4.4.1 Formulation of Non-Effervescent floating tablets Formulation of Effervescent floating tablets Formulation of Hollow microspheres

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2 4.1 Materials 4.2 Instruments and Equipments 4.3 Analytical Methods 4.4 Formulations Formulation of Non-Effervescent floating tablets Formulation of Effervescent floating tablets Formulation of Hollow microspheres 4.5 Evaluation 66

3 4.1 MATERIALS Sl. No. Drugs SOURCE 1 Verapamil Hydrochloride Glenmark Pharmaceutical Ltd., Mumbai, India 2 Rosiglitazone maleate Matrix Lab, Hyderabad, India 3 Losartan Potassium Suven life sciences, Hyderabad, India Polymers and Chemicals 4 Polyvinyl pyrrolidone (PVP) SRL, Mumbai, India 5 Chitosan Sigma Aldrich, USA 6 Hydrochloric acid Reachem lab, Chennai, India 7 Accurel MP 1000 Membrana, Obernburg, Germany 8 Karaya gum Sigma Aldrich, USA 9 Lactose Loba Chemie, Mumbai, India 10 Sodium bicarbonate Loba Chemie, Mumbai, India 11 Ethyl cellulose 7cps Loba chemie, Mumbai, India 12 Polyethylene oxide Aldrich, Mumbai 13 Hydroxy propyl methyl cellulose Ranbaxy, Baddi, India 14 Eudragit L-100 Evonik, Mumbai, India 15 Potassium chloride Lobachemie, Mumbai, India 14 Tween 80 Lobachemie, Mumbai, India 15 Dichloromethane Lobachemie, Mumbai, India 16 Ethanol Lobachemie, Mumbai, India 17 Barium sulphate Lobachemie,, Mumbai, India 67

4 4.2 INSTRUMENTS AND EQUIPMENTS Sl. No. NAME OF THE EQUIPMENT MODEL/ MANUFACTURER 1 Digital balance Shinko Sansui, Japan 2 Hot air oven Tempo, India 3 Magnetic stirrer Remi equipments, India 4 Tablet punching machine Rimek, Minipress- 1(model-1674), Karnavati, India 5 Micrometer screw gauge Mitutoyo, Japan 6 Dissolution apparatus (8 basket) Electrolab, India 7 UV-Visible spectrophotometer Shimadzu-1800, Japan 8 FT-IR spectrophotometer Shimadzu-8400 S, Japan 9 KBr Press Techno search instruments, India 10 Scanning electron microscopy (SEM) 11 Differential scanning calorimetry (DSC) Joel SEM analysis Instrument, Model JSM 840A, Japan Shimadzu DSC-60, Japan 12 Tablet hardness tester Inweka, IHT 100, Ahmedabad, India 13 Digital ph meter Elico-LI120pH(type003),Hyderabad 14 X- Ray Machine Bharat Electronics Ltd. Pune, India 15 Accu- Chek Glucometer Roche Diagnostics India Pvt Ltd, India 68

5 REAGENTS N Hydrochloric acid (HCl) 8.5 ml of concentrated hydrochloric acid solution was diluted with distilled water upto 1000 ml to give 0.1 N HCl. 0.2 M Potassium chloride g of potassium chloride was dissolved in 1000 ml of distilled water. 0.2 M Potassium dihydrogen phosphate Accurately weighed g of potassium dihydrogen orthophosphate was dissolved in 1000 ml of distilled water. Hydrochloric acid buffer (ph 1.2) 50.0 ml of 0.2 M potassium chloride was placed in a 200 ml volumetric flask, to this 85.0 ml of 0.2 M hydrochloric acid was added and then made up to the volume with water. 69

6 4.3 ANALYTICAL METHODS Verapamil hydrochloride: The method described by Florey K was followed. 81 Stock solution: Verapamil hydrochloride in ph 1.2 hydrochloric acid (HCl) buffer (100 g/ml). Scanning: From the stock solution, a suitable concentration (10 g/ml) was prepared with ph 1.2 Hydrochloric acid buffer solution and UV scan was taken between nm. The spectrum is given in figure The absorption maxima of 278 nm was selected and utilized for further studies. Figure 4.01: UV-Spectra of Verapamil hydrochloride in ph 1.2 hydrochloric acid buffer 70

