6.1 IntroductionGlyceryl Monostearate has been used in preparation of hollow microspheres by emulsification solvent diffusion technique. It works as wall membrane reinforcing agent1. Acryflow is hydrogenated castor oil which has been used in the novel pharmaceutical composition containing an active ingredient which is retained in the stomach or upper part of gastrointestinal tract for controlled delivery of medicament2. It has been shown that the nature of the drug formulation can influence the dissolution process. Solubility of glipizide increases by addition of HPMC which act as release modifying ingredient of the formulation3. It has also been shown that by incorporating polymers such as HPMC, Eudragit L100 and Ethyl cellulose within the shell of microballoons, the release rate of riboflavin from the microballoons could be controlled while maintaining high buoyancy1.Present work involves attempts to improve floating by incorporating Glyceryl Monostearate and acryflow. Further studies were done for the improvement of dissolution of the Glipizide by addition of HPMC and other polymers like Eudragit L100 and Ethyl cellulose. 6.2 Experimental6.2.1 Preparation of standard curve of Glipizide Glipizide (100 mg) was dissolved in Simulated Gastric Fluid containing 0.02% Tween 20 and volume is made up to 100 ml in volumetric flask. The UV maxima of Glipizide solution was found to be 276 nm. The standard curve was generated for entire range from 10 to 35 mcg/ml. Five ml of stock solution (1 mg/ml) was further diluted to 50 ml. This solution (100 mcg/ml) was further diluted to obtain solution of 10 mcg/ml to 35 mcg/ml. Absorbance of each solution was measured at 276 nm using Shimadzu 1700 UV/Vis double beam spectrophotometer Simulated Gastric Fluid containing 0.02% Tween 20 as a reference standard. The standard curve was generated for entire range from 10 to 35 mcg/ml. The experiment was preformed in triplicate and based on average absorbance; the equation for the best line was generated. Standard curve was also generated similarly in Phosphate buffer pH 6.8. Results of Standard Curve preparation are shown in Table 6.4, 6.5 & Figure 6.1, 6.2 6.2.2 Theoretical release profile of Glipizide Calculation of the Immediate Release Dose4 VdFCssIRP -----6.1 Calculation of Dose4 ( )1]1

+1/2 t t 0.693 1 IRP Dose -----6.2Where, IR = Immediate release,CSS= Concentration at steady state,Vd= Volume of distribution, F = Fraction bioavailable,t = time up to which sustain release is required and t1/2 = half life.The pharmacokinetic parameters of Glipizide were utilized for the calculation of theoretical drug release profile for 12 hours dosage form. The immediate release part for sustained release Glipizide was calculated using equation 6.1 and was found to be 3.5mg and dose was calculated using equation 6.2. Here, the formulation should release 35% of drug in 1 hour like conventional tablet and 5.9 % per hour up to 12 h thereafter.Table 6.1 Important pharmacokinetic parameters of glipizide5,6,7Fraction of drug absorbedElimination half-life(t1/2)Terminal disposition rate constant (kel or )Apparent volume of distribution (Vd)Minimum effective concentration (Css min)Maximum effective concentration (Css max)Clearance 13.3 h0.21 h-10.17 l/kg20 ng/ml300 ng/ml0.52 0.18 ml min-1 kg-16.2.3 Similarity between Phosphate Buffer pH 7.4 and Simulated Gastric Fluid containing 0.02% Tween 20 The similarity factor, f2, given by SUPAC guidelines for modified release dosage form was used as a basis to compare dissolution profiles8. The dissolution profiles are considered to be similar when f2 is between 50 and 100. The method was first reported by Moore and Flanner9. To check the similarity between Phosphate Buffer pH 7.4 and Simulated Gastric Fluid containing 0.02% Tween 20, dissolution of Glipizide sustained release tablet GLYTOP SR (10mg) were taken in two dissolution media i.e. 900 ml Phosphate Buffer pH 7.4 and 500 ml Simulated Gastric Fluid containing 0.02% Tween 20. Similarity factor for these dissolution profiles was calculated using equation 6.3. ;'11]1

,_

