FORMULATION AND EVALUATION OF MICROEMULSION BASED GEL OF CLOTRIMAZOLE

Transcription

1 268 P a g e International Standard Serial Number (ISSN): International Journal of Universal Pharmacy and Bio Sciences 3(3): May-June 2014 INTERNATIONAL JOURNAL OF UNIVERSAL PHARMACY AND BIO SCIENCES IMPACT FACTOR 1.89*** Pharmaceutical Sciences ICV 5.13*** RESEARCH ARTICLE!!! FORMULATION AND EVALUATION OF MICROEMULSION BASED GEL OF CLOTRIMAZOLE Vaibhavi M. Chaudhari*, Dr. M. S. Patel, Dr. M. R. Patel Department of pharmaceutics, B. M. Shah College of Pharmaceutical Education and Research, Modasa, Gujarat, India KEYWORDS: Clotrimazole; Microemulsion Based Gel; Ternary phase diagram; Topical Drug Delivery, Antifungal Activity. For Correspondence: Vaibhavi M. Chaudhari* Address: Department of pharmaceutics, B. M. Shah College of Pharmaceutical Education and Research, Modasa, Gujarat, India. vaibhavipatel717@gmail. com ABSTRACT The aim of present investigation was to formulate and evaluate microemulsion based gel of clotrimazole by increasing its solubility for the treatment of dermatophytosis. Before the formulation of microemulsion based gel, solubility study was performed in different excipients and select excipient on the basis of solubility of clotrimazole. Microemulsion region was decided by preparing ternary phase diagram. Drug excipient interaction study performed using FTIR. After preliminary study, microemulsions were prepared in Capmul MCM NF (oil), Acrysol K-140 (surfactant), and Transcutol P (co-surfactant) by phase titration method with water. Parameters evaluated included Drug content, visual assessment, Transmittance test, Particle size determination, and Zeta potential. Optimized microemulsion system containing drug was converted into gel employing various amount of carbopol 974 in which optimized batch containing 1.2%w/w carbopol 974 and evaluated for Viscosity, Spreadability, Extrudability, ph, Drug Content, In-Vitro diffusion Study, Antifungal activity, Stability study etc. From the solubility study, better solubility was seen in Capmul MCM NF (oil), Acrysol K- 140 (surfactant), and Transcutol P (co-surfactant). No any drug excipients interaction was seen. Particle size of optimized batch F4 was nm. Formulation was clear and near to 100% Transmittance after dilute with water. The zeta potential optimized batch F4 was Incorporation of clotrimazole in microemulsion based gel had drug release profile 96.15% for 10 hr and increases the antifungal activity when compared to marketed cream. In nut shell, it was concluded that microemulsion based gel was successfully prepared and evaluated for obtaining the desirable drug release, excellent antifungal activity and improvement in stability of the dosage form.

2 269 P a g e International Standard Serial Number (ISSN): INTRODUCTION Advancements in drug delivery strategies are taking place at much prompt pace than the last decade. Currently 74% of drugs are taken orally but not found to be as effective as desired because of various obstacles coming in path of oral delivery. Now-a-days, topical drug delivery system is one of the most capable modes of drug application. Topical drug delivery system is the drug delivery via skin to achieve systemic effect of drug. Topical application of the drugs offer many advantages as increased patient acceptability, avoidance of gastrointestinal disturbances, bypass of first pass hepatic metabolism 1 and sustained delivery of drugs and reduced systemic side effects 2. Recent attention has been sharply focused on microemulsions which have additional advantages of stability. Microemulsion represent as novel vehicle having potential of increasing percutaneous delivery of both hydrophilic and lipophilic drugs 3 and their use to modify penetration rate of drug across skin. The word Microemulsion was originally proposed by Hoar and Schulman in the earliest of the 1940s. They were generated a clear single-phase solution by titrating a milky emulsion with hexanol. Microemulsions are thermodynamically stable, isotropically clear mixture of oil, water and surfactant frequently in combination with co-surfactant 4. These homogeneous systems have gained wide acceptance because of their enhanced drug solubilization, longer shelf life due to thermodynamic stability, easy formation because of zero interfacial tension, ability to be sterilized by filtration 5. Microemulsion can be considered as small scale version of emulsion i.e. droplet type dispersion. There significant difference between emulsions and microemulsions. In particular, in emulsions, the average drop size grows continuously with time so that phase separation ultimately occurs under gravitational force, i.e. they are thermodynamically unstable. The drops of the dispersed phase are generally large ( 0.1 m) so that they take a milky appearance. On the other side, for microemulsion, spontaneous formation occurs they are clear or translucent in size range of nm. Microemulsions are vibrant system in which interface is continuously fluctuating. According to structure they are divided into oil-in-water (o/w), water-in-oil (w/o) and bicontinuous microemulsions. In these entire microemulsions interface is stabilized by appropriate combination of surfactant and co-surfactant 6. In fact, the presence of gelling agent in the water phase converts a microemulsion into microemulsion gel. Both oil-in-water (o/w) and water-in-oil (w/o) microemulsion are used as vehicle to deliver various drugs to the skin. Microemulsion gels for dermatological use have several favorable properties such as being thixotropic, greaseless, easily spreadable, easily removable, emollient, non-staining, long self life, bio-friendly, transparent and pleasing appearance.