7 Standard Plot: From the stock solution, 10, 20, 30, 40, 50, and 60 g/ml solutions of Verapamil hydrochloride were prepared in ph 1.2 hydrochloric acid buffer solution. The absorbance was measured at 278 nm and a graph of concentration versus absorbance was plotted. Standard plot data of Verapamil hydrochloride in ph 1.2 hydrochloric acid buffer solution is reported in table 4.01 and graph in figure Table 4.01: Standard plot data for Verapamil hydrochloride in ph 1.2 hydrochloric acid buffer Concentration (µg/ml) Absorbance at 278 nm (mean ± SD*) ± ± ± ± ± ± *Standard deviation, n = 3 Figure 4.02: Standard plot for Verapamil hydrochloride in ph 1.2 hydrochloric acid buffer 71

8 4.3.2 Rosiglitazone Maleate: Method described by Shiva SK et al. was followed. 105 Stock Solution: Rosiglitazone maleate in ph 1.2 hydrochloric acid buffer solution (100 g/ml). Scanning: From the stock solution, a suitable concentration (10 g/ml) was prepared with ph 1.2 Hydrochloric acid buffer solution and UV scan was taken between the wavelengths of nm. The spectrum is given in figure The absorption maxima of 228 nm was selected and utilized for further studies. Figure 4.03: UV-Spectra of Rosiglitazone maleate in ph 1.2 hydrochloric acid buffer 72

9 Standard Plot: From the stock solution, 4, 8, 12, 16 and 20 g/ml solutions of Rosiglitazone maleate were prepared in ph 1.2 hydrochloric acid buffer solution. The absorbance was measured at 228 nm and a graph of concentration versus absorbance was plotted. Standard plot data of Rosiglitazone maleate in ph 1.2 hydrochloric acidbuffer solution is reported in table 4.02 and graph in figure Table 4.02: Standard plot data for Rosiglitazone maleate in ph 1.2 hydrochloric acid buffer Concentration Absorbance at 228 nm (µg/ml) (mean ± SD*) ± ± ± ± ± *Standard deviation, n = 3 Figure 4.04: Standard plot for Rosiglitazone maleate in ph 1.2 hydrochloric acid buffer 73

10 4.3.3 Losartan potassium: Method described by Kalyani et al 106 was followed. Standard solution: Accurately weighed 50 mg of Losartan potassium was dissolved in 100 ml of ph 1.2 hydrochloric acid buffer to get a solution containing 500 μg/ml of drug. Scanning: From the standard solution, a solution was prepared to give a concentration of 8 μg/ml in ph 1.2 hydrochloric acid buffer and UV scan was taken between the wavelengths of nm. The spectrum is reported in the figure The absorption maxima of 248 nm was selected and utilized for further studies. Figure 4.05: UV-Spectra of Losartan potassium in ph 1.2 hydrochloric acid buffer 74

11 Standard Plot: From the standard solution, a stock solution was prepared to give a concentration of 50 μg/ml in ph 1.2 Hydrochloric acid buffer. Aliquots of 0.4, 0.6, 0.8, 1.0, 1.2 and 1.4 ml of stock solution was pipetted out into 10 ml volumetric flasks. The volume was made up to the mark with ph 1.2 hydrochloric acid buffer. These dilutions gave 2, 3, 4, 5, 6 & 7 μg/ml concentration of losartan potassium respectively. The absorbances of prepared solutions of losartan potassium in ph 1.2 hydrochloric acid buffer were measured at 248 nm spectrophotometrically against ph 1.2 Hydrochloric acid buffer as blank. Standard plot data of losartan potassium in ph 1.2 Hydrochloric acid buffer is reported in table 4.03 and graph in figure Table 4.03: Standard plot data for Losartan potassium in ph 1.2 hydrochloric acid buffer Concentration (μg/ml) *Standard deviation, n=3 Absorbance at 208 nm (Mean ± S.D * ) ± ± ± ± ± ± Figure 4.06: Standard plot of Losartan potassium in ph 1.2 hydrochloric acid buffer 75

