Figure 4 DSC Thermogram of Paracetamol

Transcription

1 Preformulation studies DRUG IDENTIFICATION TESTS Determination of melting point(s) Differential scanning colorimetry (DSC) was performed to determine the melting point of the dicyclomine and paracetamol. Accurately weighed samples (2 mg) were transferred to aluminum pans and sealed. All samples were run at a heating rate of 20 o C/min over a temperature range o C using Shimadzu DSC-60 Thermal Analyzer. The thermograms are represented in Figure 3 & 4. DSC mw Thermal Analysis Result C C C C Temp [C] Figure 3 DSC Thermogram of Dicyclomine DSC mw Thermal Analysis Result C C C Temp [C] Figure 4 DSC Thermogram of Paracetamol School of Pharmaceutical Sciences 42

2 Preformulation studies Determination of absorption maxima (λmax) Dicyclomine 10 mg of dicyclomine was accurately weighed and transferred to 100 ml volumetric flask. The drug was dissolved in 0.1 N hydrochloric acid and the volume was made up to 100 ml to obtain a stock solution of 100 μg/ml. One ml of this stock solution was added to 5 ml of methyl orange solution and was extracted with chloroform (3x1.5 ml). Organic layers were separated and pooled. The volume of organic layer was made up to 10 ml with 0.5 % sodium acetate solution (Sethi 2008). This solution was scanned between 400 nm to 500 nm in a double beam UV/ Visible spectrophotometer (Shimadzu 1700). The λmax of the dicyclomine is shown in Table 8. Paracetamol 10 mg of paracetamol was accurately weighed and transferred to 100 ml of volumetric flask. The drug was dissolved in methanol and the volume was made up to 100 ml to obtain a stock solution of 100 µg/ml. One ml of this stock solution was again diluted with methanol up to 10 ml to obtain a solution of 10 µg/ml (Pharmacopoeia of India 1996). The resulting solution was scanned between 200 nm to 400 nm in a double beam UV/ Visible spectrophotometer (Shimadzu 1700). The λmax of the paracetamol is shown in Table 8. Loss on Drying Dicyclomine Loss on drying was determined by accurately weighing 1 gm of the drug and drying at 105 ο C for three hours. It lost gm (NMT 1.0 %) of its weight (Pharmacopoeia of India 1996). The results are presented in Table 8. Paracetamol Loss on drying was determined by accurately weighing 1 gm of the drug and drying at 105 ο C for three hours. It lost gm (NMT 0.5 %) of its weight (Pharmacopoeia of India 1996). The results are presented in Table 8. School of Pharmaceutical Sciences 43

3 Preformulation studies Table 8 Comparative values of respective parameters used to identify the drug(s) S. Drug(s) Melting point λ max Loss on Drying No. ( 0 C) (nm) (%) 1. Dicyclomine 174 ( ) 2. Paracetamol 176 ( ) 420 (420) 249 (249) 0.69 (NMT 1%) 0.23 (NMT 0.5%) Infra Red Spectroscopy The Infra red spectroscopy of the sample was carried out to ascertain identity of the drugs. A pellet of approximately 1 mm diameter of each drug was prepared by compressing 3-5 mg of the drug with mg of potassium bromide in KBr press (Model M-15, Techno Search Instruments). The pellet was mounted in IR compartment and scanned between wave number cm -1 using a Shimadzu Model 8400 FTIR. The FTIR spectra are represented in Figure 5 & 6 and their interpretation is presented in Table 9. Figure 5 FTIR Spectrum of Dicyclomine School of Pharmaceutical Sciences 44

4 Preformulation studies Figure 6 FTIR Spectrum of Paracetamol Table 9 Interpretation of FTIR spectra of drugs S. No. Drug Reported Peaks (cm -1 ) Observed Peak (cm -1 ) Inference 1. Dicyclomine C-N stretching C-O stretching C-H stretching C=O (ester) stretching 2. Paracetamol O-H stretching N-H stretching C=O (amide) stretching Amide II band C-N-H group Para-disubstituted aromatic ring School of Pharmaceutical Sciences 45

5 Preformulation studies CALIBRATION CURVES Calibration Curve of Dicyclomine Preparation of Stock Solution 100 mg of dicyclomine was accurately weighed and transferred to 100 ml volumetric flask. The drug was dissolved in 0.1 N hydrochloric acid to get a solution of 1000 μg/ml (stock solution I). 10 ml of stock solution I was diluted to 100 ml with 0.1N HCl (Stock solution II). Further, 10 ml. of stock solution II was diluted up to 50 ml with methyl orange solution (1%w/v) and extracted with chloroform (3x15 ml). Organic layers were separated and pooled. The volume of pooled organic layer was made up to 100 ml with sodium acetate solution (Stoke solution III). This stock solution III was used to prepare a series of standard dicyclomine solutions as discussed below. Procedure From stock solution III aliquots of 1, 2, 3, 4, 5 6, 7 & 8 ml were transferred to a series of 10 ml volumetric flasks. The volume was made up with 0.1 N HCl to give 10, 20, 30, 40, 50, 60, 70 & 80 μg/ml of dicyclomine. The absorbance of these solutions was measured at 420 nm against blank. The same procedure was followed for the preparation of standard curve of dicyclomine in phthalate buffer ph 4.5, phosphate buffer ph 6.8, and phosphate buffer ph 7.4. The standard curve of dicyclomine in phosphate buffer ph 6.8 with pectinex ultra-spl was also prepared by this method, where the drug was dissolved in mixture of 99 ml of buffer and 1 ml of pectinex ultra-spl for the preparation of stock solution III. The data are recorded in Tables 10 & 11 and the curves are plotted Figure 7-Figure 11. School of Pharmaceutical Sciences 46

