College Papers

RESULTS AND DISCUSSION P

RESULTS AND DISCUSSION P. Rami Reddy Memorial College of Pharmacy Page 89 5. RESULTS A successful attempt was made to formulate the Fenoprofen ethosomal gel. In the present work QbD was used for optimization six formulations were prepared whose composition was mentioned. 5.1. Drug-compatability studies: Compatability studies of pure drug Fenoprofen with different polymers were carried out prior to the formulation of Ethosomal gel. IR spectra of Fenoprofen and polymer were taken, which are depicted in the figure. All the characteristics peaks of Fenoprofen were present in spectra at respective wavelengths. Thus, indicating compatability between drug and polymer. It shows that there was no significant change in chemical integrity of drug. Figure 18: FTIR spectra of pure drug Fenoprofen Figure 19: FTIR spectra of pure drug Fenoprofen with Lectin
E:IR DATAOFLOXACIN PURE.0 OFLOXACIN PURE PELLET25/09/2012
3421.513042.472932.752837.952788.001714.821621.921550.191523.211463.651399.721288.291199.481143.661053.451008.14954.09878.09802.99709.21667.51
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E:IR DATAOFLOXACIN+CHITOSAN.0 OFLOXACIN+CHITOSAN Instrument type and / or accessory11/04/2017
3974.433928.053900.343822.033800.453420.723092.933042.172924.892859.972788.052754.652685.202366.812202.622160.862063.811990.781935.071869.551715.091621.831550.781523.381461.351399.231288.701241.231198.741143.201053.371008.27977.81953.53878.79849.95829.45802.30778.31743.53708.60666.72629.71599.95
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RESULTS AND DISCUSSION P. Rami Reddy Memorial College of Pharmacy Page 90 Figure 20: FTIR spectra of pure drug with Cholesterol Figure 21: FTIR spectra of pure drug with Carbopol Figure 22: FTIR spectra of pure drug with Polyethylene glycol
E:IR DATAOFLOXACIN+BETA CYCLODEXTRIN.0 OFLOXACIN+BETA CYCLODEXTRIN Instrument type and / or accessory11/04/2017
3973.703926.863898.833818.493797.793101.473042.502969.292929.072866.192787.842754.892684.962370.962259.062234.602157.202125.672063.611990.551935.031869.481720.761622.691550.971522.781462.371350.341287.511241.711198.931146.251089.081052.26981.09952.44827.75778.62745.59707.98666.67
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E:IR DATAOFLOXACIN+CARBOPOL.0 OFLOXACIN+CARBOPOL Instrument type and / or accessory11/04/2017
3838.743417.803042.242969.372931.102865.532787.712754.482684.632364.572061.281988.831935.091715.621622.091550.451523.511460.041399.991371.201287.261242.401198.921144.311052.641007.88976.96953.29877.64849.22826.66802.06744.07708.09665.95630.59566.73
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E:IR DATAOFLOXACIN+POLYETHYLENE GLYCOL.0 OFLOXACIN+POLYETHYLENE GLYCOL Instrument type and / or accessory15/04/2017
3839.203413.212873.482097.081961.601644.061459.731352.631296.461249.831105.84949.47885.33841.68582.57
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RESULTS AND DISCUSSION P. Rami Reddy Memorial College of Pharmacy Page 91 Figure 23: FTIR spectra of pure drug with all excipients Table 13: Functional groups of Infrared spectroscopy MATERIALS GROUPS ASSIGNED C=O C-O-C C-F O-H CH3 N-H Fenopofen 1714.82 1288.29 1053.45 1339.72 2788.00 1621.92 Fenopofen + Carbopol 1715.82 1287.26 1052.64 1371.20 2787.71 1622.09 Fenopofen + Lectin 1715.09 1288.70 1053.37 1399.23 2788.05 1621.83 Fenopofen + Propylene glycol 1647.79 1289.19 1040.18 1333.97 2932.75 1522.10 Fenopofen + Alcohol 1644.06 1296.46 1105.84 1459.73 2873.48 1644.06 5.2 Compatibility studies by DSC method Figure 24: DSC Thermogram of Fenoprofen
E:IR DATAOFLOXACIN+ALL EXCIPIENTS.0 OFLOXACIN+ALL EXCIPIENTS Instrument type and / or accessory15/04/2017
3450.522877.662124.461961.681647.891456.961353.321295.921250.491104.89992.93948.22840.31804.77675.40
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RESULTS AND DISCUSSION P. Rami Reddy Memorial College of Pharmacy Page 92 Figure 25: DSC Thermogram of Soya Lectin Figure 26: DSC Thermogram of Propylene glycol Figure: 27. DSC Thermogram of Fenoprofen + Lectin

RESULTS AND DISCUSSION P. Rami Reddy Memorial College of Pharmacy Page 93 5.2.1. Compatibility study by DSC technique Table 14: Summary of enthalpy, peak temperature and Crystallinity index of DSC thermograms S.No Name of test sample Ratio Enthalpy (J/g) Peak temperature (oc) % Crystallinity index (CI) Inference 1. Pure Drugs 125.3, 12.9 247.8, 244.2 Not applicable (NA) 3 Fenoprofen+LEC 1:10 149.9 248.0 Compatible 3 Fenoprofen+PLG 1:10 145.2 238.3 Compatible 5.3. Analytical method for estimation of fenoprofen by RP-HPLC The method of analysis was carried out as per the reported method in literature.30, 31, 32,33 5.3.1. Mobile phase preparation: Mobile phase was prepared by mixing 60% of HPLC grade Acetonitrile and 30% of Phosphate Buffer. This solution was filtered using a 0.45 micron Millipore filter paper and was sonicated for 10mins. The total volume of the mobile phase prepared was 1500ml. 5.3.2. Sample preparation: 1ml of fenoprofen was taken in 10ml volumetric flask and make up the volume to 10 ml with methanol (the concentration of this solution is 1000 mg/ml). From this above solution working solution 0.1ml was pipetted into 10ml volumetric flask and volume was made up to the mark with acetonitrile (the concentration of this solution is 1000µg/ml). This is a working solution. From this different concentration ranging from 10 µg/ml, 20 µg/ml, 40µg/ml, 60µg/ml and 80 µg/ml, 100 µg/ml was prepared by transferring required aliquotes of solution to 10ml volumetric flask and make up the volume up to the mark by methanol. This was sonicated for 8 mins then the solution was filtered using 0.45 micron Millipore filters. The following parameters were validated Ø Linearity: The linearity range maintained was 20 µg/ml, 40 µg/ml, 60µg/ml, 80µg/ml and 100 µg/ml Ø Precision: 60µg/ml concentration is selected for carrying out precision and robustness Ø Accuracy: In this parameter the selected concentration 80%, 100% and 120% is done

RESULTS AND DISCUSSION P. Rami Reddy Memorial College of Pharmacy Page 94 5.3.3. Optimized Chromatographic conditions: Detector : Shimadzu spd10A uv-vis, Japan Pump : Shimadzu LC-10ATVP, Japan Software : Baseline chromatography Data System N2000 Injection valve : 7725i Rheodyne 20µl, USA Syringe : 50µl Hamilton, Switzerland, Column : Phenomenex Gemini-NX-5 µm C18(2) 110 Å, LC Column 250 x 4.6 mm, Ea Part No : 00G-4041-EO Dimensions : 250 x 4.6 mm ID Elution Type : Isocratic Elution A : Methanol Elution B : Acetonitrile: Phosphate buffer (60:40) Flow Rate : 1mL/min Col. Temp : ambient Detection : UV-Vis Abs.-Variable Wave. (UV) @ 275nm 5.3.4. Standard plot of fenoprofen The standard plot of fenoprofen was prepared in methanol using a concentration range 0 to 100 µg /ml. Acetonitrile:Phosphate Buffer (60:40) pH 3 was used as mobile phase and absorbance was measured for each solution at ?max of 275 nm using RP-HPLC and the graph was plotted for absorbance versus concentration of fenoprofen. All the readings were taken in triplicate to minimize error and standard deviation was determined. Table 15: Calibration curve data of Fenoprofen S.NO CONCENTRATION (µg/ml) ABSORBANCE 1. 0 0 2. 20 95227.305 3. 40 192930.25 4. 60 278097.563 5. 80 363711.375 6. 100 462635.438 Good linearity was observed in the concentration range of 5 to 30 µg /ml as R2 value was found to be 0.99841.

