
Accumulation of oxidative stress elevates physiological aberrances such
as aging, atherosclerosis, hypertension, obesity, myocardial infarction,
and stroke. It is desired to reduce the oxidative stress in blood as much
Paper No.: 2141

HEALTH AND DISEASE
CNS PERICYTES ORIGINATE FROM THE CIRCULATING
HEMATOPOIETIC PROGENITORS
Seiji Yamamoto(1), M Muramatsu(2), E Azuma(3), M Dohmoto(3), S
Kita(4), T Iwamoto(4), I Komuro(5), K Takano(1), S Niida(6), M Shibuya(2), N Matsuda(7), Y Hattori(1)
(1) University of Toyama, Department of Molecular and Medical Pharmacology, Toyama,J apan
(2) Tokyo Medical and Dental University, Department of Molecular
Oncology, Tokyo, Japan
(3) Genome Biotechnology Laboratory, Kanazawa Institute of Technology, Ishikawa, Japan
(4) Fukuoka University, Department of Pharmacology, Fukuoka, Japan
(5) Chiba University, Department of Cardiovascular Science and Medicine, Chiba, Japan
(6) National Center for Geriatrics and Gerontology, Laboratory of Genomics and Proteomics, Aichi, Japan
(7) Nagoya University, Department of Emergency and Critical Care
Medicine, Nagoya, Japan
Introduction: Pericytes play a pivotal role in the blood ow regulation at
the capillary level in the brain. It is believed that pericytes are originally
derived from mesenchymal cells which reside in the connective tissues
surrounding blood vessels. However, little is known about the recruitment of pericytes to cerebral blood vessels. We examined the development of cerebral blood vessels during the period of neurogenesis, with a
major emphasis on the pericyte recruitment under the physiological condition. Materials/Results: In E10.5 mouse brain, a neurogenesis stage,
CD31 + F4/80 + cells inltrated neuroepithelium around the vascular
front of the inside vasculature and dorsal mid-line area. CD31 + F4/80 +
cells were merged with NG2 and CD45. Matrigel in vivo assay and
embryo brain explant culture showed that GFP-CD31 + F4/80 + cells,
fractionated from GFP-embryos, were associated with newly formed capillaries and expressed pericyte markers. Myeloid decient mice exhibited
very few NG2 + pericyte progenitors that inltrated the dorsal mid-line,
with insufcient NG2 + pericyte coverage formation. Moreover, micro
array analysis corroborated scanty pericyte coverage of the myeloid decient mouse embryo. Ncx1-/- embryos clearly showed that blood ow
were important in recruiting NG2 + pericyte progenitors to brain dorsal
mid-line. Conclusion: This study represents the rst report that CD31 +
F4/80 + CD45 + cells inltrating in E10.5 neuroepithelium can adhere to
cerebral blood vessels and may serve as NG2 + pericytes. These data
strongly suggest that circulating hematopoietic cells are the origin of
recruited NG2 + pericyte progenitors.
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Paper No.: 2221
DISEASE AND THERAPY
MOLECULAR ASSEMBLY IN FUCTIONAL CA2 +
MICRODOMAIN IN VASCULAR SMOOTH MUSCLE CELLS
Hisao Yamamura, S Ohya, Y Imaizumi
Nagoya City University, Department of Molecular & Cellular Pharmacology, Nagoya, Japan
In smooth muscles, Ca2 + sparks occur as spontaneous Ca2 + release from
subplasmalemmal sarcoplasmic reticulum (SR) through ryanodine receptors (RyRs). Ca2 + sparks activate large-conductance Ca2 + -activated K+
(BK) channels nearby and elicit spontaneous transient outward currents
(STOCs), which cause the decrease in muscle tone via membrane hyperpolarization. In the present study, the imaging of molecular assembly in
Ca2 + spark microdomain was performed and analyzed using confocal
and total internal reection uorescence (TIRF) microscopes with uoresence-labeled molecules in single smooth muscle cells isolated from rabbit portal vein. At a holding potential of -40 mV under whole-cell
conguration, Ca2 + sparks observed in a TIRF zone triggered STOCs
and consequent membrane hyperpolarizations under current-clamp. Clusters of RyRs were accumulated in Ca2 + spark sites. BK channels were
densely distributed within 500 nm from the center of a Ca2 + spark sites,
and it was estimated that local Ca2 + increase during a spark (approximately 1 um in radius) reached sufcient Ca2 + concentration for BK
channel activation in the sites. A subset of voltage-dependent Ca2 + chan2 +
nels (VDCCs) was also distributed around Ca spark sites. In addition,
caveolin 1 (Cav1) was spatially associated with BK channel and localized around Ca2 + spark sites. Fluorescence resonance energy transfer
(FRET) analyses suggest the direct molecular interaction of BK channel
with Cav1. Spatiotemporal imaging of molecular assembly and Ca2 +
dynamics in functional Ca2 + microdomain of spark sites enables us to
elucidate its physiological impact in the regulation of membrane excitability and myogenic tone in vascular smooth muscle cells.
Paper No.: 1115

DISEASE AND THERAPY
STUDIES ON EPILEPTIC ANIMAL MODEL BEARING A
GENETIC ABNORMALITY IDENTIFIED IN AUTOSOMAL
DOMINANT NOCTURNAL FRONTAL LOBE EPILEPSY
(ADNFLE) AND BENIGN FAMILIAL NEWBORN BABY
CONVULSIONS (BFNC) OF HUMAN
Sayaka Yamashita(1), M Nagano(1), Y Tanaka(1), M Deshimaru(2),
M Okada(3), S Kaneko(4), S Hirose(5), R Saito(1), K Honda(1),
Y Takano(1)
(1) Fukuoka University, Faculty of Pharmaceutical Sciences, Department
of Physiology and Pharmacology, Fukuoka City, Japan
(2) Mie University Graduate School of Medicine, Division of Neuroscience, Tsu City, Japan
(3) Hirosaki University School of Medicine, Departments of Neuropsychiatry, Hirosaki, Japan
(4) Fukuoka University School of Medicine, Department of Paediatrics,
Fukuoka City, Japan
(5) Fukuoka University, Faculty of Science, Department of Chemistry,
Fukuoka City, Japan
Epilepsy is a common psychiatric disorder, and in the last decade,
genetic abnormalities of gene encoding ion channels expressed in the
brain have been identied. In this study, therefore, we examined nocturnal frontal lobe epilepsy (NFLE) in human using model animals. We
generated transgenic rat strains that harbor a missense mutation S284L,
which had been identied in CHRNA4 in NFLE. The methods have

been described our previous paper (J. Neurosci. 2008, 19, 12465,). The
transgenic rats were not observed biological and behavioral abnormalities, and morphological changes in the CNS. Mutations of genes encoding a 4, b 2 or a 2 subunits (CHRNA4, CHRNB2, orCHRNA2, of
neuronal nicotinic acetylcholine receptor) caused NFLE epilepsy in
human. S284L transgenetic rats developed three distinct spontaneous epileptic seizure types during slow-wave sleep phase: paroxysmal arousals,
nocturnal paroxysmal dystonia and episodic nocturnal wandering. All
three types of seizures occurred frequency in the same individual rat.
The therapeutic response of these rats to conventional antiepileptic drugs
also resembled that of NFLE patients with the S284L mutation. The rats
exhibited two major abnormalities in neurotransmission: (1) attenuation
of synaptic and extra synaptic GABAergic transmission and (2) abnormal
glutamate release during slow-wave sleep. In addition, to examine epilepsy of human BFNC, we generated two lines of knock-in mouse with
mutations of Y284C and A306T in the voltage dependency potassium
channels (KCNQ 2). Thus, our transgenic rats offer another dimension to
the epilepsy research eld.
Paper No.: 1283

ANTINOCICEPTION OF SYNTHETIC OMEGA - CONOTOXIN
SO-3, A SELECTIVE N-TYPE NEURONAL VOLTAGESENSITIVE CALCIUM CHANNEL BLOCKER, AND ITS
EFFECTS ON MORPHINE ANALGESIA IN CHEMICAL
STIMULUS TESTS AFTER ACUTE OR CHRONIC
INTRATHECAL ADMINISTRATION IN RODENT
Ling-Di Yan(1), Y-L Liu(2), L Zhang(3), H-J Dong(1), P-L Zhou(1),
R-B Su(1), Z-H Gong(1), P-T Huang(4)
(1) Beijing Institute of Pharmacology and Toxicology, Department of
(2) Soochow University, College of Pharmacy, Suzhou, PR China
(3) China-Japan Friendship Hospital, Beijing, PR China
(4) Beijing Institute of Biotechnology, Beijing, PR China
SO-3, a novel Omega-superfamily conotoxin derived from Conus striatus, selectively inhibits N-type neuronal voltage-sensitive calcium channels. In current study, antinociception of SO-3 compared with MVIIA or
morphine and its effects on morphine analgesia were investigated in
rodent chemical stimulus tests after acute or chronic intrathecal administration. In mice acetic acid writhing test, similar to MVIIA, SO-3 caused
dose- and time-dependent spinal antinociception with ED50 of 0.25 lg/
kg and t1/2 of 4 h, which was more potent and longer-acting than morphine. In rat formalin test after intrathecal bolus injection, SO-3 produced
dose- and time-dependent antinociception by suppressing acute (ED50,
1.79 lg/kg) and tonic phases (ED50, 0.41 lg/kg), which was similar to
MVIIA and approximately 10-fold potency and twice longer-acting of
morphine in blocking tonic phase responses. After repeated intrathecal
injections twice daily for 5 consecutive days, SO-3 produced analgesia
without loss of potency whereas morphine produced analgesia tolerance
in rat formalin test; further, SO-3 still produced potent analgesia in morphine-tolerant rats. SO-3 co-administrated with morphine left-shift the
dose-response curve of morphine in mice acetic acid writhing test and
signicantly potentiated morphine analgesia in rat formalin test. No
changes in motor function were seen in mice or rats receiving antinociceptive doses of SO-3 whereas MVIIA caused motor dysfunction at
doses of 1.0-2.0 lg/kg in rats. This study showed that (1) novel SO-3
produced potent and long-acting spinal antinociception without observable motor dysfunction, (2) SO-3 signicantly potentiated morphine analgesia, (3) After chronic intrathecal administration, SO-3 produced neither
tolerance nor cross-tolerance to morphine analgesia.
669
Paper No.: 1647
DISCOVERY
STIMULATION OF NORADRENALIN TRANSPORTER AND
SEROTONIN TRANSPORTER ACTIVITY BY SOY
PHYTOESTROGEN GENISTEIN
Nobuyuki Yanagihara(1), Y Toyohira(1), S Ueno(1), M Tsutsui(2),
K Takahashi(3)
(1) University of Occupational & Environmental Health, School of
Medicine, Department of Pharmacology, Kitakyushu, Fukuoka, Japan
(2) University of Ryukyu, School of Medicine, Department of Pharmacology, Japan
(3) University of Occupational and Environmental Health, Deparment of
Hospital Pharmacy, Kitakyushu, Fukuoka, Japan
Genistein, an isoavone, is a major natural phytoestrogen found in soybeans. Much attention has been focused on the high dietary intake of soy
isoavones because of their potentially benecial effects associated with
reduced risks of developing menopausal symptoms and some forms of
cancer. Previously, we reported that bisphenol A, an environmental estrogenic pollutant, inhibits noradrenaline transporter (NAT) activity. These
results prompted us to investigate the effects of phytoestrogens on NAT
or serotonin transporter (SERT) function. Treatment with genistein (10
nM-10 microM) for 20 min stimulated [3H]NA uptake by SK-N-SH
cells. Genistein also stimulated [3H]NA uptake and [3H]serotonin (5HT) uptake by NAT and SERT transiently transfected COS-7 cells,
respectively. Kinetics analysis of the effect of genistein on NAT activity
revealed that genistein signicantly increased the maximal velocity of
NAT with little change in the afnity. Scatchard analysis of [3H]nisoxetin
binding to NAT-transfected COS-7 cells showed that genistein increased
the maximal binding without altering the dissociation constant. Although
genistein is also an inhibitor of tyrosine kinases, daidzein, another soy
phytoestrogen and an inactive genistein analogue against tyrosine kinases, had little effect on [3H]NA uptake by SK-N-SH cells. The stimulatory effects of genistein on NAT activity were observed by treatment of
tyrphostin 25, an inhibitor of epidermal growth factor receptor tyrosine
kinase, whereas orthovanadate, a protein tyrosine phosphatase inhibitor,
suppressed [3H]NA uptake. These ndings suggest that genistein upregulates NAT and SERT functions probably through processes involving
protein tyrosine phosphorylation.
Supported by Grant-in-Aids (20611020 and 20590129) for Scientic
Research (C) from JSPS.
Paper No.: 950

CURCUMIN INHIBITS RENAL CYST FORMATION AND
ENLARGEMENT THROUGH REGULATING
INTRACELLULAR SIGNALLING PATHWAYS
81% in an embryonic kidney cyst model. Curcumin did not induce cytotoxicity and apoptosis, nor alter the cell proliferation rate in MDCK cells
at testing concentrations. Curcumin failed to affect the chloride transporter CFTR expression and function. Interestingly, curcumin promoted
the tubule formation in MDCK cells, which indicates curcumin promotes
MDCK cell differentiation. Furthermore, we found that curcumin downregulated intracellular signalling proteins Ras, B-raf, p-MEK, p-ERK, cfos, Egr-1 signalling proteins, but up-regulated Raf-1 and NAB2 in
MDCK cells exposed to forskolin. These data suggest curcumin may
execute its anti-cyst activity primarily by regulating MAPK signalling
pathway in the renal epithelial cells. These results provide proof-of-concept for possibly applying curcumin or its analogues as drug candidates
to reduce cyst growth in polycystic kidney disease.
Paper No.: 1046

DISEASES
TLR4-DEPENDENT INDUCTION OF VCAM-1 IN
RHEUMATOID ARTHRITIS SYNOVIAL FIBROBLASTS:
ROLES OF CPLA2/COX-2
Chuen-Mao Yang(1), H-L Hsieh(2)
(1) Chang Gung University, Department of Pharmacology, Tao-Yuan,
Taiwan
(2) Chang Gung Institute of Technology, Tao-Yuan, Taiwan
Lipopolysaccharide (LPS)/Toll-like receptor 4 (TLR4)-mediated signalling pathways have caught the attention of strategies designed for rheumatoid arthritis. In this study, we identied that cPLA2 acted as a
modulator of LPS-induced VCAM-1 expression and THP-1 adherence.
Treatment of rheumatoid arthritis synovial broblasts (RASFs) with LPS,
a TLR4 agonist, promoted the VCAM-1 expression and THP-1 adherence which were decreased by pretreatment with a selective cytosolic
phospholipase A2 (cPLA2) inhibitor (AACOCF3), implying the involvement of cPLA2 in these responses. This notion was further conrmed by
knockdown of cPLA2 expression by transfection with cPLA2 small interfering RNA (siRNA) leading to a decrease in VCAM-1 expression and
THP-1 adherence induced by LPS. Subsequently, the LPS-stimulated
cPLA2a phosphorylation was attenuated by pretreatment with a MEK1/2
inhibitor (U0126), suggesting that LPS-stimulated cPLA2a phosphorylation and activity are mediated through an ERK-dependent mechanism.
Moreover, COX-2-derived PGE2 production appeared to involve in LPSinduced VCAM-1 expression which was attenuated by pretreatment with
selective COX-2 inhibitors (NS-398 and celecoxib), transfection with
COX-2 siRNA, or PGE2 receptor antagonists. In addition, pretreatment
with ecosapentaenoic acid, a substrate competitor of arachidonic acid,
also blocked LPS-induced VCAM-1 mRNA and protein expression, and
THP-1 adherence. Collectively, these results suggest that LPS-induced
VCAM-1 expression and adhesion of THP-1 cells are mediated through
the TLR4/ERK/cPLA2a phosphorylation and COX-2 expression/PGE2
synthesis in RASFs.
Baoxue Yang, J Gao, H Zhou, T Lei, W Li, X Li

Peking University, Department of Pharmacology, Beijing, PR China
Curcumin, a polyphenol natural product of the plant Curcuma longa, has
garnered great interest as a potential therapeutic agent for the treatment
and/or prevention of various disease processes and regulates multiple cell
signalling pathways. The purpose of this study was to elucidate if curcumin inhibits renal cyst formation and growth. MDCK cyst model and
embryonic kidney cyst model were used to observe the effect of curcumin on cyst development. The effects of curcumin on cell viability, proliferation, apoptosis, CFTR function and expression, and signalling
pathways in MDCK cells were determined to explore the mechanisms
curcumin acts in. The results showed curcumin signicantly inhibited
MDCK cyst formation up to 61.57% with dose-response relationship
(IC50 was 1.175-10-7M). Curcumin also reduced cyst enlargement up to
Paper No.: 2277

ERYTHROPOIETIN-INDUCED SONIC HEDGEHOG
EXPRESSION CONTRIBUTES TO BRAIN-DERIVED
NEUROTROPHIC FACTOR-MEDIATED NEUROPROTECTION
AGAINST MITOCHONDRIAL DYSFUNCTION
Ding I Yang, CL Wu
National Yang-Ming University, Institute of Brain Science, Taipei, Taiwan
Brain-derived neurotrophic factor (BDNF) is a neurotrophin with neuroprotective effects. Sonic hedgehog (SHH) is a morphogen involved in
670
embryonic development. We have reported that BDNF induces SHH,
thereby contributing to its neuroprotective effects against 3-nitropropionic acid (3-NP) that is a mitochondrial inhibitor (Wu CL et al, Biochem
Biophys Res Commun 2009; 385: 112-117). Herein we demonstrated
that BDNF enhancement of SHH expression requires induction of erythropoietin (EPO). Primary cortical cultures were prepared from fetal SD
rats at embryonic day 18. Real-time RT-PCR and Western blotting were
respectively used to determine the mRNA and protein levels. Hoechst
staining and propidium iodide (PI)/Hoechst double staining were applied
to assess the extent of cell survival and death, respectively. We found
BDNF enhanced EPO expression at earlier time points than its induction
of SHH. BDNF-induced SHH were abrogated by soluble EPO receptor
(sEPO-R), an EPO inhibitor. In contrast, BDNF-induced EPO was not
affected by cyclopamine (CPM), a SHH pathway inhibitor. Recombinant
rat EPO (rEPO) alone was sufcient to induce SHH. These results thus
established a BDNF-EPO-SHH axis. BDNF-induced 3-NP resistance
was abrogated by the sEPO-R. Preconditioning of cortical cultures with
rEPO and puried human EPO (pEPO) both neutralized 3-NP toxicity.
The rEPO-dependent 3-NP resistance was abolished by CPM but the
EPO inhibitor sEPO-R has no effects on SHH effects, despite its inhibition on BDNF neuroprotection. In conclusion, our results establish a signaling cascade of BDNF-EPO-SHH-3-NP resistance in rat cortical
neurons.
Paper No.: 1678

HEALTH AND DISEASE
AVE3085 INHIBITS HOMOCYSTEINE-INDUCED ENDOTHELIAL DYSFUNCTION: ROLE OF PHOSPHATIDYLINOSITOL
3-KINASE / PROTEIN KINASE AKT
Qin Yang(1), H-M Xue(1), J-H Huang(1), G-W He(1,2)
(1) The Chinese University of Hong Kong, Department of Surgery, Hong
Kong, PR China
(2) Nankai University, Medical College, TEDA International Cardiovascular Hospital, Tianjin, PR China
Introduction: Hyperhomocysteinemia is an independent risk factor for
various cardiovascular diseases and is believed to cause vascular lesion
formation and endothelial dysfunction. In this study, we tested the efcacy of AVE3085, a newly developed transcription enhancer of endothelial NOS (eNOS), in inhibiting homocysteine-induced endothelial
dysfunction with the underlying mechanisms explored. Materials &
Methods: Porcine coronary small arteries (diameter 600 to 800 lm) were
incubated for 24 hours with homocysteine (50 lM), AVE3085 (10 lM),
or homocysteine plus AVE3085. Endothelium-dependent relaxation to
bradykinin (-10~-6.5 LogM) and endothelium-independent relaxation to
sodium nitroprusside (-11~-4.5 LogM) were studied in a myograph. Protein expression of eNOS and the phosphorylation of eNOS at serine1177 (p-eNOSSer1177) were determined by Western blot. Results: Incubation with homocysteine for 24 hours did not alter the vessel response
to sodium nitroprusside but signicantly decreased the vasorelaxation to
bradykinin (60.9 5.4% vs. 93.1 2.0% in control, p < 0.0001; EC50: 6.64 0.39 vs. -7.73 0.26 LogM, P < 0.05). Protein expressions of
eNOS and p-eNOSSer1177 were markedly inhibited by homocysteine.
Cotreatment with AVE3085 restored bradykinin-induced relaxation (90.5
1.6% vs. 60.9 5.4% p < 0.0001) and enhanced eNOS and p-eNOSSer1177 expression. Pretreatment with either protein kinase Akt inhibitor
or inhibitors of phosphatidylinositol 3-kinase (PI3-kinase), wortmannin
and LY294002, attenuated the protective effect of AVE3085 on vasorelaxation. Inhibition of PI3-kinase/Akt also decreased eNOS phosphorylation at serine-1177 that was enhanced by AVE3085. Conclusions:
AVE3085 treatment attenuates homocysteine-induced coronary endothelial dysfunction in an eNOS-dependent manner that involves the activation of PI3-kinase/Akt.
This study was supported by Hong Kong RGC grants (CUHK4651/07M
& 4789/09M) and CUHK direct grant 2041457.
Paper No.: 1679