6 Preformulation studies Table 10 Calibration curves data of Dicyclomine Concentration Absorbance± S.D. µg/ ml 0.1 N HCl Phthalate buffer Phosphate buffer Phosphate buffer Phosphate buffer ph 4.5 ph 6.8 ph 7.4 ph 6.8 with Pectinex ultra SPL ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.013 * Mean±S.D. (n=3) School of Pharmaceutical Sciences 47

7 Preformulation studies Absorbance (nm) y = x R 2 = Concentration (mcg/ml) Figure 7 Calibration curve of dicyclomine in 0.1 N HCl y = x R 2 = Absorbance (nm) Concentration (mcg/ml) Figure 8 Calibration curve of dicyclomine in phthalate buffer ph 4.5 School of Pharmaceutical Sciences 48

8 Preformulation studies y = x R 2 = Absorbance (nm) Concentration (mcg/ml) Figure 9 Calibration curve of dicyclomine in phosphate buffer ph y = x R 2 = Absorbance (nm) Concentration (mcg/ml) Figure 10 Calibration curve of dicyclomine in phosphate buffer ph 7.4 School of Pharmaceutical Sciences 49

9 Preformulation studies y = x R 2 = Absorbance (nm) Concentration (mcg/ml) Figure 11 Calibration curve of dicyclomine in phosphate buffer ph 6.8 and pectinex ultra SPL School of Pharmaceutical Sciences 50

10 Preformulation studies Table 11 Characteristic of calibration curves of dicyclomine Parameters Values 0.1 N HCl Phthalate buffer Phosphate buffer Phosphate buffer Phosphate buffer ph 4.5 ph 6.8 ph 7.4 ph 6.8 with Pectinex ultra SPL λ max (nm) Beer s law limit (mcg/ml) Slope (b) Intercept (a) Regression equation x x x x x (y= a+bx) Correlation coefficient School of Pharmaceutical Sciences 51

11 Preformulation studies Calibration Curve of Paracetamol Preparation of Stock Solution Accurately weighed 100 mg of the drug was transferred to 100 ml volumetric flask. The drug was dissolved in 5 ml methanol. The volume was made up to the mark with methanol (stock solution I) to make a solution of 1000 μg/ml. One ml of stock solutions I (1000 μg) diluted to 50 ml with methanol to give a stock solution of concentration 20 μg/ml (Stock solution II). Stock solution II was used to prepare a series of standard drug solutions. Procedure From stock solution II aliquots of 1, 2, 3, 4, 5, 6, 7 & 8 ml were transferred to a series of 10 ml volumetric flasks and the volume was made up to the mark with 0.1 N hydrochloric acid. The absorbance of standard solutions was measured at 249 nm. Standard curves in phthalate buffer ph 4.5, phosphate buffer ph 6.8, and phosphate buffer ph 7.4 were prepared by same method as described earlier. The standard curve of paracetamol in phosphate buffer ph 6.8 containing pectinex ultra-spl was also prepared where the drug was dissolved in mixture of 99 ml of simulated intestinal fluid of ph 6.8 and 1 ml of pectinex ultra-spl for the preparation of stock solution II. The calibration curves data is shown in Table 12 and 13 and the curves are plotted in Figure 12-Figure 14. School of Pharmaceutical Sciences 52

12 Preformulation studies Table 12 Calibration curves data of Paracetamol Concentration Absorbance± S.D. µg / ml 0.1 N HCl Phthalate buffer Phosphate buffer Phosphate buffer Phosphate buffer ph 4.5 ph 6.8 ph 7.4 ph 6.8 with Pectinex ultra SPL ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.003 * Mean±S.D. (n=3) School of Pharmaceutical Sciences 53

13 Preformulation studies y = x R 2 = Absorbance (nm) Concentration (mcg/ml) Figure 12 Calibration curve of Paracetamol in 0.1N HCl y = x R 2 = Absorbance (nm) Concentration (mcg/ml) Figure 13 Calibration curve of Paracetamol in phthalate buffer ph 4.5 School of Pharmaceutical Sciences 54

14 Preformulation studies y = x R 2 = Absorbance (nm) Concentration (mcg/ml) Figure 14 Calibration curve of Paracetamol in phosphate buffer ph y = x R 2 = Absorbance (nm) Concentration (mcg/ml) Figure 15 Calibration curve of Paracetamol in phosphate buffer ph 7.4 School of Pharmaceutical Sciences 55

15 Preformulation studies y = x R 2 = Absorbance (nm) Concentration (mcg/ml) Figure 16 Calibration curve of Paracetamol in phosphate buffer ph 6.8 with pectinex ultra SPL School of Pharmaceutical Sciences 56

16 Preformulation studies Table 13 Characteristic of calibration curves of paracetamol Values 0.1 N HCl Phthalate buffer Phosphate buffer Phosphate buffer Phosphate buffer Parameters ph 4.5 ph 6.8 ph 7.4 ph 6.8 with Pectinex ultra SPL λ max (nm) Beer s law limit (mcg/ml) Slope (b) Intercept (a) Regression equation x x x x x (y= a+bx) Correlation coefficient School of Pharmaceutical Sciences 57

