Studies of in vitro Evaluation and Formulation of Aceclofenac Loaded PLGA...

[Full title: Studies of in vitro Evaluation and Formulation of Aceclofenac Loaded PLGA Microspheres]

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Life International Journal of Pharmacology ISSN 1811-7775 Life science alert ansinet Asian Network for Scientific Information

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International Journal of Pharmacology 6 (5): 726-731, 2010 ISSN 1811-7775 2010 Asian Network for Scientific Information Studies of in vitro Evaluation and Formulation of Aceclofenac Loaded PLGA Microspheres 12V. Gupta, S.K. Shukla, 'S.M. Shrivastava, S. Shukla, K. Kumar. 4D.P. Saxena, B. Shrivastava and 'M. Chaudhary 'Venus Medicine Research Centre, Hill Top Industrial Estate, Jharmajri EPIP, Phase 1 (Extn) Bhatoli Kalan, Baddi - 173, 205, India Department of Pharmaceutical Sciences, Jaipur National University, Jaipur, India ³I.C. Bose Institute of Life Science, Bundelkhand University, Jhansi, LP, India Kumaun University, Nainital, Uttrakhand, India Abstract: The aim of this research was to study the influence of formulation parameters in the preparation of sustained release aceclofenac loaded PLGA microspheres by emulsion solvent diffusion technique. The methods used in this components and their concentration necessary for organogels formation were evaluated using phase diagram Solubility of aceclofenac was determined, Characterization of Poly (DL-lactide)-coglycolide (PLGA) polymer, solubility assessment of aceclofenac, drug-excipients compatibility studies, in vitro analytical method development, preparation of aceclofenac-loaded PLGA microspheres, characterization of the formulations. Prepared microspheres were optimized and evaluated for different parameters and best formulation was subjected to in vitro drug release studies. The prepared microspheres were while, free-flowing and almost. spherical in shape. In vitro drug release studies were carried out up to 24 h in three different pH media, i.e.. 0.1 N HCl (pH 1.2), phosphate buffer (pH 6.8) and phosphate buffer (pH 7.4). The drug-polymer concentration of dispersed phase influences the particle size and drug release properties. Thru shell it may be concluded that. sustained release aceclofenac microspheres can be successfully prepared and used parenterally with increased therapeutic value and reduced side effects. Key words: Tween80, solvent evaporation, toxicity, biodegradable, excipients Aceclofenac, INTRODUCTION amino] 2-(2.6-dichlorophenyl) phenylacetoxyacetic acid, a novel anti-inflammatory drug of choice in the treatment of osteoarthritis, rheumatoid authritis and ankylosing spondylitis. Researchers have attempted development of oral drug delivery systems for aceclofenac (Yong et al., 2005). The chronic oral administration of aceclofenac tends to cause severe gastric irritation (Tessari et al., 1995). Topical administration of accclofenac offers the advantage of enhanced drug delivery to the affected areas, by passing gastric irritation. The clinically efficiency have evaluated of topical accclofenac cream (1.5% w/w) in patients (Yang et al., 2002). The formulation showed improved therapeutic efficacy. In the formulated micro-emulsion containing aceclofenac (3%w/w) for topical delivery (Yamazaki et al., 1997). This action is supposed to mediate via its intracellular conversion to active metabolites namely diclofenac, the cyclooxygenase 1 and 2 inhibitor and 4-hydroxydiclofenac, the cyclooxygenase-2 inhibitor (Akimoto el al.. 2000). IL also stimulates glycosaminoglycan synthesis in human osteoarthritic cartilage by inhibition of interleukin-1 beta (IL-1SS) and suppresses cartilage degeneration by inhibiting IL-1SS mediated promatrix metalloproteinase production and proteoglycan release (Gowda et al., 2006). Aceclofenac is especially well-tolerated. among the non-steroidal anti-inflammatory drugs and have lower incidence of gastrointestinal (gi) adverse effects (Raber et al., 2007). It also have lower incidence of gi bleeding, abdominal pain and arterial hypertension than meloxicam and a lower incidence of gi bleeding, abdominal pain, liver toxicity. thromboembolic cardiovascular events, arterial hypertension and edema than rofecoxib (Brogden and Wiseman, 1996). Aceclofenac is indicated and widely used for the treatment of rheumatoid arthritis, osteoarthritis. ankylosing spondylitis and scapulohumeral periarthritis (El-Kousy, 1999; Najib et al., 2004). The usual dose of aceclofenac is 100 mg twice daily (Kim et al., 2001) and it is absorbed rapidly when taken orally and its analgesic effects begin within 30 min of administration Corresponding Author: Varun Gupta, Department of Pharmaceutical Sciences, Jaipur National University, Jaipur, India 726

