Fabrication and Characterization of Film-Forming Voriconazole

Bulletin of Faculty of Pharmacy, Cairo University xxx (2017) xxx–xxx

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Bulletin of Faculty of Pharmacy, Cairo University

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Original Article Fabrication and characterization of ﬁlm-forming voriconazole transdermal spray for the treatment of fungal infection ⇑ Nitin Merubhai Mori a, , Priya Patel a, Navin R. Sheth b, Lalji V. Rathod c, Kalpesh Chhotalal Ashara d a Department of Pharmaceutical Science, Saurashtra University, Rajkot 360005, Gujarat, India b GTU, Ahmedabad, India c Maharaja Sayajirao University, Vadodara, Gujarat, India d School of Pharmacy, RK University, Rajkot 360020, Gujarat, India article info abstract

Article history: Voriconazole is second-generation triazole used for the treatment of fungal infections but has serious Received 30 August 2016 unwanted adverse effects, which could be reduced by topical semisolid dosage form. Major drawbacks Received in revised form 8 January 2017 of topical semisolid products are poor patient compliance, cross contamination; gels are easily rubbed Accepted 12 January 2017 off by clothing and during day-to-day activities, physical instability. The purpose of the present work Available online xxxx was to fabricate 0.5% w/w voriconazole transdermal spray for fungal infection. The transdermal spray was generated by using a film forming polymers like Eudragit RLPO and ethyl cellulose (1:2 ratios) along Keywords: with eutectic camphor: menthol (1:1) mixture used as a penetration enhancer. The formulation opti- Eudragit mized by constrained 32 factorial design. Regression analysis and response surface methodology were Ethyl cellulose Dermatol dynamics used to optimize the effect of polymers and formulate checkpoint batch based on overlay plots. The trans- Design of the experiment dermal spray was subjected to evaluate parameters related to formulation and containers. The concen- Film forming transdermal spray tration of Eudragit RLPO and ethyl cellulose was showed influence on viscosity as well as t50. Diffusion Voriconazole study was showed 75% of voriconazole transport with 65.8 lgcm 2 h 1 fluxes. Penetration enhancers’ had shown an increase in 1.68 fold of the penetration of voriconazole through the formulation. The study was concluded that fabricated film forming voriconazole transdermal spray formulations penetrate to the deep layer of the skin and was feasible to treat the dermatological fungal infection. This delivery platform is opened a wide range of treatment of fungal infection as compared to conventional formulations. Ó 2017 Publishing services provided by Elsevier B.V. on behalf of Faculty of Pharmacy, Cairo University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc- nd/4.0/).

1. Introduction paid on transdermal effective antifungal treatments to counter against fungal pathogens. Administration of drugs through the As per dermatological sciences, the skin is the largest human human skin has several advantages such as reduced side effects, sense organ and structurally it a defense against the external envi- steady drug delivery profile, non-invasive means of drug delivery, ronment. Various skin diseases are observed in human skin. Among easy to apply and easy to termination of medication. However, one is fungal infection which is one of the most common diseases topical drug delivery is challenging since skin behaves as a natural into Asian and African country affects approximately 15% of the barrier to drug transport and the transport of drugs through the population, e.g. dermatophytes, candidacies, etc. [1]. There has been skin is a complex process. an increasing search for novel antifungal therapy due to the lack of Clinical efficacy of drug depends on its ability to penetrate into efficacy, poor MIC (minimum inhibitory concentration) values, SC (Stratum corneum) [2,3]. SC is a large and easily accessible more side effects and development of resistance associated with organ apparently offers ideal and multiple sites to administer ther- some of the existing antifungal drugs. Much attention has been apeutic agents for both local and systemic actions. Human skin is a highly efficient self-repairing barrier designed to keep insides in and the outside out’ [4,5]. Peer review under responsibility of Faculty of Pharmacy, Cairo University. Voriconazole is a second-generation triazole antifungal agent ⇑ Corresponding author at: MIL Laboratories Pvt. Ltd, Vadodara, Gujarat, India. indicated for use in the treatment of fungal infections including E-mail addresses: [email protected] (N.M. Mori), priyapatel5@gmail. invasive aspergillosis, oesophageal candidiasis, and serious fungal com (P. Patel), [email protected] (N.R. Sheth), [email protected] infections. It works principally through inhibition cytochrome (L.V. Rathod), [email protected] (K.C. Ashara). http://dx.doi.org/10.1016/j.bfopcu.2017.01.001 1110-0931/Ó 2017 Publishing services provided by Elsevier B.V. on behalf of Faculty of Pharmacy, Cairo University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