17 Preformulation studies DETERMINATION OF SOLUBILITY Dicyclomine An excess of drug was dissolved in 10 ml buffer of different ph values (1.2, 4.5, 6.8 and 7.4) in conical flask and was continuously shaken for 24 hours at room temperature with the help of conical flask shaker. After 24 hours sample was filtered through Whatman filter paper no.1, diluted appropriately and the drug was estimated using UV spectroscopy (Shimadzu 1700) (Bhatia and Seedher 2003). The value of solubility studies are shown in Table 14. Paracetamol An excess of drug was dissolved in 10 ml buffer of different ph values (1.2, 4.5, 6.8 and 7.4) in conical flask and was shaken for 24 hours at room temperature with the help of conical flask shaker. After 24 hours sample was filtered through Whatman filter paper no.1, diluted appropriately and the drug was estimated using UV spectroscopy (Shimadzu 1700) (Bhatia and Seedher 2003). The value of solubility studies are shown in table 14. Table 14 Solubility values of drugs Solvents Phthalate Phosphate Phosphate S. No. Drugs 0.1 N HCl buffer buffer buffer ph 4.5 ph 6.8 ph Dicyclomine gm/ml gm/ml gm/ml gm/ml 2 Paracetamol gm/ml gm/ml gm/ ml gm/ml DRUG-EXCIPIENTS COMPATIBILITY STUDIES FTIR spectra were recorded to assess the compatibility of the drugs and excipients. Drug(s) and excipients in the ration of 1:1 were mixed thoroughly and stored at 40 C and 75% RH for and room temperature for 1 month (Mukherjeea et al., 2005). The FTIR spectra are represented in Figure 17 Figure 20. School of Pharmaceutical Sciences 58

18 Preformulation studies Figure 17 FTIR Spectra of drug(s), Eudragit RS100 and PVA alone and in combination kept at 40 o C and 75% RH for 1 month School of Pharmaceutical Sciences 59

19 Preformulation studies Figure 18 FTIR Spectra of drug(s), Eudragit S100 and PVA alone and in combination kept at 40 o C and 75% RH for 1 month School of Pharmaceutical Sciences 60

20 Preformulation studies Figure 19 FTIR Spectra of drug(s), Eudragit RS100 and PVA alone and in combination kept at room temperature for 1 month School of Pharmaceutical Sciences 61

21 Preformulation studies Figure 20 FTIR Spectra of drug(s), Eudragit S100 and PVA alone and in combination kept at room temperature for 1 month School of Pharmaceutical Sciences 62

22 Preformulation studies RESULTS & DISCUSSION Dicyclomine and paracetamol were identified using different methods viz. melting point determination, determination of absorption maxima (λmax), loss on drying, and FTIR spectroscopy. The thermogram of differential scanning colorimetry showed sharp endothermic peaks of dicyclomine and paracetamol at C and C, respectively corresponding to the melting range of dicyclomine ( C) and paracetamol ( C) in the crystalline form. Absorption maxima (λ max ) of dicyclomine and paracetamol were found to be at wavelength 420 nm and 249 nm corresponding to the values reported in literature (dicyclomine nm and paracetamol nm). The loss on drying for dicyclomine and paracetamol was found to be 0.69% (limit NMT 1.0 %) and 0.23 % (limit NMT 0.5 %), respectively. FTIR spectra of the dicyclomine, showed characteristic C-N, C-O, C-H, C=O (ester) stretching bands at cm -1, cm -1, cm -1, cm -1, respectively. FTIR spectra of paracetamol, showed characteristic O-H, N-H, C=O (amide) stretching bands at cm -1, cm -1, cm -1, respectively. Whereas, amide II band, C-N-H group and para-disubstituted aromatic rings at cm -1, cm -1 and cm -1, respectively were also observed. The observed FTIR specta of both the drugs were matched with reference spectra. The study confirmed that the test samples were dicyclomine and paracetamol. All the tests confirmed the identity and purity of both the drugs. Calibration curves of both the drugs were prepared in 0.1N HCl, phthalate buffer ph 4.5, phosphate buffer ph 6.8, phosphate buffer ph 7.4 and phosphate buffer ph 6.8 with pectinex ultra SPL. Calibration curve data of both the drugs were subjected to linear regression analysis. Beer and Lambert s law was found to be obeyed in the concentration range of mcg/ml and 2-16 mcg/ml for dicyclomine and paracetamol, respectively in all the media. R-values were found to be , , , & for dicyclomine and , , , and for paracetamol in 0.1N HCl, phthalate buffer ph 4.5, phosphate buffer ph 6.8, phosphate buffer ph 7.4 and phosphate buffer ph 6.8 with pectinex ultra SPL, respectively which indicate linearity. School of Pharmaceutical Sciences 63

23 Preformulation studies The solubility of both the drugs was determined in different media. Both drugs were found to be sparingly soluble in acidic medium and slightly soluble in basic medium. The solubility of dicyclomine in 0.1N HCl, phthalate buffer ph 4.5, phosphate buffer ph 6.8, phosphate buffer ph 7.4 was found to be gm/ml, gm/ml, gm/ml, and gm/ml, respectively. The solubility of paracetamol in 0.1N HCl, phthalate buffer ph 4.5, phosphate buffer ph 6.8, phosphate buffer ph 7.4 was found to be gm/ml, gm/ml, gm/ml, and gm/ml, respectively. FTIR spectra were recorded to assess the compatibility of the drugs and excipients. The compatibility of drugs with eudragit RS100, eudragit S100, and PVA was assessed by FTIR spectroscopy of the samples kept at 40 C and 75% RH and at room temperature for 1 month. FTIR spectra of drug (s), physical mixture of drug (s), physical mixture of drug (s) & eudragit RS-100, physical mixture of drug (s) & eudragit S-100, physical mixture of drug (s) & PVA, physical mixture of drug (s), PVA & eudragit RS100, and physical mixture of drug (s), PVA & eudragit S100 were recorded and examined. In FTIR spectra of paracetamol, characteristic N-H stretching band at cm 1, O-H stretching band at cm 1, and carbonyl stretching band at cm 1 were noted and in case of dicyclomine, characteristic C=O stretching band was observed at cm -1 which are in agreement with the reported values. Eudragit RS 100 showed an ester C=O stretching peak around cm 1 and eudragit S 100 showed carbonyl stretching at cm -1 and bond characteristic to carboxylic group in the range cm -1 as reported in the literature. All characteristic peaks of drug(s) were observed in the FTIR spectra of physical mixture of drug (s) & eudragit S-100, physical mixture of drug (s) & PVA, physical mixture of drug (s), PVA & eudragit RS100, and physical mixture of drug (s), PVA & eudragit S100. The results showed no chemical interaction and changes took place in FTIR spectra of both the drugs and various excipients alone or in combination exhibiting compatibility of the drugs with all excipients. School of Pharmaceutical Sciences 64