HEALTH AND DISEASE
ROLE OF CANONICAL TRANSIENT RECEPTOR POTENTIAL
CHANNELS IN ENDOTHELIAL FUNCTION AND THE EFFECT
OF HYPOXIA-REOXYGENATION
Qin Yang(1), J-H Huang(1), H-M Xue(1), X-Q Yao(2), G-W He(1,3)
(1) The Chinese University of Hong Kong, Department of Surgery, Hong
Kong, PR China
(2) The Chinese University of Hong Kong, School of Biomedical Sciences, Hong Kong, PR China
(3) Nankai University, Medical College, TEDA International Cardiovascular Hospital, Tianjin, PR China
Introduction: Canonical transient receptor potential channels (TRPCs) are
important Ca2 + -permeable cation channels in vascular endothelium
physiology. In this study, we investigated the role of TRPCs in vasorelaxation and the effect of hypoxia-reoxygenation (H-R) on these channels. Materials & Methods: Endothelium-dependent relaxation to
bradykinin (-10~-6.5 LogM) was studied in porcine coronary small arteries (diameter 600 to 800 lm) in a myograph. Primary cultured porcine
coronary endothelial cells (PCECs) were used for patch-clamp study.
Results: Pre-incubation with TRPCs inhibitor SKF96365 (10 lM)
decreased bradykinin-induced vasorelaxation in coronary arteries (86.9
4.1% vs. 94.4 2.8%, P < 0.05). Similar reduction (P < 0.05) was also
observed in the vessels pre-incubated with the specic TRPC3 inhibitor,
Pyr3 (3 lM). H-R (60-30min, PO2 < 10 mmHg) markedly reduced the
vasorelaxation to bradykinin (70.3 6.4% vs. 88.9 3.5% in normoxia
control, P < 0.05) and such reduction was restored (96.4 1.8%) by
pre-incubation with TRPC3/6/7 activator 1-oleoyl-2-acetyl-sn-glycerol
(OAG, 100 lM). In primary cultured PCECs, the cation current elicited
by bradykinin was inhibited by exposure to H-R (8.6 0.4 vs. 14.0
1.5 pA/pF in control, P < 0.01). Pyr3-sensitive TRPC3 current was
reduced from 6.3 0.6 pA/pF to 3.8 0.3 pA/pF after H-R exposure (P
< 0.01). Conclusions: We conclude that TRPCs, including TRPC3, are
involved in bradykinin-induced endothelium-dependent relaxation in porcine coronary arteries. The impairment of coronary endothelial function
caused by H-R is associated with the inhibition of the channel activity of
TRPCs. Targeting on TRPCs may become a promising strategy for endothelium protection in ischemic coronary disease.
This study was supported by Hong Kong RGC grants (CUHK4651/07M
& 4789/09M) and CUHK direct grant 2041457.
Paper No.: 1545

COCAINE INDUCES NITRIC OXIDE PRODUCTION AND
APOPTOSIS IN FETAL RAT MYOCARDIAL CELLS
Shumei Yang(1), G Li(2), L Zhang(2)
(1) California State University, San Bernardino, CA, USA
(2) Loma Linda University, Center for Perinatal Biology, Loma Linda,
CA, USA
Cocaine abuse during pregnancy is associated with numerous adverse
perinatal outcomes and predisposes the fetus and neonate to various cardiovascular dysfunctions. The present study tested the hypothesis that
cocaine increases nitric oxide (NO) production and promotes apoptosis
in fetal rat myocardial cells (FRMCs). Primary culture of FRMCs was
prepared from hearts of near-term fetal rats. Cocaine increased soluble
NOS activity at 15 and 30 min and caused acute and prolonged increases
in NO production in ERMCs, which were inhibited by L-NAME and
geldanamycin. Consistent with increased NOS activity, cocaine elevated
intracellular Ca2 + concentrations. Additionally, cocaine produced a concurrent increase in peroxide production in FRMCs. In accordance,
cocaine induced apoptosis of FRMCs, which was inhibited by L-NAME.
671
Consistent with its inhibition of apoptosis, L-NAME inhibited cocaineinduced cytochrome c release and the activation of caspase-9 and caspase-3. In addition, the cocaine-mediated reduction of Bcl-2 levels in
FRMCs was blocked by L-NAME. In contrast to L-NAME, the selective
iNOS inhibitor AMT had no signicant effect on the cocaine-mediated
decrease in cell viability of FRMCs. The results demonstrate that cocaine
increases NO production and oxidative stress through stimulating nitric
oxide synthase activity in FRMCs, which play a key role in cocaineinduced apoptosis in these cells.
(Supported in part by NIH grants GM073842 and DA025319).
Paper No.: 2211

STRUCTURAL ALTERATIONS OF PKCE GENE PROMOTER
BY CYTOSINE METHYLATION DECREASES SP1 BINDING
AFFINITY
CYP2C19, and N-desmethylclobazam is inactivated by the metabolism

of CYP2C19. Low dose clobazam was effective to patients with refractory epilepsy who had higher blood levels of N-desmethylclobazam. In
this study, we quantitatively evaluate the effects of genetic polymorphisms of CYP2C19 and CYP3A5 and other antiepileptic agents on the
pharmacokinetics of clobazam and N-desmethylclobazam in Japanese
adults with refractory epilepsy by the polulation analysis. Data were collected from 77 adult refractory epileptic patients (269 concentrations).
The apparent clearance of clobazam was increased 2.3-fold by the coadministration of carbamazepine and decreased 52% in patients heterozygous for the CYP2C19 variants. The apparent clearance of N-desmethylclobazam was increased 1.3-fold and 1.5-fold by the co-administration of
carbamazepine and valproic acid, respectively, and decreased 21% in
patients homozygous for the CYP2C19 variants. The CYP2C19 variants
rather than co-administered anticonvulsants had a major impact on the
N-desmethylclobazam concentration. In order to optimize the treatment
regimen, it could be useful to obtain genetic information on CYP2C19
before the start of clobazam therapy.
Shumei Yang(1), J Tenayuca(1), L Zhang(2)

(1) California State University, Department of Chemistry and Biochemistry, San Bernardino, CA USA
(2) Loma Linda University, Loma Linda, CA, USA
Previous studies have demonstrated that adverse in utero environment
causes an increase in CpG methylation of the SP1 binding sites at -268
and -346 of protein kinase Ce (PKCe) gene promoter in the heart. Electrophoretic mobility shift assay (EMSA) was employed in the present
study to evaluate the impact of single and full methylation of CpGs at 268 and -346 sites on transcription factor binding afnity. For the both
sites, methylation of single CpG partially inhibited SP1 binding and
methylation of both CpGs caused further decreases in SP1 binding. Computation of van der Waals (VDW) energy of molecular models of SP1 in
complex with the promoter binding sites conrmed these observations in
that VDW system energy values increased progressively with the number
of CpG sites methylated. Further molecular modeling analysis has indicated that the reorientation of the phosphate groups from steric crowding
of methylated cytosine bases appears to be a key facilitator of the
observed structural alterations. Methylation of both CpG sites caused an
increase in the helical twisting of the SP1 binding sites, leading to a signicant change in the conformation of the major and minor grooves of
the binding sites and thereby impairing the extent to which SP1 can bind.
These ndings provide a possible mechanism underlying the inhibition
of SP1 binding by cytosine methylation at the promoter region.
(Supported in part by USA NIH grants GM073842 and DA025319)
Paper No.: 2295

POPULATION PHARMACOKINETICS AND PHARMACOGENETICS OF CLOBAZAM AND ITS ACTIVE METABOLITE IN
JAPANESE ADULTS WITH REFRACTORY EPILEPSY
Ikuko Yano(1,2), M Shibata(1), S Yasuda(2), A Yokomasu(2), K Hosohata(2), S Masuda(2), A Ikeda(3), R Takahashi(3), K Inui(2),
Department of Clinical Pharmacy and Education, Kyoto, Japan
(2) Kyoto University Hospital, Department of Pharmacy, Kyoto, Japan
(3) Kyoto University Graduate School of Medicine, Department of Neurology, Kyoto,Japan
Paper No.: 994

DISEASE AND THERAPY
MECHANISMS OF AMPHOTERICIN B-INDUCED RENAL
TUBULAR CELL INJURY
Takahisa Yano(1), Y Itoh(2), E Kawamura(1), A Maeda(1), Y Arimura(1),
N Egashira(1), M Nishida(3), H Kurose(3), R Oishi(1),
(1) Kyushu University Hospital, Department of Pharmacy, Fukuoka,
Japan
(2) Gifu University Hospital, Department of Pharmacy, Gifu, Japan
(3) Kyushu University Graduate School of Pharmaceutical Sciences,
Department of Pharmacology and Toxicology, Fukuoka, Japan
Amphotericin B (AmB) is one of the most effective antifungal agents,
however, its use is often limited by the occurrence of adverse events,
especially nephrotoxicity. In the present study, we investigated the cellular mechanisms underlying AmB-induced renal tubular cell injury in cultured LLC-PK1 cells. AmB caused concentration- and time-dependent
cell injury, as determined by WST-8 assay, leakage of lactate dehydrogenase and tissue ATP depletion. AmB caused an increase in the propidium
iodide (PI) uptake but no marked change in the number of annexin Vpositive cells, while a lot of other antimicrobial agents such as aminoglycosides and vancomycin caused increases in the numbers of apoptotic
cells (annexin V-positive and PI-negative). These results suggested a lack
of involvement of apoptosis in AmB-induced LLC-PK1 cell injury. The
AmB-induced cell injury was inhibited by the depletion of membrane
cholesterol, the lowering extracellular Na+ or the chelation of intracellular Ca2 + . The elevation of intracellular Ca2 + concentration may be
mediated through activation of ryanodine receptor (RyR) on endoplasmic
reticulum (ER) and mitochondrial Na+/Ca2 + exchanger, since the cell
injury was attenuated by dantrolene (RyR antagonist) or CGP37157
(Na+/Ca2 + exchanger inhibitor). Moreover, mitogen-activated protein
(MAP) kinase inhibitors reversed the mitochondrial membrane depolarization as well as the cell injury induced by AmB, suggesting the
involvement of MAP kinases in AmB-induced cell injury. These ndings
suggest that Na+ entry through membrane pores formed by the association of AmB with membrane cholesterol leads to activation of MAP kinases and elevation of intracellular Ca2 + , leading to renal tubular cell
injury.
Clobazam is mainly used as an add-on therapy for refractory epilepsy.

The conversion of clobazam to an active metabolite N-desmethylclobazam is carried out mainly by cytochrome P450 (CYP) 3A4 and partly by
672
Paper No.: 2144
DISEASES
PROSTAGLANDIN E2-EP4 SIGNALLING PROMOTES IMMUNE
INFLAMMATION THROUGH TH1 CELL DIFFERENTIATION
AND TH17 CELL EXPANSION
decreased signicantly. Whereas, the number of Bcl-2 positive cells in

the CIG 60 and 180 mg/kg groups as compared with model groups
increased signicantly. The protein expression of BDNF, TrkB, PI-3K
and p-Akt in the CIG groups as compared with model groups signicantly increased. The results suggest that CIG could promotes cell survival in the brain of focal cerebra ischemic rats, and the mechanism may
be related to the activation of PI-3K/Akt signalling pathway in the brain.
Chengcan Yao(1), D Sakata(1), Y Esaki(1), Y Li(1), T Matsuoka(1),

K Kuroiwa(2), Y Sugimoto(2), S Narumiya(1)
(1) Kyoto University Faculty of Medicine, Department of Pharmacology,
Kyoto, Japan
(2) Kyoto University Faculty of Pharmaceutical Sciences, Department of
Physiological Chemistry, Kyoto, Japan
Two distinct helper T (Th) subsets, Th1 and Th17, mediate tissue damage and inammation in animal models of various immune diseases such
as multiple sclerosis and allergic disorders. Prostaglandin E2 (PGE2) is
long known to exert suppression on T cells by elevating the intracellular
cAMP level through receptor subtypes EP2 and EP4. However, while the
EP2/EP4-mediated immunosuppression is clearly seen in vitro, such
action is rarely detected in intact animals, leaving the function of EP2
and EP4 a mystery. By means of pharmaceutical and genetic approaches,
we found that under strengthened T cell receptor stimulation, PGE2
induces IL-12-drived Th1 differentiation and IL-23-drived Th17 expansion in vitro. EP2- and EP4-selective agonists mimic these PGE2 actions,
which are absent in cells decient in the respective receptor. Notably,
while the EP2/EP4-promoted Th17 expansion is mediated by cAMPPKA signalling, the EP2/EP4-promoted Th1 differentiation results from
up-regulation of IL-12 receptor b2 chain mediated by both cAMP-PKA
and PI3K-Akt signalling. In addition to cAMP, both EP2 and EP4 activate the PI3K-Akt signalling and additively to CD3/CD28 stimulation in
T cells. Furthermore, induction of IL-23 in dendritic cells requires the
PGE2-EP4 signalling and depends on the cAMP-Epac pathway. Administration of an EP4-selective antagonist in vivo decreases accumulation of
both Th1 and Th17 cells in regional lymph nodes, and suppresses the
disease progression in mice subjected to experimental autoimmune
encephalomyelitis or contact hypersensitivity. Thus, the PGE2-EP4 signalling promotes immune inammation through Th1 differentiation and
Th17 expansion, and the EP4 antagonism may be therapeutically useful
for various immune diseases.
Paper No.: 3258

CORNEL IRIDOID GLYCOSIDE PROMOTES CELL SURVIVAL
VIA THE ACTIVATION OF PI-3K/AKT SIGNALLING
PATHWAY AFTER STROKE IN THE RAT
Ruiqin Yao(1), X Wang(1), X Liu(1), L Zhang(2), C Ye(2), W Wang(2),
H Ai(2), L Li(2)
(1) Xuzhou Medical College, Department of Neurobiology, Xuzhou, PR
China
(2) Xuanwu Hospital of Capital Medical University, Xuzhou, PR China
To investigate whether Cornel Iridoid Glycoside promotes cell survival
via PI-3K/Akt signalling pathway in the brain of focal cerebral ischemic
rat model. Adult male Sprague Dwaley rats were randomly allocated into
sham operation, model and CIG treatment groups (20, 60 and 180 mg/
kg, respectively).The model was carried on middle cerebral artery occlusion MCAO with suture embolus. CIG was intragastrically administered
to rats starting from 3 h after the onset of MCAO. The cell apoptosis
was detected by TUNEL staining. The expression of Bcl-2 and Bax were
detected by immunohistochemistry staining. The protein expression of
BDNF, TrkB, PI-3K and p-Akt were detected according to western blot.
Seven days after MCAO, the number of TUNLE and Bax positive cells
in the CIG 60 and 180 mg/kg groups as compared with model groups
Paper No.: 2772

HEALTH AND DISEASE
VASODILATATION TO NO- IN RAT SMALL MESENTERIC
ARTERIES; A ROLE FOR POTASSIUM CHANNELS
Polina Yarova, C Garland, K Dora
Nitric oxide in several redox forms, including uncharged free radical
state (NO) and nitroxyl anion (NO-) contributes to endothelium-dependent relaxation. This study proved the contribution of potassium channels
in local and distant or spreading dilatation in resistance arteries to NO-.
Arterial diameter recordings were obtained from pressurized small mesenteric arteries; arterial contraction was generated with phenylephrine.
Angelis salt (NO- donor) in concentrations 100 nM - 100 lM caused
complete vasodilatation, similar to the effect of NO-gassed solutions.
The free radical scavenger, carboxy-PTIO, did not alter the vasodilatation, but addition of the NO- scavenger L-cysteine markedly inhibited
the response. 4-AP (150 lM) and TEA (1 mM) attenuated relaxation to
Angelis salt and in combination had a small additive effect. The coapplication of 10 lM ODQ further inhibited the vasodilatation. Iberiotoxin (100 nM, BKCa inhibitor) attenuated the vasodilatation only when
applied together with an NO scavenger 100 lM hydroxocobalamin.
Depolarization (and contraction) with 45 mM KCl largely inhibited and
addition of ODQ blocked the vasodilatation. In triple-cannulated mesenteric arteries, Angelis salt evoked spreading dilatation similar to the acetylcholine. 1 mM TEA and 150 lM 4-AP alone did not modify this
dilatation, but together they enhanced the spread. 45 mM potassium
blocked the spreading response. This study suggests that NO- targets
both KV and BKCa channels, as well as guanylyl cyclase in resistance
arteries. KV and KCa channels are crucial for local vasodilatation and
inhibition of the channels improves spreading dilatation, probably by
reducing dissipation of the hyperpolarizing current activated by guanylyl
cyclase.
Paper No.: 952

DISEASES
CARBOXYAMIDOTRIAZOLE INHIBITED THE SECRETION
OF PRO-INFLAMMATION CYTOKINES AND THE
EXPRESSION OF INOS IN MACROPHAGES OF ADJUVANT
ARTHRITIS RATS
Caiying Ye, D Zhang, R Zheng, L Guo
Chinese Academy of Medical Sciences, Institute of Basic Medicine,
Carboxyamidotriazole (CAI) is an inhibitor of both proliferation of certain cancer cells and tumor angiogenesis. Its effects on acute and chronic
inammation including rat adjuvant arthritis have been reported (L Guo
et al, JPET 2008 325:10-16,). The behavior of peritoneal cavities macrophages from long term CAI treated (20mg/kg/day for 21days) adjuvant
arthritis rats is reported here. The secretion of TNF-a,IL-6 from the macrophages of CAI treated groups were signicantly lower than that form
controls, whiles IL-10 was signicantly higher. The phosphorylation of
673
IL-b in the macrophages from CAI treated group was signicant lower
than that from the control. CAI also inhibited iNOS express in the macrophages. However, it had no effect on the express of COX2 and the
secretion of PGE2. Furthermore, the phagocytosis function of the macrophages was not affected by CAI. These results indicate that mechanisms
underlying the anti-inammatory effect of CAI are different from that of
NSAID.
This work is supported by National Natural Science Foundation
30873075.
Paper No.: 935

POLYPEPTIDE FROM CHLAMYS FARRERI PROTECT
NEUROBLASTOMA (SH-SY5Y) CELLS FROM OXIDATIVE
STRESS BY ENDOPLASMIC RETICULUM STRESS AND JNK
ACTIVATION
Junli Ye(1), X Liu(2), J Yu(3), J Tong(4), C Wang(4)
remains to be claried. In rat primary cortical neurons, the oxygen-glucose deprivation (OGD) causes an increase of H2O2 as well as Sp1 in
the early phase of the treatment. We discovered hereby for the rst time
that there is a longer 5-UTR in the Sp1 mRNA that contains an internal
ribosome entry site (IRES) which is well-known to render a fast and efcient translation of existing mRNAs. Importantly, by using the polysomal
fragmentation and bicistronic luciferase assays, we found that H2O2 activates the IRES-dependent translation. In rat models of stroke, while the
Sp1 level is increased in the cortex, blocking the Sp1 binding to DNA
by mithramycin enhanced cortical injuries. These results demonstrate that
neurons can use H2O2 as a signalling molecule to activate the IRESdependent translation of Sp1 mRNA to increase Sp1.
Paper No.: 2832

MORINDA CITRIFOLIA LEAVE EXTRACT PROLONGS
HUMAN BLOOD COAGULATION IN VITRO
Peng Nam Yeoh(1), JK Kumar(2), JW Mak(2), SK Cheong(3)
(1) Department of Pathophysiology, Medical College, Qingdao University, Qingdao, Shandong Province, PR China
(2) Department of Pharmacy, Dezhou Peoples Hospital, Shandong
Province, PR China
(3) Department of Pharmacology, Laiyang medical school, Shandong
Province, PR China
(4) Department of Pharmacology, Medical College, Qingdao University,
Qingdao,Shandong Province, PR China
Oxidative stress and endoplasmic reticulum stress (ERS) has been suggested to be involved in some human neuronal diseases, such as Parkinsons disease, Alzheimers and prion disease, as well as other disorders.
Treatment with antioxidants is a promising approach for slowing disease
progression. Polypeptide from Chlamys farreri (PCF) is a natural marine
antioxidant and our previous studies have reported that PCF could effectively protect neuroblastoma SH-SY5Y cells against hydrogen peroxide
(H2O2)-induced oxidative injury in vitro. In this study, we examined the
role of the ERS and mitogen-activated protein (MAP) kinase pathway
for the protective effect of PCF. Pretreatment of the human neuroblastoma cell line SH-SY5Y with H2O2 activates the endoplasmic reticulum
(ER) chaperones, grp78 and grp94, and the transcription factor, gadd
153. These effects correlate with the activation of JNK and ERK. Treatment with PCF inhibits the activation of grp78, grp94 and gadd 153,
reduces the phosphorylation of JNK and prevents cell apoptosis. These
results indicate that ERS plays an important role in the oxidative stess
injury and PCF cytoprotection. ER stress may modulates the balance signalling pathways for PCF to prevent cell death after oxidative injury.
JNK inactivation is an important downstream effector mechanism for cellular protection by ER stress.
Keywords: Polypeptide from Chlamys farreri; H2O2; Oxidative stress;
endoplasmic reticulum stress; SH-SY5Y cells; Apoptosis; JNK
Paper No.: 1731

FOCUSED CONFERENCE GROUP: SOCIETY WORKSHOP SW08
TRANSLATIONAL AND TRANSCRIPTIONAL CONTROL OF
SP1 AGAINST ISCHEMIA VIA A HYDROGEN PEROXIDEACTIVATED IRES PATHWAY
S-H Yeh(1), Li-Chin Ou(1), P-W Gean(2), W-P Yang(2), C-Y Hsu(2),
JT Tseng(2), T-P Su(2), W-C Chang(2), J-J Hung(2)
(1) National Health Research Institutes, Zhunan, Taiwan
(2) National Cheng-Kung University, Zhunan, Taiwan
The mechanism underlying the increase of Sp1 (specic protein 1) and
the physiological consequence during hypoxia and oxidative stress
(1) International Medical University, School of Pharmacy & Health Sciences, Kuala Lumpur, Malaysia
(2) International Medical University, School of Medical Sciences, Kuala
Lumpur,Malaysia
(3) International Medical University, Clinical School, Kuala Lumpur,
Malaysia
Morinda citrifolia (Mc), known as Mengkudu in Malaysia or Noni in the
western world has many uses traditionally including to treat asthma, diabetes, high blood pressure, promote menstruation. It is claimed to have
anticoagulant, analgesic, antiinammatory, antioxidant, antiseptic, antibiotic, anticancer and immunomodulating activities. This study determines
the effect of the hot water extract (McLE) of the leaves of Mc on clotting
of blood drawn from human volunteers. A total of 25 volunteers participated in the study. Blood drawn (10ml) from each volunteer was put into
various tubes containing either the control or a certain dose of McLE.
Various doses of Mc lyophilized extract was used to study the effect on
clotting time, coagulation prole, prothrombin time (PT), activated
thromboplastin time (aPTT) and thrombin time (TT) with PBS (phosphate buffer) and EDTA used as the negative and the positive controls
respectively. The data was analyzed using paired t test. Mc (10 to 62.5
mg/ml signicantly (P < 0.05) increased meanSE clotting time compared to control (PBS) 342.9 7.8sec in a dose dependent manner, varying from 419.1 18.4sec, to 3020.1 476.8sec respectively from the
lowest to the highest dose. Blood did not clot even after 24 hours with
doses of 100 to 125 mg/ml. The coagulation prole assays show Mc to
affect the intrinsic coagulation pathway, with signicant prolongation (P
< 0.05) of the aPTT. Mc also affects the extrinsic coagulation pathway
by signicantly prolonging the PT. It affects brin formation as the TT
was signicantly (P < 0.05) prolonged. All ndings were dose dependent.
Paper No.: 3297