RESULTS AND DISCUSSION P. Rami Reddy Memorial College of Pharmacy Page 95 Figure 28: Calibration curve of Fenoprofen Linearity was observed in the concentration range of 0 to 100 µg /ml as R2 value was found to be 0.9994, readings were taken in triplicate and standard deviation was calculated which was found to be under limits. 5.4. Linearity, precision and accuracy of analytical method 5.4.1. Linearity and range The linearity was determined by analyzing 6 independent levels of standard curve in the range of 0-100 µg/ml. Table 16: Results of Linearity and range Concentration (µg/ml) Concentration as % of analyte Target Peak are (mean of three injections) Peak Area % RSD 20 20 32879.68333 0.818390452 40 40 65443.682 0.730706314 60 60 101185.065 0.955970165 80 80 132242.37 0.268945 100 100 165547.8543 0.801383311 Y=4,576.8524x + 3,257.6995 Equation for regression line Correlation coefficient (r2) = 0.9994 The values of coefficient of linearity were found to be nearer to 1 for all 3 stock solutions which indicates the linearity of the range selected. y = 4,576.8524x + 3,257.6995R² = 0.99940100000200000300000400000500000020406080100120Areaconcentration in mcgLinearity of Fenoprofen

RESULTS AND DISCUSSION P. Rami Reddy Memorial College of Pharmacy Page 96 5.4.2. Precision: Intra-day precision was determined by analyzing drug (0-100 µg/ml) at three different time points of the same day and inter day precision was determined by analyzing fenoprofen at three different time points on different days and % RSD was calculated. Table 17: Interday precision of analysis of fenoprofen Sno. Concentration 60µg/ml Retention Time Peak area 1 100528.602 5.492 2 101888.445 5.52 3 99627.797 5.492 4 101862.2 5.495 5 100751.398 5.498 6 101963.898 5.493 Mean 101103.7233 5.498333333 StDev 955.4299174 0.010856642 %RSD 0.944999735 0.197453318 Table 18: Intermediate Precision of analysis of fenoprofen Spike Day-1 Day-2 Day-3 1 102295.547 102185 101700.898 2 101803.195 100681.398 103389 3 102200.305 101185.602 102838.672 Mean 102099.6823 101350.6667 102642.8567 StDev 261.1442394 765.2708682 860.9180125 %RSD 0.255773802 0.755072358 0.838751025 The % RSD (Relative Standard Deviation) is less than 1 for intraday precision and for interday precision suggests that the method is precise and shows reproducibility.

RESULTS AND DISCUSSION P. Rami Reddy Memorial College of Pharmacy Page 97 5.4.3. Accuracy of analytical method The prepared samples were spiked and % recovery was calculated to confirm the accuracy of analytical method. Table 19: Accuracy of analysis of fenoprofen Sample Percentage nominal (mean of three injections) Amount of standard (µg) Recovery (%) spike Found 1 80 48 49.19 102.48 2 100 60 60 100 3 120 72 72.40 100.55 Percentage 80 100 120 Mean 115343.5267 133735.7133 160843.4897 StDev 489.3058001 161.6309399 806.3138053 %RSD 424216091 0.120858472 0.501303352 The method showed % recovery of more than 98%, thus confirming the accuracy of method of determining fenoprofen by RP-HPLC. 5.5. Process variable optimization 5.5.1. Probe sonication The probe sonicator was operated using 13 mm probe at an amplitude of 60%. The effect of sonication cycles on the transparency of vesicular dispersion and average size of vesicles was studied. The % transmittance was measured using UV spectrophotometer and size was measured using trinocular microscope. Table 20: Process optimization of Probe Sonication Probe Amplitude Time and Pulse Temperature 13 mm 60 % Standard 2 minutes 2 sec on, 2 sec off 4 0C Table 21: Effect of sonication cycles on vesicle size of ethosomes Cycles Observation Mean vesicle Size 2 Vesicles dispersion was slightly Hazy 3.5 µ 3 Vesicles dispersion was almost transparent 2.0 µ 4 Vesicles dispersion was almost transparent 0.6 µ 5 Vesicle dispersion was transparent, Process repeated thrice % Transmittance was 90% in every analysis 0.4 µ It was found that sonication for 2 cycles of 2 minutes produced vesicles dispersion which was

RESULTS AND DISCUSSION P. Rami Reddy Memorial College of Pharmacy Page 98 slightly hazy and mean vesicle size was found to be 3.5 µm whereas at 3 and 4 cycles for 2 minutes produced vesicles dispersion which was almost transparent with mean vesicle size of 2 µm and 0.6 µm. The sonication for 5 cycles of 2 minutes produced vesicle dispersion which was transparent and mean vesicle size observed was 0.4 µ. 5.5.2. Probe sonication: 5 sonication cycle each of 2 minutes at amplitude of 60% for ethosomes. 5.6. Preliminary studies for screening of excipients 5.6.1. Selection of excipients by formulation of trial batches of carrier system: The preliminary trial batches of ethosomes were prepared using drug and phospholipid in ratio of 1:1. Preparation was carried out at selected process parameters. 1. Soya lecithin 2. Egg lecithin Other excipient used in the preparation of ethosomes was cholesterol as stabilizers and chloroform and methanol as solvents cold method. The ethosomes prepared were observed under trinocular microscope (Carl Zeiss) to determine the possibility of preparation by using the above mentioned phospholipids. 5.7. Preliminary studies on formulation of ethosomes by 32 full factorial design The selected excipients in the preliminary studies were put in 3 factors, 2 levels factorial design and screened on the basis of outcomes of size and entrapment efficiency. Table 22: 32 full factorial design of variables Variables Factors X,Y Levels used, Actual (Coded) Low (-1) Medium (0) High (+1) Independent variables Percentage of alcohol (%w/w) X1 20 30 40 Percentage of cholesterol, (%w/w) X2 0.1 0.5 1 Percentage of phospholipid, (%w/w) X3 2 3.5 5 Dependent variables ___ Constraints Particle size (nm) Y1 Minimize Entrapment efficiency (%) Y2 Maximize

RESULTS AND DISCUSSION P. Rami Reddy Memorial College of Pharmacy Page 99 5.7.1. Preparation of trial batches of ethosomes based on factorial design The quantity of phospholipids was varied as 20 mg and 40 mg and cholesterol were varied as 0.1 mg and 1 mg for preparation of factorial batches of ethosomes. The batches were named as EF1 to EF12. 5.7.2. Factorial design for factors screening in ethosome preparation Table 23: 32 factorial design of ethosomal formulation Run X1 % of Alcohol (%W/W) X2 % of cholesterol (%W/W) X3 % of Phospholipid (%W/W) Y1 Vesicle Size (nm) Y2 Entrapment Efficiency (%) 1 30 0.55 6.02269 668 97.07 2 30 -0.206807 3.5 162 96.41 3 40 1 2 769 95.4 4 20 0.1 2 138 96.02 5 30 0.55 0.977311 196 96.3 6 20 0.1 5 159 95.46 7 40 1 5 816 99.09 8 30 0.55 3.5 295 96.3 9 46.8179 0.55 3.5 948 99.02 10 30 1.30681 3.5 348 96.19 11 40 0.1 5 878 99.37 12 20 1 2 319 95.11 13 40 0.1 2 785 95.4 14 20 1 5 172 95.64 15 30 0.55 6.02269 668 97.07 Highest drug entrapment was observed in the batch containing 5% of phospholipid and 1% of cholesterol but the size of vesicles was found to be higher than other formulations. 5.7.3. Analysis of factorial batches The factorial batches were analyzed by main effect plot, interaction plot and cube plot to determine the effect and influence of factors on responses of % drug entrapment and size of ethosomes.

RESULTS AND DISCUSSION P. Rami Reddy Memorial College of Pharmacy Page 100 5.7.3.1. Main effect plot to study the effect of factors and their levels on the responses Figure 29: Normal plot of Vesicle size Figure 30: Normal plot of Entrapment Efficiency Higher quantity of phospholipid and surfactant results in higher drug entrapment as observed in main effect plot. Higher quantities of phospholipid and cholesterol results in larger size of ethosomes as observed in the main effect plot.
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RESULTS AND DISCUSSION P. Rami Reddy Memorial College of Pharmacy Page 101 5.7.3.2. Interaction Plots of factors on the responses Figure 31: Interaction plot of Vesicle size Figure 32: Interaction plot of Entrapment Efficiency Interaction plot indicate less interaction between quantities of phospholipid and cholesterol in affecting size of ethosomes. Almost parallel lines indicate no interaction between quantities of phospholipid and cholesterol in affecting % drug entrapment of ethosomes.

