International Immunopharmacology 40 (2016) 443–451

Contents lists available at ScienceDirect

International Immunopharmacology

journal homepage: www.elsevier.com/locate/intimp

Discovery and preclinical development of a novel prodrug conjugate of

mesalamine with eicosapentaenoic acid and caprylic acid for the
treatment of inflammatory bowel diseases
Mahesh Kandula ⁎, Sunil KumarKB, Sivanesan Palanichamy, Ashok Rampal
Cellix Bio Pvt Ltd, Plot No 1177B, Road Number 56, Jubilee Hills, Hyderabad 500033, India

a r t i c l e i n f o a b s t r a c t

Article history: Mesalamine (5-ASA) is one of the drugs indicated as first line therapy in ulcerative colitis for induction and main-
Received 29 June 2016 tenance of remission. Sulfasalazine and formulations of 5-ASA (pH-dependent and controlled release formula-
Received in revised form 12 September 2016 tions) were developed to minimize the systemic absorption and maximize the delivery of the mesalamine to
Accepted 15 September 2016
colon. Although, its efficacy and safety is well documented there are approximately 30% nonresponders to 5-
Available online xxxx
ASA therapy. This necessitates the need for novel therapeutic options to improve the efficacy and safety of the
Keywords:
currently available 5-ASA therapy. CLX-103 is a novel, patented prodrug molecular conjugate of mesalamine,
CLX-103 eicosapentaenoic acid and caprylic acid designed to offer incremental benefits over the currently approved 5-
Mesalamine ASA formulations. Results of in vitro and in vivo studies showed that CLX-103, was stable in simulated gastric
Fatty acid conjugate fluid, but undergoes enzymatic hydrolysis in the small/large intestines to release the active moiety. Our data
Inflammatory bowel diseases also showed that the active moiety is retained in the targeted intestinal tissues (ileum and colon) over a longer
Ulcerative colitis period of time in relation to sulfasalazine. The in vitro data supports the observed in vivo plasma kinetics of 5-
ASA characterized by longer Tmax, low Cmax after the oral administration of CLX-103. Efficacy study of CLX-103
in acute dextran sodium sulfate (DSS) mouse colitis model showed improved potency measured as Disease Ac-
tivity Index (DAI) and histological scores in the colon as compared to sulfasalazine. These findings indicate that
CLX-103 could offer a differentiated drug product which is more efficacious and safer than the currently approved
5-ASA formulations in the treatment of inflammatory bowel diseases.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction
Inflammatory bowel disease (IBD), including ulcerative colitis (UC)
and Crohn's disease (CD), represents disorders that are characterized
by chronic inflammation of the gastrointestinal tract [1]. In ulcerative
colitis, inflammation is usually confined to the colonic mucosa, whereas
in Crohn's disease, inflammation involves all the layers of the intestinal
wall (transmural inflammation) and can affect the gastrointestinal tract,
from the mouth to anus [2]. Long-standing ulcerative colitis is an impor-
tant risk factor for colorectal cancer. The risk of colorectal cancer in-
creases depending upon the duration and extent of colitis, yet the
etiopathogenesis of ulcerative colitis is still unclear and seems to grow
in Western countries [3,4]. Globally, approximately 1.86 billion patients
have been diagnosed with ulcerative colitis [5]. Prevalence of ulcerative
colitis was estimated to be 37–246 cases per 100,000 persons, and the
⁎ Corresponding author.
E-mail addresses: kmahesh@cellixbio.com (M. Kandula), kbsunil@cellixbio.com
(K.B. Sunil Kumar), sivanesanp@cellixbio.com (S. Palanichamy), rampala@cellixbio.com
(A. Rampal).
incidence rate was estimated to be 2.2–14.3 cases per 100,000 person-
years [6].
Current therapeutic strategies for the treatment of IBD are aimed to
reduce the inflammatory response that is activated during the relapse of
the disease, as well as to prevent further disease flare-ups. Conventional
therapies for ulcerative colitis and Crohn's disease include the 5-
aminosalicylic acid (5-ASA), sulfasalazine, corticosteroids, thiopurines,
and methotrexate. 5-ASA is the recommended first-line therapy for
mild to moderate ulcerative colitis [2]. 5-ASA is also used as the mainte-
nance therapy in patients with moderate to severe ulcerative colitis,
who are initially treated with corticosteroids [7]. The mechanism of ac-
tions of 5-ASA class of drugs includes inhibition of cyclooxygenase and
lipoxygenase pathways, arachidonic acid metabolism, prostaglandin
production, and PPAR-γ agonism in the colon [2].
5-ASA drugs are generally considered to be safe and effective for the
induction and maintenance of remission in mild to moderate ulcerative
colitis. Extensive data support the use of 5-ASA drugs in active ulcerative
colitis with the highest tolerated dose in its oral, rectal, or combination
dosage form [8].
Meta-analysis of clinical trials for remission of ulcerative colitis re-
vealed that the mean remission rate was only 40% in the 5-ASA

