Chapter - V RESULTS AND DISCUSSION

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1 Chapter - V RESULTS AND DISCUSSION ANALYTICAL STUDY SCANNING OF DRUG Pure Ketoconazole was scanned in phosphate buffer saline (PBS) ph 7.4 and 10% methanol between 200 nm and 400 nm using uv-visible spectrophotometer. Ketoconazole was identified by its light absorption pattern which follows the absorption of light in the range 225 to 375 nm 28,29. Ketoconazole spectrum gave the highest peak at nm (Fig No.3) and same was selected as λ max for further evaluation. Figure No. 3: UV spectrum of KETO in PBS ph 7.4 at nm Pure Miconazole was also scanned in similar PBS ph 7.4 and 10% methanol between 200 nm and 400 nm using uv-visible spectrophotometer. Miconazole showed absorption of light in the range of 200 to 260 nm 28,29.The Kannur University 77

2 highest peak in the Miconazole spectrum was observed at nm (Fig No.4)and same was selected as λ max for further evaluation. Figure No. 4: UV spectrum of MICO in PBS ph 7.4 at nm CALIBRATION CURVE IN PHOSPHATE BUFFER ph 7.4 Standard solutions of different concentrations (Table No.4&5) were prepared and their absorbance was measured at nm, nm respectively for Ketoconazole and Miconazole. Drug concentrations versus absorbance curve was plotted as given in the Fig No.5&6. Table No. 4: Calibration data of KETO in PBS ph 7.4 at nm. Concentration (µg/ml) Absorbance* ± ± ± ± ± ± 0.02 *Values are represented as mean ±SD(n=6) Kannur University 78

3 Figure No. 5: Standard plot of KETO in PBS ph 7.4 at nm Table No.5: Calibration data of MICO in PBS ph 7.4 at nm. Concentration (µg/ml) Absorbance* ± ± ± ± ± ±0.03 *Values are represented as mean ±SD (n=6) Kannur University 79

4 Figure No. 6: Standard plot of MICO in PBS ph 7.4 at nm PREPARATION OF ETHOSOMES CONTAINING KETOCONAZOLE AND MICONAZOLE Ethosomal carriers is a system containing soft vesicles and composed mainly of phospholipids, ethanol in relatively high concentration and water. The ethosomes were prepared by United States patented hot method developed by Touitou E et al with required modifications after optimizing process and formulation variables 93,100. The ethosomal vesicles contained KETO,MICO in 2% concentration. The phospholipid concentration in ethosome was fixed at 2%.The ethanol concentration was varied between 20-40%. Propylene glycol at 10% concentration was included in the formulation 75. The compositions of different ethosomal formulations prepared in this investigation are recorded in Table No.6 Kannur University 80

5 Table No.6: Composition of different ethosomal formulations of KETO and MICO. Formulation Code %Phospholipids %Ethanol %Propylene glycol %Drug KET KET KET MET MET MET Ethosomal suspensions obtained were slight yellowish in colour and creamy in appearance. Different characteristics of ethosomes and the effect of varying concentrations of ethanol were further evaluated and results reported. Optimized ethosomal vesicles of Ketoconazole and Miconazole were formulated into water miscible cream base with simple trituration method. Similar water miscible creams were prepared using only pure drug (KETO and MICO).The concentration of drug in final formulation was 2%. The release profiles of drug from these ethosomal and non ethosomal creams were evaluated and results were reported. CHARACTERIZATION OF ETHOSOMES Since the physical characterization is meant for explaining the physical integrity of the dosage form, the results were pooled at one place. Discussion on the results for prepared ethosomal formulations and their comparisons were made under same heading. Kannur University 81

6 SIZE AND SHAPE ANALYSIS The ethosomal dispersion was spread on glass slide. Formation of multilamellar vesicles was confirmed by examining the ethosomal dispersion under an optical microscope with magnification power of 100X. The examination of prepared formulations revealed the predominance of spherical shaped vesicles. The vesicles were uniform in size and appeared to be multilayered. Visual observation of ethosomes confirmed the multi-lamellar vesicular structure; this confirms the existence of vesicular structure in ethosomes in spite of having high concentrations of ethanol. Photomicrographs (Fig No.7-12) were taken using video camera connected to microscope with the help of computer installed with special software proved the multi-lamellar vesicular structure and spherical shape of ethosomes of Ketoconazole as well as Miconazole. The Ketoconazole ethosomal formulation KET 1 with 20% ethanol and 2% phospholipids was found to be within the size range of µm and the average diameter was found to be µm (Table No.7) Table No.7: Size distribution of Ketoconazole ethosomes of formulation KET 1 Eye piece micrometer division Size range In micrometer Average size (d) No.of vesicles (n)* % vesicles (n) x (d) *Each value is an average of 3 replications. Kannur University 82

7 nd = Average diameter (d ave ) = = µm n 150 KET 1 = Ethosomal formulation containing 20% w/w ethanol Figure No.7: Photomicrograph of ethosomal formulation KET 1 (15 x 100X) Kannur University 83

8 The Ketoconazole formulation KET 2 with 30% ethanol was found to be within the size range of µm and the average diameter was found to be µm (Table No.8). Table No.8: Size distribution of Ketoconazole ethosomes of formulation KET 2 Eye piece micrometer division Size range In micrometer Average size (d) No.of vesicles (n)* % vesicles (n) x (d) *Each value is an average of 3 replications. nd Average diameter (d ave ) = = = µm n 150 KET 2 = Ethosomal formulation containing 30% w/w ethanol Kannur University 84

9 Figure No.8: Photomicrograph of ethosomal formulation KET 2 (15 x 100X) Ketoconazole ethosomes of formulation KET 3 with 40% ethanol concentration were found to be with in the range of µm and the average diameter was found to be µm (Table No.9) Table No.9: Size distribution of Ketoconazole ethosomes of formulation KET 3 Size range Eye piece micrometer In micrometer division Average size (d) No.of vesicles (n)* % vesicles (n) x (d) *Each value is an average of 3 replications. Kannur University 85