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Int. J. Pharmacol., 6 (5): 726-731, 2010 (Dashora et al., 2006). Following oral dose, it reaches a peak plasma concentration within 1-3 h (Patil and Kasture, 2007). Since, aceclofenac is indicated for various pains and inflammatory conditions, its accelerated onset of action and sustained release along with decreased incidence of dose related toxicity is desirable. Keeping in mind the above requirements we prepared accelofenac loaded Poly (DL-lactide-co-glycolide (PLGA) microspheres, characterized and evaluated for drug release pattern. PLGA is biodegradable and biocompatible polymer and widely used in controlled release drug delivery systems. Usual therapeutic dose is 100 mg twice daily and half life is 3-4 h; thus it is necessary to be administered frequently in order to maintain the desired concentration. Therefore. accclofenac 21.1 ideal candidate for sustained release formulation, resulting in more reproducible drug absorption and reducing the risk of local irritations compared to single dosage forms. 'w The solvent evaporation method is popularly used for microsphere preparation because of its simplicity. reproducibility and fast processing with minimum controllable process variables that can be easily implemented at the industrial level (Kim et al., 2002; Dashora et al., 2006). But it is frequently used for water-insoluble drugs, as the entrapment efficiency of water-soluble drugs is low due to drug loss from the organic emulsified polymeric phase before solidification of polymer in The microspheres Niwa et al., 1993, 1991). The objective of this study was to encapsulate aceclofenac with biodegradable. Polymer rosin, the effect of different formulation variables such as concentration of drug, Polymer, polyvinyl alcohol and solvent, the effect of these variables on particle size distribution, encapsulation efficiency and its in vitro release behavior. The drug was targeted to the colon and their aligned area for their local cffect. MATERIALS AND METHODS Chemicals: Aceclofenac and Poly (DL-lactide)-coglycolide (PLGA) were provided (2009) by Venus Medicine Research Center (VMRC), Baddi, H.P.. India. Acetonitrile was IIPLC grade and all other chemicals used were of analytical grade. Characterization of Poly (DL-lactide)-co-glycolide (PLGA) polymer PLGA polymer was characterized by gel permeation chromatography for its molecular weight and molecular weight distribution with mixed bed column (SDV 10, 8X300, PSS polymer standardservice GmbH) using chloroform (Injection volume: 100 µL) as mobile phase at 1 ml min flow rate for 15 min and 30°C using Refractive index detector and GPC for Class-Vp software as analyser. Solubility assessment of aceclofenac: The solubility of the drug was assessed in Ethyl acetale, Dichloromethane and Benzyl alcohol by serial addition of the known amount of drug into these solvents and observing visually for complete solubilisation. Drug-excipients compatibility studies: Excipients are integral components of almost all pharmaceutical dosage forms thus it is mandatory to delect any possible physical or chemical interaction of the drug with the excipients since the excipient can affect the bioavailability and stability of the drug. The drug and the excipients must be compatible with one another to produce a product that is stable, efficacious, attractive, easy to administer and safe. If the excipients are new and have not been used in formulations containing the active substance, the compatibility studies have a considerable importance. In vitro analytical method development: Different concentrations of aceclofenac were prepared