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Journal of Drug Delivery and Therapeutics

Open Access to Pharmaceutical and Medical Research

Copyright   © 2021 The  Author(s): This is an open-access article distributed under the terms of the CC BY-NC 4.0 which permits unrestricted use, distribution, and reproduction in any medium for non-commercial use provided the original author and source are credited

Open Access  Full Text Article                                                                                                                                       Research Article 

Formation development and evaluation of microsphere of sulfasalazine for the treatment inflammatory bowel diseases

Tanu Tiwari, Vivek Jain, Sunil Kumar Jain, Pushpendra Kumar Khangar*

Adina Institute of Pharmaceutical Science, NH86A, Lahdara, Sagar, MP 470001

INTRODUCTION

Pharmaceutical creation and investigate are progressively more focusing on delivery systems which increase attractive therapeutic objectives while diminishing side effects. Oral drug delivery system represents one of the border regions of drug delivery systems. Such a dosage form supervises widespread concern which exists in region of cost-efficient treatment, patient compliance, most favorable drug delivery and bioavailability¹. The last two decades there has been a remarkable improvement in the field of novel drug delivery systems. Carrier skill presents a smart approach for drug delivery by pairing the drug to a carrier particle such as microspheres, nanoparticles, liposomes, etc, which adjusts the release and absorption traits of the drug². Microspheres comprise a significant part of this particulate drug delivery system by virtue of their small size and proficient carrier characteristics. However, the achievement of this new drug delivery system is limited due to their short residence time at the site of absorption. It would so be beneficial to have means for providing an intimate contact of the drug delivery system with absorbing gastric mucosal membranes³. Microspheres are typically free powders consisting of proteins or synthetic polymers that are biodegradable in nature and preferably having a particle size less than 200µm⁴. Inflammatory bowel disease (IBD) is a chronic relapsing and remitting inflammatory disorder of the small intestine and colon. Ulcerative colitis (UC) and Crohn’s disease (CD) are the two main types of IBD^5-7. Colon-targeted drug delivery has been the center of many studies in current years due to its potential to progress treatment of local diseases affecting the colon, while minimizing systemic side effects. A number of instances of disease states which impact the colon include CD, UC and irritable bowel syndrome (IBS) ⁸. A number of the regularly used drugs for the treatment of this illness comprise hydrocortisone, metronidazole, sulfasalazine, dexamethasone, prednisolone and others⁹. The delivery of these drugs purposely to the colon without being absorbed first in the upper (GI) tract allows for an elevated concentration of the drug to arrive at the colon with negligible systemic absorption¹⁰. The colonic substances have a longer retention time (up to 5 days), and the colonic mucosa is known to make easy the absorption of numerous drugs, making this organ an perfect site for drug delivery^10,11. Sulfasalazine (SLZ) is the anti-inflammatory drugs used to treat various IBD such as UC and CD due to induction of T-lymphocyte apoptosis modulates inflammatory mediators. It is poorly absorbed drug with approximately 5-19 hr elimination half-life. Absolute bioavailability of SLZ is <15% when administered orally shown by in vivo. SLZ is a derivative of mesalazine and also a prodrug of 5-aminosalicylic acid that is covalently linked to the antibiotic sulfapyridine by an azo bond^12,13. The reason of the current study was to prepare, optimize and evaluate the colon-targeted microspheres of SLZ for the treatment and management of IBD.

MATERIAL AND METHODS

Reagents and chemicals

Sulfasalazine was kindly provided as a gift sample from Syntho Pharmaceuticals, Lucknow, India. Chitosan,  light   liquid  paraffin,  heavy   liquid  paraffin,  Span   85, lsoproyl  alcohol  and glutaraldehyde was purchased from Hi-Media laboratories Mumbai, India. Double distilled water was prepared freshly and used whenever required. All other ingredients and chemicals used were of analytical grade.

Preformulation studies

Physical characteristics

By visual examination, the drug was identified for physical characters like colour, texture, odour etc.

Solubility

Solubility of the drug was determined by taking some quantity of drug (about 1-2 mg) in the test tube separately and added the 5 ml of the solvent (water, ethanol, methanol, 0.1N HCL, 0.1N NaOH, chloroform and 7.4 pH buffer) Shake vigorously and kept for some time. Note the solubility of the drug in various solvents (at RT).