P-450 mediated 14 a-lanosterol demethylation, which is an essen- 2.2.2. Determination of solubility of voriconazole and polymers in tial step in fungal ergosterol biosynthesis. It is designated chemically various solvents as (2R, 3S)-2-(2,4-difluorophenyl)-3-(5-fluoro-4-pyrimidinyl)-1- As voriconazole is a hydrophobic drug, to solubilize it in organic (1H-1,2,4-triazol-1-yl)-2 butanol and has a molecular weight of solvent mixture alcohol and acetone mixture was taken. The ratio 349.31 g/mol. Lipophilicity (log p-value) of it is 1.65. It is BCS class of alcohol to acetone was taken 80:20 only because regulatory II drug. It has a melting range of 127–130 °C. It has 0.25 lg/ml of body bound to take the other ratio. The solubility of voriconazole the MIC90 and 61 lg/ml of the MIC99. was determined in a mixture containing 80 parts (80% v/v) of alco- Voriconazole is available in intravenous and oral formulations. hol and 20 parts of acetone. Eutectic mixture of camphor and men- The most commonly reported adverse effects of it are hepatotoxi- thol ratio for clear liquid form was also determined. The solubility city, skin rashes, and visual disturbances. Hepatotoxicity could be of EC and different grade of EudragitÒ was determined in the eutec- reduced by formulating topical semisolid dosage form. A major tic mixture. drawback of topical semisolid products is poor patient compliance, cross contamination, easily rubbed off by the clothing and during 2.2.3. Voriconazole-excipient compatibility studies day-to-day activities, and the biphasic semisolid topical product Voriconazole-excipient compatibility studies were carried out may show physical instability. The film forming transdermal spray by FTIR (Fourier transform Infra-Red) absorption spectroscopy retains the same efficacy of drug but with a significant reduction in and DSC (Differential scanning calorimetry). dose, even elimination of more side effects and reduce the drawback related to semisolid dosage form. In transdermal spray filled 2.2.3.1 FTIR analysis. The physicochemical compatibility must be solution delivers directly to the site of application [6]. The spray established between voriconazole and the other excipient to pro- formulation is available for self-medication, rapid action, and duce a stable, efficacious, attractive, and safe product. The sample patient compliance dosage form. It improves therapy, maximizing was consisted of pure voriconazole, pure excipients, and physical availability with a minimum dose. The safety margin of high mixture of formulation in equal proportion were mixed geometri- potency drugs can be increased with use of it. The drug transport cally and analyzed by FTIR spectroscopy [Thermo scientific USA]. can be modulated by the eutectic mixture. The formation of the The scanning range was 450–4000 cm1 and the resolution was film is depending on evaporation rate, type of solvents used, tem- 1cm1 [12]. perature [7], and concentrations of excipients [8]. The melting point of a drug influences the solubility and hence the skin pene- 2.2.3.2. DSC. It was carried out using aluminum pans contain 3 mg tration. It had been postulated that the lower the melting point sample of pure voriconazole and physical mixture of voriconazole- of the permeant, the greater was the solubility in a given solvent, excipients, in equal proportion were mixed geometrically and ana- including the skin lipids [1]. Menthol affects skin permeation by lyzed at scanning speed of 10 °C/min over a temperature range of a dual mechanism: by forming a eutectic mixture with the pene- 25–300 °C using DSC 60, (TA-60WS, Shimadzu, Japan) [13]. trating compound, thereby increasing its solubility and by altering the barrier properties of the SC [9]. 2.2.4. Preparation of formulation The aim of the present study was formulation, evaluation, and Several different types of EudragitÒ were commercially avail- clinical investigation of modified transdermal spray of voricona- able. They could be obtained as the dry powder, granules or aque- zole using ethyl cellulose (EC) and eudragit as a film formers along ous dispersion. These were used for preliminary study along with with penetration enhancers [10]. EC. Film formers (EC: EudragitÒ:1:2 ratio) and 0.5% w/w plasticizer (PEG-400) were sequentially dissolved in the eutectic mixture consisting of the equal proportion of camphor and menthol. 0.5% w/w 2. Materials and methods voriconazole was separately dissolved in vehicle blend consisting of 80 parts of alcohol and 20 parts of acetone. As per US Depart- 2.1. Materials ment of Health and Human Services-US FDA IIG (inactive ingredient guideline), limit of alcohol and acetone in topical solutions is Voriconazole was received as a gift sample from MSN labora- more than 83% and 12%, respectively so used safely [14]. The solu- tory Hyderabad, India. The different types of eudragit polymers Ò tion of EC/Eudragit in the eutectic mixture was gradually added to of various grades EudragitÒ RS100, EudragitÒ RL100, EudragitÒ the solution of voriconazole and mixed for 30 min at 200–250 rpm S100, EudragitÒ RLPO, EudragitÒ RSPO, EudragitÒ L100 were (revolution per minute), than solution was sonicated for 20 min received as a gift sample from Evonik Degussa Pvt. Ltd. Mumbai, into ultrasonic cleaner (Equitron Ltd, Japan). The resulting solution India. Ethocel 7 LV (EC standard 7 cps low viscosity grade), PEG- was filled into refillable container assembly. The polymers screen- 400 (Polyethylene glycol-400) and Camphor were purchased from ing did by physicochemical parameters [15–18]. Molychem, Mumbai India. Menthol was purchased from Suvidhi- nath lab Ahmedabad, India. The other chemicals and reagents used 2.2.5. Factorial design were of analytical grade. A32 full factorial design was used for optimization of formulated product. According to literature review, the concentration Ò 2.2. Methods of Eudragit RLPO (X1) and EC (X2) were selected as independent variables, Viscosity (Centi Poise: cP) as (Y1) and the time required 2.2.1. Spectrophotometric analysis to transport 50% of the drug (t50%,Y2) were selected as dependent The analysis for voriconazole was carried out using a UV/VIS variables. A mathematical model relates the response (degrada- (Ultraviolet/Visible) spectrophotometer (Model-1800, Shimadzu, tion) to composition [19]. It was included variables corresponding

Japan) at the absorbance maximum (kmax) of voriconazole which to each factor with qualitative levels corresponding to each excip- was 256 nm using a quartz cuvette cell and against an appropriate ient. Optimized formulation was carried by Design-Expert soft- blank. The solvents used were the same as the dissolution media ware. The optimized formulation was filled into the aerosol pH 7.4 phosphate buffer contain and 0.9% w/v Sodium Chloride container. The valve was placed and sealed in the container before (NaCl). The dilution was necessary in order to get UV absorbance filling the propellant manually by using the aerosol filling machine. readings below 1. The absorbance readings were converted to con- The density of propellant was 0.55 g/ml and weight of propellant centration (lg/ml) by using an appropriate calibration curve [11]. filled in the container was 17 g. The sprays were analyzed for

Please cite this article in press as: N.M. Mori et al., Fabrication and characterization of film-forming voriconazole transdermal spray for the treatment of fungal infection, Bulletin Facult Pharmacy Cairo Univ (2017), http://dx.doi.org/10.1016/j.bfopcu.2017.01.001 N.M. Mori et al. / Bulletin of Faculty of Pharmacy, Cairo University xxx (2017) xxx–xxx 3 pH, viscosity, the volume of solution delivered upon each actua- from the receptor compartment and voriconazole was estimated tion, spray angle, ex vivo, in vivo physical characteristics, and spectrophotometrically at 256 nm. An equal amount of fresh disso- in vitro voriconazole transport. Table 1. displayed the composition lution medium was replaced after each withdrawal. The time of the formulated batches (B1–B9) for transdermal spray [20]. Pres- required to transport 50% of the voriconazole (Y2) was found. UV surise spray filling carried using propellant as LPG (liquefied petro- spectrum of voriconazole was observed for voriconazole trans- leum gas) at Vimsons Aerosol Ltd. Gamdi, Anand, India [21]. ported through the membrane at different time period.

