Chitosan Loaded Mucoadhesive Microspheres of Gliclazide - Journal

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14 Thanoo et al . Twenty preliminary trial batches (F1 to F20) were prepared, 1.5 gm of Polymer was dissolved in 150 mL of 1% v/v aqueous acetic acid solution and 500 mg of drug was dispersed in the polymer solution. The resulting mixture was extruded through a syringe (No. 20) in 1 L of liquid paraffin heavy and light (1:1 ratio) containing 0.2% dioctyl sodium sulfosuccinate. The stirring was performed using a propeller stirrer 1000 rpm at constant for all the batches. After 15 minutes, glutaraldehyde was added and stirring was continued. The amount of glutaraldehyde and cross linking time was found varying for all the batches. On the basis of the 2 preliminary trials 3 full factorial designs were employed. The polymer-to-drug ratio (1:1, 3:1 and 6:1) and stirring speed (500, 1000 and 1500 rpm) were varied in all the nine factorial design batches. All other variables were used as mentioned in preliminary trial batches. Microspheres thus obtained were filtered and washed several time with petroleum ether (80:20) to remove traces of oil. They were finally washed with water to remove excess of glutaraldehyde. The microspheres were then dried at 25°C and 60% RH for 24 hours. Evaluation of Microspheres Drug Content According to literature review the assay for gliclazide was estimated by uv spectrophotometric method. Aqueous solution of drug was prepared in phosphate buffer (pH 6.8) and absorbance was measured on uv spectrophotometer at 229 nm the method is validated for linearity, accuracy and 22 precision . The method obeys Beer's law in the concentration range of 5 to 50 mµg/mL, a standard drug solution was analyzed repeatedly, the accuracy and precision were determined. Drug Entrapment Efficiency 50 mg of microspheres were crushed in a glass mortar and pestle, and the powdered microspheres was suspend in 10 mL of phosphate buffer solution (pH 6.8). After 24 hours, the solution filtered and the filtrate was analyzed for the drug content. The drug entrapment efficiency was calculated using the following formula; Practical drug content / Theoretical drug content x 100. Surface Morphology Surface morphology of the microspheres was determined by using scanning electron photomicrograph. Particle Size A Senthil et al./ Chitosan Loaded Mucoadhesive Microspheres of Gliclazide: In vtro and In vivo Evaluation The particle size of the microspheres was determined by using 23 optical microscopy method . 164 Swelling Index The swelling ability of microspheres in physiological media was determined by optical microscopy method. The 100 microspheres were suspended in 5 mL of stimulated gastric fluid USP (pH 1.2). The particle size was monitored by microscopy technique every 1 hour up to 8 hours using an 25 optical microscope . In vitro Wash-off Test for Microspheres A rat stomach mucosa was tied onto a glass slide (3 inch by 1 inch) using thread. Microspheres were spread onto the wet rinsed tissue specimen, and the prepared slide was hung onto one of the groves of a USP tablet disintegrating test apparatus containing the simulated gastric fluid (pH 1.2). The disintegrating test apparatus were operated such that the tissue specimen was given regular up and down movements. At the end of 30 minutes, 1 hour, and at hourly intervals up to 10 hours, the number of microspheres still adhering onto the 26 tissue was counted . Drug release Study The drug release study will perform using USP XXIV basket apparatus at 37°C±0.5°C and 100 rpm using 900 mL of phosphate buffer (pH 6.8) as dissolution medium. Microspheres equivalent to 10 mg of gliclazide were used for the test. Sample 5 mL was withdrawn at predetermined time intervals and filtered through a 0.45 micron membrane filter, diluted suitably and analyzed. Percentage drug dissolved at different time intervals was calculated using the Lamberts- 27 Beer's law equation . Release Kinetics and Mechanism To know the release mechanism and kinetics of gliclazide, optimized formulation was attempted to fit in to 2 mathematical models and n, r values for zero order, first order, higuchi and peppas models. Factorial Design A statistical model incorporating interactive and polynomial terms was utilized to evaluate the responses. 2 2 Y=b 0+b1X 1+b2X 2+b12X1X 2+b11X 1 +b22X2 Where, Y is the dependent variable, b 0 is the arithmetic mean response of the nine runs, and b 1 is the estimated coefficient for the factor X 1. The main effects (X 1 and X 2) represent the average result of changing one factor at a time from its low to high value. The interaction terms (X 1 X 2) show how the response changes when two factors are simultaneously 2 2 changed. The polynomial terms (X 1 and X 2 ) are included to investigate non-linearity. On the basis of the preliminary trials RJPS, Jul - Sep, 2011/ Vol 1/ Issue 2
