Development and in vivo evaluation of chitosan beads for the colonic delivery of azathioprine for treatment of inflammatory bowel disease

Accepted Manuscript Development and in vivo evaluation of chitosan beads for the colonic delivery of azathioprine for treatment of inflammatory bowel disease

Abdelrahman M. Helmy, Mahmoud Elsabahy, Ghareb M. Soliman, Mahmoud A. Mahmoud, Elsayed A. Ibrahim PII: DOI: Reference:

S0928-0987(17)30469-4 doi: 10.1016/j.ejps.2017.08.025 PHASCI 4183

To appear in:

European Journal of Pharmaceutical Sciences

Received date: Revised date: Accepted date:

19 May 2017 31 July 2017 18 August 2017

Please cite this article as: Abdelrahman M. Helmy, Mahmoud Elsabahy, Ghareb M. Soliman, Mahmoud A. Mahmoud, Elsayed A. Ibrahim , Development and in vivo evaluation of chitosan beads for the colonic delivery of azathioprine for treatment of inflammatory bowel disease, European Journal of Pharmaceutical Sciences (2017), doi: 10.1016/j.ejps.2017.08.025

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ACCEPTED MANUSCRIPT Development and in vivo Evaluation of Chitosan Beads for the Colonic Delivery of Azathioprine for Treatment of Inflammatory Bowel Disease

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Mahmoud6 and Elsayed A. Ibrahim1

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Abdelrahman M. Helmy1,2, Mahmoud Elsabahy1,3,4*, Ghareb M. Soliman1,5, Mahmoud A.

Department of Pharmaceutics, Faculty of Pharmacy, Assiut University, Assiut, Egypt

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Department of Pharmaceutics, Faculty of Pharmacy, Deraya University, Minia, Egypt

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Assiut International Center of Nanomedicine, Assiut University, Assiut, Egypt

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Laboratory for Synthetic-Biologic Interactions, Department of Chemistry, Texas A&M

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University, College Station, Texas, USA

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Department of Pharmaceutics, Faculty of Pharmacy, University of Tabuk, Tabuk, Saudi Arabia

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Department of Anatomy and Histology, Faculty of Veterinary Medicine, Assiut University,

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*

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Correspondence:

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Assiut, Egypt

Prof. Mahmoud Elsabahy ([email protected]) Department of Pharmaceutics, Faculty of Pharmacy, Assiut University, Assiut 71515, Egypt Tel.: +201000607466 Fax: +20882080711

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ACCEPTED MANUSCRIPT Abstract Azathioprine is a highly efficient immunosuppressant drug used for treatment of inflammatory bowel disease (IBD). Systemic administration of azathioprine results in delayed therapeutic effect and serious adverse reactions. In the current study, we have developed, for the first time,

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colon-targeted chitosan beads for delivery of azathioprine in colitis rabbit model. Several

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characterizations were performed for the azathioprine-loaded beads (e.g. drug encapsulation

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efficiency, drug loading capacity, yield, size, shape and compatibility with other ingredients). The in vitro release profiles of acid-resistant capsules filled with azathioprine-loaded beads

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showed that most of azathioprine was released in IBD colon simulating medium. The therapeutic

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effects of azathioprine-loaded beads and azathioprine crude drug were examined on acetic acidinduced colitis rabbit model. Improved therapeutic outcomes were observed in the animals

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treated with the azathioprine-loaded beads, as compared to the untreated animal controls and the

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animals treated with the azathioprine free drug, based on the clinical activity score, index of tissue edema, mortality rate, colon macroscopic score and colon histopathological features. In the

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animals treated with the azathioprine-loaded beads, the levels of the inflammatory mediators,

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myeloperoxidase enzyme and tumor necrosis factor-α, were significantly reduced to levels similar to those observed in the normal rabbits. Furthermore, the activities of the antioxidant

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enzymes, superoxide dismutase and catalase, were restored considerably in the animals treated with the drug-loaded beads. The azathioprine-loaded beads developed in the current study might have great potential in the management of IBD.

Keywords: Azathioprine, chitosan, inflammatory bowel disease, colon targeting

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ACCEPTED MANUSCRIPT 1. Introduction Inflammatory bowel disease (IBD) is an idiopathic autoimmune inflammatory disorder of the colonic mucosa. Ulcerative colitis (UC) and Crohn’s disease (CD) are the most common two forms of IBD. Both UC and CD are chronic diseases characterized by alternating periods of

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remission and relapse. The relapse of IBD is characterized by the incidence of abdominal

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spasms, rectal bleeding, anemia, fever, fatigue, nausea, and weight loss [1, 2]. Treatment of IBD

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is varied according to the disease site and activity. Aminosalicylates, corticosteroids, anti-tumor necrosis factor agents and immunosuppressive drugs are commonly used either for management

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of acute relapse or for maintenance of remission [3, 4].

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Azathioprine (AZA) is one of the most widely used immunosuppressant drugs for the treatment of IBD. AZA is rapidly converted to 6-mercaptopurine (6-MP) and finally to 6-

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thioguanine nucleotides (6-TGN) via a series of non-enzymatic and enzymatic steps [5]. The 6-

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TGN is a purine antagonist which accumulates within T lymphocytes and modifies the immune response by inhibiting ribonucleotide synthesis and inducing T cell apoptosis, and thus inhibiting

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the proliferation of T and B lymphocytes and reducing the numbers of cytotoxic T cells and

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plasma cells [6, 7]. AZA is effective for both induction and maintenance of remission in CD and UC [4]. However, the systemic use of AZA for the treatment of acute IBD relapse is limited

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because of its slow onset of action, which is due to the slow intracellular accumulation of 6TGN. Hence, AZA is usually utilized as an adjunctive therapy [4]. In addition, systemic administration of AZA results in non-dose related allergic reactions (nausea, fever, arthralgia and rash), and dose related profound leukopenia, bone marrow depression and some forms of hepatitis. In addition, it increases the risk of malignancy incidence, particularly lymphoma [4].

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ACCEPTED MANUSCRIPT Targeted delivery of AZA to the disease site (i.e. IBD Colon) might accelerate 6-TGN accumulation in the inflamed tissues and hasten the drug onset of action. Enhanced AZA therapeutic efficiency has been reported previously after topical application for the treatment of immune-mediated chronic oral inflammatory conditions [8]. In addition, the systemic absorption

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of AZA and 6-MP from colon is much lower than from stomach and small intestine, and thus

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colonic localization of AZA might decrease its systemic absorption and allow the administration

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of higher doses while minimizing the dose-related adverse reactions [9]. During IBD, the intraluminal colonic pH is lowered to 2.3–5.5, depending on the activity of the disease, due to the

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disturbance of the colonic pH controlling factors (e.g. microbial fermentation processes,

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secretions of lactate, etc.) [10, 11]. In the current study, for the first time, we utilized chitosanbased delivery system for disease-specific delivery of AZA, taking the advantage of the

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difference in pH between the healthy colonic tissues and inflamed disease sites [12, 13].

