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1 Research Article ISSN: M. K. Raval et al. / Journal of Pharmacy Research 010, 3(3), Available online through Preparation and Evaluation of Sustained Release Nimesulide Microspheres Using Response Surface Methodology M. K. Raval *, A. A. Bagda, J. M. Patel, J. S. Paun, K. R. Chaudhari, N. R. Sheth. 1 Department of Pharmaceutical Sciences, Saurashtra University, Rajkot , Gujarat, India. Received on: ; Revised on: ; Accepted on: ABSTRACT A sustained release microparticulate dosage form was prepared by emulsion solvent evaporation method by taking Nimesulide as a model drug using Eudragit RS100 (low water permeable) and RL100 together in a single matrix. 3 full factorial experiment was designed to study the effect of Eudragit RS100 ( ) and Eudragit RL100 ( ) combination on the % cumulative release in first hour (Y 1 ), time to achieve 60% drug release (Y 3 ), time to achieve 90%drug release (Y ). % yield, % Drug entrapment and particle size were also found. In vitro drug release profiles showed that a suitable combination of Eudragit RS100 and Eudragit RL100 can exhibit sustained release profile for of a drug. Differential scanning calorimetric analysis showed no interaction of drug with the polymers. Model dependent method was used to decide the release mechanism of optimized batch. Keywords: Microspheres, Surface Response Methodology, Sustained Release, Nimesulide. INTRODUCTION NSAIDs are amongst the most commonly prescribed medications in the world. However, numerous spontaneously reported adverse drug reactions, case control, cohort, and post marketing surveillance studies have revealed that NSAIDs are associated with extensive side effects. A trend of dosage form development for NSAIDs development has been an attempt to improve therapeutic efficacy and reduce severity of side effects through modified release such as enteric-coating (EC) or sustained release (SR) formulations 1. The non-steroidal anti-inflammatory drug, Nimesulide, was selected as a model drug in this study. Nimesulide or N-(4-Nitro-- phenoxyphenyl) methane sulphonamide is administered orally or rectally. It is highly effective in reducing pain associated with osteoarthritis, rheumatoid and other degenerative joints disorders, low back pain, dysmenorrhoea, gynaecological condition, thrombophlebitis, dental pain and inflammations etc. This drug is also associated with some severe side effects due to higher doses such as epigastric pain, heartburn, nausea, diarrhoea, vomiting, peptic ulcer and hepatic impairments. Nimesulide is having half-life 1.56 to 4.95 hr which requires frequent dosing to maintain plasma concentration 3-5. Because of the above mentioned drawbacks, it was considered a good candidate for sustained drug delivery. *Corresponding author. Mihir K. Raval, Asst. Prof. Department of Pharmaceutical Sciences, Saurashtra University, Rajkot, Gujarat, India. Tel.: Telefax: rmihir@yahoo.com Nimesulide microspheres spread out more uniformly in the GI tract, thus avoiding exposure of high concentration of drug to the mucosa at once and ensuring more reproducible drug absorption. The risk of dose dumping and side effect due to NSAID also seems to be lower than with a single unit dosage form 6. The purpose of this study by taking Nimesulide as a model drug is to understand the usefulness of microparticulate dosage forms in order to reduce the dosing frequency as well as side effects associated with single unit dosage forms of NSAIDs. Eudragits are also called as methacrylic acid co-polymers. A combination of Eudragit RS100 (Low water Permeability) and Eudragit RL100 (High water Permeability) in a single matrix was used to sustain the release of drug from microspheres. Both are showing ph independent drug release properties. Eudragit RL100 and Eudragit RS100 are Poly (ethyl acrylate, methyl methacrylate, trimethylammonioethyl methacrylate chloride) in the ratio of 1::0. and 1::0.1 respectively 7. MATERIALS AND METHODS Nimesulide was received as a gift sample from Atul Pharmaceuticals, Rajkot. Eudragit RS100 and Eudragit RL100 were received by courtesy Evonik Degussa Ltd., Mumbai. Light liquid paraffin and Span-80 were purchased from Lobachem Private Ltd., Mumbai. Acetone, Ethanol, Methanol, n-hexane and Petroleum ether were purchased from Merck Specialties Private Ltd., Mumbai. All other chemicals and solvents were of analytical grade and used as received. Journal of Pharmacy Research Vol.3.Issue 3.March

