Accepted Manuscript

Cytoprotection of pancreatic β-Cells and hypoglycemic effect of 2-hydroxypropyl-β-

cyclodextrin: Sertraline complex in alloxan-induced diabetic rats

Vyacheslav Buko, Ilya Zavodnik, Oxana Lukivskaya, Elena Naruta, Bartlomiej Palecz,
Silwia Belica-Pacha, Elena Belonovskaya, Robert Kranc, Vladimir Abakumov

PII: S0009-2797(15)30116-2
DOI: 10.1016/j.cbi.2015.11.014
Reference: CBI 7522

To appear in: Chemico-Biological Interactions

Received Date: 16 July 2015

Revised Date: 30 October 2015
Accepted Date: 10 November 2015

Please cite this article as: V. Buko, I. Zavodnik, O. Lukivskaya, E. Naruta, B. Palecz, S. Belica-Pacha, E.
Belonovskaya, R. Kranc, V. Abakumov, Cytoprotection of pancreatic β-Cells and hypoglycemic effect of
2-hydroxypropyl-β-cyclodextrin: Sertraline complex in alloxan-induced diabetic rats, Chemico-Biological
Interactions (2015), doi: 10.1016/j.cbi.2015.11.014.

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 ACCEPTED MANUSCRIPT

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CYTOPROTECTION OF PANCREATIC β-CELLS AND

HYPOGLYCEMIC EFFECT OF 2-HYDROXYPROPYL-β-
CYCLODEXTRIN: SERTRALINE COMPLEX IN ALLOXAN-INDUCED
DIABETIC RATS

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Vyacheslav Bukoa,b*, Ilya Zavodnika,c, Oxana Lukivskayaa, Elena Narutaa,
Bartlomiej Paleczd, Silwia Belica-Pachad, Elena Belonovskayaa, Robert Krancb,

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Vladimir Abakumove

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a
Division of Biochemical Pharmacology, Institute of Biochemistry of Biologically
Active Compounds, National Academy of Sciences, BLK-50, 230030 Grodno,

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Belarus;
b
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Department of Biotechnology, School of Medical Sciences, Krakowska 9, 15-875
Bialystok, Poland;
c
Department of Biochemistry, Yanka Kupala Grodno State University, BLK-50,
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230030 Grodno, Belarus;
d
Department of Physical Chemistry, Faculty of Chemistry, University of Lodz,
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Pomorska 165, 90-236 Lodz, Poland;

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e
Department of Pathological Anatomy, Grodno State Medical University, Gorkogo
str., 80, 20009 Grodno, Belarus.
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*Corresponding author: Prof. V. Buko, Institute of Biochemistry of Biologically

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Active Compounds, National Academy of Sciences, BLK-50, 230030 Grodno,

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Belarus, buko@bioch.basnet.by; phone: +375152438411; fax: +375152434121

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Abstract

Sertraline, a selective inhibitor of serotonin reuptake, is widely used as

antidepressant in diabetic patients for improvement of depression and glycemic

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control. Sertraline is poorly soluble in water, which limits its oral applicability. In
this work we tried to improve the pharmaceutical properties of sertraline by

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complexation with 2-hydroxypropyl-β-cyclodextrin (HPβCD) and evaluated the
efficacy of the HPβCD: sertraline complex in prevention of alloxan-induced

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lesions in rats. The solubility of sertraline increased in the presence of HPβCD and
the association constant for sertraline and HPβCD was equal to 4000 ± 1000 M-1.

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Two-week treatment of diabetic animals with the HPβCD:sertraline complex

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improved pancreatic islet morphology and β-cell survival, which, in turn, reduced
the severity of diabetes, as evidenced by lowering of blood glucose and glycated
hemoglobin contents as well as normalization of serum insulin level and insulin
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sensitivity (HOMA-IR). The effect of the HPβCD:sertraline complex was strongly
expressed in comparison with the antidiabetic effect of both the monopreparations,
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HPβCD and sertraline. It is suggested that the cyclodextrin derivative increased the
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pharmacological effect of sertraline, probably due to enhanced drug bioavailability.