12 4.4. FORMULATIONS Formulation of Non-Effervescent floating tablets Floating matrix tablets were prepared by direct compression method. All the ingredients were blended together to get homogenous mixture. Accurel MP1000 as low density polypropylene foam powder, karaya gum as release retardant, chitosan as swellable polymer, lactose as diluent and magnesium stearate as lubricant were used. Powder mass was compressed into tablets using a 10 station rotary tablet punching press with 12 mm punch and die set. Each tablet contained 50 mg of losartan potassium. Composition of each tablet is given in table Table 4.04: Formulation chart of non-effervescent floating Losartan potassium tablets Ingredients (mg) G-I G-II G-III G-IV G-V G-VI G-VII G-VIII G-IX Losartan Potassium Accurel MP Karaya Gum Chitosam Lactose Magnesium Stearate Total Weight

13 4.4.2 Formulation of Effervescent floating tablets The floating tablets of verapamil hydrochloride were prepared by direct compression technique. For each tablet formulation, drug, HPMC-K15M, karaya gum, sodium bicarbonate, and diluents were blended homogeneously for 10 min followed by addition of magnesium stearate. The total weight of each tablet was 300 mg. The amount of karaya gum used was in the range of mg, whereas HPMC was used in the range of mg. The powder mixture was further mixed for 5 min in a mortar. The resultant mixture was compressed into tablets using a Rimek rotary tablet machine. Thirteen formulations were prepared by changing the amount of the ingredients as shown in table Table 4.05: Formulation chart of effervescent floating Verapamil hydrochloride tablets Ingredients F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 F13 (mg) Verapamil Hydrochloride Karaya Gum HPMC K15 M Sodium Bicarbonate PVP K Magnesium Stearate Lactose Total weight

14 71,107, Formulation of Hollow microspheres Floating microspheres with a central hollow cavity were prepared by using a modified Quasi-emulsion diffusion technique. Weighed quantities of Rosiglitazone maleate (RSM), ethyl cellulose, polyethylene oxide and hydroxy propylmethyl cellulose (HPMC K15M) were dissolved in a mixture of ethanol and dichloromethane (1:1 solvent ratio) at room temperature in a magnetic stirrer at 50 rpm for 50 min. This solvent was poured drop wise into 100 ml distilled water containing 2 ml of Tween 80 maintained at a temperature of 50 ± 2 C. The resultant solution was stirred with a pitched-blade-type impeller type agitator at 1100 rpm for 3 h to allow the volatile solvent to evaporate. This resulted in the formation of microspheres. Different ratios of polymers were used to prepare the microspheres. Eleven formulations were prepared by changing the amount of ingredients as shown in table 4.06 and diagrammatic representation of preparation of hollow microspheres is shown in the figure Table 4.06: Formulation chart of Rosiglitazone maleate hollow microspheres INGREDIENTS F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 Rosiglitazone maleate (gm) Ethyl cellulose (gm) Polyethylene (gm) oxide HPMC K15M (gm) Eudragit S100 (gm) Solvent ratio * (ml) 1:1 1:1 1:1 1:1 1:1 1:1 1:1 1:1 1:1 1:1 1:1 Tween 80(ml) * Ethanol and dichloromethane of 30 ml each. 78

15 Figure 4.07: Diagrammatic representation of preparation of hollow microspheres by Quassi-emulsion technique 79

16 4.5 EVALUATION All the formulations were evaluated for the following parameters Non-Effervescent floating tablets and Effervescent floating tablets Weight variation Friability Hardness Diameter & thickness Uniformity of drug content Fourier transform infrared spectroscopy (FT-IR) Differential scanning calorimetry (DSC) In vitro buoyancy studies Water uptake studies In vitro drug release studies Mathematical model fitting of obtained drug release data In vivo studies Stability studies 80