http://dx.doi.org/10.1016/j.intimp.2016.09.013
1567-5769/© 2016 Elsevier B.V. All rights reserved.
444 M. Kandula et al. / International Immunopharmacology 40 (2016) 443–451
treatment groups versus 20% in placebo group, and meta-analysis for
prevention of relapse indicates the relapse rate of 40% of patients on
5-ASA therapy as compared to 63% of patients taking placebo over 6–
12 months [4]. The adverse events associated with 5-ASA formulations
(such as nausea, indigestion, headache, vomiting and abdominal pain,
flatulence, and diarrhea) does not differ between the various oral for-
mulations of 5-ASA [9]. This necessitates the need for novel therapeutic
options to improve the efficacy (remission rate, maintenance of remis-
sion and reduction of relapse rates in refractory patients) and safety of
the currently available 5-ASA therapy. CLX-103 is a novel molecular
conjugate of 5-ASA with eicosapentaenoic acid and caprylic acid. CLX-
103 is a patented prodrug molecule that is designed for the efficient re-
lease of 5-ASA at the target site (ileum and colon) as well as to provide
improved efficacy contributed by the known anti-inflammatory/antimi-
crobial effects of eicosapentaenoic acid and caprylic acid.
Eicosapentaenoic acid is an omega-3 fatty acid known to have a
broad spectrum of anti-inflammatory properties that include inhibition
of leukocyte chemotaxis, adhesion molecule expression, leukocyte–en-
dothelial adhesive interactions and cytokines' production, activation of
the anti-inflammatory transcription factor NR1C3, inhibition of the acti-
vation of the pro-inflammatory transcription factor NF-κB and well-
studied in inflammatory bowel diseases [10–14]. Caprylic acid is an
eight-carbon saturated fatty acid and a GRAS-listed molecule and is
found naturally in the milk of various mammals, and as a constituent
of coconut oil. Caprylic acid has potential anti-inflammatory, antifungal
(more specifically against Candida) activity. Caprylic acid is reported to
suppress the secretion of IL-8, a key cytokine believed to be involved in
the induction and progression of inflammatory bowel disease [15]. Ad-
ditionally, high level of Candida colonization occurs with diseases of the
gastrointestinal tract such as gastritis and colitis. This suggests that pro-
biotic and antifungal therapy could be considered in the treatment of
chronic inflammatory bowel diseases [16]. Therefore, CLX-103 has the
potential to address multiple components involved in the complex
pathogenesis of inflammatory bowel diseases and thus bring an effec-
tive cure as well as longer remission than the currently used first-line
salicylate class of drugs.
In this manuscript, we report the synthesis, physico-chemical char-
acterization, preclinical pharmacokinetics, and efficacy of CLX-103 in
an animal model of ulcerative colitis.
2. Materials and methods
2.1. Chemical synthesis
(5Z,8Z,11Z,14Z,17Z)-Ethyl icosa-5,8,11,14,17-pentaenoate (50 g,
0.151 mol) was dissolved in methanol (1000 mL) and added to NaOH
aqueous solution (60.5 g, 1.513 mol, 200 mL of H2O). Then the reaction
mixture was stirred for 2 h at room temperature. After completion of the
reaction, volatiles were evaporated under reduced pressure on a
rotavap. The obtained crude material was diluted with water
(200 mL) and acidified with 3 N HCI, and later extracted with ethyl ac-
etate (2 × 300 mL). The combined organic extracts were washed with
brine (2 × 100 mL), dried over anhydrous sodium sulfate, filtered, and
concentrated to dryness. Purification was carried out on silica gel (20%
EtOAc in hexane). To a solution of 5-amino-2-hydroxybenzoic acid
(5.06 g, 0.033 mol) in CH2Cl2 (20 mL) was added triethyl amine
(13.8 mL, 0.099 mol) and compound at 0 °C. The mixture was continued
to stir for 1 h at the same temperature. After completion of the reaction,
it was acidified with 2 N HCl; the organic layer was separated, dried over
anhydrous sodium sulfate, filtered, and concentrated to dryness. Purifi-
cation by recrystallization in hexane yielded the pre-final intermediate
(~5 g, 34.7% yield); mass (m/z): 438.5 (M + H). Then, the reaction mix-
ture temperature was raised to 50 °C and was kept for stirring over-
night. After completion of the reaction (monitored by TLC), it was
diluted with ethyl acetate and water. The organic layer was separated
and dried over anhydrous sodium sulfate, filtered, and concentrated to
dryness. Purification on silica gel (10% EtOAc in hexane) gave CLX-103
(CLX-SYN-G3-C03, 5.2 g, 75.3% yield with 98.18% purity by LC-MS) as
a brown liquid. The chemical structure of CLX-103 (Fig. 1) was con-
firmed by NMR.
1
H NMR (CDCl3): δ 10.4 (s, 1H), 8.00 (S, 1H), 7.50 (d, 1H), 7.18–7.05
(m, 2H), 6.88 (d, 1H), 5.40 (m, 10H), 2.82 (m, 8H), 2.38 (m, 4H), 2.20 (m,
2H), 2.10 (m, 2H), 1.82 (m, 2H), 1.40–1.20 (m, 12H), 1.00 (t, 3H), 0.9 (m,
6H); mass: 608.5 [M + H].
2.2. In vitro kinetic solubility in phosphate buffered saline (PBS)
In vitro solubility of CLX-103 was evaluated in phosphate buffered
saline (at pH 3.0, 6.0, 6.8, 7.4, and 9.0) and 0.1 N HCl (pH 1.2) by kinetic
method. Calibration standards (1, 5, 10, 50, 100, 200, and 300 μM) were
prepared in DMSO from a 20 mM primary DMSO stock solution of CLX-
103. The test compound (200 μM) was incubated in the respective
buffers for 90 min. The solubility of the test compound was determined
using HPLC-PDA and was calculated from the standard curve.
2.3. In vitro kinetic solubility in simulated gastric fluid (SGF) and simulated
intestinal fluid (SIF)
In vitro solubility of CLX-103 was evaluated in simulated gastric fluid
(pH: 1.2; with and without 0.5% sodium dodecyl sulfate (SDS)) and sim-
ulated intestinal fluid (pH: 6.8; with and without 0.5% SDS) by kinetic
method. Calibration standards (1, 5, 10, 50, 100, 200, and 300 μM)
were prepared in DMSO from a 20 mM primary stock solution (in
DMSO) of CLX-103. The study was conducted at 200 μM final concentra-
tion. The incubation was performed in duplicate for 90 min for the re-
spective fluids. The samples were filtered post incubation, the filtrates
were analyzed for CLX-103 using the HPLC-UV method, and the solubil-
ity was estimated from the standard curve.
2.4. In vitro stability in simulated gastric fluid (SGF) and simulated intesti-
nal fluid (SIF)
In vitro stability of CLX-103 was evaluated in simulated gastric fluid
(pH: 1.2) and simulated intestinal fluid (pH: 6.8). The final concentra-
tion of CLX-103 used in the study was 1 μM for simulated gastric fluid
and 5 μM for simulated intestinal fluid. The samples were incubated at
37 ± 1 °C in a water bath. The reactions were terminated at 0, 15, 30,
and 60 min in SGF media and at 0, 60, 120, and 180 min in SIF media
by addition of 5 vol% of acetonitrile containing internal standard. The su-
pernatant from the SGF and SIF samples was analyzed for CLX-103 using
LC-MS/MS.
2.5. In vitro stability in plasma
In vitro stability of CLX-103 was determined in mouse, rat, dog, and