2 Preparation of Microspheres (Solvent Evaporation Method) M. K. Raval et al. / Journal of Pharmacy Research 010, 3(3), The microspheres were prepared by non-aqueous emulsion solvent evaporation technique 8. The polymers Eudragit RS100 and Eudragit RL100 (In different ratios) were co-dissolved in 50 ml internal organic phase (Acetone: Ethanol (3:)) and drug (1.0 g) was dissolved in this solution. The resulting solution was emulsified within the oil phase, comprising of light liquid paraffin (500 ml) using span-80 (1%) as an emulsifier and continuously agitated using Remi Magnetic Stirrer at 700 RPM. Then 00 ml n-hexane was added drop wise at 3 ml/ min. The formation of microspheres was checked periodically by using light microscope, following which they were separated and washed three times with n-hexane to remove residual organic liquid. Finally microspheres were dried (air dried) for 4 hrs and stored in desiccators. Scanning Calorimeter (Tokyo, Japan)). The instrument was calibrated using indium as a standard. Samples weighing 3-5 mg were heated in sealed aluminum pans from 30 to 300 C at a scanning rate of 10 C/min under nitrogen purge, with an empty aluminum pan as reference. Differential scanning calorimetry was conducted first with Nimesulide pure drug, microspheres, Eudragit RS100, Eudragit RL100 and compared for possible drug-polymer interactions. Surface Morphology Surface characteristics of Nimesulide microspheres were studied by Scanning Electron Microscopy (SEM-JEOL, JSM, 5610LV, Tokyo, Japan) at 10X. The sample was mounted on double sided adhesive carbon tape on brass stubs and analyzed. The accelerating voltage was 15 kilo volts. Percentage Yield Percentage yield was calculated for each batch using following equation. Particle Size and Size Distribution Simple optical microscope was used for particle size measurement of individual microsphere in all the factorial batches. Optical micrometer was calibrated using standard stage micrometer. According to microscopic method of particle size analysis, slides of various batches of microspheres were prepared using dilute suspension of microspheres in liquid paraffin. Particle size of 100 numbers of microspheres from each batch was measured for calculating size distribution and average particle size. The study was done in triplicate. Encapsulation Efficiency Accurately weighed quantity (50 mg) of microspheres were dissolved in 100 ml methanol and assayed spectrophotometrically for Nimesulide at 96.5 nm, using a calibration curve based on standard solutions of the drug in methanol. Eudragit did not interfere with assay at that wavelength. (1) Criteria for Optimized Batch Three limits were arbitrarily selected; i) Y 1 : Percentage drug release in first hr between 18 to %, ii) t90: time for 90 % drug release between 10 to 1 hrs and iii) t60: time for 60 % drug release between 6 to 7.5 hrs. In Vitro Drug Release Study Drug release study (n = 3) was carried out for first hrs in 0.1 N HCl and then after in Phosphate Buffer ph 6.8 using USP dissolution apparatus type-i (basket type). Stirring speed was taken as 100 rpm. Accurately weighed microspheres (equivalent to 100 mg of Nimesulide) were placed into the basket containing media. At different time intervals 5 ml of aliquots of dissolution medium were withdrawn and analyzed at 97.0 nm for 1. ph and 393 nm for ph 6.8 for drug content using UV visible spectrophotometer. An equal volume of media was immediately replaced to maintain a constant volume of 900 ml within