2.2.6. Regression analysis 2.7.1.5. Ex vivo studies through biological membrane. Ex vivo studies The responses were measured for each trial and then the quad- were carried out as per the guidelines compiled by the Committee ratic model was fitted by carrying out multiple regression analysis for the purpose of control and supervision of experiments on ani- (Eq. (1)) [22]. mals (CPCSEA, Ministry of Culture, and Government of India). All the study protocols were approved by the Animal Ethical Commit- ¼ þ þ þ þ 2 þ 2 þ e ð Þ tee of the department of pharmaceutical sciences. (Protocol num- Y b0 b1X1 b2X2 b12X1X2 b11X1 b22X2 1 ber: IAEC/DPS/SU/1311)). The abdominal hair of Wister male e where, is a practical error. Y was the dependent variable, while bo albino rats was shaved using an electric razor after sacrificing with was the intercept and b1,b2,b11,b22,b12 were regression coeffi- excess ether inhalation. The abdominal skin was surgically cients. Response surface plot and counter were selected by feasibil- removed and adhered subcutaneous fat was carefully cleaned. ity and grid searches. The model should be validated by ANOVA and The skin was thoroughly washed with water, use for further study preparation of checkpoint batch/ formulation. The practical [23]. For ex vivo release studies, skins were allowed to hydrate for response observed in the checkpoint batch/ formulation must be 1 h before being mounted on the Franz diffusion cell with the SC same as the response calculated SS, MS, F value and relative error facing the donor compartment because of uniform diffusion study. by the proposed model to show the validity. The receptor compartment was filled with PBS (pH 7.4) [24] and receptor phase was maintained at 37 ± 0.5 °C. 1 ml solution of 2.2.7. Evaluations the formulation was placed on the SC side in the donor compart- 2.2.7.1. Formulation related evaluations. ment. The amount of voriconazole release was determined spec- 2.2.7.1.1 Voriconazole content. 1 ml of prepared formulation was trophotometrically at 256 nm by removing 1 ml aliquot through mixed with PBS (Phosphate buffer Saline) (pH 7.4) for 4 h at a hypodermic syringe fitted with a 0.22 mm membrane filter, at 250 rpm for complete extraction. The samples were appropriately designated time intervals. The volume was replenished with the diluted after sonication, filtration of the stock solution, and ana- same volume of PBS (pH 7.4) to maintain sink conditions [25]. Skin lyzed spectrophotometrically at 256 nm in UV spectrophotometer. retention study was done at the end of the permeation experiments after 24 h, the skin surface in the donor compartment was 2.7.1.2 Viscosity. The viscosity of the solutions was measured at rinsed with ethanol to remove excess voriconazole from the sur- 25 ± 1 °C using Brookfield viscometer (Digital viscometer model face and analyzed by spectrophotometer [26]. DV-II+, Stoughton, MA, USA). The ULA S00 spindle was rotated at 4 rpm and sample size 16 ml in ULA cylinder. The torque reading was always greater than 10%. The average of three readings taken 2.7.1.6. Permeation data analysis. Flux was the amount of voricona- in one minute was noted as the viscosity of gels [15]. zole crossing a membrane per unit area into the circulating system per unit time, and for in vitro permeation, this ‘‘system” was the 2.7.1.3. pH of the solution. pH of the formulation was measured by receptor chamber, expressed in units of mass/area/time. If the per- digital pH meter (Elico, L1612, India). The buffer solutions of pH 4, meant was applied in a finite dose, then only calculate a steady 7 and 9 were prepared. pH meter was calibrated using these 4, 7 state flux by Eq. (2): [27] and 9 pH buffer solutions. Now rod of pH meter immersed in the Q solution whose pH was measured, then pH was directly noted from J ¼ ð2Þ ss ðA tÞ the meter. where Q was the quantity of compound traversing the membrane in 2.7.1.4. In vitro voriconazole transport. A nylon membrane with time t, and A was the area of exposed membrane in cm2. Unit of flux pore rating equal to 0.22 mm was mounted in a Franz diffusion is quantity/cm2/min. The permeability enhancement ratio was also cell. The surface area of membrane available for voriconazole calculated based on permeability coefficient (K ) by Eq. (3): transport was 2.8 cm2. One ml of the formulation put in donor p compartment and 30 ml of phosphate PBS (pH 7.4) was filled in J K ¼ ð3Þ receptor compartments. The temperature of media present in the p C receptor compartment was maintained at 37 ± 1 °C using a hot- water jacket (37 ± 1 °C) throughout the experiment. Aliquots of where, J = steady state flux, C = concentration of drug in donor 1 mL samples were withdrawn at different time intervals for 10 h compartment

Table 1 Composition of voriconazole spray by layout of 32 full factorial design.

% ingredient w/w B1 B2 B3 B4 B5 B6 B7 B8 B9 B10* B11* Voriconazole 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 Eudragit RLPO 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 and acetone blend (q. s.) 100 100 100 100 100 100 100 100 100 100 100

* In above batch B11 was checkpoint batch of the formulation with a penetration enhancer, B10 was without penetration enhancer. q. s.: quite sufﬁcient.

The penetration enhancing effect of penetration enhancer was (a) Preparation of the microorganisms calculated in terms of enhancement ratio (ER) and was calculated A clinical isolate of Candida albicans (C. albicans, MTCC Code 1637) by using the Eq. (4) [28] was used to infect the animals. A working culture of the candida was grown for 48 h at 30 °C on SDA. The cells were then collected, ¼ Kpwith penetration enhancer ð Þ ER 4 washed and re-suspended to a ﬁnal concentration of 1 107 Kpwithout penetration enhencer CFU/ml, then C. albicans was suspended in sterile saline [32].