24 Preparation and Characterization of Microsponge Formulations PREPARATION OF MICROSPONGES Microsponges preparation using Eudragit RS-100 Eudragit RS-100 based paracetamol and dicyclomine loaded microsponges were prepared by quasi-emulsion solvent diffusion method. The internal phase consisted of eudragit RS-100 (200mg) and triethylcitrate (1% v/v, as plasticizer) dissolved in 5 ml dichloromethane. The drug was added to this with gradual stirring (500 rpm). The internal phase was then poured into 0.5 % w/v polyvinyl alcohol (PVA, molecular weight 30,000-70,000) solution in water, the external phase. After 8 hour of stirring the microsponges were formed due to removal of dichloromethane from the system. The microsponges were filtered and dried at 40 C for 12 hours (Orlu et al., 2006). The same method was used for the preparation of microsponges with eudragit S-100 except the stirring rate which was kept at 1000 rpm. The compositions of various microsponge formulations are given in Table 15 & 16. Table 15 Composition of Eudragit RS-100 based microsponge formulations Components Formulation code/amount FDRS1 FDRS2 FDRS3 FDRS4 FPRS1 FPRS2 FPRS3 FPRS4 Dicyclomine (mg) Paracetamol (mg) Eudragit RS-100 (mg) Triethylcitrate (%v/v) Dichloromethane (ml) PVA (% w/v) School of Pharmaceutical Sciences 65

25 Preparation and Characterization of Microsponge Formulations Table 16 Composition of Eudragit S-100 based microsponge formulations Components Formulation code/amount FDS1 FDS2 FDS3 FDS4 FPS1 FPS2 FPS3 FPS4 Dicyclomine (mg) Paracetamol (mg) Eudragit S-100 (mg) Triethylcitrate (%v/v) Dichloromethane (ml) PVA (% w/v) OPTIMIZATION OF FORMULATION Effect of drug to polymer ratio on the size of microsponges The drug and polymer in the ratios 3:1, 6:1, 9:1, 12:1 were taken to prepare different microsponge formulations. In each formulation, the amounts of polymer (200 mg), dichloromethane (5 ml), PVA (0.5% w/v) were kept constant. The microsponge formulations were prepared using mechanical stirrer (Remi RQ1217-D) at a stirring rate of 500 rpm for eudragit RS-100 based microsponges and 1000 rpm for eudragit S-100 based microsponges for 8 hours. Effect of the volume of internal phase on the production of microsponges Two different volumes 5 and 10 ml were taken to study the effect of volume of internal phase solvent (dichloromethane) on the microsponge formulations FDRS1, FPRS1, FPS1 and FDS1. Effect of stirring speed on the size of microsponges The effect of stirring speed on the average size of microsponges was studied using different stirring speeds (300, 400, and 500 rpm for formulations FDRS1 & FPRS1 and 500 & 1000 rpm for formulations FDS1 & FPS1). The effect of stirring rate on the size of microsponges is presented in Table 17 and depicted in Figure 21- Figure 24. School of Pharmaceutical Sciences 66

26 Preparation and Characterization of Microsponge Formulations Table 17 Effect of stirring speed on the size of microsponge formulations Stirring Speed Size (µm) (rpm) FPRS1 FDRS1 FPS1 FDS ± ± ± ± ± ± ± ± ± ±5.24 * Mean±S.D. (n=3) Figure 21 Photomicrographs of dicyclomine loaded microsponges (FDRS1) prepared at different stirring rates (a) 300 rpm; (b) 400 rpm; (c) 500 rpm Figure 22 Photomicrographs of paracetamol loaded microsponges (FPRS1) prepared at different stirring rates (a) 300 rpm; (b) 400 rpm; (c) 500 rpm School of Pharmaceutical Sciences 67

27 Preparation and Characterization of Microsponge Formulations Figure 23 Photomicrographs of dicyclomine loaded microsponges (FDS1) prepared at different stirring rates (a) 500 rpm; (b) 1000 rpm Figure 24 Photomicrographs of paracetamol loaded microsponges (FPS1) prepared at different stirring rates (a) 500 rpm; (b) 1000 rpm Effect of the amount of emulsifying agent on the production yield and size of microsponge Two different concentrations viz. 0.5 % and 1.0 % w/v were taken to study the effect of amount of emulsifying agent (PVA) on the microsponge formulations (FDRS1, FPRS1, FPS1 and FDS1). The effect of emulsifying agent on microsponge formulations is presented in Table 18. School of Pharmaceutical Sciences 68

