REFERENCES 1. Calvo MCR, GIL 1, Ocariz 1R, Benito RR and Benito AR. in-vitro activity of fluconazole, voriconazole and posaconazole against Candida spp. Rev Esp Quimioterap, 2003, 16(2), 227-32. 2. Inamdar NN and Waugh MP. GPAT: A Companion. 9th Edn., Career Publications, Pune, 201], pp. A-42-3. 3. Highlights of prescribing information of Vefend, Pfiezer Inc US, November 2013, www.pfizerpro.com. 4. El-Hadidy GN, Ibrahim HK, Mohamed Ml and El-Milligi MF. Micro-emulsion as vehicle for topical administration of voriconazole: formulation and in-vitro evaluation, drug dev ind pharm, 2012, 38(1), 64-72. 5. Benjamin M. method and composition for transdermal delivery of caffeine in the form of solutions or suspensions, US application No.: 20140256749, 2014. 6. Gohel, M and Nagori S, "Fabrication of modified transport fluconazole spray containing EC & Eudragit RS100 as film formers", AAPS Tech, 2009, 10(2), 684-91. 7. Bajaj A, Bakshi, A and Malhotra, G, A novel meter dose transdermal spray formulation of oxybutynim Ind JPharm Sci, 2012, 70(6), 733-9. 8. Kapadia, M, Solanki, ST, Parmar V, Thosar, MM and Pancholi, SS, "Preliminary investigation tests of novel antifungal topical aerosol", ./ Pharm Bio all Science, 2013,4,74-6. 9. Setiawan K, and Watkinson A. Transdermal delivery system. US patent US, 2013/0317122 Al, 2013. 10. Lull a A and Malhotra G. Transdermal Pharmaceutical spray formulations comprising a VP/VA copolymer and non-aqueous vehicle, WIPO patents, CA 2543245Al, 2005. 11. Sevgi G, Erdal MS and Aksu B, "New Formulation Strategies in Topical Antifungal Therapy ", J Cosmetics, Dermato logical Sci Applications, 2013, 3, 56-65. 12. Scctt LJ and Simpson D, "Voriconazole: a review of its use in the management of invasive fungal infections", Drugs, 2007, 67(2), 269-298. 13. Voriconazole, August, 2013, http://en.wikipedia.org/wiki/Fluconazole. 14. Ulhas DR, Bharnre NB, Satpute RM and Mahajan NT, "Stable topical formulation comprising Voriconazole", WIPO patents, WO/201/084505 A2, 2010. 15-Vonaz Tab/Inj, in Leave behind litraturc, United Biotech Ltd., Solan (HP), India, August, 2013, 2-6, www.unitetedbiotechindia.com. 16. Williams AC, Barry BW, "Terpenes and the lipid-protein-partitioning theory of skin penetration enhancement", Pharm Res, 1991, 8(1), 17-24. 17. Parthibarajan R, Gowrishankar NL and Rajitha M, "Formulation and evaluation of voriconazole floating tablets", Asian j pharm din Res, 2012, 5(3), 180-4. 18. Pal T K, Kharat RB, "Differential thermal analysis", IndJChem, 1989, 28(A), 55-8. 19. IIG, FDA, Janl3, http://www.accessdata.fda.gov/scripts/cder/iig/ getiigweb.cfin. 20. Gohel, M and Nagori S, "Fabrication of modified transport fluconazole spray containing EC & Eudragit RS100 as film formers", ^PS Tech, 2009, 10(2), 684-91. 21. 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El-Maghraby GM, Williams AC and Barry BW, "Oestradiol skin delivery from ultradeformable liposomes: refinement of surfactant concentration" Int, J. Pharm., 2000, 196(l),63-74. 29. Sathali AAH and Rajlaxmi G "Evaluation of Transdermal Targeted Niosomal Drug Delivery of Terbinafine Hydrochloride", Int J of PharmTech Res, 2010, 2(3), 2081-9. 30. Gupta M, Goyal AK, Paliwal SR, Paliwal R, Mishra N etc. "Development and characterization of effective topical liposomal system for localized treatment of cutaneous candidiasis",./. Liposome Res., 2010, 20(4), 341-50. 31.Godin B and Touitou E, "Ethosomes new Prospects in transdermal Delivery", Critical review in the rap drug carrier systems, 2003, 20(1), 63-102. 32. Higuchi T, "Method for in-vitro determination of transdermal absorption", US Patent 4771004,1986. 33. 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Maghraby G, Barry,BW, Williams AC, "Liposomes and Skin from Drug Delivery to Model Membranes", EurJPharm Sci, 2008, 34(4-5), 203-22. TOPICAL ANTIFUNGAL FILM FORMING TRANSDERMAL SPRAY COMPOSITION AND METHOD OF PREPARATION THEREOF FIELD OF THE INVENTION The present invention is related to topical pharmaceutical formulation containing Voriconazole film forming transdermal spray and the method of preparation of the same which is suitable for the topical fungal infection. BACKGROUND OF THE INVENTION AND PRIOR ART Human body mostly host of many microorganisms like bacteria and fungi. Some of them may grow rapidly and cause infection. Fungal skin infection is produced by fungi. They may be yeast fungus (Candida) that cause candidiasis and mould-like fungi (dermatophytes) that cause ring worm or tinea. Fungal infections are caused by microscopic