International Journal of Advances in Pharmaceutical Sciences 1 (2010) 156-166 http://www.arjournals.org/ijoaps.

html Research Article

ISSN: 0976-1055

Design and Evaluation of Microemulsion Based Drug Delivery System

K.R. Jadhav , S.L. Shetye , V.J. Kadam

*Corresponding author: S.L. Shetye

Department of Pharmaceutics, Bharati Vidyapeeths College of Pharmacy, CBD Belapur, Sector-8, Navi- Mumbai 400614, India. E-mail: sulabhashetye@gmail.com.

Abstract
The present study was conducted to investigate the microemulsion based topical drug delivery system of antifungal drug Fluconazole (FLZ) in order to bypass its gastrointestinal adverse effects and to improve patient compliance. The pseudo ternary phase diagrams were developed for combinations of Isopropyl Palmitate (IPP) or Light Liquid Paraffin (LLP) as the oil phase, Aerosol OT as surfactant and Sorbitan Monooleate as cosurfactant using water titration method. Microemulsions obtained were analyzed for transdermal permeability of fluconazole using Keshary-Chien diffusion cell through an excised rat skin. Higher invitro permeation was observed from IPP based microemulsion. Thus it was selected for further formulation studies. The developed microemulsion was characterized for optical birefringence, globule size and polydispersibility index. The average globule size of the microemulsion was found be less than 100m. Centrifugation studies were carried out to confirm the stability of the developed formulation. The formulation was thickened with a gelling agent carbopol 940, to yield a gel with desirable properties facilitating the topical application. The developed microemulsion based gel was characterized for pH, spreadability, refractive index and viscosity. Optimized formulation was then subjected to invitro antifungal screening in comparison to currently available marketed gel formulation of fluconazole (Flucos gel). Optimized microemulsion based gel formulation was found to exhibit significant antifungal activity as compared to marketed formulation. The safety of gel formulation for topical use was evaluated using skin irritation test. Thus the present study indicates that microemulsion can be a promising vehicle for the topical delivery of fluconazole. Keywords: Fluconazole, microemulsion, microemulsion based gel, antifungal, Candida albicans and topical delivery.

Introduction
Topical and transdermal products are important classes of drug delivery systems and their use in therapy is
doi:10.5138/ijaps.2010.0976.1055.01019

becoming more widespread. Although topical formulations to treat ailments have existed from ancient times, transdermal products, for which the skin

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Jadhav et al. International Journal of Advances in Pharmaceutical Sciences 1 (2010) 156-166 is used as an alternative route for systemic and regional therapy, are relatively new entities1. The purpose of topical dosage forms is to conveniently deliver drugs to a localized area of the skin2. Although microemulsions can be used to deliver drugs via several routes, these versatile systems have been extensively studied as vehicles for topical administration. Their composition and structure enables them to incorporate greater amount of drug than other topical formulations such as ointments, creams, gels and lotions. These systems are comparatively thermodynamically stable systems because they contain surfactant, cosurfactant, and oil3. Microemulsion-based colloidal drug delivery systems have gained wide acceptance because of their enhanced drug solubilization, thermodynamic stability, and ease of manufacture4-7. Delivery of drugs using these microemulsions through skin increases the local/systemic delivery of the drug by different mechanisms that make them suitable vehicles for the delivery of Antifungals. Fungal infections are common in human beings, which are either topical or severe systemic infections. Invasive fungal infections are being identified with an everncreasing frequency in prematured infants, immuno-compromised hosts, and patient s receiving immunosuppressive agents and in those with acquired immuno- deficiency syndrome (AIDS). The prevention of fungal infections has been improved by the antifungal agent such as Fluconazole. Fluconazole,, a recent synthetic triazole antifungal drug widely used for the treatment of superficial and systemic fungal infections. The drug has slight solubility in water (8mg/mL at 370C) and a melting point of 1380C to 1400 C. It is widely available as tablets and IV infusion. Oral administration of fluconazole often produces gastric irritation, heartburn and vomiting and sometimes patient can develop ulceration, and there is less patient compliance with long term therapy. Topical drug delivery system localizing the drug at skin will be much favorable for the treatment of skin infections and symptomatic relief. The purpose of the present study was to investigate the microemulsion based formulations for topical delivery of fluconazole in order to by pass its gastrointestinal adverse effects and improved patient compliance.

