Iraqi J Pharm Sci, Vol.23(1) 2014 Mucoadhesive clotrimazole vaginal hydrogel 19 Preparation and in-Vitro Evaluation of Mucoadhesive Clotrimazole Vaginal Hydrogel Basma I. Abdul –Aziz * and Nawal A. Rajab **,1 ⃰ National Center for Drug Quality Control and Research, Ministry of Health, Baghdad, Iraq. ⃰⃰ ⃰ Department of Pharmaceutics, College of Pharmacy, University of Baghdad, Baghdad, Iraq. Abstract Clotrimazole (CLO) is an antimycotic imidazole derivative applied locally for the treatment of vaginal yeast infections. In this study, CLO was formulated as vaginal mucoadhesive hydrogel, using different types of mucoadhesive polymers to ensure prolonged contact between active ingredient and vaginal mucosa. Physicochemical properties of the prepared formulas were evaluated as a visual inspection, pH, swelling index, spreadability, and mucoadhesive characteristics, in addition to an in-vitro drug release. The influence of type and concentration of polymers as CMC-Na (1.5, 2.5, and 3.5%w/w), carbopol 940( 0.25, 0.5, and 1 %w/w) and poloxamer 407 (15, 25, 30%w/w) on CLO release from the prepared gels were also investigated. All the different concentrations of each polymer gave a percent of drug release profile inversely proportional with the polymer concentration. The lowest concentration showed faster release within the first half an hour of 99± 2% of drug release , while CMC-Na at 3.5% (F2) demonstrated the least release of 40 % after three hours. On the other hand, poloxamer 407 of 25% gel (F7) produced the highest release of 98%, while carbopol 1% (F5) and CMC-Na (F1) produced 85% and 80% ,respectively. All the prepared formulas were following Higuchi kinetic model. Based on overall result, CLO can be formulated as mucoadhesive vaginal hydrogel using 25%poloxamer as the best prepared formula. Key words: Hydrogel, Clotrimazole, Mucoadhesive. كهىتريًازول بشكم هالو يائي باستخذاو قىاعذ نًادة تحضير و تقييى خارج انجسى انحي هبهيًيخاطيت االنتصاق نالستخذاو ان 1،** نىال عياش رجبو *عًاد عبذ انعسيسبسًت * .انًشكض انٕطُي نهشقابت ٔانبحٕد انذٔائيت ,انعشاق , بغذاد ** . قسى انصيذالَياث, كهيت انصيذنت , صايعت بغذاد, انعشاق , بغذاد انخالصت فطشياث انًٓبم . في ْزِ انذساست حى يعخبش عقاس انكهٕحشيًاصٔل ٔاحذا يٍ يشخقاث االيًيذاصٔل يسخخذو يٕضعيا في عالس ححضيش انكهٕحشيًاصٔل بصيغت ْالو يائي نّ انقابهيتعهٗ االنخصاق بانضذساٌ انًخاطيت . االَخفاخ,قابهيت االَخشاس َسبت ،انكيًيائيت يزم)انٕصف انخاسصي ,االط انٓيذسٔصيُي-حى حقيى انصيغ يٍ حيذ انخٕاص انفيضيأيت االنخصاق(باالضافت انٗ حقيى ححشس انذٔاء خاسس انضسى .حًج دساست انعٕايم انًؤرشة في ححشسانذٔاء كُٕع ٔحشكيض انقٕاعذ ٔقابهيت - 25-15) ٔ 040 (% يٍ انكاسبابٕل 1 - 25,0 -5,0(% يٍ انكاسبٕكسي يزيم سيهيهٕصصٕديٕو ٔ)5,3 - 5,2 -5,1انضالحيُيت ) .404 (% يٍ انبٕنٕكساييش30 في صًيع انصيغ انًحضشة باخخالف يع صيادة حشكيض انبٕنيًشعكسيا حخُاسب بصٕسة عايتكهٕحشيًاصٔل ت ححشس كًيٌ ا ثٔقذ ظٓش % خالل َصف انساعت االٔنٗ نهضيغ رٔاث انخشاكيض انقهيهت نكافت انبٕنيًشاث 2±00حشاكيضْا, حيذ اٌ ححشس انذٔاء كاٌ بًعذل % خالل رالد ساعاث . يٍ انُاحيت االخشٖ , اٌ 40بًقذاس %5,3م سهيهٕص صٕديٕو ,بيًُا ابطئ ححشس في حشكيبت كاسبٕكسي يزي 040انكاسبابٕل ى%( بعذ يشٔس ساعخيٍ ٔيٍ ر09اظٓشث اعهٗ ححشسنهذٔاء) %(25) 404انبٕنٕكساييش انخشكيبت انحأيت عهٗ %(.اٌ ححشسانذٔاءيٍ انٓالو انًائي نكافت انصيغ انًحضشة حخضع نقإٌَ 90صٕديٕو) يزيم سيهيهٕص انكاسبٕكسي ٔاخيشا %(95) انعانى ْيضٕشي . % انبٕنٕكساييش25بُاءا عهٗ انُخائش انًسخحصهت اربج باٌ دٔاء انكهٕحشيًاصٔل يًكٍ حشكيهّ بصيغت ْالو يائي يٓبهي باسخخذاو 404. هالو يائي ، كهىتريًازول ، االنتصاق انًخاطي :انكهًاث انًفتاحيت Introduction Vaginal delivery as a route of drug administration is currently of a great interest to scientists and pharmaceutical industry (1) . Many different approaches have been tested to develop vaginal drug delivery system (DDS) that can meet clinical and patient requirements (2) . Considerable attention has been focused on the development of vagina l (DDS) that providing a gradual release of drug over time. One interesting group of auxiliary agent is the mucoadhesive