the dissolution beaker (USP) at every withdrawal of aliquot. Full Factorial Design To study possible combinations of all the factors at every levels, a two-factor, three- level full factorial design constructed and conducted in fully randomized order. Independent variables selected Table 1: Formulation and Dissolution Characteristics of Batches in a 3 Full Factorial Design Fourier Transformed Infrared (FT-IR) Spectroscopy The sample powder was dispersed in KBr powder and analyzed after converting into pallet. FT-IR spectra were obtained by powder diffused reflectance on a FT-Infrared spectrophotometer. Differential Scanning Calorimetry (DSC) DSC curves were recorded on a scanning calorimeter equipped with a thermal analysis data system (Shimadzu, Differential Batch Variables Response Values X1 X Cumulative % Time for 90 % Time for 60 % Release at First Drug Drug Hour (Y1) Release (t90) Release (t60) E E E E E E E E E E Journal of Pharmacy Research Vol.3.Issue 3.March

3 Coded Actual Values + Value X1 is amount of Eudragit RS100 in milligram; + X is amount of Eudragit RL100 in milligram M. K. Raval et al. / Journal of Pharmacy Research 010, 3(3), were concentration of Eudragit RS100 ( ) and Eudragit RL100 ( ). While dependent variables measured were cumulative percentage release at first hour (Y 1 ), time of 60% release (Y ) and time for 90% drug release (Y 3 ). The drug: polymer ratio 1:5 was selected from preliminary trials and kept constant in whole design; the polymer to polymer ratio was altered. Thus as per 3 design, total 9 batches were prepared. High and low levels of each factor were coded as 1 and -1, respectively, and the mean value as zero. The composition and responses of the 3 design are shown in Table 1. Kinetic Modeling of Drug Release The drug: polymer ratios were tried from 1:1 to 1:5 by keeping both the Eudragit ratios in equal quantity. It was seen that the drug: polymer ratio (1:5) only gave encouraging result. Thus the drug: polymer ratio (1:5) was selected for microsphere preparation. Dissolution study was carried out for first hours in 0.1 N HCl and then in 6.8 ph Phosphate buffer in USP dissolution apparatus type-i for the drug loaded microspheres prepared from Eudragit RS100 as well as Eudragit RL100 separately. The release was delayed in case of RS100, whereas from RL100, the drug was released completely within few hours. These release data showed a need of combination of both the polymers in order to control the rate of drug release. This required changing polymer ratios to obtain desired release of drug. Factorial design was one of the promising approaches to solve these objectives. Percentage Yield A perusal from Table showed that batch E 7 gave the highest % yield which was 94. The remaining batches were having % yield in the range of 70% to 93%. Dissolution of all the batches was fitted to zero-order, firstorder, Higuchi, Hixon-Crowell, Korse-meyer and Peppas and Weibull to ascertain the kinetic modeling of drug release 9. Model fitting was done using FORTRAN software. The least value of sum of square of residuals (SSR) and Fisher s ratio (F) were used to select the most appropriate kinetic model 10. RESULTS AND DISCUSSION Various organic solvent systems alone as well as in combination were tried as an internal phase and liquid paraffin with 1% span as an external phase for preparation of blank microspheres containing polymer Eudragit RS100 and Eudragit RL100. It was finalized to select Acetone: Ethanol (3:) as an internal phase for preparing microspheres as it was not possible to formulate the microspheres with the other solvent systems. Moreover, different stirring speeds from 500 to 900 RPM were tried for the preparation of microspheres. Microspheres were not possible with the stirring speed less than 700 RPM. Furthermore, there was no considerable difference in drug entrapment with stirring speed 700 RPM to 900 RPM. The particle size reduction was also not significant as the RPM was increased. Finally, 700 