2.7.1.7. Kinetics of model fitting. In vitro voriconazole transport data (b) Cutaneous infection of optimized batches was Q = f (t). Some analytical definitions of Each animal’s back was shaved with an electric clipper and an the Q (t) function were commonly used, were analyzed by zero- approximately 3.0 cm2 area was marked on each animal’s back. order, first-order, Higuchi, Hixson–Crowell, Korsemeyer–Peppas. The marked area was infected with 1 107 CFU/ml suspensions FORTRAN software, developed in-house, was used. The highest R2 by gently rubbing onto the skin with the help of a sterile, cotton- value and least value of SSR (sum of square of residuals) and Fish- tipped swab until no more visible fluid was observed [32]. Control er’s ratio (F) were used to select the most appropriate kinetic animals were infected in the same manner. However, they did not model [29–31]. receive any voriconazole spray formulation.

2.7.1.8. Antifungal studies. The Antifungal activity of optimized (c) Treatment of the infection batch, eutectic blend with solvent, and solvent alone were determine using Candida albicans (MTCC Code 1637) as representative A total of 10 animals were taken, 5 served as control (treated fungi, by cup and plate method with previously prepared Sabour- with placebo) and the remaining 5 were treated with voriconazole aud’s dextrose agar (SDA) dried plates. Freshly prepared culture containing spray formulation. The inoculation of animals was done 2 loops were streaked across the agar at the right angle from the under general anesthesia. An area of 3 cm , on the rats back, was, ditch to the edge of the plate. 1 ml of the formulation were placed clipped and made hair free. The area was marked with a marker in the well and the plate was incubated aerobically at 25 °C. After and abraded with sterile fine grit sandpaper. A cell suspension con- incubation for 24 h at 25 °C, the fungal growth was observed and taining Candida albicans was applied on skin during lag phase. The zone of inhibition was measured using a ruler [32,33]. animals were observed on the daily basis for signs of infection. The topical treatment was started after 7 days growth of fungus examination by visualization, appearance, and confirmation of fungal 2.7.1.9. Skin irritation studies. The studies were carried out as per hyphae on the skin of animals’ hair follicle grown in SDA media the guidelines compiled by the CPCSEA, Ministry of Culture, and for overnight. The clinical, as well as mycological parameters, were Government of India. All the study protocols were approved by also evaluated after 2 weeks from initiation of topical treatment. the Animal Ethical Committee of the department of pharmaceu- For histopathological examination, skin biopsy samples (Sanjivani tical sciences. (Protocol number: IAEC/DPS/SU/1311). These stud- path lab and PDU medical college, Rajkot, Gujarat, India) were ies were undertaken with an aim to evaluate any irritant obtained from one animal per group after completion of the treat- potential of the developed formulation in vivo on rat skin after ment period. The tissue was fixed in 10% neutral buffered formalin, its application. The hair on the dorsal side (3 cm 3 cm) of the embedded in paraffin, and processed for histopathological exami- male Wister rat weighing between 300 and 350 g was removed nation. The fungal elements were visualized using hematoxylin- with electric clipper in the direction of the tail to head without eosin (HE) and Periodic acid-Schiff (PAS) staining [34]. damaging the skin. Two groups with one rat each was made. One group rat was treated as control (1 g placebo spray) and the other was treated with optimized 1 g voriconazole spray for- 2.7.2. Container related evaluations mulation were applied uniformly on the dorsal region. The study The prepared formulation was filled into the metal container by was carried for three times priming in each a day for two days pressures filling using LPG as a propellant that was evaluated for consecutively. The animals were observed for the signs of itching following parameters. or any change in the skin such as erythema, papule, flakiness, and dryness [34]. 2.7.2.1. Volume of solution delivered upon each actuation. The volume of solution delivered upon each actuation was calculated using Eq. (5): 2.7.1.10. In vivo dermatol dynamics studies. In vivo studies were carried out as per the guidelines compiled by the CPCSEA, Ministry of ðWo WtÞ AL ¼ ð5Þ Culture, and Government of India. All the study protocols were Dn approved by the Animal Ethical Committee of the department of where A was the volume of solution delivered upon each actuation, pharmaceutical sciences. (Protocol number: IAEC/DPS/SU/1311). L the Wt was weight of formulation after actuation, Wo was the ini- The male Wister rats each weighing 250–280 g were divided into tial weight of the formulation before actuation, and Dn was the den- two group as shown in Table 2, housed in individual cages, sity of the formulation [15]. received food and water ad libitum [35].

2.7.2.2. Spray angle. The sprays were actuated in the horizontal direction onto a white paper mounted at a distance of 10 cm from Table 2 the nozzle. The radius of the circle, formed on the paper, was In vivo dermatol dynamic study groups. recorded in triplicate from different directions. Spray angle (h) Groups I II was calculated by Eq. (6): Treatment Control Test L Dose(mg/kg) 5 mg/ml (0.5% VRZ) Placebo (No drug) h ¼ tan 1 ð6Þ No of animals 5 5 r Route Duration of treatment Topical 14 days Topical 14 days where L was the distance of paper from the nozzle, and r was the VRZ: voriconazole. average radius of the circle [15].

2.7.2.3. Spray pattern test. The method of impingement of spray on 3.3. Determine solubility a piece of paper was used for the study. 10 mg Sudan red was dissolved in the formulation to facilitate visualization. Voriconazole is freely soluble in acetone, ethanol, and metha- nol, very slightly soluble in water (0.7 mg/ml). Among all most suitability, good characteristic was observed in EudragitÒ RLPO. 2.7.2.4. Container seal efficiency test/leak test. Physical or chemical An optimized grade of EudragitÒ RLPO was also shown good solu- container and closure system integrity test (e.g., bubble tests, pres- bility into the eutectic blend. Based on that EudragitÒ RLPO and EC sure/vacuum decay, trace gas permeation/leak tests, dye penetra- (7cps) were used for further study. tion tests, seal force test etc.) were used for the examination of the packaging of the container. Dye penetrant method is a tech- 3.4. Evaluation nique used to find cracks in metals and defects in welds. The test is simple, low cost, it leaves records, and the sensitivity can be as 3.4.1. Evaluation parameters related to formulation high as 10–6 bar/s. It was used a low viscosity fluid that exhibits 3.4.1.1. Viscosity (Y1). The viscosity of all Batches B1 to B9 was from a high rate of surface migration. This fluid was painted on one side 8.8 cps to 101.3 cps at different rpm 1, 2, 4, 6, 10, 12, and 20. Vis- of a suspected leak site and after a time, it was detected on the cosity was increased with increased in polymers concentration other side of the wall [8]. EudragitÒ RLPO and EC (Table 3).