Materials and Methods

Materials Fluconazole was a gift sample from CENTAUR Pharma Ltd Mumbai, India. Isopropyl palmitate, Light liquid paraffin, sorbitan monooleate, docusate sodium, xanthan gum, sodium alginate, hydroxypropyl methylcellulose (Methocel K4M) and carbopol 940 were purchased from S. D. Fine Chemicals, Mumbai, India. FLUCOS GEL (0.5% w/w); manufactured by Cosme Health Care, Goa, India, was purchased from the local market. All the other chemicals were of the analytical grade. Double distilled water was used throughout the experiment. Methods In vitro inherent flux study of a drug The inherent flux of fluconazole was determined using the Keshary-Chien type diffusion cells. Full thickness abdominal skin of albino rats (125-150 g) was used. The dermal surface of skin was carefully cleaned to remove subcutaneous tissues and fats without damaging the epidermal surface. The Keshary-Chien diffusion cell assembly consisted of donor and receptor compartments. The diffusion cell has a capacity of 20 ml and effective surface area of 3.14 cm2. The receptor compartment was filled with saline phosphate buffer [pH=7.4] with 1% Sodium lauryl sulphate . The skin was cut to a suitable size and clamped between the two half cells of the cell. The stratum corneum part of the skin was exposed to the donor compartment and the dermal part of the skin was facing the receptor compartment. The cells were thermostated at 37 + 1 0C and the receptor solution was stirred with a magnetic stirrer at 200 rpm. The saturated solution of drug was placed on the skin surface in the donor compartment. Aliquots of 2 ml were withdrawn at 0, 0.5, 1, 2, 3, 4, 5, 6 and 7 hours from the receptor compartment and it was replaced by the 2 ml of fresh receptor medium. The amount of drug diffused across the skin was estimated by analyzing the drug concentration within receptor medium using HPLC method. Data Treatment The inherent flux per cm2 (Jss) of the drugs in g/cm2/h was given by the slope of the steady state portion of the line in the plot of drug amount 157

Jadhav et al. International Journal of Advances in Pharmaceutical Sciences 1 (2010) 156-166 permeated/unit area of the membrane (g/cm2) Vs time (h).From the following formula given by Aguiar et. Al8, the diffusivity coefficient was calculated. D = h2/6tL Where, D = Diffusivity coefficient in cm2/h. h = Thickness of the skin in cm tL = Lag time in hour [the intercept on the time axis in the plot of cumulative amount permeated (g/cm2) Vs time (h)] The permeability coefficient was calculated by using the following formula given by Flynn et. Al9, Kp =Jss/Cd Where, Kp = Permeability coefficient in cm2/h Jss = Steady state flux in g /cm2/h. Cd = Saturation solubility of drug in g/ml in phosphate buffer saline pH 7.4 Formulation A ratio of surfactant (S) (Aerosol OT) over cosurfactant (CoS) (Sorbitan monooleate) i. e. S/CoS was chosen and the corresponding mixture was made. The following ratios were tried - 1:1, 2:1, 3:1. At a fixed S/CoS ratio, the (S/CoS) mixture was mixed with oil phase to give Oil:(S/CoS) weight ratios of 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8 and 1:910. Each mixture was mixed thoroughly using magnetic stirrer until a homogenous dispersion/solution was obtained. The mixture was titrated with water at ambient temperature with constant stirring. Double distilled water was used in the formulation so as to prevent the incorporation of surface-active impurities. After each addition of water, the system was examined for clarity, birefringence, flow properties and stability. The end point of the titration was the point where the solution became cloudy and/or birefringent. The quantity of the aqueous phase added when the mixture become turbid was noted. The percentages of the three different pseudo-phases incorporated were calculated. Same procedure was also followed for all other S/CoS ratios. Phase diagrams were prepared after calculating the percentage of each phase required to form microemulsion. After preparing the pseudo ternary phase diagram the medicated microemulsions were formulated. The 158 formulations were then characterized by using different techniques and then evaluated for their in vitro performance. The compositions of different microemulsions for isopropyl palmitate with different S: CoS (Aerosol OT: Sorbitan monooleate) ratios are given in the Table 1 and the compositions of different microemulsions for light liquid paraffin with different S: CoS ratios are given in the Table 2. During formulation of medicated microemulsions the drug was dissolved in the mixture of oil and S: CoS. This final mixture was then titrated with water so as to get a clear and transparent microemulsion.
Table 1. Composition of Microemulsions Containing Isopropyl Palmitate as Oil Phase (S:CoS Aerosol OT: Sorbitan Monooleate) Table 1. a) S:CoS = 3:1 Formulation code IPPF1 IPPF2 IPPF3 IPPF4 IPPF5 Oil (%w/w) 55.97 31.84 22.46 17.05 12.75 S:CoS (%w/w) 6.21 7.96 9.62 11.37 12.75 Drug (%w/w) 0.5 0.5 0.5 0.5 0.5 Water (%w/w) 37.31 59.70 67.40 71.07 73.98