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Chitosan is a high molecular weight cationic and biodegradable copolymer composed of linked N-acetyl-D-glucosamine and glucosamine units. Chitosan is soluble in acidic pH due to

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the protonation of the amino groups and precipitates at pH higher than 7 [14]. Hence, upon

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circumventing the gastric acidic environment, limited drug release from the chitosan-based delivery vehicles is expected in the small intestine, whereas drug release could be accelerated at

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the colonic inflamed tissues of low pH in IBD. To bypass the gastric pH, chitosan is usually coated with acid-resistant polymers [12, 15], or mixed with other polymers to form acid-resistant complexes [16]. In this study, chitosan beads loaded with AZA were prepared by ionic cross-linking with sodium tripolyphosphate (STPP). Several characterizations and optimization steps were carried out to select formulations with high drug loading capacity and efficiency, and with suitable

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ACCEPTED MANUSCRIPT physicochemical characteristics. To minimize drug release in the gastric fluids, the beads were filled into acid-resistant capsules. The therapeutic effects of azathioprine-loaded beads and azathioprine crude drug were examined on acetic acid-induced colitis rabbits models.

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2. Materials and methods

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2.1. Materials

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AZA was kindly provided by T3A Pharmaceutical Co. (Assiut, Egypt). Low molecular weight chitosan (100,000–300,000 kDa) and STPP were purchased from Acros Organics (Geel,

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Belgium). DRcaps™ acid-resistant capsules (size 3) were purchased from Capsugel ® (Bornem,

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Belgium). Potassium dihydrogen phosphate and sodium hydroxide were purchased from El-Nasr Pharmaceutical Chemicals Co. (Cairo, Egypt). Glacial acetic acid was purchased from SDFCL

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(Mumbai, India). All other chemicals were of analytical grades.

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2.2. Beads preparation method

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The beads were prepared by ionic cross-linking technique. Chitosan solution was prepared by dissolving chitosan in acetate buffer (pH 5) under continuous stirring for 1 h. Then, AZA was

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dispersed uniformly in the chitosan solution and homogenized for 30 min. AZA dispersion (2 g)

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was dropped through 0.4 mm inner diameter needle of 5 mL-syringe into 10 mL STPP solution under continuous magnetic stirring at 200 rpm for 15 min. Beads were collected by sieving, washed three times with distilled water and dried at the ambient temperature for 24 h. Different concentrations of AZA, STPP and chitosan were examined to select the optimal composition ratio (Table 1). Unloaded beads were prepared using the same procedures utilized for the optimal formulation but without including the drug.

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ACCEPTED MANUSCRIPT 2.3. Beads characterization 2.3.1. Size and morphology Digital photographs of 20 beads of each sample mounted on a calibrated stage were captured using Canon® digital camera SX 230 HS (Japan). Size analysis (average Feret’s diameter) and

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morphological characterizations were performed using a computerized image analysis Imagej®

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software (Maryland, USA). Bead sphericity was evaluated in terms of circularity and roundness [17]. Circularity and roundness were determined based on equations (1) and (2), respectively.

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The means and standard deviations of the characterization parameters of 20 beads in each sample

(1)

(2)

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were calculated.

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The surface morphology of the optimal AZA-loaded beads was studied using a scanning electron microscope (Jeol, JSM-5200, Tokyo, Japan) at low energy (15 kV). In a vacuum

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evaporator, AZA-loaded beads and unloaded beads were mounted onto stubs, sputter coated with

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gold and photographed at magnifications of 15, 50 and 1000×. 2.3.2. Yield (%), Drug loading capacity (%) and encapsulation efficiency (%) The dried AZA-loaded beads were weighed to calculate the yield per batch. The percentage of yield was calculated using equation (3). To determine the drug content, specific weight of AZAloaded beads was placed into 100 mL volumetric flask and the volume was completed to 100 mL with 0.1 N HCl. The flasks were incubated in a shaking water bath at 100 rpm and 40 °C for 24 h and filtered. Drug concentration was then determined by UV–Vis spectrophotometer (Genway, 6

ACCEPTED MANUSCRIPT England) at wavelength of 280 nm. Drug loading capacity (DL) and encapsulation efficiency (EE) were calculated using equations (4) and (5), respectively.

(4)

(5)

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(3)

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High Performance Liquid Chromatographic System (HPLC, Agilent HP1100, Agilent

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Technologies, Santa Clara, CA) equipped with G1322A Degasser, G1311A Quaternary pump, G1313A ALS (auto sampler), G1316A column oven, G1314A 1100 variable wavelength

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detector, HP ChemStation for LC 3D Rev.A.06.03 computer software, Hypersil (C18) column

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(4.6 mm×150 mm, 5 µm), was utilized for the quantitative analysis of the drug content (as a confirmatory assay). The mobile phase constituted of methanol-water-acetic acid (20:80:1,

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v/v/v), and the flow rate was adjusted at 1.0 mL/min. UV detection was set at 280 nm. The volume of injection was fixed at 20 µL. All analyses were performed at the room temperature. Azathioprine standards were accurately weighed (25 mg) and transferred to a 100 mL volumetric flask, 10 mL of 0.1 N HCl was added, and the content of the flask was ultrasonicated for 10 min. Appropriate dilutions were made with methanol to obtain solutions containing 10, 25, 50 and 100 µg/mL of azathioprine. Each standard solution was injected and the calibration curve was constructed based on the calculated area under the curve (AUC) of each solution. Then, 7.4 mg 7

ACCEPTED MANUSCRIPT of AZA-loaded beads (B11) was accurately weighed and transferred to a 100 mL volumetric flask, 10 mL of 0.1 N HCl was added, and the content of the flask was allowed to stand for 24 h, and ultrasonicated for 20 min. The solution in the flask was diluted with methanol and ultrasonicated for additional 20 min. The sample solution was filtered (0.45 µm filter) and

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injected into the HPLC instrument. The AUC for the sample peak was used to calculate the drug

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concentration in the sample solution using the standard calibration curve. 2.3.3. In vitro release studies

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To study the release profiles in a small intestine simulating medium [18], beads were enclosed in stainless-steel baskets suspended in screw-capped vessels containing 100 mL phosphate buffer at

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pH 6.8. The vessels were placed in a shaking water bath at 50 rpm and temperature of 37.0 ± 0.5

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°C. At specified time intervals (1, 2, 3 and 4 h), 3 mL aliquot of each sample was withdrawn

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from the vessel and replaced by an equal volume of the release medium. Samples were filtered and amounts of drug released were determined spectrophotometrically at wavelength of 280 nm.

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The data were presented as mean ± SD of at least triplicates.

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The release of azathioprine from the acid-resistant capsules filled with the selected formulation was examined by the same previously described method with some modifications.

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The beads filled into the capsules were pre-incubated in simulated gastric medium (i.e. 0.1 N HCl, pH 1.2) for 2 h, followed by 4 h in phosphate buffer (pH 6.8), and then for additional 24 h in acetate buffer at pH 4.0 (simulating IBD colon) [12, 19] or phosphate buffer at pH 7.2 (simulating healthy colon). All the media were maintained at a temperature of 37.0 ± 0.5 °C.

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ACCEPTED MANUSCRIPT 2.3.4. Fourier transform-infrared spectroscopy (FT-IR) studies FT-IR analyses of samples were performed using JASCO FT-IR–4200 type A (JASCO Co., Tokyo, Japan). Samples (3–4 mg) of chitosan, unloaded beads, AZA and AZA-loaded beads were mixed with potassium bromide (IR grade) and compressed into disks under vacuum.

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Spectral scanning was performed in the range of 4000–400 cm-1.

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2.3.5. Differential scanning calorimetry (DSC) studies

A computer-interfaced Shimadzu Calorimeter (Model DSC–50, Kyoto, Japan) was used for DSC

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analysis of chitosan, unloaded beads, AZA and AZA-loaded beads. Samples (3–5 mg) were

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placed in aluminum pans, sealed and heated at a constant rate of 10 °C/min in the range of 25– 350 °C under constant flow of nitrogen gas. Indium was sealed in an aluminum pan and used for

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instrument calibration.