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Keywords: alloxan; diabetes; sertraline; 2-hydroxypropyl-β-cyclodextrin;

pancreatic islets; insulin.
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1. Introduction

Diabetes mellitus is a highly prevalent chronic disease affecting approximately 9 to

10% of the global adult population [1]. Depression is common in diabetes and it is
associated with hyperglycemia, diabetes-related complications and mortality [2].

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Sertraline (SER), (1S,4S)-4-(3,4-dichlorophenyl)-N-methyl-1,2,3,4-
tetrahydronaphthalen-1-amine, is widely used as antidepressant in patients with

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poorly controlled hyperglycemia for improvement of depression and glycemic
control [3]. This compound is a strong and selective inhibitor of serotonin reuptake

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[4]. Reports are currently available on serotonin effects on insulin secretion and
blood glucose concentration [5]. There is evidence for increased blood plasma

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serotonin level in diabetic patients and serotonin participation in translocation of

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insulin receptor substrate IRS1 in the cell cytoplasm and its subsequent
degradation [6]. These findings suggest possible involvement of the serotoninergic
system in the pathogenesis of diabetes. Search is presently carried out for novel
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antidiabetic drugs, and modulators of serotoninergic system can be quite promising
in this connection. Among antidepressants, applied in depressive diabetic patients,
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the selective serotonin reuptake inhibitor SER is a medicine of choice.

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The efficacy of diabetes-specific therapy using SER in adults with depression and
diabetes has been widely discussed. It was shown recently that 12-week SER
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treatment of depressive patients without diabetes significantly raised insulin levels

in comparison with the pretreatment values [7]. Likewise, triglyceride
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concentrations were significantly increased compared with the pretreatment values.

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The SER administration during 28 days reduced the serum glucose level in diabetic
rats but did not influence glycosylated hemoglobin level [8].
SER is poorly soluble in water, which limits its oral applicability in patients [4].
β-Cyclodextrin (β-CD)- and SER-based systems were studied to improve the drug
solubility, bioavailability and specificity of treatment. Cyclodextrins are cyclic
carbohydrates composed of 6, 7 or 8glucopyranose units (α-, β- and γ-cyclodextrin,
respectively) linked by (1 to 4) glycosidic bonds. The cyclodextrin molecule has a
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torus shape with a cavity whose size of α-, β- and γ-cyclodextrin amounts to 500,
620 and 800 pm, respectively [9]. The inclusion of the SER molecule in the host
cavity of β-CD considerably enhanced the drug solubility. The bio-accessibility of
the complex of β-CD and SER was considerably higher in relation to SER alone
[10]. It was evidenced that there is a multi-equilibrium in aqueous solutions of

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SER and β-CD. The interaction process with a high affinity binding constant was
spontaneous and exothermic [11]. Additionally, the stoichiometry coefficient (n)

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was 1.31 [12]. Multiple SER sites were verified to be included into the host cavity.
Recently we determined thermodynamic and spectroscopic parameters

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characterizing the interactions of SER with β-CD and its derivative, heptakis (2,6-
di-O-methyl)-β-CD, in water [13]. The values of the binding constants obtained for

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the binding of SER by β-CD (K = 5850±500 M-1) and heptakis (2,6-di-O-methyl)-
β-CD (K = 7960±500 M-1) indicate high affinity of the cyclodextrin for the drug
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molecules included into hydrophobic interior [13]. Experiments in vivo showed
that the host-guest ratio in the inclusion complexes was able to modify SER
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activity. Higher SER plasma concentration was determined after 30 min of mice
treatment with 1:1 inclusion of SER - β-CD complex in comparison with free SER,
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demonstrating greater drug transport efficacy of the inclusion complex [10]. Thus,
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the above results indicate that complexation of SER with β-CD and its derivatives
significantly enhanced the solubility and bioavailability of the drug.
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In this work we made an attempt to improve the pharmaceutical properties of SER

by complexation with 2-hydroxypropyl-β-cyclodextrin (HPβCD) and evaluated the
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efficacy of the complex in prevention of alloxan-induced lesions in rats. We used

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HPβCD instead of β-CD due to its higher water solubility in comparison with β-
CD [14] and lower toxicity [15]. The pharmacological effect of the HPβCD:SER
complex in experimental diabetes had never been studied before and therefore the
aim of the present work was to compare the hypoglycemic and β-cell protective
effects of SER and the HPβCD:SER complex during alloxan-induced experimental
diabetes in rats.