human plasma. The stability of the test compound was assessed at a
concentration of 3 μM. The plasma samples were diluted with
Fig. 1. Chemical structure of CLX-103 (CLX-SYN-G3-C03).
 M. Kandula et al. / International Immunopharmacology 40 (2016) 443–451
phosphate buffered saline, pH 7.4 (1:1, v/v) and pre-incubated at 37 ±
1 °C for 1 min before the addition of the test item. The test compound
spiked plasma samples were incubated at 37 ± 1 °C in a water bath.
The samples were collected at 0, 5, 15, 30, 60, and 120 min post initia-
tion of the incubation, and the reaction was terminated by adding ace-
tonitrile containing phenacetin as the internal standard. The samples
were analyzed for CLX-103 using liquid chromatography–tandem
mass spectrometry (LC-MS/MS) method. The percentage of the test
item (CLX-103) remaining at each time point was calculated.
2.6. In vitro metabolic stability in liver microsomes
In vitro metabolic stability of CLX-103 was determined in mouse, rat,
dog, and human liver microsomes. The test compound (1 μM) was incu-
bated with the respective liver microsomes (at a protein concentration
of 0.5 mg/mL), at 37 ± 1 °C in a shaking water bath. The reaction was
initiated by the addition of NADPH (final NADPH concentration:
1 mM). The reaction was terminated at 0, 5, 15, and 30 min post reaction
initiation, using a quenching solution containing the internal standard
(phenacetin). The supernatants were analyzed for CLX-103 using a liq-
uid chromatography–tandem mass spectrometry (LC-MS/MS) method.
The percent disappearance of the test compound (CLX-103) at each
time point was calculated.
2.7. In vitro metabolic stability in rat intestinal tissue homogenates
In vitro stability of CLX-103 was determined using rat (Sprague
Dawley) small intestinal and colon tissue homogenates. The test com-
pound (5 μM) was incubated with the tissue homogenates (5% w/v) of
rat small intestine and colon, at 37 ± 1 °C in a shaking water bath. Ali-
quots were withdrawn at 0, 1, 2, and 4 h post incubation, and the reac-
tion was terminated, using a quenching solution containing the internal
standard (phenacetin). The supernatants were analyzed for CLX-103
and 5-ASA using a liquid chromatography–tandem mass spectrometry
(LC-MS/MS) method. The percent disappearance of the test compound
was calculated.
2.8. In vivo intestinal tissue distribution of 5-ASA after oral gavage admin-
istration of CLX-103 in C57BL/6 mice
Male C57BL/6 mice (aged 5–6 weeks; body weight range of 25–30 g)
were randomly assigned to three groups of six animals in each. The an-
imals in Groups 1 and 2 received a single dose of CLX-103 at 457 mg/kg
(equivalent to 115 mg/kg 5-ASA), or 150 mg/kg (equivalent to 38 mg/kg
5-ASA) by oral gavage. Animals in Group 3 were treated similarly with
the reference compound, sulfasalazine at 300 mg/kg (equivalent to
115 mg/kg 5-ASA). The test and reference compounds were formulated
as a suspension in Tween 80 (0.5%) and carboxymethyl cellulose (0.5%).
The dose volume used was 10 mL/kg. Intestinal tissue (jejunum, ileum,
and colon) and blood samples were collected from 3 mice/group at 3
and 6 h post dosing. The plasma and tissue homogenate samples were
prepared and analyzed for 5-ASA and N-acetyl-5-ASA using an LC-MS/
MS method.
2.9. Single-dose oral comparative pharmacokinetic study with CLX-103 and
mesalamine (5-ASA) in Sprague Dawley rats
Two groups of five male rats in each (age 8–10 weeks; body weight
range 250–300 g) were assigned to receive CLX-103 (Group 1) or 5-ASA
(Group 2). The suspension formulations of CLX-103 and 5-ASA were
prepared using Tween-80 (0.5%) and carboxymethyl cellulose (0.5%).
The formulations of CLX-103 or 5-ASA were administered to the rats
in the respective treatment groups after overnight fasting by oral ga-
vage. The blood samples from the animals were collected at 0.25, 0.5,
1, 2, 4, 8, 10, 12, and 24 h post dosing. The plasma samples were
445
analyzed for CLX-103, 5-ASA, and N-acetyl 5-ASA using a fit for purpose
LC-MS/MS method.
2.10. Seven-day oral probe toxicity study of CLX-103 in C57BL/6 mice
Male C57BL/6 mice (age 8–10 weeks; body weight range 23–25 g;
N = 3 mice/group) were assigned to normal control, CLX-103 (50,
150, and 500 mg/kg/day) groups. The suspension formulations of CLX-
103 were prepared using Tween-80 (0.5%) and carboxymethyl cellulose
(0.5%). Animals from respective treatment groups were administered
with either vehicle or test compound once daily for seven days. Mortal-
ity, clinical signs of toxicity (daily), body weight (twice weekly), fecal
consistency (daily), and water and food consumption (twice weekly)
were recorded. On Day 8, the animals were sacrificed and gross pathol-
ogy was carried out.
2.11. Evaluation of the efficacy of CLX-103 in DSS-induced acute colitis in
C57BL/6 mice
Male C57BL/6 mice (age 8–10 weeks; body weight range 25–30 g)
were assigned (N = 12 mice/group) to normal control, disease control,
CLX-103 (50, 150, and 450 mg/kg/day) or sulfasalazine (300 mg/kg/
day) groups. Colitis was induced in mice by providing 2.25% DSS in
drinking water for 5 days (Day 1 to Day 5). The suspension formulations
of CLX-103 and sulfasalazine were prepared using Tween-80 (0.5%) and
carboxymethyl cellulose (0.5%). Animals from respective treatment
groups were administered with either vehicle or test/reference com-
pound by oral gavage, once daily from Day 1 to Day 10. Mortality, clin-
ical signs, body weight, fecal consistency, hemoccult scoring (daily),
and water and feed consumption (twice weekly) were recorded. Dis-
ease activity index (DAI) was calculated to evaluate the severity of the
disease. On Day 11, animals were sacrificed, and colon length and
weights were recorded (the colon weight/length ratio was calculated).
Colon tissues were also collected for MPO activity and histopathological
analysis.
3. Results
3.1. In vitro kinetic solubility in phosphate buffered saline (PBS)
CLX-103 showed low solubility in 0.1 N HCl (pH 1.2) and at pH 3.0 in
phosphate buffered saline. CLX-103 showed moderate solubility across
the pH range of 6–9 in phosphate buffered saline under the tested con-
ditions (Table 1).
3.2. In vitro kinetic solubility in simulated gastric fluid (SGF) and simulated
intestinal fluid (SIF)
CLX-103 was poorly soluble in the simulated gastric fluid (pH: 1.2)
and simulated intestinal fluid (pH 6.8). The solubility in simulated gas-
tric fluid was significantly higher in the presence of 0.5% SDS. In simulat-
ed intestinal fluids, the addition of SDS (0.5%) resulted in a marginal
increase in solubility under the test conditions (Table 2).
Table 1
Solubility of CLX-103 in 0.1 N HCl and phosphate buffered saline (PBS).
Test compound Solubility in 0.1 N HCl Solubility in PBS (μM/μg per mL)
(μM/μg per mL)
pH 1.2 pH 3.0 pH 6.0 pH 6.8 pH 7.4 pH 9.0
CLX-103 13/7 2/1 25/15 34/21 17/10 35/21
446 M. Kandula et al. / International Immunopharmacology 40 (2016) 443–451