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2.4. In vivo evaluation

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2.4.1. Animals

Nineteen adult male New Zealand white rabbits (Total Farm, Assiut, Egypt) with average weight

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of 1.8 kg were housed in the animal housing facility (Assiut University Faculty of Medicine).

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The rabbits were kept in individual cages in the laboratory at constant ambient temperature (25– 27 °C) with a 12-h light/dark cycle and allowed access to water and food ad libitum. The rabbits were allowed to adapt to the laboratory conditions for 3 days before experimentation. The rabbits were randomly distributed into 4 experimental groups, normal untreated group, colitis untreated group, AZA-treated group and AZA-loaded beads treated group. The animals were appropriately treated and all animal experiments were approved by the Institutional Animal Ethical Committee

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ACCEPTED MANUSCRIPT of the Faculty of Pharmacy, Assiut University, and it adheres to the Guide for the Care and Use of Laboratory Animals, 8th Edition, National Academies Press, Washington, DC. 2.4.2. Induction of colitis All rabbits were fasted 24 h prior to the experimental procedures with access to water ad libitum.

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Soft infant feeding tube (size: 10 FG) with a thin layer coat of white petroleum jelly was inserted

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carefully into the anus of rabbits at 20 cm proximal to the anus. Then, a 3-mL plastic syringe filled with 4% acetic acid solution in saline was fitted onto the feeding tube and 1.5 mL of the

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solution was administered slowly. Then, the acetic acid syringe was directly replaced with a 10mL empty plastic syringe and 7 mL air was introduced rapidly to spread acetic acid solution

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throughout the colon. The rabbits were maintained in a Trendelenburg position for 30 s to

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prevent the leakage of acetic acid solution. Finally, another 10-mL plastic syringe filled with saline was fitted onto the catheter and 7 mL saline was instilled to flush the colon. After 15 s, the

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rabbits were maintained in a reverse Trendelenburg position for 10 s to allow the withdrawal of

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the excess instilled fluids. Rabbits were returned back to cages with free access to water and food. In the case of the normal group, the same procedures were followed except of using saline

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instead of the 4% acetic acid solution [20, 21].

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2.4.3. Treatment protocol and sample collection After induction of colitis, the rabbits of the normal group (n = 4) and the colitis group (n = 7) did not receive treatment. The AZA-treated rabbits (n = 4) were administered one capsule daily of acid-resistant capsules filled with AZA (10 mg/Kg/day) for three days. The AZA-loaded beads treated rabbits (n = 4) were administered one capsule daily of acid-resistant capsules filled with AZA-loaded beads (equivalent to AZA dose of 10 mg/Kg/day) for three days. The first dose was administered 1-h after colitis induction. The animals were sacrificed by cervical decapitation 2410

ACCEPTED MANUSCRIPT h after the last dose and the distal colons were resected directly. For each rabbit, the colon was opened by longitudinal incision to score stool consistency and stool bleeding. Then, the colon was rinsed with normal saline solution to wash out the fecal contents for macroscopic scoring. The part of the colon that had major gross pathologic changes was cut into two pieces. One piece

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was frozen by immersion into liquid nitrogen and then stored at - 20 °C for biochemical assays.

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The second piece was fixed immediately in 10% formalin for histologic examination [21].

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2.4.4. Treatment assessment

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2.4.4.1. Colitis severity assessment

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The severity of colitis was evaluated using a clinical activity score system that assesses weight loss, stool consistency and stool bleeding as reported by Hartmann et al. with minor

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modifications [22]. Weight loss was scored as 1 point for weight gain > 1%, 2 points for weight

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loss or weight gain < 1%, 3 points for weight loss between 1–5% and 4 points for weight loss > 5%. Stool consistency was scored as 1 point for well-formed pellets, 2 points for semi-formed

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pellets, 3 points for pasty stool and 4 points for watery stool. For stool bleeding, 1 point was

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given for normal color stool, 2 points for brown color stool, 3 points for reddish color stool and 4 points for bloody stool. The summation of these scores ranged from 3 (healthy) to 12 (severe

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colitis). For assessment of colon tissue edema, a 25-cm segment of distal colon was cut and weighed. The index of tissue edema was calculated based on the ratio between the colon wet weight and the body weight [23]. The number of rabbits died in the different groups was monitored and the mortality rate was calculated.

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ACCEPTED MANUSCRIPT 2.4.4.2. Macroscopic assessment of the colonic damage The macroscopic damage of the colonic mucosa was assessed using the grading scale of Morris et al [24]. The arbitrary scale ranges from 0–5: 0: no damage, 1: localized hyperemia with no ulcers, 2: single site of ulceration with no inflammation, 3: single site of ulceration with

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inflammation, 4: more than one site of ulceration and inflammation and the size of ulcers < 1 cm,

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2.4.4.3. Histological evaluation

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ulceration extending > 1 cm along the length of the colon.

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and 5: two or more sites of ulceration and inflammation or one major site of inflammation and

The formalin-fixed tissues were trimmed, dehydrated in serial concentrations of alcohol and

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embedded in paraffin blocks. The paraffin blocks were sectioned (5 µm thickness) and stained

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with hematoxylin and eosin (H&E) stain, periodic acid Schiff and hematoxylin (PAS&H) stain

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and acridine orange (AO) stain. The samples were evaluated for colon damage by light microscope (Olympus BX51, Japan) and the photos were taken by a camera (Olympus DP72,

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Japan) adapted to the microscope.

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2.4.4.4. Biochemical assays

One gram of colon tissue was homogenized into 10 mL 50 mM phosphate buffered saline (PBS,

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pH 7.0). Samples were then centrifuged for 30 min at 10,000 g at 4 °C and the supernatant was removed for analysis. Myeloperoxidase enzyme (MPO) activity was determined by colorimetric assay using NWLSS™ Myeloperoxidase Activity Assay Kit (AMS Biotechnology, Abingdon, UK). Tumor necrosis factor-α (TNF-α) concentration was determined using a rabbit TNF-α MyBiosource® ELISA kit (San Diego, USA). Superoxide dismutase (SOD) and catalase (CAT) enzymes activities were determined by colorimetric assays using SOD and CAT Biodiagnostic ®

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ACCEPTED MANUSCRIPT research kits (Giza, Egypt). All assays were carried out according to the manufacturers' instructions. 2.5. Statistical analysis All statistical analyses were performed using GraphPad Prism® version 5.00 for Windows (San

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Diego, California, USA). All experimental data were expressed as the mean ± standard deviation

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(SD). For multiple comparisons between formulations in terms of EE, DL, diameter, roundness, circularity, and the amount of drug released, one-way analysis of variance (ANOVA) with Tukey

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post-hoc test was performed. Regarding the treatment assessment, one-way ANOVA with Dunnett post-hoc test was performed to compare the different groups of animals vs. the normal

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Statistical significance was set at p < 0.05.

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group, and Tukey post-hoc test was performed to compare between the different animal groups.

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ACCEPTED MANUSCRIPT 3. Results and Discussion 3.1. Beads preparation and optimization Different formulations of AZA-loaded beads (B1–B12) were prepared and evaluated to select the beads with the optimal composition. The evaluation criteria were the yield, EE, DL,

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morphological characters, and the release profiles in simulated intestinal buffer of pH 6.8.