3.4.1.2 In vitro voriconazole diffusion study. The voriconazole diffu- 2.7.2.5. Flammability test. In the pressurized gas system, the con- sion data obtained for formulations B1 to B9 were tabulated in tainer was filled with LPG gas so flammability test was done to Table 4. The total amount of voriconazole transported in batches evaluate flame projection. Flame projection (cm) was kept a gas B1 to B9 were varied from 87.01 to 61.15% and observed at differ- flame as medium and from 50 cm distance spray applies on paper ent time intervals for a period of 10 h. In all the cases, starting time to evaluate flame flashback (cm) of LPG gas [7]. interval voriconazole released was remained nearest to each other but it was changed with the time interval. In first voriconazole transport due to the diffusion of alcohol from present solution. In 2.7.3. Short-term stability study diffusion study of batch B1, B2, B3, B4, B5, B6, B7, B8, B9 were Short term stability of the optimized batch was carried out for shown transport of voriconazole 87.01%, 83.62%, 79.25%, 81.59%, one month at 25 ± 2 °C and RH 60 ± 50. The purpose of stability 77.13, 71.25%, 78.82%, 69.09%, and 63.15% respectively at 10 h. testing was provided evidence on how the quality of a formulation However, time require to 50% voriconazole transport various from varies with time under the influence of a variety of environmental 183, 221, 256, 275, 294, 381, 272, 365, and 463 min respectively. In factors such as pH, viscosity, volume of solution delivered upon all the cases voriconazole transport was reduced due to increased actuation, spray angle, and ex vivo physical characteristics of opti- level of concentration of EC, also due to that time interval change mized batch remained unchanged during the study [36]. for 50% voriconazole transported. In this medium level (0, 0) of both polymer gave constant sustain release effect. Here in all the batches rate of the polymer concentration of Eudragit and EC 3. Results and discussion increased their voriconazole transport through nylon membrane was reduced. 3.1 Spectrophotometric estimation of voriconazole

3.4.1.3. Ex vivo physical evaluations. Here ex vivo film formation Calibration curve of voriconazole in PBS (pH 7.4) was analyzed time, mucoadhesive, dermal flexibility, appearance, and water in range 200–400 nm and found to be a linear regression of absor- washability of the films were studies (Table 5). EudragitÒ RLPO bance vs. concentration, y = 0.0213x + 0.0117 with a correlation 2 was most suitable for film formation. Film formation rate was coefficient (R ) 0.9979. depended on evaporation of solvents, which leave the residue of film that gave effective voriconazole release concentration for the 3.2. Voriconazole-excipient compatibility studies predetermined time period of 600 min. B1, B2, and B4 had poor dermal adhesion flexibility of film, film formation in the shorter 3.2.1 FTIR study period of time 120, 165 and 212 due to a lower concentration of Entire characteristic peak broad due to –OH group 3194 cm1, Eudragit and EC. B7 to B9 had the high concentration of Eudragit 1496 cm1 of C–H stretching, 1616 cm1 of C–N stretching, and EC, the higher mucoadhesive film, which was difficult to wash 666 cm1 of N(2), N(4), coordination mode of 1H,1,2,4-triazole, from the skin by water. Batches B4 to B6 were showed moderate 1007 cm1of C–F stretching band appeared in the spectra of mucoadhesive property, appearance of the film indicated that in voriconazole as well as in physical mixture of voriconazole and low concentration 5% eudragit and 2.5% EC give transparent film excipients, which was confirmed voriconazole purity (spectra for while in higher concentration 10%, 15% and EC 5%, 7.5% respec- all formulations were shown in Fig. 1). There was no appearance tively appearance of the film was translucent to opaque. The opa- or disappearance of any characteristics peaks of voriconazole and que film was difficult to wash from the skin. Most suitability was in the physical mixture of voriconazole and polymer, which confound in Batch B5. In the study for assessment of dermal adhesion firms the absence of chemical interaction between voriconazole of the film, the palms of each volunteer were rotated for 10 min in and polymer. an anti-clockwise direction with occasional opening and closing of palm, after 8 min of actuation of spray. During the study (600 min), the nature of the film was carefully evaluated for any fracture, sep- 3.2.2. DSC analysis aration or removal. After 600 min, water washability of the film Voriconazole and mixture of voriconazole-excipients sample was checked on the nylon membrane. were exhibited an endothermic peak at 132.53–135.53 °C and

131.76–134.3 °C respectively. The melting point of voriconazole 3.4.1.4. Effect of the independent variable on Y1 viscosity (cps) and Y2 is 130 °C, which was indicated that sample purity of voriconazole time (minute) require to 50% voriconazole transport. The full model and almost no interaction between voriconazole and excipients. represents all signiﬁcant and less signiﬁcant data whereas reduce

Fig. 1. Overlay of FT-IR spectra (1) Drug: Voriconazole, (2) Polymer: EC, Eudragit RLPO, PEG 400, Camphor, Menthol (3) Physical mixture: voriconazole and excipient.

Table 3 Results design layout for factorial design responses.

Batch code Real values Dependent variables

y y y y Eudragit RLPO (X1) EC X2 t50 (min) (Y2)t50 (min) (Y2) B1 5 2.5 183 183 B2 5 5 221 221 B3 5 7.5 256 256 B4 10 2.5 275 275 B5 10 5 294 294 B6 10 7.5 381 381 B7 15 2.5 272 272 B8 15 5 365 365 B9 15 7.5 463 463 y X1 and X2 are the concentration of Eudragit RLPO and ethyl cellulose (%w/w) respectively, response Y1 is the viscosity (cps), t50% = Time require for 50% drug transport from 2.8 cm2 area of nylon membrane in Franz diffusion cell.

Table 4 In vitro diffusion studies.