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2. Materials and methods

2.1. Chemicals.

Alloxan, sertraline hydrochloride and trichloroacetic acid were from Sigma-

Aldrich (St. Louis, MO, USA). HPβCD was obtained from CycloLab Ltd

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(Budapest, Hungary). Hydroxypropyl methylcellulose (hypromellose) was
produced by Shin-Etsu Chemical Co (Tokyo, Japan). All other chemicals were of
reagent grade, purchased from Sigma (USA) and from ICN (USA). All the water

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solutions were made with water purified in the Milli-Q system.

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2.2. Animal model

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Female albino Wistar rats weighing 230-250 g were used. A standard balanced diet
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and tap water were provided ad libitum. Four groups of animals were treated with a
single injection of alloxan (170 mg kg-1 b.w., intraperitoneally), whereas the
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control animals received a single intraperitoneal injection of the same volume of
saline. Each group consisted of eight animals.
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Only rats having blood glucose concentrations over 12.0 mmol/l after the alloxan
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treatment were used in further studies. One of the diabetic groups received HPβCD
(300 mg/kg b.w.), the second group – SER (30 mg/kg b.w.) and the third group
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received the complex of HPβCD (300 mg/kg b.w.) and SER (30 mg/kg b.w.). The
test substances were suspended in 0.8% aqueous hypromellose gel to the
appropriate concentrations. All the compounds were administered by intragastric
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intubation between 10 and 11 a.m. every day starting two weeks after alloxan
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administration throughout the last two weeks of the experimental period. The
control and the diabetic groups untreated with the test compounds received an
equivalent amount of hypromellose intragastrically during the last two weeks of
the experiment.
Rats were killed via terminal bleeding under pentobarbital anaesthesia (40 mg/kg,
intraperitoneally) on the next day after the last treatment. Blood samples were
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taken immediately and centrifuged at 3000 rpm. Serum was separated for further
measurements. The pancreas was removed from each animal and placed in a 4%
formaldehyde solution for histological investigations.
The care, use, and procedures performed on these rats were approved by the Ethic
Committee of the National Academy of Sciences, Belarus, and complied with the

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European Convention for the Protection of Vertebrate Animals Used for
Experimental and Other Scientific Purposes and NIH guidelines.

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2.3. Preparation of the HPβCD:SER complex

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The HPβCD:SER complex was prepared by mixing SER and HPβCD aqueous

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solutions as described earlier [13, 14, 16]. The water solution of HPβCD (0.27 M,

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pH 6.8) was mixed with SER at a SER:HPβCD molar ratio 1:2 with a mechanical
stirrer for 4 h at the temperature 25 ᵒC. The complex formation was monitored by
the methods of isothermal titration calorimetry (VP-ITC MicroCal calorimeter,
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USA) [13] and UV–Vis spectrophotometry (Specord 50 spectrophotometer,
Analytic Jena, Germany). The stability of the complex HPβCD:SER was evaluated
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by using an association constant. The freshly prepared solution was then filtrated
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and used for the animal treatments.

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2.4. Histological and morphometric studies

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The pancreatic tissue samples were fixed in 4% formaldehyde, dehydrated in a

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graded series of ethanol concentrations and embedded in paraffin. Five-micrometer

sections were cut and stained with haemotoxilin and eosin. Staining with aldehyde
and fuchsin was used for selective detection of pancreatic β-cells.
Tissue sections were imaged with an Olympus CX-41 light microscope using a 40
objective and the digital images were captured with an OlympusС-5060 camera
(Olympus, Japan). Analyses were performed with the Image J morphometric
analysis software (NIH, USA).
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The point counting method was used for measurements of islet size and islet cell
[17] counting. The formula of Fullman was applied to calculate the mean islet
diameter, mean area and the mean islet volume [18].