EFFECT OF MORINDA CITRIFOLIA HOT WATER EXTRACT
ON PLATELET AGGREGATION
Peng Nam Yeoh(1), V Ramachandran(2), JW Mak(2), SK Cheong(3), CF
Leong(4)
(1) International Medical University, School of Pharmacy & Health Sciences, Kuala Lumpur, Malaysia
(2) International Medical University, School of Medical Sciences, Kuala
Lumpur, Malaysia
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(3) International Medical University, Clinical School, Kuala Lumpur,
Malaysia
(4) University Kebangsaan Malaysia, Faculty of Medicine, Kuala Lumpur, Malaysia
Morinda citrifolia (Mc) known in Malaysia as Mengkudu or Noni in the
western world is a medicinal plant used traditionally for the relief of
many diseases. Earlier studies in this laboratory showed the fruit extract
could reduce blood coagulation in rabbits (in vivo) and in human blood
(in vitro). This investigation determined the effect of the hot water extract
of Mc fruits on platelet function in human whole blood (in vitro) with
phosphate buffer as the control. The assay was carried out using Multiplate analyzer (Dynabyte, Germany), with different activators / agonists
(ristocetin, collagen, adenosine diphosphate and arachidonic acid), which
were used for assessing different aspects of platelet function. The data
was analyzed using paired t test against phosphate buffer as the control. The results showed that Mc extract reduced platelet aggregation signicantly (P < 0.05) with all agonists. The lowest dose, 0.5mg/ml Mc
decreased platelet aggregation signicantly (P < 0.001) with collagen
only, by 38%. Higher doses, 5mg/ml and 25mg/ml Mc decreased platelet
aggregation by 58% to 80% signicantly (P < 0.001) with agonists, ristocetin, collagen and arachidonic acid. With adenosine diphosphate, Mc
(5 and 25 mg/ml) produced a decrease of 39% (P < 0.05), 78% (P <
0.01) of platelet aggregation, respectively. The results suggest that Mc
could interrupt the ADP, GPIb and the collagen receptors as well as inhibit the cyclooxygenase pathway to affect platelet aggregation.
Paper No.: 2243

MITOCHONDRIA AND DIHYDROTANSHINONE-INDUCED
APOPTOSIS IN HEPG2 CELLS
JHK Yeung(1), WYW Lee(1), Peter Xuelin Zhou(1), YW Kwan(1), SK
Kong(1), HP Ho(2)
(1) The Chinese University of Hong Kong, Faculty of Medicine, School
of Biomedical Sciences, Hong Kong, PR China
(2) The Chinese University of Hong Kong, Faculty of Engineering,
Department of Electronic Engineering, Hong Kong, PR China
Dihydrotanshinone is one of the abietane type-diterpene quinones (tanshinones) isolated from the dried roots and rhizome of Radix Salvia miltiorrhiza (Danshen), a Chinese herbal medicine. Reactive oxygen species
(ROS) has been suggested to play an important role in its anti-cancer
property. This study investigated the mechanism(s) by which mitochondria, the major site of ROS generation in mammalian cells, modulate the
ROS-mediated p38 mitogen-activated protein kinase in dihydrotanshinone-induced apoptosis in HepG2 cells (Lee WYW et al., Cancer Letters
2009;285: 46-57). Using confocal microscopy, mitochondrial membrane
potential and mitochondrial ROS after dihydrotanshinone loading was
monitored in HepG2 cells pre-probed with oxidised Mitotracker Red
CMXRos and reduced Mitotracker Red CM-H2XRos, respectively.
Compared to tanshinone IIA, a weaker pro-oxidative tanshinone, dihydrotanshinone more signicantly reduced mitochondrial membrane
potential (by 65% vs 26%) and induced mitochondrial ROS depletion
(by 44% vs 24%). N-acetyl cysteine reduced the depletion of mitochondrial ROS (P < 0.001), but not the loss of mitochondrial membrane
potential, which could partly explain its protective effect on dihydrotanshinone-induced apoptosis. A simultaneous rise in nitric oxide (NO),
monitored by diuorouorescein diacetate, was also observed. However,
blocking NO generation increased cellular susceptibility towards dihydrotanshinone-induced cell death in MTT assay. This is the rst evidence
which indicates the importance of mitochondrial ROS level in regulating
cellular response toward tanshinones treatment.
Paper No.: 1653

VASCULAR EFFECTS OF PROTEASE INHIBITORS AND
NON-NUCLEOSIDE REVERSE TRANSCRIPTASE INHIBITORS,
THE MAJOR COMPONENTS OF HIGHLY ACTIVE
ANTIRETROVIRAL THERAPY [HAART]
Yuen Ting Yeung(1), SS Lee(2), PM Vanhoutte(1), SWS Leung(1)
(1) The University of Hong Kong, Li Ka Shing Faculty of Medicine,
Department of Pharmacology and Pharmacy, Hong Kong, PR China
(2) The Chinese University of Hong Kong, PR China
The number of patients with human immunodeciency virus (HIV)
infection increase every year worldwide. Highly active antiretroviral therapy (HAART), including protease inhibitors (PIs) and non-nucleoside
reverse transcriptase inhibitors (NNRTIs), has shown promise for the
treatment of HIV. However, previous experimental studies suggest that
certain PIs affect vascular regulation, leading to the development of cardiovascular disease (CVD), which becomes one of the major causes of
morbidity and mortality in HIV-infected patients. Therefore, it is possible
that HAART imposes adverse effects on vascular reactivity. The present
study was designed to study the acute effects of various clinically used
PIs (indinavir, lopinavir, nelnavir, ritonavir, saquinavir and tipranavir)
and NNRTIs (efavirenz and nevirapine) on isolated rat mesenteric arteries suspended in organ chambers for the measurement of isometric force.
Among the antiretroviral drugs tested, all PIs and NNRTIs, except nevirapine, caused full relaxation of mesenteric arteries during contractions to
phenylephrine. These relaxations were not affected by the presence of
nitro-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthases. The present data suggested that most clinically used PIs and NNRTIs relax mesenteric arteries by activating a signalling pathway that is
independent of the release of nitric oxide. Since the relaxation occurred
when the antiretroviral drugs are present at high concentrations, care
should be taken with the prescription of high doses of these drugs to
avoid unwanted vasodilatation.
Paper No.: 2002

DIFFERENCES IN EFFICACY AMONG FOLFOX FOR
ADVANCED GASTRIC CANCER INVOLVE 14-3-3D
Bo Yi(2), QY Li(2), OP Huang(1), D Liu(3), D Yin(4), M He(1)
of Pharmacology& Molecular Therapeutics, Nanchang, PR China
(2) Jiangxi Tumor Hospital, Nanchang, PR China
(3) Jiangxi Maternal and child Health Hospital, Nanchang, PR China
Medicine at SecondAfliated Hospital, Nanchang, PR China
(5)
(6) Nanchang University School of Pharmaceutical Science
14-3-3 d, a member of a highly conserved family of 14-3-3 proteins, regulates many cellular processes that are implicated in cancer development.
Since the publication of the MOSAIC test results in 2004, the FOLFOX
regimen has been established as an adjuvant treatment which is recommended in advanced gastric cancer. However, FOLOX on the efcacy of
advanced gastric cancer patients varies considerably and its underlying
mechanism is unknown. We hypothesize that differences in efcacy
among FOLFOX for advanced gastric cancer may be related with 14-3-3
d. In present study we have selected 47 patients with advanced gastric
cancer and 36 patients with peptic ulcer. 47 patients treated with FOLFOX regimen divided into 27 therapeutic void patients and 20 therapeutic effective patients. After the end of treatment, Paired samples of
gastric cancer and peptic ulcer were obtained and submitted to reverse
transcription polymerase chain reaction (RT- PCR) and western blot. The
675
results show that 14-3-3 d protein is signicant decrease in advanced
gastric cancer, compared to peptic ulcer. Moreover, 14-3-3 d is methylated in patients with effective FOLOX treatment, while not in patients with
void FOLOX treatment. Therefore, we can drew a conclusion whether
14-3-3 d methylation or not is main reason for differences in efcacy
among FOLFOX for advanced gastric cancer.
This study was supported by the National Science Foundation of China
(30460048) and the the 973 Program(2009CB526405).
Paper No.: 2008

THE ROLE OF THE REACTIVE OXYGEN SPECIES (ROS)INDUCED ROS RELEASE MEDIATED BY MITOCHONDRIAL
PERMEABILITY TRANSITION PORE IN IRON OVERLOAD
RESULTED IN HEPATIC DAMAGE
Dong Yin(2), D Liu(1), A Que(1), Q Huang(1), M He(1)
(1) JNanchang University School of Pharmaceutical Science, Department
Chronic iron overload can result in hepatic brosis and even neoplastic
transformation due to the burst of reactive oxygen species(ROS). Mitochondria are proposed to play a prominent role in the ROS production.
The purpose of this study was to investigate the the role of mitochondrial
permeability transition pore (mPTP) in the burst of ROS, and futher to
clarify the mechanism of the ROS indued by iron overload resulting in
hepatic damage. It was found that when iron Cdextran-induced iron overload, mice were fed cyclosporin A (CsA), a specic inhibitor of the
mPTP, diet (10 mg/kg) for 50 days, liver-to-body weight ratio, serum
levels of ALT and AST, ROS production, mitochondrial swelling, loss of
mitochondrial membrane potential, and hepatocyte apoptosis were
decreased; while total antioxidant status including SOD, GSH-Px and
catalase activities increased. The protective effect of CsA on liver of iron
overload mous e may due to both the inhibition of ROS burst and successive antioxidant effect. These data provide support that a phenomenon
termed ROS-induced ROS release (RIRR) may be involved in the burst
of ROS in the liver and greatly contributed to the hepatic damage initiatede by iron overload for the rst time.
Acknowledgement: Project supported by the National Science Foundation of China(No,30860271) and the 973 Program(No 2009CB526405).
Paper No.: 2834

SEROTONIN 5-HT2C RECEPTORS ALTERS GENE
EXPRESSION OF AMYLOID PRECURSOR PROTEIN
Ming Yin, LC Guo, J Zhang
method, selected using Zeocin and clonal cell lines developed by limit
dilution. Agonist-induced secretion of APP, APPs and Ab was investigated by pre-incubating the cells with different concentrations (0.001
lM, 0.01 lM, 0.1 lM and 1 lM) of the agonist meta-chlorophenylpiperazine (m-CPP). Immunoblotting and ELISA methods were used to detect
the APP, APPs and Ab. Results: Compared with non-edited 5-HT2CINI
isform, Abproduction decreased in 5-HT2CVNI and 5-HT2CVSV isforms,
and increased in 5-HT2CVNV and 5-HT2CVGV isforms. APP expression
and APPs secretion also displayed the same tendency for down- or upregulation. Conclusion: The different effects of 5-HT2C receptors on APP
processing may due to the APP gene expression difference.
Acknowledgement: Supported by the National Natural Science Foundation of China (30672443).
Paper No.: 2929

PSYCHOTROPIC DRUG UTILIZATION IN OLD-ADULT
COMMUNITY
N Yodu, C Pena, Rene Delgado-Hernandez
10 de Octubre Medical School, Department of Pharmacology, Havana,
Cuba
There are biopsychosocial problems typical of ageing that involve the
medical prescription or self medication of psychotherapeutic agents in
the elderly. To learn about the consumption habits of psychotherapeutic
agents among the elderly community, an observational and descriptive
study was done. A sample of 100 old adults was randomly selected and
surveyed from a population that used psychotropic drugs, had 65 or more
years of age and was under the care of family doctors in Havana City,
from March 2006 to 2007. The highest psychotropic drug consumption
was observed in women (63%), with anxiolytics being the most used
(71.2%). They were daily used in 86.1%. Diazepam had the rst place
with 23.4% followed by Nitrazepam and Meprobamate with 18.9% each.
The referred causes of use were anxiety-nervousness (46.8%) and insomnia (26.6%). Although these treatments coincide with the medical recommendations, it can be questioned if they were used rationally, after
learning that 77.3% used these drugs for a very long time, 45.6% were
self medicated and 43% of this last group got the medication though
ways out of the control of the Health National System. The inadequate
use of psychotherapeutic drugs could affect the satisfactory longevity of
older adults and it is necessary to search for ways to solve this health
problem.
Paper No.: 1313

URINARY CORTISONE/CORTISOL RATIO CORRECTED
WITH THE CORTISOL CONCENTRATION SERVES AS AN
INDEX FOR THE ACTIVITY OF HUMAN 11B-HSD2 IN VIVO
Akitomo Yokokawa, H Shibasaki, T Furuta
Shanghai Jiaotong University, School of Pharmacy, Shanghai, PR China

Introduction: Serotonin 5-HT2C receptors underwent RNA editing events
lead to produce decades of variants with different constitutive activities
and agonist or antagonist potency. 5-HT2C receptor in stably transfected
mouse 3T3 broblast cells by dexnorfenuramine increased secretion of
APPs, one of the metabolite derived from the amyloid precursor protein
(APP). It is not known whether stimulation of RNA-edited receptor isoforms can also affect APP metabolism in vitro. Materials and Methods:
The nonedit 5-HT2CINI receptor underwent site-specic mutation by
overlapping extension PCR to create four edited isforms: 5-HT2CVNI, 5HT2CVNV, 5-HT2CVSV and 5-HT2CVGV. HEK293APPSwe cells were
stably transfected with each receptor transcript using the lipofectamine
Tokyo University of School of Pharmacy and Life Sciences, Department

of Medicinal Chemistry and Clinical Pharmacy, Tokyo, Japan
11b-Hydroxysteroid dehydrogenase 2 (11b-HSD2) is expressed in aldosterone target tissues, primarily the kidney in humans. The enzyme converts physiologically active cortisol into inactive cortisone. Of the two
hormones, only cortisol has afnity to mineralocorticoid receptors (MRs)
and thus induces mineralocorticoid effects. A normal activity of 11bHSD2 is crucial for prevention of mineralocorticoid activity of cortisol.
Absent or decreased 11b-HSD2 activity results in apparent mineralocorticoid excess (AME) and cortisol-mediated hypermineralocorticoid hypertension. Urinary free cortisone/cortisol ratio has been used as a measure
676
of 11b-HSD2 activity. However, large within-day variations in the ratio
make it difcult to evaluate 11b-HSD2 activity for individuals. The aim
of the present study was to re-examine the urinary free cortisone/cortisol
ratio as the index for the activity of 11b-HSD2 in vivo. We measured the
cortisone/cortisol ratio in 60 urine samples obtained from 15 healthy volunteers. A signicant correlation of the urinary cortisone/cortisol ratio
(E/F) and the urinary cortisol concentration (F) was observed (r =
0.8582). The correlation between log (E/F) and log F shows a linear
regression equation (y = -0.4487x + 2.7475). The cortisone/cortisol ratio
below the standard straight line indicates the decreased activity of 11bHSD2. Administration of glycyrrhetinate acid to 3 normal adults clearly
demonstrated the decreased ratios below the standard line. The urinary
cortisone/cortisol ratio corrected with the cortisol concentration serves as
an index for assessing the activity of human 11b-HSD2 in vivo.
Paper No.: 1630

NASAL INSTILLATION OF NANOPARTICLE-RICH DIESEL
EXHAUST PARTICLE AFFECTS EMOTIONAL BEHAVIOR
AND LEARNING CAPABILITY IN RATS
Syunji Yokota(1), H Takashima(1), T Miyahara(1), Y Yoshida(1), T Negura(1), Y Saito(1), N Hirabayashi(1), T Watanabe(1), R Ohta(1), S Horiuchi(1), Y Fujitani(2), S Hirano(2), H Fujimaki(2)
1) Hatano Research Institute, Food and Drug Safety Center, Kanagawa,
Japan
(2) National Institute for Environmental Studies, Tsukuba, Japan
Recent studies have indicated that intranasal instillation (iNI) of ultrane
particles (UFP) may cause inammation in the brain, although none of
the studies demonstrated any effects of UFP on the central nervous system (CNS) and the animal behavior yet. In the present study, the effects
of nanoparticle-rich diesel exhaust particle (NRDEP; mean count mode
diameter, 21.45 nm) on emotional behavior and learning capability were
studied in rats by administered with iNI of NRDEP (10 or 50 microgram/rat) in 2-week old male infant by once a week for 4 weeks. Spontaneous motor activity measured at 6-week of age was higher in low doseNRDEP group during initial 3-h of 24-h measurement. In active avoidance test using shuttle box, almost all the animals indicated low avoidance ratio (within rst quartile) were NRDEP-treated animals. 24-h after
the nal iNI, histopathological examination was performed in the animals
treated with 50 microgram-NRDEP and increased number of lamella in
microglia of olfactory bulb was observed. Dopamine and its metabolite
(DOPAC) contents in medial mammillary nucleus of the brain tended to
decrease in NRDEP-treated animals at 14-week of age. From the present
results, we suggest that iNI of NRDEP to the infant rats may affect emotionality and learning capability of animals in later life via modulation in
monoamine neurotransmitter level in CNS. However, the changes were
very slight.
Paper No.: 516

INVOLVEMENT OF GLUTAMATE RECEPTORS EXISTING IN
PERIPHERAL ENDING OF PRIMARY AFFERENT FIBERS IN
NOCICEPTION
Norifumi Yonehara, Y Koike
Ohu University School of Pharmaceutical Sciences, Department of Pharmacology, Koriyama, Japan
the supercial dorsal horn. However, the role of Glu and its receptors
(GluRs) in the peripheral nervous system is not well understood. In the
present study, we determined Glu levels released in the subcutaneous
(s.c.) perfusate of the rat hind instep using a microdialysis catheter, and
the thermal withdrawal latency using the Plantar Test and the expression
of c-Fos in the dorsal horn following injection of drugs associated with
GluRs into the hindpaw. Antidromic stimulation of the sciatic nerve and
s.c. injection of capsaicin (Cap) caused an increase in Glu level in the
s.c. perfusate. Cap also remarkably decreased withdrawal latency to irradiation. These effects of Cap were inhibited by pretreatment with capsazepine. Cap-induced Glu release was also inhibited by combination
with each antagonist of iGluRs and group1 mGluR (mGluR1), but not
group II and group III mGluRs. Furthermore, these GluRs antagonists
showed remarkable inhibition against capsaicin-induced thermal hyperalgesia. Cap-induced expression of c-Fos in the dorsal horn was also inhibited by pretreatment with iGluRs and mGluR1 antagonists. These results
suggest that Glu is released from the peripheral endings of Cap-sensitive
afferent C-bers through a mechanism such as axon-reex following
noxious stimuli, and then binds to iGluRs and mGluR1 in unmyelinated
axons reinforcing their activity.
Paper No.: 1638

ROLE OF HEPARAN SULFATE IN GLUCOSE-INDUCED
INSULIN SECRETION IN MOUSE PANCREATIC BETA-CELL
LINE MIN6 CELLS
Takeo Yoshikawa(1), S Takayanagi(1), N Noguchi(2), D Zhang(1),
Y Ariyama(1), A Uruno(3), H Okamoto(3), A Sugawara(3), K Yanai(1)
Phamacology, Sendai, Japan
Otorhinolaryngology, Head and Neck Surgery, Sendai, Japan
Biochemistry, Sendai, Japan
Heparan sulfate (HS) is a highly sulfated glycosaminoglycan widely distributed on the cell surface and in the extracellular matrix. HS has been
implicated in a variety of biological events through the interactions with
numerous functional proteins including growth factors, morphogens, and
extracellular matrix components. We have recently demonstrated that HS
played an essential role to maintain normal glucose-induced insulin
secretion (GIIS) in pancreatic islets, although the precise mechanism of
GIIS regulation by HS remained to be elucidated. Here, we rst examined GIIS in mouse pancreatic b-cell line MIN6 cells after the heparitinase (an HS degrading enzyme) digestion. GIIS was decreased to about
60% in heparitinase-treated MIN6 cells compared to control MIN6 cells
treated without the enzyme. However, insulin secretion induced by glybenclamide, which closed ATP-sensitive K+ (KATP) channels, elicited
Ca2 + inux from extracellular spaces and induced insulin secretion, was
unaffected in heparitinase-treated MIN6 cells. Next, we investigated the
intracellular Ca2 + concentration [Ca2+]i) by fura-2 uorescence because
an increase in [Ca2+]i was essential for GIIS. The enzymatic removal of
HS in MIN6 cells attenuated the increase in [Ca2+]i in response to glucose. On the other hand, the increase in [Ca2+]i induced by glybenclamide was again unaffected. These results thus demonstrate that HS
contributes to ensure normal GIIS probably by regulating glucoseinduced [Ca2+]i increase in MIN6 cells, independently of the events
downstream of KATP channels. Therefore, HS might be involved in GIIS
at the level of ATP production upstream of KATP channels and/or Ca2 +
mobilization from intracellular Ca2 + stores.
Glutamate (Glu) is the predominant excitatory neurotransmitter in the

central nervous systems, and is implicated in nociceptive transmission in
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Paper No.: 1796
GRAVITY-CHANGING STRESS INCREASES SEROTONINRELATED GENE EXPRESSION IN THE MOUSE BRAIN
Mitsuhiro Yoshioka(1), T Yamaguchi(1), H Ohta(2), J Gyotoku(3), T
Ochiai(3)
(1) Hokkaido University School of Medicine, Department of Neuropharmacology, Sapporo, Japan
(2) Sato Pharmaceutical Co. Ltd, Japan
(3) Mitsuhishi Heavy Industry, Japan
A number of parabolic ight experiments have shown that processes
within the central nervous system (CNS) are affected by weightlessness
in human. It is, however, likely that changes in the CNS observed during
parabolic ights are not solely due to the repeated changes in gravity
experienced during the ight. Instead, these changes may be related to
secondary psycho-physiological reactions to the emotional and physical
stress during these ights. The purpose of the present experiment is to
elucidate whether gene expression levels, especially serotonin-related
genes, are altered in the mouse brain exposed to gravity-changing stress
using a RT-PCR method. Mice were exposed to gravity-changing stress
during 8 times repeated by parabolic ights performed by an airplane.
Serotonin transporter, tryptophan transporter and tryptophan hydroxylase-2 mRNA levels in the midbrain 6 h after the ight were signicantly
increased compared with pre-parabolic ight control. In contrast, those
of monoamine oxidase-A, 5-HT1A receptor and GAD65/67, synthases
for GABA, were not altered by the ight. The results of the present study
suggest that the serotonergic system, particularly synthetic pathway,
might be activated in the CNS by gravity-changing stress.
Paper No.: 1082