from drug stock solution (50 µg m1. ') prepared in phosphate buffer pH 7.1 and analytical parameters were determined using Reverse Phase High-performance liquid chromatography (RP-HPLC, Waters) system consisting of a photodiode array detector. The column was a reverse phase (Inertsil ODS C18, 250×4.6 mm, 5 µm). The mobile phase was acetonitrile/10 mM phosphate buffer (pII 7.0) (10:60, v/v) and the flow rate was 1 mL/1 min. The temperature of the system was maintained at room temperature and the detection wavelength was 271 nm. PLGA Preparation of Accclofenac-loaded Microspheres PLGA was first dissolved in ethyl acetale (2 mL) and aceclofenac was further dissolved in polymer solution. This polymer-drug solution was then added to Poly Vinyl Acetate (PVA) surfactant solution under continuous stirring at different speeds to obtain a primary emulsion in aqueous phase. Ethyl acetate was then allowed to evaporate. The solid microspheres were washed thoroughly using distilled water, passed through sieve (BSS #120), freeze dried and evaluated. Characterization of the formulations Drug content in microspheres: Microspheres (5 mg) were dissolved in 5 mL of Dichloromethane and then evaporated under stream of nitrogen. To this 10 mL acetonitrile was added. One milliliter of this solution was then diluted to 3 mL, and filtered through 0.22 µ filter paper. The amount of drug in the test sample was determined by RP-HPLC method. 727

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Int. J. Pharmacol., 6 (5): 726-731, 2010 The drug loading capacity and encapsulation efficiency of the microspheres were calculated according to the following equations: Loading capacity Total drug in microsphere Total weight of microsphere Entrapment Ef ficiency = Total drug in microsphere Total weight of microsphere Particle size measurements: Particle sizes of microspheres were determined using scanning optical microscopy technique. Drug content analysis: Ten milligram accurately weighed portion of microspheres were taken in a clean 100 mL volumetric flask and dissolved in about 2 mL of acetone and the volume was made up to the mark with buffer pII 7.1. After filtration and dilution, samples were analyzed spectrophotometrically and the amounts of drug encapsulated in the microspheres were calculated. The drug content of each sample was determined in triplicate and the results were averaged. The entrapment efficiency of microspheres was calculated by dividing the actual drug content to the theoretical drug content of microspheres. Redispersability: Prepared sodium carboxymethyl cellulose (0.5% w/v) in buffer pH 7.0 To 20 mL of sodium CMC solution SPAN 80 solution (10 mL, 1% in buffer pH 7.0) and NaCl (170 mg) was added and volume was made 100 mL in buffer pII 7.1. 300 mg of microspheres were taken and to this 1 mL of dispensing fluid was added. The microspheres were hand shaken and a small amount of sample was taken on a slide and observed. under microscope. Injectability: Microspheres were redispersed in the dispensing fluid (30% w/v) and passed through 22 G needle to determine the injectability behaviour of the microspheres. In vitro drug release studies: In vitro release of STP from microspheres was evaluated in phosphate buffer (pII 7.4). Amount of microspheres equivalent to 20 mg of STP were transferred to the prewarmed dissolution media (20 mL) and maintained at 37±0.5°C under stirring at 50 rpm. Samples were withdrawn every hour up to 6 hours and the volume was replaced immediately by fresh phosphate buffer. The sample withdrawn was centrifuged (3000 rpm, 15 min). The supermalant solution was filtered and analyzed for STP content by measuring absorbance in a LV-spectrophotometer (Shimadzu UV-1700, Pharmaspec) at 229.5 mm by the first-derivative spectrophotometric method (Saudagar et al., 2007) using phosphate buffer (pH 7.4) as blank. Results were expressed as mean±SD of 3 experiments. Statistical models are extensively used in diversified areas to strengthen the art of drug formulation (Dubey and Parikh, 2004; Govender et al., 2005). RESULTS Characterization of poly (DL-lactide)-co-glycolide (PLGA) polymer: Unimodal and symmetrical GPC trace was obtained (Fig. 1) thus confirming the