10 nd Average diameter (d ave ) = = = µm n 150 KET 3 = Ethosomal formulation containing 40% w/w ethanol Figure No.9: Photomicrograph of ethosomal formulation KET 3 (15 x 100X) The size of Miconazole ethosomes from formulation MET 1 prepared with 20% ethanol concentration was found to be within µm and showed an average vesicle diameter of µm (Table No.10) Kannur University 86

11 Table No.10: Size distribution of Miconazole ethosomes of formulation MET 1 Eye piece micrometer division Size range In micrometer Average size (d) No.of vesicles (n)* % vesicles (n) x (d) *Each value is an average of 3 replications. nd Average diameter (d ave ) = = = µm n 150 MET 1 = Ethosomal formulation containing 20% w/w ethanol Kannur University 87

12 Figure No.10: Photomicrograph of ethosomal formulation MET 1 (15 x 100X) The Miconazole ethosomal formulation MET 2 with 30% ethanol concentration and 2% phospholipids had vesicle size range of µm.the average vesicle diameter was found to be µm (Table No.11) Table No.11: Size distribution of Miconazole ethosomes of formulation MET 2 Eye piece micrometer division Size range In micrometer Average size (d) No.of vesicles (n)* % vesicles (n) x (d) *Each value is an average of 3 replications. Kannur University 88

13 nd Average diameter (d ave ) = = = µm n 150 MET 2 = Ethosomal formulation containing 30% w/w ethanol Figure No.11: Photomicrograph of ethosomal formulation MET 2 (15 x 100X) Kannur University 89

14 The size of Miconazole ethosomes of MET 3 was found to be with in the range of µm and average diameter was found to be µm (Table No.12) Table No.12: Size distribution of Miconazole ethosomes of formulation MET 3 Size range Eye piece micrometer In micrometer division Average size (d) No.of vesicles (n)* % vesicles (n) x (d) *Each value is an average of 3 replications. nd Average diameter (d ave ) = = = µm n 150 MET 3 = Ethosomal formulation containing 40% w/w ethanol Kannur University 90

15 Figure No.12: Photomicrograph of ethosomal formulation MET 3 (15 x 100X) The results obtained by vesicular size analysis showed the effect of ethanol concentration on vesicle size. The effect of ethanol concentration on ethosomes containing KETO as well as MICO is found to be similar. The size of ethosomal vesicles decreased as the concentration of ethanol increased 83,93 with largest vesicle size µm, µm with 20% ethanol and smallest µm, µm with 40% ethanol for both KETO and MICO based ethosomes. As per the findings, there was a reduction in average vesicular size of ethosomes with increase in ethanol concentration from 20% to 40% (Table No.13). But ethanol concentration above 50% may increase vesicular diameter along with loss of its lamilarity. This kind of phenomenon was observed by number of 83, 93,103,104 scientific groups and it was discussed through their scientific literatures. Kannur University 91

16 Figure No.13: Effect of ethanol concentration on KETO ethosome vesicle size The decrease in vesicle size with increase in ethanol concentration, may be due to formation of a phase with interpenetrating hydrocarbon chain that may lead to decrease in size of ethosomal vesicle on increasing ethanol concentration 13,99,103. Another possibility, ethanol may cause a modification in net charge of the system and confer it some degree of stearic stabilization that may lead to decrease in average vesicle diameter 84. Both the drugs (KETO, MICO) do not show any proved surface active property and hence so the reason for reduction in average vesicle diameter is due to presence of ethanol (Fig No.13&14). Kannur University 92

17 Figure No.14: Effect of ethanol concentration on MICO ethosome vesicle size During literature review it was found that liposomes prepared using same phospholipids by film hydration technique but without ethanol had large vesicle size than ethosomes. The results obtained on vesicle size of ethosomes are in conformity with findings of other investigators who concluded that, such generalized reduction in average vesicle diameter is due to the presence of ethanol in the vesicles 26. Table No. 13: Effect of ethanol concentration on ethosome vesicle size Ethanol concentration %w/w Average size of vesicles (µm) Ethosomal preparation with Ketoconazole Ethosomal preparation with Miconazole Kannur University 93

18 Even though there is no significance in this investigation, upon comparison of ethosomal vesicles comprising of the KETO and MICO, the average vesicle diameter of KETO based ethosomes was slightly smaller than the MICO (Table 13). The actual reason may not be explained for this, but may be it has to do with molecular structural difference between drugs 105. ENTRAPMENT EFFICIENCY Size and entrapment efficiency are always considered as parameters for optimizing vesicular formulations.once the presence of bilayers of vesicle was confirmed in ethosomal system, the ability of vesicle for entrapment of drug was investigated using ultracentrifugation method 20. Using ultracentrifugation method ethosomal vesicles containing drug and un-entrapped or free drug were separated to find out the entrapment efficiency. Results obtained are given in table. Table No.14: Drug entrapment efficiency of Ketoconazole ethosomes Formulation % Ethanol %Entrapment ± SD Code content Efficiency* KET KET KET *Values are represented as mean ±SD (n=6) From the data obtained (Table No.14), the maximum entrapment efficiency of Ketoconazole ethosomal vesicle as determined by ultracentrifugation was 62.41±0.89% for KET 2 formulation containing 30% ethanol concentration. This was much higher than ethosomal formulation KET 3 with 40% ethanol. The entrapment efficiency was 37.28±0.84% for KET 3. As the ethanol concentration Kannur University 94