THERAPEUTIC TARGETS
NORADRENALINE ATTENUATES HYDROGEN
PEROXIDE-INDUCED CELL DEATH OF ASTROCYTE
THROUGH THE INCREASE IN THE LEVEL OF
INTRACELLULAR GLUTATHIONE
Yasuhiro Yoshioka, H Kadoi, A Yamamuro, A Kasai, A Maeda,
Setsunan University, Department of Pharmacotherapeutics, Osaka, Japan
Noradrenaline (NA) has been known to modulate the functions of astrocytes. However, the effect of NA on the antioxidative capacity of astrocytes has been poorly investigated. In this study, we investigated the
effect of NA on hydrogen peroxide (H2O2) toxicity in human astrocytoma U-251 MG cells. Cell viability was estimated by a colorimetric
MTT assay, and intracellular glutathione (GSH) level was determined by
DTNB recycling assay. Treatment of U-251 MG cells with H2O2 induced
cell death in a concentration-dependent manner. Pretreatment with NA
(1-100 lM) for 24 h concentration dependently attenuated the H2O2
induced cell death. Cytoprotective effects of NA were inhibited by a badrenoreceptor antagonist propranolol, and pretreatment with a b-adrenoreceptor agonist isoproterenol (30-100 lM) attenuated the H2O2induced
cell death. Treatment with NA (1-100 lM) for 24 h concentration-dependently increased intracellular GSH level. DL-buthionine-[S,R]-sulfoximine, a glutathione (GSH) synthesis inhibitor, inhibited the increase in
the level of intracellular GSH and negated the cytoprotection by NA.
The NA-induced increase in the level of intracellular GSH was inhibited
by propranolol. Western blot analysis revealed that NA increased the
level of c-glutamylcysteine synthetase (c-GCS), the rate-limiting enzyme
in GSH synthesis. The induction of c-GCS protein by NA was inhibited
by propranolol. These results indicate that NA protects U-251 MG cells
against H2O2-induced death through the increase in the level of intracellular GSH, and that NA increases the intracellular GSH level through the
induction of c-GSC protein via b-adrenoreceptor stimulation.
Paper No.: 2017

REGULATION OF NMDA RECEPTORS BY COPPER IONS
Haitao You(1), S Tsutsui(2), PK Stys(2), GW Zamponi(1)
(1) University of Calgary, Hotchkiss Brain Institute, Departments of
Physiology & Pharmacology, Calgary, ALB, Canada
(2) University of Calgary, Hotchkiss Brain Institute, Departments of
Clinical Neurosciences, Calgary, ALB, Canada
Copper is an essential trace metal element that is present at up to low
micromolar levels throughout the mammalian central nervous system.
Dysfunction of copper homeostasis in CNS leads to several neurological
disorders, including Wilsons disease and Menkes disease, with the latter
having been linked to NMDA receptor hyperactivity. Previous ndings
have suggested that copper ions can inhibit NMDA receptor activity.
NMDA receptor recordings are typically performed in the presence of
high (non-physiological) levels of the receptor co-agonist glycine (i.e.,
tens of micromolar), and a little known fact is that glycine chelates copper ions. Hence, the role of endogenous copper in regulating NMDA
receptor activity under physiological conditions has remained poorly
understood. To investigate the role of copper in modulating NMDA
receptor activity, we performed electrophysiological recordings in medium containing low concentrations of glycine (300 nM) in the presence
and the absence of the copper chelator bathocuproine sulphonate (BCS).
In the absence of BCS, NMDA currents rapidly desensitized, whereas
BCS induced a nonCdesensitizing current component that amounted to
approximately 20% of peak NMDA current. Similar results were
obtained with another copper chelator (cuprizone). Overall, our data indicate that copper ions are important physiological regulators of NMDA
receptor function. Furthermore, our data suggest that the well known
slowing of NMDA receptor desensitization by high levels of glycine
may at least in part be due to copper chelation.
Paper No.: 3328

CYP2C9*3 AND *13 ALLELES WERE SIGNIFICANTLY
ASSOCIATED WITH THE DECREASED METABOLISM OF
LORNOXICAM
So-Jung Youn, J-W Bae, C-G Jang, S-Y Lee
Lornoxicam has analgesic and antipyretic effects in part through the nonselective inhibition of cyclooxygenase (COX)-1 and COX-2. It is indicated for the treatment of osteoarthritis, rheumatoid arthritis and for the
management of postoperative pain. It is reported that CYP2C9 is the primary enzyme responsible for the biotransformation of the lornoxicam to
its major metabolite, 5-hydroxylornoxicam. We evaluated the effects of
genetic polymorphism of the CYP2C9 on the pharmacokinetics of lornoxicam. Thirteen healthy Korean volunteers were selected for this study.
They were grouped according to CYP2C9 genotype, group1
(CYP2C9*1/*1, n = 5), group2 (CYP2C9*1/*3, n = 5) and group3
(CYP2C9*1/*13, n = 3). They were administered a single oral dose of 8
mg lornoxicam and plasma concentrations of lornoxicam and 5-hydroxylornoxicam were determined by using HPLC-UV system. Cmax of
lornoxicam in group2 and group3 (815.3 94.0 ng/mL and 935.1
73.31 ng/mL, respectively) were signicantly higher than that in group1
(655.7 143.1 ng/mL, P = 0.0187). AUC0- of lornoxicam in group2
and group3 (5463 1279 nghr/mL and 5312 1267 nghr/mL, respectively) was also higher than that in group1 (2863 371 nghr/mL, P =
0.0031). Elimination half-life (t1/2) of lornoxicam and 5-hydroxylornoxicam in group2 and group3 were signicantly longer than those in group1
(P < 0.0001). Plasma AUC ratio (AUCLornoxicam/AUC5-OH) in group1,
group2 and group3 were 2.9 0.3, 3.6 0.3 and 3.8 0.8, respectively.
678
In conclusion, the CYP2C9*3 and CYP2C9*13 alleles are associated
with the decreased metabolism of lornoxicam.
Paper No.: 3329

IMPACT OF GENETIC VARIATIONS IN CYP450 ENZYMES ON
THE PHARMACOKINETICS OF CILOSTAZOL
So-Jung Youn, C-I Choi, Jung-WooJ-W Bae, C-G Jang, S-Y Lee
Cilostazol is a cyclic nucleotide phosphodiesterase type III inhibitor,
which is approved for the treatment of intermittent claudication. The metabolic pathway of cilostazol is so complex and many of cytochrome
P450 isozymes are involved in the biotransformation of cilostazol,
including CYP3A, CYP2C19, CYP2C8, CYP2D6, CYP1A2 and so on.
It is considered that co-administration of CYP3A or CYP2C19 inhibitors
with cilostazol causes an increase in plasma concentration of cilostazol
and possibly, an increase in the risk of adverse drug events of this agent.
We investigated the effects of CYP2C19, CYP2D6 and CYP3A5 genetic
polymorphism on the pharmacokinetics of cilostazol. After genotyping
for each isozyme using PCR-RFLP methods, 33 healthy Korean volunteers were selected for this study. They were classied as extensive metabolizers (EMs) or poor metabolizers (PMs) for each isozyme according
to genotype. A single oral dose of 100 mg cilostazol was administered to
each subject and plasma concentration of cilostazol was measured by
using HPLC-UV system. Although relatively higher Cmax and AUC and
lower CL/F were observed in CYP2C19/CYP3A5 PM/PM group compared to CYP2C19/CYP3A5 EM/EM group, these differences were not
signicant. In addition, there were no signicant changes in other pharmacokinetic parameters of cilostazol between different CYP2C19/
CYP2D6, CYP2C19/CYP3A5, or CYP2D6/CYP3A5 genotype groups.
Genetic polymorphism of cytochrome P450 enzymes involved in the
metabolism of cilostazol did not signicantly affect the pharmacokinetic
changes of cilostazol.
Paper No.: 3330

PARTIAL EFFECTS OF UGT1A1*28 ALLELE ON THE
PHARMACOKINETICS OF EZETIMIBE AND ITS
GLUCURONIDE IN HEALTHY SUBJECTS
So-Jung Youn, C-I Choi, J-W Bae, M-J Kim, C-G Jang, S-Y Lee
Ezetimibe is a lipid-lowering compound that selectively inhibits the
intestinal absorption of cholesterol and related phytosterols by binding of
Niemann-Pick C1-Like 1 (NPC1L1) protein. The major metabolic pathway of ezetimibe is hepatic glucuronidation to the pharmacologically
active phenolic glucuronide by phasec enzyme UDP-glucuronosyltransferases (UGTs), including UGT1A1. UGT1A1 is one of
highly polymorphic drug metabolizing enzyme, and UGT1A1*28 allele
is known to be associated with lower UGT1A1 expression. So we investigated the effects of UGT1A1*28 allele on the pharmacokinetics of ezetimibe and its glucuronide. Genotyping for UGT1A1*28 was performed
using direct sequencing methods, and observed allelic frequency of
UGT1A1*28 was 14.1%. Thirty-four healthy Korean volunteers were
selected and were divided by three groups according to the number of
UGT1A1*28 allele, group1 (UGT1A1*1/*1, n = 12), group2
(UGT1A1*1/*28, n = 16) and group3 (UGT1A1*28/*28, n = 6). A single
oral dose of 10 mg ezetimibe was administered to each subject and
plasma concentration of ezetimibe and ezetimibe glucuronide were measured by LC-MS/MS system. Cmax of ezetimibe in group3 was 118%
and 141% higher than that in group1 and group2 (P < 0.001 and P <
0.0001, respectively). AUC0-48 of ezetimibe in group3 was 77% and
90% higher than that in group1 and group2 (P < 0.001 and P < 0.01,
respectively). Other pharmacokinetic parameters of ezetimibe were not
signicantly different between three genotype groups. Also, Pharmacokinetic differences of ezetimibe glucuronide were statistically signicant
changed between three genotype groups. In conclusion, UGT1A1*28
allele may partially affect the pharmacokinetics of ezetimibe.
Paper No.: 3365

DISCOVERY
UDCA-INDUCED HNF-1CA INHIBITION AND
PHARMACOKINETIC CHANGES OF PITAVASTATIN IN
RELATION TO OATP1B1 TRANSPORT ACTIVITY
So-Jung Youn, C-I Choi, J-W Bae, M-J Kim, C-G Jang, S-Y Lee
Pitavastatin is an inhibitor of 3-hydroxymethylglutaryl-coenzyme A
reductase (HMG-CoA reductase) and is used for the treatment of hypercholesterolemia. Pitavastatin is known as a substrate of OATP1B1, one
of hepatic uptake drug transporters. Ursodeoxycholic acid (UDCA) is
known to be associated with lower expression of OATP1B1 by inhibition
of the activity of hepatocyte nuclear factor-1ca (HNF-1ca). We investigated the effects of UDCA on the pharmacokinetics of pitavastatin. After
OATP1B1 genotyping by PCR-RFLP method, 13 healthy Korean volunteers with OATP1B1*1a/*1b genotype selected in this open-labeled, twophase parallel study. In control phase, each subject received a single oral
dose of 2 mg pitavastatin. After washout period (UDCA phase), subjects
administered UDCA 300 mg twice daily for 14 days. On day 15, a single
oral dose of 2 mg pitavastatin was administered to each subject. Plasma
concentration of pitavastatin and its lactone were measured by using LCMS/MS system. There were no signicant differences in the pharmacokinetics of pitavastatin between two phases. Although slightly increased
Cmax, AUC0-48 and AUC0- of pitavastatin lactone in UDCA phase compared with control phase were observed, these parameters were also not
statistically signicant. Inhibition of HNF-1ca by UDCA doesnt inuence the transport capacity of OATP1B1 enough to affect the pharmacokinetics of pitavastatin and pitavastatin lactone.
Paper No.: 641

POLYPRENOLS FACILITATE TRANSPORTING OF
ADRIAMYCIN ACROSS BRAIN-BLOOD BARRIER VIA
ANTI-ANGIOGENESIS AND P-GP INHIBITION
Shenghua Yuan(1), W Ge(1), H Li(2), H Zhu(1), C Wang(3)
(1) The Afliated Drum Tower Hospital of Nanjing University Meidical
School, Nanjing, PR China
(2) Nanjing University of Chinese Traditional Medicine, Nanjing, PR
China
(3) Institute of Chemical Industry of Forest Products, Nanjing, PR China
Polyprenols (PPs) are natural long-chain isoprenoid alcohols of general
formula H-(C5H8)n-OH and metabolized into dolichols in living creatures. Published researches have demonstrated its wide range bio-activity
and extremely low toxicity. Our previous work showed that PPs orally
co-administrated with Adriamycin (ADM) to human cerebroma SF763
cells loaded nude mice could averagely prolong life spans from30 to 50
days. Since ADM can barely get through brain-blood barrier (BBB), we
hypothesized that the improved ADM therapeutic effects by PPs might
ascribe to its ability to facilitate drug delivery through BBB. Firstly we
679
observed an approximately 40% drop in proliferation of cultured rat
brain microvascular endothelium cells (rBMECs) after 24h exposure to
100lg/ml PPs by MTT method and relatively limited vasoformation in
embryonated eggs after 72h exposure to (5 ~100)lg/ml PPs in a concentration dependent manner as well. Later we detected a doubled uptaking
of ADM on cultured rBMECs monolayer after 100lg/ml PPs co-incubation for 24h or 48 h by HPLC assay, indicating that in addition to antiangiogenesis, more factors participated. P-glycoprotein (p-gp) is recognized essential for BBB function, followed this we found a down regulation of p-gp expression on membrane of rBMECs by western blotting
and elevated Rhodamine123 (a potent substrate for p-gp) levels in
rBMECs after 24h incubation with different concentrations of PPs by
ow cytometer. These results support our hypothesis concerning BBB
opening activity of PPs in terms of anti-angiogenesis and p-gp inhibition,
thus conrm PPs as a promising option in co-chemotherapy.
Key words: polyprenols, BBB, angiogenesis, p-gp, co-chemotherapy.
Paper No.: 588

THE INFLUENCE OF CYP2A6 POLYMORPHISM ON
ARTESUNATE AND AMODIAQUINE TOLERABILITY
due to an adverse drug reaction (ADR) since 1997. Data of this database
of the years 1999 to 2008 were used for evaluation. Cases were all
admissions due to antibiotic associated CDAD, non-cases all other ADR
related admissions. Exposure was evaluated for the use of antibiotics,
PPI and statins at the time of admission. Disproportionality was assessed
by reporting odds ratios (ROR). Results: 3672 ADR reports were evaluated, 56 (1.5%) were due to CDAD. 16 (28.6%) cases with CDAD and
713 (19.7%) controls used PPI; 4 (7.1%) cases with CDAD and 662
(18.3%) of the controls used statins. PPI use was associated with an odds
ratio (OR) of 1.63 (95% CI 0.91-2.93), while statin use resulted in an
OR of 0.34 (95% CI 0.12-0.95). Conclusion: Using a local database with
serious adverse drug reactions a trend towards an increased risk of antibiotic associated CDAD associated with PPI use was identied. Statin use,
however, was not associated with an increased risk.
Supported by BfArM: V-11337/68605/2008-2010
Paper No.: 2838

ENDOMORPHINS ARE POTENTIAL ENDOGENOUS
REGULATORS OF THE GASTRIC MUCOSAL INTEGRITY
Zoltan Zadori, N Shujaa, A Ronai, K Gyires
Wardah Yusof, SH Gan

University Sains Malaysia School of Medical Sciences, Department of
Pharmacology, Kubang Kerian, Kelantan, Malaysia
Artesunate (AS) combined with amodiaquine (AQ) is one of the most
widely used antimalarials. Currently, the drugs are administered as loose
formulations. A xed-dose combination (AS/AQ) is benecial in improving compliance. AQ is extensively metabolized to desethylamodiaquine
by CYP2C8 while AS is metabolized by CYP2A6 to dihydroartemisinin.
The objective of this study is to investigate the inuence of both genes
on artesunate and amodiaquines tolerability for both loose and AS/AQ.
In this randomized crossover study, loose and AS/AQ formulations were
administered to normal healthy volunteers (n = 24) over two study phases.
Its tolerability (the incidence of facial ushing, giddiness, headache, nausea, abdominal discomfort, the progression of liver enzymes and neutrophil
count) were compared between the treatment groups. Volunteers were
genotyped for the CYP2C8 and CYP2A6 variants. The age, sex and gender
between the two treatment groups were comparable. The frequency of the
alleles were 14.6%, 16.7%, 4.2% and 10.4% for CYP2A6*1B, *4, *8 and
*9 respectively. However, no mutations for CYP2C8 were detected. Subjects (25%) having the CYP2A6*1B variants responsible for ultra rapid
metabolism of AS were found to suffer from a higher incidence of
adverse effects. Gender also plays a role where females suffered more
incidences of adverse effects such as nausea. It is included that genetic
polymorphisms of CYP2A6 as well as gender may inuence the incidence of adverse effects following administration of AS and AQ.
Paper No.: 2431

PROTON PUMP INHIBITOR (PPI) OR STATIN ASSOCIATED
RISK FOR CLOSTRIDIUM DIFFICILE ASSOCIATED DISEASE
(CDAD)?
Semmelweis University, Department of Pharmacology and Pharmacotherapy, Budapest, Hungary

Introduction: Endomorphin-1 and endomorphin-2, the endogenous agonists of the l-opioid receptor were identied in 1997. They are widely distributed in the brain, and central injection of the endomorphins has been
shown to induce analgesia, reduced anxiety, impaired spatial learning or
increased food intake. Since the l-opioid receptors play particularly
important role in the regulation of gastrointestinal functions, we investigated the effect of endomorphins on the gastric mucosal defense. Materials: Gastric mucosal damage was induced by acidied ethanol in male
Wistar rats. Endomorphins were injected intracerebroventricularly (i.c.v.).
Results: 1. Both endomorphin-1 (0.03 - 20 pmol) and endomorphin-2
(0.03 - 3 pmol) exerted a dose-dependent gastroprotective effect after
i.c.v. administration. 2. Inhibition of dipeptidyl peptidase IV, a key
enzyme in the degradation of endomorphins with diprotin A (0.5-1 lmol)
resulted also in a reduced mucosal damage. 3. Pretreatment with the
respective antisera abolished the effect of the endomorphins, and the antiserum against endomorphin-2 partially antagonized the protective effect
of diprotin A. 4. The effect of both endomorphins and diprotin A was
antagonized by naloxone (27 nmol i.c.v.). 5. Pretreatment with LNNA
(27,4 lmol/kg i.v.), CGRP8-37 (32 nmol/kg i.v.) or indomethacin (42
lmol/kg per os) markedly reduced the gastroprotective effect of endomorphin-2. Conclusion: Endomorphins induce gastroprotection in the picomolar dose range, which is far below the nanomolar doses required for
the other central effects. Inhibiting the degradation of the endomorphins
resulted also in a gastroprotection, which implies that these two peptides
are important endogenous regulators of the mucosal integrity.
Anita Zachow(1), S Sonnenberg(1), G Haase(1), S Koenig(1), B Drewelow(1), S C Mueller(1)
Paper No.: 1590

CHRONIC HYPERINSULINEMIA INDUCES HYPERTENSION
RESULTED FROM ABNORMAL INNERVATION OF
PERIVASCULAR NERVES IN RAT MESENTERIC RESISTANCE
ARTERIES
University of Rostock, Institute of Clinical Pharmacology, Rostock, Germany
Yoshito Zamami(1), S Takatori(1), N Yabumae(1), M Hosoda(1),

T Koyama(1), N Hobara(2), K Sasaki(1), H Kawasaki(1)
Introduction: While antibiotic therapy is a known risk factor for CDAD,

it was recently hypothesized that use of PPI or statins could present additional risk factors. Methods: All admissions to the internal medicine of 2
general hospitals in Rostock are prospectively screened for admissions
(1) Okayama University, Department of Molecular Design for Medicine,