2 a 3 full factorial design was employed to study the effect of independent variables i.e. polymer-to-drug ratio (X 1) and the stirring speed at rpm (X 2) on dependent variables % mucoadhesion, drug entrapment efficiency and particle size. In-vivo Anti-diabetic Study In-vivo evaluation studies for gliclazide mucoadhesive microspheres were performed on normal healthy wistar rats weighing 250 to 300 g each. The approval of the Institutional Animal Ethics Committee was obtained before starting of the study. Two groups of Wistar rats (5 in each group) that were fasted with water at least 12 hours before the experiments were used for the study. Before drug administration, a blood sample as a control was taken for each rat from behind the eyeball through the angle of ocular cavity using small capillary tubes. The blood glucose level for the control and test sample was determined using the glucose measuring instrument. The instrument was self calibrated, and the samples were allowed to dry before the results were read to avoid contamination of the lens. Pure gliclazide and mucoadhesive microspheres of gliclazide were administered orally to each group using stomach intubations. A dose of 800 g/kg of gliclazide was administered in suspension form for each rat. Blood samples were collected at predetermined time at 1 hour intervals up to 24 hours, and the blood glucose level was performed as per method described earlier. The 31-32 percentage reduction in blood glucose level was measured . Stability Testing Optimized formulations of microspheres were tested for stability studies. Both the formulations were divided into 3 sample sets and stored at 4±1°C, 25±2°C and 60±5% RH and 37±2°C and 65±5% RH. After 30 days, in vitro drug release studies and percentage entrapment efficiency were determined. RESULT AND DISCUSSION A Senthil et al./ Chitosan Loaded Mucoadhesive Microspheres of Gliclazide: In vtro and In vivo Evaluation The gliclazide mucoadhesive microspheres were prepared by simple emulsification phase separation technique using chitosan as natural polymer. Acetic acid from 1% to 6% v/v was used to prepare polymer solution. But there was no effect in concentration of acetic acid was observed on percentage mucoadhesion or drug entrapment efficiency, therefore 1% v/v of acetic acid was used. Based on viscosity of polymers solution three different concentrations 0.5, 1 and 2% v/v were selected for trial batches, from this 1% concentration showed a maximum sphericity was observed so we select 1% w/v of polymer in 1% v/v acetic acid was found to be the optimum concentration and 1:1 heavy and light paraffin was used as dispersion medium and 0.2% dioctyl sodium sulfosuccinate 165 surfactant to dispersion medium was found to be essential to minimize aggregation of microspheres. Cross-linking agent glutaraldehyde was selected due to its high rate of crosslinking and increased in glutaraldehyde concentration caused highly cross-linked spheres and become dense by hardening process. The long term exposure to 100 ppb glutaraldehyde vapour cause respiratory tract lesions including hyperplasia of squamous epithelium therefore, it is important to remove excess of glutaraldehyde from the microspheres to avoid any toxic reactions. The chitosan microspheres are a useful tool to improve the uptake of hydrophilic substance across epithelial layer. The glutaraldehyde was deposited on the surface of microspheres so easy removal of the unreacted free 33 glutaraldehyde as reported by Sahin et al . Preliminary trail batches of microspheres were prepared by using chitosan as polymers, the volume of cross-linking agent 10 to 70 mL and stirring speed were varied from 500, 1000 and 1500 rpm. From these batches F13, 60 mL of crosslinking agent and 1 hour cross-linking time was the optimum amount and time used for the preparation of mucoadhesive microspheres. Increase in the cross-linking time (1 to 4 hours) was inversely affected the percentage mucoadhesion. The cross-linking chitosan mucoadhesive polymer probably becomes more rigid and thus mucoadhesiveness decreases. The cross-linking time did not have a significant effect on the percentage drug entrapment efficiency were shown in (Table 1). 2 On the basis of the preliminary trials 3 full factorial design were employed, to study the effect of independent variable X 1 (polymer-to-drug ratio 1:1, 3:1 and 6:1) and the stirring speed X 2 (500, 1000 and 1500 rpm) on dependent variables percentage mucoadhesion, drug entrapment efficiency and particle size. The results depicted in (Table 2) clearly indicate that all the dependent variables are strongly dependent on the selected independent variable as they show a wide variation among the nine batches. Factorial Equation for Drug Entrapment Efficiency and Particle Size The drug entrapment efficiency was an important variable for assessing the drug loading capacity of microspheres and their drug release profile, thus suggesting the amount of drug availability at site. The following polynomial equation was derived by multiple regression analyses of the data. 2 2 Y = 69.11 + 10.01 X1 - 2.91 X2 - 0.484 X1 -7.03 X2 - 0.38 X1 X2 The drug entrapment efficiency of chitosan loaded mucoadhesive microspheres varied from 49% to 54%, 66% to 72%, and 70% to 77% at lower, medium and higher levels of RJPS, Jul - Sep, 2011/ Vol 1/ Issue 2
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