19 increased from 20% to 30% there was improvement in entrapment efficiency (Fig No.15). The formulation KET 1 with 20% ethanol concentration was having entrapment efficiency of 51.78±0.67%, beyond 30% ethanol concentration the entrapment efficiency was found to be declining. Figure No.15: Effect of ethanol concentration on entrapment efficiency of KETO ethosomes Table No.15: Drug entrapment efficiency of Miconazole ethosomes Formulation Code % Ethanol content %Entrapment Efficiency* ± SD MET MET MET *Values are represented as mean ±SD (n=6) The entrapment efficiency of Miconazole based ethosomal formulations were also determined by ultracentrifugation technique. Like Ketoconazole Kannur University 95

20 ethosomal formulations, entrapment efficiency of vesicles increased with increase in ethanol concentration (Fig No.16), but further increase of (> 30%) ethanol concentration brought decrease in entrapment efficiency. From the data collected (Table No.15), the maximum entrapment efficiency in Miconazole ethosomal formulations observed was 57.51±0.44% and lowest entrapment efficiency was 32.77±0.39%. The Miconazole ethosomal vesicles with 20% ethanol concentration had entrapment efficiency of 50.83±0.42%. Figure No.16: Effect of ethanol concentration on entrapment efficiency of MICO ethosomes As per entrapment efficiency data both drugs (KETO, MICO) showed greater retentivity in ethanol present in ethosomal core. The data indicates that entrapment efficiency depends on ethanol concentration, as an increase in ethanol concentration results in increase in entrapment efficiency of ethosomal formulations. With further increase in ethanol concentration, entrapment efficiency decreases owing to increase in fluidity and permeability of membrane 13,93,99. Kannur University 96

21 The presence of ethanol at optimum concentration may exert a stabilizing effect in the formulation preventing or at least delaying the formation of vesicle aggregation because of the electrostatic repulsions 83. There are two factors governing the stability and hence the entrapment efficiency of vesicles. The first is ethanol concentration, the vesicles containing high ethanol concentration have thinner membranes corresponding to the formation of the phase with interpenetrating hydrocarbon chains 83.The second factor is the presence of phospholipids which contributes to the vesicle stability as it provides greater rigidity to the lipid layers and confers to the system a higher stability, reduced likelihood of vesicle fusion and greater resistance to the high rotational energy exerted by ultracentrifugation 83.This result also suggests that 2% phospholipid is optimal concentration along with 30% ethanol concentration for better entrapment efficiency and any increase or decrease in concentration of phospholipids or ethanol reduces the entrapment efficiency of ethosomes. These results are further supported by observations made by Jain N K et al 79,84. In an investigation made by Patel et al, the liposomal vesicles containing Ketoconazole showed highest entrapment efficiency of 54.41% 92.Where as ethosomal vesicles of Ketoconazole had entrapment efficiency of 62.41% which was higher than the liposomal vesicles. This further proves the superiority of ethosomal vesicles over liposome which was always under investigations. Kannur University 97

22 Table No.16: Effect of ethanol concentration on entrapment efficiency of ethosomes. Ethanol concentration % w/v Ethosomal preparation with Ketoconazole %Entrapment Efficiency* Ethosomal preparation with Miconazole ± ± ± ± ± ±0.39 *Values are represented as mean ±SD (n=6) The data obtained for percentage entrapment efficiency proves the importance of concentration of ethanol in ethosomes (Table No.16). As concentration of ethanol increased percentage entrapment efficiency of ethosomes increased up to 30% and further increase in the ethanol concentration significantly decreased the percentage entrapment efficiency. The reason for this may be the increase in solubility of drug at optimum ethanol concentration (up to 30%) which may entrap the drug with in the core of vesicles. But as concentration of ethanol increases above 30% there may be a leakage of drug from bilayers of vesicles which might have caused the decrease in percentage entrapment efficiency. 103 Data were analyzed statistically by one-way analysis of variance (ANNOVA) using Danniel soppers software as well as Microsoft excel 2003.The level of significance was p< 0.05.The results obtained after one-way analysis of variance (ANNOVA) proved the significance of the study. The highest entrapment value obtained for ethosomes containing 30% ethanol concentration with Ketoconazole as well as Miconazole statistically proved the superiority of the formulation over the rest, and the comparisons were statistically significant. Kannur University 98

23 STABILITY STUDIES All regulatory bodies accept only real time data for any drug or pharmaceutical for the purpose of assessing the shelf-life and accelerated stability studies may only serve as a tool for formulation screening and stability issues related to shipping or storage at room temperature. The accelerated stability studies were carried out in accordance with the ICH guidelines. The ability of vesicles to retain the drug was assessed by keeping the ethosomal suspension at different temperature. Optimized ethosome formulations were selected for stability studies of vesicles. Ethosomal formulations consisting of drugs (KETO and MICO) were observed for any change in appearance of colour for a period of eight weeks. There was no change in appearance of ethosomal vesicles of Ketoconazole as well as Miconazole.To avoid any interaction with container, ethosomal vesicles were stored in borosilicate containers. When ethosomes were observed under microscope, there was no significant physical change or increase in average vesicle size was observed. As they were insignificant, data are not reported in the thesis. Since the stability of drug and stability of vesicles are the major determinant for the stability of formulations, studies were carried out to evaluate total drug content and drug entrapment at room temperature (30±2 o C) and refrigeration temperature (4±2 o C).Stability study could not be carried out at higher temperature (>60 o C) because phospholipid used as one of the major component for ethosomes would get detoriated at higher temperature 94. Ketoconazole based ethosomal formulations were evaluated for loss in percentage drug content (Table No.17).The maximum loss in percentage drug content was 4.39% that too at room temperature for ethosomal formulation KET 3.The loss in percentage drug content in refrigerated condition was 2.52% from the initial drug Kannur University 99