RESULTS AND DISCUSSION P. Rami Reddy Memorial College of Pharmacy Page 102 5.7.3.3. Cube plots to study effects of levels of factors on responses Figure 33: Cube plot of Vesicle size Figure 34: Cube plot of Entrapment Efficiency As observed from the cube plot, Quantity of phospholipid has positive influence on size Quantity of cholesterol has positive influence on size Quantity of alcohol has positive influence on size Quantity of phospholipid has positive influence on % drug entrapment Quantity of cholesterol has positive influence on % drug entrapment Quantity of alcohol has greatest influence on % drug entrapment

RESULTS AND DISCUSSION P. Rami Reddy Memorial College of Pharmacy Page 103 5.8. Shape and homogeneity of ethosomes The shape and homogeneity of factorial design batch EF7 was studied by trinocular microscope (Carl Zeiss) at 40 X magnification. 5.9. Formulation of drug carriers incorporated gel 5.9.1. Preparation of transdermal gel: A 1% carbopol gel was prepared as per standard methods by dispersing carbopol 934 in distilled water as a base for incorporation of drug loaded carriers for transdermal delivery. 5.9.2. Formulation batches of drug carriers incorporated gel based on experimental design (Central composite design): The experimental batches based on central composite design were further prepared by varying the level of phospholipids, Alcohol and cholesterol Table 24: Formulation batches of drug carriers incorporated gel Formulation Percentage of Alcohol (% w/v) Percentage Of cholesterol (% w/w) Percentage of Phospholipid (% w/w) EFG1 46.8179 0.55 3.5 EFG 2 40 0.1 5 EFG 3 20 0.1 5 EFG 4 13.1821 0.55 3.5 EFG 5 30 0.55 3.5 EFG 6 30 0.55 6.02269 EFG 7 30 1.30681 3.5 EFG 8 20 1 2 EFG 9 30 0.55 0.977311 EFG 10 40 1 2 EFG 11 20 0.1 2 EFG 12 30 -0.206807 3.5 EFG 13 40 1 5 EFG 14 20 1 5 EFG 15 40 0.1 2 The responses measured for batches were % drug entrapment and entrapment efficiency the effect of factors on the responses were studied. Ethosomes batches based on central composite design

RESULTS AND DISCUSSION P. Rami Reddy Memorial College of Pharmacy Page 104 5.10. Evaluation of ethosomes incorporated gel: Table 25: Evaluation of ethosomal gel on the basis of gel characteristics Formulation pH Refractive Index Spread ability (gm.cm/sec) Gel strength (gm) Extrudability (gm) Press 1 Press 2 Press 3 EFG1 6.7 1.42 1.4199 20.18 3.114 2.187 1.96 EFG 2 6.2 1.39 1.3614 22.92 3.467 2.114 1.650 EFG 3 6 1.38 1.3812 21.55 3.737 2.008 1.275 EFG 4 6 1.44 1.3101 24.50 2.898 2.009 1.197 EFG 5 6.1 1.36 1.3980 22.70 3.567 2.886 1.854 EFG 6 5.9 1.45 1.3417 20.46 3.786 2.002 1.713 EFG 7 6.3 1.48 1.2698 23.11 3.641 1.996 1.118 EFG 8 6.2 1.39 1.4143 20.18 3.487 2.775 1.729 EFG 9 6 1.43 1.3977 21.87 3.773 2.087 1.684 EFG 10 6 1.39 1.2928 22.34 2.644 2.113 1.883 EFG 11 6.7 1.42 1.3249 22.76 3.179 2.005 1.480 EFG 12 6.6 1.41 1.3287 22.09 3.583 2.505 1.860 Plain drug gel 6 1.34 1.3595 17.23 2.880 1.996 1.234 Figure 35: Ethosomal Tropical formulation Table 26: Physical evaluation of clarity and homogeneity Formulation code Clarity Homogeneity EFG1 ++ Good EFG 2 ++ Good EFG 3 +++ Good EFG 4 ++ Good EFG 5 +++ Good EFG 6 ++ Good EFG 7 ++ Good EFG 8 + Good EFG 9 + Good EFG 10 ++ Good EFG 11 + Good EFG12 + Good

RESULTS AND DISCUSSION P. Rami Reddy Memorial College of Pharmacy Page 105 From above table, based on evaluation of pH, refractive index, spreadability, gel strength and extrudability of all the formulations, it was observed that drug loaded ethosomes incorporated in gel are better than plain drug gel. 5.11. In-vitro and ex-vivo membrane permeation studies and determination of permeation flux for ethosomal gel: In-vitro and ex-vivo permeation studies were performed as per the ethical guidelines approved by institutional animal ethics committee. The in-vitro permeation studies were carried out for all the experimental design batches whereas ex-vivo studies were performed for optimized batch. The permeation flux for experimental batches of ethosomal gel and plain drug gel were determined. 5.12. Evaluation of experimental design batches of ethosomes of fenoprofen: The experimental design batches were evaluated for in-vitro drug release characteristics using modified Franz diffusion cell using dialysis membrane. The invitro drug release studies were performed for all the fifteen batches of ethosomal gel and also for the plain drug gel for comparison. 5.13. In-vitro permeation studies of ethosomes: Studies were carried out for all the experimental batches of ethosomes and the cumulative drug release as well as the permeation flux was determined Figure 36: Permeation studies of Ethosomal gel of Fenoprofen

RESULTS AND DISCUSSION P. Rami Reddy Memorial College of Pharmacy Page 106 Table 27: Cumulative drug release (µg) by experimental batches of ethosomal gel of Fenoprofen Time (Hrs) % Drug Release EF1 EFF2 EFF3 EFF4 EFF5 EFF6 EFF7 EFF8 EFF9 EFF10 EFF11 EFF12 Plain gel 1 26.86 25.28 23.89 25.88 27.98 26.57 21.06 23.05 26.69 24.08 23.65 22.88 31.49 2 35.98 36.98 34.25 34.67 34.23 35.77 35.76 35.24 34.98 31.68 32.66 33.78 41.64 3 43.16 45.36 43.67 45.44 45.90 46.88 44.56 43.18 42.86 42.68 41.23 42.57 56.87 4 52.18 51.46 52.86 56.80 55.60 55.26 56.24 52.87 53.45 54.66 52.68 52.88 61.51 5 62.86 61.93 61.66 65.29 64.38 63.38 66.22 61.45 62.12 65.76 64.46 63.68 74.68 6 72.56 71.50 70.88 72.66 73.14 74.18 78.90 70.24 73.24 76.34 75.56 74.34 83.17 7 80.46 80.34 81.45 81.66 82.98 83.06 89.34 81.09 81.34 82.28 83.28 85.24 89.47 8 89.86 90.14 91.06 92.08 90.60 91.89 98.53 92.64 90.97 91.77 92.86 93.88 91.78

RESULTS AND DISCUSSION P. Rami Reddy Memorial College of Pharmacy Page 107 5.14. Kinetic Models Data Analysis The results of dissolution data fitted to various drug release kinetic equations like Zero order, First order, Higuchi model and Korsemeyer-Peppas. The kinetic values obtained for all formulations F1, F2, F3, F4, F5, F6, F7, F8, F9, F10, F11, F12 were tabulated in table 28 to table 35 respectively, Graphs are Plotted for Zero order, First order, Higuchi model and Korsemeyer-Peppas against cumulative % drug release Vs Time (Hrs), Log cumulative % drug remaining Vs Time (Hrs), cumulative % drug release Vs Square root of Time, Log cumulative % drug release Vs Log Time are shown in the figure 14 to figure 61 respectively. Table 28: In-vitro drug release kinetics data for Formulation F1 Zero order First order Higuchi’s data Korsmeyer-Peppas data Time (h) % CDR Time (h) Log % CD Remaining SQR Time % CDR Log Time Log % CDR 1 26.86 1 1.864155 1 26.86 0 1.429106 2 35.98 2 1.8063157 1.414 35.98 0.301 1.5560612 3 43.16 3 1.7546541 1.732 43.16 0.477 1.6350814 4 52.18 4 1.6796096 2 52.18 0.602 1.7175041 5 62.86 5 1.5698419 2.236 62.86 0.698 1.7983744 6 72.56 6 1.4383841 2.449 72.56 0.778 1.8606973 7 80.46 7 1.2909246 2.645 80.46 0.845 1.90558 8 89.86 8 1.006038 2.828 89.86 0.903 1.9535664 Figure 37: Zero order Plot for F1 Formulation Figure 38: First order plot for F1 Formulation 0204060801000510Cumulative % drug releaseTime(Hrs)Zero order plot for F1 Formulation00.511.520510Log cumulative % drug remainingTime(Hrs)First order plot for F1 Formulation

RESULTS AND DISCUSSION P. Rami Reddy Memorial College of Pharmacy Page 108 Figure 39: Higuchi plot for F1 Formulation Figure 40: Korsmeyer Peppas plot for F1 Formulation Table 29: In-vitro drug release kinetics data for Formulation F2 Zero order First order Higuchi’s data Korsmeyer-Peppas data Time (h) % CDR Time (h) Log % CD Remaining SQR Time % CDR Log Time Log % CDR 1 25.28 1 1.8734369 1 25.28 0 1.4027771 2 36.98 2 1.7994784 1.414 36.98 0.301 1.5679669 3 45.36 3 1.7375107 1.732 45.36 0.477 1.656673 4 51.46 4 1.6860998 2 51.46 0.602 1.7114698 5 61.93 5 1.5805829 2.236 61.93 0.698 1.7919011 6 71.5 6 1.4548449 2.449 71.5 0.778 1.854306 7 80.34 7 1.2935835 2.645 80.34 0.845 1.9049318 8 90.14 8 0.9938769 2.828 90.14 0.903 1.9549176 0204060801000123Cumulative % drug releaseSquare Root TimeFigure: Higuchi plot for F1 Formulation00.511.522.500.51Log cumulative % drug releaseLog TimeKorsmeyer-peppas plot for F1 Formulation