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3.1.1. Effect of STPP:chitosan ratio (B1–B4)

Beads were formed via ionic cross-linking of the positively charged chitosan and the negatively

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charged STPP. By increasing STPP:chitosan ratio from 0.4:1 to 4:1 (B1–B3), the size of beads

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decreased significantly and the sphericity increased significantly, indicating a denser crosslinking of chitosan due to the higher STPP content [17, 25]. The size and shape of the beads

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prepared at the highest ratio (8:1, B4) were not significantly different from the beads prepared at

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4:1 ratio (Table 2).

Generally, EE was high for all formulations due to the poor aqueous solubility of AZA

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and thus limited diffusion of the drug from the beads during cross-linking and washing processes

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[26]. The increase of STPP:chitosan ratio from 0.4:1 to 4:1 resulted in significant increase in EE. No further significant increase in the EE was achieved upon increasing the ratio to 8:1. DL did

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not increase upon increasing the STPP:chitosan ratio due to the increased content of STPP in the beads (i.e. relative to the drug content) (Table 3). Beads prepared at higher ratios (i.e. 4:1 and 8:1) released lower amounts of AZA after 4 h in the simulated intestinal medium, as compared to the beads prepared at lower ratios (i.e. 0.4:1 and 0.8:1), probably due to the stronger crosslinking of chitosan at the higher ratios (Figure 1a) [26].

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ACCEPTED MANUSCRIPT Beads prepared at ratios 4:1 (B3) and 8:1 (B4) had similar characteristics (size, sphericity, EE, DL and drug release profile). However, regarding the yield, B3 had almost twice the yield of B4. Therefore, the ratio 4:1 (B3) was selected as the optimal STPP:chitosan ratio for further studies.

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3.1.2. Effect of chitosan concentration (B3, B5–B8)

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Beads prepared from different concentrations of chitosan had similar diameters of 1.29–1.55 mm. The shape of 2–3.5% chitosan beads (B3, B6–B8) was almost spherical, whereas the beads

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prepared at lower chitosan concentration (1.5%) (B5) formed irregular spheres probably due to the lower polymer content of the beads (Table 2). High and similar EE and DL were observed

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for the beads prepared from 2–3.5% chitosan solutions, and lower EE and DL were found for the

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beads prepared at lower polymer content (B5). The yields of beads of different compositions were similar (Table 3). The amounts of drug released from the beads prepared from 1.5% and

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2% chitosan (B5 and B3) were significantly higher than those released from the beads prepared

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from chitosan of higher concentrations (2.5–3.5%) (Figure 1b). Chitosan solutions of higher concentrations might form thicker bead walls and stronger cross-linked matrix, and thus resulting

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in lower swelling and drug release over time. Hence, beads prepared from chitosan concentration

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of 2.5% (B6) was selected for further studies because it was difficult to prepare beads from chitosan of higher concentrations due to the high viscosity of the formed chitosan solutions. 3.1.3. Effect of AZA:chitosan ratio (B6, B9–B12) Beads prepared at low AZA:chitosan ratio of 0.2:1 (B9) had the smallest diameter and the most spherical shape, whereas significant increase in size and irregularity in sphericity were observed upon increasing the AZA:chitosan ratio to 1:1. Further increase of AZA:chitosan ratio above 1:1 resulted in a sharp increase in size and formation of irregular spherical beads (Table 2). 15

ACCEPTED MANUSCRIPT Increasing AZA:chitosan ratio above the 0.2:1 increased the DL and EE. Generally, the yield increased upon increasing the drug to chitosan ratio (Table 3). The percentages of drug released after 4 h in simulated intestinal medium (pH 6.8) from the beads prepared at different AZA:chitosan ratios were similar except for the beads prepared at AZA:chitosan ratio of 0.2:1

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which demonstrated a faster release kinetics (Figure 1c). Based on the morphological and

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physicochemical evaluations, AZA-loaded beads (B11) prepared from 2.5% chitosan at 4:1

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STPP:chitosan ratio and 1:1 AZA:chitosan ratio was selected as the optimal formulation for further studies. Moreover, we have carried out a simple comparative study between the UV-

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spectrophotometric method and HPLC analysis for determination of the AZA content in the

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beads (i.e. the optimal beads formulation B11 was selected for this study). The drug loading efficiency calculated based on the HPLC method was 36.9%, similar to that calculated based on

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the UV-spectrophotometry (36.3%). Hence, determination of drug content in the beads by UV-

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spectrophotometry using 0.1 N HCl as a solvent was accurate and precise method for

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quantitative analysis of the drug.

3.2.1. SEM

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3.2. Characterizations of the optimal formulation

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Scanning electron microscopy images of the unloaded beads showed small sized nearly spherical beads with dense and smooth surface (Figures 2a–2c). On the other hand, the AZA-loaded beads were larger in size and relatively less spherical (Figures 2d and 2e). The differences in size and sphericity between unloaded and loaded beads were attributed to the entrapment of the suspended AZA particles that may have partially disrupted the cross-linked chitosan network. Images at higher magnification (Figure 2f) demonstrate a porous and rugged surface with the drug particles embedded in the cross-linked matrix. 16

ACCEPTED MANUSCRIPT 3.2.2. Fourier transform-infrared spectroscopy studies FT-IR spectrum of chitosan showed a broad stretching vibration band (3000–3700 cm-1) of OH groups overlapped with the stretching vibration of NH groups [27]. The spectrum showed also a characteristic stretching vibration band of amidic C=O at 1651 cm-1 and bending vibration band

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of amidic NH group at 1595 cm-1 (Figure 3a) [17]. The spectrum of the unloaded beads showed

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the same broad band of OH groups (3000–3700 cm-1). The two characteristic bands of amidic

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C=O and NH groups were shifted to 1649 cm-1 and 1556 cm-1, respectively, indicating the cross-

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linking of chitosan [25, 28]. Furthermore, a characteristic band of P=O (i.e. corresponds to the STPP cross-linker) appeared at 1092 cm-1 (Figure 3b) [29]. On the spectrum of AZA, bands of

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N–H stretching of purine at 3191 cm-1, C–H stretching of imidazole groups at 3108 cm-1, C–H

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stretching of pyrimidine groups at 2976 cm-1 and C–H stretching of CH3–N group at 2807 cm-1 were observed (Figure 3c) [30]. The characteristic bands of chitosan and AZA were observed on

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the spectrum of the AZA-loaded beads almost at the same frequencies, and thus indicating the

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absence of chemical interactions between the drug and the bead components (Figure 3d).

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3.2.3. DSC

DSC thermogram of chitosan showed a broad endothermic peak at 124.2 °C due to water

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evaporation (i.e. absorbed water on the surface) and a second exothermic decomposition peak at 305.6 °C (Figure 4a) [29]. The thermogram of the unloaded beads showed the water evaporation endothermic peak at 121.2 °C of chitosan, but more intense due to the increased water holding capacity of the cross-linked chitosan. The second exothermic peak of polymer decomposition also appeared but at lower temperature (243 °C) as compared to chitosan alone (Figure 4b). Chitosan might have consumed higher energy for degradation due to the presence of the unsubstituted amine groups [29, 31]. The thermogram of AZA showed a characteristic sharp 17

ACCEPTED MANUSCRIPT melting endothermic peak at 259 °C and a sharp exothermic peak of AZA decomposition at 266.5 °C (Figure 4c) [30]. The thermogram of the AZA-loaded beads demonstrated the same pattern of chitosan beads, whereas the endothermic peak of AZA melting was not detected. Disappearance of the drug characteristic peak after loading into carriers is commonly observed

phase (Figure 4d) [32].