3 270 P a g e International Standard Serial Number (ISSN): Many widely used topical agents like ointment cream, lotion many disadvantages. They have very sticky causing uneasiness to patient when applied. Moreover they also have lesser spreading coefficient and need to apply with rubbing. And they exhibit the problem of stability also. Due to all these factors within the major group of semisolid preparations, the use of transparent gels has expanded both in cosmetics and in pharmaceutical preparations. A gel is colloid that is typically 99%wt liquid, which is immobilized by surface tension between it and a macromolecular network of fibers built from a small amount of a gelatin substance present. In spite of many advantages of gels a major limitation is in the delivery of hydrophobic drugs. So to overcome this limitation an emulsion based approach is being used so that even a hydrophobic therapeutic moiety can be successfully incorporated and delivered through gels MATERIALS AND METHOD: 2.1 Materials Clotrimazole was obtained from Zydus Cadila Ltd., Ahmedabad, Capmul MCM NF was obtained from Abitec Corporation, USA. Acrysol K-140 was obtained from Corel chem., Ahmedabad, India. Transcutol P was obtained from Gatteforsse, France. 2.2 Methods: Drug dissolved in the Capmul MCM NF. Then add Acrysol K-140 and Transcutol P as surfactant and Co-surfactant respectively, mixed well on magnetic stirrer. Then water was added drop by drop and mixed well on magnetic stirrer. Transparent microemulsion was formed. Now for preparation of Gel base, Carbopol 974 was suspended in water and hydrated it for overnight. ph was adjusted using Triethanolamine. For preparation of Microemulsion based gel; Microemulsion dispersed into Carbopol 974 gel. 2.3 FTIR of clotrimazole Identification of Clotrimazole by Fourier Transform Infrared (FTIR): Identification of Clotrimazole was done to ensure that available sample was clotrimazole or not. Infrared spectrum of pure drug was determined by KBr press pellet technique using FTIR instrument. KBr pellets were prepared by mixing pure drug with KBr in 1:10 ratios Drug Excipient Compatibility Study: Drug-Excipient interactions play a vital role in the release of drug from formulation. Fourier transform infrared spectroscopy has been used to study the physical and chemical interactions between drug and the excipient used. Fourier transforms infrared (FTIR) spectra of clotrimazole and excipients were recorded using KBr mixing method on FTIR instrument.

4 Absorbance 271 P a g e International Standard Serial Number (ISSN): Analytical Methodology 8, Preparation of Stock Solution of Methanol Weighed accurately 100 mg of clotrimazole and transferred to 100 ml volumetric flask. Then volume was made up to 100 ml with Methanol to get the concentration of drug 1000 µg/ml (Stock A) Preparation of Calibration Curve in Methanol From stock A, different dilutions were prepared in the concentration range of 50, 100, 150, 200, 250, 300, 350 µg/ml and absorbance was recorded in shimadzu-1700 UV/Visible spectrophotometer at λmax, 261 nm. Table 1: Calibration Curve data of Clotrimazole in Methanol Sr. No. Concentration Absorbance Mean (µg/ml) Absorbance ± S.D ± ± ± ± ± ± ± y = x R² = Abs Concentration µg/ml Linear (Abs.) Figure 1: Calibration Curve of Clotrimazole in Methanol Method for Preparation of Phosphate Buffer ph Preparation of 0.2M NaOH Weight accurately 8gm of sodium hydroxide using digital weighing balance. Dissolve it into 1000ml distilled water in 1000ml of volumetric flask to get 0.2M NaOH.

5 272 P a g e International Standard Serial Number (ISSN): Preparation of 0.2M Potassium Dihydrogen Phosphate Weight accurately 27.22gm of Potassium Dihydrogen Phosphate using digital weighing balance. Dissolve it into 1000ml distilled water in 1000ml of volumetric flask to get 0.2M Potassium Dihydrogen Phosphate Preparation of Phosphate Buffer ph 7.4 Measure 50ml of the 0.2M Potassium Dihydrogen Phosphate using measuring cylinder, transfer into 1000ml volumetric flask. Add 39.1ml of 0.2M NaOH and then make up the volume up to 1000ml using distilled water to get ph 7.4 Phosphate buffer Preparation of Calibration Curve in Phosphate Buffer ph 7.4 Preparation of Calibration Curve of clotrimazole in Phosphate Buffer ph 7.4 using Phosphate Buffer ph 7.4: Methanol (6:4). Weighed accurately 100 mg of clotrimazole and transferred to 100 ml volumetric flask. Then volume was made up to 100 ml with Phosphate Buffer ph 7.4: Methanol (6:4) to get the concentration of drug 1000 µg/ml (Stock A). From stock A, different dilutions were prepared in the concentration range of 50, 100, 150, 200, 250, 300, 350 µg/ml and absorbance was recorded in shimadzu-1700 UV/Visible spectrophotometer at λmax, 261 nm Measurement of Absorbance Maxima of Clotrimazole Clotrimazole test solution of concentration of 200μg/ml was scanned for determination of absorbance maxima (λ max ) in a spectrophotometer. Scanning was carried out in a range of nm. The λ max was found to be 261 nm as shown in figure 2. Table 2: Calibration Curve data of Clotrimazole in Phosphate Buffer ph 7.4 Sr. Concentration Absorbance Mean Absorbance No. (µg/ml) ± S.D ± ± ± ± ± ± ± 0.001

6 Absorbance 273 P a g e International Standard Serial Number (ISSN): Figure 2: U.V. Maxima of Clotrimazole at 261 nm in Phosphate Buffer ph y = 0.002x R² = Concentration µg/ml Abs. Figure 3: Calibration Curve of Clotrimazole in Phosphate Buffer ph Solubility study of clotrimazole in various excipients Solubility of clotrimazole was determined in different modified oils, surfactant and co-surfactants. Two ml of each component was taken in screw cap vials with known quantity (250mg) of excess drug. A vortex mixer was used to facilitate the solubilization. Vials are sealed with cap. Sealed vials were kept on isothermal mechanical shaker at 40 ± 2 C for 72 hours. After equilibrium each test tube was centrifuged at 6000 rpm for 20 min using a centrifuge. Supernatant was filtered through membrane filter using 0.45μm filter disk. Filtered solution was appropriately diluted with methanol and UV absorbance was measured at 261 nm. Concentration of dissolved drug was determined using stander equation.