2.5. UV–Vis spectrophotometry - solubility studies

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Solubility studies were performed by adding the same amount of solid sertraline

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hydrochloride to 2 ml of aqueous HPβCD solutions of varying concentrations
(from 0 to 0.85 mM). The samples were mixed in an ultrasonic bath for 30 minutes

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and kept at 25oC for 1 week in order to achieve solubility equilibrium. The
solutions were then filtrated and after dilution the absorption spectra of sertraline

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hydrochloride were measured with the use of a Specord 50 one-beam

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spectrophotometer (Analytic Jena).The concentrations of the drug were calculated
with a previously established calibration curve [13].
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2.6. Biochemical parameters
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Glucose concentration in the fasting blood obtained from the tail vein in dynamics
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of diabetes was measured with an AccuCheck glucometer (Hoffmann-La Roche,

Germany). The blood glucose level was measured twice per week. At the end of
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the experiment, fasting blood glucose was measured enzymatically using o-tolidine
as the final oxygen acceptor. The glycosylated hemoglobin content in blood was
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measured by using a commercial kit from Pliva-Lachema (Czech Republik). The

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serum level of fasting insulin was measured by using an ELISA commercial kit
from LDN GmbH & Co. KG (Nordhorn, Germany) according to the instruction of
the manufacturer. The degree of insulin resistance was estimated according to the
method described by Matthews et al. [19]. Homeostasis model assessment - insulin
resistance (HOMA-IR) score was calculated with the formula: fasting plasma
glucose (mmol/l) times fasting serum insulin (pmol/l) divided by 22.5; higher
HOMA-IR values indicate low insulin sensitivity (insulin resistance).
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2.7. Statistics

Values are expressed as means ± standard error of the means (M ± SEM). Data
were analyzed statistically by two-way analysis of variance (ANOVA) (GraphPad

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software v5.0; Prism). The level of significance was determined when P<0.05.

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3. Results

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3.1. Complex formation between SER and HPβCD

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Figure 1A shows the absorption spectra of SER for the formation of a complex

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between sertraline and HPβCD. For the maximum at wavelength of 282.0 nm the
changes in absorbance of SER solutions with increasing HPβCD concentrations
were determined in order to draw a phase solubility diagram (Fig 1B). The aqueous
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solubility of SER increased linearly with the slope 0.939 and the intercept
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0.004207 as a function of HPβCD concentration. This phase solubility diagram

(Fig. 1B), according to the Higuchi and Connors concept [20, 21], could be
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classified as an AL type diagram with the assumption that the increase in solubility
observed is due to the formation of a 1:1 inclusion complex. In consequence, the
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solubility of the SER increases with addition of HPβCD (at the HPβCD
concentration of 0.85 mM the SRT solubility increased by ca. 33%). The
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quantitative analysis of types AL diagram allows estimation of the association

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constant from equation (1), where So is the drug solubility in the absence of
HPβCD and K1:1 is the association constant:

K1:1 = Slope/S0 (1 – Slope) (1)

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From the linear plot of the phase solubility diagram (Fig. 1B) and according to
equation (1) the value of the association constant for SER and HPβCD is equal to
4000 ± 1000 M-1.The value confirms rather high affinity of the SER molecule to
the HPβCD cavity.

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3.2. Morphology of pancreatic islets

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The histological evaluation revealed no pathological abnormalities in the
pancreatic islets from control rats. These islets were round or oval with well-

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defined boundaries (Fig. 2A). However, the pancreatic islets from the untreated
diabetic animals showed substantial changes in islet morphology, including

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degenerative alterations and disrupted islet architecture (Fig. 2B). The round islet
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boundary was destroyed and islet shrinkage, frequently with finger-like
protuberances into the surrounding exocrine tissue, was observed in this group.
Islet β-cells of diabetic rats showed irregular outlines with marked vacuolation,
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degranulation, pycnotic cell nuclei. The treatment of diabetic rats with HPβCD did
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not practically affect morphological changes of the islets, whereas the

administration of SER partially restored β-cells granulation (Fig. 2C, D). The
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beneficial effect of the HPβCD:SER combination on the islet morphology was

more pronounced and partially normalized degenerative changes, as well as
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increased β-cells granulation (Fig. 2E). Vacuolation was reduced or absent in many
islets.
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The morphometric studies revealed that the volume of Langerhans islets was
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significantly lower in both the diabetic group and all the treated groups as
compared to the control animals. The administration of the tested compounds alone
did not change this parameter in alloxan-treated rats. However, the volume of
pancreatic islets was higher with statistical significance in animals treated with the
combination of HPβCD and SER in comparison with the group treated with SER
alone. Similar changes were observed in other parameters characterizing the size of
pancreatic islets, as well as the area and diameter of these islets (Table 1). The
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number of β-cells per 1000 µm2 of the pancreatic square was markedly lower in
alloxan-treated animals in comparison with the control rats. The treatment with the
tested compounds elevated the amount of pancreatic β-cells in the order :
HPβCD<SER< HPβCD:SER complex where only the effect of the HPβCD:SER
complex was statistically significant. A similar result was shown when the number

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of β-cells was calculated per each islet (Table 1).