+ 1000.5n1 i2Ti Rin11 log 50 2F -----6.3Where n is the number of dissolution time and Ri and Ti are the reference and test dissolution values at time t. Two dissolution profiles are considered similar when the F2 value is 50 to 100.6.2.4 Preparation and Optimization of Acrycoat S100 microspheres of Glipizide Glipizide , Acrycoat S100, Acrycoat L100, Ethyl Cellulose, Hydroxy propyl methyl cellulose ,Glyceryl Monostearate and acryflow in quantities shown in Table 6.2 and 6.3 were dissolved in a mixture of Ethanol, IPA and Dichloromethane (4:6:5). This mixture was poured in a 500 ml water containing 1% PVA maintained at a temperature of 300 C with stirring at 250 rpm. Stirring was continued for 1 hr. to allow the volatile solvent to evaporate. The microspheres formed were filtered, washed with D.M.water and dried overnight at 400C in oven. 6.2.5 In vitro drug release studyDrug release study from the hollow microspheres is complicated because the hollow microspheres float and hence adhere to the inside surfaces of the dissolution basket while the dissolution experiments are in progress, which leads to the nonparticipation of the hollow microspheres or their surface in the release study. Hollow microspheres have the propensity to exhibit a buoyancy effect in vivo, but the development of a dissolution method as a quality control tool with the simulated buoyant condition is difficult.The drug release rate from floating microspheres was determined using USP XXIII basket type dissolution apparatus. A weighed amount of floating microspheres equivalent to 10 mg glipizide was taken for dissolution study. The microspheres were placed in a non reacting mesh nylon bolting cloth that had a smaller mesh size(200 #) than the microspheres. The mesh was tied with a nylon thread to avoid the escape of any microspheres, and glass marble was used in the mesh to help induce any possible sinking of the microspheres in the dissolution medium10. Simulated gastric fluid (SGF, pH 2.0) (500 ml) containing Tween 20 (0.02 w/v %) was used as the dissolution medium11 and maintained at 370C at a rotation speed of 100 rpm. 10 ml sample was withdrawn at 1 hr interval and analyzed spectrophotometrically at 276 nm to determine the concentration of drug present in the dissolution medium. The initial volume of the dissolution fluid was maintained by adding 10 ml of fresh dissolution fluid after each withdrawal. The dissolution studies were repeated using pH 6.8 All experiments were conducted in triplicate. Results of Drug release study are shown in Table 6.9 to 6.11 and Figure 6.7 to 6.96.2.6 Kinetics modeling of drug dissolution profiles12The dissolution profile of all the batches was fitted to Zero order, First order and Higuchi to ascertain the kinetic modeling of the drug release. The method of Bamba et al. was adopted for deciding the most appropriate model.Zero orderIn many of the modified release dosage forms, particularly sustained or controlled release dosage forms (those dosage forms that release the drug in planned, predictable and slower than the normal manner), is zero-order kinetic.t k m ----- 6.4Where, k is zero-order constant, m is the % drug unreleased and t is the time. The plot of % drug unreleased (released) versus time is the linear.First orderMost conventional dosage forms exhibits this dissolution mechanism. Some modified release preparation, particularly prolonged release formulations, adheres to this type of dissolution pattern.ln (100-Q) = ln100-k1t ----- 6.5Where Q is the percent of drug release at time t, and k1 is the release rate constant. It assumes that the drug molecules, diffuses out through a gel like layer formed around the drug during the dissolution process. A plot of log % drug release versus time is the linear.Higuchi Model:A large number of modified release dosage form contain some sort of matrix system. In such instances, the drug dissolves from the matrix. The dissolution pattern of the drug is dictated by water penetration rate (diffusion controlled) and thus the following relationship applies: Q = k2t 1/2 ----- 6.6Where Q is the percent of drug release at time t, and k2 is the diffusion rate constantIn higuchi model, a plot of % drug unreleased (released) versus square root of time is linear.The correlation coefficient values of the zero-order, first order and higuchi kinetics are shown in Table 6.12.6.3 Results and Discussion6.3.1 Preparation of standard curve of GlipizideStandard curve was prepared according to procedure given in 6.2.1.The method obeys Beer's Law in the concentration range of 10 to 35 mcg/mL Standard drug solution was analyzed repeatedly (n =3). The results of standard curve preparation are shown in Table 6.4, 6.5 and Figure 6.1, 6.2.Table 6.4 Standard curve of Glipizide in Simulated Gastric Fluid containing 0.02% Tween 20Sr. No.Concentration(mcg/ml)AbsorbanceI