Melting point determination

Melting point of drug was indomitable by Open capillary method. 

Determination of partition coefficient

50 mg of drug was taken in 3 separating funnels. The separating funnels were shaken for 2 hrs in a wrist action shaker for equilibration. 2 phases were alienated and the quantity of the drug in aqueous phase was analyzed spectrophotometrically. The partition coefficient of the drug in phases was calculated by using formula:

K_PC = Concentration of Drug in Oil Phase/ Concentration of Drug in Water Phase

Determination of λ _max of SLZ

Accurately weighed 10 mg of drug was dissolved in 10 ml of phosphate buffer pH 6.8 solutions in 10 ml of volumetric flask. The resulted solution 1000µg/ml and from this solution 1 ml pipette out and transfer into 10 ml volumetric flask and volume make up with phosphate buffer pH 6.8 solution. Prepare suitable dilution to make it to a concentration range of 2-20μg/ml. The spectrum of this solution was run in 400-800 nm range in U.V. spectrophotometer (ShimadzuUV-1600, Japan). A graph of concentration Vs absorbance was plotted.

FTIR spectroscopy 

Identification of SLZ was done by FTIR spectroscopy with respect to marker compound. Sulfasalazine was obtained as white to brownish powder. It was identified from the result of IR spectrum as per specification. FTIR spectra recorded on KBr disk method using Brukers Alpha Spectrophotometer with IR solution software. Sample powder was systematically mixed by triturating with KBr in a glass mortar with pestle and compressed into disks in a hydraulic press. FTIR spectra of all the samples were recorded over a spectral region from 4700 to 400 cm-1 using 20 scans with 4 cm-1 resolution.

Preparation of SLZ microspheres

The SLZ loaded microspheres were prepared by easy emulsification method followed by cross-linking method. Chitosan solution was prepared by dissolving the 100 mg of chitosan 1% v/v acetic acid (50 ml). The SLZ (100 mg) was added to the disperse phase (chitosan solution). The drug-chitosan solution was extruded through a syringe (No. 20) in liquid paraffin (100 ml, heavy and light, 1 : 1 ratio) containing Span 85 (0.5%), and it was stirred at 1500 rpm using mechanical shaker. After 15 minutes, cross linking agent (v/v aqueous solution) was added and stirring was continued for next 3 hours. The obtained microsphere were filtered and washed with isopropyl alcohol to eliminate traces of oil. They were finally washed with water to eliminate excess of cross linking agent. The microspheres were then dried at 25°C and 60% relative humidity for 24 hrs¹⁴.

Optimization of SLZ microspheres

The SLZ microspheres were optimized by preparing six formulations (Table 1) using different variables such as drug: polymer ration, stirring speed, volume of glutaraldehyde. The resultant particle size, entrapment efficiency and drug release studies were considered for optimization process.

Table 1: Optimization of SLZ microspheres

Characterization of microspheres 

Percentage yield

The prepared microspheres with a size range of 200-300nm were collected and weighed from dissimilar formulations. The measured weight was divided by the total amount of all non-volatile components which were used for the preparation of the microspheres.

Particle size analysis

The particle size was determined by microscopic method.  For each batch of the microsphere, 100 particles were randomly selected using an optical microscope fitted with a camera (Yoko CDD camera, Taiwan) and Medical Pro software (Version 3.0).   

Determination of encapsulation efficiency

Weighed  amount  of   microspheres  were  triturated   with  100  ml   of  phosphate  buffer (pH   6.8).  The resulting mixture was stirred by magnetic stirrer for 2h.  The solution was filtered through a membrane filter (0.45 mm pore size). 1 ml of the filtrate was suitably diluted using phosphate buffer (pH 6.8) and analyzed spectrophotometrically at 359 nm using Shimadzu UV‐Visible spectrophotometer (UV-1600). The EE was calculated using the formula.

In-vitro drug release study

 A  weighed   quantity  of  the   microspheres  was  suspended   in  200  ml   of  phosphate buffer pH 6.8 for 24 hrs using United States Pharmacopoeia basket-type dissolution rate  test   apparatus.  Sample  solution   (5ml)  was  withdrawn   at  predetermined  time intervals  and   filtered  through  whatman   filter  paper.  The samples were diluted suitably and analyzed spectrophotometrically with UV-Visible spectrophotometer (ShimadzuUV-1600, Japan) at 359 nm. 