yeasts and moulds that live on the skin, hair (Tinea Capitis) or nails (Onychomycosis). Some of current semisolid antifungal preparations for topical fungal infections are Zocon transgel (Fluconazole 0.5% gel) and Nizral® Cream (2% Ketoconazole Cream) and Voriconazole tablet and IV preparations. Fluconazole and Ketoconazole (and other imidazole derivatives) have less effective MIC, MIGxv90%, values than Voriconazole(1), while Fluconazole a first generation triazole is also resistant to some of Candida species and more chances of recurrences while treatment with them. Voriconazole is potent second generation triazole with broad-spectrum anti-fungal activity against clinically significant and emerging pathogens. However, Voriconazole is CYP34A inhibitor so it may has substantial toxicity and some patients may develop severe toxicity when taken concurrently.(2) Voriconazole tablet and IV may cause serious side effects including, liver problems, nausea or vomiting, vision changes, blurred vision, photophobia, serious heart problems, cardiac arrest, tachycardia, chest tightness, nausea, itching, skin rash, kidney problems like kidney failure, hallucinations.(3) Voriconazole loaded micro-emulsion showed significantly better antifungal activity against Candida albicans than Voriconazole super saturated solution but had shorter shelf-life as more chances of cracking of micro-emulsion so less physical stability.(4) Major drawbacks of topical semisolid products are poor patient compliance, cross contamination, gels are easily rubbed off by the clothing and during day-to-day activities and biphasic semisolid topical product may show physical instability. These formulations are typically in continuous contact with the skin. Creams and ointments can cause skin irritation and sensitization. A significant proportion of patch users suffer from skin irritation and sensitization due to adhesives used in the patch. Film foming transdermal spray minimize pain and irritation experience during the application, still retain the same efficacy but with a significant reduction in dose, even elimination or lower incidence of side effects and reduce the drawback related to semisolid dosage form and patch(5). In transdermal spay filled solution delivery directly on the site of application. Infection covers by extensive area of transdermal film. Film forming transdermal spray is better choice than topical patch and gel as is easy to use, quick drying, well tolerated and non-occlusive. Film forming transdermal spray provides long durability in the superficial iayer of the fungal infected skin. (6"8) Administration of Voriconazole through film fomiing transdermal spray provides controlled route of release and simple regimen of treatment(9,10) OBJECT OF THE INVENTION 1. Low dose of Voriconazole required for drug action so reduces side effects. 2. Local as well as transdennal drug delivery through skin would give better treatment in fungal infection. 3. Long term drug delivery due to adhesive & flexible Film. 4. Increased contact time for local activity. 5. Easy to apply & remove. 6. Film forming Transdennal spray offers aesthetic properties, ease of application and maintainability of integrity, tamperproof system and prevention of contamination of unused content. SUMMARY OF INVENTION Voriconazole is 2nd generation triazole with broad spectrum anti-fungal activity against clinically significant and emerging pathogens and is successfully used as antifungal drug. Drawbacks of oral and IV preparations of Voriconazole are adverse effects, so required to administered through alternative route of administration so topical route of administration selected to treat topical fungal infections mostly candidiasis, ring worm etc. The formulation excipients didn't show absorbance at 256nm. The UV spectrum remains unchanged during in-vitro drug transport study, indicating stability of voriconazole during study. Optimization of formulation based on the study effect of selected dependent variable on the concentration of Eudragit RLPO & EC. It was observed that increasing the polymer concentration, (Eudragit RLPO, EC) there increase viscosity, t 50%, mucoadhesive, flexibility of film, water washability & opaqueness of the film, whereas retard the drug diffusion & decreases transparency of film. As per ANOVA, both testing modules that regression analysis of viscosity & t1/2, Fcalculated. Pressurise Spray filling carried using propeilant as LPG at Vimsons Aerosol Ltd. Gamdi, Anand.