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3.3. Biochemical findings

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After 1 and 2 weeks, the blood glucose levels were significantly higher in all the
groups treated with alloxan (Fig. 3). However, after 3 and 4 weeks (the 1st and the

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2nd weeks after treatment with the test substances), the rats receiving the substances

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showed a hypoglycemic effect in the order: HPβCD<SER< HPβCD:SER complex,
whereas the blood glucose concentration remained high, of about 20 mmol/l, in
non-treated diabetic rats. The hypoglycemic efficacy of the HPβCD:SER complex
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was confirmed by the final fasting glucose concentration which was significantly
lower in the blood of rats treated with the HPβCD:SER complex as compared to
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the HPβCD- and SER-administered groups (Fig. 4A). Glycated hemoglobin was
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dramatically raised in diabetic rats and lowered after treatments with SER and the
HPβCD:SER complex, whereas the complex decreased this parameter significantly
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as opposed to the SER-treated group (Fig. 4B). The serum insulin concentration
was 1.5- fold decreased in alloxan-induced diabetic rats compared to healthy non-
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diabetic animals (Fig. 4C). All the tested substances elevated this parameter up to
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the control value where the effects of SER and the HPβCD:SER complex were
statistically significant in comparison with the diabetic group. On the other hand,
there was a significant increase in HOMA-IR in the diabetic group compared with
the control group, and the treatment with HPCD did not change this parameter,
whereas the treatment with SER and the HPβCD:SER complex prevented the
development of insulin resistance in diabetic rats as was evaluated by the HOMA-
IR parameter (Fig 4D).
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4. Discussion

Numerous clinical findings indicate that treatment of depression in diabetic

patients considerably improves psychological outcomes and glycemic control [3, 7,
22]. Animal experiments confirm hypoglycemic efficiency of sertraline which

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neutralized the increase in glycemia after glucose overload both in diabetic and
non-diabetic rats [23].

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In the present study, we compared the hypoglycemic and pancreas-protective
effects of the HPβCD:SER complex with its compounds, monopreparations of both

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HPβCD and SER, in rats with alloxan-induced diabetes. It was found that the
alloxan administration significantly increased the concentrations of blood glucose

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and glycosylated hemoglobin and decreased the insulin level in the serum of

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alloxan-treated rats. The histological findings in these rats were also typical for
diabetes and characterized by degenerative changes of pancreatic islets and
destruction and, consequently, reduction of the number of insulin-producing β-
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cells.
Hyperglycemia is the main manifestation of diabetes. It demonstrates not only a
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high level of blood glucose but an elevated amount of non-enzymatic glycation

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products. In our experiment, alloxan-treated animals showed a significant increase

in blood glucose and glycated hemoglobin contents. Simultaneously, the serum
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insulin concentration was significantly decreased. These data, combined with the
results of histological studies clearly suggest development of insulin-dependent
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diabetes in rats administered with alloxan.

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We found that 2-week treatment with the tested compounds reduced the severity of
diabetes as evidenced by lowering of blood glucose and glycated hemoglobin
contents and improvement of pancreatic islet morphology and β-cell survival. If
the impact of HPβCD on the above parameters was negligible, the effect of the
HPβCD:SER complex was strongly pronounced and larger than the antidiabetic
effect of both the monopreparations, HPβCD and SER, with statistical
significance. The level of serum insulin was significantly increased in the groups
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treated with both SER and the HPβCD:SER complex, whereas the elevation of this
parameter in rats treated with HPβCD alone only tended to statistical significance.
Alloxan administration induces diabetes by destruction of pancreatic β-cells with
changes in metabolic parameters and the toxic diabetogenic action of alloxan is the
sum of several processes, oxidation of SH groups, inhibition of glucokinase,