Table 2 Table 5
Solubility of CLX-103 in simulated gastric fluid and simulated intestinal fluid. In vitro metabolic stability of CLX-103 in liver microsomes.

Test compound Solubility (μM/μg per mL) Test compound Species % metabolized (CLX-103) after 30 min incubation

SGF SGF SIF SIF (pH 6.8) CLX-103 Mouse 34.8

(pH 1.2) (pH 1.2) with 0.5% SDS (pH 6.8) with 0.5% SDS Rat 45.8
Dog 41.9
CLX-103 b1/b1 71/42 13/8 20/12
Human b5

3.3. Stability in simulated gastric fluid (SGF) and simulated intestinal fluid
(SIF) with sulfasalazine, the tissue levels of 5-ASA showed a marked reduc-
tion with increase in time. CLX-103 showed less inter-animal variability
CLX-103 was stable in simulated gastric fluid (N95% remaining at the in the tissue levels of 5-ASA in relation to that observed with
end of 60 min incubation period) and was moderately stable in simulat- sulfasalazine (Tables 7 and 8 and Fig. 2).
ed intestinal fluid (54% remaining at the end of 180 min incubation pe-
riod) (Table 3).
3.8. Single dose oral comparative pharmacokinetic study of CLX-103 with
mesalamine (5-ASA) in Sprague Dawley rats
3.4. In vitro stability in plasma
CLX-103 was not detected in most of the plasma samples from the
CLX-103 was stable (percentage remaining at 120 min was N70) in
animals dosed with CLX-103. Systemic exposure (AUC0-t and Cmax) to
mouse, rat, and human plasma. CLX-103 was moderately stable in dog
5-ASA and its metabolite, N-acetyl-5-ASA were significantly lower
plasma (percentage remaining at 120 min was 59) (Table 4).
after the administration of CLX-103, when compared to that observed
in 5-ASA-dosed animals (Table 9 and Fig. 3).
3.5. In vitro metabolic stability in liver microsomes

CLX-103 was moderately stable (percentage metabolized was be-
tween 30 and 50) in mouse, rat, and dog liver microsomes. CLX-103
was stable (percentage metabolized was b5) in human liver micro-
somes (Table 5).
3.6. In vitro stability in rat intestinal tissue homogenates
CLX-103 was unstable in the rat small intestinal and colon tissue ho-
mogenates (b10% remaining at the end of 4 h incubation period). Ap-
pearance of 5-ASA from CLX-103 was approximately 1.1 μg/mL in both
rat small intestinal and colon tissue homogenate after 4 h of incubation
(Table 6).
3.9. Oral probe toxicity study (7-day) of CLX-103 in C57BL/6 mice
Once-daily administration of CLX-103 to male C57BL/6 mice at 50,
150, and 500 mg/kg/day for 7 days showed no clinical signs of toxicity
or mortality. There were no test item-related changes on body weight,
food consumption, water consumption, and fecal consistency scores
throughout the study period. Repeated oral (gavage) administration of
CLX-103 to male C57BL/6 mice was clinically well tolerated up to 500
mg/kg/day.
3.10. Evaluation of the efficacy of CLX-103 in DSS-induced acute colitis in
C57BL/6 mice