II III Mean 1234561015202530350.225 0.3350.4130.5210.6300.729 0.2290.3430.4200.5290.628 0.7200.2400.3510.4270.5200.6250.7140.231t 0.0070.343t 0.0080.420t 0.0070.523t 0.0050.628t 0.0030.721t 0.007Absorption = 0.0203X + 0.017 Correlation Coefficient = 0.9975 Table 6.5 Standard curve of Glipizide in Phosphate Buffer pH 7.4Sr. No.Concentration(mcg/ml)AbsorbanceI II III Mean 1234010152000.2150.3380.45700.2210.3480.47000.2290.3540.47900.222t 0.0070.347t 0.005672530350.5290.6620.7320.5380.669 0.7470.5470.6800.75680.469t 0.0110.538t 0.0090.670t 0.0090.745t 0.012Absorption = 0.0215 X + 0.0124Correlation Coefficient = 0.9957Figure 6.1 Calibration Curve of Glipizide in Simulated Gastric Fluid Containing 0.02% Tween 2000.10.20.30.40.50.60.70.80 10 20 30 40Conc. (mcg/ml)Abs.Figure 6.2 Calibration Curve of Glipizide in Phosphate Buffer pH 7.400.10.20.30.40.50.60.70.80.90 10 20 30 40Conc. (mcg/ml)Abs.6.3.2 Theoretical release profile of Glipizide Theoretical release profile of Glipizide was calculated by using pharmacokinetic parameters shown in Table 6.1. Theoretical release profile of Glipizide is shown in Table 6.6.6.3.3 Similarity between Table 6.6 Theoretical Dissolution Profile of GlipizideTime in min Cumulative % drug release0601201802403003604204805406006607200.003540.946.852.758.664.570.476.382.288.194.0099.99Phosphate Buffer pH 7.4 and Simulated Gastric Fluid containing 0.02% Tween 20F2 value of Dissolution Profile of Glipizide Tablet in Phosphate Buffer pH 7.4 and Simulated Gastric Fluid containing 0.02% Tween 20 was found to be 76.654. Cumulative % Drug Release of Glipizide Tablet (GLYTOP SR 10 mg) in Phosphate Buffer pH 7.4 and Simulated Gastric Fluid containing 0.02% Tween 20 is shown in Table 6.7Table 6.7 Cumulative % Drug Release of Glipizide Tablet (GLYTOP SR 10 mg)Time hrPhosphateBuffer 7.4SGF containing 0.02% Tween 200123456789101112036.25841.39846.59052.26658.56563.78969.63776.28081.07587.09093.16398.432033.00038.44443.94449.50054.26460.30566.94974.83179.97986.81890.95495.1266.3.4 Preparation and optimization of Acrycoat S100 microspheres of GlipizideFormulation 1 is the formulation of optimized composition prepared with optimized condition selected in Chapter 5. It was good with Morphology, Yield and Drug Entrapment and thus it is satisfying the objective and criteria of Chapter 5. But %floating of the Formulation 4 was between 50 60% which is not satisfactory so attempts have made to improve the floating of the microspheres.Table 6.8 Evaluation of Acrycoat S100 microspheres of Glipizide batches F1 to F7Batches Mean particle size (m) %Yield % Floating Incorporation EfficiencyF1F2F3F4F5F6F73002541025395313008037020340504103592%91%90%96%94%89%97%50%87%85%85%81%60%58%80%91%92%90%85%92%89%Figure 6.3 Mean Particle Size of Acrycoat S100 Microspheres Batches F1 to F7050100150200250300350400450500F1 F2 F3 F4 F5 F6 F7BatchMean Particle Size Figure 6.4 %Yield of Acrycoat S100 Microspheres Batches F1 to F7 84%86%88%90%92%94%96%98%F1 F2 F3 F4 F5 F6 F7Batch%YieldFigure 6.5 % Floating of Acrycoat S100 Microspheres Batches F1 to F7 0%10%20%30%40%50%60%70%80%90%100%F1 F2 F3 F4 F5 F6 F7Batch%Floating Figure 6.6 Incorporation Efficiency of Acrycoat S100 Microspheres Batch F1 to F774%76%78%80%82%84%86%88%90%92%94%F1 F2 F3 F4 F5 F6 F7BatchIncorporation EfficiencyIn Formulation 2, in addition to glipizide and Acrycoat S100, Glyceryl Monostearate was also added which acts as a wall membrane Reinforcing Agent. In Formulation 3, Glyceryl Monostearate was replaced with Acryflow. Acryflow is Hydrogenated Castor Oil, which is having low density and it helps the microspheres to float. Formulation 2 and 3 were evaluated for Mean particle size (m), %Yield%, Floating and Incorporation Efficiency as shown in Table 6.8. It is comparable to Formulation 1 with respect to all parameters except %floating. Floating is reasonably good in Formulation 2 and 3 as compared to Formulation 1.In dissolution study, due to their floating nature, the microspheres were forcibly immersed into the dissolution media to avoid adherence to the surface of the dissolution jar, thus leading to nonparticipation in the dissolution process. The drug release was extended to more than 12 hr.From Table 6.9 it can be seen that drug release rate of the formulation 2 and 3 is much less as compared to formulation 1. So in formulation 4 and 5, we decreased the concentration of Glyceryl Monostearate and acryflow, respectively. As a result there is some