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3.2.4. In vitro release study of AZA-loaded beads filled capsules

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when the drug is molecularly dispersed in the carrier in an amorphous or disordered crystalline

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The release profiles of AZA from the drug-loaded beads filled into acid-resistant capsules were studied in 0.1 N HCl (pH 1.2, simulating gastric pH) for 2 h, followed by phosphate buffer (pH

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6.8, simulating small intestine pH) for 4 h and finally in acetate buffer (pH 4.0, simulating IBD

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colonic pH) or phosphate buffer (pH 7.2, simulating healthy colonic pH) for additional 24 h (Figure 5). During the first 90 min, the capsules were still intact and no drug was released,

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inferring excellent acid-resistance capacity of these capsules [33]. By the end of the two hours,

PT

the capsules swelled slightly and only ca. 4% of the drug was released. The use of acid-resistant capsules was efficient in overcoming the premature drug release at the gastric pH, and simpler

CE

than the other enteric coating techniques. After replacement of the dissolution medium with

AC

phosphate buffer (pH 6.8), the beads were leaked from the opened capsules during the first hour, and AZA was released slowly due to the limited swelling of chitosan at this pH [34]. By the end of the six hours in the 0.1 N HCl and phosphate buffer pH 6.8, the cumulative released percentage of AZA was ca. 22.2 ± 1.7%. At pH 7.2, the release pattern from the beads was similar to that at pH 6.8. The overall amount of drug released after 30 h was 74.3 ± 1.5%. On the contrary, at pH 4.0, swelling of chitosan beads could be observed visually, and the drug release rate sharply increased (i.e. ca. 92.4 ± 2.2% of the drug was released after 10 h), and the drug was 18

ACCEPTED MANUSCRIPT completely released after 14 h. The capsules filled with the beads could protect the drug from the harsh gastric conditions and minimize the amount lost in the small intestine, and could release the drug completely and rapidly in conditions simulating the pathological colonic conditions (i.e. IBD-targeted delivery system).

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3.3. Treatment evaluation

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Colitis results in intensive mucosal damage and inflammatory cell infiltration, and the disease is characterized by body weight loss, watery diarrhea and rectal bleeding [1, 20, 35]. Several

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pathological and biochemical parameters (clinical activity score, index of tissue edema, mortality rate, macroscopic score, histological features and biochemical analysis of inflammatory

AN

mediators and antioxidants) have been assessed to confirm the development of the disease model

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3.3.1. Colitis severity assessment

M

in the rabbits and to evaluate the therapeutic efficacy of the AZA-loaded beads vs. the free drug.

The clinical activity scores (depends on body weight loss, stool consistency and stool bleeding)

PT

of the colitis group and the AZA-treated group were significantly higher (p < 0.001 and p < 0.01,

CE

respectively) than the score of the normal group. The clinical activity score of the AZA-loaded beads treated group was not significantly different from that of the normal group (p > 0.05)

AC

(Figure 6a). The indices of tissue edema of the colitis group and the AZA-treated group were significantly higher (p < 0.05) than the index of tissue edema of the normal group. The index of tissue edema of the AZA-loaded beads treated group was as low as that of the normal group (Figure 6b). High mortality rate (43%) was observed in the colitis group as compared to the mortality rate (25%) in the AZA-treated group. No animals died in the normal group and in the group treated with the AZA-loaded beads. Based on the clinical activity score, index of tissue

19

ACCEPTED MANUSCRIPT edema and mortality rate, the AZA-loaded beads have demonstrated a better therapeutic effect than the free drug. 3.3.2. Macroscopic assessment of the colonic damage Morphologically, the colons of the colitis group and the AZA-treated group had wide areas of

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ulceration with intensive inflammation as compared to the normal group (i.e. significantly higher

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macroscopic score, p < 0.001 and p < 0.01, respectively). The colons of the animals in the AZAloaded beads treated group showed hyperemia, sometimes with a single small area of ulceration,

US

and, had a macroscopic score slightly higher than the score of the normal group (p > 0.05)

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(Figures 7 and 8). 3.3.3. Histological evaluation

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Normal histology and architecture were observed in the colons of the normal group. The lamina

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epithelialis was formed of simple columnar epithelium with PAS positive goblet cells and with no or few number of apoptotic cells. The submucosa was formed of loose connective tissue free

CE

10a and 11a) [36].

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from leukocyte infiltration and contained crypts of lieberkühn (intestinal glands) (Figures 9a,

The histopathological examination of the colons of the colitis group revealed intensive

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mucosal injury characterized by ulcerative colitis, surface epithelium erosion, cryptitis, crypt abscesses and degenerating of intestinal glands, and degenerated and thickened muscularis mucosae [36, 37]. Depletion and degeneration of the PAS positive goblet cells were observed. Large number of AO stained apoptotic cells (i.e. exposed to oxidative DNA damage) was found [38]. Leukocyte infiltration and edema in the submucosa was also observed in the colitis group due to the release of inflammatory mediators and the increased vascular permeability (Figures 9b, 10b and 11b). 20

ACCEPTED MANUSCRIPT The colons of the AZA-treated group exhibited moderate to severe mucosal injury characterized by ulcerative colitis, crypt abscesses, cryptitis, and degeneration of the intestinal glands. Distinctive depletion and degeneration of the PAS positive goblet cells and increased number of apoptotic cells were observed. Degeneration and thickening of the muscularis

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mucosae were also recognized. The detected leukocyte infiltration in the submucosa was

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moderate (Figures 9c, 10c and 11c).

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Histologically, AZA-loaded beads had an ameliorative effect on acetic acid-induced colitis appeared as a restoration of the normal histology and architecture of the colons of the

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animals in the AZA-loaded beads treated group. PAS positive goblet cells were distributed

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normally in the lamina epithelialis and within the intestinal glands, which reached normally to the thin muscularis mucosae. Few apoptotic cells and limited leukocyte infiltration and edema in

3.3.4. Biochemical assays

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the submucosa were observed (Figures 9d, 10d and 11d).

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The activity of MPO enzyme and the concentration of TNF-α in the colitis group were significantly higher (p < 0.001 and p < 0.001, respectively) than their levels in the normal group

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(Figures 12a and 12b). MPO enzyme is found predominantly in neutrophils and it catalyzes the

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reaction between chloride and hydrogen peroxide to form hypochlorous acid which is extremely pathogenic in IBD causing impairment in cell membrane stability and death of the cells by lipid peroxidation [39, 40]. The inflammatory mediator TNF-α is overproduced via activation of leukocytes in IBD and plays an important signaling role in the subsequent inflammation reactions which result in the production of peroxide anions and oxygen/nitrogen radicals causing colonic tissue damage [35]. The significantly higher levels of the inflammatory mediators, MPO

21

ACCEPTED MANUSCRIPT enzyme and TNF-α, indicates the incidence of acute inflammation and confirms the histologically observed leukocyte infiltration in the colitis group. The levels of MPO and TNF-α in the AZA-treated group were significantly higher (p < 0.05 and p < 0.01, respectively) than their levels in the normal group. However, as compared to

T

the colitis group, MPO had significantly lower level (p < 0.05), whereas the difference between

IP

the levels of the TNF-α in the AZA-treated group and the colitis group was not statistically

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significant (p > 0.05) (Figures 12a and 12b). This corroborates the moderate leukocyte infiltration observed in the submucosa of the colons of this group and indicates presumably the

US

delayed onset of anti-inflammatory action of the systemically absorbed AZA (i.e. high intestinal

AN

absorption of the drug) [9, 41].