3.7. In vivo intestinal tissue distribution in C57BL/6 mice Once-daily oral administration of CLX-103 at 50, 150, and 450 mg/
kg/day showed statistically significant reduction in the disease activity
The systemic exposure (plasma) to 5-ASA and N-acetyl-5-ASA was index (DAI) (Fig. 4) and in colon weight/length ratio (p b 0.05 at
low in animals treated with CLX-103 in relation to sulfasalazine. The 50 mg/kg and p b 0.01 at 450 mg/kg) (Table 10 and Fig. 5). Additionally,
levels of 5-ASA in the intestinal tissues (jejunum, ileum, and colon) in the CLX-103 had shown lower mean MPO activity in colon tissue ho-
the mice treated with CLX-103 did not show any dose-related increase mogenates as compared to disease control (Fig. 6).
from 150 to 457 mg/kg. In mice treated with CLX-103, the intestinal Histologically, the disease control group showed moderate to
levels of 5-ASA were sustained from 3 to 6 h post dosing. In mice treated marked degree of colitis in the colon tissue. CLX-103 showed a dose-re-
lated reduction in the colitis in relation to the disease control group
(Figs. 7 and 8). Notably, mild degree of histological improvement in co-
litis was evident even at the lowest dose of 50 mg/kg/day, which is con-
Table 3
Stability of CLX-103 in simulated gastric and intestinal fluids.
sistent with the disease activity score. The histological improvement in
the colitis noted at the highest dose of 450 mg/kg/day of CLX-103 was
Media Time % test compound (CLX-103) remaining comparable to that noted in positive control group (sulfasalazine; 300
(min) at the end of incubation period
mg/kg/day). The effect was biologically significant even at the lowest
Simulated gastric fluid (SGF) 60 Approximately 95% dose of 50 mg/kg/day tested. The reference compound sulfasalazine at
Simulated intestinal fluid (SIF) 180 Approximately 54%
300 mg/kg/day demonstrated significant efficacy in this model, as
expected.

Table 6
Table 4 In vitro stability of CLX-103 in rat intestinal tissue homogenates.
In vitro plasma stability of CLX-103 in various species.
Test Time % test compound (CLX-103) % test compound (CLX-103)
Matrix % test compound (CLX-103) remaining at the end compound point remaining in small intestinal remaining in colon tissue
of incubation period (120 min) tissue homogenate homogenate

Mouse plasma 81.5 CLX-103 0h 100 100

Rat plasma 82.5 1h 46.8 11.8
Dog plasma 59.4 2h 24.4 6.01
Human plasma 77.2 4h 10.1 3.63
 M. Kandula et al. / International Immunopharmacology 40 (2016) 443–451 447

Table 7
Mean concentration of 5-ASA following oral administration of CLX-103 at 150 mg/kg (equivalent to 5-ASA dose of 38 mg/kg), CLX-103 at 457 mg/kg, and sulfasalazine at 300 mg/kg
(equivalent to 5-ASA dose of 115 mg/kg). All the values are expressed as mean ± SEM. NC: not calculated as the concentration in most of the samples were bLLOQ (Lower Limit of Quan-
tification (50 ng/mL)).

Test compound Plasma (ng/mL) Jejunum (ng/g) Ileum (ng/g) Colon (ng/g)
(Dose)
Time points (h) Time points (h) Time points (h) Time points (h)

3 6 3 6 3 6 3 6

CLX-103 NC NC 11,636 ± 525 7189 ± 1564 10,301 ± 2060 11,705 ± 4272 10,900 ± 1086 17,289 ± 4670
(150 mg/kg)
CLX-103 NC NC 8960 ± 3495 10,596 ± 368 10,817 ± 3642 12,805 ± 1214 12,171 ± 1477 10,702 ± 1445
(457 mg/kg)
Sulfasalazine 771 ± 199 624 ± 92 13,264 ± 967 10,955 ± 745 11,496 ± 841 16,397 ± 2974 71,680 ± 46,310 34,960 ± 13,400
(300 mg/kg)

Table 8
Mean concentration of N-acetyl-5-ASA following oral administration of CLX-103 at 150 mg/kg (equivalent to 5-ASA dose of 38 mg/kg), CLX-103 at 457 mg/kg, and sulfasalazine at 300 mg/
kg (equivalent to 5-ASA dose of 115 mg/kg). All the values are expressed as mean ± SEM.

Test compound Plasma (ng/mL) Jejunum (ng/g) Ileum (ng/g) Colon (ng/g)
(Dose)
Time points (h) Time points (h) Time points (h) Time points (h)

3 6 3 6 3 6 3 6

CLX-103 637 ± 302 247 ± 44 755 ± 590 152 ± 27 1873 ± 1046 445 ± 140 1025 ± 292 1229 ± 292
(150 mg/kg)
CLX-103 2997 ± 268 1064 ± 551 1251 ± 210 1336 ± 645 15,159 ± 1224 1528 ± 38 3271 ± 1788 3665 ± 205
(457 mg/kg)
Sulfasalazine 5555 ± 482 4293 ± 250 4302 ± 1321 1607 ± 592 3050 ± 1071 2322 ± 660 27,114 ± 13,247 14,452 ± 2593
(300 mg/kg)