Table 1. b) S:CoS = 2:1 Formulation code IPPF1 IPPF2 IPPF3 IPPF4 IPPF5 Oil (%w/w) 52.67 34.60 24.87 18.65 13.81 S:CoS (%w/w) 5.85 8.65 10.66 12.43 13.81 Drug (%w/w) 0.5 0.5 0.5 0.5 0.5 Water (%w/w) 40.97 56.24 63.96 68.40 71.86

Table 1. c) S:CoS = 1:1 Formulation code IPPF1 IPPF2 IPPF3 IPPF4 IPPF5 Oil (%w/w) 63.96 44.22 31.65 22.96 16.58 S:CoS (%w/w) 7.10 11.05 13.56 15.30 16.58 Drug (%w/w) 0.5 0.5 0.5 0.5 0.5 Water (%w/w) 28.42 44.22 54.27 61.23 66.33

Jadhav et al. International Journal of Advances in Pharmaceutical Sciences 1 (2010) 156-166

Table 2. Composition of Microemulsions Containing Light Liquid Paraffin as Oil Phase (S:CoS Aerosol OT: Sorbitan Monooleate) Table 2. a) S:CoS = 3:1 Formulation code LLPF1 LLPF2 LLPF3 LLPF4 LLPF5 Oil (%w/w) 63.96 41.89 29.02 22.11 16.58 S:CoS (%w/w) 7.10 10.47 12.43 14.74 16.58 Drug (%w/w) 0.5 0.5 0.5 0.5 0.5 Water (%w/w) 28.42 47.13 58.04 62.64 66.33

transparent or translucent11. The microemulsion systems were inspected for optical transparency and homogeneity by usual observation against strong light. The systems were also checked for the presence of undissolved drug or other solid ingredient. Globule Size Analysis of the Microemulsion The average globule size and polydispersity index of the medicated microemulsion were determined by the photon correlation spectroscopy. Measurements were carried at an angle of 90 at 25 C. Microemulsion was diluted with double distilled water to ensure that the light scattering intensity was within the instruments sensitivity range. Double distilled water was filtered through 0.45 membrane filters prior to globule size determination. Optical Birefringence Microemulsion was placed between two polarizing plates in a series and then observed for light transmittance. After this, one of the plates was rotated relative to the other through 90o (crossed polarizers) and then examined. Centrifugation This technique helps to determine behaviour of small particles in gravitational field i.e., their separation rate is quite simple and inexpensive providing a rapid fullproof identification of the system as microemulsion. Microemulsion systems were subjected to centrifugation at 3000 rpm for 30 minutes and then examined for any phase separation. Solubility Analysis An excess amount of drug was added in test tube containing 5 ml of IPP and IPP-microemulsion. The tubes were kept on mechanical water bath shaker (Neolab) at 320C for 72 h. The suspension was filtered through membrane filter [0.45] .The filtrate was diluted with methanol and drug concentration was determined spectrophotometrically at 261 nm. Identical method was followed to determine solubility of drug in LLP and LLP microemulsion. In Vitro Evaluation for Screening of Microemulsion The diffusion of fluconazole from the microemulsion was investigated across the excised rat skin using the same diffusion cell model (Keshary-Chien type 159