7 274 P a g e International Standard Serial Number (ISSN): Pseudo-ternary phase diagram study On the basis of the solubility study of drug, oil, surfactants, co-surfactants and aqueous phase were used for construction of phase diagram. Oil, surfactant, and co-surfactant are grouped in four different combinations for phase studies. Surfactant and co-surfactant (Smix) in each group were mixed in different weight ratio (1:0, 1:1, 1:2, 2:1, 1:3, 3:1, 1:4, 4:1 etc.). These Smix ratios are chosen in increasing concentration of surfactant with respect to co-surfactant and in increasing concentration of co-surfactant with respect to surfactant for detail study of the phase diagram for formulation of microemulsion. For each phase diagram, oil, and specific Smix ratio are mixed thoroughly in different weight ratio from 1:9 to 9:1 (0.5:9.5, 1:9, 1.5:8.5, 2:8, 2.5:7.5, 3:7, 3.5:6.5, 4:6, 4.5:5.5, 5.0:5.0, 5.5:4.5, 6:4, 6.5:3.5, 7:3, 7.5:2.5, 8:2, 8.5:1.5, 9:1, 9.5:0.5) in different glass vials. Different combination of oils and Smix were made so those maximum ratios were covered for the study to delineate the boundaries of phase precisely formed in the phase diagrams. Pseudo-ternary phase diagram was developed using aqueous titration method. Slow titration with aqueous phase is done to each weight ratio of oil and Smix and visual observation is carried out for transparent and easily flowable o/w microemulsion. The physical state of the microemulsion was marked on a pseudo-three-component phase diagram with one axis representing aqueous phase, the other representing oil and the third representing a mixture of surfactant and co-surfactant at fixed weight ratios. 2.7 Formulation of Microemulsion The formulations were prepared by initially dissolving required quantity of clotrimazole in oil. Then Surfactant and Co-surfactant mixer were added and the final mixture was mixed by vortexing until a clear solution was obtained. Finally, an appropriate amount of water was added to the clotrimazole solution mixture drop by drop to get microemulsion. The different composition and different ratio like 2.5:1, 3:1, 3.5:1 of microemulsion formulated shown in table no To Performed different evaluation parameter of microemulsion like % transparency, Drug Content, Droplet size, zeta potential and thermodynamic stability. From result of the evaluation parameter of microemulsion select one optimized batch. Table 3: Formulation of Microemulsion in Different Ratio Formulation code F1 F2 F3 F4 F5 F6 Drug %w/w Smix (ratio) 2.5:1 2.5:1 3:1 3:1 3.5:1 3.5:1 Components of microemulsion %w/w Oil Surfactant Co-Surfactant Water

8 275 P a g e International Standard Serial Number (ISSN): EVALUATION OF MICROEMULSION FORMULATIONS Transmission test 10 Stability of optimized microemulsion formulation with respect to dilution was checked by measuring transmittance through U.V. Spectrophotometer. Transmittance of samples was measured at 650 nm and for each sample three replicated assays were performed Visual assessment 11 The quality of microemulsion was assessed by visual inspection and it was graded in various grades. Clotrimazole microemulsion (approximately 0.2 ml) was diluted with purified water (100 ml) and gently stirred with magnetic stirrer at temp.37 ºC. Table 4: Different Grade and Appearance of Microemulsion GRADE APPEARANCE Grade I Having clear or bluish appearance Grade II Having a bluish white appearance Grade III Fine milky emulsion Grade IV Dull, grayish white emulsion having slight oily appearance Grade V Formulation exhibiting poor appearance Drug Content 12 Clotrimazole from microemulsion formulation was extracted in methanol using sonication technique. The solution was filtered, using watt man filter paper. The methanolicextract was analyzed for the clotrimazole content spectrophotometrically (UV-1800, Shimadzu, Japan) at 261 nm using calibration curve Droplet size determination 13 It is a precise method for evaluation of stability. Size of droplet is measured by photon correlation spectroscopy (PSC) with Zetasizer. All measurements are carried out at scattering angle of 90 and 25 C temperatures. Prior to measurement, microemulsion is diluted in two-steps with pure water then it is filtered through a 0.22um filter just before it is added to cuvette. In first step it is diluted with equal amount of water. In second step the mixture is further diluted to appropriate concentration for the measurement. That depends on droplet size (Usually diluted times) Zeta potential measurement 14 Zeta potential for microemulsion was determined using Zetasizer HSA 3000 (Malvern Instrument Ltd., UK). Samples were placed in clear disposable zeta cells and results were recorded. Before putting the fresh sample, cuvettes were washed with the methanol and rinsed using the sample to be measured before each experiment.

9 276 P a g e International Standard Serial Number (ISSN): Thermodynamic Stability Heating cooling cycle: Six cycles between refrigerator temperature 4 ºC and 45 ºC with storage at each temperature of not less than 48 wash studied. Stable formulation at these temperatures, were subjected to centrifugation test. 2. Centrifugation: Passed formulations were centrifuged at 3500 rpm for 30 min. formulation that did not show any phase separation was taken for the freeze thaw stress test. 2.9 PREPARATION OF MICROEMULSION BASED GEL FORMULATION Different formulations were prepared using varying amount of gelling agent. Carbopol 974 as a gelling agent was used to construct the microemulsion based gel for improving viscosity of microemulsion for topical administration. The gel was slowly mixed with microemulsion under stirring by dispersion method. The dispersion was neutralized by using triethanolamine to obtain the gel. As the microemulsion added to the gel viscosity of system was decreased. Hence, Sufficient viscosity microemulsion based gel was obtained, the gels were prepared at various concentration (0.6, 0.8, 1, 1.2, 1.4, 1.6%w/w) and final concentration was selected on the basis of viscosity, transparency, drug content, In-Vitro diffusion study. Table 5: Formulation of Microemulsion Based Gel in Different Ratio of Carbopol 974 in %w/w Ingredients F1 F2 F3 F4 F5 F6 Clotrimazole 1% 1% 1% 1% 1% 1% Capmul MCM NF Acrysol K Transcutol P Carbopol % 0.8% 1% 1.2% 1.4% 1.6% Oleic acid 1% 1% 1% 1% 1% 1% Triethanolamine 0.1% 0.1% 0.1% 0.1% 0.1% 0.1% Water q.s. q.s. q.s. q.s. q.s. q.s EVALUATION OF MICROEMULSION BASED GEL FORMULATIONS Physical appearance: The prepared clotrimazole microemulsion gel formulations were inspected for their colour, homogeneity, consistency and ph. The ph values of 1 % aqueous solutions of the prepared microemulsion gel were measured by a ph meter Spreadability measurement: To determine the spreadability of microemulsion based gel, 0.5 g of gel was placed within a circle of 1 cm diameter pre-marked on a glass plate, over which a second glass plate was placed. A weight of 1000 g was allowed to rest on the upper glass plate for 5 min. The increase in the diameter due to gel spreading was noted 17.