Okayama, Japan
(2) Okayama University of Science, Okayama, Japan
680
Many clinical studies have shown a possible relationship between hypertension and insulin resistance, since patients with type 2 diabetes are frequently associated with hypertension. Our previous in vivo study showed
that chronic hyperinsulinemia and insulin resistance induced by 15%
fructose drinking elicit abnormal neuronal regulation of vascular tone,
which may partly contribute to the development of hypertension. Therefore, in this study, to clarify further mechanisms of malfunctioned neuronal regulation, we investigated the neurogenic vascular responses, and
perivascular innervation using isolated mesenteric vascular beds in fructose-drinking rats (FDR). Male Wistar rats received 15% fructose as
drinking solution for 10 weeks, which resulted in signicant increases in
plasma levels of insulin, glucose-insulin index and systolic blood pressure, but not blood glucose levels. In perfused mesenteric artery of FDR,
adrenergic nerve-mediated vasoconstriction was enhanced without changing vasoconstriction induced by exogenously injected noradrenaline. The
density of adrenergic neuropeptide Y- like immunoreactive (LI) bers
innervation in FDR mesenterc areteries was signicantly increased, compared with control. Futhermore, serum noradrenaline levels in FDR was
greater than that in control. In the FDR perfused mesenteric vascular bed
precontracted with methoxamine, calcitonin gene-related peptide-containing nerves (CGRPergic nerves)-mediated vasodilation and the density of
CGRPergic nerve innervation were signicantly decreased, and the contents of CGRP in the dorsal root ganglia of FDR were smaller than those
in control. These ndings suggest that abnormal innervation of perivascular nerves resulted from hyperinsulinemia contributes to development
of hypertension in FDR.
Paper No.: 2658

HEALTH AND DISEASE
MECHANISM FOR THE DIFFERENTIAL REGULATION OF
INOS EXPRESSION IN J774 MACROPHAGES AND VASCULAR
SMOOTH MUSCLE CELLS BY THE C-JUN N-TERMINAL
KINASE INHIBITOR SP600125
Marzieh Zamani, S Thakur, A Baydoun
University of Hertfordshire, School of Life Sciences, Hateld, UK
Induced nitric oxide synthase (iNOS) expression is regulated by complex
signalling mechanisms which may include the c-Jun NH2-terminal kinases (JNKs). The role of the latter is however equivocal due in part to species and/or cell type differences and in part to the use of pharmacological
agents which may produce effects beyond JNK inhibition. We have
therefore investigated whether JNK activation is critical for iNOS expression in rat cultured aortic smooth muscle cells (RASMCs) and in J774
macrophages using SP600125 and correlated effects of this JNK inhibitor
with changes in the activation of AP-1 subunits. Effects of SP600125
(0.1-10 lM) on NO production in bacterial lipopolysaccaride (LPS;
100`g/ml) and interferon-c (100 U/ml) activated RASMCs or in LPS
(1`g/ml) activated J774 macrophages was determined by the Griess assay.
iNOS was detected by western blotting. Activation of AP-1 subunits was
monitored using the TransAM kit. SP600125 failed to alter iNOS expression or NO synthesis in RASMCs but caused a biphasic effect on both in
J774 macrophages, potentiating these processes at concentrations of 0.31 lM and inhibiting thereafter. SP600125 blocked c-Jun activation in
both cells but stimulated JunD selectively in RASMCs. Since both these
subunits positively regulate iNOS expression, it is likely that upregulation of JunD compensates for the suppression of c-Jun in RASMCs.
These results suggest a critical requirement of the JNK/AP-1 pathway
that may not always be apparent in studies using pharmacological interventions because of the induction of potential compensatory mechanisms
that could mask their inhibitory actions in certain cell systems.
Paper No.: 773

BIOCHEMICAL ANALYSIS OF THE CLEAVED N-TERMINAL
OF THE PAR1 RECEPTOR
Dimitrios Zampatis(1,2), N Tsopanoglou(2), R Schuelein(1)
(1) Leibniz Institute for Molecular Pharmacology, Berlin, Germany
(2) University of Patras Medical School, Department of Pharmacology,
Greece
Protease-activated receptors or PARs are a subfamily of G-protein coupled receptors (GPCRs) that are activated by cleavage of their extracellular domain. PARs are activated by the action of serine proteases such as
thrombin (acts on PARs 1, 3 and 4) and thrypsin (PAR 2). These
enzymes cleave the N tail of the receptor, which in turn acts as a tethered
ligand. In the cleaved state, part of the receptor itself acts as the agonist,
causing a physiological response. PARs are highly expressed in platelets,
but also in endothelial cells, myocytes and neurons. There are four (4)
known PARs numbered from one to four (PAR1, PAR2, PAR3 and
PAR4). In the case of the PAR1 receptor, it is known that the receptor
has a cleavage site in position 41-42 in its N tail where thrombin cleaves
and activates the receptor. According to prediction programs, the PAR1
receptor also possesses a cleavable signal peptide in its N tail (residues
1-21) which might mediate targeting of the receptor to the membrane of
the endoplasmic reticulum (ER), the rst step of the intracellular transport of the receptor. However, it was unknown whether this signal peptide is indeed functional. We have addressed this question using marker
protein secretion studies and immunoprecipitation experiments. Our initial results indicate that the signal peptide mediates ER targeting and
insertion of the receptor. Moreover, the signal peptide seems to be
cleaved off following the ER insertion process.Taken together, these
results indicate that the signal peptide is indeed functional.
Paper No.: 1029

MECHANISM OF IMPROVEMENT IN VAGAL ACTIVITY
AGAINST MYOCARDIAL ISCHEMIC INJURY
Wei-Jin Zang, L Sun, D-L Li, S-S Kong, H-K Jiang, Y-H Wang, X-J Yu
PR China
Autonomic nervous system imbalance, especially the impaired vagal
activity was related to the ischemic heart disease. How to improve vagus
for treatment of ischemic injury is a severe problem in pharmacological
research. Adult male rats were subjected to coronary artery ligation treatment with adenosine, exercise training or vagus stimulation respectively.
In vitro H9c2 cells were stimulated by ischemia-mimetic solution with
vagal neurotransmitter acetylcholine. Immunohistochemical staining
shows that the muscarinic M2 receptor and cholinesterase-positive nerve
are down-regulated after myocardial infarction (MI). Adenosine reverses
the ischemia-induced reduction of receptor and nerve to improve cardiac
function. Adenosine demonstrates a cardioprotective effects via cholinergic system-dependent mechanisms in part and which is likely due to
receptor crosstalk. In addition, exercise training meliorates the mitochondrial morphology, structure and function in the margin of infrac zone.
Heart rate variance analysis indicates it enhances the vagal activity as
well. Exercise training also attenuates MI induced mesenteric artery dysfunction and restores the morphosis of endothelial cells, which is related
to the increase in the expression of PI3K, Akt, eNOS phosphorylation.
Vagus stimultion ameliorates post-infracted cardiac function and inhibits
the tumor necrosis factor-a (TNF-a). Meanwhile, acetylcholine inhibits
hypoxia-mediated TNF-a production in a concentration-dependent manner in H9c2 cells. Activation of M2 receptor and changes of MAPKs
681
phosphorylation are involved acetylcholine-induced down-regulation of
TNF-a in a post-transcriptional manner. In conclusion, there are several
effective approaches to increase vagal activity which upregulates muscarinic receptor to activate signal pathway and inhibit inammatory cytokine. To improve vagus may have important therapeutic implications
against ischemic injury.
Paper No.: 1050

MECHANISM OF IMPROVEMENT IN VAGAL ACTIVITY
AGAINST MYOCARDIAL ISCHEMIC INJURY
with moxonidine, a centrally- acting antihypertensive agent that

decreases the sympathetic nervous systems activity and improves insulin
resistance. Results: the mean plasma resistin concentration before treatment was signicantly higher in HT than in 17 healthy persons (C), aged
from 46 to 67 years (12,01 2,20 vs. 8,99 2,98 ng/ml; P < 0,01), and
signicantly higher in women than in men in both investigated groups
(for HT: 14,20 1,86 vs. 9,87 1,59 ng/ml, P < 0,05; for C: 10,93
2,15 vs. 7,05 1,96 ng/ml, P < 0,05). After treatment with moxonidine
the resistin levels in HT group went down not signicantly and were still
higher in women than in men (12,68 3,45 vs. 9,46 2,67 ng/ml, P <
0,05).Conclusion: Although both increased sympathetic activity and elevated resistin concentrations are linked to the metabolic syndrome, an 8week treatment with moxonidine- the drug which decreases both sympathetic drive and insulin resistance- doesnt inuence the plasma resistin
levels in hypertonics.
Wei-Jin Zang, L Sun, D-L Li, S-S Kong, H-K Jiang, Y-H Wang, X-J Yu
China
Autonomic nervous system imbalance, especially the impaired vagal
activity was related to the ischemic heart disease. How to improve vagus
for treatment of ischemic injury is a severe problem in pharmacological
research. Adult male rats were subjected to coronary artery ligation treatment with adenosine, exercise training or vagus stimulation respectively.
In vitro H9c2 cells were stimulated by ischemia-mimetic solution with
vagal neurotransmitter acetylcholine. Immunohistochemical staining
shows that the muscarinic M2 receptor and cholinesterase-positive nerve
are down-regulated after myocardial infarction (MI). Adenosine reverses
the ischemia-induced reduction of receptor and nerve to improve cardiac
function. Adenosine demonstrates a cardioprotective effects via cholinergic system-dependent mechanisms in part and which is likely due to
receptor crosstalk. In addition, exercise training meliorates the mitochondrial morphology, structure and function in the margin of infrac zone.
Heart rate variance analysis indicates it enhances the vagal activity as
well. Exercise training also attenuates MI induced mesenteric artery dysfunction and restores the morphosis of endothelial cells, which is related
to the increase in the expression of PI3K, Akt, eNOS phosphorylation.
Vagus stimultion ameliorates post-infracted cardiac function and inhibits
the tumor necrosis factor-a (TNF- a). Meanwhile, acetylcholine inhibits
hypoxia-mediated TNF-a production in a concentration-dependent manner in H9c2 cells. Activation of M2 receptor and changes of MAPKs
phosphorylation are involved acetylcholine-induced down-regulation of
TNF- a in a post-transcriptional manner. In conclusion, there are several
effective approaches to increase vagal activity which upregulates muscarinic receptor to activate signal pathway and inhibit inammatory cytokine. To improve vagus may have important therapeutic implications
against ischemic injury.
Paper No.: 3283

HEALTH AND DISEASE
THE INFLUENCE OF MOXONIDINE ON PLASMA RESISTIN
LEVELS IN PATIENTS WITH ESSENTIAL HYPERTENSION
Paper No.: 829

INHIBITION OF ACETYLCHOLINESTERASE ACTIVITY IN A
RAT MODEL OF WERNICKES ENCEPHALOPATHY: IN VIVO
AND IN VITRO EFFECTS OF THIAMINE
A Zarros(1,2), C Liapi(1), Hussam Al-Humadi(1), M Almpani(1),
V Stolakis(1,2), N Skandali(1), E Katsouni(1), S Tsakiris(2)
Department o f Pharmacology, Athens, Greece
Department of Physiology, Athens, Greece
Wernickes encephalopathy (WE) is a serious acute neuropsychiatric syndrome that is caused by thiamine-deprivation. Rats were placed on ethanol
consumption (20% v/v) for a total of 5 weeks. By the end of the third
week, rats were also fed with thiamine-decient diet (TDD) and were treated with pyrithiamine (0.25mg/kg) for the remaining 2 weeks. Several
intermediate groups of rats were also studied, while control rats were also
studied. Moreover, half of the WE group and the control group rats were
treated with three consecutive intraperitoneal injections of thiamine
(100mg/kg) once every 8h, right after the induction of the WE symptomatology. Acetylcholinesterase (AChE) activity was measured spectrophotometrically. Untreated WE caused a signicant inhibition of AChE
activity (-14%, P < 0.001, vs untreated Control) that was not reversed by
thiamine administration (-20%, P < 0.001, vs untreated Control; +4%, p >
0.05, vs treated Control). That in vivo inhibition of AChE activity due to
the experimentally-induced WE was also observed at the groups receiving
TDD with ethanol or pyrithiamine (P < 0.01 and P < 0.001, respectively).
Moreover, in vitro incubation of the untreated WE brain homogenates
with thiamine resulted in non-signicant changes in their activity, while,
in vitro incubation of pure eel Electroforus electricus AChE resulted in a
signicant decrease in its activity (-34%, P < 0.001, vs Control pure
AChE). Our study concludes that AChE is inhibited by thiamine-deprivation and probably this inhibition could participate in the WE pathophysiology. However, thiamine-administration cannot reverse this inhibition (at
least under the examined in vivo experimental conditions).
Iwona Zaporowska-Stachowiak(1), A Targonska(2), T BobkiewiczKozowska(1)

(1) Poznan University of Medical Sciences, The Departament of Pharmacology, Poznan, Poland
(2) Polyclinic Salutaris, Poznan, Poland
Hypothesis/aims: resistin, an adipokine which induces insulin resistance
in rodents, has been suggested to be correlated with both markers of
inammation and metabolic syndrome in humans. The metabolic disturbances, in essential hypertonics, are probably linked to the inuence of
the sympathoadrenal system. The aim of his study was to compare the
plasma resistin levels in 43 patients with stage 1 essential hypertension
(HT), aged from 44 to 68 years, before and after 8-week of treatment
Paper No.: 2950

PROSTATOPROTECTIVE ACTIVITY OF BEE POLLEN
LIPOPHILIC EXTRACT
Ganna V Zaychenko, S Gevoyan
National University of Pharmacy, Department of Pharmacology,
Kharkov, Ukraine
682
Prostatitis is the most wide-spread disease among the diseases of male
genitals. The chronic prostatitis leads to deterioration of the life quality,
both sexual and reproductive male dysfunctions. To treat prostatitis medicines of natural origin are widely used, among them the medicines containing apiculture products attract a great attention to. The aim of our
research was in studying the pharmacological activity of suppositories
containing 5 mg of bee pollen lipophilic extract (BPLE) on the various
prostatitis models in rats. The reference medicine was suppositories with
pumpkin seeds oil. The medicines were introduced per rectum in the
therapeutic and prevention scheme for 15-22 days. The male white nonlinear rats with the body weight of 200-220 g were used in the experiment. The rst model (traumatic prostatitis) was caused by stitching
of the prostate with the help of silk thread, the second (abacterial
prostatitis) - by rectal introduction of the turpentine mixture with dimexide in the ratio of 3:1. Under the effect of BPLE and the reference medicine the regress of clinical manifestations of prostatitis,
normalization of the biochemical indexes, restoration of the morphological structure of tissues and functional activity of the prostate have
been noted. The analysis of the results obtained testies the presence of
the marked prostatoprotective action of suppositories of BPLE on models
in rats; this action exceeds the efciency of suppositories with pumpkin
seeds oil. BPLE can become a promising object for developing a new
prostatoprotector.
Paper No.: 3186

GRAVIDOPROTECTIVE ACTIVITY OF GLUCOSAMINE
Paper No.: 2085

DISCOVERY
IN VIVO STUDY OF PENETRATION OF
ACETYLCHOLINESTERASE REACTIVATORS THROUGH
BLOOD-BRAIN BARRIER
Jana Zdarova Karasova, K Kuca, M Pohanka, L Novotny
Faculty of Military Health Sciences, Department of Toxicology, Hradec
Many specic and nonspecic effects have been demonstrated after
intoxication by organophosphorus inhibitors (OPI) in central nervous
system (CNS). They are caused by cholinesterase inhibition with subsequent changes of the neurotransmitters including acetylcholine and catecholamines, changes in membrane permeability, metabolic imbalances,
and changes in brain energy metabolism, lipid peroxidation and increase
ATP level. The key role in treatment of these intoxications plays acetylcholinesterase (AChE) reactivators (also called oximes). They are effective causal antidotes. The main question dealing with the reactivators
effect on the central nervous system was discussed formerly. Due to their
quaternary structure and crucial hydrophilic functional groups (oximes),
the blood-brain barrier (BBB) remains almost intact for the mono- or bisquaternary molecules where penetration of the reactivator is very slow
or do not occur. In our study we bring direct evidence about penetration
of oximes into the CNS in low (therapeutic) doses. The concentration
levels in CNS was low (10-8 - 10-7 g) and also was changed in different
brain regions. It is conrmed that access by relatively small quantities of
oximes to some parts of the CNS might be enough to elicit pharmacological effects and reactive a small percentage of the blocked AChE, and
thus save the life of intoxicated patients.
Ganna V Zaychenko
National University of Pharmacy, Department of Pharmacology,
Kharkov, Ukraine
Introduction: One of the actual problems of modern obstetrics is the
search of new effective and safe medicines for preventing placental dysfunction (PD) and its complications such as intrauterine growth retardation and fetal hypoxia, premature tocus and development of respiratory
distress-syndrome in newborns. The aim of our research was to study the
gravidoprotective action of glucosamine hydrochloride (GH) in the
experimental placental dysfunction in rats. Materials: GH (Sigma, US)
was introduced to pregnant female rats in our research from the 11 to the
20 day of gestation in the dose of 90 mg/kg intragastrically in the therapeutic and prevention scheme in PD caused by different toxicants (tetrachlormethane, serotonine, N -nitro-L-agrinine). Results and Conclusion:
It has been found that GH decreases the effect of toxicants on the organism of the pregnant female rats, prevents development and progressing
of PD. It is conrmed by improvement of the blood circulation and
decrease of hemorheological disorders, reduction of intensity of the free
radical oxidation processes in the placental tissues, restoration of integrity and functions of the endothelium of the placental and uterine vessels.
The post-implantation fetal death decreases under the effect of GH, the
processes of fetogenesis are standardized, and it is accompanied by
increase of the body weight and its size, as well as improvement of
maturing of fetal lungs. The presence of antioxidant, antiaggregate, fetoprotective action and the ability to stimulate the synthesis of the surfactant in fetus are the basis for creating a new gravidoprotector based on
GH.
Paper No.: 1357

INFLUENCE OF CALCIUM ANTAGONISTS ON DNA-DAMAGE
INDUCED BY CYCLOPHOSPHAMIDE AND DIOXIDINE
Aliy K Zhanataev, AV Kulakova, EA Anisina, AD Durnev, SB Seredenin,
State Zakusovs Research Institute of Pharmacology of RAMS,
Department of Pharmacological Genetics, Moscow, Russian Federation
Purpose: In the course of experiments it was stated that calcium antagonists (CA) nifedipine, diltiazem and verapamil possessed co-mutagenic
activity, manifested in their ability to potentiate clastogenic effects of
cyclophosphamide (CP) and dioxidine (DN) in bone marrow cells of
mice. The purpose of present study was to evaluate the inuence of CA
on DNA-damage induced by CP and DN. Materials and methods: Experiments were carried out on male C57BL/6 mice. DNA-damage was estimated by the alkaline DNA comet assay. Results: Nifedipine (5 mg/kg)
depending on the term of application (6 and 18 hours) reduced the level
of DNA damage (%DNA in the tail) induced by CP by 36-70%, and DN
effects (1 and 3 hours) by 19-37%. Diltiazem (5 mg/kg) and verapamil
(2.5 mg/kg) reduced CP-induced DNA- damage by 48-64% and 58-74%,
respectively. Reduction of DN-induced DNA-damage by 20-46% and
41-42% was observed respectively for diltiazem and nifedipine. Conclusion: Experiments showed that CA did not increase DNA-damaging
activity of the mutagens, but reduced it considerably, that disprove the
earlier suggested accumulation theory, which explained CA co-mutagenic
effects by the ability to accumulate mutagens in cells and, as supervening, lead to the enhancement of their damaging effect. Reduction of DNinduced DNA damage in a 1 hour and in 6 hours after CP administration
presupposed CA inuence on the early stages of apoptosis induced by
mutagens. A more detailed study of the problem is supposed to be the
subject of further research.
683
Paper No.: 913
DISEASES
THE EFFECT OF CARBOXYAMIDOTRIAZOLE ON DEXTRAN
SULPHATE SODIUM INDUCED MOUSE EXPERIMENTAL
ULCERATIVE COLITIS
Dechang Zhang, C Ye, X Hao, R Zheng
Institute of Basic Medical Sciences, Chinese Academy Medical Sciences,
Carboxyamidotriazole (CAI) is an anticancer drug in clinical trails. Its
effects on acute and chronic inammation including rat adjuvant arthritis
have been reported (L Guo et al, JPET 2008 325:10-16,). In this work
we reported that CAI possesses protective and therapeutic action on
mouse ulcerative colitis induced by dextran sulphate sodium (DSS) in a
dose dependent manner (10, 20 and 30mg/kg, p.o.). The drug improves
the symptoms of diarrhea and hemafecia and weight loss caused by
DSS. Microscopy examination shows alleviated hyperemia, edema and
mucous membrane damages in CAI treated group. CAI inhibits the
increase of TNF-a IL-1b IL-6 levels caused by DSS treatment in serum,
colon homogenate and peritoneal macrophage of the mouse. CAI also
decreases the content of TGF-b 1 in colon homogenate, which is a mediator of collagen accumulation in UC. The results indicated that CAI is an
anti-ulcerative colitis drug. Combined with its effects on adjuvant arthritis, CAI may be developed as a small molecule pro-inammation cytokines inhibitor for treatment of autoimmune diseases.
This work is supported by China National Natural Science Foundation
30873075.
Paper No.: 1345