In the AZA-loaded beads treated group, the levels of MPO and TNF-α were decreased to

M

comparable levels as in the normal group, explaining the rapid onset of anti-inflammatory effect

ED

of the targeted AZA-loaded beads vs. the free drug (Figures 12a and 12b). SOD and CAT enzymes play an important role in the cellular antioxidant defense

PT

mechanism against reactive oxygen species, which can initiate extensive pathological changes in

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the cellular lipids, proteins and DNA [40, 42]. The antioxidant enzymes SOD and CAT were consumed considerably due to the oxidative stress in the colons of the animals in the colitis

AC

group and their activities were significantly lower (p < 0.001 and p < 0.01, respectively) than those in the normal group (Figures 12c and 12d). The activities of SOD and CAT in the AZA-treated group were significantly lower than their levels in the normal group (p < 0.001 and p < 0.05, respectively). The activity of SOD was significantly higher as compared to the colitis group (p < 0.01), whereas no significant difference was observed for the CAT enzyme (p > 0.05) (Figures 12c and 12d). Thus, the administration of

22

ACCEPTED MANUSCRIPT free AZA drug had minimal curative effect on acetic acid-induced acute ulcerative colitis in the present short term study [7, 43]. In the AZA-loaded beads treated group, CAT activity was raised to a comparable level as in the normal group (p > 0.05). The SOD activity was significantly lower than the level in the

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normal group (p < 0.01), but higher (p < 0.001) than the enzyme level in the colitis group

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(Figures 12c and 12d). The restored activity of the antioxidant enzymes indicates the ability of

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the AZA-loaded beads to minimize the oxidative damage of colonic tissues as a consequence of

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the acetic acid-induced inflammation cascade.

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4. Conclusions

Azathioprine was loaded into chitosan beads, and the beads were filled into acid-resistant

M

capsules for selective drug delivery to the disease sites of acetic acid-induced colitis rabbit

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model. Uniform beads with high drug loading efficiency were prepared, and the beads have demonstrated a pH-sensitive release pattern to minimize drug loss in the non-targeted

PT

gastrointestinal segments. In vivo, improved therapeutic outcomes were observed in the animals

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treated with the azathioprine-loaded beads, as compared to the untreated animal controls and the animals treated with azathioprine free drug, based on the clinical activity score, index of tissue

AC

edema, mortality rate, colon macroscopic score, colon histopathological features, and levels of the inflammatory mediators and antioxidant enzymes. The AZA-loaded beads developed in the current study might have a great potential in the management of IBD.

23

ACCEPTED MANUSCRIPT 5. References [1] G.-T. Ho, C. Lees, J. Satsangi, Ulcerative colitis, Medicine, 35 (2007) 277-282. [2] I. Ordás, L. Eckmann, M. Talamini, D.C. Baumgart, W.J. Sandborn, Ulcerative colitis, Lancet, 380 (2012) 1606-1619.

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[3] A. Dignass, J.O. Lindsay, A. Sturm, A. Windsor, J.-F. Colombel, M. Allez, G. D'Haens, A.

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D'Hoore, G. Mantzaris, G. Novacek, T. Öresland, W. Reinisch, M. Sans, E. Stange, S. Vermeire,

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S. Travis, G. Van Assche, Second European evidence-based consensus on the diagnosis and management of ulcerative colitis Part 2: Current management, J.Crohns Colitis, 6 (2012) 991-

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[4] C. Mowat, A. Cole, A. Windsor, T. Ahmad, I. Arnott, R. Driscoll, S. Mitton, T. Orchard, M. Rutter, L. Younge, Guidelines for the management of inflammatory bowel disease in adults, Gut,

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[5] B.I. Eklund, M. Moberg, J. Bergquist, B. Mannervik, Divergent Activities of Human Glutathione Transferases in the Bioactivation of Azathioprine, Mol. Pharmacol. 70 (2006) 747-

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[6] S. Schroll, A. Sarlette, K. Ahrens, M.P. Manns, M. Göke, Effects of azathioprine and its metabolites on repair mechanisms of the intestinal epithelium in vitro, Regul. Peptides, 131

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[7] C.G. Su, R.B. Stein, J.D. Lewis, G.R. Lichtenstein, Azathioprine or 6-mercaptopurine for inflammatory bowel disease: do risks outweigh benefits?, Digest. Liver Dis. 32 (2000) 518-531. [8] J.B. Epstein, M. Gorsky, M.S. Epsteinc, S. Nantel, Topical azathioprine in the treatment of immune-mediated chronic oral inflammatory conditions: A series of cases, Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod. 91 (2001) 56-61.

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ACCEPTED MANUSCRIPT [9] E.C. Van Os, B.J. Zins, W.J. Sandborn, D.C. Mays, W.J. Tremaine, D.W. Mahoney, A.R. Zinsmeister, J.J. Lipsky, Azathioprine pharmacokinetics after intravenous, oral, delayed release oral and rectal foam administration, Gut, 39 (1996) 63-68. [10] J. Fallingborg, L.A. Christensen, B.A. Jacobsen, S.N. Rasmussen, Very low intraluminal

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disease: possible determinants and implications for therapy with aminosalicylates and other drugs, Gut, 48 (2001) 571-577.

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[12] K. Kaur, K. Kim, Studies of chitosan/organic acid/Eudragit® RS/RL-coated system for

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[13] E.L. McConnell, H.M. Fadda, A.W. Basit, Gut instincts: explorations in intestinal

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[14] J. Berger, M. Reist, J.M. Mayer, O. Felt, N.A. Peppas, R. Gurny, Structure and interactions in covalently and ionically crosslinked chitosan hydrogels for biomedical applications, Eur. J.

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Pharm. Biopharm. 57 (2004) 19-34.

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[15] W.S. Omwancha, R. Mallipeddi, B.L. Valle, S.H. Neau, Chitosan as a pore former in coated beads for colon specific drug delivery of 5-ASA, Int. J. Pharm. 441 (2013) 343-351.

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[16] T. Cerchiara, A. Abruzzo, C. Parolin, B. Vitali, F. Bigucci, M.C. Gallucci, F.P. Nicoletta, B. Luppi, Microparticles based on chitosan/carboxymethylcellulose polyelectrolyte complexes for colon delivery of vancomycin, Carbohyd. Polym. 143 (2016) 124-130. [17] H.H. Gadalla, G.M. Soliman, F.A. Mohammed, A.M. El-Sayed, Development and in vitro/in vivo evaluation of Zn-pectinate microparticles reinforced with chitosan for the colonic delivery of progesterone, Drug Deliv. 23 (2016) 2541-2554.

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ACCEPTED MANUSCRIPT [18] M. Drechsler, G. Garbacz, R. Thomann, R. Schubert, Development and evaluation of chitosan and chitosan/Kollicoat® Smartseal 30 D film-coated tablets for colon targeting, Eur. J. Pharm. Biopharm. 88 (2014) 807-815. [19] N. Shimono, T. Takatori, M. Ueda, M. Mori, Y. Nakamura, Multiparticulate chitosan-

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dispersed system for drug delivery, Chem. Pharm. Bull. 51 (2003) 620-624.

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[20] S.A. Nour, N.S. Abdelmalak, M.J. Naguib, Novel chewable colon targeted tablets of

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bumadizone calcium for treatment of ulcerative colitis: Formulation and optimization, J. Drug Deliv. Sci. Technol. 35 (2016) 172-183.