improvement in drug release and floating was also satisfactory. So we further decreased the amount of Glyceryl Monostearate and acryflow in formulation 6 and 7 but there is no significant increase in dissolution profile while % floating was decreased. So in further experiment, we have to use at least 0.25 mg of Glyceryl Monostearate or acryflow. Table 6.9 Drug Release Profile of Acrycoat S100 Microspheres of Glipizide of Batches F1 to F7Time (hr) F1 F2 F3 F4 F5 F6 F70123456789101112027.78530.57533.57135.93638.79940.68243.40947.44050.87753.27957.73661.557012.69216.52619.47722.45925.71527.59230.19532.68235.93338.15640.40042.66409.92311.87915.24117.71520.40822.74125.29228.60030.93834.03336.23638.459014.07717.92620.89224.35127.18730.44433.53837.02139.82142.08545.29247.608011.76914.20819.44122.42324.75626.62329.67733.53837.26739.04442.22144.505016.84619.34122.32326.25930.05933.32636.45140.91843.72846.49549.28752.105013.61516.07418.09721.06423.82826.62329.21532.60536.32840.40344.05646.362In formulation 4 and 5, little Glipizide was released from microspheres in 0.1N HCl containing 0.02% Tween 20. Thus, in order to enhance the drug release rate from the microspheres, they were prepared by mixing hydrophilic or hydrophobic polymer in Acrycoat S100. (Batches F8 to F16)The microspheres prepared upon mixing Acrycoat L100 in Acrycoat S100 (batches F8 & F9) was having rough surface. The amount of Glipizide released from microspheres prepared by mixing Acrycoat L100 was high, probably due to facilitated penetration of dissolution media in the microspheres. Increased amount of Acrycoat L100 (0.2 gm) was used in formulations F10 & F11 for further improvement of dissolution rate but it didnt improve dissolution rate significantly. Figure 6.7 Drug Release Profile of Glipizide from Microspheres of Batches F1 to F7 0204060801001200 2 4 6 8 10 12 14Time (hr)% CDRF1 F2 F3F4 F5 F6F7 Theoritical profile Table 6.10 Evaluation of Acrycoat S100 microspheres of Glipizide batches F8 to F16Batches Mean particle size (m) %Yield % Floating Incorporation EfficiencyF8F9F10F11F12F13F14F15F1638645429672565651624276364174233515350354008072%91%69%91%96%92%95%92%93%72%74%85%85%90%91%78%77%76%83%90%91%82%87%89%93%87%90%In the case of ethyl cellulose (EC), (batches F12 &F13) release profiles of the microspheres exhibited a burst (6.8%) of glipizide during the initial stage (for 20 min), followed by a plateau pattern for 12 h. In addition, buoyancy appeared to be high as a consequence of hydrophobic properties of the EC polymer.In the case of hydroxypropyl methyl cellulose (HPMC) batches F14 to F16 , buoyancy was high. HPMC was considerably soluble and gelled in dissolution media. Additionally, microspheres are having smooth surfaces. Thus, buoyancy appeared to be high due to the difficulty in penetration of dissolution media through the rigid smooth surfaces.It has been shown that presence of HPMC improves the solubility of Glipizide in the formulation. This was based on the assumption that polymer dissolution during the time course of study changes the surface tension of the medium and increases drug solubility. This can be attributed to the surface activity of the polymer. The surface tension of water (at 20C) is ~72 mN/m and that of HPMC polymer at the same temperature ranges from 42 to 64 mN/m.20 This reduction in surface tension can increase the wetting of the drug particles and as a result, increase the solubility3. It was found that the drug release rate and buoyancy of microspheres prepared by co formulating HPMC was relatively improved due to gelation in dissolution media. Therefore, the effect of HPMC mixing ratio on physicochemical properties and drug releasing behaviors of the microspheres were investigated as shown in Table 6.11 and Fig 6.9. Although the recovery of microspheres appeared unchanged by HPMC ratio, the buoyancy decreased with increasing HPMC ratio. These results were attribuTable to the conversion of spherical microspheres to needle-like particles possessing no hollow structure. In addition, the dissolution media can readily penetrate into microspheres the increased dissolution of HPMC in the solution. The amount of Glipizide released from microspheres in 0.1N HCl containing 0.02% Tween 20 (pH 1.2) increased with increasing HPMC ratio. This behavior was explained by the increased contact area of particles with the medium due to the poor buoyancy associated with increased HPMC ratio. The amount of Glipizide released from microspheres in dissolution media significantly increased in association with increased HPMC ratio. In conclusion, a recommendable preparation formulation of microspheres to increase the bioavailability of Glipizide is formulation F14 due to their desired drug release and floatable properties. Formulation F15 containing 0.25 gm acryflow still need improvement in drug release. So quantity of HPMC was increased to 0.2 gm. Similarity values between drug release profile of formulation F14 and F16 to that of theoretical profile are 86.509 and 85.335, respectively. 