24 content for KET 3. The loss in percentage drug content for KET 1 under refrigerated as well as room temperature was found to be 2.39%, 4.14% respectively after eight week study. The Ketoconazole ethosomal vesicles with 30% ethanol concentration KET 2 was showing loss in percentage drug content of 1.35%,2.35% respectively under refrigerated and room temperature, which was lesser compared to the findings made on other Ketoconazole formulations. Table No.17: Loss in percentage drug content during stability study for KETO ethosomes Formulation code 4±2 0 C Initial 27± 2 0 C After 2 weeks 4±2 0 C 27±2 0 C Drug content in % After 4 weeks 4±2 0 C 27±2 0 C After 6 weeks 4±2 0 C 27±2 0 C 4±2 0 C After 8 weeks 27±2 0 C KET ± ± ± ± ± ± ± 0.21 KET ± ± ± ± ± ± ± 0.25 KET ± ± ± ± ±0.16 *Values are represented as mean ±SD (n=6) ± ± 0.21 Loss in percentage drug content was not more than 5% incase of Miconazole based ethosomal formulations (Table No.18).The highest Miconazole loss was observed at room temperature after eight weeks as compared to refrigeration temperature.similar to the findings of Ketoconazole formulations, MET 2 with 30% ethanol concentration showed lowest percentage loss in drug content out of other two formulations MET 1 and MET 3. The percentage loss in drug content found in MET 2 was only 1.66%, 3.06% under refrigeration and room temperature. There was no significant loss observed in any of the formulations containing either Ketoconazole or Miconazole.The drug content in different Kannur University 100

25 formulations indicating the stability of drugs (KETO, MICO) even after eight weeks. Table No.18: Loss in percentage drug content during stability study for MICO ethosomes Formulation code 4±2 0 C Initial 27±2 0 C After 2 weeks 4±2 0 C 27±2 0 C Drug content in % After 4 weeks 4±2 0 C 27±2 0 C After 6 weeks 4±2 0 C 27±2 0 C After 8 weeks 4±2 0 C 27±2 0 C MET ± ± ± ± ± ± ±0.19 MET ± ± ± ± ± ± ±0.23 MET ± ± ± ± ±0.17 *Values are represented as mean ±SD (n=6) ± ±0.18 Entrapment efficiency is the integral part of stability of vesicles.according to the data, there was no significant change observed with the ethosomal formulations containing Ketoconazole (Table No.19). During the eight weeks of study fewer than two different conditions of refrigeration temperature and room temperature the entrapment efficiency was not changed to a great extent in any of the Ketoconazole ethosomal formulations. The initial entrapment efficiency of KET 2 formulation was 62.41±0.89%, after eight weeks of evaluation entrapment efficiency was calculated as 57.42±0.69% and 54.56±0.61% respectively under refrigeration and room temperature. The maximum drop of entrapment efficiency in KET 2 was only 7.85% under room temperature, where as under refrigeration the drop was only 4.99%. Among Ketoconazole ethosomal formulations the maximum drop of entrapment efficiency was observed in KET 3 with 11.77% under room temperature. Kannur University 101

26 Table No.19: Decrease in entrapment efficiency for Ketoconazole During stability study Formulation code 4±2 0 C Initial 27±2 0 C After 2 weeks 4±2 0 C %Entrapment Efficiency* 27±2 0 C After 4 weeks 4±2 0 C 27±2 0 C After 6 weeks 4±2 0 C 27±2 0 C After 8 weeks 4±2 0 C 27±2 0 C KET ± ± ± ± ± ± ± ± ± ±0.67 KET ± ± ± ± ± ± ± ± ± ±0.61 KET ± ± ± ± ± ± ± ± ± ±0.57 *Values are represented as mean ±SD (n=6) The finding of Miconazole based ethosomal vesicles was no different (Table No.20). The maximum drop in entrapment efficiency during eight weeks of study on MET 3 was observed to be 12.99% under room temperature and 9.55%. under refrigeration. In case of Miconazole based ethosomal formulations, maximum stability in terms of entrapment efficiency was observed with MET 2 where the initial entrapment efficiency was 57.51±0.44% and after eight weeks of study it was 52.40±0.39%,51.52±0.31% respectively under refrigeration and room temperature. Kannur University 102

27 Table No.20: Decrease in entrapment efficiency for Miconazole during stability study Formulation code 4±2 0 C Initial 27±2 0 C After 2 weeks 4±2 0 C %Entrapment Efficiency* 27±2 0 C After 4 weeks 4±2 0 C 27±2 0 C After 6 weeks 4±2 0 C 27±2 0 C After 8 weeks 4±2 0 C 27±2 0 C MET ± ± ± ± ± ± ± ± ± ±0.51 MET ± ± ± ± ± ± ± ± ± ±0.31 MET ± ± ± ± ± ± ± ± ± ±0.67 *Values are represented as mean ±SD (n=6) The data obtained, indicates that the formulations stored at refrigeration temperature and containing 30% ethanol were found to have higher entrapment efficiency. During the storage stability evaluations of liposomes, acceleration in drug leakage was observed. This may be due to the effect of temperature on gel to liquid transition of lipid bilayers together with possible chemical degradation of phospholipids which may lead to defects in membrane packing 13,107. But in case of ethosomal formulations, the loss in percentage drug content as well as drop in entrapment efficiency were negligible during eight weeks of stability study. Irrespective of Ketoconazole or Miconazole incorporation in ethosomes, the stability found to be intact under different temperature conditions. The possible reasons for the stability of ethosomal formulations may be the presence of ethanol which leads to the formation of phase with interpenetrating hydrocarbon chains 83, also the presence of phospholipids at optimum 2% concentration contributes to vesicle stability as it provides greater rigidity to lipid bilayers and provides ethosomal systems a higher stability 13,83. Eventhough it may not be carrying much significance, but ethanol concentration in ethosomes has its role since in both formulations KET 2 and MET 2 the ethanol concentration was 30% and both these Kannur University 103