HANGOVER EFFECTS OF ORALLY, NOCTURNALLY ADMINISTRATED ANTIHISTAMINES: A DOUBLE-BLINDED, PLACEBO-CONTROLLED, CROSSOVER POSITRON EMISSION
TOMOGRAPHY (PET) STUDY IN HEALTHY SUBJECTS
Dongying Zhang(1,2,3), M Tashiro(4), K Shibuya(0), Y Funaki(5),
S Watanuki(4), M Kato(2), K Yanai(0)
Pharmacology, Sendai, Japan
Anesthesiology, Sendai, Japan
(3) Chinese Medical University, Department of Anesthesiology, Shenyang, PR China
(4) Tohoku University, Division of Cyclotron Nuclear Medicine, Cyclotron and Radioisotope Center, Sendai, Japan
(5) Tohoku University, Division of Radiopharmaceutical Chemistry,
Cyclotron and Radioisotope Center, Sendai, Japan
Introduction: Antihistamines are known to induce sedation. This sedative
property of antihistamines can be objectively evaluated in terms of brain
histamine H1 receptor occupancy (H1RO) by positron emission tomography (PET) with 11C-doxepin. Instead of the well studied acute sedative
property of antihistamines, the next-day residual property, so-called
hangover effect, is concerned in this study basing on the fact that sedating antihistamines are always taken at night. Subjects and methods: Eight
healthy male adult subjects received the PET measurement in the morning (11:00) after orally taking diphenhydramine 50 mg (Drewell), bepotastine 10 mg (Talion, a second-generation product) or placebo in
the previous night (23:00) in a random, double-blinded, crossover style
with a 7-day-long wash-time. H1ROs were calculated in different brain
regions such as cingulate gyrus, fronto-temporal cortex and cerebellum.
Subjective sleepiness and plasma concentration of antihistamines were
measured and their correlations to H1RO were examined. Results:
Diphenhydramine remained occupied 45% of H1 receptors across all the

cortical regions at 12 hr post-dose, whereas bepotastine gave a signicantly lower value as 17% (paired-t test, P < 0.01). Subjective sleepiness
was not difference among three treatments. A moderate positive correction was found between plasma concentration and H1RO only in diphenhydramine condition (r = 0.43, Spearman correlation test). Conclusion:
Diphenhydramine, usually available in over-the-counter (OTC) preparations either alone or combined, may have predominant next-day hangover effects whereas bepotastine serves well as a non-sedating
antihistamine at its recommended dosage. Clinical benets from antihistamines will have to be weighed against their possible residual sedation
risks in various clinical settings.
Paper No.: 1758

GLYCOSAMINOGLYCANS MIMETICS REGULATION OF
LYSOSOMAL DISTURBANCES AND TAU HYPERPHOSPHORYLATION INDUCED BY OXIDATIVE STRESS
Ganlin Zhang(1,2), S Lehri(2), X X Zhu(1), MB Huynh(2), L Sissoeff(2),
YJ Li(1), X Liu(3), R Raisman(4), C Morin(2), P Li(3), D Papy-Garcia(2)
(1) China Academy of Traditional Chinese Medicine, Institute of Chinese
Materia Medica, Beijing, PR China
(2) Laboratory CRRET-EAC CNRS 7149, France
(3) Beijing Institute of Chinese Medicine, PR China
(4) INSERM UPMC UMRS 975-CRICM, France
In the neurodegenerative processes particularly in Alzheimers disease
(AD), Tau hyperphosphorylation and apoptosis are known to play important roles. Recently, cathepsin and/or caspase 3 inhibitions were shown
to stabilize or even diminish Tau (truncated at Asp421) suggesting links
between apoptosis and Tau pathology. Glycosaminoglycans (GAGs), a
family of ionic polysaccharides, have been suggested to have tight relationship not only with Tau abnormal phosphorylation and aggregation
but also with the regulation of apoptosis. Here, we report that PC12 cells
response to oxidative stress with clear lysosome morphologic changes
and cathepsins release, and this response are accompanied by Tau hyperphosphorylation, caspase activation, and apoptosis. GAGs mimetics Fucoidan and the synthetic OTR4120 protect lysosome from membrane
disruption and cathepsins release. These GAGs mimetics also decreased
Tau hyperphosphorylation and increased cell survival. These results support the theory that oxidative stress is involved in AD, and suggest that
GAGs and their mimetics can regulate death cascade at lysosome level,
while hinder Tau hyperphosphorylation. This opens a new therapeutic
approach to AD.
Paper No.: 1985

PRELIMINARY STUDIES ON THE RELATIONSHIP BETWEEN
STUB1 AND THE KEY PATHOLOGICAL PROTEINS IN AD
ANIMAL MODEL SAMP8
Gui-Rong Zhang(1,2), X-R Cheng(2), W-X Zhou(2), Y-X Zhang(2),
D-L Gao(3), X-D Xia(1), X-P Jiang(1), L-Y Nie(1), Y Ji(1), S-P Zhou(1),
(1) The Institute for Drug and Instrument Quality Control, Department of
(3) 302 Hospital, Beijing, PR China
Alzheimers Disease (AD) is pathologically characteristic by abnormal
changes such as aggradation of abnormal peptides within special brain
areas and progressive degeneration of cognitive functions. STUB1 is a
684
newly discovered muti-functional protein, which plays a key role in degradation of aberrant proteins. Since AD is a progressive degeneration, the
detailed expression relationship between STUB1 and the key pathological proteins in AD progress is of great value, yet which is still unclear till
now. Senescence-accelerated mice (SAM) prone/8 (SAMP8), together
with its matched control SAM resistant /1 (SAMR1), is an excellent animal model for AD. This study further investigated the relationship
between STUB1 and the two key pathological proteins, APP and Tau, in
AD process using SAMP8 and SAMR1 by Real-Time Quantitative PCR,
Western blot and immunohistochemisty. Results showed compared with
SAMR1, the expression of both APP and Tau were increased in aged
SAMP8 while STUB1 decreased at the same time in SAMP8 but could
be up-regulated by cholinesterase inhibitor Huperzine A. Predictive analysis by bioinformation software showed that STUB1 may indirectly act
with APP and Tau. Preliminary immunouorescence experiments
showed that STUB1 and APP may colocalized within frontal cortex in
six-month-old mice brain. This research further demonstrates our former
hypothesis that STUB1 is closely linked to aging cognitive fuctions and
might be a potential target for anti-AD drugs.
(Acknowledgments: This work was supported by the National Natural
Science Foundation of China (No. 30901581, 30973541, 30701073).
Paper No.: 3207

BIMODALITY OF PLASMA GLUCOSE DISTRIBUTIONS IN
CYNOMOLGUS MACAQUES
Guodong Zhang(1), D Yi(1), C Zhou(1,2), C Zhu(1), P Chan(1,2)
(1) Wincon Theracells Biotechnologies Co., Ltd., Nanning, PR China
(2) Capital University of Medical Science, Beijing, PR China
Type 2 diabetes mellitus (T2D) animal models are of importance in
understanding the pathogenesis and in developing potential therapeutic
agents for this disease in human. Old World nonhuman primates (NHPs),
especially cynomolgus macaques, can be a valuable animal model of
T2D because they have biological and genetic similarities to humans and
the disease is also common in older, obese populations. However the criteria for diagnosis of T2D in non-human primates still remains obscure.
In this study, we present the results of fasting plasma glucose concentration (FPG) distribution of 190 cynomolgus macaques, age 9~18 year old,
by utilizing the statistic method of bimodality. The results of this study
demonstrate that the distribution of the plasma glucose level among the
c. macaques is highly comparable to the early ndings in human studies
by others, which might indicate some similarities of T2D pathogenesis
and prevalence between cynomolgus macaques and humans. The bimodality found in the FPG distributions also provides a reference value that
could be used to separate individual c. macaques into normal or abnormal glycemia as T2D models and would provide adequate evidence in
developing T2D diagnostic criteria for non-human primate diabetes.
Paper No.: 867

CONNECTION BETWEEN COGNITION AND SEXUAL
FUNCTION
Juntian Zhang
Chinese Academy of Medical Sciences, Institute of Materia Medica,
Ginsenoside Rg1 is a pure compound isolated from panax ginseng,
which shares many pharmacological effects of ginseng. Aim of this study
is investigating its noontropic and sexual enhancing effects and mechanism, revers connection between cognition and sexual function. Behavioral eletrophysiological methods, biochemical and molecular techniques
such as western blot, immunohistochemistry, confocal mircroscopy etc.

were used in experiment. Results showed that Rg1 could improve cognition in ten models of memory impairment and sexual function in male
mice. The noontropic mechanism include: rstly, increase neural plasticity in both effecacy and structure. Secondly, activated learning and memory signal transduction pathway. Thirdly accelerate hippocampal
neurogenesis. The sexual- enhancing mechanism is mainly by activating
NO/cGMP signalling pathway in corpus cavernosum. Further study indicated that Rg1 could increase serum testosterone level, inhibit PDE4 and
PDE5 to produce cAMP and cGMP, activate NOS to generate NO,
which are elementary bio-moleculars underlying improvement of noontropic and sexual behaival. In conclusion, Rg1 improve cognition having
new mechanism that different from the available drugs, sexual-enhancing
mechanism of Rg1 is the same as that of sildenal, but the former
showed many advantages compairing with sildenal. Interestingly, Rg1
induced some biochemical changes can be served as commom mechanism of both cognition and sexual function improvement.
Paper No.: 1742

EFFECTS OF SALVIANOLIC ACID A ON RENAL INJURY IN
TYPE 2 DIABETIC KKAY MICE
Li Zhang, G Qiang, L Shi, H Zhang, B Chen, G Du
Chinese Academy of Medical Sciences and Peking Union College, Institute of Materia Medica, National Center for Pharmaceutical Screening,
Beijing, PR China
Introduction: Salvianolic acid A (SalAA) is one of the main active components in Salvia miltiorrhiza. In this study, we investigated the effects
of SalAA on the prevention of diabetic nephropathy in type 2 diabetic
KKAy mice. Methods: KKAy mice were randomly divided into model
control group and four treatment groups. The KKAy mice were orally
given valsartan or SalAA twice per day consecutively for 16 weeks.
C57BL mice were used as the non-diabetic control and were given the
drinking water twice per day for 16 weeks. Body weight, blood glucose,
blood lipid, blood uric nitrogen, serum creatinine, urine N-acetyl-jB-glucosaminidase (NAG) and urine protein were measured. Pathologic examination and scanning microscope observation were performed to analyze
renal injury. Results: Increased weight, hyperglycemia, hyperlipidemia,
evaluated UAE and NAG/Cr were observed in the KKAy model group.
SalAA could reduce the level of NAG/Cr. The urinary protein level in
SalAA groups decreased signicantly, compared with that of the model
control group. The kidney of type 2 diabetic KKAy mice were pathologically examined hypertrophy, mesangial expansion and glomerular sclerosis. Administration of SalAA could attenuate histopathological injury. The
results of scanning microscope showed that the width of glomerular basement membrane in SalAA groups was less than that in the model control
group. Conclusion: Our results suggest that SalAA might be a new therapeutic agent for the prevention of nephropathy in type 2 diabetes.
Acknowledgements: This study was supported by the 11th Five-Year
Program of MOST, No.2009ZX09102-123 and Basic Research Project
of IMM, No.2009ZHD-LCH02.
Paper No.: 1344

DOSE-DEPENDENT DRUG-DRUG INTERACTION BETWEEN
PARACETAMOL AND WARFARIN IN ADULTS RECEIVING
LONG-TERM ORAL ANTICOAGULANTS
Qian Zhang(1), G Simoneau(1), L Drouet(2), C Bal-dit-Sollier(2),
J-C Alvarez(3), I Mahe(1), J-F Bergmann(1), S Mouly(1)
(1) Lariboisiere Hospital, Therapeutic Research Unit, Department of
Internal Medicine, Paris, France
685
(2) Lariboisie`re Hosipital, Experimental Thrombosis and Atherosclerosis
Laboratory, Paris, France
(3) Raymond Poincare Hospital, Department of Pharmacology, Garches,
France
Background: Paracetamol (P) is recommended as the analgesic and antipyretic drug of choice in patients receiving long term oral anticoagulation
with warfarin (W). Objective: To determine whether an interaction exists
between P (2g/day and 3 g/day) and W. Methods: 45 adult patients on
stable W therapy, enrolled in this prospective, randomized, double-blind,
parallel (3 arms), placebo-controlled study, received a 10-day regimen of
P (2 g/day or 3 g/day) or placebo. Blood samples were collected at day
0, 3, 5, 8, 10 to determine INR, R- and S-W, P and factors II, V, VII
plasma concentrations. Results: Mean maximum increase in INR
observed was 0,7 0,5 and 0,7 0,6 in patients receiving P 2 g/day and
3 g/day, respectively (P = 0,01 for the respective comparisons versus placebo). The INR started to increase on Day 3 and Day 8 at 2g/day and 3
g/day of P, respectively. No relation was observed between age and the
extent of INR increase (R2 = 0,002, P = 0,72). Conversely, INR increase
was independently and signicantly correlated to a decrease in factor II
(R2 = 0,39, P < 0,01) and factor VII (R2 = 0,44, P < 0,01) and an
increase of P concentration (R2 = 0,13, P = 0,03), while R-, S-W and
factor V concentrations remained constant. Conclusion: Paracetamol,
starting at 2g/day, signicantly increased INR in patients on stable W
therapy, thus requiring close monitoring. This interaction may be
explained by a decrease in vitamin K c-carboxylase activity, as ascribed
by the lack of factor V or R- and S-W changes in our patients.
Paper No.: 1463

AMOXICILLIN/CLAVULANIC ACID DID NOT INCREASE INR
IN PATIENTS TREATED WITH WARFARIN
Qian Zhang(1), G Simoneau(1), C Verstuyft(2), L Drouet(3), C Bal-ditSollier(3), J-C Alvarez(4), J-F Bergmann(1), L Becquemont(2), S Mouly(1),
(1) Lariboisiere Hospital, Therapeutic Research Unit, Department of
Internal Medicine, Paris, France
(2) University of Paris Sud, Department of Pharmacology, Le Kremlin
Bicetre, France
(3) Lariboisie`re Hosipital, Experimental Thrombosis and Atherosclerosis
Laboratory, Paris, France
(4) Raymond Poincare Hospital, Department of Pharmacology, Garches,
France
Background: Increased INR was previously observed in patients simultaneously treated with warfarin and amoxicillin/clavulanic acid (AM/CL).
However it is difcult, in this infectious context, to determine whether
AM/CL or the infection itself resulted in this INR increase. Objectives:
We aimed to investigate whether an interaction exists between AM/CL
and warfarin in patients outside of any infectious or inammatory context. Methods: In a prospective, double-blind, randomized, cross-over,
placebo-controlled study, 12 patients (6 men & 6 women), aged 22 to 68
year-old on stable warfarin therapy since at least one month received a
7-day AM/CL regimen (1g bid) or matching placebo. INR, factor II, Rand S- warfarin plasma concentrations were measured before and then
on day 3, 5, 6, 7 and 10 upon treatment administration. Results: Mean
baseline INR values before AM/CL and placebo intake were similar
(2.35 0.4 vs 2.43 0.5, respectively, P = 0.66). The mean maximum
INR observed over the study period was 2.57 0.4 versus 2.67 0.5 in
the AM/CL and placebo treatment period, respectively (P = 0.70), corresponding to a maximum increase from baseline of 0.22 0.3 with AM/
CL versus 0.24 0.6 with placebo (P = 0.92). Likewise, the Day 7
Day 1 difference in factor II, R- and S- warfarin plasma concentrations
were similar between AM/CL and placebo (P = 0.79, P = 0.45, P = 0.75,
respectively). Conclusion: AM/CL did not increase INR in stable patients
treated with warfarin without any infectious or inammatory syndrome,
suggesting that the previously observed INR increase in these patients

may not be attributable to a classical pharmacokinetic/dynamic drug-drug
interaction.
Paper No.: 1769

DISEASES
ANTI-INFLAMMATORY ACTIVITY OF A NOVEL NATURAL
SALICYLATE DERIVATIVE FROM GAULTHERIA
YUNNANENSIS (FRANCH.) REHDER
Tiantai Zhang, Lan Sun, Rui Liu, Xi Lan, JH Sun, GH Du
(Chinese Academy of Medical Sciences & Peking Union College, Institute of Materia Medica, National Center for Pharmaceutical Screening,
Beijing, PR China
Gaultheria yunnanensis (FRANCH.) REHDER is a member of the Ericaceae family growing in the southwest and southern regions of China,
and is widely used in these districts as a folk medicine for the treatment
of rheumatoid arthritis, swellings, pain, trauma, chronic tracheitis, cold
and vertigo. Our previous study showed that DL0309, a natural salicylate
derivative from Gaultheria yunnanensis, has anti-inammatory effects
and absence of gastric ulcerogenic property, and the metabolism characters of DL0309 in mice and rats indicated that it could be metabolically
converted to salicylate, which produced the pharmacological effects and
provided effective concentrations for an extended period. This study
investigated the effect and underlying mechanism of DL0309 against
LPS-induced toxicity in Raw264.7 cells. NF-jB, phos-NF-jB, I-j a,
phos-I-j a were determined by western blotting; TNF-a, IL-1b and IL-6
were assayed by ELISA; NF-jB activation was assessed by gel shift
assay. The results demonstrated that DL0309 exerted anti-inammatory
effects against LPS-induced toxicity in Raw264.7 cells. In these effects,
NF-jB DNA binding activity was inhibited; expressions of phosphor-Ija and NF-jB were up-regulated; the production of TNF-a, IL-1b and
IL-6 from Raw264.7 cells were suppressed. In conclusion, DL0309
exerted anti-inammatory effects, partly resulting from the inhibition of
NF-jB activation in macrophages. These will help to understand the
pharmacological effects and potential mechanism of DL0309 on antiinammation.
(The project was supported by the National Natural Science Foundation
of China, NO.30772749; and Major Scientic and Technological Special
Project for Signicant New Drugs Creation, NO.2009ZX09102-034.)
Paper No.: 871

ASTHMA DRUG REGIMENS AND HEALTH SERVICE UTILIZATION: WHAT IMPACT DO CHANGES IN THE OPTIMAL
USE OF MEDICATION HAVE ON CHILDREN WITH ASTHMA?
Tingting Zhang(1), A Smith(2), R Prosser(1,2), B Carleton(1,2,3)
(1) University of British Columbia, Faculty of Pharmaceutical Sciences,
Vancouver, BC, Canada
(2) British Columbia Childrens Hospital, Vancouver, BC, Canada
(3) Child & Family Research Institute (CFRI), Vancouver, BC, Canada
Suboptimal medication use is associated with a high risk of asthma exacerbations, yet many children receive suboptimal therapy. This study
determined the association between changes of asthma drug therapy and
patients health service utilization. 13,738 asthma patients 5-18 years
were identied using provincial data (including all prescription medications, physician and hospital visits) 1996-2000. Canadian national asthma
guideline recommendations for optimal drug therapy were used to cate-
686
gorize patient-specic regimens of inhaled short-acting b-agonists
(SABAs) and inhaled corticosteroids (ICSs) as optimal, suboptimal or
indeterminable optimality. Analyses focused on patterns of drug use: (1)
suboptimal therapy in 1996 that became optimal by 2000; (2) optimal
therapy in 1996 that became suboptimal by 2000; (3) suboptimal therapy
1996-2000, and (4) optimal therapy1996-2000. Outcomes were rate
changes of asthma-related emergency department (ED) visits or hospitalizations between 1996 and 2000. Children who used asthma medication
suboptimally the entire time were seven times more likely to use ED or
hospital services for asthma compared to children who used medications
according to published guidelines (OR 6.8, 95%CI 5.0-9.3). Children
who changed their drug therapy from suboptimal to optimal were signicantly less likely to be admitted to ED or hospital for asthma compare to
patients who used their medications suboptimally (OR 0.6, 95%CI 0.40.8). Changing from suboptimal to optimal therapy results in reductions
in healthcare service use by children with asthma. Findings suggest the
need to reinforce to clinicians the importance of close monitoring of their
asthma patients therapy and encouraging adherence to specic therapeutic objectives.
Paper No.: 2143

PHARMACOEPIDEMIOLOGICAL STUDIES IN CHINESE AND
CANADIAN CHILDREN - LACK OF COMPREHENSIVE
PRESCRIPTION DATA A MAJOR LIMITATION
Tingting Zhang(1), Z Li(2), A Smith(3), Y Wang(4), D Cao(2),
B Carleton(1)
(1) University of British Columbia, Faculty of Pharmaceutical Sciences,
Vancouver,BC, Canada
(2) Childrens Hospital of Fudan University, Department of Pharmacy,
Shanghai, PR China
(3) British Columbia Childrens Hospital, Pharmaceutical Outcome Programme (POPi), Vancouver, BC, Canada
(4) Childrens Hospital of Fudan University, Department of Paediatrics,
Shanghai, PR China
(5) University of British Columbia, Faculty of Medicine, Department of
Paediatrics, Vancouver, BC, Canada
Comprehensive population-based assessments of pharmaceutical utilization are important in medication safety and effectiveness research in children. Databases that capture an entire populations drug utilization are
scarce. The objective of this study is to illustrate challenges in conducting pharmacopidemiological studies in paediatric patients through comparing psychostimulant use by British Columbia Childrens Hospital
outpatients with that of Fudan Childrens Hospital outpatients. BC and
Fudan Childrens Hospitals are the largest tertiary paediatric hospitals in
each province, with 142 and 600 beds respectively. Cohorts of patients
0-18 years who visited outpatient clinics in BC Childrens Hospital
(2003-2005) and Fudan Childrens Hospital (2006-2008) were identied
(N = 31,072, 1,263,437 respectively). Psychostimulants dispensing
records were extracted from the Fudan and BC outpatient prescription
dispensing databases. We compared proportions of children who were
dispensed psychostimulants from outpatient clinics in each hospital. 5%
of children who visited outpatient clinics in BC Childrens Hospital
received psychostimulants in 2003-2005 compared to 0.04% to 0.07% in
2006-2008 who were outpatients of Fudan Childrens. The estimated rate
of psychostimulant use is far lower than expected given the prevalence
of ADHD (2-3%) in Chinese children. We believe the low rates of psychostimulant use are a result of lack of comprehensive prescription dispensing data. Personal identication numbers are not available to link
patients multiple outpatient visits and prescription dispensings in other
psychiatric centers or paediatric outpatient clinics in Shanghai. Until
comprehensive population-based prescription utilization databases are
developed comparative pharmacoepidemiological research using data
from paediatric patients in Fudan will be unable to produce results of
clinical value.
Paper No.: 1896