10 277 P a g e International Standard Serial Number (ISSN): The calculation of spreadability is as follows; S=M.L/T Where M = Weight tide the Upper Slide (g) L = Length moved on the glass slide (cm) T = Time taken Rheological Study: The viscosity of the microemulsion gel formulations is determined at 25 C using a cone and plate viscometer with spindle 2 (Brookfield Engineering laboratories) and connected to a thermostatically controlled circulating water bath Drug content determination: Clotrimazole content in microemulsion based gel was measured by dissolving 1 g gel in methanol by sonication. Absorbance was measured after suitable dilution at 261 nm using UV/VIS spectrophotometer (UV-1800, Shimadzu, Japan) Extrudability (Tube Test): It is a usual empirical test to measure the force required to extrude the material from tube. The method adopted for evaluating microemulsion based gel formulation for extrudability is based upon the quantity in percentage of gel and gel extruded from aluminum collapsible tube on application of weight in grams required to extrude at least 0.5 cm ribbon of microemulsion gel in 10 seconds. More quantity extruded better is extrudability 20. The extrudability is than calculated by using the following formula: Extrudability = Applied weight to extrude microemulsion gel from tube (in gm)/area (in cm 2 ) In vitro Diffusion study (a) Apparatus: The modified diffusion cell was fabricated from borosilicate glass and consisted of two compartments i.e. receptor and donor. The cell and effective receptor surface area of 3.14cm 2. The two halves of the cell were secured in place with the help of strong metallic clips. The receptor solution was stirred by a Teflon bead of 12mm length on a magnetic stirrer. The temperature of the cell contents were maintained at 37±1 o C with the help of thermostat attached to the magnetic stirring assembly. (b) Procedure for Drug diffusion Studies: A modified Diffusion Cell was used for evaluating drug release profiles cellophane membrane. The receptor compartment was filled with 65ml of PBS ph 7.4 containing 10% methanol stirred by the use of the Teflon coated bead on a magnetic stirrer. The whole assembly was kept tightened with the screws for the cell setup. The whole assembly was kept on

11 278 P a g e International Standard Serial Number (ISSN): the magnetic stirrer and the temperature was maintained at 37±1 o C with the water jacket and at 100rpm speed of magnetic bead. The withdrawal port was covered with the glass cork which prevents air entrapment. The amount of (1gm) gel was applied between to the donor compartment to receptor compartment. The amount of drug released in to the receptor solution was determined by 5ml of sample withdrawal at one hr intervals for 10hrs. The withdrawn volume was replaced with an equal volume of fresh buffer solution. The drug was release/diffuse was determined by analyzing the samples at 261nm. The result of In-Vitro released study is represented by following graphs. Cumulative percentage released versus time Similarity & Dissimilarity Comparison of Optimized formulation with Marketed Formulation The prepared optimized formulation was compared with marketed formulation of microemulsion based gel of clotrimazole. In-Vitro release profile was considered for comparison. The In-Vitro release profile of optimized formulation was compared with marketed formulation for similarity factor (f 2 ) and dissimilarity factor (f 1 ). The similarity factor (f 2 ) was defined by CDER, FDA, and EMEA as the logarithmic reciprocal square root transformation of one plus the mean squared difference in percent drug dissolved between the test and the reference products. Moore and Flanner give the model independent mathematical approach for calculating a similarity factor (f 2 ) for comparison between dissolution profiles of different sample. The similarity factor was calculated by following formula. Where, n= no. of time points f2 = 50 log n Rt = the reference profile at the time point t Tt = the test profile at the same point n t=1 (Rt Tt) Table 6: The similarity factor (f 2 ) and its significances Similarity Factor (f 2 ) Significance < 50 Test and reference profiles are dissimilar Test and reference release profiles are similar 100 Test and reference release profiles are identical >100 The equation yields a negative value The difference factor calculates the percentage difference between two profiles i.e. marketed product. Dissolution profile and optimized batch dissolution profile at each sampling points and corresponds to a relative error measure between the two profiles.

12 279 P a g e International Standard Serial Number (ISSN): f1 = n t=1 n t=1 Rt Tt Rt 100 R-T = absolute difference of % drug released at each time points between reference products and test product. R = % drug released of reference product at each time point. F 1 value should be < 15 as close as possible to 0 to shows similarity Stability studies The prepared Microemulsion based gel were packed in aluminium collapsible tubes (5gm) and subjected to stability studies at 25 0 C/60% RH, and 40 0 C/75% RH for a period of 21 days. Samples were withdrawn at 21 day and evaluated for physical appearance, ph, rheological properties, drug content, and drug release profiles by UV- Visible spectrophotometer Antifungal activity: Antifungal activity of formulation was checked by cup-plate method. A definite volume of the Candida albicans suspension (inoculum) was poured into the sterilized dextrose agar media (cooled at 40ºC) and mixed thoroughly. About 20 ml of this suspension was poured aseptically in the petri plates and kept till the solidification. The surface of the agar plates was pierced using a sterile cork borer. The prepared wells were filled with equal volume of optimized batch F4 of microemulsion based gel and marketed gel (1% clotrimazole gel) after that it was incubated at 18 to 24ºC, for 72 hr. The fungal growth was observed and the zone of inhibitions was measured using antibiotic zone reader In vitro Skin Irritation test The aim of the study was to determine the time of irritation of selected MBG, to investigate the acceptability of different polymers for use in MBG and to determine any redness/pink colour of skin or side effects produced by the formulation. The study was conducted in three healthy human volunteers (aged 21 to 28 yrs). MBG without drug were used in the study. Food was prohibited from 4 hr before till the end of the study. Water was provided as and when required. The volunteers were given different coded MBG, along with written instruction sheets. They were instructed to press the Gel against the outer side of the skin for about 30 seconds. The volunteers asked to record the time of MBG applied and the time and circumstances of the penetrate and were asked to complete a questionnaire after trial period for scoring irritancy, non- irritant, irritant, highly irritant at the place of attachment produced formulations.