17 Floating Hollow microspheres Percentage yield Drug loading and Entrapment efficiency Fourier transform infrared spectroscopy (FT-IR) Differential scanning calorimetry (DSC) Scanning electron microscopy Sphericity of the microspheres Micromeritic properties In vitro buoyancy of microspheres In vivo floating behaviour In vitro drug release studies Mathematical model fitting of obtained drug release data In vivo drug release studies Stability studies 81

18 Technological characteristics of floating tablets Weight variation test 20 tablets from each formulation were randomly picked up and weighed individually and the average weight was calculated. The individual weights were then compared with the average weight. For the tablets of average weight 350 mg, the % deviation allowed is ± 5 % Friability Ten tablets were weighed and placed in a Roche friabilator and rotated at 25 rpm for 4 min. The tablets were taken out, dedusted, and reweighed. The percentage friability of the tablets was calculated using the equation: % F = {1-(W t /W)} 100 Where, % F is percentage friability, W is the initial weight of tablet and W t is the final weight of tablets after revolutions. Compressed tablets with a loss of less than 1 % are generally considered acceptable Hardness The hardness of core tablets was measured using Inweka hardness tester. A total of five tablets from each formulation were taken for the study and the average of the three is reported. It is expressed in kg Thickness and diameter Thickness and diameter of the tablets were determined by using Mitutoyo micrometer screw gauge. The average of five tablets from each formulation was taken. It is expressed in millimeter. 82

19 Uniformity of drug content Drug content uniformity was determined by randomly selecting 5 tablets were powdered. The quantity equivalent to single dose of the drug was dissolved in HCl buffer solution, ph 1.2 for 5 hours with occasional shaking and diluted to 100 ml with buffer. After filtration to remove insoluble residue, 1 ml of the filtrate was diluted to 10 ml with the buffer. The absorbance was measured at the required λ max using a UV visible spectrophotometer. The experiments were carried out in triplicate for all formulations and average values were recorded. The drug content was calculated using the following equation: % Drug content = conc. (μg/ml) Dilution factor 100/ Drug-excipient compatibility studies Fourier transform infra red spectroscopy (FT-IR) In order to evaluate the integrity and compatibility of the drug in the formulation, drug-excipient interaction studies were performed. Pure drug and optimized formulations were analyzed by Fourier transform infra-red (FTIR) spectroscopy. FTIR spectra of pure drug and its formulations were obtained by a FT-IR Shimadzu 8400S (Japan) spectrophotometer using the KBr pellet method. The samples were scanned from 400 to 4,000 cm 1 wave number Differential scanning calorimetry (DSC) Differential scanning calorimetry was performed on pure sample of drug and its formulation. Calorimetric measurements were made with empty cell (high purity alpha alumina discs) as the reference. The dynamic scans were taken in nitrogen 83

20 atmosphere at the heating rate of 10 C min -1. The energy was measured as Joules per kilocalorie In vitro floating studies 110 The in vitro buoyancy was characterized by floating lag time and total floating time. The test was performed using a USP dissolution apparatus type-ii (basket) using 900 ml of 0.1 N HCl buffer solution at 100 rpm at 37 ± 0.5 C. The time required for the formulation to rise to the surface of the dissolution medium and the duration for which the formulation constantly floated on the dissolution medium were noted as floating lag time and total time, respectively Water uptake studies 111 The swelling of the polymers was measured by their ability to absorb water and swell. The water uptake study of the tablet was done using a USP dissolution apparatus type-ii (basket) in 900 ml of ph 1.2 Hydrochloric acid buffer at 100 rpm. The medium was maintained at 37 ± 0.5 C throughout the study. At regular time intervals, the tablets were withdrawn, blotted to remove excess water, and weighed. Swelling characteristics of the tablets were expressed in terms of water uptake (WU) as: WU (%) =Weight of Swollen tablet- Initial weight of tablet X 100 Initial weight of tablet Percentage drug entrapment efficiency 112 Floating microspheres equivalent to 4 mg of drug was dissolved in 10 ml ethanol. The samples were assayed for drug content using UV spectrophotometer at 228 nm after suitable dilution. No interference was found due to the other components of floating microspheres at 228 nm. The percentage drug entrapment efficiency and yield were calculated as follows 84