IN VITRO ANTIOXIDANT AND IN VIVO
ANTI-INFLAMMATORY AND ANTINOCICEPTIVE
ACTIVITIES OF OPHIOGLOSSUM THERMALE
Xue-Qiong Zhang(1), Y-H Zhang(1), J-H Kim(2), J-T Bae(2), J-Y Oh(2),
G-S Lee(2), H-B Pyo(2)
(1) Peking University, School of Basic Medical Science, Department of
Pharmacolog,Beijing, PR China
(2) Hanbul Cosmetics Co. Ltd.,R&D Center, Chungbuk, Korea
Ophioglossum thermale is a Chinese medicinal plant traditionally used
for the treatment of inammation including pediatric pneumonia, boils
swollen poison etc.. This study was performed to provide evidence of its
antiinammation activity in order to conrm its popular use that could
be related to inammation. Fractions from Ophioglossum thermale were
extracted with ve different polar solvents using Soxhlet type extractor.
EtOAc fraction exhibited the best performance in DPPH assay, NBT
assay and lipid peroxidation assay, while crude extract, BuOH, water and
MC fraction also showed good antioxidant effects. The order of antioxidant capacity of each fraction is EtOAc > BuOH > MC > water > hexane and this order is consistent with the phenolic contents of each
fraction. Furthermore, all the fractions of Ophioglossum thermale showed
more potent activity than EGCG, a well known antioxidant. After the
treatment of EtOAc fraction at 0.005 and 0.01% (g/100), we observed
signicant decrease of ROS induction after UVB irradiation in HDFs. In
carrageenan-induced edema model, EtOAc fraction showed inhibitory
effect. These results suggested that the antioxidant activity of Ophioglossum thermale correlates closely with their phenolic contents and demonstrated that extracts of Ophioglossum thermale have excellent antioxidant
activities and thus it has great potential as a source for natural health
products.
This study was supported by grants from The Ministry of Knowledge
Economy of Korea (70004326), national scientic & technological major
special project (2009ZX09103-124), the National Natural Science Foundation of China (no. 30640070 and 30772556) and the 985 project from
Peking University.
Paper No.: 2050

DISEASES
UP-REGULATION OF KININ RECEPTORS IN RAT BRONCHI
VIA IKB KINASE-MEDIATED INFLAMMATORY SIGNALLING
PATHWAY
Yaping Zhang(1), Y Lei(1), Y-X Cao(2), L Edvinsson(1), C-B Xu(1)
(1) Lund University, Institute of Clinical Sciences, Division of
Experimental Vascular Research, Lund, Sweden
(2) Xian Jiaotong University College of Medicine, Department of
Pharmacology, Xian, Shaanxi, PR China
IkB kinase (IKK)-mediated intracellular signaling mechanisms are
believed to be involved in airway hyperresponsiveness through up-regulation of kinin receptors. The present study was designed to examine if
organ culture of rat bronchial segments induces airway hyperresponsiveness to kinins and if inhibition of IKK can abrogate the airway hyperresponsiveness to kinins via suppressing the expression of kinin B1 and B2
receptors. Rat bronchi were isolated and cut into ring segments. The segments were then organ cultured in the presence or absence of IKK inhibitors, BMS-345541 or TPCA-1. The airway contraction induced by desArg9-bradykinin (B1 receptor agonist) and bradykinin (B2 receptor agonist) were monitored by a sensitive myograph. The expression of kinin
B1 and B2 receptors, inammatory mediators and phosphorylated IKK
were studied by real-time PCR and/or immunohistochemsity using con-
687
focal microscopy. Organ culture of the bronchial segments induced a
time-dependent up-regulation of kinin B1 and B2 receptors. The IKK
inhibitors abolished the organ culture-induced up-regulation of kinin B1
and B2 receptor-mediated contractions in a concentration-dependent manner. This was paralleled with inhibition of IKK activity (phosphorylation), reduced mRNA and protein expressions of kinin B1 and B2
receptors and decreased mRNA expression of inammatory mediators
(interleukin-6, inducible nitric oxide synthase, cyclooxygenase 2 and
matrix metalloproteinase 9). Our results show that organ culture induces
IKK-mediated inammatory changes in airways which subsequently
results in airway hyperresponsiveness to kinins via the up-regulated kinin
receptors. Thus, IKK inhibition might be a promising approach for treatment of airway inammation and airway hyperresponsiveness that are
often seen in asthmatics.
Paper No.: 1926

DISCOVERY
CALCIUM SIGNAL PASSWAY-BASED ON TO RESEARCH THE
NEUROPROTECTION OF EFFECTIVE COMPONENTS OF
TRADITIONAL CHINESE MEDICINE
Yi Zhang, X Lai, J Zhang
Chengdu University of Traditional Chinese Medicine, National Medical
College, Chengdu, PR China
Glaucoma is the main blinding disease which foremost pathological
change is optic nerve injure. Erigeron breviscapus, Chuanxiong and
Radix peucedani are the objects of our project.We screen out the active
parts or components which have Ca2 + antagonistic property by 45Ca
transmembrane inux technique, which are Seopoletin, Caffeic acid, Scutellarin, 3-O- dicaffeoylquinate, Quercetin, Apigenin, Erigeside, Chuanxiongzine hydrochloride, imperatiorin, isoimperatorin, Praeruptorin B, etc.
The results of retinal neurons cultivated in normal atmosphere in vitro
indicate that the dicaffeoyl extract from Erigeron breviscapus and isoimperatorin can promote retinal neurons alive in vitro. Ferulic acid, Verapamil and Chuanxiongzine hydrochloride can inhibit the apoptosis of retina
gangliocyte caused by high pressure in vitro. The total avonoids of erigeron breviscapus, breviscapine,imperatiorin,Praeruptorin B and Nimodipine can promote retinal neurons alive in vitro as well as inhibit the
apoptosis of retina gangliocyte caused by high pressure in vitro. Combined Ca2 + antagonistic experiments, effects on animals and relative references, we have found the optical neuroprotection and Ca2 +
antagonistic effect of erigeron breviscapus,chuanxiong and radix peucedani are corrrelated obviously. Furthermore, we have determined the
optical neuroprotectional components of erigeron breviscapus by HPLCDAD ngerprints and phytochemistrical methods. Conclusively, our
project provides new ideas for exploring the physical basis of Chinese
medicine for glaucoma prevention and treatment, to develop innovative
drugs based on the machanism of the optical neuroprotection.
Paper No.: 1881

INVOLVEMENT OF SEROTONERGIC SYSTEM IN
GANODERMA LUCIDUM EXTRACTION-INDUCED HYPNOSIS
Yong-He Zhang, X-Y Cui
Peking University, School of Basic Medical Science, Department of
Pharmacolgy, Beijing, PR China
GLE in normal rats. Serotonergic (5-HT) system is one of the most

important mediators of sleep. Since then, this study was focused on the
serotonergic system. The present results showed that GLE showed no
effects on sleep in normal rats at lightperiod (9:00~15:00). In contrast, at
night time (21:00 ~ 3:00) GLE signicantly increased total sleep time by
enhancement of light sleep during NREM sleep. Flumazenil, a benzodiazepine receptor antagonist, showed a signicant antagonistic effect on
the hypnotic effect of GLE. On the other hand, diazepam showed synergistic effects with GLE on the sleep parameters in normal rats. These
results suggested that the hypnotic effect of GLE may be related to
GABAA/BDZ receptor. Pretreatment with PCPA, an inhibitor of triptophanhydroxylase, also signicantly decreased the hypnotic effect of GLE
and thishypnotic effect was promoted by 5-HTP. In addition, 5-HT1A
and 5-HT2 antagonists signicantly inhibited the hypnotic effects of
GLE. Furthermore, GLE signicantly enhanced the concentrations of 5HT in the regions that were implicated in the regulation of sleep-wake
behaviour, notably in the hypothalamus and DRN. These results suggested that GLE may exert hypnotic effect through benzodiazepine
receptors and serotonergic system..
Paper No.: 3274

DISCOVERY
MOLECULAR MECHANISMS OF HERG K+ CHANNEL
BLOCKADE BY FLUOROQUINOLONE ANTIBIOTIC AGENT
CHINFLOXACIN HYDROCHLORIDE
Zhao Zhang, X Zhang, J Guo, J Liu, Z Zhu, Z Hu, Z Zhao
College of Life Science Nanjing Normal University, Jiangsu the Key
Laboratory for Molecular and Medical Biotech, Nanjing, PR China
Human ether-go-go-related gene (hERG) encodes the b subunit of the
delayed rectier potassium channel that plays an important role in the
process of repolarization of the cardiac action potential. It has been well
established that QT prolongation induced by a number of drugs application is due to the inhibition of cardiac hERG K+ channel. In the present
study, effects of chinoxacin hydrochloride, a novel uoroquinolone
antibiotic agent, on the hERG K+ channel heterologously expressed in
HEK293 cells were rst investigated using the whole-cell patch clamp
technique. The hERG potassium channel homology model was built
based on the KcsA and MthK channel crystal structures and ligand-docking were used to know the mechanisms of the interaction thoroughly.
Moreover, the role of two canonical sites, Y652 and F656 which are proposed to be key determinants of blockade by a range of compounds, in
drug binding was determined by molecular biological and molecular
Modelling analysis. Limited data through whole-cell patch clamp recording indicated that the hERG K+ current was inhibited by chinoxacin in
a concentration-dependent manner. However, inhibitory potency of chinoxacin to hERG K+ current was less than that of its positive control,
moxioxacin hydrochloride. Therefore, in terms of tail current, application of chinoxacin with 7 different concentrations gave an IC50 of
127.7 lM while moxioxacin gave an IC50 of 62.5 lM, respectively,
through Logistic t to percentage of drugs inhibition. Characteristics of
chinoxacin in hERG K+ channel and molecular mechanisms will be further investigated..
Paper No.: 2154
HEALTH AND DISEASE
PROTECTIVE EFFECT OF CELECOXIB AGAINST ACUTE
MYOCARDIAL ISCHEMIA ON CORONARY ARTERY OF
RABBITS
M Zhao, X He, H-L Zhang, X-J Yu, Wei-Jin Zang
In East Asia, Ganoderma lucidum has been used as a tranquilizing agent

to treatinsomnia for thousands of years. In our previous study, Extract of
Ganoderma Lucidum (GLE) showed benzodiazepine-like hypnotic activity. This study was performed to investigate the hypnotic mechanism of

PR China
688
Selective cyclooxygenase (COX)-2 inhibitors (celecoxib, rofecoxib and
valdecoxib) were widely used in the treatment of rheumatoid arthritis.
However, cardiovascular events have been regarded as key limitation for
clinical application of COX-2 inhibitors. Therefore, rofecoxib and valdecoxib were revoked by the FDA in September 2004 and April 2005,
respectively. Fortunately, celecoxib at the usual doses does not result in
an elevated risk of vascular occlusion. Our aim was to assess the role of
celecoxib in rabbit coronary arterial injury associated with acute myocardial ischemia (AMI). The vasomotion was recorded by the Multi Myograph System; Serum ferment activity and myocardial infarct size were
measured by biochemical analysis. The results showed that celecoxib (3
mg/kg) signicantly reduced the infarct size from 18.6%2.7% to
4.4%1.3% in AMI (P < 0.01). Serum ferment levels of creatine kinase,
malondialdehyde and tumor necrosis factor-a were markedly reduced
and nitric oxide, nitric-oxide synthase were signicantly increased in celecoxib (3 mg/kg) group compared with AMI group. Our study also
revealed that coronary arteries were the most seriously impaired blood
vessels when compared to cerebral and mesenteric arteries in AMI. Celecoxib (3 mg/kg) could inhibit the vasoconstriction of coronary arteries
caused by AMI. The Emax values induced by noradrenaline, acetycholine,
5-hydroxytryptamine and CaCl2 (66.53%5.42%, 10.98%4.43%,
60.31%9.19% and 92.14%5.33%) were lower than those of AMI
(80.15%7.13%, 57.10%5.76%, 93.05%6.02% and 107.94%3.72%).
Overall these results suggest that celecoxib protect coronary arteries
against AMI. The mechanism underlying this effect could be linked to
inhibition of coronary arterial contraction and acceleration of nitric oxide
production.
Paper No.: 3205

CREATION OF A NEW CLASS OF MEDICINAL AGENTS ON
THE BASIS OF NANOTECHNOLOGIES
Vadim Zhdanov(1), A Dygai(1), A Artamonov(2), A Bekarev(2), E Vereschagin(2), A Churin(1)
(1) Siberian Branch of Russian Academy of Medical Sciences, Research
Institute of Pharmacology, Pathophysiology Laboratory, Tomsk, Russian
Federation
(2) Joint-Stock Company Siberian Center of Pharmacology and Biotechnology, Novosibirsk, Russian Federation
Nanoparticle drug delivery systems can improve current disease therapies
because of their ability to overcome biological barriers, to decrease of
clearance, directional delivery of medical products and absence of toxic
action. The polymer-drug conjugates obtained by covalent adsorption of
biological molecules on polyethylenglycole the basic interest are presents. In JSC SCP&B used electron-beam immobilisation has conclusive advantage before traditional chemical, increasing efciency of
nanocomplexes synthesis. The rst thrombolytic agent Trombovasim for
oral administration was created with use of electron beam technology.
This preparation with vasoprotective properties has passed preclinical
researches and state registration. Preclinical tests and clinical researches
of Imocept, a recombinant human C-peptide immobilized on PEG (for
treatment of complications of type 1 diabetes) are nished. Its high efciency and absence of toxic action have been shown. Immobilisation of
G-CSF allows to use it for therapy of intimate insufciency, a liver cirrhosis, illnesses of blood system. Action of a preparation is shown at its
intragastric and parenteral administration and is comparable to effects of
G-CSF made on traditional technology. Immobilized hyaluronidase has
been developing as a remedy for regenerative medicine. It increases the
amount of mesenchymal and hemopoietic stem cells. Native hyaluronidase lacks the ability because of the rapid destruction in organism. As an
immunomodulator are investigated the immobilized form of Interferon-a
2 b and the preparation of immobilized oligonucleotides. Analogues to
the oral form does not exist. Thus, it is possible to consider perspective
development of new medical products with the use of biomolecules

immobilization on PEG.
Paper No.: 855

DISEASES
ARTESUNATE POTENTIATES ANTIBACTERIAL EFFECTS OF
ANTIBIOTICS IN VITRO AND IN VIVO BY INCREASED
ACCUMULATION OF ANTIBIOTICS VIA AN INHIBITION ON
DRUG EFFLUX PUMPS, ACRAB-TOLC AND MDTABC
Hong Zhou, BL Li, Q Yao, R Zhang, X Pan, J Li, G Ding, X Liu, C Wu,
J Zheng
The Third Military Medical University, Department of Pharmacology,
Chongqing, PRChina
Infections lead to death by large bacterial growth and excessive inammatory response. In our previous study, we have found that ART and AS
could protect sepsis model mice challenge with a heat-killed E. coli by
reduction of proinammatory cytokine release and endotoxin levels via a
mechanism involving a decrease in TLR4, TLR9 mRNA expression and
NF-jB activation. Here, we demonstrated AS could potentiate antibacterial effects of antibiotics in vitro and in vivo. In vitro, AS could synergize
with antibiotics against E.coli although AS had no directly antibacterial
effect. AS could increase the accumulation of daunomycin (DNR) within
E.coli ATCC35218 in dose- and time- dependent manners. Antibacterial
potentiation of AS on antibiotics against E.coli was associated with
reduced AcrAB-TolC and MdtABC mRNA expressions, a multi-drug
efux. In vivo, AS in combination with antibiotics decreased sepsis mice
mortality both in mice challenged by lethal live E.coli or cecal ligation
and perforation (CLP) sepsis model. Importantly, AS could potentiate
ampicillin sodium-sulbactam sodium (AMPS) further decrease bacterial
numbers in blood of CLP mice. Additionally, AS in combination with
AMPS decreased the serum endotoxin and IL-6 levels although AMPS
increased the serum endotoxin and IL-6 levels. In summary, our results
demonstrated that AS-mediated protection on sepsis model mice was not
only tightly related to a reduction in proinammatory cytokine release
and endotoxin levels due to anti-inammatory effects of AS, but also
tightly related to its antibacterial potentiation on antibiotics against bacteria via an inhibition on drug efux pump, AcrAB-TolC.
Paper No.: 1858

ROLES OF ANTIOXIDANTS AND HEAT SHOCK PROTEINS AS
AN ANTIFATIGUE EFFECT INDUCED BY MEIRUSENJYU ON
AGED MICE
Jianrong Zhou, K Yokomizo, T Miyata
Sojo University, Faculty of Pharmaceutical Sciences, Kumamoto, Japan
The antifatigue properties of meirusenjyu (MRSJ), which is composed of
extract essence of Lycium chinense Miller, Crataegus cuneata Siebold et
Zuccarini, Phyllanthus emblica L., Chrysanthemum morifolium, Zizyphus jujuba Miller var. inermis Rehd, Ganoderma lucidum, and Coix lacryma-jobi L. var. mayuen Stapf, were studied using aged mice, and the
roles of antioxidant enzymes and heat shock proteins were detected with
mice tissues and Human Gastric Cancer (MKN-45) cell line, respectively.
Mice were chronically (one month) administered MRSJ (1%) in the
drinking water. Mice pre-treated MRSJ showed a good appetite; however, they exhibited a lower rate ofbody weight increase compared to
control mice. In mice subjected to the rotarod test, MRSJ treatment provided effective adaption to fatigue and signicantly increased the duration of mice on the rotarod. In locomotor activity tests, MRSJ
potentiated mice motility and signicantly increased rearing behavior. In
689
the antioxidant experiments, MRSJ treatment signicantly increased
superoxide dismutase (SOD) activity in spleen and glutathione (GSH)
level in liver. 5% MRSJ-treated MKN-45 cells increased the expressions
of HSP 72 and GRP 78 by 1.7 and 2.6 times, respectively. These ndings suggested that MRSJ may be utilized as an antifatigue agent, which
may be function through antioxidant activity and cell-protection via
induction of HSP72 and GRP78.
Paper No.: 2078

STUDY ON ANTI-TUMOR ACTIVITY AND MECHANISM OF
METHYLACRYLOYLSHIKONIN IN VITRO AND IN VIVO
Liming Zhou, Y Wu, X Zheng, Z Shao
CYP2E1 activity were measured using a model CYP2E1 probe substrate

chlorzoxazone and its metabolite, 6-hydroxy-chlorzoxazone determined
by HPLC. Danshen extract (60 lg/ml to 1 mg/ml) increased rat hepatocytes viability by 1.8-5.7-fold and APAP-induced glutathione depletion
in rat hepatocytes was reduced by Danshen extract, Sal B and Danshensu
dose-dependently. Danshen extract inhibited CYP2E1 activity with an
IC50 of 1.03 mg/ml in rat liver microsomes. Protection of rat hepatocytes
from APAP-induced toxicity by Danshen extract may involve CYP2E1
inhibition, decrease of glutathione depletion and decrease in ROS.
Paper No.: 1541

DISCOVERY AND VALIDATION OF THE COMBINATIONAL
THERAPEUTIC TARGETS FOR ALZHEIMERS DISEASE: A
PRELIMINARY STUDY
Sichuan University, Hua Xi Medical Center, Department of Pharmacology, Chengdu, PR China
Wenxia Zhou, X-R Cheng, N Jiang, Y Zhang
Objective: To investigate anti-hepatocarcinoma effects and mechanism of

Methylacryloylshikonin in Vitro and in vivo. Methods: The inhibitory
effect of Methylacryloylshikonin was evaluated by MTT assay and
growth curve analysis. DNA ladder Hoechst 33258 were used to assess
cell apoptosis; Hepatic cancer H22 model in mice was performed to
study anti- hepatocarcinoma activity of Methylacryloylshikonin in vivo.
The growth curve and inhibitory rate of tumor growth was measured.
Immunohistochemical assay was used to observe expression of NF-jB
P65. Results: After treated for 48 hours, MTT assay shows Methylacryloylshikonin can inhibit the proliferation of SMMC-7721 cells and have
obvious dose-effect relationship. IC50 of Methylacryloylshikonin to
SMMC-7721 cells was (21.16 0.96) lg /ml. The dose-effect relationship and time-effect relationship were described in growth curve. DNA
ladder Hoechst 33258 indicated the antitumor activity of Methylacryloylshikonin was related to apoptosis. Under the treatment of 0.38, 0.75
and 1.50mg /kg of Methylacryloylshikonin the inhibitory rates of H22
transplanted tumor in mice were 22.98%(P < 0.05),39.08%(P <
0.05),47.13%(P < 0.01), respectively. Immunohistochemistry assay
shows p65 was down regulated. Conclusion: Methylacryloylshikonin has
signicant antitumor effects both in vitro and in vivo, the mechanism
may be related to apoptosis.
Beijing Institute of Pharmacology and Toxicology, Department of Neuroimmunopharmacology, Beijing, PR China
Paper No.: 2239

PROTECTION OF A WATER-SOLUBLE DANSHEN (SALVIA
MILTIORRHIZA) EXTRACT AND ITS ACTIVE INGREDIENTS
ON PARACETAMOL-INDUCED TOXICITY IN RAT
HEPATOCYTES
Xuelin Zhou, WYW Lee, CM Cheung, PMY Or, JHK Yeung,
The Chinese University of Hong Kong Faculty of Medicine, School of
Biomedical Sciences, Hong Kong, PR China
Paracetamol (APAP)-induced hepatotoxicity is caused by a CYP2E1mediated chemically reactive intermediate N-acetyl-p-benzoquinoneimine. In this study, a water-soluble Danshen extract, a widely used Chinese herbal medicine, and its active ingredients Salvianolic acid B (Sal
B) and Danshensu, were investigated for their protective effects on
APAP-induced toxicity. Primary rat hepatocytes, isolated from acetonetreated male rats with 3.5-fold over-expression of CYP2E1, were used to
determine the effects of Danshen extract and its active ingredients on
APAP-induced hepatocytes injury as measured by LDH and MTT assay.
Glutathione was measured as described previously (Food and Chemical
Toxicology 2008; 46: 328-338), and oxidative stress was evaluated by a
reaction of the reactive oxygen species (ROS) with diacetyldichlorouorescein. The effects of Danshen extract, Sal B and Danshensu on rat
Introduction To discover and validate series candidate molecules, i.e.