Table 2. b) S:CoS = 2:1 Formulation code LLPF1 LLPF2 LLPF3 LLPF4 LLPF5 Oil (%w/w) 68.88 44.22 30.28 22.96 17.15 S:CoS (%w/w) 7.65 11.05 12.97 15.30 17.15 Drug (%w/w) 0.5 0.5 0.5 0.5 0.5 Water (%w/w) 22.96 44.22 56.24 61.23 65.19

Table 2. c) S: CoS = 1:1 Formulation code LLPF1 LLPF2 LLPF3 LLPF4 LLPF5 Oil (%w/w) 71.64 49.75 34.82 25.95 19.13 S:CoS (%w/w) 7.96 12.43 14.92 17.30 19.13 Drug (%w/w) 0.5 0.5 0.5 0.5 0.5 Water (%w/w) 19.90 37.31 49.75 56.24 61.23

Characterization of Microemulsion The formulated microemulsions were then recognized and characterized on the basis of their physical properties, which can not only explain the performance of the system but also help in modifying their performance attributes. The optical properties of the microdroplets, their behaviour in a gravitational field and rheological behaviour easily differentiate them from macrodroplets. Transparency/Translucency The droplets of the microemulsions being smaller than th the wavelength of visible light, permit white light to pass through the dispersed system making it

Jadhav et al. International Journal of Advances in Pharmaceutical Sciences 1 (2010) 156-166 diffusion cells) and the same method that was used for in vitro inherent flux study. Full thickness abdominal skin of albino rats (125-150 g) was used. The dermal surface was carefully cleaned to remove subcutaneous tissues and fats without damaging the epidermal surface. One-gram of drug formulation was placed on the skin surface in the donor compartment. The amount of drug diffused across the skin was estimated by analyzing the drug concentration within receptor medium using HPLC method. Average values of three readings of in vitro permeation data were calculated and the average cumulative amount of drug permeated per unit surface area of the skin was plotted versus time. The slope of the linear portion of the plot was calculated as flux Jss (g/cm2/h)12 and the permeability coefficient was calculated using following formula: Kp = Jss Cv Where Kp is permeability coefficient and Cv is total amount of drug. The drug fluxes from IPP microemulsions were compared with the fluxes from LLP microemulsions. Formulation Development of Microemulsion Based Gel Various gelling agents namely, xanthan gum, sodium alginate, hydroxypropyl methylcellulose (Methocel K4M) and Carbopol 940 were evaluated for their ability to gel medicated microemulsion. Gelling agent was dispersed slowly in the medicated microemulsion with the help of overhead stirrer. In case of Carbopol 940, the dispersion was neutralized by using triethanolamine to obtain the gel. Characterization of Microemulsion Based Gel Drug Content For determination of drug content, about 1 g of the gel was weighed in a 100-ml volumetric flask and dissolved in methanol; it was diluted appropriately and drug content was determined spectrophotometrically (261 nm). Measurement of pH The pH of microemulsion based gel was measured on digital pH meter standardized using pH 4.0 and 7.0 standard buffers before use. 160 Refractive Index The refractive index of plain formulation and medicated formulation was determined using an Abbetype refractometer. Spreadability An apparatus suggested by Mutimer et al13 to determine spreadability of the formulation, was modified suitably in laboratory and used for the study. It consists of a wooden block to which a pulley is attached to one end. A rectangular ground glass plate is fixed on the same end. An excess of microemulsion (3g) under study was placed on ground glass plate. The microemulsion is than sandwiched between glass plate and another glass plate having a hook to which a pan is attached at one end with the help of string. The top glass plate was subjected to a weight of 50gm by putting weight in the pan and the time (in sec) required by the top plate to travel a distance of 10cm is noted. A shorter time interval indicates better Spreadability Rheological Behaviour Rheology is less precise but simpler way to identify anisotropic aggregates in the system. In microemulsion, formation of liquid crystalline stage coincides with formation of nonspherical aggregates (cylindrical or lamellar aggregates), which obstructs the flow in the dispersion medium. This produces high yield value. Microemulsions being isotropic (spherical) systems offer less resistance to flow and exhibit low viscosity as compared to macroemulsions also. Rheological properties (study of deformation and flow of matter) are required in various pharmaceutical areas. It helps to monitor the effect of vehicles consistency on release of drug from the preparations and subsequent percutaneous absorption. Also it is important from the manufacturing point of view. Viscosity measurements were carried out using a Brookfield viscometer (LVT Model). 20 ml of microemulsion was filled in the cylindrical tube and the dial reading was noted at 0.3, 0.6, 1.5, 3, 6, 12, 30, and 60 rpm. The speed was then successively lowered and the corresponding dial readings were noted. Direct multiplication of the dial readings with factors given in the Brookfield viscometer catalogue gave the viscosity in centipoises. Comparison with Marketed gel of Fluconazole