28 formulations shared higher stability than other formulations. Hence the ethosomal formulations containing 30% ethanol concentration can be considered highly stable than the rest. IN-VITRO SKIN PERMEATION STUDIES In-vitro skin permeation study or in vitro skin diffusion study have been extensively studied using franz diffusion cell and epidermal surface of rat skin.the study may be used as an indirect measurement of drug solubility especially in preliminary assessment of formulation factors and manufacturing methods that are likely to influence bioavailability.all formulations had propylene glycol as skin penetration enhancer in a definite concentration. 75 The objectives in the developments of in-vitro diffusion tests are to show the release rates and extent of drug release from dosageform.skin permeation study was carried out for 72 hours duration, all results were shown on table and represented graphically 10. For the formulation KET 1 with 20% ethanol concentration,the percentage drug release after one hour was only 1.45±0.04%, which was improved to 22.39±0.17% after 4 hours of diffusion. The percentage drug release from KET 1 was almost doubled to 44.40±0.51% after 12 hours of study. After 24 hours of diffusion, the percentage drug release was 55.57±0.60%. There was an increase of nearly 11 % in drug release during the twelve-hour duration. The percentage drug release on 48 th, 60 th hours of study was 65.85±0.25% and 71.04±0.22% respectively for KET 1. At the end of diffusion study after 72 hours, the percentage drug release from KET 1 was 78.03±0.18% and 21.97% was remaining with in the KET 1 (Table No.21 &Fig No.17). Kannur University 104

29 Table No.21: In-vitro drug release for Ketoconazole entrapped ethosomes KET 1 Time in Hours Dilution factor % Drug unreleased % Drug released* ± SD *Values are represented as mean ±SD (n=6) Kannur University 105

30 Fig No.17: Percentage drug release from KET 1 Kannur University 106

31 Diffusion study carried out with Ketoconazole ethosomal formulation KET 2, which has 30% concentration of ethanol showed an improved release profile.in the first hour of diffusion study, the percentage drug release from KET 2 was 6.72±0.13% which has increased almost five times to 29.52±0.16% at the end of fourth hour. The percentage drug release from KET 2 after 12 hours was 54.02±0.17%.On the 24 th, 48 th and 60 th hour of diffusion, the percentage drug release from KET 2 were 60.43±0.18%, 77.46±0.16% and 84.26±0.18% respectively.at the end of the study, the percentage unreleased was only 10.95% where as 89.05±0.17% of drug was released from KET 2 formulation (Table No.22 & Fig No.18) Kannur University 107

32 Table No.22:In-vitro drug release for Ketoconazole entrapped ethosomes KET 2 Time in Hours Dilution factor % Drug unreleased % Drug released* ± SD *Values are represented as mean ±SD (n=6) Kannur University 108

33 Fig No.18 Percentage drug release from KET 2 Kannur University 109

34 When KET 3 with 40% ethanol concentration was subjected to diffusion study,the results were less impressive when compared to KET 1 & KET 2. Surprisingly after one hour of diffusion, no release was observed with KET 3. The percentage drug release was 1.88±0.15% after two hours and 6.03±0.23% after four hours of diffusion. On 12 th, 24 th and 48 th hour the release of Ketoconazole was 30.66±0.17%, 37.56±0.23% and 46.77±0.18% from KET 3. Upon completion of 72 hours of diffusion study, the percentage drug release from KET 3 was only 55.27±0.17% and 44.73% of drug was unreleased from the vesicles (Table No.23 & Fig No.19). Kannur University 110

35 Table No.23: In-vitro drug release for Ketoconazole entrapped ethosomes KET 3 Time in Hours Dilution factor % Drug unreleased % Drug released* ± SD *Values are represented as mean ±SD (n=6) Kannur University 111

36 Fig No.19 Percentage drug release from KET 3 Kannur University 112

37 Table No.24: Comparisons of percentage drug release from Ketoconazole ethosome vesicles Time in Hours % Drug released* KET 1 % Drug released* KET 2 % Drug released* KET ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.17 *Values are represented as mean ±SD (n=6) Kannur University 113

38 Fig No 20 Comparisons of percentage drug release from Ketoconazole ethosome vesicles Kannur University 114

39 In-vitro skin permeation study was conducted on all Miconazole based ethosomal formulations using similar methodology applied for Ketoconazole ethosomes.the Miconazole ethosomal formulations containing 20% ethanol concentration MET 1 was subjected to diffusion studies and percentage drug release observed after a hour of diffusion was 4.03±0.09% which was increased more than six times to 25.49±0.10% after four hours of diffusion. The percentage drug release from MET 1 after 12,24 and 48 th hours of diffusion was 48.69±0.25%,56.03±0.10% and 65.09±0.09% respectively, which was further increased to 69.09±0.10% after 60 hours. The diffusion study was completed after 72 hours of diffusion and percentage drug release observed from MET 1 was 74.95±0.13% and 25.05% was unreleased (Table No.25& Fig No.21). Kannur University 115

40 Table No.25: In-vitro drug release for Miconazole entrapped ethosomes MET 1 Time in Hours Dilution factor % Drug unreleased % Drug released* ± SD *Values are represented as mean ±SD (n=6) Kannur University 116

41 Fig No. 21 Percentage drug release from MET 1 Kannur University 117

42 For MET 2 with 30% ethanol concentration, the percentage drug release was 5.25±0.14% after one hour of diffusion, which was improved to more than five times after 4 hours of diffusion to 27.25%.Being the best among the MICO based ethosomal formulations in terms of stability and entrapment, a similar impressive profile was observed during the diffusion studies. After 12, 24 and 48 hours of diffusion, the percentage drug release from MET 2 was 51.05±0.10%, 60.13±0.14% and 74.17±0.12% respectively. After 72 hours of diffusion study, the percentage drug release was 85.43±0.12% and only 14.57% of Miconazole was found to be unreleased from the MET 2 formulation (Table No.26& Fig No.22). Kannur University 118