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generation of free radicals and disturbances in intracellular calcium homeostasis
[24]. Consequently, destruction of pancreatic β-cells responsible for production

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and release of insulin leads to hypoinsulinemia and hyperglycemia. Our study
revealed significant pathological changes in pancreatic islets and β-cells of diabetic

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rats as compared to control animals. The administration of SER attenuated and that
of its complex with HPβCD almost completely prevented destruction of insulin-

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secreting cells in diabetic rats, which caused increase of insulin level and, in turn,

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reduction of blood glucose concentration. Moreover, after treatment with the
HPβCD:SER complex the pancreatic islet volume was ameliorated compared to
the SER group.
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Experiments in vitro showed that SER inhibited insulin secretion by Min6 β cell
culture and pancreatic islets, but did not affect glucose-independent secretion,
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induced by KCl and arginine [25]. The treatment of non-diabetic rats with a single
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injection of SER caused an elevation of plasma insulin concentration in animals

overloaded with glucose but did not change this parameter in rats not submitted to
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glucose overload [26]. Depressive patients in the absence of diabetes treated with
sertraline over prolonged periods exhibited permanently raised blood serum insulin
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levels by 2-fold as early as on the 4th week of the treatment, and this level was
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maintained till completion of the study (12 weeks) [7].

Most researchers indicate a pronounced hypoglycemic effect of sertraline in
diabetic patients and experimental animals. The improvement of glycemic control
in depressive patients with diabetes both type 1 and type 2 was described in a
number of clinical [3, 27] as well as in experimental observations in animals with
diabetes type 1 [8, 23], where sertraline reduced the levels of fasting glucose. In
our experiment, both sertraline monopreparate and its HPβCD complex decreased
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the level of fasting glucose and raised blood serum insulin level with statistical
significance. However, the other study carried out on mice with alloxan-induced
hyperglycemia showed that SER administration did not alter blood insulin level
either in diabetic or normoglycemic animals significantly decreasing blood glucose
level [28]. Earlier Khanam and Pillai (2006) showed sertraline-induced

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hypoglycemia in diabetic and non-diabetic rats and proposed that the effect of SER
is insulin-independent and one of possible mechanism of SER hypoglycemic effect

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can be realized via a closing of potassium channels in the pancreatic β-cell
membrane [29]. However, in this mice experiment only mild diabetes was

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observed where blood glucose level was only 1.75-fold increased in streptozotocin-
treated animals whereas in our rat study this parameter was increased by 4.4-fold.

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In the light of our own results and literature findings [7] we suggest that the

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insulin-stimulating effect of SER can depend on the blood glucose concentration
and the duration of the treatment.
Insulin resistance is usually accompanied by alloxan-induced diabetes in
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experimental animals [30]. In our experiment, increased insulin resistance
(HOMA-IR index) was observed in the alloxan-treated group but not in the groups
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treated with SER and the HPβCD:SER complex. Therefore, SER and its complex
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with HPβCD have not only insulin stimulating but also insulin-sensitizing
properties.
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Since sertraline exhibits poor solubility in water, this limits its peroral application
due to low absorption [12, 31]. At the same time complexation of sertraline with β-
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cyclodextrins demonstrates high solubility of the drug and considerably enhances

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its bioavailability [10].

The solubility of SER increases on addition of HPβCD and for 0.85 mM
concentration of cyclodextrin, the SER solubility increased by ca. 33%, and this
can lead to enhancement of drug biological activity. In our experiments, the
binding constant of SER incorporated into cyclodextrins was almost the same for
HPβCD (4000±1000 M-1) and for β-CD (5850±550 M-1) [13]. But it may be
difficult to estimate the proper value of the association constant from the solubility
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phase diagram. That is why we are going, in the near future, to compare that result
to the values obtained from other experimental techniques, like ITC or NMR
measurements [13].
Complexation of drugs with cyclodextrins is widely used in pharmacology to
enhance solubility of low-soluble complexed compounds, which is important in

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peroral drug application. Such complexation increases their absorbability,
bioavailability and pharmacological effect, which enables to decrease the doses of

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the drugs applied and to minimize their side effects [32]. Cyclodextrins are most
physiologic nanoparticles used for drug transfer since, being cyclic