13 280 P a g e International Standard Serial Number (ISSN): RESULT AND DISCUSSION: 3.1 FTIR spectra of Clotrimazole 110 %T clotrimazole /cm Figure 4: FTIR Spectrum of Clotrimazole Table 7: Interpretation of FTIR spectrum bands of clotrimazole Functional Group Frequencies (in cm -1 ) C-H Stretch , C-C Stretch C-N Stretch Aromatic (in ring) C-C Stretch Aromatic (in ring) , C-N Stretch Aromatic (in ring) , Aromatic C-H out of plane bending 765.4, 741.2, Aromatic ring C-H out of plane bending 707.8, 696.4, These peaks can be considered as characteristic peaks of clotrimazole. 3.2 DRUG EXCIPIENTS COMPATIBILITY STUDY 110 %T clotrimazole clotrimazole formulation clotrimazole /cm Figure 5: FTIR Spectrum of Clotrimazole Formulation: The FTIR Spectrum of clotrimazole formulation showed absorption band at cm -1 (C-H, str.), cm -1 (C-C, str. In aromatic ring), cm -1 (C-N, str. In aromatic ring), ,

14 mg/gm 281 P a g e International Standard Serial Number (ISSN): cm -1 (Aromatic C-H, out of plane bending), , , cm -1 (Aromatic ring C-H, out of plane bending), it is near to FTIR of pure clotrimazole So, drug and excipients is compatible. 3.3 SOLUBILITY STYDY AND SELECTION OF EXCIPIENTS Selection of Oil Solubility studies were aimed at identifying a suitable oil phase for development of clotrimazole microemulsion. Identifying the suitable oil having the maximal solubilizing potential for the drug under investigation is very important to achieve optimum drug loading. The solubility of clotrimazole in different oil depicted in table 8 and figure 6. Table 8: Solubility of Clotrimazole in different Oil Oil Solubility (mg/gm) Seasam oil Castor oil Linseed oil Olive oil Soyabean oil Cottonseed oil 9.78 Sunflower oil Penut oil Capmul MCM NF Capmul PG-8-NF Capmul PG Labrafac Captex 2P Solubility in Oil Oil Figure 6: Schematic diagram of drug solubility in different Oil As per solubility data of Clotrimazole in different oils, maximum amount of drug dissolved in Capmul MCM NF ( mg/gm). So, it was selected for further study.

15 mg/gm 282 P a g e International Standard Serial Number (ISSN): Selection of Surfactant The surfactant involved in the formulation of microemulsion should have a relatively high HLB and hydrophilicity so that immediate formation of o/w droplets and rapid spreading of the formulation in the aqueous media can be achieved. Surfactants are amphiphilic in nature and they can dissolve or solubilized relatively high amounts of hydrophobic drug compounds. As per the solubility method described in solubility of Clotrimazole in different surfactant is described in table 9 and figure 7. Table 9: Solubility of Clotrimazole in different Surfactant Surfactant Solubility (mg/gm) Tween Tween Span Acrysol EL Acrysol K Acrysol K Acrysol K Labrasol Tween 40 Tween 80 Solubility in Surfactants Span 80 Acrysol Acrysol EL-135 K-140 Surfactants Acrysol K-150 Acrysol K-160 Lbrasol Figure 7: Schematic diagram of drug solubility in different Surfactants Nonionic surfactants are generally considered less toxic than ionic surfactants. They are usually accepted. As per solubility data of Clotrimazole in different surfactant, maximum amount of drug dissolved in Acrysol K-140 (152.65mg/gm). The oily phase Capmul MCM exhibited the highest emulsification efficiency with Acrysol K-140 for the homogenous emulsion formation. On the other hand, Capmul MCM showed poor emulsification properties with other surfactants. Therefore, this surfactant was selected for Microemulsion formulation Selection of Co-Surfactant The co-surfactant help to further the surface tension and fluidize the surfactant film, which increases the entropy of the system leading to its thermodynamic stability co-surfactant, increase the flexibility

16 mg/gm 283 P a g e International Standard Serial Number (ISSN): of the surfactant film around the microemulsion droplet the relatively small co-surfactant molecules at the oil/water interface. This releases the bending stress and allows for easier dispersion. As per the solubility method described in the solubility of Clotrimazole in different Co-surfactant is described in table 10 and figure 8. Table 10: Solubility of Clotrimazole in different Co-surfactant Co-surfactants Solubility(mg/gm) Propylene glycol PEG PEG Transcutol P Transcutol CG Propylene glycol Solubility in Co-Surfactnts PEG 200 PEG 400 Transcutol Co-Surfactnts P Transcutol CG Figure 8: Schematic diagram of drug solubility in different Co-Surfactant As per solubility data of Clotrimazole in different Co-surfactant, maximum amount of drug dissolved in Transcutol P ( mg/gm). Acrysol K-140 exhibited good emulsification with Transcutol P. Hence, Transcutol P was selected. Microemulsion consists of oil, surfactant and co-surfactant as constructing component of the system. Drug should be completely soluble in all three components and their mixture. Therefore solubility of drug should be one of the criteria for selection of oil, surfactant and co-surfactant. Moreover solubility of drug is also important to decide the dose of drug. Hence microemulsion should consist of such oil, surfactant and co-surfactant that accommodate dose of drug. Another factor which can be affected by solubility is partitioning effect. If drug is not soluble and stable in mixture it will be diffused towards water at the time of formation of Microemulsion and as drug is water insoluble, it will precipitate out in the formulation. Considering both these facts, selection of excipients is crucial factor for successful formulation. The Table shows that clotrimazole has good solubility in synthetic oils in comparisons to vegetable oils. So, Capmul MCM NF was selected as oil phase. Acrysol K- 140 which can act as surfactant due to its high HLB values showed good solubility of clotrimazole.