*"^ Table 1: composition of voriconazole spray by layout of 32 full factorial designs % Ingredient vv/w Bl B2 B3 B4 B5 B6 B7 B8 B9 B10* Bll* Voriconazole 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 EudragitRLPO 5 5 5 10 10 10 15 15 15 10.0 10.05 EC (7 cPs) 2.5 5 7.5 2.5 5 7.5 2.5 5 7.5 5.0 5.02 Eutectic blend 10 10 10 10 10 10 10 10 10 - 10 PEG 400 0.45 0.45 0.45 0.45 0.45 0.45 0.45 0.45 0.45 0.45 0.45 Alcohol & acetone blend (q.s.) 100 100 100 100 100 100 100 100 100 100 100 TB ■ ■ ■ ' ■ ■ ■ ■ ■ ■ ■ ' Bl 1 check point batch of formulation with penetration enhancer, BIO without penetration enhancer. Optimization of formulation batch from 3" factorial by using two dependent variable viscosity (Y1) and time require to 50% drug transport from the 2.8 cm" area of nylon membrane (Y2). Responses were measured for each trial and then quadratic model was fitted by carrying out multiple regression analysis. (26,27) Y = b0+b1X]1+ b2X2 + b 12X1X2-t-b11X 12 + b22X22... Where, bo was intercept and b1, b2, bn, b22, t>12 were regression coefficients. Response surface plot and counter were selected by feasibility and grid searches. Model should be validated by ANOVA and preparation of CHECK-POINT batch/formulation. Practical response observed in check-point batch/formulation must be same as response calculated SS, MS, F value and relative error by proposed model to show validity. Formulation reiated evaluations Drug content: A 1 ml of prepared formulation mixed 7.4 phosphate buffer contain (0.9% NaCl and 1% SLS) for 4 hr. at 250 RPM for complete extraction. Sample dilution after sonication and filtering stock solution and absorbance was measured after appropriate dilution was analyzed spectrophotometrically at 256 nm in UV spectrophotometer. Viscosity: Viscosity of solutions was measured at 25±1°C using Brookfield viscometer (Digital viscometer model DV-I1+, Stoughton, MA, USA). ULA S00 spindle was rotated at 4 rpm and sample size 16 ml in ULA cylinder. Torque reading was always greater than 10%. Average of three readings taken in one minute was noted as viscosity(20). Viscosities of all Batches Bl to B9 were various 8.8 cps to 101.3 cps at different RPM 1, 2, 4, 6, 10, 12, and 20. All formulated batches result data presented in fig. 4. Viscosity was increase with increase polymers concentration Eudragit RLPO and EC. pH of the solution: pH of formulation was evaluated using digital pH meter (Elico, L1612, India). pH was in the range. In-vitro drug transport: A nylon membrane with pore rating equal to 0.22 urn was mounted in a Franz diffusion cell. Surface area of membrane available for drug transport was 2.8 cm . 1 ml of drug formulation in and 30 ml of solution pH 7.4(contain 0.9 % NaCl and 1% SLS) were filled in donor and receptor compartments, respectively. Throughout experiment, temperature of media present in receptor compartment was maintained at 37±1°C using a hot-water jacket. SLS was used to provide sink condition. AJiquots of 1 ml samples were withdrawn at different time intervals for lOhr from the receptor compartment, and Voriconazole was estimated spectrophotometrically at 256 nm. Equal amount of fresh dissolution medium was replaced after each withdrawal. Time required to transport 50% of drug (Y2) were found. UV spectrum of Voriconazole was observed for drug transported through membrane at different time period. In-vitro diffusion study of Batch Bl to B9 show plots of cumulative percent ding transported through nylon membrane (0.22(μmX45mm size) as a function of time for different formulations respectively. Total amount of drug transported in batches Bl to B9 various from 87.01% to 61.15% was observed at different time intervals for a period of 10 hours. From the results of experimental design result tabulated in table 2, Table 2: Results design layout for factorial design responses Batch code %Concentration Dependent Variables Eudragit RLPO(X,)t EC(X2) f Viscosity(Y|)(cps) f t50(min) |(Y2) Bl 5 2.5 8.8±0.63 183 B2 5 5 15.3±0.2 221 B3 5 7.5 24.0±0.09 256 B4 10 2.5 12.1±0.35 275 B5 10 5 31.5±0.26 294 B6 10 7.5 51.5±0.72 381 B7 15 2.5 21.1±0.5 272 B8 15 5 72.8±1.22 365 B9 15 7.5 101.3±1.72 463 t5o%=Time require for 50% drug transport from 2.8 cm area of nylon membrane in franz diffusion cell.