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[21] H.S. Farghaly, R.H. Thabit, L-arginine and aminoguanidine reduce colonic damage of acetic

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acid-induced colitis in rats: potential modulation of nuclear factor-kappaB/p65, Clin. Exp. Pharmacol. P. 41 (2014) 769-779.

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[22] G. Hartmann, C. Bidlingmaier, B. Siegmund, S. Albrich, J. Schulze, K. Tschoep, A. Eigler,

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H.A. Lehr, S. Endres, Specific type IV phosphodiesterase inhibitor rolipram mitigates experimental colitis in mice, J. Pharmacol. Exp.Ther. 292 (2000) 22-30.

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[23] A. Lamprecht, U. Schäfer, C.-M. Lehr, Size-Dependent Bioadhesion of Micro- and

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Nanoparticulate Carriers to the Inflamed Colonic Mucosa, Pharmaceut. Res. 18 (2001) 788-793. [24] G.P. Morris, P.L. Beck, M.S. Herridge, W.T. Depew, M.R. Szewczuk, J.L. Wallace,

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Hapten-induced model of chronic inflammation and ulceration in the rat

colon,

Gastroenterology, 96 (1989) 795-803. [25] M. Nand Singh, H. KS Yadav, M. Ram, H. G Shivakumar, Freeze dried chitosan/poly(glutamic acid) microparticles for intestinal delivery of lansoprazole, Curr. Drug Deliv. 9 (2012) 95-104.

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ACCEPTED MANUSCRIPT [26] J. Ko, H.J. Park, S. Hwang, J. Park, J. Lee, Preparation and characterization of chitosan microparticles intended for controlled drug delivery, Int. J. Pharm. 249 (2002) 165-174. [27] S.M. Silva, C.R. Braga, M.V. Fook, C.M. Raposo, L.H. Carvalho, E.L. Canedo, Application of infrared spectroscopy to analysis of chitosan/clay nanocomposites, In: T. Theophile (Ed.),

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Infrared spectroscopy-materials science engineering and technology, InTech, Rijeka , 2012, pp.

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[28] A.F. Martins, D.M. de Oliveira, A.G.B. Pereira, A.F. Rubira, E.C. Muniz, Chitosan/TPP microparticles obtained by microemulsion method applied in controlled release of heparin, Int. J.

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Biol. Macromol. 51 (2012) 1127-1133.

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[29] D.R. Bhumkar, V.B. Pokharkar, Studies on effect of pH on cross-linking of chitosan with sodium tripolyphosphate: a technical note, Aaps Pharmscitech, 7 (2006) E138-E143.

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[30] W.P. Wilson, S.A. Benezra, Azathioprine, In: K. Florey (Ed.), Analytical Profiles of Drug

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Substances, Academic Press, New York, 1981, pp. 29-53. [31] F. Kittur, K.H. Prashanth, K.U. Sankar, R. Tharanathan, Characterization of chitin, chitosan

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and their carboxymethyl derivatives by differential scanning calorimetry, Carbohyd. Polym. 49

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(2002) 185-193.

[32] Y. Dong, S.-S. Feng, Poly (d, l-lactide-co-glycolide)/montmorillonite nanoparticles for oral

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delivery of anticancer drugs, Biomaterials, 26 (2005) 6068-6076. [33] M. Marzorati, S. Possemiers, A. Verhelst, D. Cadé, N. Madit, T. Van de Wiele, A novel hypromellose capsule, with acid resistance properties, permits the targeted delivery of acidsensitive products to the intestine, LWT - Food Sci. Technol. 60 (2015) 544-551. [34] X. Shu, K. Zhu, Controlled drug release properties of ionically cross-linked chitosan beads: the influence of anion structure, Int. J. Pharm. 233 (2002) 217-225.

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ACCEPTED MANUSCRIPT [35] C. Zeng, J.-H. Xiao, M.-J. Chang, J.-L. Wang, Beneficial Effects of THSG on Acetic AcidInduced Experimental Colitis: Involvement of Upregulation of PPAR-γ and Inhibition of the NfΚb Inflammatory Pathway, Molecules, 16 (2011) 8552-8568. [36] K. Geboes, Histopathology of Crohn’s disease and ulcerative colitis, In: J. Satsangi, L.R.

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Sutherland (Eds.), Inflammatory bowel disease, Churchill Livingstone, London, 2003, pp. 255-

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[37] T.C. DeRoche, S.-Y. Xiao, X. Liu, Histological evaluation in ulcerative colitis, Gastroenterol. Rep. (Oxf), 2 (2014) 178-192.

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[38] K. Liu, P.-c. Liu, R. Liu, X. Wu, Dual AO/EB staining to detect apoptosis in osteosarcoma

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cells compared with flow cytometry, Med. Sci. Monit. Basic Res. 21 (2015) 15-20. [39] A. Cetinkaya, E. Bulbuloglu, E.B. Kurutas, H. Ciralik, B. Kantarceken, M.A. Buyukbese,

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Beneficial Effects of N-Acetylcysteine on Acetic Acid-Induced Colitis in Rats, Tohoku J. Exp.

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Med. 206 (2005) 131-139.

[40] J.C. Fantone, P.A. Ward, Role of oxygen-derived free radicals and metabolites in leukocyte-

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dependent inflammatory reactions, Am. J. Pathol. 107 (1982) 395-418.

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[41] C. Cuffari, S. Hunt, T. Bayless, Enhanced bioavailability of azathioprine compared to 6‐mercaptopurine therapy in inflammatory bowel disease: correlation with treatment efficacy,

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Aliment. Pharm. Therap. 14 (2000) 1009-1014. [42] W. Beyer, J. Imlay, I. Fridovich, Superoxide dismutases, Prog. Nucleic acid Re. 40 (1991) 221-253. [43] I.D.R. Arnott, D. Watts, J. Satsangi, Azathioprine and anti-TNFα therapies in Crohn’s disease: a review of pharmacology, clinical efficacy and safety, Pharmacol. Res. 47 (2003) 1-10.

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ACCEPTED MANUSCRIPT Figures

30

0.4:1 0.8:1

20

4:1

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10

8:1

0 0

1

2

3

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30

20

10

0

2

3

1.5% 2% 2.5% 3% 3.5%

4

Time (h)

PT Drug release (%)

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(c)

1

ED

0

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Drug release (%)

(b)

4

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Time (h)

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Drug release (%)

(a)

0.2:1

30

0.5:1 20

0.8:1 1:1

10

1.4:1 0 0

1

2

3

4

Time (h)

Figure 1. Release profiles of AZA-loaded beads prepared at different STPP:chitosan ratios (a), chitosan concentrations (b) and AZA:chitosan ratios (c). Release medium was phosphate buffer (pH 6.8). Data are presented as mean ± SD. 29

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ACCEPTED MANUSCRIPT

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Figure 2. Scanning electron microscopy micrographs of the unloaded beads at 15× (a), 50× (b)

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PT

ED

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and 1000× (c), and AZA-loaded beads at 15× (d), 50× (e) and 1000× (f).

30

ED

M

AN

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ACCEPTED MANUSCRIPT

AC

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PT

Figure 3. FT-IR spectra of chitosan (a), unloaded beads (b), AZA (c) and AZA-loaded beads (d).

31

ED

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AN

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ACCEPTED MANUSCRIPT

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Figure 4. DSC thermograms of chitosan (a), unloaded beads (b), AZA (c) and AZA-loaded

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beads (d).