purity of the polymer. The polymer PLGA was having weight average molecular weight of 130 kDa, number average molecular of 76 kDa and polydispersity of 1.7 which was sufficient enough for current application. Optimization of drug: Polymer concentration, PVA concentration and speed are shown in Table 1. Solubility studies of accclofenac revealed soloubility of aceclofenac in following order Benzyl alcohol Ethyl acetate Dichloromethane and are shown in Table 2. In vitro analytical method development by validation parameters for calibration over a range of 1 to 50 mg mL is summarized. The rapid diffusion of acetone into the aqueous phase causes a remarkable decrease in interfacial tension between the organic and aqueous phase and hence finer microspheres are obtained Niwa et al., 1991). At least one stabilizer is necessary for microsphere formation and suspension stabilization. Tween 20, Tween 40 and Tween 80 were used as surfactants to prepare blank microspheres; however, using Tween 20 and Tween 40, microsphere formation was not achieved successfully independent of their concentration. Microspheres with superior topographical characteristics were obtained when Tween 80 was used as the emulsifier (Fig. 2). This might be due to better emulsification capability of Tween 80 as compared with Tween 20 or Tween 40 (Kibbe, 2000) The pH of Tween 80 aqueous solution is in the range of 6.0 to 6.5 and hence did not warrant. pH adjustment. 0.005- Retension 0.000- -0.005- -0.015 -0.020 0.025 -0.030_ -0.035- -0.040- -0.045- 0 I u 122 ༣ A 5 6 7 8 9 10 11 12 13 14 15 Time (min) Fig. 1: Representative gel permeation chromatogram of PLGA (75: 25) 728

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Int. J. Pharmacol., 6 (5): 726-731, 2010 Table 1: Optimization of drug: polymer concentration, PVA concentration and speed Formularion code Drug (mg) Polymer (mg) Ethyl-acctate PVA Speed Particle size (mL) (%) (rpm) Mcan loading (average diameter, um) capacity (%) Mean entrapment efficiency (%) VI 10 10 2 0.5 500 28.22 22.08 55.12 V 2 10 80 0.5 500 53.28 21.61 50.23 V 3 10 120 0.5 500 98.56 25.26 53.17 V 1 80 10 0.5 500 38.21 26.38 56.11 V 5 80 80 0.5 500 15.21 27.18 57.89 V 6 80 120 0.5 500 78.67 32.12 79.72 V7 120 10 0.5 500 36.26 27.11 67.11 V 8 120 80 0.5 500 98.23 29.18 73.18 V9 120 120 0.5 500 109.10 32.19 79.85 V 10 80 120 0.2 500 101.23 3.1.01 VII 80 120 0.5 500 78.67 32/12 V 12 80 120 0.8 500 69.86 31.91 V 13 80 120 1.0 500 60.93 31.26 V 11 80 120 1.0 500 60.86 31.11 V 15 80 120 1.0 1000 13.65 30.28 V 16 80 120 1.0 1500 32.15 27.31 Table 2: Solubility of aceclofenac in different solvents 70Solvents Solubility (mgm) Dichloromethane <50 60Ethyl acetate <130 Benzyl alcobol <150 Cumulative release (%) 50- 40- 30- 20- 10- 1278 11kV X7, Pm HD39 Fig. 2: Scanning electron micrograph (SEM) of cellulose acetate microspheres % of particles 2- 12- 18- 16- ས་༠idཋ 14- 20-30 30-40 50-60 40-50X Fig. 3: Particle microspheres 60-70X 70-80 Size range (um) 80-90 90-100 100-110 si ze distribution of drug loaded 729 $ 10 15 20 25 30 35 40 Time (h) Fig. 4: In vitro release of acelofenac from microsphere (Salamone and Wodzinski, 1997). In vitro studies showed in this batch of Aceclofenac Loaded PLGA Microsphere showed cumulative percentage release of 10 to 65% respectively for 10 h (Fig. 1). Evaluation of formulation variables: As mentioned previously, the effect of different formulation variables on microspheres properties including entrapment efficiency and particle size distribution were evaluated. Variables studied in this investigation were as follows. Drug polymer ratio, volume of dichloromethane and polyvinyl alcohol concentration in the external phase of the emulsion. Particle size measurements: All the particles of the selected batch V15 were spherical in shape (Fig. 2) and the particle size was in the range of 20- 110 μm (Fig. 3), of which 53% particles were in the range of 30-60 um and