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polysaccharides, they can be subjected to enzyme degradation to monosaccharides
and serve a supplementary energy source [33]. HPβCD, a hydroxypropylated

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derivative of β-CD is very convenient for drug complexation, which explains its

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improved inclusion complexing ability, high water solubility, and low toxicity
[15]. The bioavailability of different antidiabetic drugs was significantly enhanced
by complexation with HPβCD [34, 35].
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The preference of the HPβCD:SER complex over sertraline is confirmed by the
effect of the HPβCD: SER complex on the amount of pancreatic β-cells, blood
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glucose and glycated hemoglobin contents, as well as on the serum insulin level. It
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is suggested that the cyclodextrin derivative increase the pharmacological effect of

SER, probably due to increased bioavailability by HPβCD as a drug permeation
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enhancer [36]. We cannot also exclude a contribution of an additive effect of the

HPβCD component, whose monopreparation developed a slight hypoglycemic
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effect, normalized serum insulin level and tended to improve the pancreatic islet
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morphology. It is well-known, that β-CDs lower cholesterol level, removing

cholesterol from cell membranes [37]. Studies on experimental animals and cell
cultures showed that treatment with β-CD and its derivatives, HPβCD being among
them, develop a hypoglycemic effect, improve defective glucose uptake, insulin
sensitivity and insulin secretion via their cholesterol-lowering effect [38-40].

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5. Conclusions

In this study we clearly demonstrated that the protective effect of the HPβCD: SER
complex on pancreatic β-cells survival after toxic impact of alloxan was stronger
than those of SER alone. This is supported by the results of histological studies

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(sizes of pancreatic islets and the amount of β-cells in them) and confirmed by the
biochemical data (levels of glucose, insulin and glycosylated hemoglobin).

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The acceleration of antidiabetic activity of the HPβCD:SER complex as compared
to the monopreparations, can be attributed to improved solubility of SER, which

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results in enhanced bioavailability of the low-soluble SER molecule and, probably,
higher permeability after inclusion in the cyclodextrin cavity. From the SER phase

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solubility diagram we calculated the association constant for SER and HPβCD
which was 4000 ± 1000 M-1. Thus, the HPβCD:SER complex exerted a
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pronounced beneficial effect in alloxan-induced diabetes which consisted in
protection of the pancreatic β-cell structure and morphology, normalization of
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pancreatic secretory function, insulin sensitivity and, as a consequence, in a
hypoglycemic effect. We believe that the HPβCD-complexed SER appears to be an
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interesting candidate for oral delivery of SER.

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Conflict of interest
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The authors do not disclose any actual or potential conflict of interest, including
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any financial, personal or other conflicts.

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Acknowledgement

This study was supported by grant № 2.5.23 from the State Program of Scientific
Research "Functional and Composite Materials, Nanomaterials," the subprogram
"Nanomaterials and Nanotechnologies" (the Republic of Belarus).

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Figure legends

Figure 1. A. Absorption spectra of SER (0.15 mM) in the absence and in the
presence of HPβCD at 298 K in the range from 279 to 295 nm, where the
concentrations of HPβCD are 0.85 mM (○), 0.45 mM (■), 0.39 mM (□), 0.365 mM

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(×), 0.129 mM (►) and 0 mM (solid line); B. Phase solubility diagram of SER in
aqueous HPβCD solution measured by the absorption at 282 nm; the coefficients

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of the equation St = S0 + Slope·x determined from the linear regression are: S0 =
(4.2 ± 1.5) mM and the Slope = (0.939 ± 0.1), where S0 and St are the drug

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solubility in the absence and the presence of HPβCD, respectively.