RPM stirring speed was selected for preparation of all the batches of microspheres. Table : Evaluation Parameters for Microspheres of Factorial Batches. Batch % Drug Entra- %Yield ± S. D. Particle size pment ± S. D. (µm) ± S. D. E ± ± ± E 76.6 ± ± ± E ± ± ± E ± ± ± E ± ± ± 0.15 E ± ± ± 0.59 E ± ± ± 0.15 E ± ± ± 0.08 E ± ± ± Particle Size and Size Distribution A perusal from Table showed that batch E 7 gave lowest particle size, which was 9.53 µm. The remaining batches were having particle size in the range of 30 to 69 µm. Encapsulation Efficiency A perusal from Table indicated that batch E 9 was showing highest % entrapment of The remaining batches were having % entrapment in the range of 56 % to 76 %. Fourier Transformed Infrared (FT-IR) Spectroscopy The stability of Nimesulide in microspheres was investigated by infrared spectroscopy study (IR). The study of IR spectra of Nimesulide (Figure 1) demonstrated that the characteristic absorption bands for aromatic rings, SO anti-symmetric stretch, Aryl nitro group stretching and N-H stretching vibration appeared at 1589, 115, 1343, 1519 and 386 cm -1, respectively. The almost identical absorption bands were obtained from Nimesulide loaded microspheres, but with lower intensity as shown in Figure 1. The above observed absorption bands were similar to the reported value 11. Thus, the IR study indicated a stable nature of Nimesulide in the microspheres. Differential Scanning Calorimetry (DSC) The DSC thermogram of Nimesulide, Eudragit RS100, Eudragit RL100 and Nimesulide-loaded microspheres are shown in Figure. The DSC thermogram of pure drug and drug loaded microspheres produced almost similar melting endotherms at 150 o C and 158 o C, respectively. However, the intensity of the endotherm in microspheres was comparatively less than that of the pure drug. DSC of polymer Eudragit RS100 and Eudragit RL100 did not show any peak in this range. These results were an indication of absence of any chemical interaction between drug and excipients 1. Journal of Pharmacy Research Vol.3.Issue 3.March

4 M. K. Raval et al. / Journal of Pharmacy Research 010, 3(3), Table 3: ANOVA Results (P Value) of the effect of the Variables on Release Characteristics of Microspheres Factors Drug released at First Hour Time for 90% drug release Time for 60% drug release Coefficient P Coefficient P Coefficient P < a < a < a 4.89 < a < a < a < a Constant < a < a < a r a Regression Coefficients, Statistically significant (p < 0.05) Table 4: Mathematical Models Function Surface Morphology The SEM photomicrograph of the microspheres is shown in Figure 3. The photographs indicated a spherical shape of microspheres, but some amount of drug was recrystallized on the surface of the microspheres. This quantity might have been a supplement in initial drug release. In Vitro Drug Release Study A perusal from Table 1and Figure 4, it was shown that Batch E 7 released the drug up to 4 hours, but the release was only 6.7% in First hour. The probable reason was the highest amount of Eudragit RS100 and lowest amount of Eudragit RL100 in its formulation. Other batches like E 1, E, E 4, E 5, E 8 and E 9 showed first hour drug release less than 18 %, which was not satisfactory to the desired criteria. Batches E 3 and E 6 showed Y 1 7 % and 19 %, respectively. Moreover, batch E 3 released more than 90 % drug within 6 hrs which was not acceptable. Only batch E 6 fulfilled the desired criteria. By looking at the dissolution profile of all batches in Table 1, it was concluded that in all the levels of Eudragit RS100, Y 1 increased as the amount of Eudragit RL100 increased. Moreover, in medium level of, the amount of drug released in first hour was decreased with compare to the batches of low level. The same observation was again made in the case of comparison of medium and high level. This might be due to increase in the amount of Eudragit RS100 in the formulation. Factorial Design Equation Zero-order % disso * = kt First-order % disso * = 100 (1-e -kt ) Hixson-Crowell % disso * = 100 [1-{1-(kt/4.6416)} 3 ] Higuchi % disso * = kt 0.5 Korysmeyer-Peppas % disso * = kt n Weibull % disso * = 100 [1-e -(t/td)ß ] % disso * is the percent dissolved at time t, k is the dissolution rate constant, N is the release component, ß is the shape parameter and Td is the time at which 63.