4. Discussion Delivering effective therapeutic concentrations in the distal gut,

Ulcerative colitis (UC) is a chronic inflammatory condition of unclear
etiology affecting the large bowel, most commonly the rectum and ex-
tending proximally in a continuous fashion. Sulfasalazine and 5-
aminosalicylate (5-ASA) drugs are used as first-line therapy for the
treatment of mild to moderate ulcerative colitis. The mechanism of ac-
tion of 5-ASA is not fully understood, but generally it is proposed to
have an anti-inflammatory effect on the colonic epithelial cells (locally
acting). 5-ASA reduces the inflammation by blocking cyclooxygenase
and inhibits the prostaglandin production in the colon. Various other
mechanisms of action have been proposed for 5-ASA, including inhibi-
tion of the activity of the nuclear factor-kappa B (NF-κB) and conse-
quently the production of key pro-inflammatory cytokines. In
addition, there is also recent evidence that the 5-ASA mediates its
anti-inflammatory effects by acting through direct activation of peroxi-
some proliferator-activated receptor gamma (PPARγ) in the colon or
rectal epithelium [17].
which is the target site of the disease, for sufficient duration is an impor-
tant determinant for the efficacy of 5-ASA in the treatment of ulcerative
colitis. Oral administration of 5-ASA might result in rapid and nearly
complete absorption from the upper GI and its conversion to the inac-
tive metabolite N-acetyl-5-ASA, which prevents the delivery of thera-
peutically sufficient concentrations to act locally in the colonic
mucosa. Based on this key factor in the development of 5-ASA, pH-de-
pendent and controlled release formulations (Asacol HD®, Apriso®,
Pentasa®, Lialda®) or as various prodrugs were developed to minimize
the systemic absorption of 5-ASA from the small intestine and thus
maximize the delivery of the active 5-ASA to the colon, which is the
site of inflammation [17].
5-ASA is currently available in various formulations for the treat-
ment of ulcerative colitis. Olsalazine (Dipentum; Celltech Pharmaceuti-
cals, Inc., Rochester, NY, USA) and balsalazide (Salix Pharmaceuticals,
Inc., Morrisville, NC, USA) are prodrugs of 5-ASA. Two 5-ASA are cova-
lently linked through an azo linkage in olsalazine, and a 5-ASA molecule

5-ASA concentration in mice intestinal tissues

Concentration (ng/g) Mean±SE

(3 and 6 h post dosing: ng/g)