28 Preparation and Characterization of Microsponge Formulations Table 18 The effect of emulsifying agent on microsponge formulations Formulation Code PVA (% w/v) Yield (%) Mean Diameter (µm ± S.D.) FPS ± ±7.89 FPS ± ±6.73 FPRS ± ±6.89 FPRS ± ±3.15 FDS ± ±5.24 FDS ± ±5.64 FDRS ± ±5.67 FDRS ± ±4.28 * Mean±S.D. (n=3) CHARACTERIZATION OF MICROSPONGES Angle of repose Angle of repose was determined using funnel method. 5 gm microsponges were allowed to pass through a funnel that was raised vertically until a maximum cone height, h, was obtained. Diameter of heap, D, was measured. The repose angle,θ, was calculated by formula (Rao and Patil 2005). The standard value and experimental value of angle of repose are shown in Table 19 and 20. School of Pharmaceutical Sciences 69

29 Preparation and Characterization of Microsponge Formulations Carr s Index and Hausners ratio The Carr s Index and Hausners ratio were calculated using formula: Tapped density was determined by placing 5 gm of the microsponges in a graduated cylinder tapping it for 100 times. Poured density was determined by placing 5 gm of microsponges into a graduated cylinder and measuring the volume (Rao and Patil 2005). The standard value and experimental value of Carr s Index and Hausners ratio are shown in Table 19 and 20. Table 19 Standard values of Angle of repose, Carr s index and Hausner s ratio S.No. Angle of Repose Carr s Index Hausner s Ratio Type of Flow (Inference) 1 < Excellent < 1.25 Good Passable >1.25 Poor Very Poor 6 >40 >40 Extremely Poor School of Pharmaceutical Sciences 70

30 Preparation and Characterization of Microsponge Formulations Table 20 Characterization of microsponges Formulation Evaluation Parameters Code Angle of Repose (θ) Poured density Tapped Density Carr s Index (%) Hausners Ratio ( 0 ) (gm/cm3) (gm/cm3) FPRS ± ± ± ± ±0.01 FPRS ± ± ± ± ±0.28 FPRS ± ± ± ± ±0.01 FPRS ± ± ± ± ±0.25 FPS ± ± ± ± ±0.54 FPS ± ± ± ± ±0.25 FPS ± ± ± ± ±0.06 FPS ± ± ± ± ±0.26 FDRS ± ± ± ± ±0.47 FDRS ± ± ± ± ±0.33 FDRS ± ± ± ± ±0.11 FDRS ± ± ± ± ±0.16 FDS ± ± ± ± ±0.06 FDS ± ± ± ± ±0.07 FDS ± ± ± ± ±0.04 FDS ± ± ± ± ±0.11 * Mean±S.D. (n=3) Determination of production yield The production yield of the microsponges was determined by calculating the initial weight of the raw materials and the final weight of the microsponges obtained. All the experiments were performed in triplicate and the mean of the each value was reported (Jelvehgari et al., 2006). The results of production yield of the microsponge are shown in Table 21. School of Pharmaceutical Sciences 71

31 Preparation and Characterization of Microsponge Formulations Actual drug content and encapsulation efficiency For paracetamol microsponges The weighed amount of drug loaded microsponges (100 mg) was suspended in 100 ml phosphate buffer ph 6.8 for 12 h and subjected to intermittent stirring. The sample was filtered using 0.45_m membrane filter and analyzed at 249 nm against blank using UV spectrophotometer (UV 1700, Shimadzu, Japan). All analyses were carried out in triplicate. The results of actual drug content and encapsulation efficacy are shown in Table 21. For dicyclomine microsponges The weighed amount of drug loaded microsponges (100 mg) was suspended in 100 ml phosphate buffer ph 6.8 for 12 h (sample-1) and subjected to intermittent stirring. 10 ml of sample-1 was diluted with 10 ml of 0.1N HCl (sample-2). 10 ml of sample-2 was further diluted with 50 ml of methyl orange (1%w/v) and extracted with chloroform (3x1.5 ml). The organic phase was separate and pooled and the volume of sample was made up to 100 ml with methylated sodium acetate. The solution was filtered using 0.45_m membrane filter. The absorbance was taken at 420 nm against blank using UV spectrophotometer (UV 1700, Shimadzu, Japan). The drug content and encapsulation efficiency were calculated using the following formula. Actual drug content (%) =M act /M ms 100 Encapsulation efficiency (%) =M act /M the 100 Where M act is the actual drug content in microsponges, M ms is the total amount of the microsponges and M the is the amount of drug added to the microsponges. All analyses were carried out in triplicate. The results of actual drug content and encapsulation efficacy are shown in Table 21. Particle size analysis Particle size was determined by photomicroscope (RXLr-3T, Radical). Microsponges were suspended in glycerol, and the particle size was determined using the software, Biowizard. The results of particle size analysis are shown in Table 21. School of Pharmaceutical Sciences 72

32 Preparation and Characterization of Microsponge Formulations Table 21 Characterization of various microsponge formulation (n=3) Formulation Drug:Polymer Ratio Production yield (% ± S.D.) Theoretical drug content (%) Actual drug content (% ± S.D.) Encapsulation efficiency (% ± S.D.) Mean Particle size (µm ± S.D.) FDRS1 3: ± ± ± ±5.67 FDRS2 6: ± ± ± ±7.11 FDRS3 9: ± ± ± ±6.45 FDRS4 12: ± ± ± ±6.20 FDS1 3: ± ± ± ±5.24 FDS2 6: ± ± ± ±4.89 FDS3 9: ± ± ± ±5.21 FDS4 12: ± ± ± ±4.99 FPRS1 3: ± ± ± ±6.89 FPRS2 6: ± ± ± ±5.39 FPRS3 9: ± ± ± ±7.24 FPRS4 12: ± ± ± ±5.34 FPS1 3: ± ± ± ±7.89 FPS2 6: ± ± ± ±7.67 FPS3 9: ± ± ± ±6.37 FPS4 12: ± ± ± ±5.54 School of Pharmaceutical Sciences 73