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Figure 2.Histopathological studies of pancreas in alloxan diabetic rats treated with
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the tested compounds (representative hematoxylin and eosin-stained sections). An
islet of Langerhans is in the centre of the picture and stained β-cells can be seen.
A. Control; B. Alloxan treatment; C. Alloxan and HPβCD treatment; D. Alloxan
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and SER treatment; E. Alloxan and HPβCD:SER complex treatment. Original
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magnification х 40.
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Figure 3. Effect of alloxan-induced diabetes and treatment with the tested

compounds over 2 weeks on the dynamics of blood glucose concentration
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(mmol/l) in rats. 1. Control; 2. Alloxan treatment; 3.Alloxan and HPβCD

treatment; 4.Alloxan and SER treatment; 5. Alloxan and HPβCD:SER complex
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treatment.
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Figure 4. Fasting blood glucose, mmol/l (A), glycosylated hemoglobin, % of total

hemoglobin (B), serum insulin, ng/ml (C) levels, and HOMA-IR index (D) in
alloxan diabetic rats treated with HPβCD, SER and their combination. The results
are shown as the mean ± standard error. Designations: a - p<0.05 compared to the
control group; b - p<0.005 compared to the alloxan group; c - p<0.005 compared to
the group treated with alloxan and SER.
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Table 1. Histomorphometric parameters of pancreatic islet of rats with alloxan-

induced diabetes treated with 2-hydroxypropyl-β-cyclodextrin (HPβCD), sertraline

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and their combination

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Control Alloxan Alloxan + Alloxan + Alloxan +
HPβCD sertraline combination

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Area of islets,
µm2 17616±1225 7350±998 a 8685±1118 a 6977±892 a 9869±858 ac

Diameter of

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islets, µm 201,6±7,25 143,6±10,30 a 150,1±11,72 a 120,9±7,98 a 144,6±7,65 ac

Volume of
islets, µm3 4,76±0,59
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1,31±0,29 a 1,52±0,34 a 1,05±0,21 a 1,95±0,29 ac

Number of β-
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cells/islet 58,3±8,37 19,4±3,24 a 25,9±5,01 a 32,6±9,96 42,0±3,22 b

Number of β-
cells/1000 µm2 3,04±0,229 1,74±0,175 a 2,10±0,339 2,58±0,569 3,00±0,294 b
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Designations: a – P<0.05 as compared to the Control group;

b
– P<0.05 as compared to the Alloxan group;
c
– P<0.05 as compared to the Alloxan + sertraline group.
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Figure 1. A. Absorption spectra of SER (0.15 mM) in the absence and in the
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presence of HPβCD at 298 K in the range from 279 to 295 nm, where the
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concentrations of HPβCD are 0.85 mM (○), 0.45 mM (■), 0.39 mM (□), 0.365 mM
(×), 0.129 mM (►) and 0 mM (solid line); B. Phase solubility diagram of SER in
aqueous HPβCD solution measured by the absorption at 282 nm; the coefficients
of the equation St = S0 + Slope·x determined from the linear regression are: S0 =
(4.2 ± 1.5) mM and the Slope = (0.939 ± 0.1), where S0 and St are the drug
solubility in the absence and the presence of HPβCD, respectively.

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Figure 2. Histopathological studies of pancreas in alloxan diabetic rats treated with

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the tested compounds (representative hematoxylin and eosin-stained sections). An

islet of Langerhans is in the center of the picture and stained β-cells can be seen.
A. Control; B. Alloxan treatment; C. Alloxan and HPβCD treatment; D. Alloxan
and SER treatment; E. Alloxan and HPβCD:SER complex treatment. Original
magnification х 40.

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Figure 3. Effect of alloxan-induced diabetes and treatment with the tested

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compounds over 2 weeks on the dynamics of blood glucose concentration
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(mmol/l) in rats. 1. Control; 2. Alloxan treatment; 3.Alloxan and HPβCD
treatment; 4.Alloxan and SER treatment; 5. Alloxan and HPβCD:SER complex
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treatment.
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Figure 4. Fasting blood glucose, mmol/l (A), glycosylated hemoglobin, % of total
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hemoglobin (B), serum insulin, ng/ml (C) levels, and HOMA-IR index (D) in
alloxan diabetic rats treated with HPβCD, SER and their combination. The results
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are shown as the mean ± standard error. Designations: a - p<0.05 compared to the
control group; b - p<0.005 compared to the alloxan group; c - p<0.005 compared to
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the group treated with alloxan and SER.

C
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Highlights

1. Sertraline complexation with hydroxypropyl-β-cyclodextrin increased its

solubility
2. Treatment with the complex alleviate signs of alloxan-induced diabetes in

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rats
3. Complexation with the cyclodextrin increased sertraline pharmacological

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effect

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