( ' Ex-vivo film formation property: In study of physical evaluation ex-vivo film formation time, mucoadhesive and dermal flexibility, appearance of film and water washability were most import. Table 3: Ex-vivo film formation property All the value SD ±3 (n=3), indicates + = Poor, ++ = Moderate, +++= Good, * and **. ** Shiny and transparent (+) or shiny and translucent (++) or dull and opaque (+++), Batch code Ex- vivo film formation time (sec.) Mucoadhesive flexibility of the film* Water washability* Appearance of the film** Bl 120 + +++ + B2 165 +++ + B3 212 ++ + ++ B4 186 + +++ + B5 247 +++ ++ B6 304 +++ + +++ B7 229 ++ ++ B8 360 +++ + ++ B9 435 +++ + +++ Experiments after 24 hr, skin surface in donor compartment was rinsed with ethanol to remove excess drug from surface and analysed by spectrophotometric analysis. Property ,(28) of film was depicted in table 3. Figure 5: Film formation remains residue film Eudragit RLPO was most suitable for film formation. Film formation rate was depend on evaporation of solvents, which leave residue of film that give effective drug release concentration for predetermine time period of 600 min and film formation on nylon membrane and ex-vivo application site (Placebo) shown in Fig.5 Effect of independent variable on viscosity (cps) & time (minute) require to 50% drug transport: Figure 7: (a) Counter plot and (b) Surface plot of Eudragit (Xi) and EC (X2) effect on time require to 50% drug transport (Y2) A statistical model incorporating interactive and polynomial terms was used to evaluate the responses, where Y\ and Y2 were dependent variable, relationship between variables was further elucidated using contour plot shown in Fig. 6(a), surface plot shown in fig. 6(b) & contour plot shown in fig. 7(a), surface plot in Fig.7 (b) respectively. Y,= 33.96 + 24.51 Xl+22.46 X2 + 16.25 X,X2+ 8.85X,2-3.4 X22 1 R" for Full model and reduce model was 0.988 and 0.964 respectively. Y2 = 304.77 + 66.5 X, + 64.166X2 +19.25X,X2 -17.16 X12 + 7.83 X22 ........2 R for Full model and reduce model was 0.993 and 0.973 respectively. Checkpoint batch analysis: Check point batch formula was formulated based on coded value of overlay plot. Experimental value & predicted value were 31.9 & 33.09 for viscosity where time required to 50% drug transport ware 297 & 300.49 respectively. Criteria for selection of optimized batch: i) Yj: viscosity of the formulation should be less than 80 cps and more than 20 cps require for good mucoadhesive film formation. ii) Y2 (t5o%): Time require to 50% drug transport should be equal to 300 min±5%. Optimized batch should form film within 8 min. Film formed should be mucoadhesive and flexible in nature to allow day to day activity yet should be water washable. Spray angle should be less than 85°. Criteria were arbitrarily selection of optimized batch. Batch Bll showed desirable drug release (Nearly 23% drug release in first hr and sustained release thereafter), so results of this batch used for further optimization by regression analysis. Model analysis carried using ANOVA and formulating check point batch. Overlay plots of two responses could be used to determine desired concentration of eudragit RL.PO and EC. In below fig.: 8 show yellow region of overlay plot show desired range of responses. By choosing flagon line any concentration of eudragit RLPO and EC in this region, desired responses could be achieved that consider as check point batch and all characterization of transdermal spay was carried for further study of characterization. Figure 8: Overlay plot showing combined effects of factors Xi and X2 on Yi and Y2 Evaluation parameters of optimize batch Drug transport through biological membrane (Rat skin): Rat skin was considered as biological membrane and drug diffusion experiments were done in Franz diffusion cell. For both batches evaluate effect of penetration enhancers by drug diffusion pattern for without penetration enhancer (BIO) and with penetration enhancer (Bll) result was depicted in figure 9, indicate the effect of eutectic mixture on drug transport was checked using rat skin as a membrane in Franz diffusion cell. In Batch Bll with penetration enhancer drug transport was 1.68 fold compare to without BIO penetration enhancer eutectic mixture (camphor: menthol) significantly favors drug transport. Enhance penetration through skin penetration pathway of intracellular & transcellular pathways within a deep layer of skin. Camphor and menthol causes leaching of lipids present in skin and thus causes pore formation. Figure 9: Ex- vivo drug transport study through biological membrane Flux calculation: Flux is the amount of permeant crossing a membrane per unit area into the circulating system per unit time and for in-vitro permeation this "system" is the receptor chamber, expressed in units of mass/area/time. If the permeant was