43 Table No.26: In-vitro drug release for Miconazole entrapped ethosomes MET 2 Time in Hours Dilution factor % Drug unreleased % Drug released* ± SD *Values are represented as mean ±SD (n=6) Kannur University 119

44 Fig No. 22 Percentage drug release from MET 2 Kannur University 120

45 In-vitro skin permeation study conducted on MET 3 was showing less impressive reports compared to MET 1 and MET 2. It required more than 1 hour to initiate the release of Miconazole from MET 3. After 6 hours of diffusion, the percentage drug release from MET 3 was less than 15%. After 12 hours of diffusion study, more than 73% of drug was found to be unreleased from the MET 3 formulation. When percentage drug release was calculated after 24 th, 48 th and 60 th hours of diffusion, it was 33.96±0.09%, 42.45±0.12% and 46.63±0.11% respectively for MET 3. When diffusion study was completed after 72 hours only little more than 50% Miconazole was released and nearly 50% was found to be unreleased from MET 3 containing 40% ethanol concentration (Table No.27 & Fig No.23). Kannur University 121

46 Table No.27: In-vitro drug release for Miconazole entrapped ethosomes MET 3 Time in Hours Dilution factor % Drug unreleased % Drug released* ± SD *Values are represented as mean ±SD (n=6) Kannur University 122

47 Fig No. 23 Percentage drug release from MET 3 Kannur University 123

48 Table No.28: Comparisons of percentage drug release from Miconazole ethosome vesicles Time in Hours % Drug released* MET 1 % Drug released* MET 2 % Drug released* MET ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.11 *Values are represented as mean ±SD (n=6) Kannur University 124

49 Fig No. 24 Comparisons of percentage drug release from Miconazole ethosome vesicles Kannur University 125

50 The release data were obtained for all the ethosomal formulations containing Ketoconazole as well as Miconazole with different levels of ethanol. Spectrophotometric results were obtained and consideration was given to sampling loss to calculate actual cumulative percentage drug released. The results for percentage drug released were plotted as function of time. The percentage drug released from ethosomal formulations KET 1, KET 2 and KET 3 of Ketoconazole were found to be 78.03±0.18%,89.05±0.17% and 55.27±0.17% respectively (Fig No.20).Similarly, for MET 1, MET 2 and MET 3 with Miconazole was 74.95±0.13%,85.43±0.12% and 50.01±0.11% (Fig No.24) after 72 hrs of diffusion. From the data, it can be predicted that the percentage drug diffused from ethosomal formulations containing 30% ethanol concentration was showing nearly 12% greater release than ethosomes with 20% ethanol and up 35% greater release than ethosomes containing 40% ethanol. Based on skin permeation study,ethosomes with 30% ethanol concentration containing Ketoconazole as well as Miconazole was having only 10.95% and 14.57% drug unreleased after 72 hrs of release study, which shows the superiority of these formulations over other. During the literature review 92, it was found that Ketoconazole encapsulated as liposomes had percentage drug release of 45% after 12 hrs where as Ketoconazole formulated as KET 2 was showing percentage drug release of 54.02±0.17% after 12 hrs, which definitely proves the potential of ethosomes as better vesicular drug delivery system. When soft, small vesicles of ethosomes were applied on to the skin a number of concomitant processes may take place involving stratum corneum and pilosebaceous path ways 70. Ethanol has long been known to have penetration enhancing properties. However, the permeation enhancement from ethosomes observed in the past studies are much greater than would be expected from ethanol alone, suggesting that some kind of synergisting mechanism between ethanol, vesicles and skin lipids 12,72,75. Kannur University 126

51 The data obtained during studies also suggest that value of percentage drug released depend on the ethanol concentration 11,12. As concentration of ethanol increased, percentage drug release of Ketoconazole as well as Miconazole increased up to 30% and further increase in the ethanol concentration significantly decreased the percentage drug release. The possible reason for this may be the detoriating effect of ethanol on lipid bilayers at higher concentrations 83,84. The significant difference in percentage drug release between ethosomal formulations containing different concentrations of ethanol (20,30& 40%) clearly indicates that the ethosomal system (KETO, MICO) with 30% ethanol concentration has better permeability through rat skin than formulation containing higher or lower concentrations of ethanol (Fig No.20&24). DIFFERENTIAL SCANNING COLORIMETRY (DSC) STUDIES The possible interaction between drug and excipients were studied by DSC method 103,104.DSC study was carried out for samples of pure drugs, phospholipids and drug loaded ethosomes.since KET 2 and MET 2 proved to be better formulations in terms of size, stability, entrapment efficiency and release profile. They were selected for DSC study. The method for DSC study 102 has been described previously under methodology chapter. The DSC thermograms of Ketoconazole, Miconazole, phospholipids, KET 2 and MET 2 were taken for further interpretation. Figure No.25: DSC thermograph of pure Ketoconazole Kannur University 127

52 Figure No.26: DSC thermograph of pure Miconazole Figure No.27: DSC thermograph of phospholipid The DSC thermograms of pure Ketoconazole and Miconazole showed endotherm at o C, o C respectively (Fig No.25&26). The DSC thermogram of phospholipids showed endotherm at o C (Fig No.27).The DSC thermogram of drug loaded ethosomes comprising same phospholipids interestingly showed a disappearance of melting endotherm of Ketoconazole and Miconazole.The major endotherm of KET 2 and MET 2 was observed at o Kannur University 128