polymers, which are the basis of newly designed system. The most widely used mucoadhesive vaginal (DDS) is (hydrogel) (3, 4) .Hydrogel is one of the 1 Corresponding author E-mail:nawalayash@yahoo.com Received:24 /11/2013 Accepted:4 /3/2014 Iraqi J Pharm Sci, Vol.23(1) 2014 Mucoadhesive clotrimazole vaginal hydrogel 20 upcoming classes of polymer-based controlled release (DDS) due to several factors among them are their hydrophilicity, biocompatibility mucoadhesive characteristics (5) . Also gels are easy to manufacture, comfortable, and have ability to spread onto the surface of mucosa to achieve an intimate contact with vaginal mucosa. Moreover, because of their high water content and their rheological properties, they provide hydration and lubrication action (6) . Clotrimazole is an imidazole derivative with a broad spectrum of antimycotic activity, the drug is a weak base described as a white to pale yellow crystalline powder, practically insoluble in water, freely soluble in chloroform and methanol (7, 8) .The objective of this study is to formulate 1% Clotrimazole vaginal mucoadhesive hydrogel using three different types of polymers with different concentration of each.The physicochemical characteristics of gel and mucoadhesive property of selected formulas were also evaluated. Materials and Method Materials Clotrimazole and methyl paraben were supplied by (Samara Drug Industry), Sodium carboxy-methylcellulose (BDH chemicals Ltd, Pool, England), Cabopol 940(Hi Media lab.,Ltd, Mumdai, India), Poloxamer 407. (Sigma, Germany ), monobasic potassium phosphate (Fluka , Switzerland), tween 80 (Merk-Schuncherdt, Germany). Method Preparation of Clotrimazole vaginal gels: Nine formulas of CLO hydrogel were prepared by cold mechanical method (9) as shown in (table 1). Pre weighed amount of polymers was dispersed in sufficient quantity of distilled water (containing 1% tween 80), the dispersion was homogenized using magnetic stirrer for 1 hour and then left to for 24 hours for complete swelling and equilibration of polymer. Weighted amount of CLO and methyl paraben were added with continuous stirring. In case of carbopol 940 containing gel, [ specific amount of triethanolamine (TEA) drop by drop was added with continuous mixing] and the final weight was completed to 100g with the aqueous solution. The formulas were placed in refrigerator to complete the formation of hydrogel (10) . Table (1) Composition of clotrimazole vaginal hydrogels formulas (%w/w). * Sodium carboxymethyl cellulose ** Triethanol-amine Evaluation of the prepared hydrogel formulas Visual examination The examination considered a series of visual characteristics (consistency, homogeneity) (11) . pH determination The pH of the prepared gel was measured using pH – meter by putting the tip of the electrode into the gel and after 2 minutes the result was recorded (12). Swelling study One gram sample from each formula was soaked into 5ml of phosphate buffer pH 4.0 and left for a specific time, and then the excess buffer was removed and reweights the samples again. This test done for time intervals after one and three hours. The following formula will be used to calculate the swelling ratio. Swelling ratio= Ws-W0 / W0 × 100 Where Ws is the weight of the swollen hydrogel at time t and Wº is the initial weight (13) . Distilled Water Methyl- paraben Tween 80 TEA* * Poloxamer 407 Carbopol 940 CMC-Na* Clotrimazol e Formula No. Up to 100 0.4 1 --- --- --- 3.5 1 F1 Up to 100 0.4 1 --- --- --- 2.5 1 F2 Up to 100 0.4 1 --- --- --- 1.5 1 F3 Up to 100 0.4 1 2.0 --- 1.0 --- 1 F4 Up to 100 0.4 1 1.0 --- 0.5 --- 1 F5 Up to 100 0.4 1 0.5 --- 0.25 --- 1 F6 Up to 100 0.4 --- --- 30 --- --- 1 F7 Up to 100 0.4 --- --- 25 -- -- 1 F8 Up to 100 0.4 --- --- 15 --- -- 1 F9 Iraqi J Pharm Sci, Vol.23(1) 2014 Mucoadhesive clotrimazole vaginal hydrogel 21 Spreadability measurement Spreadability was measured on the basis of“Slip” and “Drag” characteristics