Jadhav et al. International Journal of Advances in Pharmaceutical Sciences 1 (2010) 156-166 The release of drug from optimized IPP microemulsion based gel was compared with that of marketed gel containing 0.5% w/w of fluconazole and the flux (Jss) and permeability coefficient (Kp) were calculated. The enhancement of drug penetration due to microemulsion formulation was noted as enhancement factor (EF),14 which was calculated using following formula: EF = Kp (microemulsion) Kp (marketed gel ) In Vitro Anti-fungal Studies In vitro anti-fungal activity studies of optimized IPP formulation were carried out using fungus Candida albicans. The antifungal activity of fluconazole from the optimum formula as well as the reference standard (marketed gel) was determined using candida albicans (ATCC No.: 10231) as a representative fungi, using saboured dextrose agar as culture medium adopting cup plate method. The mean inhibition zone was calculated for each plate, and this value was taken as an indicator of the antifungal activity. A single well isolated colony of candida albicans of atleast 1mm diameter was picked from the culture plate and was streaked aseptically to agar slant. The slant was incubated for 24 hrs at 37oC. After incubation, the inhibition zone diameter around each well was measured using a ruler. Histopathological investigation microemulsion formulation of skin using
Table 4. Parameters Calculated from In Vitro Inherent Flux Study Sr. No. 1 2 3 4 5 Parameters Jss (g /cm /h) Cd (g/ml) Kp (cm/h) tL (h) D (cm2/h)
2

A plot of cumulative amount permeated (g/cm2) vs. time (h) is shown in Figure 1.The results of regression analysis are depicted in Table 3. The values for Jss, Cd, Kp, tL and D are indicated in Table 4.Thus the in vitro inherent flux study helps to compare the above parameters with the results obtained for optimized formulation.
120 Cumu.Amt.Released (g/cm 2) 100 80 60 40 20 0 0 2 4 Time (hrs) 6 8

Figure 1. Plot of Cumulative Amount Permeated (g /cm2) Vs Time (h). Table 3. Regression Parameters from In Vitro Inherent Flux Study Regression Parameters Correlation coefficient Slope Y- intercept Observed Values 0.9195 13.999 6.509

The rat abdominal skin region measuring approximately 4 cm2 was mounted on modified Keshary Chien diffusion cell. The microemulsion (3 g) was applied identical to diffusion study and the effects were compared against water as control. The skin was fixed in 10% neutral formalin for 24 hours and then cut vertically against the surface at the central region (4mm width). Each section was dehydrated using graded solutions of ethanol and then embedded in paraffin wax. Tissues were divided into small pieces and stained with haematoxylin and eosin. The sections were observed under 100 x magnifications and photographed15.