53 C, o C respectively (Fig No.28&29). The melting endotherm of phospholipids shifted from o C to o C, o C for KET 2 and MET 2 ethosomes.this signified that, all components in ethosomes interact with each other to a great extent while forming phospholipids bilayers.the incorporated KETO as well as MICO associated with phospholipids bilayers and interacted to a large extent with them. Figure No.28: DSC thermograph of formulation KET 2 Figure No.29: DSC thermograph of formulation MET 2 Kannur University 129

54 In an investigation made by Misra et al, the DSC results suggested that the ethosomes are comparatively more in fluid states than the liposomes 93.The difference may be attributed to the presence of ethanol in ethosomes. The absence of melting endotherm of drugs and shifting of phospholipids bilayers component endotherm suggested significant interaction of Ketoconazole as well as Miconazole with bilayers leading to enhanced entrapment of drug 92,93. The DSC results of ethosomes suggest enhanced entrapment efficiency of Ketoconazole as well Miconazole in the prepared ethosomal vesicles. IN-VITRO DRUG RELEASE STUDIES FROM CREAM BASED FORMULATIONS All formulations of ethosomes were evaluated extensively and based on the data obtained ethosomal formulation with 30% ethanol concentration was found to be optimum.hence the ethosomes with 30% ethanol concentration was selected and incorporated in to water miscible cream base which is commonly used in all topical antifungal creams available in the market. Ethosomal formulation with 30% ethanol concentration (KET 2, MET 2 ) was incorporated in to water miscible cream base with simple trituration, the final concentrations of drugs (KETO,MICO) were fixed at 2.0% w/w in cream base.similarly pure drugs at 2.0%w/w concentration were directly incorporated in to cream base to formulate cream with 2.0% drug concentration (which is normally available in the market).these prepared formulations were subjected to in vitro release studies across a regenerated cellulose acetate membrane using PBS ph 7.4 medium 10. Kannur University 130

55 Table No.29: In-vitro drug release from Ketoconazole ethosomes based cream formulation (KETC) Time in Hours Dilution factor % Drug unreleased % Drug released* ± SD *Values are represented as mean ±SD (n=6) Kannur University 131

56 Fig No.30 Percentage drug release from KETC Kannur University 132

57 The cream containing 2% Ketoconazole (KTC) had a delayed drug release in comparison with KET 2 based cream (KETC) for the initial two hour duration. The percentage drug release was 14.23± 0.18%, 4.03±0.16% respectively for KETC and KTC. The possible reason for this may be the presence of ethanol at optimum level which may help the better release of Ketoconazole from ethosome based cream 104. At the end of 6 hrs, KETC had 37.99±0.21% (Table No.29) of drug released from the formulation where as KTC only managed 18.47±0.18% (Table No.30) of drug release. Throughout the duration of the release study, percentage drug released across the membrane from KETC was impressive. At the end of 72 hrs of release, only 15.50% of drug was found to be unreleased from KETC where as percentage unreleased from KTC was 46.49%.The results obtained were plotted graphically between percentage cumulative drug release and time, which clearly shows the improvement in percentage drug release by ethosome based cream in comparison to normal creams (Fig No.30-31). Kannur University 133

58 Table No.30: In-vitro drug release from Ketoconazole based cream (KTC) Time in Hours Dilution factor % Drug unreleased % Drug released* ± SD *Values are represented as mean ±SD (n=6) Kannur University 134

59 Fig No.31 Percentage drug release from KTC Kannur University 135

60 The results obtained from MET 2 incorporated cream (METC) were similar to that of KETC. During the study, METC released 12.51±0.10% of Miconazole after two hrs in comparison to 1.53±0.09% from normal cream containing 2% w/w Miconazole (MCC).The release from MCC was 0% during the early hrs of study. The release was found to be improving in case of METC, which was 32.95± 0.10% at the end of six hrs (Table No.31), during the same period it was only 11.17±0.11% for MCC (Table No.32).At the end of 72 hrs of study, percentage drug release was 80.33±0.11%, 44.21±0.11% respectively for METC and MCC. The percentage cumulative drug release and time was plotted for METC and MCC, which clearly showed the improved release profile for ethosome encapsulated cream (Fig No.32-33) Kannur University 136

61 Table No.31: In-vitro drug release from Miconazole ethosomes based cream formulation (METC) Time in Hours Dilution factor % Drug unreleased % Drug released* ± SD *Values are represented as mean ±SD (n=6) Kannur University 137

62 Fig No.32 Percentage drug release from METC Kannur University 138

63 Table No. 32: In-vitro drug release from Miconazole based cream formulation (MCC) Time in Hours Dilution factor % Drug unreleased % Drug released* ± SD *Values are represented as mean ±SD (n=6) Kannur University 139

64 Fig No.33 Percentage drug release from MCC Kannur University 140

65 From the data obtained, the percentage drug release from KETC and METC was nearly three times greater than normal creams for the first two hrs (Fig No.34-35). Improved release data were observed in case of ethosomal creams during the 72 hrs of the study. The possible reason for high initial drug release from ethosomal creams may be due to the presence of ethanol and other penetration enhancers at optimum concentration. Propylene glycol used in ethosome vesicles are widely incorporated as penetration enhancer in topical formulations either alone or in combination with other fatty acids, which may enhance the solubility and partitioning of drug across the membrane and there by improve activity of drug 75,99. They may also increase the permeability of vesicles through biological membrane due to synergistic effect with ethanol on bilayers of the vesicle 13,103,107. In case of KTC and MCC, significantly lower percentage of drug release at all points of time may be due to the absence of any penetration enhancers. Based on present study it was clearly evident that cream containing ethosomal vesicles had clear edge over simple drug based creams, since more than 50% of incorporated drugs are found to be unreleased, which suggest the importance of drug encapsulation in ethosomal vesicles. Kannur University 141

66 Table No.33: Comparisons of percentage drug release from formulations KETC& KTC Time in Hours % Drug released* KETC % Drug released* KTC ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.19 *Values are represented as mean ±SD (n=6) Kannur University 142