21 % Drug entrapment efficiency = Calculated drug concentration 100 Theoretical drug concentration Yield of floating microspheres The yield was determined by weighing the microspheres and then the percentage yield was calculated with respect to the weight of the input materials, i.e., weight of rosiglitazone maleate and polymers used. The formula for calculation of percentage yield is as follows % Yield= Total weight of floating microspheres 100 Total weight of drug and polymer Scanning electron microscopy (SEM) The surface morphology of the microspheres was examined by scanning electron microscopy (SEM; JSM-5200, Jeol, Tokyo, Japan) operated at 15 KV on samples, gold-sputtered for 120 s at 10 ma, under argon at low pressure Sphericity of the microsphere 112 The sphericity of the prepared microspheres can be confirmed using a camera lucida by taking the tracings of the microspheres on a black paper. The tracings help to calculate the circulatory factor and confirm the sphericity of microspheres if the obtained values are nearer to 1. To determine the sphericity, the tracings of prepared microspheres (magnification 45x) were taken on a black paper using camera lucida, (Model-Prism type, Rolex, India). Circulatory factor (S) was calculated using S= P 2 /12.5xA Where A is area (cm 2 ) and, P is the perimeter of the circular tracing. 85

22 113, Micromeritic properties of microsphere The microspheres were characterized by their micromeritic properties, such as particle size, bulk density, compressibility index and angle of repose (values useful in prediction of flowability) Particle size The particle size of the microspheres was measured using an optical microscopic method and the mean particle size was calculated by measuring 425 particles with the help of a calibrated ocular micrometer with stage micrometer Angle of repose The flow characteristics of microspheres are measured by angle of repose. Improper flow of microspheres is due to frictional forces between the microspheres. These frictional forces are quantified by an angle of repose. Angle of repose is the maximum angle possible between the surface of a pile of the microspheres and the horizontal plane. Relationship between angle of repose and powder flow is given in table Fixed funnel method was employed. A funnel that was secured with its tip at a given height above the graph paper was placed on a flat horizontal surface. Microspheres were carefully poured through the funnel until the apex of the conical pile just touches the tip of the funnel. The radius and height of the pile were then determined. The angle of repose ( ) for samples were calculated using the formula Tan θ = Height Radius 86

23 Table 4.07: Relationship between angle of repose and powder flow Angle of repose ( ) Flowability <25 Excellent Good Passable >40 Very poor Tapped bulk density The tapping method was used to determine the tapped density of the microspheres using tapped density testing apparatus (Electrolab tapped density tester ETD-1020) and percent compressibility index as follows Tapped density = Mass of microspheres Volume of microspheres after tapping Compressibility (Carr s) index Carr s index is a dimensionless quantity, which proved to be useful to the same degree as the angle of repose values for predicting the flow behavior. Apparent bulk density was determined by pouring the samples in bulk into a graduated cylinder. Tapped density was determined by placing a graduated cylinder containing a known mass of powder on a mechanical Electrolab tap density tester. Samples were tapped until no further reduction in volume of the sample was observed. Relationship between powder flowability & % compressibility is shown in table Carr s index is calculated using the formula % Compressibility index = 1- V/Vo 100 Where Vo and V are the volumes of the sample before and after the standard tapping. 87

24 Table 4.08: Relationship between powder flowability & % compressibility % Compressibility range Flow description 5-15 Excellent (free flowing granules) Good (free flowing powder granules) Fair (powdered granules) Poor (very fluid powders) Poor (fluid cohesive forces) Very Poor >40 Extremely poor Floating Characteristics In vitro buoyancy of microspheres 71 The floatation study was carried out to ascertain the floating behaviour of the microspheres prepared with various polymer combinations. Floating behaviour of hollow microspheres was studied using a USP dissolution test apparatus II by spreading the microspheres (100 mg) on 900 ml of 0.1 N HCl containing 0.02 % v/v tween 80 as surfactant. The medium was agitated with a paddle rotating at 100 rpm and maintained at 37 ± 0.5 C for 12 h. Both the floating and the settled portions of microspheres were collected separately. The microspheres were dried and weighed. The percentage of floating microspheres was calculated using the following equation % Floating capability = Weight of floating hollow microspheres 100 Initial weight of hollow microspheres 88