Fig. 2. Single oral dose administration of CLX-103 at 150 and 457 mg/kg/day in C57BL/6 mice showed sustained intestinal levels of 5-ASA from 3 to 6 h post dosing. In mice treated with
sulfasalazine, the tissue levels of 5-ASA showed a marked reduction with increase in time.
448 M. Kandula et al. / International Immunopharmacology 40 (2016) 443–451
Table 9
Summary of pharmacokinetic parameters of 5-ASA and N-acetyl-5-ASA following oral administration of 5-ASA and CLX-103 (equivalent to 5-ASA dose of 100 mg/kg). All the values are
expressed as mean ± SD.
Test compound Pharmacokinetic parameters for 5-ASA
(Dose)
Cmax Tmax
(μg/mL) (h)
5-ASA 56.3 ± 9.32 0.70 ± 0.27
(100 mg/kg)
CLX-103 6.39 ± 2.30 0.90 ± 0.22
(397 mg/kg equivalent to 100 mg/kg of 5-ASA)
is azo-bonded to a benzoic acid derivative in balsalazide. These drugs re-
lease 5-ASA by bacterial azo-reduction in the colon, and the therapeutic
effect is dependent only on its release of active drug in the colon [17,18].
Despite the large number of formulations available, there are about
30% of patients treated with 5-ASA who are considered to be non-re-
sponders, due to various factors such as allergy, intolerance,
incompliance, and refractoriness to the treatment, and are in need of
switching to more potent but toxic therapy [19]. Also, there are large
inter-individual variations in measured intraluminal concentrations of
5-ASA. The release profile and the concentrations of 5-ASA in the
colon lumen could be affected by the oro-caecal transit time, the pH of
gut lumen, which can vary between healthy and diseased individuals,
and also the severity of disease. Until now, there is no convincing scien-
tific evidence to show that there are significant differences between var-
ious delivery systems of 5-ASA in efficacy or safety [20,21]. The
systemically absorbed 5-ASA is also inactivated by acetylation in the
gut epithelium and liver. In addition, the topical formulations of 5-ASA
are considered to be less convenient, and it is largely used in the distal
ulcerative colitis [20]. Hence, the goal of 5-ASA therapy should be
attaining relatively more stable concentrations of 5-ASA in the distal
gut regions than the currently available 5-ASA formulations. In addition,
the drug that could be poorly absorbed will cause relatively few system-
ic adverse effects due to 5-ASA or its metabolites.
CLX-103 (5-ASA-eicosapentaenoic acid–caprylate conjugate) is a
novel molecule that utilizes prodrug technology for efficient release of
5-ASA at the target site by slow and sustained release of the active moi-
eties (ileum and colon). The presence of two ester bonds in the mole-
cule is expected to control the release profile of 5-ASA in the intestine
and to ensure the effective delivery of the active moieties at the target
site (ileum and colon), while minimizing the systemic absorption. The
eicosapentaenoic acid and caprylic acid were selected as the molecular
partners of 5-ASA, considering their proven therapeutic effects in vari-
ous inflammatory conditions and the availability of extensive human
safety data [10–16].
Fig. 3. Plasma concentration of mesalamine (5-ASA) in Sprague-Dawley rats following
single oral dose administration of CLX-103 and 5-ASA (dose equivalent to 100 mg/kg 5-
ASA).
Pharmacokinetic parameters for N-acetyl-5-ASA
AUC0-t Cmax Tmax AUC0-t
(μg h/mL) (μg/mL) (h) (μg h/mL)
81.2 ± 8.83 61.1 ± 10.5 1.10 ± 0.55 162 ± 17.5
10.4 ± 1.80 31.7 ± 10.2 1.20 ± 0.45 83.2 ± 12.1
In agreement with the hypothesis, CLX-103 was stable in the simu-
lated gastric fluid and was moderately stable in simulated intestinal
fluid (54% remaining at the end of the 180 min incubation period). In
vitro assessment with the rat small intestine and colon tissue homoge-
nate study suggested that CLX-103 was unstable (b 10% remaining at
the end of the 4 h incubation period). The stable conjugate in the simu-
lated gastric fluid is an ideal characteristic for the delivery of the actives
to lower intestinal regions, and the intact conjugate is more likely to be
delivered in the intestinal tissues and allows the release of the actives
for a longer period of time. Hence, the active drugs are less likely to
get absorbed into the systemic circulation. This was further supported
in the intestinal tissue distribution study in mice and pharmacokinetic
study in rats.
The tissue concentrations of 5-ASA observed in C57BL/6 mice (after
single oral dosing) revealed that the levels were sustained at 3 and 6 h in
small intestine and colon tissues in the CLX-103-dosed group as com-
pared to the sulfasalazine group. There was also less variability in the in-
dividual animal concentrations as compared to sulfasalazine. Also,
sulfasalazine-dosed animals showed time-dependent reduction in the
5-ASA concentration in colon tissue. The results of these studies sug-
gested that there was improved local retention of the intact conjugate
and hydrolysis to an active moiety for a prolonged period of time. In ad-
dition, there was no systemic concentration of CLX-103 and low sys-
temic exposure to 5-ASA as compared to sulfasalazine.
The in vivo data were consistent with the drug release profile
observed in the in vitro studies. In a comparative pharmacokinetic study
with 5-ASA in rats, the systemic exposure to 5-ASA and its metabolite
(N-acetyl-5-ASA) were significantly lower with CLX-103 as compared to
that observed with an equivalent dose of 5-ASA administration. Also,
there was no detectable level of CLX-103 in plasma. These data suggest
the potential retention of the active components in the intestine when
dosed with CLX-103, which is an essential requirement for an IBD drug.
Acute dextran sodium sulfate (DSS)-induced colitis in mice is the
most frequently used preclinical model for testing efficacy of drugs for
Fig. 4. Once-daily oral administration of CLX-103 at 50, 150, and 450 mg/kg/day showed
statistically significant reduction in the disease activity index (DAI) in DSS-induced
acute colitis model in C57BL/6 mice. Data shown as mean ± SEM, *p b 0.05, **p b 0.01,
***p b 0.001, and ****p b 0.0001 vs. disease control, Two-way ANOVA followed by
Bonferroni post-test.
 M. Kandula et al. / International Immunopharmacology 40 (2016) 443–451
Table 10
Summary of colon length (cm), colon weight (mg), and colon weight: length ratio follow-
ing oral administration CLX-103 (at 50, 150, and 450 mg/kg/day) in DSS-induced acute co-
litis in C57BL/6 mice. All the values are expressed as mean ± SEM.
Treatment group Colon length Colon weight Colon weight:length
(Dose) (cm) (mg) ratio
Normal control 8.7 ± 0.1 236.7 ± 3.6 27.2 ± 0.5
Disease control 6.5 ± 0.1 288.0 ± 13.6 44.3 ± 1.5
CLX-103 7.4 ± 0.2 288.0 ± 9.6 39.1 ± 1.6
(50 mg/kg/day)
CLX-103 7.0 ± 0.2 284.8 ± 9.2 40.7 ± 1.4
(150 mg/kg/day)
CLX-103 7.8 ± 0.3 291.7 ± 6.5 38.0 ± 1.6
(450 mg/kg/day)
Sulfasalazine 7.7 ± 0.3 297.6 ± 9.2 39.1 ± 1.3
(300 mg/kg/day)
inflammatory bowel disease. DSS-induced colitis in C57BL/6 mice is a
relevant and validated model for the extrapolation of mice efficacy
data to human and also to identify and validate new therapies for treat-
ment of IBD [22]. CLX-103 showed a dose-related reduction in the colitis
in relation to the disease control group in C57BL/6 mice acute colitis
model. CLX-103 showed reduction in the DAI (disease activity index;
statistically significant at all dose levels), in myeloperoxidase activity
(a marker for neutrophil infiltration and inflammation) in the colon tis-
sue, in colon weight:length ratio (p b 0.05 at 50 mg/kg and p b 0.01 at
450 mg/kg), and in histological score (at all the dose levels tested). In
the previously reported studies using 5-ASA compounds, the recom-
mended dose for the activity of 5-ASA in DSS-induced acute colitis
model in C57BL/6 mice varied between 50 and 200 mg/kg/day [23,24].
In BALB/c mice, 5-ASA had shown only weakly activity even at
100 mg/kg [25]. Literature evidence also suggests that sulfasalazine
was used as the positive control in DSS-induced acute colitis at the
dose of 150 mg/kg/day [26]. Hence, the efficacy of CLX-103 is considered
to be biologically significant even at the lowest dose of 50 mg/kg/day
tested (dose equivalent: 13 mg/kg/day of 5-ASA) as compared to the
ED50 established for the reference compound sulfasalazine in the test
laboratory, that is, 300 mg/kg (dose equivalent: 115 mg/kg/day of 5-
ASA), and the reported efficacious doses of 5-ASA in mice colitis models.
The possible reasons for the improvement in the disease activity
index and histological scores at the low dose evaluated in a mouse
model of acute colitis were the sustained concentrations of 5-ASA as
demonstrated in the colon tissues of mice. The tissue concentrations
of 5-ASA observed in C57BL/6 mice (after single oral dosing) revealed
sustained levels of 5-ASA at 3 and 6 h in the small intestine and colon
tissues in the CLX-103 group as compared to the sulfasalazine group,
suggesting the local retention of the drug for a prolonged period of
time. In addition, eicosapentaenoic acid and caprylic acid released
from the conjugate could contribute to the improved or synergistic
pharmacology of the conjugate as compared to 5-ASA drugs. Adjunct
Fig. 5. Once-daily oral administration of CLX-103 showed statistically significant reduction
in colon weight:length ratio at 50 and 450 mg/kg/day in DSS-induced acute colitis model
in C57BL/6 mice. Data shown as mean ± SEM, *p b 0.05 and **p b 0.01 vs. disease control,
One-way ANOVA followed by Dunnett's post-test.
449
Fig. 6. Once-daily oral administration of CLX-103 showed reduction in MPO activity in
DSS-induced acute colitis model in C57BL/6 mice. Data shown as mean ± SEM, one-way
ANOVA followed by Dunnett's post-test.
therapy of n-3 fatty acids to 5-ASA has been shown to ameliorate in-
flammatory score and decreases NF-κB in rats with TNBS-induced colitis
[27]. It is also reported that omega-3 polyunsaturated fatty acids had
anti-inflammatory properties in IBD experimental models by reducing
PGE2, and LTB4, as this acts as a competitive substrate for the arachidon-
ic acid metabolism [28]. Studies had shown that there is a high level of
Candida species colonization in the inflammatory diseases of the gastro-
intestinal tract, which delays the healing of inflammatory lesions, and
again inflammation promotes colonization with elevated levels of the
pro-inflammatory cytokines [29]. Caprylic acid and medium chain tri-
glycerides were reported to suppress IL-8 secretion by Caco-2 cells at
the transcriptional level when pre-cultured together for 24 h [15]. In ad-
dition, caprylic acid has been shown to possess antibacterial and anti-
fungal properties as demonstrated against various bacterial and
Candida species in an in vitro study [30].
The efficacy observed at a low dose tested in this animal model has
also suggested that the CLX-103 could improve the treatment response
in patients (induction and prevention of relapse) with ulcerative colitis
at the much lower dose level of 5-ASA and also offer a potentially new
therapeutic opportunity for the treatment of inflammatory bowel dis-
eases. The efficacy observed at the low dose tested (50 mg/kg/day of
CLX-103 is equivalent to approximately 13 mg/kg/day of 5-ASA as com-
pared to 300 mg/kg/day of sulfasalazine equivalent to 115 mg/kg/day 5-
ASA) was correlated to the improved local retention of the active drug
with reduced systemic exposure, as demonstrated in intestinal tissue
distribution and pharmacokinetic studies. In humans, this unique pro-
file could possibly result in improved efficacy and a reduction in 5-
ASA dosage and the number of adverse events that are commonly re-
ported with this class of drugs.
5.0
Mean score of colitis grading (0 to 5)
(100%)
4.0
Mean score of colitis grading
(71%)
3.0 (58%)
(42%)
2.0 (32%)
1.0
(0%)
0.0
Normal Disease CLX-103 CLX-103 CLX-103 Sulfasalazine
control control 50 mg/kg 150 mg/kg 450 mg/kg 300 mg/kg
Fig. 7. Once-daily oral administration of CLX-103 showed notable reduction in histological
score in the 2.25% DSS-induced acute colitis in mice, at all the dose levels tested. Data
shown as mean ± SD. The values in parenthesis above each bar represents % colitis
score in each group. The effect was biologically significant even at the lowest dose of 50
mg/kg/day tested in the DSS-induced acute colitis model in C57BL/6 mice.
450 M. Kandula et al. / International Immunopharmacology 40 (2016) 443–451