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Int. J. Pharmacol., 6 (5): 726-731, 2010 75% of the particles were in the range of 20-70 μm which is very much suitable for intramuscular parenteral drug delivery system. Redispersability: The microspheres were found to be uniformly dispersed in the dispensing fluid. Further they were present as single particles when observed under microscope. It was also observed that particles were suspended in the fluid for more than 3 min which is very much sufficient for injection. Injectability: The suspension of the microsphere was easily passed through 22 G needle therefore no problem will arise due to injectability properties of the prepared formulations. DISCUSSION This research study indicates that aceclofenac loaded PLGA microspheres can be successfully formulated for parenteral drug delivery of the drug. All the preparation having ratio 1:1 showed good release properties. As the amount of polymer increased, drug release was decreased. This is because smaller the particle size, larger the surface area available for drug release. This may be attributed to the film forming ability of the lipospheres pellet which reduces transepidermal water loss similar to solid lipid nanoparticles (Mei et al., 2003). The major finding of this study can be concluded as aceclofenac loaded PLGA microspheres with mean size of about 54 µm can be prepared using solvent-evaporation technique with PVA as stabilizer and ethyl acetate as solvent. Microspheres belong to the above category and are utilized for controlled drug delivery. Microspheres were prepared by the emulsion solvent evaporation method and evaluated. Accclofenac is a novel anti-inflammatory dog with multidisciplinary therapeutic activity in pain and inflammatory conditions. The loading capacity increases with increase in initial drug polymer ratio. The formulated microspheres can be used for IM delivery of the drug with sustained effect. Dose of the drug is reduced with consequently decrease in dose related toxicity of the drug. ACKNOWLEDGMENTS Authors are Thankful Lo my department of Pharmaceutical Sciences, Jaipur National University. Jaipur and CGM Domestic operations of Venus Remedies Limited for providing the samples of drug, polymer and facility for completion of this research. REFERENCES Akimoto, II., R. Yamazaki, S. IIashimoto, T. Sato and A. Ito, 2000. 4-IIydroxy aceclofenac suppresses the interleukin-1 induced production of promatrix metalloproteinases and release of sulfated glycosaminoglycans from rabbit articular chondrocytes. Eur. J. Pharmacol., 101: 129-136. Brogden, R.N. and L.R. Wiseman, 1996. Aceclofenac. A review of its pharmacodynamic properties and therapeutic potential in the treatment of rheumatic disorders and in pain management. Drugs. 52: 113-124. Dashora, K., S. Saraf and S. Saral, 2006. In-vitro studies of lizanidine controlled release microcapsullar matrices. Pak. J. Pharm. Sci., 19: 177-181. Dubey, R.R. and R.H. Parikh, 2004. Two slage optimization process for formulation of chitosan microspheres. AAPS Pharm. Sci. Tech., 5: 20-28. El-Kousy, V.M., 1999. Spectrophotometric and spectro orimetric determination of stodolac and accclofenac. J. Pharm. Biomed. Anal., 20: 185-194. Govender, S., V. Pillay, D.J. Chetty, S.Y. Essack, C.M. Dangor and T. Govender, 2005. Optimization and characterization of bioadhesive controlled release tetracycline microspheres. Int. J. Pharm.. 306: 24-40. Gowda, K.V., D.S. Rajan, U. Mandal, P.S. Selvan and W.D. Sam Solomon et al., 2006. Evaluation of bioequivalence of two formulations containing 100 mg of aceclofenac. Drug Dev. Indus. Pharm.. 32: 1219-1225. Kibbe, A.II, 2000. Handbook of Pharmaceutical Excipients 3rd Edn., American Pharmaceutical Association Press, Washington, DC. Kim, YG, Y.J. Lee, H.J. Kim, S.D. Lee and J.W. Kwon et al., 2001. Bioequivalence of two aceclofenac tablet formulations after a single oral dose to healthy male Korean volunteers. Int. J. Clin. Pharmacol. Ther., 39: 83-88. Kim, B.K., S.J. Hwang, J.B. Park and H.J. Park, 2002. Preparation and characterization of drug-loaded polymethacrylate microspheres by an emulsion solvent evaporation method. J. Microencapsul 19: 811-882. Mei, Z., H. Chen, T. Weng, Y. Yang and X. Yang, 2003. Solid lipid nanoparticles and microemulsion for topical delivery of triptolide. Eur. J. Pharm. Biopharm.. 56: 189-196. Najib, N., N. Idkaidek, M. Beshtawi, M. Bader, 1. Adinour, S.M. Alam, Q. Zaman and R. Dham, 2004. Bioequivalence evaluation of two brands of aceclofenac 100 mg tablets (Aceclofar and Brista?am) in healthy human volunteers. Biopharm. Drug Dispos, 25: 103-108. 730