Time (min) B1 B2 B3 B4 B5 B6 B7 B8 B9 0000000000 10 16.84 ± 0.1 13.32 ± 0.11 12.37 ± 0.2 11.42 ± 0.3 8.85 ± 0.4 14.96 ± 0.45 13.94 ± 0.51 11.07 ± 0.6 9.63 ± 0.35 20 18.16 ± 0.2 14.97 ± 0.3 14.97 ± 0.3 13.09 ± 0.4 9.72 ± 0.45 17.22 ± 1 14.52 ± 1.2 11.73 ± 1.1 10.54 ± 2.2 30 19.01 ± 0.11 22.34 ± 1.2 18.25 ± 2.1 16.59 ± 2.2 10.58 ± 0.8 17.66 ± 0.9 15.14 ± 0.9 13.10 ± 1.1 11.96 ± 1.2 60 24.24 ± 0.5 23.52 ± 1.2 21.66 ± 1.1 20.2 ± 1.3 13.22 ± 0.2 21.73 ± 1.1 20.65 ± 2 17.41 ± 3 14.10 ± 1 120 38.36 ± 1.1 35.40 ± 2 29.14 ± 3 28.04 ± 2.5 23.53 ± 1.5 24.25 ± 1.6 27.18 ± 1.7 25.86 ± 1.8 20.72 ± 1.8 180 49.59 ± 1.2 43 ± 1.3 37.93 ± 1.4 38.32 ± 1.5 32.45 ± 1.6 30.66 ± 1.7 36.26 ± 1.65 32.82 ± 1.25 25.17 ± 1.35 240 57 ± 2.1 53.29 ± 2.5 46.73 ± 2.6 47.94 ± 2.8 41.73 ± 3.1 36.29 ± 1.8 45.11 ± 1.9 38.49 ± 2.1 31.34 ± 3.2 300 63.93 ± 2.2 57.89 ± 2.11 52.36 ± 3.12 55.73 ± 3.18 50.93 ± 2.19 41.62 ± 2.65 54.22 ± 2.78 43.69 ± 2.28 36.63 ± 3.28 360 69.25 ± 2.3 63.12 ± 2.65 59.01 ± 2.98 61.10 ± 2.58 58.11 ± 2.39 47.23 ± 2.49 60.59 ± 2.95 49.48 ± 2.93 41.68 ± 3.35 420 71.69 ± 3.1 69.35 ± 4.29 66.18 ± 5.21 68.17 ± 4.19 65.31 ± 1.39 55.48 ± 3.29 66.77 ± 4.23 56.35 ± 3.31 47.88 ± 2.35 480 78.13 ± 2.8 74.47 ± 3.18 73.89 ± 2.81 74.19 ± 2.98 71.4 ± 3.18 61.62 ± 1.98 71.17 ± 3.09 61.27 ± 3.18 53.67 ± 1.98 540 83.68 ± 2.9 80.95 ± 4.35 78.27 ± 3.75 77.00 ± 4.38 75.81 ± 3.35 67.03 ± 3.38 76.35 ± 2.95 66.15 ± 3.45 58.57 ± 3.98 600 87.01 ± 3.2 83.62 ± 4.2 79.25 ± 5.2 81.59 ± 5.12 77.13 ± 6.22 71.24 ± 5.62 78.82 ± 3.62 69.09 ± 3.91 63.15 ± 3.19

Mean ± SD; n = 3.

¼ : þ : þ : þ : þ : 2 : 2 model gave only signiﬁcant (p < 0.05) parameters. Reduced model Y1 33 96 24 51X1 22 46X2 16 25X1X2 8 85X1 3 4X2 was given confound effect but able to screen out most signiﬁcant þ eðR2 ¼ 0:993Þð7Þ parameters that were affected the response (Eq. (7)).

Table 5 Ex vivo ﬁlm formation property.

Batch code** Ex vivo film formation time (s) Mucoadhesive flexibility of the film* Water washability* Appearance of the film** B1 120 + +++ + B2 165 + +++ + B3 212 ++ + ++ B4 186 + +++ + B5 247 ++ +++ ++ B6 304 +++ + +++ B7 229 ++ ++ ++ B8 360 +++ + ++ B9 435 +++ + +++

All the value Mean ± SD; n = 3, indicates + = Poor, ++ = Moderate, +++ = Good, * and **. ** Shiny and transparent (+) or shiny and translucent (++) or dull and opaque (+++), Measured for placebo batches.

Correlation coefﬁcient value for the full model and reduce the concentration of penetration enhancer increased voriconazole model was 0.988 and 0.993 respectively. permeation was increased.