Morphological characterization of microspheres 

 Scanning  electron   microscopy  is  the   very  adequate  method   for  the  investigation   of surface  morphology  of   the  prepared  microspheres.   The  microsphere  samples   were prepared  by  smattering   the  powder  on   a  double-sided  adhesive   tape  stuck  to  an aluminum  stub.  The   coating  of  gold   to  a  thickness   of  ~300  Å   under  an  argon atmosphere  using   a  gold  sputter   module  in  high-vacuum   evaporator.  The coated samples  were   then  randomly  scanned   and  photomicrographs  were   taken  with  a scanning electron microscope (LEO-430).  

Stability studies

Six  batches  of   optimized  formulation  F-5  were   stored  in  amber   colored  screw capped glass vials in stability chamber at 40±1⁰C and 75%±5 relative humidity, room temperature  and  4±0.51⁰C refrigerator)  for   3  months.  Samples   were  analyzed  for physical   appearance,  residual  drug   content  after  a   period  of  0,   7,  15,  30,   60  and  90 days. Initial drug content was taken as 100 % for each formulation.   

RESULTS AND DISCUSSION

λ _max of SLZ was found to be 359 nm by using U.V. spectrophotometer (Shimadzu-1600, Japan) in linearity range 2-20µg/ml Fig.1 Identification of SLZ was done by FTIR spectroscopy with admiration to marker compound. It was identified from the consequence of IR spectrum as per specification Fig.2.The melting point and partition coefficient of SLZ was found to be 240-242^oC and 2.25 respectively. It is very slightly soluble in ethanol; practically insoluble in diethyl ether, chloroform, and benzene; soluble in aqueous solution of alkali hydroxides; practically insoluble in water. Microspheres of SLZ have been effectively prepared using by easy emulsification method followed by cross-linking method due to high entrapment efficiency. A variety of  variables  (drug:   polymer  ratio,  stirring   speed,  concentration  of   cross-linking agent)  play  an  significant  role   in  the  formulation   of  microspheres  and their characteristics. Percentage yield of dissimilar formulation was determined by weighing the microspheres after drying.The percentage yield of dissimilar formulation was in range of 75.65- 81.25%. The drug entrapment efficacies of dissimilar formulations were in range of 43.47-79.54% w/w. This is because of the mucoadhesion characteristics of chitosan that could make easy the diffusion of part of entrapped drug to surrounding medium during preparation of SLZ microspheres Table 2. On the basis of the utmost percentage yield and drug entrapment was establish to be formulation F-5 in mucoadhesive microspheres so formulation F-5 was further studies. The consequences of measurement of mean particle size of optimized formulation F-5 of mucoadhesive microsphere was found 136.43µm as shown in Table 3. Shape and surface characteristic of SLZ microspheres examine by Scanning Electronic Microscopy analysis. Surface morphology of formulation examines at two different magnifications 55X which illustrate the smooth surface of microspheres Fig. 3. The in vitro drug release studies were performed in simulated colonic fluid (pH 6.8). The quantity of the drug released from the formulation in dissolution medium without rat caecal contents was found to be only 99.78 of F-5. According to ICH guidelines, 3 months accelerated stability study at 40±2°C and 75±5% RH, room temperature  and   4±0.51⁰C refrigerator)  optimized formulations (F-5) was carried out. It showed slight change over time for parameters like appearance and drug content, No noteworthy difference observed in the drug content between initial and formulations stored at 4°C and room temperature for 3 months.

Figure 1 UV spectra of SLZ in phosphate buffer (pH 6.8)

Figure 2 FT-IR spectrum of pure drug (SLZ)

Table 2: Percentage yield and drug entrapment for different formulation

Table 3:  Experimental data (Optimization of particle size, µm)

Figure 3 SEM image of optimized mucoadhesive formulation F-5

Table 4: Release study data of formulation F1-F6

CONCLUSION

Microspheres loaded SLZ have been prepared by easy emulsification method followed by cross-linking method. The variables such as drug: polymer ratio, stirring speed and concentration of glutaraldehyde were optimized on the basis of particle size, entrapment efficiency. The prepared microspheres were stable, spherical particles and showed favorable release profiles in simulated colonic fluid. However, additional evaluation of these carriers can be performed for their probable to treat colonic diseases, as a future scope.

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