17 284 P a g e International Standard Serial Number (ISSN): The third component of microemulsion i.e. co-surfactant which is not necessarily required but may help surfactant to stabilize the system is Transcutol P. Table 11: Selection of Oil, Surfactant and Co-Surfactant for Microemulsion formulation Phase Ingredient Oil Capmul MCM NF Surfactant Acrysol K-140 Co-surfactant Transcutol P 3.4 PSEUDO-TERNARY PHASE DIAGRAM Microemulsifying system from fine oil/water emulsions with only gentle agitation, upon their introduce into aqueous medium. Surfactant and co-surfactant it preferentially adsorbed at the interface, reducing the interfacial energy as well as providing a mechanical barrier to coalescence. The decrease in the free energy required for the emulsion formulation consequently improves the microemulsion formulation.therefore the selection of oil and surfactant and the mixing ratio of oils to S/CoS play an important role in the formulation of the microemulsion. After performing solubility studies, components in which drug showed more solubility put forwarded for phase behavior study. In present study, combinations of surfactant (Smix) with high and low HLB value were used. Capmul MCM NF has low HLB value and Acrysol k-140 having higher HLB value. Combination of low and high HLB surfactant leads to more rapid dispersion and finer emulsion droplet size on addition to aqueous phase. Capmul MCM NF and Acrysol k-140 in the ratio of 2.5:1 showed wider microemulsion existence area and rapid emulsification compared with 3:1 and 3.5:1 thus 3.5:1 Smix selected for formulation development. Optimization of concentration of Oil, surfactant and Co-surfactant for microemulsion formulation there was different ratio considered as per table 12. Table 12: Oil (Capmul MCM NF) and Surfactant (Acrysol K-140)/Co-Surfactant (Transcutol P) Ratio: (3.5:1) Oil Sur/Co-Sur (3.5:1) Water Observations (Parts) (Parts) (Parts) Clear solution Clear solution Clear solution Clear solution Clear solution Clear solution Clear solution Not Clear solution Not Clear solution Not Clear solution Not Clear solution Not Clear solution

18 285 P a g e International Standard Serial Number (ISSN): Figure 9: Pseudo-Ternary Phase diagram of Oil (Capmul MCM NF) and Surfactant (Acrysol K-140)/Co-Surfactant (Transcutol P) Ratio: (3.5:1) To determine concentration of oil, surfactant and co-surfactant phase diagram were constructed. Microemulsion forms when titrated with water under agitation condition. The droplet size of microemulsion is less than 100 nm and as the energy required forming microemulsion is very low it is thermodynamically spontaneous process. This process is facilitated by presence of surfactant. The surfactant forms a layer around oil globule in such a way that polar head lies. Towards aqueous and non-polar tail pull out oil and there by reduces surface tension between oil phase and aqueous phase. Since surfactant and co-surfactant adsorb at interface and providing mechanical barrier to coalescence, selection of oil, surfactant and co-surfactant and mixing ratio to S/CoS play important role in microemulsion formulation. In figure only microemulsion point are plotted (shaded area), so that there is no overcrowding of the phases in the diagram, as for formulation development only the microemulsion area of interest. 3.5 EVALUATION OF MICROEMULSION FORMULATIONS % Transmittance: The clarity of microemulsion was checked by transparency, measured in terms of transmittance (%T). Microemulsion forms o/w microemulsion since water is external phase. Formulation F5 has higher % transmittance value 99.7% these results indicate the high clarity of microemulsion. Batch F1, F3 is less clear than F5 batch. Where as batch F2, F4, and F6 has % transmittance is less which indicates less clarity of microemulsions. This may due to greater particle size of the formulation. Due to higher particle size, oil globules may reduce the transparency of microemulsion and thereby values of %T. The results of %T are shown in Table 13.

19 286 P a g e International Standard Serial Number (ISSN): Table 13: % Transmittance of F1 to F6 Formulation Formulation Code Transmittance (%) F ± 0.2 F ± 0.1 F ± 0.3 F ± 0.1 F ± 0.2 F ± Visual Assessment Clotrimazole Microemulsion concentration (approximately 0.2 ml) was diluted with purified water (100 ml) and gently stirred with magnetic stirrer. Temperature should be 37 0 C. The results of visual assessments are shown in Table 14 and Figure 10 shows different grade of microemulsion. Table 14: Visual assessments various formulation Formulation code Grade Inference F1 Grade I Clear bluish appearance F2 Grade II Bluish white appearance F3 Grade I Clear bluish appearance F4 Grade II Bluish white appearance F5 Grade I Clear bluish appearance F6 Grade II Bluish white appearance Figure 10: Visual Assessment of Microemulsion in Different Ratio Drug Content The drug content of formulations F1 to F6 was found in range of % (Table 15). Table 15: %Drug content of Microemulsion formulation Formulation code % Drug content* F ± 0.09 F ± 0.45 F ± 0.18 F ± 0.24 F ± 0.06 F ± 0.12 *Values are expressed as mean ± S.D, n=3

20 287 P a g e International Standard Serial Number (ISSN): Droplet Size Determination The droplet size of the emulsion is a crucial factor in microemulsion based gel performance because it determines the rate and extent of drug release as well as absorption. Average droplet size was found in water, which range from to nm indicating all the droplets were in the nanometer range. The result shows that the higher S mix proportion led to increase in mean droplet size. The smallest particles were observed for formulation F5 (17.08nm) and largest droplets were obtained for formulation F6. Polydispersibility Index (PDI) Polydispersibility determines size range of droplet in the system. It is expressed in terms of Polydispersibility index (PDI). Polydispersibility index (PDI) (0.3) indicates a homogenous distribution, while a PDI (>0.3) indicates a higher heterogeneous dispersion. The data are as shown in table 5.13 The results show that formulations F1, F2, F3, F4, F6 does not pass the test as they have PDI more than 0.3 whereas remaining all formulations pass the test as they have PDI less than 0.3. PDI value of formulation F5 is less compare to other Formulations. Report of particle size analysis is represents in figure 11 and table 16. Droplet Size Distribution F5: Formulation Code Figure 11: Graph of Droplet size distribution Batch F5 Table 16: Droplet size and PDI of Microemulsion in water Mean Particle size Polydispersibility ± S.D (nm) Index F F F F F F