% of drug is dissolved. A statistical model used in order to estimate the relationship between the response variables and the independent variables. A 3 design was constructed to study the effect of amount of Eudragit RS100 ( ) and Eudragit RL100 ( ) on the drug release from microspheres. The dependent variables chosen were Amount of drug release at first hour (Y 1 ), time for 90% drug release (t90) and time for 60% drug release (t60). A statistical model incorporating interactive and polynomial terms was utilized to evaluate the response. Y = b 0 + b 1 + b + b 1 + b 11 + b () Where Y is dependent variable. b 0 is the arithmetic mean response of the nine runs, b 1 and b are the estimated coefficients for factor and respectively. The main effects ( and ) represent the average result of changing one factor at a time from its low to high value. The interaction term ( ) show how the response changes when two factors are changed simultaneously. The polynomial terms ( and ) are included to investigate the nonlinearity. The statistical analysis of the factorial design batches was performed by multiple linear regression analysis using Microsoft Excel. A number of preliminary trials were taken to decide the control factors and their levels which are shown in Table 1. The ANOVA results (p value) of the effects of variables on % drug release from microspheres are shown in Table 3. The significant factors in the equation were selected using a stepwise forward and backward elimination for the calculation of regression analysis. The terms of full model had nonsignificant p value (p > 0.05) and negligible contribution in obtaining dependent variables and thus were neglected 13. The equations representing are quantitative effect of the formulation variables on the % drug release are shown below: Amount of drug released at first hour, Y 1 = (3) (R = 0.959; p < 0.05) Time for 90 % drug release, t90 = (4) (R = ; p < 0.05) Time for 60 % drug release, t60 = ) (5) (R = ; p < 0.05 Coefficients with one factor representing the effect of that particular factor, while the coefficients with more than one factor and Journal of Pharmacy Research Vol.3.Issue 3.March

5 M. K. Raval et al. / Journal of Pharmacy Research 010, 3(3), A check point batch (E 10 ) mentioned in Table 1 and Figure 4 was formulated. The microspheres of Batch E 10 were subjected to in vitro dissolution in the same manner of the earlier factorial batches. The computed and experimental values of Y 1, t90 and t60 for batch E 10 were 0.35, and 7.54; 18.94, 10.8 and 6.8 respectively. The in vitro drug release data of batch E 10 was analyzed for establishing kinetics of drug release. The models tested are shown in Table 4. Korsmeyer-Peppas model showed least sum of square (SSR = Figure 1: FT-IR Spectra of Nimesulide (a), Microspheres (b), Eudragit RS100 (c), Eudragit RL100 (d) Figure 3: Scanning Electron Microscopy (00 x and 100 x) of optimized batch Figure : DSC profile of Nimesulide (a), Microspheres (b), Eudragit RS100 (c), Eudragit RL100 (d) those with second-order terms represent the interaction between those factors and the quadratic nature of that phenomena, respectively. Positive sign in front of the terms indicates a positive effect and negative sign in front of the factor indicates a negative effect of the factors. The above equations shows that Eudragit RS100 showed positive effect on t90 and t60, while negative effect on Y 1. In the same way, Eudragit RL100 showed positive effect on Y1 but negative effect on t90 as well as t60. Figure 4: Dissolution Profile of all the factorial batches (E 1 to E 9 ) and Check Point Batch (E 10 ) Figure 5, 6 and 7 shows the Surface plots shows the