32

ACCEPTED MANUSCRIPT

100

80 pH 4.0

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60

pH 7.2

40

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Drug release (%)

pH 1.2 pH 6.8

pH 4.0

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20

0

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0 6

2

12

18

pH 7.2

24

30

ED

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Time (h)

Figure 5. Release profiles of acid-resistant capsules filled with AZA-loaded beads using 0.1 N

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HCl at pH 1.2 for 2 h, then phosphate buffer pH 6.8 for 4 h and then either acetate buffer at pH

AC

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4.0 or phosphate buffer at pH 7.2 for 24 h. Data are shown as mean ± SD.

33

ACCEPTED MANUSCRIPT (a)

***

**

9

T

6

be

ad s

ZA A

AN

A

ZA

-lo

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N

C

ol

or m

iti s

al

0

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IP

3

ad ed

Clinical activity score

12

(b)

M

PT

0.2

*

*

ED

0.3

0.1

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Index of tissue edema

0.4

ad s

ZA

be

A A

ZA

-lo

ad ed

C

ol

iti s

al m or N

AC

0.0

Figure 6. Clinical activity scores (a) and indices of tissue edema (b) of the normal group, the colitis group, the AZA-treated group and the AZA-loaded beads treated group. Data are shown as mean ± SD * p < 0.05, ** p < 0.01, *** p < 0.001 as compared to the normal group. 34

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AN

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ACCEPTED MANUSCRIPT

Figure 7. Photographs of the colons of the normal group (a), the colitis group (b), the AZA-

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treated group (c) and the AZA-loaded beads treated group (d).

35

ACCEPTED MANUSCRIPT

6

**

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IP

T

4

ds be a

A

s

ol i ti C

ed -lo ad ZA A

PT

ED

M

N

or m al

0

ZA

US

2

AN

Macroscopic score

***

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Figure 8. Macroscopic scores of the normal group, the colitis group, the AZA-treated group and the AZA-loaded beads treated group. Data are shown as mean ± SD ** p < 0.01, *** p < 0.001

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as compared to the normal group.

36

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AN

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ACCEPTED MANUSCRIPT

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Figure 9. Photomicrographs of paraffin sections in the colons of the rabbits of the normal group (a), the colitis group (b), the AZA-treated group (c) and the AZA-loaded beads treated group (d).

AC

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Original magnification, 100×, scale bar = 200 µm, hematoxylin and eosin stain.

37

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AN

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ACCEPTED MANUSCRIPT

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Figure 10. Photomicrographs of paraffin sections in the colons of the rabbits of the normal group (a), the colitis group (b), the AZA-treated group (c) and the AZA-loaded beads treated group (d).

AC

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Original magnification, 100×, scale bar = 200 µm, periodic acid Schiff and hematoxylin stain.

38

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AN

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ACCEPTED MANUSCRIPT

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Figure 11. Photomicrographs of paraffin sections in the colons of the rabbits of the normal group

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(a), the colitis group (b), the AZA-treated group (c) and the AZA-loaded beads treated group (d).

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Original magnification, 100×, scale bar = 200 µm, acridine orange stain.

39

ACCEPTED MANUSCRIPT

(a)

(b) MPO

TNF-alpha

***

25

*** **

5

0

or m al

ad ed

C

CR US

AZ Alo

ad ed

N

AZ A

s C ol

iti

al or m N

be ad s

0

be ad s

T

10

AZ A

5

15

IP

*

ol iti s

pg/g tissue

U/g tissue

20

10

AZ Alo

15

(d)

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(c)

M

SOD 0.4

CAT 15

be ad s

AZ A

AC

AZ Alo

ad ed

C

ol iti s

0

or m al

be ad s

*

5

AZ Alo

CE

ad ed

iti ol C

N

or m

al

s

0.0

AZ A

***

0.1

**

10

N

**

***

U/g tissue

ED

0.2

PT

U/g tissue

0.3

Figure 12. Determination of MPO enzyme activity (a), TNF-α concentration (b), CAT enzyme activity (c) and SOD enzyme activity (d), in the colons of the normal group, the colitis group, the AZA-treated group and the AZA-loaded beads treated group. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 as compared to the normal group.

40

ACCEPTED MANUSCRIPT Tables Table 1. Compositions of the different AZA-loaded beads. In B1–B4, the effect of STPP (sodium tripolyphosphate):chitosan ratio was studied. In B3, B5–B8, the effect of chitosan

Chitosan (%)

B1

0.4:1

2

B2

0.8:1

2

B3

4:1

2

B4

8:1

2

B5

4:1

B6

4:1

B7

4:1

B8

4:1

B9

4:1

B10

4:1

B11

4:1

B12

4:1

0.5:1 0.5:1

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CR

0.5:1

0.5:1

1.5

0.5:1

2.5

0.5:1

3

0.5:1

3.5

0.5:1

2.5

0.2:1

2.5

0.8:1

2.5

1:1

2.5

1.4:1

AN M ED

AZA:chitosan

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STPP:chitosan

AC

CE

PT

Formulation

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concentration was studied. In B6, B9–B12, the effect of AZA:chitosan ratio was evaluated.

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ACCEPTED MANUSCRIPT Table 2. Average Feret’s diameter, circularity and roundness of the different AZA-loaded beads. Average Feret’s Formulation

Circularity ± SD

Roundness ± SD

diameter (mm) ± SD 1.89 ± 0.17

0.83 ± 0.09

0.60 ± 0.13

B2

1.45 ± 0.07

0.84 ± 0.07

0.58 ± 0.11

B3

1.29 ± 0.07

0.92 ± 0.03

B4

1.34 ± 0.06

0.93 ± 0.02

B5

1.44 ± 0.19

0.79 ± 0.07

B6

1.35 ± 0.05

0.94 ± 0.02

0.76 ± 0.07

B7

1.42 ± 0.08

0.95 ± 0.02

0.76 ± 0.06

B8

1.56 ± 0.17

0.87 ± 0.08

0.79 ± 0.10

B9

1.19 ± 0.08

0.96 ± 0.02

0.88 ± 0.06

B10

1.31 ± 0.06

B11

1.55 ± 0.06

B12

2.06 ± 0.11

0.73 ± 0.08 0.79 ± 0.10 0.69 ± 0.14

0.92 ± 0.03

0.70 ± 0.06

0.93 ± 0.02

0.74 ± 0.07

0.83 ± 0.05

0.75 ± 0.09

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ACCEPTED MANUSCRIPT Table 3. Yield (%), drug loading capacity (%) and encapsulation efficiency (%) of the different AZA-loaded beads. Encapsulation Formulation

Yield (%)

Drug loading (%) ± SD

97.5

23.9 ± 0.3

B2

82.1

24.3 ± 0.4

B3

37.2

23.2 ± 0.5

B4

21.9

23.7 ± 0.4

98.6 ± 1.8

B5

41.6

19.9 ± 0.6

90.9 ± 2.9

B6

36.9

24.3 ± 0.5

98.8 ± 1.9

B7

34.3

25.2 ± 0.6

95.3 ± 2.3

B8

39.2

23.0 ± 0.8

99.4 ± 3.5

B9

35.1

8.4 ± 0.1

77.1 ± 0.7

B10

40.6

30.8 ± 1.4

90.7 ± 4.1

B11

45.0

36.3 ± 0.5

98.0 ± 1.3

B12

53.5

40.4 ± 0.7

98.8 ± 1.7

88.6 ± 1.3 91.7 ± 1.4 94.9 ± 2.0

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efficiency (%) ± SD

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ACCEPTED MANUSCRIPT

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Graphical abstract

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