RESULTS AND DISCUSSION P. Rami Reddy Memorial College of Pharmacy Page 109 Figure 41: Zero order Plot for F2 Formulation Figure 42: First order plot for F2 Formulation Figure 43: Higuchi Plot for F2 Formulation Figure 44: Korsmeyer Peppas plot for F2 Formulation 0204060801000510Cumulative % drug releaseTime(Hrs)Zero order plot for F2 Formulation00.511.520510Log cumulative % drug remainingTime(Hrs)First order plot for F2 Formulation0204060801000123cumulative % drug ReleaseSquare Root TimeHiguchi plot for F2 Formulation00.511.522.500.51Log cumulative % drug releaseLog TimeKorsmeyer-peppas plot for F2 Formulation

RESULTS AND DISCUSSION P. Rami Reddy Memorial College of Pharmacy Page 110 Table 30: In-vitro drug release kinetics data for Formulation F3 Zero order First order Higuchi’s data Korsmeyer-Peppas data Time (h) % CDR Time (h) Log % CD Remaining SQR Time % CDR Log Time Log % CDR 1 23.89 1 1.8814417 1 23.89 0 1.3782161 2 34.25 2 1.8178958 1.414 34.25 0.301 1.5346606 3 43.67 3 1.7507398 1.732 43.67 0.477 1.6401832 4 52.86 4 1.6733896 2 52.86 0.602 1.7231272 5 61.66 5 1.5836521 2.236 61.66 0.698 1.7900035 6 70.88 6 1.4641914 2.449 70.88 0.778 1.8505237 7 81.45 7 1.2683439 2.645 81.45 0.845 1.9108911 8 91.06 8 0.9513375 2.828 91.06 0.903 1.9593276 Figure 45: Zero order Plot for F3 Formulation Figure 46: First order Plot for F3 Formulation Figure 47: Higuchi plot for F3 Formulation Figure 48: Korsmeyer Peppas plot for F3 Formulation 0204060801000510Cumulative % drug releaseTime(Hrs)Zero order plot for F3 Formulation00.511.520510Log cumulative % drug remainingTime(Hrs)First order plot for F3 Formulation0204060801000123Cumulative % drug releaseSquare Root TimeHiguchi plot for F3 Formulation00.511.522.500.51Log cumulative % drug releaseLog TimeKorsmeyer-peppas plot for F3 Formulation

RESULTS AND DISCUSSION P. Rami Reddy Memorial College of Pharmacy Page 111 Table 31: In-vitro drug release kinetics data for Formulation F4 Zero order First order Higuchi’s data Korsmeyer-Peppas data Time (h) % CDR Time (h) Log % CD Remaining SQR Time % CDR Log Time Log % CDR 1 25.88 1 1.8699354 1 25.88 0 1.4129643 2 34.67 2 1.8151127 1.414 34.67 0.301 1.5399538 3 45.44 3 1.7368744 1.732 45.44 0.477 1.6574383 4 56.8 4 1.6354837 2 56.8 0.602 1.7543483 5 65.29 5 1.5404546 2.236 65.29 0.698 1.8148467 6 72.66 6 1.4367985 2.449 72.66 0.778 1.8612954 7 81.66 7 1.2633993 2.645 81.66 0.845 1.9120094 8 92.08 8 0.8987252 2.828 92.08 0.903 1.9641653 Figure 49: Zero order Plot for F4 Formulation Figure 50: First order plot for F$ formulation Figure 51: Higuchi plot for F4 Formulation Figure 52: Korsmeyer Peppas plot for F4 Formulation 0204060801000510Cumulative % drug releaseTime(Hrs)Zero order plot for F4 Formulation00.511.520510Log cumulative % drug remainingTime(Hrs)First order plot for F4 Formulation0204060801000123Cumulative % drug releaseSquare Root TimeHiguchi plot for F4 Formulation00.511.522.500.51Log cumulative % drug releaseLog TimeKorsmeyer-peppas plot for F4 Formulation

RESULTS AND DISCUSSION P. Rami Reddy Memorial College of Pharmacy Page 112 Table 32: In-vitro drug release kinetics data for Formulation F5 Zero order First order Higuchi’s data Korsmeyer-Peppas data Time (h) % CDR Time (h) Log % CD Remaining SQR Time % CDR Log Time Log % CDR 1 27.98 1 1.8574531 1 27.98 0 1.4468477 2 34.23 2 1.8180278 1.414 34.23 0.301 1.5344069 3 45.9 3 1.7331973 1.732 45.9 0.477 1.6618127 4 55.6 4 1.647383 2 55.6 0.602 1.7450748 5 64.38 5 1.5516939 2.236 64.38 0.698 1.808751 6 73.14 6 1.429106 2.449 73.14 0.778 1.864155 7 82.98 7 1.2309596 2.645 82.98 0.845 1.9189734 8 90.6 8 0.9731279 2.828 90.6 0.903 1.9571282 Figure 53: Zero order Plot for F5 Formulation Figure 54: First order Plot for F5 Formulation Figure 55: Higuchi plot for F5 Formulation Figure 56: Korsmeyer Peppas plot for F5 Formulation 0204060801000510Cumulative % drug releaseTime(Hrs)Zero order plot for F5 Formulation00.511.520510Log cumulative % drug remainingTime(HrsFirst order plot for F5 formulation0204060801000123Cumulative % drug releaseSquare Root TimeHiguchi plot for F5 Formulation00.511.522.500.51Log cumulative % drug releaseLog TimeKorsmeyer-peppas plot for F5 Formulation

RESULTS AND DISCUSSION P. Rami Reddy Memorial College of Pharmacy Page 113 Table 33: In-vitro drug release kinetics data for Formulation F6 Zero order First order Higuchi’s data Korsmeyer-Peppas data Time (h) % CDR Time (h) Log % CD Remaining SQR Time % CDR Log Time Log % CDR 1 26.57 1 1.8658735 1 26.57 0 1.4243916 2 35.77 2 1.8077379 1.414 35.77 0.301 1.5535189 3 46.88 3 1.7252581 1.732 46.88 0.477 1.6709876 4 55.26 4 1.650696 2 55.26 0.602 1.7424109 5 63.38 5 1.5637183 2.236 63.38 0.698 1.8019522 6 74.18 6 1.4119562 2.449 74.18 0.778 1.8702868 7 83.06 7 1.2289134 2.645 83.06 0.845 1.9193919 8 91.89 8 0.9090209 2.828 91.89 0.903 1.9632683 Figure 57: Zero order Plot for F6 Formulation Figure 58: First order Plot for F6 Formulation Figure 59: Higuchi plot for F6 Formulation Figure 60: Korsmeyer Peppas plot for F6 Formulation 0204060801000510Cumulative % drug releaseTime(Hrs)Zero order plot for F6 Formulation00.511.520510Log cumulative % drug remainingTime(Hrs)First order plot for F6 Formulation0204060801000123Cumulative % drug releaseSquare Root TimeHiguchi plot for F6 Formulation00.511.522.500.51Log cumulative % drug release Log TimeKorsmeyer-peppas plot for F6 Formulation

RESULTS AND DISCUSSION P. Rami Reddy Memorial College of Pharmacy Page 114 Table 34: In-vitro drug release kinetics data for Formulation F7 Zero order First order Higuchi’s data Korsmeyer-Peppas data Time (h) % CDR Time (h) Log % CD Remaining SQR Time % CDR Log Time Log % CDR 1 21.06 1 1.8972971 1 21.06 0 1.3234584 2 35.76 2 1.8078055 1.414 35.76 0.301 1.5533975 3 44.56 3 1.7438232 1.732 44.56 0.477 1.6489452 4 56.24 4 1.6410773 2 56.24 0.602 1.7500453 5 66.22 5 1.5286596 2.236 66.22 0.698 1.8209892 6 78.9 6 1.3242825 2.449 78.9 0.778 1.897077 7 89.34 7 1.0277572 2.645 89.34 0.845 1.9510459 8 98.53 8 0.1673173 2.828 98.53 0.903 1.9935685 Figure 61: Zero order Plot for F7 Formulation Figure 62: First order Plot for F7 Formulation Figure 63: Higuchi plot for F7 Formulation Figure 64: Korsmeyer Peppas plot for F7 Formulation 0501001500510Cumulative % drug releaseTime(Hrs)Zero order plot for F7 Formulation00.511.520510Log cumulative % drug remainingTime(Hrs)First order plot for F7 Formulation0204060801001200123Cumulative % drug releaseSquare Root TimeHiguchi plot for F7 Formulation00.511.522.500.51Log cumulative % drug releaseLog TimeKorsmeyer-peppas plot for F7 Formulation