of gels. 1.0gm of gel was placed on the center of ground slid plate and spread over an area of 1 cm diameter. The gel was then sandwiched between two slides by the application of 200 gm weight. The spread diameter was recorded after 5 minutes, and measured in cm. This measured area was taken as comparative values for spreadability, (diameter of the spread circle – initial diameter) (12) . Estimation of drug content by HPLC analysis The content of CLO in prepared formulas was determined using HPLC analysis method stated in the United State Pharmacopeia (USP) for CLO assay (8) . HPLC from Knauer ,Germany was utilized. A specified quantity of the gel equivalent to 50 mg was stirred using ultrasound with about 30 ml of the diluents [a mixture of 25% phosphate buffer (4.35mg/ml of K2HPO4) and 75% methanol] and then the volume was made up to 50 ml with same diluents. The above solution was further diluted to obtain solution having concentration of 0.2mg/ml. The chromatographic system was carried out using the following specifications:- -Column: 4.6-mm x25 –cm; 5µm packing L1. -Mobile phase: Acetonitrile and Buffer (3: 1), filtered and degassed. (Buffer: - 4.35mg/ml of dibasic potassium phosphate) -Detector: UV 254nm. -Flow rate: 1.5 ml/min. -injection size: 20 µl. Mucoadhesive strength measurement The mucoadhesive strength of selected formula was determined by measuring the weight required to detach the gel from the sheep vaginal mucosal tissue by using a modified chemical balance. A section of vaginal mucosa was cut from the sheep’s vaginal cavity and instantly fixed with mucosal side out onto each glass vial using a rubber band. The vials with vaginal mucosa were stored at 37°C for five minutes; the vial was attached to the balance instead of one of the pans by a height-adjustable hook. Next, glass plate was placed under the mucosal tissue .The gel was placed on the glass plate; then, the height of the vial was adjusted so that the gel could touch the mucosal tissue which was allowed to adhere for three minutes to the gel (preload time) (14) . On the other side of the balance, a plastic cup was placed to collect water, and balanced with the other side. Water was added drop by drop to the plastic cup until the weight of water in the cup detached the glass slide from the mucosal tissue. The detachment stress (dyne/cm 2 ), was determined from the minimal weights that detached two surfaces from each other, was calculated using the following equation F= 980 m/π r 2 Where F is the detachment force (dyne/cm 2 ) per unit area of mucosa (cm 2 ) (π r 2 in which r is the radius of the vial), (r =1.2 cm) by the balance weight m (g) and 980 is acceleration due to gravity (cm/sec 2 ). In-vitro dissolution test The in vitro release of CLO from all hydrogel formulas was performed by using dissolution apparatus type 2 (paddle type).A weighing quantity of a gel ( 10 gm that contain 100 mg CLO ) was uniformly spread on petri dish 4.5cm in diameter, and this was immersed in dissolution jar filled with 900ml dissolution media ( phosphate buffer pH 4.0 B.P. containing 2% tween 80 ) at 37˚C (15) . The paddle was placed about 2cm above the petri dish and rotated at speed of 50 rpm. Samples of 5ml were withdrawn at intervals of 30, 60, 120, and 180 minutes and were replaced with equal volume of the fresh buffer solution each time to maintain constant volume. Samples were analyzed for CLO by HPLC (16). Variable affecting release profile Effect of different polymer concentration on the release profile of clotrimazole The effect of different concentrations of the used polymer on the release profile of the drug was studied; all formulas were subjected to this study. Effect of different types of polymers on the release profile of clotrimazole One formula was selected from each polymer CMC-Na, carbapol 940 and poloxamer 407 ( the highest release profile ). Comparion between these formulas were achieved to