67 Fig No.34 Comparisons of percentage drug release from formulations KETC& KTC Kannur University 143

68 Table No.34: Comparisons of percentage drug release from formulations METC& MCC Time in Hours % Drug released* METC % Drug released* MCC ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.11 *Values are represented as mean ±SD (n=6) Kannur University 144

69 Fig No.35 Comparisons of percentage drug release from formulations METC& MCC Kannur University 145

70 IN-VIVO STUDIES OF ETHOSOMAL FORMULATIONS Since in-vitro studies of a formulation alone do not permit its administration to humans, it is essential to prove its pharmacological efficacy that too without much toxicological effects. In-vivo antifungal activities of developed ethosomal creams (KETC, METC) were evaluated and compared with standard cream (KTC, MCC), normal control and placebo control. The in-vivo studies of KETC were conducted on healthy male New Zealand rabbits separated into four groups with 3 rabbits each (Table No. 35). Similar experimental protocols were followed for METC formulation (Table No. 36). Experiments were performed with the approval of institutional animal ethical committee (Reg No. APTUS/IAEC/234) Table No.35: Experimental protocols for KETO based formulations Groups Title Formulations Group I Normal control Not treated Group II Placebo control Treated with cream base Group III Test Treated with KETC Group IV Reference Treated with KTC Kannur University 146

71 Table No.36: Experimental protocols for MICO based formulations Groups Title Formulations Group I Normal control Not treated Group II Placebo control Treated with cream base Group III Test Treated with METC Group IV Reference Treated with MCC All tested creams with and without ethosomes demonstrated a clear antifungal activity, although to different degrees. The lesions showed a marked clinical improvement at site of application. ie a rapid decrease in the inflammation and diameter of lesions. The results reported in Table No 37 show progressive recovery of infected area treated with test formulations of both drugs. After six days from assessed infection (48 hrs from inoculam), all animals treated with KETC showed nearly 25% cure. Assesment on 12 th day (48 hrs from inoculum) showed 50% of cure in animals treated with KETC when compared with 42% cure with KTC. On 18 th day of evaluation the cure with KETC was impressive 83% where as KTC had 75% animals cured after making re-isolation of c.albicans from the site of treatment (Fig No. 36). Graphical representation for the findings has been shown in Fig No. 37. Kannur University 147

72 Table No.37: Topical efficacy of ethosomal formulations in experimental model for cutaneous candidiasis in male New zealand white rabbits. Sample Normal control Placebo (Vehicle) KETC (Test) KTC (Standard) METC (Test) MCC (Standard) % Conc Cure rate at day post infection, no (% animals) of drug Nil 0/12 (0%) 1/12 (8%) 3/12 (25%) 4/12 (33%) Nil 0/12 (0%) 1/12 (8%) 2/12 (16%) 3/12 (25%) 2% 0/12 (0%) 3/12 (25%) 6/12 (50%) 10/12 (83%) 2% 0/12 (0%) 2/12 (16%) 5/12 (42%) 9/12 (75%) 2% 0/12 (0%) 2/12 (16%) 6/12 (50%) 9/12 (75%) 2% 0/12 (0%) 2/12 (16%) 4/12 (33%) 7/12 (58%) Kannur University 148

73 Fig No. 36 Wound healing effect of Ketoconazole formulations Kannur University 149

74 Fig No. 37 Graphical representation for wound healing effect of Ketoconazole formulations The treatment with Miconazole formulation also showed considerably higher cure rate than the controls. After 12 days from assessed infection (48 hrs from inoculum) animals treated with METC had good mycological cure rate of 50% in comparison with 33% cure rate with animals treated by MCC.Upon completion of evaluation ie 16 days (48 hrs from inoculum) the cure rate with METC improved to 75% where as with MCC it was only 58% (Fig No. 38).Meanwhile the mycological cure rate with normal control and placebo control were only 33% and 25% respectively after completion of study (Table No.37).Graphical representation (Fig No. 39) of the findings shows the clear improvement in mycological cure rate in animals treated with ethosomal cream of Miconazole. Kannur University 150

75 Fig No. 38 Wound healing effect of Miconazole formulations Kannur University 151

76 Fig No. 39 Graphical representation for wound healing effect of Miconazole formulations The results obtained during the antifungal study were further substantiated by measuring the microbial load for C. albicans at the site of inoculum using plate count method. The microbial load on the initial day was too numerous to count on all samples (Fig No. 40), which has reduced significantly to 3cfu/plate at 10 6 dilution for KETC and for KTC it was 26 cfu/plate at 10 6 dilution on 18 th day of the study (Table No. 38).For placebo and normal control these values were at considerably higher side and healing was poor. Hence based on microbial load count KETC showed superiority over KTC which was non- ethosomal formulation. Kannur University 152

77 Table No.38: Microbial load determination by plate count method Sample % Concn of drug Load of Candida albicans (cfu/plate at 10 6 dilution) Normal control Nil TNTC Placebo (Vehicle) KETC (Test) KTC (Standard) METC (Test) MCC (Standard) Nil TNTC % TNTC % TNTC % TNTC % TNTC * TNTC Too Numerous To Count Kannur University 153

78 Fig No. 40 Plate count method for Ketoconazole formulation Kannur University 154

79 Microbial load measurement for Miconazole based samples also showed superior results for ethosomal formulation. On 18 th day of study METC was able to reduce the microbial load to 12 cfu/plate in comparison to 34 cfu/plate reduction of MCC at 10 6 dilution (Table No. 38) & (Fig No. 41). At same stage microbial load for normal and placebo control were counted as 325 and 286 cfu/plate at 10 6 dilution, which clearly proves the improved antifungal activity of KETC and METC formulations against selected C.albican strains. Kannur University 155

80 Fig No. 41 Plate count method for Miconazole formulation Kannur University 156