RESULTS AND DISCUSSION P. Rami Reddy Memorial College of Pharmacy Page 115 Table 35: In-vitro drug release kinetics data for Formulation F8 Zero order First order Higuchi’s data Korsmeyer-Peppas data Time (h) % CDR Time (h) Log % CD Remaining SQR Time % CDR Log Time Log % CDR 1 23.05 1 1.8862086 1 23.05 0 1.3626709 2 35.24 2 1.8113068 1.414 35.24 0.301 1.5470359 3 43.18 3 1.7545012 1.732 43.18 0.477 1.6352826 4 52.87 4 1.6732974 2 52.87 0.602 1.7232093 5 61.45 5 1.5860244 2.236 61.45 0.698 1.7885219 6 70.24 6 1.4736329 2.449 70.24 0.778 1.8465845 7 81.09 7 1.2766915 2.645 81.09 0.845 1.9089673 8 92.64 8 0.8668778 2.828 92.64 0.903 1.9667985 Figure 65: Zero order Plot for F8 Formulation Figure 66: First order Plot for F8 Formulation Figure 67: Higuchi plot for F8 Formulation Figure 68: Korsmeyer Peppas plot for F8 Formulation 0204060801000510Cumulative % drug releaseTime(Hrs)Zero order plot for F8 Formulation00.511.520510Log cumulative % drug remainingTime(Hrs)First order plot for F8 Formulation0204060801000123Cumulative % drug releaseSquare Root TimeHiguchi plot for F8 formulation00.511.522.500.51Log cumulative % drug releaseLog Timekorsmeyer-peppas plot for F8 Formulation

RESULTS AND DISCUSSION P. Rami Reddy Memorial College of Pharmacy Page 116 Table 36: In-vitro drug release kinetics data for Formulation F9 Zero order First order Higuchi’s data Korsmeyer-Peppas data Time (h) % CDR Time (h) Log % CD Remaining SQR Time % CDR Log Time Log % CDR 1 26.69 1 1.8651632 1 26.69 0 1.4263486 2 34.98 2 1.813047 1.414 34.98 0.301 1.5438198 3 42.86 3 1.7569402 1.732 42.86 0.477 1.6320522 4 53.45 4 1.6679197 2 53.45 0.602 1.7279477 5 62.12 5 1.57841 2.236 62.12 0.698 1.7932314 6 73.24 6 1.4274861 2.449 73.24 0.778 1.8647483 7 81.34 7 1.2709116 2.645 81.34 0.845 1.9103042 8 90.97 8 0.9556878 2.828 90.97 0.903 1.9588982 Figure 69: Zero order Plot for F9 Formulation Figure 70: First order Plot for F9 Formulation Figure 72: Higuchi plot for F9 Formulation Figure 71: Korsmeyer Peppas plot for F9 Formulation 0204060801000510Cumulative % drug releaseTime(Hrs)Zero order plot for F9 Formulation00.511.520510Log cumulative % drug RemainingTime(Hrs)First order plot for F9 Formulation0204060801000123Cumulative % drug releaseSquare Root TimeHiguchi plot for F9 Formulation00.511.522.500.51Log cumulative % drug releaseLog TimeKorsmeyer-peppas plot for F9 Formulation

RESULTS AND DISCUSSION P. Rami Reddy Memorial College of Pharmacy Page 117 Table 37: In-vitro drug release kinetics data for Formulation F10 Zero order First order Higuchi’s data Korsmeyer-Peppas data Time (h) % CDR Time (h) Log % CD Remaining SQR Time % CDR Log Time Log % CDR 1 24.08 1 1.8803562 1 24.08 0 1.3816565 2 31.68 2 1.8345479 1.414 31.68 0.301 1.5007852 3 42.68 3 1.7583062 1.732 42.68 0.477 1.6302244 4 54.66 4 1.6564815 2 54.66 0.602 1.7376696 5 65.76 5 1.5345338 2.236 65.76 0.698 1.8179618 6 76.34 6 1.3740147 2.449 76.34 0.778 1.8827522 7 82.28 7 1.2484637 2.645 82.28 0.845 1.9152943 8 91.77 8 0.9153998 2.828 91.77 0.903 1.9627007 Figure 73: Zero order Plot for F10 Formulation Figure 74: First order Plot for F10 Formulation Figure 75: Higuchi plot for F10 Formulation Figure 76: Korsmeyer Peppas plot for F1o Formulation 0204060801000510Cumulative % drug releaseTime(Hrs)Zero order plot for F10 Formulation00.511.520510Log cumulative % drug remainingTime(Hrs)First order plot for F10 Formulation0204060801000123Cumulative % drug releaseSquare Root TimeHiguchi plot for F10 Formulation00.511.522.500.51Log cumulative % drug releaseLog TimeKorsmeyer-peppas plot for F10 Formulation

RESULTS AND DISCUSSION P. Rami Reddy Memorial College of Pharmacy Page 118 Table 38: In-vitro drug release kinetics data for Formulation F11 Zero order First order Higuchi’s data Korsmeyer-Peppas data Time (h) % CDR Time (h) Log % CD Remaining SQR Time % CDR Log Time Log % CDR 1 23.65 1 1.882809 1 23.65 0 1.3738311 2 32.66 2 1.8282731 1.414 32.66 0.301 1.5140162 3 41.23 3 1.7691557 1.732 41.23 0.477 1.6152133 4 52.68 4 1.6750447 2 52.68 0.602 1.7216458 5 64.46 5 1.5507174 2.236 64.46 0.698 1.8092903 6 75.56 6 1.3881012 2.449 75.56 0.778 1.8782919 7 83.28 7 1.2232363 2.645 83.28 0.845 1.9205407 8 92.86 8 0.8536982 2.828 92.86 0.903 1.9678287 Figure 77: Zero order Plot for F11 Formulation Figure 78: First order Plot for F11 Formulation Figure 79: Higuchi plot for F11 Formulation Figure 80: Korsmeyer Peppas plot for F11 Formulation 0204060801000510Cumulative % drug releaseTime(Hrs)Zero order plot for F11 Formulation00.511.520510Log cumulative % drug remainingTime(Hrs)First order plot for F11 Formulation0204060801000123Cumulative % drug ReleaseSquare Root TimeHiguchi plot for F11 Formulation00.511.522.500.51Log cumulative % drug releaseLog TimeKorsmeyer-peppas plot for F11 Formulation

RESULTS AND DISCUSSION P. Rami Reddy Memorial College of Pharmacy Page 119 Table 39: In-vitro drug release kinetics data for Formulation F12 Zero order First order Higuchi’s data Korsmeyer-Peppas data Time (h) % CDR Time (h) Log % CD Remaining SQR Time % CDR Log Time Log % CDR 1 22.88 1 1.887167 1 22.88 0 1.359456 2 33.78 2 1.8209892 1.414 33.78 0.301 1.5286596 3 42.57 3 1.7591388 1.732 42.57 0.477 1.6291037 4 52.88 4 1.6732053 2 52.88 0.602 1.7232914 5 63.68 5 1.5601458 2.236 63.68 0.698 1.8040031 6 74.34 6 1.4092567 2.449 74.34 0.778 1.8712226 7 85.24 7 1.1690864 2.645 85.24 0.845 1.9306434 8 93.88 8 0.7867514 2.828 93.88 0.903 1.9725731 Figure 81: Zero order Plot for F12 Formulation Figure 82: First order Plot for F12 Formulation Figure 83: Higuchi plot for F12 Formulation Figure 84: Korsmeyer Peppas plot for F12 Formulation 0204060801000510Cumulative % drug releaseTime(Hrs)Zero order plot for F12 Formulation00.511.520510Log Cumulative % drug remainingTime(Hrs)First order plot for F12 Formulation0204060801000123Cumulative % drug releaseSquare Root TimeHiguchi plot for F12 Formulation00.511.522.500.51Log cumulative % drug releaseLog TimeKorsmeyer-peppas plot for F12 Formulation

RESULTS AND DISCUSSION P. Rami Reddy Memorial College of Pharmacy Page 120 Figure 85: In-vitro drug release Profiles of Formulations F1-F6 Figure 86: In-vitro drug release Profiles of Formulations F7-F12 0204060801001200246810Cumulative % drug releaseTime(Hrs)F1F2F3F4F5F601020304050607080901000246810Cumulative % drug releaseTime(Hrs)F7F8F9F10F11F12