6.3.5 Kinetics modeling of drug dissolution profilesThe in vitro release data obtained were fitted in to various kinetic equations. Correlation coefficients of individual batch with applied equation are given in Table 6.12. All batches showed higher correlation with Higuchi plot than zero order and first order so predominant drug release mechanism is Diffusion controlled release.Table 6.12 Correlation Coefficients of Drug Release Curves For Acrycoat S100 Microspheres Batches F14 to F16 Based on Three ModelsModelr2F14 F15 F16Zero order0.9368 0.9409 0.939First order0.8607 0.9691 0.8681Higuchi0.9875 0.9852 0.9768Release mechanismDiffusion controlledDiffusion controlledDiffusion controlledFigure 6.8 Drug Release Profile of Glipizide From Microspheres of Batches F8 to F130204060801001200 2 4 6 8 10 12 14Time (hr)%CDRF8 F9 F10 F11 F12 F13 Theoritical profile Figure 6.9 Drug Release Profile of Glipizide from microspheres of Batches F14 to F160204060801001200 5 10 15Time (hr)% CDRF14 F15 F16 Theoritical profile 6.4 ConclusionsThe microspheres prepared by emulsification solvent diffusion technique, have lower densities, exhibits buoyancy and retain in the gastric environment for more than 12 h. Even though the CR systems released the drugs for a longer time, once these passed through the upper portion of the small intestine, the released drug cannot be utilized because of a gastric retention time less than 812 h. Therefore, it is not possible to deliver the drug from the oral route for more than 12 h. The present study demonstrated that the hollow microspheres developed floated for more than 12 h, so we can deliver drugs like Glipizide for a longer time (>12 h) in body. Thus, major advantages of the system include: (i) Ease of preparation, (ii) Good buoyancy, (iii) High encapsulation efficiency, and (iv) Sustained drug release over several hours.6.5 References 1. Sato, Y., Kawashima, Y., Takeuchi H.,Yamamoto, H., 2004. In vitro evaluation of floating and drug releasing behaviors of hollow microspheres (microballoons) prepared by the emulsion solvent diffusion method. Eur. J. Pharm. Biopharm. 57,235-243. 2. Bhushan, B., 2006. Novel floating dosage form. US Patent Application, 20060013876, 19 January.3. Jamzad, S., Fassihi, R., 2006. Role of Surfactant and pH on Dissolution Properties of Fenofibrate and Glipizide: A Technical Note, AAPS Pharm Sci. Tech. 7 Article.2. 4. Patel, S., Patel, J., 2005. Studies in Design and development of chitosan microspheres using different techniques. M. Pharm. Thesis. North Gujarat University.5. US pharmacopeia 27, 2004.First Supplement. US Pharmacopeial Convention, Rockville, MD, pp.8676. Benet, L.Z., Oie, S., Schwartz, J.B., 1996. Design and optimization of dosage regimens, pharmacokinetic data, In: Hardman, J.G., Limbird, L.E., Goodman and Gilmans The Pharmacological Basis of Therapeutics, Ninth ed., McGraw Hill, New York.7. Verma, R.K., Garg, S., 2004. Development and evaluation of osmotically controlled oral drug delivery system of glipizide. Eur. J. Pharm. Biopharm. 57, 5135258. Guidance for Industry SUPAC-MR. Modified Release Solid Oral Dosage Forms Scale-Up and Postapproval Changes: Chemistry, Manufacturing, and Controls. In vitroDissolution Testing and In Vivo Bioequivalence Documentation. 9. Moore, J., Flanner, H., 1996. Mathematical comparison of dissolution profiles. Pharm Tech. 20, 64-74. 10. Soppimath, K., Kulkarni, A., Development of Hollow Microspheres as Floating Controlled Release Systems for Cardiovascular Drugs, Preparation and Release Characteristics, Drug Dev. Ind. Pharm. 27, 507 515.11. Jain, S.K., Awasthi, A.M., Jain, N.K., 2005. Calcium silicate based microspheres of repaglinide for gastroretentive floating drug delivery: Preparation and in vitro characterization. J. Control. Rel. 107, 300 309.12. Costa, P., 2001. Modeling and comparision of dissolution profiles. Eur. J. Pharm. Sci. 13, 123-133.