33 Preparation and Characterization of Microsponge Formulations Fourier transform infrared (FTIR) analysis FTIR spectra were recorded to assess the chemical interaction or changes during microsponge preparation. FTIR spectra of the drug (s), physical mixture of drug (s) with different polymers, and different microsponge formulations were recorded in potassium bromide disc using a Shimadzu Model 8400 FTIR spectrometer. The FTIR spectra of different microsponge formulations are shown in Figure 25- Figure 28. Figure 25 FTIR Spectra of dicyclomine, physical mixture of drug & Eudragit RS-100, and microsponge formulations FDRS1 FDRS4 School of Pharmaceutical Sciences 74

34 Preparation and Characterization of Microsponge Formulations Figure 26 FTIR Spectra of dicyclomine, physical mixture of drug & Eudragit S- 100, and microsponge formulations FDS1 FDS4 School of Pharmaceutical Sciences 75

35 Preparation and Characterization of Microsponge Formulations Figure 27 FTIR Spectra of paracetamol, physical mixture of drug & Eudragit RS-100, and microsponge formulations FPRS1 FPRS4 School of Pharmaceutical Sciences 76

36 Preparation and Characterization of Microsponge Formulations Figure 28 FTIR Spectra of paracetamol, physical mixture of drug & Eudragit S- 100, and microsponge formulations FPS1 FPS4 School of Pharmaceutical Sciences 77

37 Preparation and Characterization of Microsponge Formulations Differential scanning calorimetric (DSC) analysis DSC provides information about the physical properties of the drugs and demonstrates a possible interaction between drug and other compounds in microsponges. Thermal analysis using DSC was carried out on drug (s), physical mixture of drug (s) with different polymers and different microsponge formulations using Shimadzu DSC-60 Thermal Analyzer. 2 mg of samples were loaded into aluminum pans and sealed. All samples were run at a heating rate of 20 o C/min. over a temperature range o C. The DSC thermograms of different microsponge formulations are shown in Figure 29- Figure 32.. Figure 29 DSC thermograms of dicyclomine, physical mixture of drug & Eudragit RS-100, and microsponge formulations FDRS1 FDRS4 School of Pharmaceutical Sciences 78

38 Preparation and Characterization of Microsponge Formulations Figure 30 DSC thermograms of dicyclomine, physical mixture of drug & Eudragit S-100, and microsponge formulations FDS1 FDS4 School of Pharmaceutical Sciences 79

39 Preparation and Characterization of Microsponge Formulations Figure 31 DSC thermograms of Paracetamol, physical mixture of drug & Eudragit RS-100, and microsponge formulations FPRS1 FPRS4 School of Pharmaceutical Sciences 80

40 Preparation and Characterization of Microsponge Formulations Figure 32 DSC thermograms of Paracetamol, physical mixture of drug & Eudragit S-100, and microsponge formulations FPS1 FPS4 Morphology The morphology of the microsponges was studied using scanning electron microscopy (SEM). All the samples were coated with gold palladium alloy under vacuum. Coated samples were then examined using LEO 430 SEM analyzer. The SEM micrograph of different microsponge formulations are shown in Figure 33 - Figure 36. School of Pharmaceutical Sciences 81

41 Preparation and Characterization of Microsponge Formulations Figure 33 (a-h) SEM photograph of microsponge formulations (dicyclomine: eudragit RS-100). The photograph coded A represents whole image; B represents surface photograph School of Pharmaceutical Sciences 82

42 Preparation and Characterization of Microsponge Formulations Figure 34 (a-h) SEM photograph of microsponge formulations (dicyclomine: eudragit S-100). The photograph coded A represents whole image; B represents surface photographs. School of Pharmaceutical Sciences 83

43 Preparation and Characterization of Microsponge Formulations Figure 35 (a-h) SEM photograph of microsponge formulations (paracetamol: eudragit RS-100). The photograph coded A represents whole image; B represents surface photographs. School of Pharmaceutical Sciences 84

44 Preparation and Characterization of Microsponge Formulations Figure 36 (a-h) SEM photograph of microsponge formulations (paracetamol: eudragit S-100). The photograph coded A represents whole image; B represents surface photographs. School of Pharmaceutical Sciences 85