RESULTS AND DISCUSSION P. Rami Reddy Memorial College of Pharmacy Page 121 Table 40: In-vitro drug release kinetics Correlation coefficient data and diffusion exponent data of F1-F12 formulations Formulation code Correlation Coefficient values (R2) Diffusion Exponent value (n) Zero order First order Higuchi Korsmeyer-Peppas F1 0.9708 0.9240 0.9747 0.9781 0.5902 F2 0.9700 0.9135 0.9781 0.9881 0.6021 F3 0.9795 0.9076 0.9805 0.9910 0.6439 F4 0.9720 0.9093 0.9861 0.9892 0.6206 F5 0.9689 0.9326 0.9802 0.9763 0.5911 F6 0.9711 0.9154 0.9833 0.9885 0.6052 F7 0.9883 0.7922 0.9858 0.9968 0.7380 F8 0.9799 0.8730 0.9769 0.9916 0.6546 F9 0.9743 0.9139 0.9737 0.9768 0.6036 F10 0.9807 0.9349 0.9827 0.9830 0.6759 F11 0.9850 0.9115 0.9772 0.9828 0.6806 F12 0.9865 0.8933 0.9790 0.9901 0.6884 Table 41: Best fitting model for all Formulations Formulation code Best fit Model F1 Peppas F2 Peppas F3 Peppas F4 Peppas F5 Peppas F6 Peppas F7 Zero and Peppas F8 Peppas F9 Peppas F10 Peppas F11 Peppas F12 Peppas F1, F2, F3, F5, F6, F7, F8, F9, F10, F11 and F12 formulations were followed Korsemeyer-Peppas with correlation coefficient R2= 0.9781, 0.9881, 0.9910, 0.9763, 0.9885, 0.9892, 0.9916, 0.9768, 0.9830, 0.9828 and 0.9901, respectively. F7 formulation follows both Zero order and Korsemeyer-Peppas models, It indicates diffusion release mechanism followed by non-fickian transport.

RESULTS AND DISCUSSION P. Rami Reddy Memorial College of Pharmacy Page 122 Permeation flux is the slope of percentage drug release v/s time. It is expressed as µg.cm-2/hr-1 Table 42: % Drug entrapment and in-vitro permeation flux (µgcm-2 hr-1) of ethosomal gel batches. Formulation % Drug entrapment Permeation Flux (µg.cm-2 hr-1 ) EF1 97.07 74.15 EF2 96.41 76.11 EF3 95.4 74.50 EF4 96.02 78.14 EF5 96.3 78.96 EF6 95.46 76.64 EF7 99.09 71.26 EF8 96.3 72.82 EF9 99.02 75.80 EF10 96.19 73.10 EF11 98.37 74.61 EF12 95.11 75.04 EF13 95.4 69.12 EF14 95.64 68.58 EF15 97.07 64.79 Plain drug gel — 71.69 The drug entrapment for EF7 were found to be high i.e. 99.09% with permeation flux of 71.26 µg.cm-2 hr-1 respectively. The average size of ethosomes formulations EF7 were 816 nm respectively, whereas the zeta potential values were also found to be -25.4 indicating moderate stability.

RESULTS AND DISCUSSION P. Rami Reddy Memorial College of Pharmacy Page 123 5.15. Analysis of experimental design batches based on response surface methodology and formulation optimization: The batches of ethosomes prepared using experimental design were analyzed by statistical tools of response surface methodology to investigate the probable combinations of factors and their levels to achieve the closest target responses. The purpose was to find out an optimized formula of ethosomes which can provide maximum drug entrapment and permeation through skin. 5.15.1. Analysis of batches by contour plot: Figure 87: Contour plot of vesicle size Figure 88: Contour plot of Entrapment Efficiency From the contour plot of maximum % drug entrapment, the desired quantity of components in

RESULTS AND DISCUSSION P. Rami Reddy Memorial College of Pharmacy Page 124 formulation will be phospholipid 3.5 mg. The contour plots showed the design space with some probable levels of factors which can provide maximum desired responses of drug entrapment and permeation flux. 5.15.2. Probability Plots: Figure 89: Probability Plot for % drug entrapment Figure 90: Probability plot for permeation flux

RESULTS AND DISCUSSION P. Rami Reddy Memorial College of Pharmacy Page 125 The probability plot showed that the drug entrapment and permeation flux data of all the experimental batches were normally distributed at 95% confidence interval. 5.16. Optimization plot for determining the formula which can provide maximum desired responses: Table 43: Optimization formulation Sl. No % of Alcohol (%W/W) % of cholesterol (%W/W) % of Phospholipid (%W/W) Vesicle Size (nm) Entrapment Efficiency (%) EOF1 37.649 0.451 4.448 685.932 98.185 The ethosomes of fenoprofen were prepared as per the predicted formula for optimization and evaluated further for size, shape, zeta potential, % drug entrapment, ex-vivo permeation flux using excised rat skin, viscosity, pH, spreadability, gel strength and rheological properties. 5.16.1. Analysis of Ethosomal design batches by response surface methodology: The ethosomes batches were further analyzed by contour plot to screen the suitable quantities of excipients for maximum responses Figure 91: Response surface methodology of vesicle size

RESULTS AND DISCUSSION P. Rami Reddy Memorial College of Pharmacy Page 126 Figure 92: Response surface methodology of Entrapment Efficiency 5.16.2. Optimization plot based on response surface methodology for ethosomes: The optimization plot provided the levels of factors in formulation which has the possibility to achieve the set target responses of size and entrapment efficiency. The desirability of 1.00 predicted the possibility of achieving responses of % drug entrapment and permeation flux same to set target values. Figure 93: Desirability of optimized formulation

RESULTS AND DISCUSSION P. Rami Reddy Memorial College of Pharmacy Page 127 Figure 94: All responses of optimized formulation Figure 95: 3D Surface of desirability of optimized formulation

RESULTS AND DISCUSSION P. Rami Reddy Memorial College of Pharmacy Page 128 Figure 96: 3D Surface of Vesicle size of optimized formulation Figure 97: 3D Surface of Entrapment efficiency of optimized formulation

RESULTS AND DISCUSSION P. Rami Reddy Memorial College of Pharmacy Page 129 5.16.3. Overlay plot: Figure 98: overlay plot of optimized formulation 5.16.4. Multiple response optimization and optimum desirability This step helps determine the combination of experimental factors which simultaneously optimize several responses. This achieved by maximizing a desirability function. Among the design points, maximum desirability is achieved at run 1. To find the combination of factors which achieves the overall optimum desirability, select Optimization from the Tabular Options. The combination of factor levels which maximize the desirability function over the study aim indicated that the optimum levels for X1, X2 and X3 are 37.649, 0.451 and 4.448 %, respectively. An optimized formulation that contains the above mentioned optimum levels of the studied variables was prepared and characterized as previously stated. The observed values for Y1 and Y2 were found to be 685.93 nm, 98.18% respectively.

RESULTS AND DISCUSSION P. Rami Reddy Memorial College of Pharmacy Page 130 5.16.5. Pertubation Plot: Figure 99: Pertubation plot of optimized formulation Figure 100: All responses of optimized formulation

RESULTS AND DISCUSSION P. Rami Reddy Memorial College of Pharmacy Page 131 5.16.6. Interaction plots: Figure 101: Interaction plot of all responses of optimized formulation Figure 102: Cube plot of all responses of optimized formulation

RESULTS AND DISCUSSION P. Rami Reddy Memorial College of Pharmacy Page 132 5.17. Size and Zeta potential of optimized ethosomes formulation as determined by dynamic light scattering (Zeta Sizer): The size and stability of ethosomal dispersion was determined using Zeta Sizer (Malvern Instruments Ltd. Malvern, UK MAL10020 Figure 103: Size and zeta potential of optimized batch of ethosomes of fenoprofen The optimized batch of ethosomes shows size of 485.93 nm and a zeta potential of -21.4 indicating lesser size and uniform vesicle size distribution.