A:Normal control B:Disease control

C: CLX-103(50 mg/kg/day) D: CLX-103 (150 mg/kg/day)

E:CLX-103 (450 mg/kg/day) F: Sulfasalazine (300 mg/kg/day)

Fig. 8. The effect of CLX-103 on colon tissue histology in 2.25% DSS-induced acute colitis in C57BL/6 mice. (A) Normal control with normal architecture, and intact mucosa and glands. (B)
Disease control showing moderate to marked degree of colitis. (C) CLX-103 (50 mg/kg/day): mild inflammation with occasional extension into the submucosa, focal erosions, minimal to
mild mucosal hyperplasia, and minimal to moderate mucin depletion. (D) CLX-103 (150 mg/kg/day): mild inflammation with occasional extension into the submucosa, focal erosions,
minimal to mild mucosal hyperplasia. (E) CLX-103 (450 mg/kg/day): minimal to mild inflammatory infiltrates with or without mild mucosal hyperplasia, reduction or absence of
ulceration. (F) Sulfasalazine (300 mg/kg/day): minimal to mild inflammatory infiltrates with or without mild mucosal hyperplasia, reduction or absence of ulceration (H&Ex4).

5. Conclusions offer potentially a new drug for the treatment of inflammatory bowel
diseases.
The data from various in vitro and in vivo studies showed that CLX-
103, a novel molecular conjugate of 5-ASA with eicosapentaenoic acid
and caprylic acid, is stable in simulated gastric fluid and releases the ac- Conflict of interest
tive 5-ASA in the intestinal tissues, systemic exposure to the conjugate
and 5-ASA is low, and the local retention of 5-ASA in the intestinal tis- The authors declare that they have no conflict of interest.
sues (small intestine and colon) is sustained over a long period of
time. CLX-103 improves the DAI (disease activity index), and reduces
myeloperoxidase activity and histological scores in the colon tissues at Acknowledgements
much lower dose as compared to sulfasalazine in the acute colitis
model in mice. These findings indicate that CLX-103 could be more effi- This research did not receive any specific grant from funding agen-
cacious and safer than the currently approved 5-ASA formulations and cies in the public, commercial, or not-for-profit sectors.
 M. Kandula et al. / International Immunopharmacology 40 (2016) 443–451
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