combinational targets for screening and evaluation of anti-Alzheimers
disease (AD) drugs. Materials The substrain of senescence-accelerated
mouse (SAM)/prone 8 (SAMP8) and its control SAM/resistant
1(SAMR1). Results A total number of 81 related molecules were
obtained from the hippocampus and cerebral cortex of SAMP8 and all
the molecules were changed regularly with ageing process. Some of the
molecule expressions were changed in SAMP8 when treated with different traditional Chinese medicinal prescriptions (TCMP) which have the
improving effects on AD patients in clinical trials or in animal models.
However, the changes of the molecule expressions varied with the prescriptions. The validation of 9 typical molecules, such as AMFR,
STUB1, NF-L, NDUFS2, CAMK2A, EphB6, CSTN1, NRXN1 and
NGRN was conducted. The results revealed that both the expressions of
genes and proteins of the molecules showed age-related changes in
SAMP8, treatment with TCMP affects the expressions of genes and proteins. Conclusion The candidate molecules for the combinational therapeutic targets for AD are closely related with pathophysiological
processes of AD, and may become the members of the combinational
therapeutic targets, which is worthy of further studies.
(Supported by grants from the National Natural Science Foundation of
China 30701073, 90709012 and the National High Technology Research
and Development Program of China 2008AA02Z423)
Paper No.: 1220

TREATING DIABETIC COMPLICATIONS? MULTITARGETING
EFFECTS OF SCUTELLAREIN
Banghao Zhu
Sun Yat-Sen University, Department of Pharmacology, Guangzhou, PR
China
In treating diabetic complications, is there any good drug besides hypoglycemics? Scutellarein(Scu) with Mr 462.21, one of the avonoides isolated from Chinese herb (erigeron breviscapus, handmazz) we found that
Scu has manifold effects in treating diabetic complications: 1. Scu prevented the diabetic-associated decit of endothelium-dependent relaxation in streptozotocin Cinduced diabetic rats. 2. Scu blocked protein
kinase C (PKC) translocation which caused by diabetic conditions. 3.
Scu blocked voltage-dependent Ca2 + channels in smooth muscle cells of
rat thoracic aortic artery, also increased [Ca2+]i by blocking sarcoplasmic
690
reticulum Ca2 +/ATPase. 4. Scu inhibited hypoxia- and moderate high
glucose-induced proliferations and VEGF expression in human retinal
endothelial cells. These results suggest that Scu will be a potent preventer of diabetic complications and the clinic effect will be investigated
further.
Paper No.: 1716

HIGHER SPECIFICITY OF THE ACTIVITY OF LOW
MOLECULAR WEIGHT FUCOIDAN FOR THROMBININDUCED PLATELET AGGREGATION
Z Zhu(1), Q Zhang(2), L Chen(1), S Ren(1), Dali Luo(1)
(1) Capital Medical University, Department of Pharmacology, Beijing,
PR China
(2) Chinese Academy of Sciences, Institute of Oceanology, Qingdao, PR
China
Algal fucoidans possess a wide variety of biological activities. We
assessed the activities of antiaggregation, anticoagulation and antithrombosis and the underlying mechanism of the low molecular weight fucoidan from the seaweed Laminaria japonica of Qingdao, China (F-Q). In
the platelets of rats and humans, F-Q, like the commercially purchased
fucoidan (F-S) and heparin, showed an inhibitory effect on thrombininduced aggregation with an IC50 of 8 lg/mL, approximately ve times
lower than those of F-S and heparin. In anticoagulation tests, lower anticoagulant effect for F-Q than heparin and F-S was observed in the activated partial thromboplastin time test (<160 s vs. >250 s at 40 lg/mL, P
< 0.01) and in the thrombin catalyzed brinogen cleavage test (IC50 10
lg/mL vs. 2.8 lg/mL) in vitro, and also in rat thrombosis inhibition at -3
mg/kg in vivo. The inhibitory effects of F-Q and heparin on thrombin
activity were strikingly enhanced by either antithrombin (AT) or heparin
cofactor II (HCII). A direct interaction of F-S with thrombin, or F-Q or
heparin with AT was displayed in both uorescence quenching and
PAGE analysis. Additionally, a pro-aggregation effect and an enhancement of thrombin activity were also observed with F-S, but not with F-Q
or heparin, treatment. These results indicate that F-Q inhibits thrombin
via activation of AT and HCII, whereas F-S mainly interacts directly with
thrombin, and its higher specicity for hypoaggregation and a weaker
effect for anticoagulant proles than heparin and F-S might make it a
promising candidate for the treatment of thrombosis-related diseases.
Paper No.: 834

IN VITRO ANTIFUNGAL ACTIVITY OF THE ESSENCE OF
THYMUS VULGARIS AND MYRTUS COMMUNIS L AGAINST
CANDIDA ALBICANS STRAINS ISOLATED FROM THE
PATIENTS WITH ORAL CANDIDIASIS IN COMPARISON
WITH NYSTATIN
tus communis L and the mix of these essences on the growth of Candida
albicans in solid media, and also their MIC was compared with the MIC
of Nystatain in the same conditions. Materials and Methods: Thirty-two
strains of Candida albicans isolated from patients with oral candidiasis
were studied in this research. We provided a yeast suspension of candida
yeast cells, and also a serial dilution from these essences and Nystatin in
Sabouraud Dextrose Agar (SDA) medium. A loop of candida suspension
was cultured on all of the solid media and was incubated at 35C for 48
hours. The obtained results were recorded during 7 days. Results: Our
nding showed that the MIC of Thymus vulgaris, Myrtus communis L,
mix of these essences and Nystatin was 0.2 ll/ml, 12.5 ll/ml, 0.78 ll/ml
and 7.9 ll/ml respectively. Conclusion: It seems that Thymus vulgaris
has antifungal activity against Candida albicans. This plant products are
inexpensive and their side effects probably are very lower than Nystatin.
Key words: Thymus vulgaris, Myrtus communis L, Nystatin, Candida
albicans, oral candidiasis
Paper No.: 916

MAKING CREAM FROM BEE PROPOLIS FOR TREATMENT
OF DERMATOPHYTIC INFECTIONS
Mohammadali Zia(1), M Rouholamin(1), R Mannani(2), S Gavanji(1)
(1) Islamic Azad University Khorasgan Branch, Department of
Pharmacy, Isfahan, Iran
(2) Islamic Azad University Khorasgan Branch, Department of Food
Science & Technology, Isfahan, Iran
Introduction: Popolis (bee glue) is a resinous or sometimes wax-like bee
hive product that has been used by man since ancient times for its pharmaceutical properties. The antibacterial and antifungal activities are the most
popular and among the most extensively investigated biological actions of
propolis. ermatophytosis refers to the disease of the skin, hair and nail
which caused by dermatophytes. Topical or systemic treatments have been
empirically administered, although such drugs are widely used, some of
them have been reported as ineffective and even toxic to the host and sometimes it takes time to be cured. Recently, propolis has been attracted the
attention of researchers due to its various biological activities and therapeutic properties. The aim of this study was making topical cream from propolis for treatment of dermatophytic infections. Material and methods: For
making of different formulations, we used from a base formulation, and
then with adding the other materials and some variations established in the
base formulation, nally 25 formulations were provided. All of formulations were examined in terms of consistency, stability, PH average, appearance, microbial studies and some other features of cream and the best
formulation with the best efcacy was selected. Results: The selected formulation had a very good effect for treatment of experimental dermatophytosis in rabbits in comparison with synthetic products such as some azoles.
Conclusion: The natural products are very effective for treatment of many
fungal infections and also in comparison with synthetic drugs are more
inexpensive and have lower side effects.
Mohammadali Zia(1), M Rouholamin(2), M Bayat(3), H Khalkhali(4)

(1) Islamic Azad University Khorasgan Branch, Department of Basic
Sciences, Esfahan, Iran
(2) Food Science & Technology, Iran
(3) Islamic Azad University, Science and Research Branch, Tehran, Iran
(4) Islamic Azad University- Khorasgan Branch, Esfahan, Iran
Introduction: Candida albicans is the most frequent etiological agent of
oral candidiasis. Nystatain is a choice drug in the treatment of this infection, but it has some problems through its side effects. In recent years,
the use of plant products has been increased for treatment of different
fungal infections. The aim of this study was to determine the Minimum
Inhibitory Concentration (MIC) of the essence of Thymus vulgaris, Myr-
Paper No.: 3130

HEALTH AND DISEASE
GENERATING NOVEL HYPOTHETICAL ASSOCIATIONS
BETWEEN STATIN THERAPY AND ENDOTHELIAL ADHESION MOLECULES USING COMPUTERIZED TOOL SEMBT
Lovro Ziberna(1), M Lunder(1), G Drevensek(1), D Hristovski(2)
(1) University of Ljubljana, Faculty of Medicine, Ljubljana, Institute of
Pharmacology, Slovenia
691
(2) University of Ljubljana, Faculty of Medicine, Institute of Bioinformatics and Medical Statistics, Ljubljana, Slovenia
Statins are one of the most used drugs for treating atherosclerosis. They
inuence numerous processes in the vascular wall and endothelial adhesion molecules. In the last years abundance of literature sources on atherosclerosis were published, as well as on statins. Thus we tried to
identify some novel potential associations between statin therapy and
expression of endothelial adhesion molecules that may inuence the atherosclerosis process. We used the literature-based discovery (LBD)
approach and the computerized tool SemBT (http://sembt.mf.uni-lj.si) to
extract known facts from the literature and to generate novel hypotheses.
Semantic relations were extracted from 6,699,763 MEDLINE citations
published between 1999 and 2009. We used the number of semantic relations, the number of papers indexed in PubMed and the number of concepts as numeric variables to describe the associations. After the
computerized search, human experts evaluated the generated hypotheses.
We discarded the bad and kept the good hypotheses. Using SemBT we
narrowed the search on endothelial adhesion molecules, when combined
with statin therapy, from 65 to 19 concepts, from 108900 to 305 indexed
PubMed papers, and from 52800 to 91 semantic relations, respectively.
Furthermore, we have generated seven new hypothetical concepts; these
are endothelial adhesion molecules correlated with atherosclerosis but
not yet associated with statin therapy. These candidate adhesion molecules, if tested with in vitro approaches, can then be shown as new targets for statins or other drugs. In conclusion, SemBT computerized tool
has proven as an effective assistance in searching for novel endothelial
adhesion molecules in atherosclerosis.
Paper No.: 1076

DISEASES
INTERFERENCE OF NEWLY DEVELOPED ANTIVIRALS
WITH ACTIVITY OF IMMUNE SYSTEM
Paper No.: 1104

THE PRESENCE OF P2X RECEPTORS IN CD34-POSITIVE
CELLS OF HUMAN UMBILICAL CORD BLOOD
Airat U Ziganshin(1), RR Kazakova(1), IG Moustan(1), DV Nurullina(1), GZ Sagdieva(1), FF Kazakova(2), LE Ziganshina(3)
(1) Kazan State Medical University, Department of Pharmacology, Pharmacognozy and Botany, Kazan, Russian Federation
(2) Kazan City Hospital # 11, Kazan, Russian Federation
(3) Kazan State Medical Academy, Kazan, Russian Federation
P2 receptors have been found on the surface of many animal and human
cells including blood cells. It is suggested that P2 receptors appear at the
earliest stages of tissues development and could be involved in differentiation and maturation of the cells. The aim of our study was to determine
expression of subtypes of P2X receptors on haemopoietic stem (CD34positive) cells of human cord blood. Cord blood was taken during normal labor. The informed consent was received from each woman
involved; the study was approved by the local ethical committee. The Ficoll was used for separation of the mononuclears. CD34 + cells were isolated from fraction of mononuclears by method of immunomagnetic
separation. Further investigation was done using ow cytometry. Method
is based on staining cells with antibody conjugated with uorochrome to
subtypes of P2X receptors and to C-kit-positive cells. We also analyzed
expression of P2X receptors on lymphocytes of adult peripheral and cord
blood. The population of CD34 +/C-kit+ cells which express P2X receptors was also detected. Results of our study showed the expression of
P2X2-7 receptors on CD34 + cells of cord blood. The type of expression
was varying individually. The level of expression of P2X receptors was
one order higher in lymphocytes of cord blood in compare with cells of
adult peripheral blood. In conclusion, CD34 + cells of human umbilical
cord blood express several subtypes of P2X receptors which support the
idea of possible involvement of P2 receptors in differentiation and maturation of the cells in the developing organism
Zdenek Zdek(1), E Kmonckova(1), A Holy(2)

(1) Institute of Experimental Medicine, Acad. Sci., v.v.i., Department of
Pharmacology, Prague, Czech Republic
(2) Institute of Organic Chemistry and Biochemistry, Acad. Sci, v.v.i.,
Prague, Czech Republic
The synthetic analogues of natural nucleoside monophosphates, acyclic
nucleoside phosphonates (ANPs), are antivirals inhibiting replication of
both DNA-viruses and retroviruses. Tenofovir, 9-(R)-[2-(phosphonomethoxy)propyl]adenine is broadly used for therapy of AIDS, adefovir, 9-[2(phosphonomethoxy)ethyl]adenine has been approved for the treatment
of hepatitis B. We have investigated possible immune activities of ANPs
in cultures of mouse peritoneal cells (PEC) and human peripheral blood
mononuclear cells (2 mil/mL). Secretion of cytokines was determined
after the 5-h interval by ELISA. Production of NO was assayed at the
interval of 24 h using the Griess reagent. The compounds did not stimulate production of NO in mouse PEC if they were applied on their own.
However, a number of them did signicantly augment the NO formation
triggered by the mouse recombinant IFN-c. These ANPs also activated
the secretion of anti-HIV effective chemokines RANTES and MIP-1a
and cytokines TNF-a and IL-10. The ANP effects were expressed within
the interval of 2-4 h, at as low concentrations as 2 to 5 uM, and
depended on the activation of NF-jaB. Highly signicant correlation
was found to exist between the NO production by mouse cells and levels
of cytokines produced by both mouse and human cells. It may be concluded that ANPs represent a new generation of antivirotics with combined antimetabolic and immunostimulatory properties. The data
demonstrate that the animal NO platform is a rapid and economical
screening approach allowing reliable prediction of cytokine-stimulatory
potential of drugs in human cell system.
Acknowledgements. The work was supported by the grant no. 1M0508.
Paper No.: 2330

INVESTIGATING POTENTIAL IMMUNOMODULATORY
ACTIVITY IN NATIVE FLORA
James Ziogas(1), L Ng(1), L Bennett(2), R Williams(2), K Robinson(2),
S Sudharmarajan(2), AG Stewart(1)
(1) University of Melbourne, Department of Pharmacology, Melbourne,
VIC, Australia
(2) CSIRO Food and Nutritional Sciences, 671 Sneydes Rd, Preventative
Health National Research Flagship, Werribee, VIC, Australia
Bioactivity screening of a library of processed dietary plants by CSIRO,
identied anti-inammatory activity in a selection of plants from the
Myrtaceae family, including Syzygium aromaticum (Clove), Eugenia
Reinwardtiana (Cedar Bay Cherry) and Syzygium Luehmannii (Riberry).
Anti-inammatory activity was detected as down-regulation of IFNgamma production in IL-12 and IL-7 stimulated human polymorphonuclear blood cells. Further investigations were undertaken in the current
study, using the lipopolysaccharide (LPS)-stimulated J774 murine macrophage model of inammatory mediator production. The effects of the
extracts on nitrite, tumour necrosis factor-a (TNF-a) and interleukin-6
(IL-6) levels in the culture medium 24h after LPS stimulation were determined. Pre-treatment for 30 min prior to LPS stimulation with Clove
extract showed a concentration-dependent inhibition of nitrite and IL-6
production, with a maximal effect of 45-50% inhibition seen at 200 lg/
mL. Cedar Bay Cherry and Riberry extracts did not signicantly affect
nitrite and IL-6 release (up to 200 lg/mL). TNF-a release was unaffected
by any of the three extracts. In a pattern similar to that for the clove
692
extract, the inhibitor Curcumin (3 lM) had no effect on TNF-a but did
inhibit nitrite and IL-6 levels by 30-45%. Partially puried clove extract
and curcumin each inhibited NFjB phosphorylation. Inhibition of other
signaling pathways, p38 MAP kinase (SB 202190, 0.3-10 lM), MEK1/2
(U 0126, 1-10 lM) or c-Jun (SP 600125, 3lM) elicited different patterns
of inhibition of mediator release to those generated by clove extract or
curcumin. Clove may diminish inammation through inhibition of the
NFjB transcriptional activity.
Paper No.: 1255

HEALTH AND DISEASE
VITAMIN D3 DOES NOT AUGMENT EDHF-MEDIATED
RELAXATIONS TO BRADYKININ
Qian Zou, PM Vanhoutte
The University of Hong Kong, Li Ka Shing Faculty of Medicine, Department of Pharmacology and Pharmacy, Hong Kong, PR China
The most active metabolite of vitamin D, 1, 25-dihydroxyvitamin D3
acutely reduces endothelium-dependent contractions to acetylcholine by
reducing the increase in calcium concentration caused by the muscarinic
agonist in the endothelial cells. The release of endothelium-derived hyperpolarizing factor involves calcium-dependent potassium channels.
Thus, vitamin D3 may also affect endothelium-dependent relaxations by
regulating EDHF-mediated responses. The present experiments were
designed to investigate the effect of acute treatment of vitamin D3 on the
EDHF pathway. Rings of porcine coronary arteries, with endothelium,
were suspended in organ chambers for isometric tension recording. To
study the EDHF mediated relaxation, quiescent rings were incubated
with L-NAME (nitric oxide synthase inhibitor, 10-4 M) and indomethacin (cyclooxygenase inhibitor, 10-5 M) for 40 minutes. They were contracted with prostaglandin F2a and then relaxed with cumulatively
increasing concentrations of bradykinin. The experiments were carried
out in the presence or absence of 10-7 M 1,25-dihydroxyvitamin D3.
The results show that the relaxation induced by bradykinin was not
altered signicantly by the prior incubation of 1,25-dihydroxyvitamin
D3, suggesting that vitamin D3 does not have an regulatory effect on
EDHF-mediated responses. Thus the vascular protective effect of vitamin
D is probably due to a reduction of endothelium-dependent contractions.
Paper No.: 2331

GENDER FACTORS IN PHARMACOKINETICS OF
CIPROFLOXACIN IN HEALTHY VOLUNTEERS
Jana Zoulova(1), J Kopecky(1), Z Vybiralova(1), J Chladek(1),
K Macek(2), J Kvetina(1)
(2) University Hospital, Hradec Kralove, Czech Republic
effect of gender on the pharmacokinetics of ciprooxacin (second generation uoroquinolone antibacterial agent). Ciprooxacin was given to 14
healthy men (20-38 years, 61-85 kg) and 10 healthy women (20-36
years, 50-74 kg) in a single oral dose of 500 mg (one tablet). Blood samples (5 ml) were drawn 0-24 hours after the administration. Plasma ciprooxacin concentrations were determined by HPLC with uorometric
detection. Calculated pharmacokinetic parameters (mean and standard
deviation except for Tmax data which are given as median and range)
are presented below. Men: AUC = 29.55 (8.39) nmol.h/ml, AUCt =
28.19 (8.01) nmol.h/ml, Cmax = 8.41 (2.11) nmol/ml, Tmax = 0.67
(0.67-1.50) h, T = 6.04 (0.82) h. Women: AUC = 40.81 (9.47)
nmol.h/ml, AUCt = 39.73 (8.95) nmol.h/ml, Cmax = 9.63 (2.00) nmol/
ml, Tmax = 1.00 (0.67-3.00) h, T = 4.95 (0.90) h. Tendency to higher
plasma concentrations was observed in women. Statistical analysis of
parameters (t-test) revealed signicant differences between men and
women in the case of AUCs parameters; however after adjusting the
AUCs for body weight the signicance disappeared. Thus, the differences in AUCs seem to be caused by body weight. T was signicantly
lower in women. Interindividual variability was moderate both in men
and women.
Paper No.: 2723

SESSION - ONCOLOGY
DIRECT ANTIPROLIFERATIVE EFFECT OF NOVEL
NONSTEROIDAL 17BETA-HYDROXYSTEROID
DEHYDROGENASE TYPE 1 INHIBITORS
Istvan Zupko(1), A Berenyi(1), S Marchais-Oberwinkler(2),
M Frotscher(2)
(1) University of Szeged, Department of Pharmacodynamics and
Biopharmacy, Szeged, Hungary
(2) Saarland University, Department of Pharmaceutical and Medicinal
Chemistry, Saarbrucken, Germany
17b-estradiol (E2), the most important estrogen in humans, is involved
in many proliferative disorders including breast cancer and endometriosis. 17b-Hydroxysteroid dehydrogenase type 1 (17betaHSD1) is responsible for the conversion of the much less potent estrogen estrone into E2.
The inhibition of this enzyme is therefore considered as an innovative
approach to treat estrogen-dependent diseases. The aim of the present
study is to characterize the direct antiproliferative action of a set of
recently synthesized nonsteroidal 17bHSD1 inhibitors. Inhibitors have
been tested on human adherent cell lines (HeLa, MCF7) by MTT assay.
Compounds exhibiting IC50 value lower than 10 lM were subjected to
cell cycle analysis. Hela cells were exposed to the test compounds for 24
hours and the cell phase distribution was determined by ow-cytometry
after propidium staining. One compound out of 6 displayed increased
subG1 phase while two others caused a substantial decrease in S-G2/M
phase. The increase of membrane permeability has been additionally evidenced by uorescent microscopy. Based on the presented result apoptosis induction and cell cycle inhibition at G1-S transition is suggested as
mechanism of direct antiproliferative action which is most likely not
linked to 17bHSD1 inhibitory activity. It is speculated that the combination of estrogen synthesis inhibition by blockade of 17bHSD1 and direct
antiproliferative effects offers a promising approach for the treatment of
estrogen-dependent cancers.
Gender differences in pharmacokinetics have been described in many

drugs (Harris RZ et al., Drugs 1995; 50: 222-39; Schwartz JB, Clin Pharmacokinet 2003; 42: 107-21). The aim of our study was to nd out the

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162

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RS79948 in non-specic binding wells) in 50 mM glycylglycine buffer

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dent vasodilatation. 4-aminopyridine or glibenclamide inhibited acidotic

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(1) Kaunas University of Medicine, Kaunas, Lithuania

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