RESULTS AND DISCUSSION P. Rami Reddy Memorial College of Pharmacy Page 133 Table 44: Statistical analysis Anova of Vesicle size Model 7.791E+05 3 2.597E+05 5.69 0.0133 significant A-Alcohol 7.178E+05 1 7.178E+05 15.73 0.0022 B-Cholesterol 13464.37 1 13464.37 0.2950 0.5979 C-Phospholipid 47781.93 1 47781.93 1.05 0.3282 Residual 5.021E+05 11 45646.49 Cor Total 1.281E+06 14 Table 45: Statistical analysis Anova of Entrapment Efficiency Source Sum of Squares df Mean Square F-value p-value Model 29.73 6 4.95 12.65 0.0011 significant A-Alcohol 16.26 1 16.26 41.49 0.0002 B-Cholesterol 0.1394 1 0.1394 0.3559 0.5673 C-Phospholipid 5.83 1 5.83 14.89 0.0048 AB 0.0253 1 0.0253 0.0646 0.8058 AC 7.39 1 7.39 18.86 0.0025 BC 0.0820 1 0.0820 0.2093 0.6595 Residual 3.13 8 0.3918 Cor Total 32.86 14 P-values less than 0.0500 indicate model terms are significant. In this case A, C, AC are significant model terms. Values greater than 0.1000 indicate the model terms are not significant. If there are many insignificant model terms (not counting those required to support hierarchy), model reduction may improve your model. Table 46: Statistical analysis of Point Prediction Vesicle Size 685.93 685.93 213.65 80.59 508.54 863.32 -312.16 1684.03 Entrapment Efficiency 98.18 98.18 0.62 0.26 97.57 98.79 94.93 101.43 Table 47: Statistical analysis ANOVA of the Optimized formulation Intercept A B C AB AC BC Vesicle Size 480.133 229.265 31.3991 59.1502 p-values 0.0022 0.5979 0.3282 Entrapment Efficiency 96.4747 1.09109 -0.101048 0.653517 0.05625 0.96125 0.10125 p-values 0.0002 0.5673 0.0048 0.8058 0.0025 0.6595 5.18. Development of optimized formulation of ethosomal gel The novel transdermal gel has been developed containing drug loaded ethosomes based on the optimized formula and process. The optimized batch was further evaluated to check the ability to achieve the target responses. The developed formulations were also compared with the conventional transdermal gel formulations.

RESULTS AND DISCUSSION P. Rami Reddy Memorial College of Pharmacy Page 134 5.19. Evaluation data of optimized batch of ethosomes and plain drug gel on response surface methodology The optimized formulation was evaluated and the evaluation results of optimized formula were matched with the desired target responses fixed for optimization. The optimized batch showed closeness to the target and thus found to exhibit good % drug entrapment and ex-vivo permeation flux as well as satisfactory handling characteristics and ease of application. The ethosomal gel showed better % drug entrapment and ex-vivo permeation flux. 5.20. Surface morphology of ethosomes: Surface morphology of optimized formulation of ethosomes was determined using SEM (Model: Tecnai 20, Make: Philips, Holland) at SICART, Gujarat. Figure 104: The SEM images shows the the vesicles ethosomes are well identified, spherical vesicles

RESULTS AND DISCUSSION P. Rami Reddy Memorial College of Pharmacy Page 135 5.21. Rheological studies of optimized carrier incorporated gel The rheological behavior of prepared ethosomal gel was studied to ensure the ease of application and capability to withstand the stress of handling and storage. The properties were compared with the plain drug gel also to observe any change in gel characteristics that may occur due to presence of drug carriers in place of plain drug in gel. The rheological behavior of gel was determined by Brookfield viscometer using helipath with spindle no. 96. Figure 105: Viscocity of ethosomal formulations Table 48: viscosity in centipoises of ethosomal gel and Plain drug gel RPM Viscosity in centipoises Ethosomal gel Plain drug gel Increasing RPM Decreasing RPM Increasing RPM Decreasing RPM 2 30388 27989 34890 23478 3 20219 19432 22646 20112 5 15110 13420 18766 12789 10 10119 9100 12057 11075 20 5125 4999 7645 6346 30 4228 4228 5938 5938

RESULTS AND DISCUSSION P. Rami Reddy Memorial College of Pharmacy Page 136 Figure 106: Rheological behavior of ethosomal gel Figure 107: Rheological behavior of plain drug gel Drug carrier incorporated gel showed pseudo plastic behavior. This indicated that upon application of minimum shear stress, they would thin out but ones the shear stress is removed they would regain their normal thickness. This ensures the physical integrity of the formulated gels under various stress conditions like manufacturing, handling and packaging. 05000100001500020000250003000035000123456Viscosity in cpRPMRheological behavior of ethosomal drug gelEthosomal Increasing RPMEthosomal Decreasing RPM0500010000150002000025000300003500040000123456Viscosity in cpRPMRheological behavior of plain drug gel Plain Drug Increasing RPMPlain drug Decreasing RPM

RESULTS AND DISCUSSION P. Rami Reddy Memorial College of Pharmacy Page 137 5.22. Evaluation data of optimized batch of ethosomes in gel based on response surface methodology: Table 49: Evaluation data of optimized batch of ethosomes gel Optimized Formula Size (nm) Zeta Potential (mv) % drug entrapment Ex-vivo Permeation Flux (µgcm-2 hr-1 ) Viscosity (cp) pH Spreadabi lity (gm.cm/ s) Gel strength (gm) Ethosomal gel 685.93 -21.4 98.18% 27.11 4562 6.2 1.398 22.67 Plain drug gel – – – 22.69 4557 6.0 1.363 25.34 The optimized batch of ethosomes shows size of 685.93 nm and a zeta potential of -21.4 indicating lesser size and uniform vesicle size distribution. The optimized batch showed closeness to the target and thus exhibit a good % drug entrapment and ex-vivo permeation flux as well as good handling characteristics. The ethosomal gel showed better % drug entrapment and ex-vivo permeation flux as compared to plain drug gel. 5.23. Determination of amount of fenoprofen permeated and absorbed in excised rat skin by after permeation studies: The percentage drug diffused into in acceptor compartment, percentage drug absorbed in skin and percentage drug retained on skin in donor compartment was determined by ex-vivo studies for optimized batch of ethosomal gel and plain drug gel. 5.23.1. The ex-vivo release data for optimized formulations: The evaluation outcomes of ex-vivo studies carried out for ethosomal gel and plain drug gel are as follows: Table 50: Ex-vivo studies for Ethosomal gel and plain drug gel Optimized Formula In acceptor compartment Retained On Surface Skin In skin Loss Ethosomal gel 78.40% 14.02% 4.33% 3.25% Plain gel 70.15 % 17.56% 6.24% 6.05% It was observed that, better permeation (78.40 %) of drug across rat skin takes place through ethosomal gel. The permeation through plain fenoprofen gel is significantly less than the drug carriers incorporated gel.

RESULTS AND DISCUSSION P. Rami Reddy Memorial College of Pharmacy Page 138 5.24. Pharmacokinetics studies and comparative pharmacokinetic profiles of ethosomal gel and plain gel of fenoprofen: HPLC method details for analysis of fenoprofen in plasma and preparation of standard curve of drug in plasma. Mobile phase 40 volume of Phosphate buffer and 60 volume of acetonitrile pH 3 Injection volume 10µl Flow rate 1 ml/min ?max 275 nm Column Phenomenex Gemini-NX-5 µm C18(2) 110 Å, LC Column 250 x 4.6 mm, Ea Figure 108: HPLC chromatogram of blank Plasma Figure 109: HPLC chromatogram of plasma containing standard drug concentration

RESULTS AND DISCUSSION P. Rami Reddy Memorial College of Pharmacy Page 139 Table 51: Standard curve of drug fenoprofen in plasma Concentration Peak area of analyte Peak area of internal standard Peak ratio of analyte 2 µg/ml 6.56911 26.2552 0.2850 4 µg/ml 10.7614 25.5002 0.5400 6 µg/ml 13.3273 25.6049 0.6385 8 µg/ml 21.6492 27.5956 0.8920 10 µg/ml 28.7864 27.8996 1.1676 Figure 110: Standard curve of drug in plasma Linearity was observed in the curve between peak area ratio and concentration of drug in plasma as R2 value was found to be 0.98083. 5.24.1. Plasma profile of drug administered through drug carriers incorporated transdermal gel: Approval was taken from the Institutional Animal Ethics Committee to carry out pharmacokinetic studies. (Approval no: Sunbiologicals) Table 52: Plasma concentration profile of Ethosomal gel of fenoprofen Time Peak area of analyte Peak area of internal standard Peak ratio of analyte/ Int. standard Concentration (µg/ml) 30 min 5.7643 26.1552 0.1518 1.368 1 hour 7.4432 25.4002 0.2223 2.060 2 hour 12.0198 25.5049 0.393 3.734 3 hour 18.6309 27.4956 0.5590 5.361 4 hour 18.806 26.7996 0.6135 5.896 6 hour 20.2219 28.7543 0.6142 6.002 8 hour 18.7621 27.2324 0.5304 5.080 y = 0.2117x + 0.0695R² = 0.9808300.20.40.60.811.21.40123456Peak Area RatioConcentration ?g/mlPeak area ratio vs Concentration

RESULTS AND DISCUSSION P. Rami Reddy Memorial College of Pharmacy Page 140 Table 53: Plasma concentration profile of plain gel of fenoprofen Time Peak area of analyte Peak area of internal standard Peak ratio of analyte/Int. standard Concentration (µg/ml) 30 min 5.4101 27.2552 0.1985 1.946 1 hour 7.4398 26.5002 0.2807 2.752 2 hour 12.1476 26.6049 0.4565 4.476 3 hour 15.0659 28.5956 0.5268 5.165 4 hour 16.2375 27.8996 0.0582 5.711 6 hour 18.2618 29.8543 0.6117 5.998 8 hour 12.4545 29.3324 0.4246 4.163 Figure 111: Plasma concentration of drug after administration in rats