45 Preparation and Characterization of Microsponge Formulations RESULTS AND DISCUSSION Quasi-emulsion solvent diffusion method was used for preparation of microsponges because of its simplicity and reproducibility. Moreover, it has advantage of avoiding solvent toxicity (Orlu et al., 2006). The drug and polymer in the ratios 3:1, 6:1, 9:1, 12:1 were taken to prepare different microsponge formulations. In each formulation, the amounts of polymer (200 mg), dichloromethane (5 ml), PVA (0.5% w/v) were kept constant. The microsponge formulations were prepared using mechanical stirrer (Remi RQ1217-D) at a stirring rate of 500 rpm for eudragit RS-100 based microsponge and 1000 rpm for eudragit S- 100 based microsponge for 8 hours. The various microsponge formulations namely FDRS1, FDRS2, FDRS3, FDRS4 & FPRS1, FPRS2, FPRS3, FPRS4 containing drug:eudragit RS-100 in the ratios 3:1, 6:1, 9:1, 12:1, respectively and FDS1, FDS2, FDS 3, FDS4 and FPS1, FPS2, FPS3, FPS4 containing eudragit drug:s-100 in the ratios 3:1, 6:1, 9:1, 12:1, respectively were prepared. The effect of various variables like drug to polymer ratio, stirring rate, volume of internal phase, amount of emulsifying agent on the nature of microsponges was studied. Effect of drug-polymer ratio on the size of microsponges The morphology of the microsponges was studied by scanning electron microscopy (SEM). The microsponges were observed to be spherical and uniform with no drug crystals on the surface. It was noted that drug-polymer ratio has considerable effect on the morphology and size of microsponges. It was observed that as the ratio of drug to polymer was increased, the particle size decreased. The mean particle size of formulations FDRS1-FDRS4, FPRS1-FPRS4, FDS1-FDS4, and FPS1- FPS4 in the ratios of 3:1, 6:1, 9:1 and 12:1 were found to be between 60-44µm, µm, µm and µm, respectively. This could probably be due to the fact that in high drug to polymer ratios, the amount of polymer available per microsponge was comparatively less. Hence less polymer surrounded the drug resulting in smaller microsponges (Chaurasia and Jain.2004). School of Pharmaceutical Sciences 86

46 Preparation and Characterization of Microsponge Formulations Effect of stirring rate on the size of microsponges The effect of stirring rate on the size of microsponges was studied by photo microscope (RXLr-3T, Radical, India). The formulation with the lower drug to polymer ratio (i.e., 3:1) was chosen to investigate the effect of stirring rate on the morphology of microsponges. The stirring rate was varied in the range of 300 to 500 rpm for eudragit RS-100 based formulations and 500 to 1000 for eudragit S-100 based formulations. The dispersion of the drug and polymer into the aqueous phase and the formulation of microsponge were found to be dependant on the agitation speed. As the speed was increased, smaller spherical microsponges with uniform size were formed (Perumal 2001). When the rate of stirring was increased rpm eudragit RS-100 based microsponges, the spherical microsponges were formed with mean particle size of 72 µm - 60 µm and 77 µm - 62 µm for formulation FDRS1 and FPRS1, respectively. When the rate of stirring was increased rpm for eudragit S-100 based microsponges the spherical microsponges were formed with mean particle size of 74 µm - 53 µm and 79 µm - 55 µm for formulation FDS1 and FPS1, respectively. Effect of volume of internal phase on the formation of microsponges It was observed that on increasing the volume of internal phase from 5 to 10 ml microsponges were not formed. This may be due to the decrease in viscosity of the internal phase (Yang et al., 2003). As the amount of dichloromethane was increased, though the finely dispersed spherical quasi-emulsion droplets were seen in solvent under the agitation, but as the stirring was discontinued emulsion droplets adhered to each other and coalesce. Consequently, no microsponges could be formed. The result suggests that the amount of dichloromethane need to be controlled within an appropriate range to effect not only the formation of quasi-emulsion droplets at the initial stage but also the solidification of drug and polymer in the droplets. Microsponges were formed when 3 to 5 ml of dichloromethane was used. Effect of amount of emulsifying agent on the production yield and size of microsponges An increase in amount of polyvinyl alcohol (emulsifying agent) from 0.5 % to 1.0 % w/v resulted in decreased production yield and increased mean particle size. School of Pharmaceutical Sciences 87

47 Preparation and Characterization of Microsponge Formulations The amount of emulsifying agent significantly effected the production yield and mean particle size. Due to non-ionic nature of the emulsifier some hydrophobic region might have formed which dissolved some of the drug and polymer resulting in lower production yield 3. An increased amount of emulsifying agent decreased the production yield from 79% to 61%, 72% to 67%, 73% to 65%, 71% to 68% for the formulations FDRS1, FPRS1, FDS1, FPS1, respectively The increase in the amount of emulsifying agent resulted in larger microsponges, probably due to increased viscosity, wherein larger emulsion droplets formed resulting in larger microsponges (Devrim and Canefe 2006). An increased amount of emulsifying agent increased the mean particle size from 60 µm to 71 µm. 62 µm to 66 µm, 53 µm to 64 µm, 55 µm to 56 µm for the formulations FDRS1, FPRS1, FDS1, FPS1, respectively. The production yield was found to be between 72-76% for FPRS1-FPRS4, 71-77% for FPS1-FPS4, 70-79% for FDRS1-FDRS4, and 68-77% for FDS1-FDS4. The actual drug content was found to be between 74-91% for FPRS1-FPRS4, 72-89% for FPS1-FPS4, 62-81% for FDRS1-FDRS4, and 67-83% for FDS1-FDS4. The encapsulation efficiency ranged from 82-98%. The mean particle size was found to be between µm for FPRS1-FPRS4, µm for FPS1-FPS4, µm for FDRS1-FDRS4, and µm for FDS1-FDS4. The data obtained for various formulations in respect to production yield, actual drug content, and encapsulation efficiency were subjected to t-test at 95% level of significance. No significant difference in relation to these parameters was observed amongst various formulations at p <0.05. Characterization of microsponges DSC studies were carried out to confirm that no interaction took place between drug and other compounds in microsponges (Ceschel et al., 2003). According to the thermograms, drugs showed sharp endothermic peaks (dicyclomine and paracetamol at C and C, respectively) which corresponds to the melting point of drug in the crystalline form. In the DSC curve of physical mixture, FPRS1-FPRS4, FPS1-FPS4, FDRS1-FDRS4, and FDS1-FDS4 the characteristic peaks of drug(s) were seen. The result showed that drugs were compatible with polymers. It could also be conferred that microsponge preparation processes did not change the nature of drugs in microsponges. School of Pharmaceutical Sciences 88