21 288 P a g e International Standard Serial Number (ISSN): Zeta Potential Determination Zeta potential can be defined as the difference in potential between surface of the tightly bound layer (shear plane) and the electro neutral region of an emulsion. It has got practical application in the stability of emulsion since, zeta potential governs the degree of repulsion between adjacent, similarly charged, dispersed droplets. If the zeta potential is reduced below a certain value (which is depends on a particular system being used), the attractive forces exceed the repulsive forces, and the particles come together leading to flocculation. The ideal zeta potential for microemulsion is less than -30, formulation F1 and F5 were Passed the zeta potential test. Formulation F5 has lowest zeta potential (-9.82) on this basis F5 is optimize. Report of Zeta Potential Distribution is represents in fig. 12 and table 16. Zeta Potential Distribution F5: Figure 12: Graph of Zeta Potential Distribution F5 Table 17: Zeta Potential of Microemulsion formulations Formulation Code Zeta potential (mv) F F F F F F Thermodynamic stability Table 18: Temperature stability study of F1 to F6 Formulation Formulation Code Phase Separation Precipitation F1 Not Seen Not Seen F2 Not Seen Seen F3 Not Seen Not Seen F4 Not Seen Not Seen F5 Not Seen Not Seen F6 Not Seen Not Seen

22 289 P a g e International Standard Serial Number (ISSN): Table 19: Centrifugation stability study of F1 to F6 Formulation Formulation Code Phase Separation F1 F2 F3 F4 F5 F6 Not Seen Not Seen Not Seen Not Seen Not Seen Not Seen Microemulsions are thermodynamically stable systems and are formed at a particular concentration of oil, surfactant and water, without phase separation. 3.6 EVALUATION OF MICROEMULSION BASED GEL (MBG) Physical Appearance Microemulsion based gel formulations were visually transparent viscous appearance. Batch F1 to F4 found transparent on other hand F5 and F6 found in white appearance. The phase separations were not occurred. The all formulations were stable and good consistency. In the formulation F1- F2 the consistency was found less as compared to other formulations. In the formulation F3-F4 the consistency was found more as compared to F1-F2 formulations. In the formulation F5-F6 the consistency was found higher as compared to other formulations due to higher concentration of carbopol 974 compared to F1-F4 batch. Results have been depicted in table 20 Table 20: Physicochemical Characteristics of MBG formulations Formulation Appearance Phase separation Consistency code F1 Transparent None ++ F2 Transparent None ++ F3 Transparent None +++ F4 Transparent None +++ F5 white None +++ F6 white None +++ Excellent +++, Good ++, Satisfactory Determination of ph The ph of microemulsion based gel shown in table 21 and figure 13. It was in the range of 7.07 to 7.43 ph which lies in the normal ph range of the skin. There was no significant change in ph values as a function of time for all formulations. Table 21: ph of Microemulsion Based Gel formulations Formulation Code F1 F2 F3 F4 F5 F6 ph

23 Spreadability (gm.cm/sec) ph 290 P a g e International Standard Serial Number (ISSN): F1 F2 F3 F4 F5 F6 Formulation Batch Spreadability Figure 13: ph of Microemulsion based gel Spreadability of the Microemulsion based gel (MBG) was decreases as increase in concentration of the carbopol 974. The Spreadability was very much important as show the behavior of MBG comes out from the tube. In the formulation batch F1 & F2 the different concentration of carbopol 974 was used. So the Spreadability of the F1 & F2 was slightly increased with decreasing in concentration of Carbopol 974. Spreadability of F3-F6 was slightly decreased with increasing concentration of Carbopol 974. Spreadability data was shown in table 22 and figure 14. Table 22: Spreadability of Microemulsion based gel formulations Formulation code F1 F2 F3 F4 F5 F6 Spreadability 8.9± ± ± ± ± ±0.98 (gm.cm/sec.) * *Values are expressed as mean ± S.D, n= F1 F2 F3 F4 F5 F6 Formulation Batch Figure 14: Spreadability of Microemulsion based gel

24 Viscosity (centipoise) 291 P a g e International Standard Serial Number (ISSN): Rheological Study The microemulsion based gel was rotated with spindle no. 2 at 12rpm for 5-10min. Viscosity of the formulations was shown in table 23 figure 15. F1, F2, F3 were less viscous where as F5, F6 were highly viscous. So from the result, it was indicated that F4 formulation had desired viscosity compared to other formulation of microemulsion based gel. Table 23: Viscosity of Microemulsion Based Gel formulations Formulation Code F1 F2 F3 F4 F5 F6 Viscosity (centipoise) F1 F2 F3 F4 F5 F6 Formulation Batch Figure 15: Viscosity of Microemulsion based gel Drug Content Determination The drug content was determined for all formulations by U.V. Spectrophotometric method at 261 nm. It was found uniform in all the formulations which are shown in table 24 and figure 16. The data obtained from triplicate study were analyzed for mean and standard deviation. The mean drug content was found range between to 99.60%. It was considered that drug was dispersed in uniformly throughout the microemulsion gel. Table 24: Drug Content of Microemulsion Based Gel formulations Formulation Code F1 F2 F3 F4 F5 F6 Drug content*% ± 0.12 ± 0.18 ± 0.23 ± 0.14 ± 0.27 ± 0.16 *Values are expressed as mean ± S.D, n=3