Y1 (Viscosity) was influenced with increased concentration 3.4.1.6.3. Kinetics of model fitting. The Ex vivo voriconazole trans- polymers as a positive effect (Eq. (8)). port data of optimized batch were further analyzed. Model fitting was done using an in-house program developed by excel sheet program. The cumulative amount of voriconazole permeated per ¼ : þ : þ : þ : : 2 Y2 304 77 66 5X1 64 166X2 19 25X1X2 17 16X1 square centimeter of film through rat skin was plotted against þ : 2 þ eð 2 ¼ : ÞðÞ time. It was tested to zero order; first order, Higuchi, Hixson- 7 83X2 R 0 973 8 Crowell, Korsmeyer-Pappas, and Weibull models. The best-fit Correlation coefficient value for the full model and reduce model was selected on the basis of relatively high correlation coef- model was 0.993 and 0.973 respectively. Increased concentration ficient values, correlation coefficient value as indicated in Table 6. of Eudragit RLPO and EC increased time require to 50% voricona- Higher R2 value lower fisher’s ratio was observed (0.9981 and zole transport (Y2). 0.940 respectively). That indicated that the best suitable fit model for prepared formulation was Hixon-Crowell model. It should be 3.4.1.5. Check-point batch analysis. The checkpoint batch formula more than 0.8 regression coefficient data for correlation of data. was formulated to check the adequacy of the response. The exper- Hixon-Crowell physical principles one can expect that the rate of imental value and the predicted values as per Eqs. (6) and (7) were dissolution depends on the surface of solvent - the larger is area 31.9 and 33.09 for viscosity were times require to 50% voriconazole the faster is dissolution [37]. transport was 297 and 300.49 respectively. The % relative error for 3.4.1.6.4. Antifungal activity. The studies were carried for the best the checkpoint batch was in the range of 1.16 to 3.59 % respec- formulation and zone of inhibition observed at B11 (27 mm), the tively, which was less than 8%, hence statically acceptable. It was results were satisfactory as indicate in Table 7. concluded that the experimental values and predicted values show 3.4.1.6.5. Skin irritation study. After a 24 h exposure, the film good agreement between each other. formed was removed. The test sites were wiped with tap water to remove any remaining test article residue shown in Table 8. The itching was observed at 4 h in both formulations due to the 3.4.1.6. Evaluation parameters of the optimized batch. eutectic mixture that induces the soothing and cooling effect of 3.4.1.6.1. Voriconazole transport through biological membrane (Rat- formulation on the skin and after 24 h no observations of any visu- skin). Rat skin was considered as biological membrane and alized sign of edema, erythema. Papule and flakiness of test formu- voriconazole diffusion experiments were done in Franz diffusion lation observed the complete absence of reaction. cell. Voriconazole transport studies for B10 and B11 were carried 3.4.1.6.6. In vivo dermatol dynamics study. (a) Cutaneous infection: out. B11 with penetration enhancer had voriconazole transport Cutaneous candidiasis-related skin fungal infections are mostly was 1.68 fold compare to without B10 penetration enhancer eutec- reoccurred and are rarely cured; hence patients receive therapy tic mixture (camphor:menthol) significantly favors voriconazole over a long time. The cutaneous infection showed Fig. 2 (A) was transport. The B11 batch was enhanced penetration through the induced in Wister rat by applying Candida albicans and the com- skin through intracellular and transcellular pathways within a plete fungal infection was induced within 7 days. Wister rats were deep layer of skin. Camphor and menthol cause leaching of the observed daily for the signs of infection. The first signs of infection lipids present in the skin and thus causes pore formation. were observed on the 5th day (Fig. 2(B)) after inoculation in all the 3.4.1.6.2. Permeation data analysis. Flux calculation: The steady animals manifested in the form of redness and scaling. These alter- state flux was determined from the slope of the linear portion of ations became more evident around the 7th day (Fig. 3(C)) with a cumulative amount permeated versus time plot. Calculated val- marked hair loss and brittle hair. The lesions progressively ues were related to ex vivo permeation data analysis for Jss, Kp, increased in diameter in the control group (treated with placebo) and ER. After using the eutectic blend as a penetration enhancer, and were found to be covered with white-yellow crusts strongly the flux value for B10 and optimized batch (B11) were found adhered to the epidermis. Redness and itching at the site of infec- 39.1 and 65.8 (lg/cm2/h) respectively. This was indicated that per- tion in the treatment group were allayed in 2–3 days. meation flux increased in presence of permeation enhancer. The (b) After treatment: After induction of infection, the formulation enhancement ratio was 1.68. Effect of penetration enhancer was was applied to the infected site twice a day. It was also observed observed when formulation incorporated with penetration enhan- that there was the shedding of the infected skin scales and appear- cer in different concentrations. This result was indicated that the ance of light pink colored skin with very fine hair growth on the formulation containing 10% eutectic mixture camphor and men- 5th day Fig. 2 (D) after the initiation of treatment. Subsequently, thol (1:1) with 0.45% w/w PEG-400 were given better penetration uniform and healthy hair growths were observed at the site of of voriconazole through rat skin. These studies indicated that as infection after 10 days (Fig. 2 (E)) treatment. The complete healing

Table 6 Kinetics model ﬁtting data

Kinetics parameter Equation R2 value F ratio SSR

Zero order Q ¼ K0t 0.9827 12.64 101.15 First order lnð1 QÞ¼K1t 0.9951 5.55 44.47 Higuchi Q ¼ K2t1 0.9618 27.87 223.01 2 1 1 Hixon-Crowell 3 3 ¼ 0.9981 0.940 7.51 Q 0 Q t kt Korsmeyer Peppas Mt ¼ Ktn 0.9976 3.95 27.69 M1 Weibull ¼ ½ ð TÞ 0.9947 2.42 16.94 M M0 1 e t a b

Table 7 Antimicrobial activity voriconazole and formulation ingredients.

%W/W ingredient y B11 C D E Voriconazole 0.5 0.5 – – Eudragit RLPO 10.05 – – – EC 5.025 – – – PEG 400 0.45 – – – Eutectic blend 10 – 10 – Ethanol and acetone bland 100 100 100 100 Zone of inhibition (mm) 27 ± 1.7 16 ± 3 13 ± 1.5 7 ± 1 y Values are expressed in Mean ± S.D. n = 3, C = voriconazole with solvent, D = eutectic blend with solvent, E = ethanol, and acetone blend.

Table 8 Skin irritation test of voriconazole transdermal spray on rat skin.

No. of Rabbity Itching Edema Erythema Papule and ﬂakiness 4 h 12 h 24 h 4 h 12 h 24 h 4 h 12 h 24 h 4 h 12 h 24 h Rat-1 (Control) + Rat-2 (Test) + y Sign indicate no reaction, + indicate some reaction, +++ sign indicate more reaction.

Fig. 2. Cutaneous infection and treatment are before treatment (A) 1st day, (B) 5th day, (C) 7th day, and 14 days treatments healing (D) 5th day, (E) 10th Day, (F)14th day after treatment show complete healing of candidiasis. of the infected site was achieved in 14 days (Fig. 2 (F)) with com- skin sections were stained with PAS stain and HE stains plete hair growth. Skin biopsies were obtained from the test areas, histopathological examination of skin sections was performed to