plots of Amount of Eudragit RS100 ( ) and Eudragit RL100 ( ) versus Y 1, t90 and t60 respectively. The plot was drawn using Design- Expert Software 7.1 Trial Program (Stat-Ease, Inc., Minneapolis, MN). The data demonstrates that both ( and ) affected the dependent variables. It may be concluded from Figure 5 that low level of and high level of increased the value of Y 1. At the same time, Figure 6 and 7 shows that, if the amount of increased and/ or amount of decreased, value of t90 as well as t60 increased. It means Eudragit RS100 works as a release retardant. It can be concluded that, the drug release pattern may be changed by appropriate selection of the Figure 5: Surface Response Plot for Cumulative Percentage Drug and levels. Release at 1 st Hour (Y 1 ) Journal of Pharmacy Research Vol.3.Issue 3.March

6 M. K. Raval et al. / Journal of Pharmacy Research 010, 3(3), Figure 6: Surface Response Plot of time required for 90 % drug release (t90) Figure 7: Surface Response Plot of time required for 60 % drug release (t60) 4.3) and Fischer s ratio (F=3.47). The mechanism of release of Nimesulide from the formulated batch was by anamolous non-fickian diffusion i.e. diffusion coupled with erosion. CONCLUSION In the present work, microspheres were formulated to provide sustained release of Nimesulide as a model drug using nonaqueous solvent evaporation method with a view to reduce frequency of dosage administration, reduce side effects generally associated with NSAIDs and better distribution in GIT. Drug release from microspheres was highly influenced by the combination of Eudragit polymers. In vitro dissolution of optimized batch exhibited optimum sustained release of Nimesulide for approximately 1 hrs following anamolous diffusion mechanism. According to the results of DSC and FT-IR proved that there was no drug-polymer interaction. This study demonstrates the use of factorial design and response surface methodology for the preparation of sustained release formulations. This statistical technique allows scientists to examine more than one independent variable at a time. REFERENCES 1. Davies N. M., Sustained Release and Enteric Coated NSAIDs: Are They Really GI Safe? J Pharm Pharmaceut Sci, 1999, (1): Bernareggi A. The pharmacokinetic profile of nimesulide in healthy volunteers. Drugs, 1993, 46, Boelsterli, U. A., Mechanisms of NSAID-induced hepato-toxicity: focus on nimesulide. Drug Saf., 00, 5 (9), Singla A.K., Chawla M., Singh A., Nimesulide: some pharmaceutical and pharmacological aspects- an update. J. Pharm. Pharmacol., 000, 5, Perucca E., Drug interactions with nimesulide. Drugs, 1993, 46 (1), Burcu, D., Kandemir C. Preparation And Evaluation Of Modified Release Ibuprofen Microspheres With Acrylic Polymers (Eudragit ) By Quasiemulsion Solvent Diffusion Method: Effect Of Variables, Acta Poloniae Pharmaceutica and Drug Research, 006, 63 (6), Rowe R.C., Sheskey P. J., Owen S. C., Handbook of Pharmaceutical Excipient, Pharmaceutical Press, 5 th Edn., 006, London (UK), Bhalerao S. S., Lalla J. K., Rane M. S., Study of processing parameters influencing the properties of Diltiazem hydrochloride microspheres, J. of Microencapsulation 001, 18 (3), Patel N., Chotai N., Patel J., Soni T., Desai J., Patel R., Comparison of In Vitro Dissolution Profiles Of Oxcarbazepine-HP B-CD Tablet Formulations With Marketed Oxcarbazepine Tablets, Dissolution Technologies, 008, Coasta, P., Sousa L., Modeling And Comparison Of Dissolution Profiles. Eur. J. Pharm. Sci., 001, 13, Singh A., Singh P., Kapoor V. K., Analytical Profiles of Drug Substances And Excipients. Academic Press, 001, (8), NY, 198Y Aulton M. E., Pharmaceutics: The Science of Dosage form Design, Churchill Livingstone, nd Edn., 001, NY, Dave B. S., Amin A. F., Patel M. M., Gastroretentive drug delivery system of Renitidine HCl: Formulation and invitro evaluation, AAPS PharmSciTech, 004, 5 (), 3-5. Source of support: Nil, Conflict of interest: None Declared Journal of Pharmacy Research Vol.3.Issue 3.March