show the effect of the polymer on the release. Release kinetics To analyze the mechanism and rate of drug release from prepared gel, the dissolution data obtained were fitted to kinetic equations including Zero order, First order, Higuchi and Korsmeyer- peppas. R 2 value was used to show the best fit model to be selected (17). Iraqi J Pharm Sci, Vol.23(1) 2014 Mucoadhesive clotrimazole vaginal hydrogel 22 Results and Discussion Macroscopic feature: Visual inspection of prepared gel indicated the homogeneity of all formulas, no phase separation, with semi-rigid texture and white to off white smooth gel. This result is similar to that defined in references (7, 18). pH The pH of all formulas were almost neutral as show in table (2 ). Table (2) pH of prepared formulas Swelling index Swelling of the polymer depends on the concentration of the polymer, degree of cross- linking, ionic strength and presence of water (18). Increasing in cross- linker concentration shows a direct negative effect due to tighter structure (19) . Tablet (3) shows that F1 and F2 started swelling rapidly and continued for three hours due to high amount of hydroxyl group in each cellulose unit which contributing in presences of hydrogen bound (20) . F4 shows less swelling index at the beginning but polymer when fully hydrated swelled in phosphate buffer and surface disintegration of the gel occurred at the end of three hours (21). The swelling ratios of ionic hydrogels were often an order of magnitude higher than those of neutral gels because of the presence of intermolecular interactions. (F7-F9) being contain neutral gel have no swelling appearance may be due to full hydration of the polymer (20, 22). Table (3) Swelling ratio of prepared formulas Swelling ratio w/w after 3 hours ± S.D. Swelling ratio w/w after 1 hour Formula no. 80% ± 1.8% 50% ± 1% F1 150% ± 0. 23% 45% ± 1. 2% F2 50% ± 0.5% 0% F3 30% ± 0.65% 8% ± 0.3% F4 -- -- F5 -- -- F6 -- -- F7 -- -- F8 -- -- F9 -- : swelling ratio is negligible because the polymer used is water soluble (20) . Spreadability measurement All prepared gels using different concentrations of different polymers were spreadable on the tissue surface as show in table (4). Spreadibilty of prepared gels was decreased as the polymer concentration increased as in formula (F1,F5,F7). It should be mentioned that the addition of polysorbate 80 improved the physical characteristics including spreadability, consistency and skin feel duo to an increase in the diameter of the spread circle of the gel (23) . Table (4) Spreadability values of prepared gels. Formula No. F1 F2 F3 F4 F5 F6 F7 F8 F9 Spread-ability cm ± S.D 1.5 ±0.1 2 ±0.08 3 ±0.14 1.5 ±0.08 2.2 ±0.1 2.8 ±0.12 0.4 ±0.02 0.6 ± 0.04 3 ±0.15 Mucoadhesive strength measurement The mucoadhesive strength of the prepared gels was affected by polymer type and it was found in the following order: poloxamer 407 >carbopol 934 > CMC Na (F8, F4, and F2) respectively as illustrated in table (5). This is due to the fact that mucoadhesive strength depends on the polymer structure, molecular weight, and other physicochemical properties which are varied according to the polymer type (24, 25) . Table (5) Result of force required for detachment Formula No. F2 F4 F8 Detachment stress (dyne/cm 2 ) ±S.D. 4009.6 ± 2.7% 5310.1 ± 3.5% 6783.8 ± 4.3% pH Formula no. 6.4 ± 0.10 F1 6.4 ± 0.11 F2 6.2 ± 0.08 F3 7.3 ± 0.06 F4 7.4 ± 0.05 F5 7.1 ± 0.08 F6 7.3 ± 0.07 F7 7.0 ± 0.10 F8 7.1 ± 0.085 F9 Iraqi J Pharm Sci, Vol.23(1) 2014 Mucoadhesive clotrimazole vaginal hydrogel 23 Content uniformity A validated HPLC method was used for determination of CLO content in the gels. Table 6 indicated that the results obtained were within the limit of USP monograph (90-110) % of the theoretical content of the prepared gel (8) . Table (6) Drug content uniformity Formula No. F1 F2 F3 F4 F5 F6 F7 F8 F9 % of original amount od drug ± S.D. 103 ± 0.20 104 ± 0.30 99 ± 0.80 95.2 ± 