Fig. 3. Histopathology of rat skin infected with Candida albicans, (A) Normal skin histogram, (B) control group (contain placebo) arrow show the presence of spore hyphae swelling of the cell, (C) after treatment with test formulation showing the complete absence of fungal elements. determine whether there was any skin tissue invasion by candida spaying formulation from the container (Fig. 4). The viscosity species or not. higher than 80 cps was showed poor spray ability. However, batch 3.4.1.6.7. Histopathology. The skin samples were examined to eval- B11 had 31.9 ± 0.2 cps that less than 80 cps hence good spray abil- uate the cutaneous irritation potential of optimized voriconazole ity of formulation from the container. There was no leaking of for- spray, after treatment of 21 days. Histopathological examination mulation from the container that evaluates using dye test. The and clearly observes voriconazole penetration through hair folli- amount of solution delivered upon each actuation and spray angle 0 cles. Photomicrographs of untreated rat skin were showed normal was wound 0.29 ml and 78.69 respectively. The two parameters skin. There was no any sign of disease with well-defined epidermal were correlated with polymer concentration and viscosity of the and dermal layers (Fig. 3(A)) However, in the animals receiving formulation. The acceptable spray angle was arbitrarily decided 0 placebo treatment fungal elements in the hair follicles were clearly as less than 85 for easy actuation of voriconazole solution from visible as a black color dot as hyphae of Candida albicans, which the container and covers the maximum surface area. Ex vivo film depicted in Fig 3(B). After treatment of 21 days by applying the formation was transparent in appearance and good. Flammability transdermal spray formulation, it was penetrated the deep layer test was required because filled propellant gas was LPG. Keeping of the skin, which indicated the complete absence of any fungal element in the skin biopsies of the animal as shown in Fig 3C.

3.4.2. Evaluation parameters related to container Container related parameters for optimized batch voriconazole transdermal spray were evaluated. The formulation with less than 80 cps showed acceptable spray pattern as per visualizes in Table 9

Table 9 Evaluation Parameters Related to container for optimizing formulation.

Test parameter Average results Amount of delivered upon each actuation 0.29 ± 0.043 ml Amount of voriconazole delivered upon each actuation 1.45 ± 0.3 mg Spray angle 78.690 ± 1.59 Leak test No leak after feeling. Appearance of the film Transparent Muco-adhesive and flexibility of film ++ Water washability +++ Ex vivo film formation time 247 ± 4.04 (s) Viscosity (cps) 31.9 ± 0.2 (cps)

Mean ± SD; n = 3; + indicate some value, ++ indicate moderate value, +++ sign indicate more value. Fig. 4. Spray pattern of the formulation. Delivery of solution from container.

flame 50 cm away from spray container and delivery of spray Patent (dated 19/11/2016). Mr. Kalpesh C. Ashara was also got observe. Flash back to spray around 12 ± 2 cm from the flame that prior permission (dated 20/12/2016) from deputy controller of indicated flammability of spray; this was due to the presence of patent and design, Mumbai, India regarding publication of the LPG gas as a propellant. Therefore, it was advised to patients that research work. container should be keep away from flame during spraying on skin. The amount of 0.29 ± 0.043 ml of spray was delivered upon each Ethical issues actuation. The amount was quite enough for film formation. All authors declare that they have no ethical issues. 3.4.3 Short-term stability studies ° Optimized formulation was stored for 6 months at 30 ± 3 C Conflict of interest away from light. At the end of six months, the formulation was subjected to various tests like viscosity, flux (Jss), the volume of All authors declare that they have no conflict of interest. solution delivered upon each actuation, pH, viscosity, solution delivered upon each actuation, spray angle, Ex vivo, in vivo physical Funding characteristics and in vitro voriconazole transport. The procedure employed for the study was identical to that described above. This research did not receive any specific grant from funding Which indicated in Table 10. agencies in the public, commercial, or not-for-profit sectors. How- ever, Mr. Nitin Merubhai Mori was received only tuition fees for his 4. Conclusions M. Pharm. study as a scholarship from united fusion study founda- tion (UFSF), Baroda, India. Mr. Nitin Merubhai Mori had also The present study was demonstrated that optimized batch was received GPAT scholarship from AICTE, India under post-graduate suitable for transdermal spray. The formulation held great poten- scholarship scheme (In pursuance of the policy framework for tial for treating the fungal infection in skin with improved patient the promotion of post-graduate education and research in techni- compliance and better ER. Thus on the basis of all the studies, it cal education as announced by the ministry of human resource could be concluded that the batch having polymer concentration development, AICTE). EudragitÒ 10.05% w/w, EC 5.02% w/w penetration enhancer eutectic mixture 10%w/w provide a transdermal spray formulation may Disclaimer be considered as a promising approach for the treatment of the fungal infection. Any oppositions, opinion, findings, and conclusions or recom- mendations expressed in this material are those of the Mr. Nitin Authors’ contributions’ Merubhai Mori, Mr. Kalpesh Chhotalal Ashara, and the owner of Patent 3386/MUM/2014A only. Mr. Nitin M. Mori carried out the study and analyzed data, Mr. Kalpesh C. Ashara wrote and edited the manuscript. M/s. Priya Acknowledgments Patel and Dr. Navin R. Sheth participated in the design of the study and research guide. Lalji V. Rathod was contributed in data collec- The authors thank all the individuals who took part in these tions. All authors read and approved the final manuscript. studies, the other healthcare providers, technicians, and adminis- trative staff who have enabled this work to be carried out. They Authors’ disclosures are thankful to Vimson Aerosols, Anand, India for allowing spray filling facility, Evonik Degussa Pvt. Ltd., India for providing the gift This was part of M. Pharm. research project of Mr. Nitin M. Mori samples of different types of eudragit polymers, teaching and non- submitted and presented at Saurashtra University, India, April- teaching staff of Saurashtra University, Rajkot, India, PDU Medical 2013. The part of this study is filled as Patent 3386/MUM/2014A College, Rajkot, India, B.T. Savani Hospital, Rajkot, India for their (filling date 24/10/2014, publication date 06/05/2016) at Indian help in research. Authors are grateful to Mr. BP Ghelani, Chief Phar- Patent office. The literature review of the study was supported macist, Saurashtra Kidney Research Institute, Rajkot, India for his by Saurashtra University, Rajkot, Gujarat, India. Mr. Kalpesh C. active motivation during research and M/s. Kruti Rathod, GTU, Ashara has already signed commercialization agreement with an India for providing English language copy editing service regarding Indian Partnership Firm (‘‘IIPRD”) for commercialization of the the article. Authors are thankful to Dr. Velichka Andonova, Chief Assistant Professor, Medical University – Plovdiv, Bulgaria for providing technical guidance in writing and editing manuscript. Table 10 Test parameters for short term Stability Study of Optimized Batch. 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