Results 13.999 + 0.238 9304.241 + 186 (1.504 + 0.08) 10-3 0.57 + 0.075 (1.7894 + 0.116) 10-4

Each Value Represents Mean + S.D., n = 3

Results and Discussion

In Vitro Inherent Flux Study of Drug 161

Characterization of Microemulsion Different compositions of formulations were tried and medicated microemulsions were formulated with 0.5%

Jadhav et al. International Journal of Advances in Pharmaceutical Sciences 1 (2010) 156-166 drug. Further these formulations were characterized for transparency/translucency, globule size analysis, optical birefringence, stability. Transparency/Translucency All the microemulsions formed were transparent and appeared like a homogenous single-phase liquid, when observed for visual clarity against strong light. No traces of undissolved drug or other solid ingredient were found in all samples. Globule Size Analysis of the Microemulsion Polydispersity indicates the uniformity of droplet size within the formulation. The higher the polydispersity, the lower the uniformity of the droplet size in the formulation. The IPP microemulsion had globule size of 66.5 nm and polydispersity index of 0.011. The blank IPP microemulsion had globule size of 67.9 nm and polydispersity index of 0.051. The LLP microemulsion had globule size of 52.7 nm and polydispersity index of 0.369. The blank LLP microemulsion had globule size of 54.2 nm and polydispersity index of 0.421. The incorporation of fluconazole did not have considerable influence on the globule size of the microemulsion. Optical Birefringence The samples were examined by ocular inspection in a cross polarizer for sample homogeneity and birefringence. The microemulsions appeared completely dark when observed under cross polarizer. The observations indicated that all the microemulsions were optically isotropic colloidal dispersions. Centrifugation None of the microemulsion systems showed signs of phase separation on centrifugation at 3000 rpm for 30 minutes. This result provided a rapid and full proof identification of the system as microemulsion. Solubility Analysis The results of the solubility study are represented in Table 7. The solubility of fluconazole in IPP microemulsion and in LLP microemulsion is almost 4 folds higher than that in plain IPP and LLP. Microemulsions have solubilising ability for drugs of diverse chemical nature. These results suggest that microemulsions were found to increase the drug 162 solubility. The increase solubility is expected to enhance the performance of formulations. Table 7. Solubility data of fluconazole in plain oils and their corresponding microemulsion system.
Solvent IPP LLP IPP-Microemulsion (IPP-ME) LLP Microemulsion (LLP-ME) Each value represents mean S.D. (n = 3). Solubility [mg/ml] 4.2490.172 3.4240.152 14.9460.18 14.0140.139

In Vitro Evaluation for Screening of Microemulsion When in vitro release studies of formulations through rat skin were carried out, the flux of drug from the formulations of IPP (Table 8) was found to be greater as compared to formulations LLP (Table 9). This was because of higher solubility of fluconazole in IPP and S: CoS mixture as found in solubility studies. Hence,
Table 8. In Vitro Skin Permeation Study for IPP Microemulsion Formulations F1 F2 F3 Flux (+ SD) (g/cm /hr) 112.35 + 3.51 153.25 + 4.79 108.98 + 2.42
2

Permeability coefficient (cm2/hr) 22.47 30.65 21.796

Table 9. In Vitro Skin Permeation Study for LLP Microemulsion Formulations F1 F2 F3 Flux (+ SD) (g/cm2/hr) 102.92 + 2.59 124.70 + 4.28 73.32 + 2.54 Permeability coefficient (cm2/hr) 20.584 24.94 14.664

IPP microemulsions were selected for further studies.