applied in a finite dose, then you can only calculate a steady state flux by equation:(29) Jss = Q/(Axt) 3 Where Q is the quantity of compound traversing the membrane in time t, and A is the area of exposed membrane in cm . Units of flux are quantity/cm /min. also calculate permeability enhancement ratio was calculated based on permeability coefficient (Kp) by Where, J= steady state flux, C = concentration of drug into donor compartment Penetration enhancing effect of penetration enhancer was calculated in terms of enhancement ratio (ER) and was calculated by using the equation (30) The steady state flux was determined from the slope of the linear portion of a cumulative amount permeated versus time plot. Calculated values related to ex-vivo permeation data analysis for steady state flux (Jss), permeability coefficient (Kp) and enhancement ratio (ER) after using eutectic blend as penetration enhancer were the flux value for BIO and optimized batch (B11) was found 39.1 and 65.8 (μg/cm7hr) respectively, however indicted that permeation flux increased in present of permeation enhancer in ratio of 1.68. Effect of penetration enhancer was observed when formulation incorporated with penetration enhancer in different concentrations. This result indicate that the formulation containing 10 % eutectic mixture camphor & menthol (1:1) with 0.45 % PEG 400 gave better penetration of voriconazole through rat skin. These studies indicated that as the concentration of penetration enhancer increased drug permeation was increased. Kinetics of model fitting: Highest R" value and least value of sum of square of residuals (SSR) and Fisher's ratio (F) were used to select most appropriate kinetic model ( '" ' Hixon-Crowell model was fitted for the study. Antifungal studies: Antifungal activity of optimized batch, eutectic blend with solvent and solvent alone was determine using Candida albicans (MTCC 1637) as representative fungi, by adopting cup and plate method. Previously prepared Sabouraud's dextrose agar dried plates were used. Freshly prepared culture loops were streaked across agar at right angle from ditch to edge of plate. 1 ml of formulation was placed in ditch and plate was incubated aerobically at 25oC for 24 hr., fungal growth and zone of inhibition was measured.(34:35) Studies were carried for best formulation and zone of inhibition observed at Bl 1 (27mm), results were in table 4. Table 4: Antimicrobial activity voriconazole and formulation ingredients t Values in Mean ± S.D, n=3, C= voriconazole with solvent, D= eutectic blend with solvent, E= ethanol and acetone blend. %W/W ingredient f Bll C D E Voriconazole 0.5 0.5 — — Eudragit RLPO 10.05 ~ — — EC 5.025 — — — PEG 400 0.45 — — — Eutectic blend 10 — 10 — Ethanol & acetone bland 100 100 100 100 Zone of inhibition (mm) 27±1.7 16±3 13±1.5 7±1 Figure 10: Displays that mean zone of inhibition by cup and plate method of Optimized Batch Bll Alcohol with Voriconazole (C), Eutectic mixture (D), Alcohol and acetone blend (E) Skin irritation studies: Table 5: Skin irritation test of voriconazole transdermal spray No. of Rabbit t Time (hr) Itching Edema Erythema Papule and flakiness 4 12 24 4 12 24 4 12 24 4 12 24 Rat -1 (Control) + - - - - - - - - - - - Rat-2(Test) + - - - - - - - - - - - | - Sign indicate no reaction, + indicate some reaction, +++ sign indicate more reaction. Figure 11: Skin irritation study (A) Control (B) Test after 24 hr Aim to evaluate any irritant potential of formulation in-vivo on rat skin. Hair on dorsal side (3cmX3cm) of male Wister rat (wt 300-350gm) was removed without damaging skin. One rat was kept as control and other was treated with test. Voriconazole spray formulation was applied uniformly on dorsal region three times priming in each a day for two consecutively. Rats were observed for signs.(j6) After a 24 hour exposure, film formed was removed. Test sites were wiped with tap water to remove any remaining test article residue, there were no any visualised signs of edema, erythema, papule, flakiness & dryness of rat skin indicating test formulation observed complete absence of reaction & the safety of formulation. In-vivo pharmacodynamics studies: In-vivo studies were carried out as per the guidelines compiled by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA, Ministry of Culture, and Government of India). All the study protocols were approved by the Animal Ethical Committee of the department of pharmaceutical sciences. (Protocol number: 1AEC/DPS/SU/1311)) The male Wistar rats each weighing 250-280 