25 In vivo floating behavior 115 Barium sulphate loaded microspheres were prepared by adopting the procedure as described earlier, except for using barium sulphate instead of drug. Healthy rabbit weighing approximately 2.3 Kg was used to assess in vivo floating behaviour. Ethical clearance for the handling of experimental animals was obtained from the institutional animal ethical committee (IAEC) of JSS College of Pharmacy, Mysore constituted for the purpose. The animal was fasted for 12 h and the first X-ray photographed to ensure absence of radio opaque material in the stomach. The rabbit were made to swallow barium sulphate loaded microspheres with 30 ml of water. During the experiment, rabbits were not allowed to eat but water was provided. At predetermined time intervals, the radiograph of abdomen was taken using an X-ray machine. 108, 116, In vitro drug release study The release rate of drug from formulations was determined using USP dissolution testing apparatus II (basket type). The dissolution test was performed using 900 ml of 0.1 N HCl, at 37± 0.5 o C and 50 to 100 rpm. Aliquots (5mL) were withdrawn at regular intervals for 12 h, sample was replaced by its equivalent volume of fresh dissolution medium to maintain the sink condition. The samples were analyzed spectrophometrically at wavelength corresponding to absorption maxima of the drugs. The release kinetics was fitted into various models using PCP dissolution v2.08 software Mechanism of drug release, 118,119,120,121 The different mathematical models may be applied for describing the kinetics of the drug release process from dosage forms the most suited being the one which best fits to the experimental results. The best models describe drug release from 89

26 pharmaceutical dosage form resulting from a simple phenomenon, or when this phenomenon, by being the rate-limiting step, conditions all the process occurring in the system. The kinetics of release from formulations were determined by finding the best fit of the release data to zero order, first order, matrix(higuchi), Hixson-Crowell, and Korsmeyer- Peppas plots. Higuchi developed several theoretical models to study release of high and low water soluble drugs incorporated in the semi-solid and/or solid matrices. According to this model, drug release was described as a square root of time-dependent diffusion process based on Fick s law. This relation can be used to describe drug dissolution from several types of modified release pharmaceutical dosage forms. Q t =K H = t where K H is Higuchi s rate constant, and Q t is the amount of drug released at time t. If a plot of square root of time versus cumulative amount of drug release yields a straight line, and the slope is 1 or more than 1, then the particular dosage form is considered to follow Higuchi kinetics of drug release. In some experimental situations the release mechanism deviates from the Fick s equation, following an anomalous behavior (Non-Fickian release). In these cases a more generic equation can be used. Korsmeyer et al. developed a simple, semi-empirical, relating exponentially the drug release to the lapsed time. Q t/ Q α =Kt n where Q t /Q α is the fraction of drug released at time t; K is the constant comprising a structural and geometric characteristics of the tablets; and n, the release exponent, is a parameter that depends on the release mechanism and is thus used to characterize it. 90