RESULTS AND DISCUSSION P. Rami Reddy Memorial College of Pharmacy Page 141 Figure 112: Comparative plasma profile of drug carriers incorporated gel Table 54: Comparative pharmacokinetics profile of drug carriers incorporated gel Formulation Cmax (µg/ml) Tmax (hr) AUC0-t µg.hr/ml AUC t-? µg.hr/ml AUC o-? µg.hr/ml Ethosomal gel 6.002 6 34.846 7.806 42.763 Plain gel 4.998 6 36.805 5.006 31.812 The blank sample of plasma was found to be free of any other component. The fenoprofen was detectable in standard solution of drug in plasma by the RP-HPLC method. The concentration of drug in plasma after transdermal application of ethosomal gel was detected and quantified. The blank sample of plasma was found to be free of any other component. The fenoprofen was detectable in standard solution of drug in Plasma by the used RP-HPLC method. The concentration of drug in plasma after transdermal application of optimized formulations of drug loaded ethosomal gel and plain gel was detected and quantified. The Ethosomal gel showed Cmax of 6.002 µg/ml and a Tmax of 6 hrs. The plain gel of fenoprofen as compared to ethosomal gel, showed lesser Cmax but the same Tmax. The bioavailability as measured by AUC was found to be highest for ethosomal gel formulation. Both the ethosomal gel were found to have better bioavailability as compared to plain gel of fenoprofen. 5.25. Analgesic activity by hot plate method in rats: The time of latency was determined as the time period between the zero point, when the animal 0123456730 min1 hour2 hour3 hour4 hour6 hour8 hourConcentration (?g/ml)TimesComparative plasma profile of drug carriers incorporated gelEthosmal gelPlain drug gel

RESULTS AND DISCUSSION P. Rami Reddy Memorial College of Pharmacy Page 142 is placed on the hot plate surface, and the time when the animal jumps off to avoid thermal pain. Figure 113: Observation of reaction time on Eddy’s hot plate in analgesic activity studies Table 55: Analgesic activity of fenoprofen transdermal formulations on rats Formulation Reaction time in seconds at time intervals (minutes ) 30 60 90 120 150 180 Control 4 3 5 4 3 4 Ethosomal gel 9 10 11 14 14 13 Plain drug gel 8 9 11 11 7 6 Figure 114: Comparison of analgesic activity The group of rats who received the application of ethosomal gel of fenoprofen showed more tolerance to pain as compared to the group of rats who received the application of plain drug gel of fenoprofen. Therefore, it can be interpreted that ethosomal gel of fenoprofen showed better 0246810121416306090120150180Reaction Times (Seconds)Time Interval (Minutes)Comparison of analgesic activityControlEthosomal gelPlain drug gel

RESULTS AND DISCUSSION P. Rami Reddy Memorial College of Pharmacy Page 143 analgesic activity than plain drug gel. 5.26. Confirmation of improvement in analgesic activity by statistical analysis by two sample T test: Figure 115: Boxplot of ethosomal gel and plain gel Figure 116: individual value of plot of ethosomal gel and plain gel Two-Sample T-Test at 95% confidence interval Table 56: Two sample T-test Mean N Mean St. Dev Ethosomal gel 6 11.83 2.14 Plain Drug gel 6 8.67 2.07

RESULTS AND DISCUSSION P. Rami Reddy Memorial College of Pharmacy Page 144 5.27. Anti-inflammatory activity determination by rat paw edema method using plethysmometer: The anti-inflammatory activity was carried out by carrageenan induced paw edema method to compare the activity of ethosomal gel and plain drug gel of fenoprofen using plethysmometer. The % inhibition of edema was calculated for each group using the following equation: % inhibition of edema= 1-(a-x / b-y) X 100 a= mean paw volume of treated animal after carageenan injection x= mean paw volume of treated animal before carageenan injection b= mean paw volume of control animal after carageenan injection y= mean paw volume of control animal before carageenan injection Table 57: Applied transdermal formulations on rat paw Rat Group Applied Formulation I Control II fenoprofen ethosomal gel III Plain Drug gel Table 58: Rat paw volume measurement data Group Paw volume in ml at time intervals 0 hr 1 hr 2hr 3 hr 4 hr 5 hr 6 hr I 0.49 0.88 1.02 1.08 1.09 1.09 1.04 II 0.48 0.74 0.76 0.72 0.72 0.66 0.64 III 0.48 0.76 0.87 083 0.79 0.74 0.72 Figure 117: Comparison of paw volume in rats 00.20.40.60.811.21234567Paw VolumeTimePaw Volume vs TimeGroup IGroup IIGroup III

RESULTS AND DISCUSSION P. Rami Reddy Memorial College of Pharmacy Page 145 Group-I Control Group Group-II Ethosomal Fenoprofen Gel Group-III Plain Fenoprofen Gel Figure 118: Paw edema observed in rats after Table 59: Anti-inflammatory activity of formulations in groups of rat Group % Anti-inflammatory activity I 1 hr 2 hr 3hr 4 hr 5 hr 6 hr 6 hr II 33% 48 50 61.6 70 71.9 71.4 III 28 26 40.8 44.6 53.6 51.8 51.1 Figure 119: Comparison of antiedema activity 010203040506070801 hr2 hr3hr4 hr5 hr6 hr6 hr% Anti-edema ActivityTime in Hrs% Anti-edema ActivityGroup IIGroup III

RESULTS AND DISCUSSION P. Rami Reddy Memorial College of Pharmacy Page 146 The paw volume in rats after topical application of ethosomal gel was found to be lower than paw volume after application of plain drug gel, so the ethosomal gel has better anti edema activity than plain drug gel. 5.28. Stability Studies The stability data were analyzed using software “R-Stab”. The observed and calculated values are given in Table 30. The residuals obtained from the calculated values are shown in Figure 64. The predicted shelf life was shown in Figure 63. The data of time versus cumulative percentage drug release profile are given in Table 31.and release pattern were shown in Figure 65. Table 60: Comparison of observed assay with calculated assay of optimized formulation subjected to stability study Sl.no Observed Assay (%) Mean ± SD Calculated Assay (%) Mean ± SD 0 100.36±0.8 99.90±0.44 1 99.30±0.2 99.37±0.71 2 98.59±0.3 98.75±0.66 3 97.79±0.6 98.13±0.77 4 97.41±0.4 97.51±0.71 5 96.91±0.4 96.90±0.71 6 96.58±0.4 96.28±0.71 Each value represents the mean ± standard deviation (n=3) Figure 120: Graph showing predicted shelf life of optimized formulation

RESULTS AND DISCUSSION P. Rami Reddy Memorial College of Pharmacy Page 147 Figure 121: Normal Q-Q plot of residuals obtained from calculated values of optamized formulation batches subjected for stability study Table 61: Comparison of dissolution data of best formulation optimized subjected to stability study with standard release Time (Hrs) Cumulative % drug release of best formulation Standard After 2 months After 4 months After 6 months 0 0 0 0 0 1 21.06 20.58 20.10 19.38 2 35.76 35.28 34.80 34.08 3 44.56 44.08 43.60 42.88 4 56.24 55.76 55.28 54.56 5 66.22 65.74 65.26 64.54 6 78.90 78.42 77.94 77.22 7 89.34 88.86 88.38 87.66 8 98.53 98.05 97.57 96.85

RESULTS AND DISCUSSION P. Rami Reddy Memorial College of Pharmacy Page 148 Figure 122: Drug release pattern of optimized formulation during stability study for every 2 month up to 6 months Overall observations from different evaluation studies such as drug-polymer interactions, evaluation of prepared formulations and In vitro drug release studies were carried out. Based on the obtained results best formulation was subjected for further stability study. The stability study was conducted as per ICH guidelines for the period of six months at various accelerated temperature and humidity conditions of 25°C/60%RH, 40°C/70%RH, 60°C/80%RH. The data of multiple batches were analyzed using linear regression, poolability tests and ANOVA statistical modeling these were amenable to analysis for quantitative attributes with upper acceptance criteria of 110% and lower acceptance criteria of 90% of label claim. There was a significant difference in intercepts (Y= 100.17, 100.26, 100.84) but no significant difference in slope –0.549 among the batches. The predicted shelf life was found to be 12.14 months. It was observed that there was no substantial change in dissolution profile after six months. The stability study revealed that the best formulation may be stable for the period of 12.14 months. 0204060801001200246810Cumulative % Drug ReleaseTime(Hrs)Comparision of in-vitro profiles of optamized formulation subjected to stability studiesStandardAfter 2 monthsAfter 4 monthsAfter 6 months