gm, which divided in two group shown in table 6 were selected and housed in individual cages and received food and water ad libitum(37). Table 6: In-vivo pharmacodynamics study groups Groups I II Treatment Control Test Dose(mg/kg) 5mg/ml(0.5%VRZ) Placebo(Nodrug) No of animals 5 5 Route Duration of treatment Topical 14 days Topical 14 days (a) Preparation of the microorganisms: Clinical isolate of Candida albicans (MTCC 1637) was used to infect the animals. A working culture of the Candida was grown for 48 hr at 30 °C on Sabouraud dextrose agar (SDA). The cells were then collected, washed and resuspended to a final concentration of lxl07 colony forming units/ml (cfu/ml, suspended in sterile saline) of C. albicans{j4K(b) Cutaneous infection: Each animal's back was shaved with an electric clipper and an approximately 3X3 cm" area was marked on each animal's back, marked area was infected with 1><107 cfu/ml suspensions by gently rubbing onto the skin with the help of a sterile, cotton-tipped swab until no more visible fluid was observed/ * Control animals were infected in the same manner; however, they did not receive any Voriconazole spray formulation. Cutaneous candidiasis related skin fungal infections most often reoccurs and is rarely cured: hence patients can receive theranv over a lon^ time. Figure 12: cutaneous infection and treatment are before treatment (a) 1st day, (b) 5th day, (c) 7th day and 14 days treatments healing (d) 5rh day, (e) 10th day, (f) 14th day after treatment show complete healing of candidiasis. The cutaneous infection shown fig. 12 (A) was induced in Wistar rat by apply Candida albicans and the complete fungal infection was induced within 7 days. Wister rats were observed daily for the signs of infections. The first signs of infection were observed on the 5th day (fig. 12 (B)) after inoculation in all the animals manifested in the form of redness and scaling. These alterations became more evident around the 7th day (Fig. 12 (C)) with marked hair loss and brittle hair. The lesions progressively increased in diameter in the control group (treated with placebo) and were found to be covered with white-yellow crusts strongly adhered to the epidermis. Redness and itching at the site of infection in the treatment group was allayed in 2-3 days. (c) Treatment of the infection: A total of 10 animals were taken, 5 served as control (treated with placebo) and the remaining 5 were treated with voriconazole containing spray formulation. The inoculation of animals was done under general anaesthesia. An area of 3cmX3cm, on the rats back was clipped and made hair free; area was marked with a marker and abraded with sterile fine grit sandpaper. A cell suspension containing Candida albicans was applied on skin during lag phase. The animals were observed on daily basis for signs of infection. The topical treatment was started after 7 days growth of fungus examine by visualization and appearance and confirmation of fungal hyphae on skin of animals hair follicle grown in SDA media for overnight. The clinical as well as mycological parameters were also evaluated after 2 week from initiation of topical treatment. For histopathological examination, skin biopsy samples (Sanjivani patholab and PDU medical college, Rajkot) were obtained from one animal per group after completion of the treatment period. The tissue was fixed in 10% neutral buffered formalin, embedded in paraffin and processed for histopathological examination. The fungal elements were visualized using haematoxylin, eosin and Periodic acid-Schiff (PAS) staining (36) After induction of infection formulation was applied to the infected site twice a day. It was also observed that there was shedding of the infected skin scales and appearance of light pink coloured skin with very fine hair growth on the 5th day 15 (D)) after the initiation of treatment. Subsequently a uniform and healthy hair growth was observed at the site of infection after 10 days (fig. 12 (E)) treatment. The complete healing of the infected site was achieved in 14 days (fig. 12 (F)) with complete hair growth. Skin biopsies were obtained from the test areas, skin sections were stained with PAS stain and HE stain histopathological examination of skin sections was performed to determine whether there was any skin tissue invasion by Candida species. Histopathology result of HE Stain: Skin samples were examined to evaluate the cutaneous irritation potential of optimized voriconazole spray, after 21 days shown was observed by Figure 13: Histological