Jadhav et al. International Journal of Advances in Pharmaceutical Sciences 1 (2010) 156-166 Formulation Development of Microemulsion Based Gel Microemulsions have lower viscosity and are difficult to apply on skin so for the ease of application they are tried to be gelled with suitable gelling agent. Various gelling agents such as xanthan gum, sodium alginate, hydroxypropyl methylcellulose and Carbopol 940 were evaluated for the gelling of fluconazole microemulsion. The fluconazole microemulsion used for this purpose contained 0.5% (w/w) fluconazole. The concentration of the fluconazole was selected to enable comparative evaluation with the currently marketed 0.5% (w/w) fluconazole formulations (Flucos gel). It was observed that sodium alginate affected the structure of the microemulsion and resulted in separation of oily phase. This observation could be attributed to that fact that salts like sodium alginate can affect the structure of the microemulsion86. Xanthan gum and hydroxypropyl methylcellulose was unable to yield gels of acceptable consistency. Only Carbopol 940 at a concentration of 1 %w/w was able to thicken the microemulsion, could yield gel consistency without disturbing the microstructure of the fluconazole microemulsion. Hence, Carbopol 940 was selected for the formulation of MBG Characterization of Microemulsion Based Gel Drug content Fluconazole content in the gel was found to be 99.36 1.06 % of the theoretical value (0.5%w/w) Measurement of pH The pH of microemulsion based gel systems was found to be in the range of 7.0 to 8.0 Refractive Index The values of the refractive index of medicated formulations and plain formulations showed that there were no significant differences between the values. (Table 5) Therefore, it can be concluded that the formulations were chemically stable and remained isotropic; thus, there were no interactions between formulation excipients and drug.
Table 5. Comparative medicated IPP MBG refractive index of plain and 1. 2. 3. 4. 5. 6. 7. 8. IPP MBG Refractive Index Medicated Formulation 1.685 0.007 1.680 0.005 Each Value Represents Mean + S.D., n = 3 Plain Formulation

Spreadability The rheological properties of topical preparations influence the performance of drug delivery systems. The spredability is important for uniform and ease of application of topical preparation from patient compliance point of view. It was found in the range of 8 to 15 sec for different formulations which indicates good spreadability. Rheological Behaviour Viscosity Rheological behavior of the microemulsion based gel systems indicated that the systems were non Newtonian in nature showing decrease in viscosity at the increasing shear rates. The viscosity data has been summarized in Table 6 There was no significant difference found between the viscosities of plain and medicated microemulsion based gels (Fig. 2).
Table 6. Comparative viscosity of plain and medicated IPP MBG Sr. no Spindle speed RPM 0.3 0.6 1.5 3 6 12 30 60 Viscosity of plain MBG centipoise 44000 27000 14000 9000 5700 3800 2320 1680 Viscosity of medicated MBG centipoise 42000 25000 12000 8000 5000 3400 2200 1650

RPM - Revolutions per Minute

163

Jadhav et al. International Journal of Advances in Pharmaceutical Sciences 1 (2010) 156-166

Table 10. Comparison of Optimized Formulation with Marketed Gel and Drug Dispersion
IPP medicated MBG IPP plain MBG

Viscosity in cp

50000 40000 30000 20000 10000 0 0 50 100 Spindle speed in rpm

Formulation

Flux (g/cm2/h)

Permeability Coefficient (cm2/h)

Enhancement Factor (Compared with Marketed gel )

Figure 2. Comparative viscosity of IPP medicated and plain MBG Composition of Optimized Formulation The results obtained from the flux studies helped to select the formula with maximum flux for further studies. The composition of the final formulation was: Oil (34.60% w/w), S: CoS (8.65%w/w), Water (56.24%w/w) and fluconazole (0.5%w/w). Comparison of Optimized Formulation with Gel Cream When the release of optimized formula was compared with the 0.5% marketed formulation (Flucos gel), the optimized microemulsion based gel showed the higher flux as compared to the marketed formulation (Fig 3). The results showed that the fluconazole has the higher flux as compared to marketed flucos gel and in vitro inherent flux of flurconazole in saline phosphate buffer (pH 7.4). The permeability enhancement factor for microemulsion based gel when compared with marketed formulation was found to be 2.196 and that of microemulsion based gel and flucos gel compared with drug dispersion in saline phosphate buffer was found to be 5.4260 and 2.4704 respectively (Table 10).
Amt.Released (g/cm )
2

Microemu - lsion Based Gel Marketed gel Drug Dispersio n (Buffer pH 7.4)

40.82 18.588 13.999

8.164 3.717 1.5046

2.196 -

Enhanc ement Factor (Compa red with Drug Dispersi on (Buffer pH 7.4 ) 5.4260 2.4704 -