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Int. J. Pharmacol., 6 (5): 726-731, 2010 Niwa, T., H. Takeuchi, T. Hino, N. Kunou and Y. Kawashima, 1993. Preparation of biodegradable nano-spheres of water soluble and insoluble drugs with D,L-lactide/glycolide copolymer by a novel spontaneous emulsification solvent diffusion method and the drug release behaviour. J. Control. Release, 25: 89-98. Niwa, T., II. Takeuchi, T. Ilino, N. Kumuo and Y. Kawashima, 1994. In vitro drug release behaviour of DL-Lactide/Glycolide co polymer (PLGA) nanospheres with nafarelin acetate prepared by a novel spontaneous emulsification solvent diffusion method. J. Pharm. Sci., 83: 727-732. Patil, S.S. and P.V. Kasture, 2007. Studies on preparation and evaluation of biodegradable Poly (Laclide-CoGlycolide) microsphere of aceclofenac, CMU J. Nat. Sci., 6: 195-205. Raber, A., J. Heras, J. Costa, J. Fortea and A. Cobos, 2007. Incidence of spontaneous notifications of adverse reactions with accclofenac, meloxicam and rofecoxib during the first year after marketing in the United Kingdom. Ther. Clin. Risk Manage., 3: 225-230. Salamone, P.R and R.J. Woxdzinski, 1997. Production, purification and characterization of a 50-kDa extracellular metalloprotease from Serratia marcescens. Appl. Microbiol. Biotechnol., 48: 317-324. Saudagar, R.B., M. Rawat, D. Singh, S. Saraf and S. Saraf, 2007. Development and validation of derivative spectrophotometric method for determination of serratiopeptidase in pharmaceutical formulation. Oriental. J. Chem., 23: 1057-1060. Tessari, L., L. Ceciliani, A. Belluati, G. Letizia and U. Martorana et al., 1995. Acecloferiae ctca Versus piroxicam cream in the treatment of patients with minor traumas and phlogistic affections of soft tissues: A double-blind study. Curr. Ther. R es.. 56: 702-712. Yamazaki, R., S. Kawai, T. Matsuzaki, N. Kaneda and S. Hashimoto et al, 1997. Aceclofenac blocks prostaglandin E2 production following its intracellular COTIV CTSTOTI i11.0 cyclooxygenase inhibitors. Eur. J. Pharmacol., 329: 181-187. Yang, J.H., K. Young and K.M. Kim, 2002. Preparation and evaluation of aceclofenac microemulsion for transdermal delivery system. Arch. Pharm. Res., 25: 534-540. Yong, CS, Y.K. Oh, K.H. Lee, S.M. Park and Y.J. Park et al., 2005. Trials of clear aceclofenacloaded son capsules with accelerated oral absorption in human subjects. Int. J. Pharm., 302: 78-83. 731

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Rheumatoid arthritis, Adverse effect, Therapeutic efficacy, Topical administration, Osteoarthritis, Analgesic effect, Particle size, Gastric irritation, Ankylosing spondylitis, Therapeutic Activity, Molecular weight, Analytical grade, Tween 80, Non steroidal anti inflammatory drug, Particle size distribution, Sustained release, Mobile phase, Therapeutic dose, Controlled drug delivery, Surfactant, Cartilage Degeneration, Encapsulation efficiency, Entrapment efficiency, Analytical method development, Pharmaceutical dosage form, Drug-polymer ratio, Solubility studies, Drug content, In vitro drug release, Oral drug delivery, Solvent evaporation technique, Dissolution media, Solvent Evaporation Method, Reverse-phase high performance liquid chromatography, Biodegradable polymer, Sustained release formulation, Drug content analysis, Cumulative percentage release, Drug loading capacity, Topical delivery, Biocompatible polymer, Statistical model, HPLC grade, Aceclofenac, Drug-excipients compatibility, Aqueous phase, Diclofenac, Polyvinyl alcohol, Active metabolite, Polymer concentration, Molecular weight distribution, In vitro release behavior, Gastrointestinal adverse effects, Bioavailability and stability, Dose related toxicity, Photodiode array detector, Solid microspheres, PLGA Microspheres, Solubility assessment, Emulsion solvent evaporation, Particle Size Measurement, Water insoluble drug, Local effect, Parenteral Drug Delivery, Gel permeation chromatography, Cyclooxygenase Inhibitor, Injectability, Blank microspheres, Reverse phase, Formulation variable, Water soluble drug, Primary emulsion, Poly Vinyl Acetate, Vitro drug release.