photographs of rat skin by the stain (A) Control and (B) After treatment with voriconazole transdermal spray Figure 14: Histopathology of rat skin infected with Candida albicans^ (A) Normal skin histogram, (B) Control group (contain placebo) arrow show presence of spored hyphae swelling of cell, (C) After treatment with test formulation showing complete absence of fungal elements Histopathological examination and clearly observes drug penetration through hair follicles. Penetration pathways show structural histological photograph (fig. 13). The photomicrographs of untreated rat skin (control) showed normal skin with well-defined epidermal and dermal layers (Fig. 12 (A)). Also, no histopathological changes were observed in rat skin treated with optimized voriconazole transdermal spray establishing the safety of prepared formulation on rat skin (Fig. 12 (B)) uniformly layered stratum corneum and loosely textured collagen in the dermis could be observed. The histopathogical results fig 14 (A) shown structure of normal skin, there was no any sign of disease. However, in the animals receiving placebo treatment fungal elements in the hair follicles were clearly visible as black colour dot as hyphae of Candida albicans, which depicted in figure 14 (B). After treatment of 21 days by applying the transdermal spray formulation it was penetrate deep layer of the skin, which indicate complete absence of any fungal element in the skin biopsies of animals as shown in figure 14 (C) Container related evaluations: The prepared formulation filled into metal container by pressures filling using LPG gas as propellant that evaluated following parameters: Volume of solution delivered upon each actuation: The volume of solution delivered upon each actuation was calculated using equation.... AL= (W0- Wt) / Da 6, Where Wt is weight of formulation after actuation, Wo is the initial weight of the formulation before actuation, and Da is the density of the formulation/2 ' The amount of solution delivered upon each actuation was wound 0.29±0.043 ml. Spray angle: The sprays were actuated in horizontal direction onto a white paper mounted at a distance of 10 cm from the nozzle. The radius of the circle, formed on the paper, was recorded in triplicate from different directions. Spray angle (6) was calculated by equation. Spray angle (6) = tan" (1/r) 7, Where 1 is the distance of paper from the nozzle, and r is the average radius of the circle.(20) The acceptable spray angle was arbitrarily decided as less than 85°for easy actuation of drug solution from the container and cover maximum surface area. Spray angle was found to be 78.69°±1.2° per spray, which illustrated based on spray pattern. Spray pattern test: The method of impingement of spray on a piece of paper was used for the study. Sudan red (10 mg) was dissolved in formulation to facilitate visualization. Figure 15: Spray pattern of formulation (A) Delivery of solution from container (B) Observed film pattern present of color solution of safranin dye Spray pattern was studied by a horizontal cut of the stained sheet of paper in 10 mm for spray formulation some chemical reactions may be used to develop the pattern, which is considered more accurately. Container seal efficiency test / Leak test: Physical or chemical container and closure system integrity test (e.g. bubble tests, pressure/vacuum decay, trace gas permeation/leak tests, dye penetration tests, seal force test etc.) Use to examine packaging of container. Dye penetrant method is used to find cracks in metals and defects in welds(7) There was no color solution pass from valve assembly without actuation, hence confirm that no leakage of container closure system. Flammability test: Flammability test was requiring because filled propellant gas was LPG. In Flame projection as flame was keep 50cm from spray container and on delivery of spray observe flash back of spray around 12±2 cm from flame that indicate flammability of spray due to presence of LPG gas as propellant, there for advise to patient container should be keep away from flame during spraying on skin. Characterization of optimizes formulation by following parameter: Following parameters are evaluated for optimized formulation more characterization of voriconazole transdermal spray. The formulation with less than 80 cps showed acceptable spray pattern from container. The viscosity higher than 80 cps showed poor sprayability. However batch Bl 1 had 31.9±0.2 cps that less than 80 cps hence good sprayability of formulation from the