Antifungal activity Results summarized in Table 11 show that mean zone of inhibition (the antifungal activity) of the tested MBG is large than the reference standard (marketed gel). It is noted that the plain MBG diluted with the diethyl ether showed no antifungal activity. The students t-test shows that there is a significant difference in the MBG zone of inhibition in comparison to the reference standard at p < 0.05 where calculated t value is higher than the tabulated t value. The increase in antifungal activity of the fluconazole in formulation may be because of the surfactant action and oil phase which may help in diffusion of drug.
Table 11 : Comparative zone of inhibition of reference and test

250 200 150 100 50 0 0 5 Time (h) 10

Marketed Gel Microemulsion Based Gel Drug Dispersion in Buffer (pH 7.4)

Sample 1 Reference 3.0 Test 3.6

Zone of Inhibition (cm) 2 2.4 3.1 3 2.5 3.2 4 2.6 3.6 5 3.1 3.8

Mean

Cumulative

2.72 3.46

Figure 3. Comparison of Optimized Formulation with Marketed Gel and Drug Dispersion

164

Jadhav et al. International Journal of Advances in Pharmaceutical Sciences 1 (2010) 156-166 isopropyl palmitate or light liquid paraffin, aerosol OT as surfactant, sorbitan monooleate as cosurfactant, water and carbopol 940 as a gelling agent. Solubility study showed fluconazole has higher solubility in microemulsion compared to in their corresponding plain oils. This leads to increased drug loading. The flux of IPP based ME was better than LLP based ME in the in vitro release studies. Thus IPP based ME was selected for gel formulation. The developed gel formulation showed higher flux as compared to marketed gel and in vitro inherent flux study. In the skin irritation study the formulation was found to be safe as indicated by histopathological findings. The invitro anti fungal activity of fluconazole from microemulsion based system was good as compared to that of marketed gel. Therefore the microemulsion based gel of fluconazole was prepared to obtain improved patient compliance. Hence an attempt was made to increase the skin permeation of fluconazole and skin tolerability.

Histopathological Investigation of Skin Using ME Formulation The histology of excised rat skin in control and treated with optimized microemulsion after 24 hours is shown in Figure 4.1 and Figure 4.2 respectively. The microscopic observations indicate that the optimized microemulsion has no significant effect on the microscopic structure of the skin. The surface epithelium lining and the granular cellular structure of the skin were totally intact. No major changes in the ultra structure of skin morphology could be seen and the epithelial cells appeared mostly unchanged. IPP is widely used in cosmetics and topical formulations and is generally regarded as nontoxic. Aerosol OT used in the formulation is GRAS (Generally Recognized as Safe) listed and it has been included in the FDA Inactive Ingredients Guide for IM injections, oral capsules, suspensions and tablets and also topical preparations.

Acknowledgement
We would like to thank Centaur pharma Ltd, Mumbai for providing gift sample of fluconazole. REFERENCES 1. 2. 3. 4. Shah, V.P.; Behl, C.R.; Flynn, G. L.; Higuchi, W. I. ;Schaefer, H. J. Pharm. Sci., 1992, 81, 1051. David, A.O.; Anton, H.A. Topical Drug Delivery Formulations. David, A.O.; Anton, H.A. Ed .Marcel Dekker: New York, 1990, 1 Rhee, Y.S.; Choi, J.G.; Park, E.S.; Chi, S.C. Int J Pharm., 2001, 228, 161170. Bourrel, M.; Schechter. R.S. Microemulsions and related systems, formulation, solvency and physical properties., Ed .Marcel Dekker: New York, 1998. Kumar, P.; Mittal, K.L. Handbook of microemulsion science and technology., Ed .Marcel Dekker: New York, 1999. Constantinides, P.P. Pharm Res., 1995, 12, 15611572. Gasco, M.R. Microemulsions in the pharmaceutical field: perspectives and applications. In: Industrial applications of microemulsions., Ed .Marcel Dekker: New York, 1997, 97122.

Figure 4.1. Histopathology Control Sample

5.
Figure 4.2. Histopathology Test Sample

Summary and Conclusion

Microemulsion based gel formulation containing fluconazole was prepared with the aim of achieving maximum release through the skin thus by passing its gastrointestinal adverse effect. Thus a microemulsion based gel formulation was successfully prepared using 165

6. 7.

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