25 Extrudability (gm/cm 2 ) Drug content % 292 P a g e International Standard Serial Number (ISSN): F1 F2 F3 F4 F5 F6 Formulation Batch Figure 16: Drug Content of Microemulsion based gel Extrudability Study The microemulsion based gel filled into collapsible tube and checked it extrudability which was shown in table 25 and figure 17. F1, F2, F3 formulation had very high extrudability due to less amount of carbopol 974 and not desired for the optimum on the other hand F5 and F6 formulation had poor extrudability hence it was not able to extrude easily from the tube. F4 formulation had the desired extrudability compared to the other formulation. Table 25: Extrudability of Microemulsion Based Gel formulations Formulation Code F1 F2 F3 F4 F5 F6 Extrudability (gm./cm2) * ± 0.16 ± 0.23 ± 0.12 ± 0.18 *Values are expressed as mean ± S.D, n= ± ± F1 F2 F3 F4 F5 F6 Formulation Batch Figure 17: Extrudability of Microemulsion based gel

26 293 P a g e International Standard Serial Number (ISSN): In-Vitro Diffusion Study The release of the clotrimazole from the microemulsion based gel was depending on the concentration of the polymer. The release of the drug from the microemulsion based gel formulations written in high to low release in order: F4>F3>F1>F2>F5>F6 where, the amounts of drug release after 10 hr were 96.15%, 92.32%, 89.98%, 86.34%, 85.12%, 84.87% respectively. Amount of the drug diffused from the formulation through the diffusion membrane was completely related to the concentration of polymer. The formulation containing less amount of carbopol 974 like F1, F2, F3, had high release compared to formulation F5 and F6 which containing high amount of the carbopol 974. Batch F4 formulation had highest diffusion profile or release of drug from the gel. It has been indicating that as if increase in the concentration of polymer, the diffusion of the drug decreases. So, finally the In-Vitro release study clearly revealed that the release of clotrimazole is depending on the concentration of the polymer. The cumulative % drug release of all the formulations were shown in the table 26. Graph was plotted cumulative % drug release versus time as shown in fig 18. Table 26: % Cumulative Drug Release of Microemulsion Based Gel formulations Time % Cumulative Drug Release (Hrs) F1 F2 F3 F4 F5 F

27 %CDR 294 P a g e International Standard Serial Number (ISSN): F1 F2 F3 F4 F5 F Time (hr) Figure 18: % Cumulative Drug Release of Microemulsion Based Gel formulations Similarity & dissimilarity Comparison of In-Vitro diffusion study for optimized formulation of microemulsion based gel with marketed gel In-Vitro diffusion study of optimized formulation was compared with marketed formulation shown in table 27 and figure 19. Table 27: In-Vitro Diffusion Study of Optimized Formulation Comparison with Marketed Gel TIME (Hr) Optimized batch of MBG (F4) % CDR Marketed Product Clotrimazole 1% gel Similarity factor (f2) % Dissimilarity factor (f1) %

28 %CDR 295 P a g e International Standard Serial Number (ISSN): Figure 19: In-Vitro Diffusion Study of Optimized Formulation Comparison with Marketed Cream In-Vitro diffusion profile of optimized formulation compared with marketed product (1% Clotrimazole Gel) for similarity factor f1 and dissimilarity factor f2. From the table 5.24, values of similarity factor was (54.52%) and dissimilarity factor was (13.65%) indicating similarity between optimized formulation and marketed product. After comparison of drug diffusion profile of optimized batch and marketed product it was clear that optimized formulation F4 is better than marketed product, because of faster diffusion than marketed product which exhibited in table 27 and fig Optimized Batch Time (hr) Stability Study The optimized formulation of microemulsion based gel was selected for stability study. The microemulsion based gel showed excellent physiochemical property and In-Vitro drug release profile of the clotrimazole. Stability study of the prepared microemulsion based gel performed at 25ºC, 40ºC temperature at 60%, 75% Relative Humidity. No any Visual changes were observed in the microemulsion based gel. The microemulsion based gel was found to stable under different temperature condition. No any significant changes were observed in spreadability, drug content under different temperature after 21 days. The results are shown in table 28 & 29 and fig.20. Table 28: In vitro Diffusion profile data of optimized batch after Accelerated Stability study. Time (hr) %CDR (Initial) %CDR (After 21 days) Similarity factor (f2) = 96.15% Dissimilarity factor (f1) = 1.24%

29 %CDR 296 P a g e International Standard Serial Number (ISSN): At Initial After 21 Days Time (hr) Figure 20: In vitro Diffusion profile data of optimized batch after Accelerated Stability study The result showed that there was no any change in physical appearance of microemulsion based gel. The spreadability and drug content were found in acceptable limit and values of similarity factor and dissimilarity factor were 96.2% and 0.99% indicating good similarity of dissolution profiles initially and after 21 days of the study. So, prepared microemulsion based gel of clotrimazole were found stable at 25ºC/65% RH and 40ºC/75% RH. Table 29: Stability Study of F4 formulation of microemulsion based gel Parameters Time Interval/ Stability 0 th Day After 21 Days Condition Drug content% 25ºC ± 2ºC and 60 ± 5% RH ± ± ºC ± 2ºC and 75 ± 5% RH ± ± 0.17 Spreadability 25ºC ± 2ºC and 60 ± 5% RH 8.4 ± ± ºC ± 2ºC and 75 ± 5% RH 8.4 ± ± 0.26 Colour change 25ºC ± 2ºC and 60 ± 5% RH Transparent Transparent In-Vitro Antifungal Studies 40ºC ± 2ºC and 75 ± 5% RH Transparent Transparent Results of antifungal activity are shown in table 30 and fig. 20 and 21. The values of mean zone of inhibition (In-Vitro antifungal activity) of optimum microemulsion based gel (F4 batch) and marketed formulation.

30 297 P a g e International Standard Serial Number (ISSN): Figure 20: Zone of inhibition of Optimized batch F4 Figure 21: Zone of inhibition of marketed product Hence the optimized formulation F4 had highest zone of inhibition in others compared to marketed product. So, it clearly indicated that the present clotrimazole in batch F4 formulation had good antifungal activity compared to the marketed product. Table 30: Mean zone of inhibition of Optimized batch (F4) and marketed formulation Sr. No. Formulation Mean zone of inhibition in (cm) ± S.D 1. Optimized formulation (F4) 4.2 ± Marketed formulation 3.1 ± 0.26 *Values are expressed as mean ± S.D, n=3