container. There was no leaker of formulation from the container that evaluate using dye test. The amount of solution delivered upon each actuation and spray angle was wound 0.29 ml and 78.69° respectively. The two parameters v/ere correlated with polymer concentration and viscosity of the formulation. The acceptable spray angle was arbitrarily decided as less than 85° for easy actuation of drug solution from the container and cover maximum surface area. Ex-vivo film formation was transparent in appearance. Table 7: Test parameters for optimized formulation Sr. no Tests parameter Average results 1 2 3 4 Amount of delivered upon each actuation Spray angle Leak test Appearance of the film 0.29 ±0.043 ml 78.69°±1.59 No leak after feeling. Transparent 5 Mucoadhesive and flexibility of film ++ 6 Water washability +++ 7 Ex-vivo film formation time 247=b4.04(sec.) 8 Viscosity (cps) 31.9±0.2(cps) Short term stability study: Optimized formulation was stored for 6 month at 30±3°C away from light. At the end of six month, the formulation was subjected to various tests. Procedure employed for study was identical to that above, which indicated in table 8:( Table 8: Test parameters for short term stability study of optimized batch Sr.No Tests parameter Before stability After stability 1 Viscosity (cps) 31.9+O.2cps 32.3±0.38 (cps) 2 volume of solution delivered upon each actuation 0.29±0.043 ml 0.301 ±0.06 ml 3 Spray angle 78.69 °±1.2. 77.49° ±1.41 4 Leak test No leak No leak after feeling. 5 Appearanceof the film Transparent Transparent 6 Mucoadhesive, flexibility of film ++ ++ 7 Water washability +++ +++ 8 Ex-vivo film formation time 247±5 (Sec.) 238 ±4.04(sec.) 9 Flux, Jss (|im/cm7hr.) 65.8±0.98 61.12±1.25(ixm/cm2/hr) 10 pH 6.2±0.2 6.2±0.2 The drug diffusion of formulation before stability & after short term (one month) stability study was 76.50% and 75.72 % and time require to tso% was 299 and 303 minute respectively, which indicate there no statically significance difference. To check the similarity & dissimilarity between two dissolution profile f2 & f1 was applied. Similarity factor f2 was found to be 93.74, which was in the range of 50-100 & dissimilarity factor f1 value is 1.5 which was in range of 0-15, which indicated that dissolution profiles before and after stability testing were almost similar and super imposable. This confirmed that voriconazole transdermal spray would be stable when packed in proper container. CLAIMS: We claim, 1. Composition and method of preparation of film forming transdermal spray of voriconazole 2. Composition of spray of claim las: a. Active pharmaceutical ingredient Voriconazole or its any salt equivalent to voriconazole in an amount between 0.05%-2%. b. Film forming polymers Eudragit in an amount between 5%-15%, c. Polymer RLPO, EC (7 cPs) in an amount between 2.5%-7.5%, d. Eutectic blend in an amount between 10%, e. Plasticizer PEG-400 in an amount between 0.4%-0.5%, f. Solvent Alcohol & acetone blend is enough to formulate as spray. Amounts being based upon the weight of the final composition 3. According to claim 1, film forming transdermal spray comprising eutectic blend composition of camphor and menthol in ratio of 1:1 4. According to claim 1, film forming transdermal spray comprising solvent mixture of alcohol and acetone not more than 83 % and 12 % respectively. 5. According to claim 1, Film forming transdermal spray having viscosity less than 80 cPs, 6. According to claim 1, film formed within lOmin of time period 7. According to claim 1, Film forming transdemial spray prepared by applying suitable design of experiment 8. According to claim 1, Film forming transdermal spray of voriconazole which had been prepared by screening of film forming polymers, eutectic composition and its ratio, solvent and its ratio on the bases of film forming capacity. 9. According to claim 8, in film forming transdermal spray of voriconazole, ethyl cellulose, Eudragit RLPO, polyethylene glycol-400 were sequentially dissolved in the eutectic mixture consisting of equal proportion of camphor and menthol. Voriconazole was separately dissolved in vehicle blend. The solution of ethyl cellulose/ Eudragit® RSI00 in eutectic mixture was gradually added to the solution of Voriconazole and mixed for .15 mm at 200-250 RPM. Than solution would be sonicited for 20 minute in to ultrasonic cieaner. The resulting solution was filled in to aerosol container and sealed. 10. According to claim 9, vehicle blend consisting of 80 parts of alcohol (80% v/v) and 20 parts of acetone.