48 Preparation and Characterization of Microsponge Formulations FTIR spectra were recorded to assess the chemical interaction or changes during microsponge preparation (Mukherjeea et al., 2005). FTIR spectra of drug (s), physical mixture of drug (s) & eudragit RS-100, physical mixture of drug (s) & eudragit S-100 and formulations FPRS1-FPRS4, FPS1-FPS4, FDRS1-FDRS4, and FDS1-FDS4 were recorded and examined. In FTIR spectra of paracetamol, characteristic N-H stretching band at cm 1, O-H stretching band at cm 1, and carbonyl stretching band at cm 1 were seen and in case of dicyclomine, characteristic C=O stretching band was observed at cm -1 which are in agreement with the reported values. All characteristic peaks of drug(s) were observed in the FTIR spectra of different microsponge formulations namely FPRS1- FPRS4, FPS1-FPS4, FDRS1-FDRS4, and FDS1-FDS4. These results indicated that no chemical interaction or changes took place during microsponge preparation. The drug was compatible with all excipients used for microsponge preparation. School of Pharmaceutical Sciences 89

49 In-vitro drug release studies of Microsponge Formulations EXPERIMENTAL WORK IN-VITRO DRUG RELEASE STUDIES In-vitro drug release studies of paracetamol loaded microsponges In-vitro release studies were carried out in USP basket apparatus with stirring rate 50 rpm at 37±0.5 o C. Initial drug release was carried out in 900 ml of 0.1N hydrochloric acid for 2 hours followed by phosphate buffer ph 6.8 for next 6 hour. Samples were withdrawn at regular intervals and analyzed spectrophotometrically at 249 nm (Orlu et al., 2006). All the readings were taken in triplicate. The same procedure was followed for in-vitro release studies of dicyclomine loaded microsponges. The samples were analyzed at 420 nm. The in-vitro release data of paracetamol loaded microsponges are given in Table 22 - Table 29 and dicyclomine loaded microsponges are given in Table 30 - Table 37. KINETICS OF RELEASE To determine the drug release mechanism and to compare the release profile amongst various microsponge formulations, the in-vitro release data was fitted to various kinetic equations. The kinetic models included zero order, first order, Higuchi model, and Korsmeyer-Peppas model (Nokhodchi et al., 2007). The plots were drawn as per the following details. Cumulative percent drug released as a function of time (zero order kinetic plots). Log cumulative percent drug retained as a function of time (first order kinetics plots). Log cumulative percent drug released as a function of log time (Korsemeyer plots). Cumulative percent drug released versus square root of time (Higuchi plots). The in-vitro release data of different kinetic models are shown in Table 38 and presented in Figure 37 Figure 44 for paracetamol microsponges and Figure 45 Figure 52 for dicyclomine microsponges. School of Pharmaceutical Sciences 90

50 In-vitro drug release studies of Microsponge Formulations Table 22 In-vitro drug release data for formulation FPRS1 Time in hrs T T Log T Cum. % drug released Log Cum % drug released Cum. % drug remained Log Cum. % drug remained ± ± ± ± ± ± ± ± Table 23 In-vitro drug release data for formulation FPRS2 Time in hrs T T Log T Cum. % drug released Log Cum % drug released Cum. % drug remained Log Cum. % drug remained ± ± ± ± ± ± ± ± School of Pharmaceutical Sciences 91

51 In-vitro drug release studies of Microsponge Formulations Table 24 In-vitro drug release data for formulation FPRS3 Time in hrs T T Log T Cum. % drug released Log Cum % drug released Cum. % drug remained Log Cum. % drug remained ± ± ± ± ± ± ± ± Table 25 In-vitro drug release data for formulation FPRS4 Time in hrs T T Log T Cum. % drug released Log Cum % drug released Cum. % drug remained Log Cum. % drug remained ± ± ± ± ± ± ± ± School of Pharmaceutical Sciences 92

52 In-vitro drug release studies of Microsponge Formulations Table 26 In-vitro drug release data for formulation FPS1 Time in hrs T T Log T Cum. % drug released Log Cum % drug released Cum. % drug remained Log Cum. % drug remained ± ± ± ± ± ± ± ± Table 27 In-vitro drug release data for formulation FPS2 Time in hrs T T Log T Cum. % drug released Log Cum % drug released Cum. % drug remained Log Cum. % drug remained ± ± ± ± ± ± ± ± School of Pharmaceutical Sciences 93

53 In-vitro drug release studies of Microsponge Formulations Table 28 In-vitro drug release data for formulation FPS3 Time in hrs T T Log T Cum. % drug released Log Cum % drug released Cum. % drug remained Log Cum. % drug remained ± ± ± ± ± ± ± ± Table 29 In-vitro drug release data for formulation FPS4 Time in hrs T T Log T Cum. % drug released Log Cum % drug released Cum. % drug remained Log Cum. % drug remained ± ± ± ± ± ± ± ± School of Pharmaceutical Sciences 94

54 In-vitro drug release studies of Microsponge Formulations Table 30 In-vitro drug release data for formulation FDRS1 Time in hrs T T Log T Cum. % drug released Log Cum % drug released Cum. % drug remained Log Cum. % drug remained ± ± ± ± ± ± ± ± Table 31 In-vitro drug release data for formulation FDRS2 Time in hrs T T Log T Cum. % drug released Log Cum % drug released Cum. % drug remained Log Cum. % drug remained ± ± ± ± ± ± ± ± School of Pharmaceutical Sciences 95