27 Peppas used this n value in order to characterize different release mechanisms. If the n value is 0.5 or less, the release mechanism follows Fickian diffusion, and higher values (0.5 < n < 1) for mass transfer follow a non-fickian model (anomalous transport). Hixson-Crowell recognized that area of the particle is proportional to the cubic root of its volume, and derived an equation as follows W o 1/3 - W t 1/3 =Ks t where W o is the initial amount of drug, W t is the remaining amount of drug in dosage form at time t, and K S is a constant incorporating the surface volume relation. 38, In vivo evaluation In vivo evaluation studies of the optimized microsphere formulation and pure drug were carried out on normal healthy male albino rats selected with average body weight of about gm. They were housed individually in polypropylene cages, maintained under standard conditions (12 h light and 12-h dark cycle; 27±2 C; 50±10% relative humidity); the animals were fed with standard rat pellet diet and water with glucose. Ethical clearance for the handling of experimental animals was obtained from the institutional animal ethical committee (IAEC) constituted for the purpose. Non-insulin dependent diabetes mellitus (NIDDM) was induced in animals which were fasted overnight by a single intraperitoneal injection of alloxan at the dose of 120 mg/kg for all group animals except the group I animals, which served as control. The blood glucose level was determined after 72 h of alloxan administration using Glucometer. The animals with blood glucose level more than 187 mg/dl were chosen for the experiment. All the animals showed hyperglycemia after 72 h of alloxan administration. Only the rats found with permanent NIDDM were used for in vivo evaluation studies. For the control (group I & II), the animals were kept fasting 91

28 overnight and water with glucose adlibitum. For group III and group IV, pure drug and hollow microspheres were administered orally with oral gauss in the morning following overnight fasting. No food and liquid except water with glucose were given to the animals during the experiment. After collection of zero-hour blood sample, F3 was administered orally through oral gauss. Blood sample was collected from the tail vein of the rat at every 1 h interval. Plasma glucose levels were determined using one touch ACCU-Chek Active. The number of animals required for the in vivo evaluation studies is given the table Table 4.09: Number of animals required for the in vivo evaluation studies Group Treatment No. of animals number 1 Positive control (normal control) 6 2 Negative control (diabetic control rat administered) 6 3 Pure drug (Rosiglitazone maleate of 4-mg/kg) 6 4 Optimized formulation ( F3 of 4-mg/kg) 6 Total 24 92

29 Figure 4.08: Administration of alloxan by I.P route Figure 4.09: Administration of microspheres by oral gauze in suspension form 93

30 Figure 4.10: Blood collection from rat tail vein In vivo studies were carried out to monitor the gastric retention property of floating tablet formulations. Barium sulfate loaded formulations were used as X- ray markers. The study was conducted after obtaining approval from the Institutional animal ethics committee of JSS College of Pharmacy, Mysore. Albino rabbits (2.5 kg) were used in the study. Before the test, the rabbits were fasted overnight and the formulations were administered orally to the rabbits with water. X- Ray pictures were taken at different time intervals after the administration of the formulations. 94

31 Stability studies 123 Stability testing of drug products begins as a part of drug discovery and ends with the demise of the compound or commercial product. FDA and ICH specifies the guidelines for stability testing of new drug products, as a technical requirement for the registration of pharmaceuticals for human use. The objective of stability testing is to investigate the effect of environmental factors on changes in product quality with time so as to establish its shelf life and recommend its storage conditions. Drug decomposition or degradation occurs during storage, because of chemical alteration of the active ingredients or due to product instability, leading to lower concentration of the drug in the dosage form, hence the stability of pharmaceutical preparation needs to be evaluated. The objective of stability studies is to predict the shelf life of a product by accelerating the rate of decomposition, preferably by increasing the temperature and relative humidity (RH) conditions. A drug formulation is said to be stable if it fulfills the following requirements: It contains at least 90% of the stated active ingredient It contains effective concentration of the added preservatives, if any It does not exhibit discoloration or precipitation, nor develops foul odour It does not develop irritation or toxicity Formulations were packed in a screw capped bottle and studies were carried out for 12 months by keeping at 25± 2 C and 60 ± 5% RH 30 ± 2 C and 65 ± 5% RH and for 6 months for accelerated storage condition at 40 ± 2 C and 75 ± 5% RH 95

32 Samples were withdrawn on 0, 3, 6 and 12 months for long term storage condition and 0, 3 and 6 months for accelerated storage condition and checked for changes in physical appearance and drug content as per ICH Q1A (R 2 ) guidelines. Graphs were plotted using Sigmaplot 12.0 to determine the statistical significance. Results obtained in the methods and the conclusions arrived from them are provided in the following chapters. 96