0.23 97.5 ± 1.00 101 ± 1.20 98.8 ± 0.43 97.6 ± 0.66 99 ± 0.50 Effect of polymer concentration on the in vitro release It was found in formulas F1, F2 and F3 that the amount of drug release from CMC-Na hydrogel was decreased with increasing polymer concentration as in figure (1). This result could be attributed to an increase in the density which leads to increase in diffusional path way of drug from more sticking cellulose matrix (26). Figure (2) illustrates the difference in percent of drug release which was attributed to the mesh size of the network. The mesh size depends on the grade of crosslinking and the chemical structure of the monomers, so the diffusivity of the incorporated drug was effected (27). The hydrogel with low degree of crosslinking (F6) was fast released. On the other hand, gradual release of drug occurred in F4 due to the tighter network (28) . Figure 1: Effect of CMC-Na concentration on the release of clotrimazole from three different formulas. Figure 2: Effect of different carbopol 940 concentration on the release of clotrimazole from three different formulas. Figure (3) showed that the increase in polyxamer 407 concentration led to decrease the release rate (F7- F9). It is hypothesized that the drug is released by diffusion through the extra micellar water channels of the hydrogels matrix, and higher concentration of polyxamer 407 causes smaller size of water channels (29). Figure 3: Effect of different poloxamer 407 concentration on the release of clotrimazole from three different formulas. Effect of polymer types on in vitro release Three formulas of different types of polymers which show best release profile were chosen for this study and illustrated in figure (4). Poloxamer 407 (F8) performed higher release with control rate when compared with other formulas. This due to the mechanism of drug delivery via poloxamer which occurred by diffusion and dissolution of the gel at administered site. Poloxamer presented as complete dissolution in three hours, and this feature can be used to prepare pharmaceutical formulation. The amount of CLO released, was found in the following order: poloxamer ˃carbopol and CMC, this may be due to higher viscosity which was responsible for hindering drug release from gel matrix (30) . Figure 4: Effect of different polymers on the clotrimazole release from different formulas. Iraqi J Pharm Sci, Vol.23(1) 2014 Mucoadhesive clotrimazole vaginal hydrogel 24 Figure 4: Effect of different polymers on the clotrimazole release from different formulas. Mathematical modelling of drug release profile According to the results obtained and shown in table (7), the release of CLO from polymer network forming hydrogel for formulas F1-F3, F4, F6 and F9 obeys higuchi release kinetic as there (R 2 )values gave high results, while F4, F7, and F8 follow zero-order kinetics. The mechanism of release depending on the composition of hydrogel, geometry, preparation technique and environmental conditions during drug release (17). Table (7) Mathematical finding of clotrimazole release from repared gels. Formula No. R 2 Krossmeyr-peppas n Zero order First order Higuchi F1 0.979 0.861 0.997 0.22 F2 0.982 0.845 0.990 0.221 F3 0.730 0.733 0.961 F4 0.999 0.861 0.973 0.1 F5 0.831 0.754 0.992 F6 0.730 0.733 0.962 F7 0.997 0.891 0.986 0.216 F8 0.999 0.876 0.974 0.167 F9 0.734 0.734 0.970 Conclusion The present study demonstrates that the CLO confirm a promising vaginal mucoadhesive hydrogel since it provided highest release profile with maximum adhesion property especially for poloxamer 407 of 25% (F8), in addition to the acceptable physiochemical parameter such as pH, swelling index, drug content, and spreadability. References 1. Hussain, A. & Ahsan, F. "The vagina as a route for systemic drug delivery". Journal of Controlled Release. 2005; 103(2): 301-313. 2. Friend, D."Advances in vaginal drug delivery". Drug Delivery and Translational Research .2011; 1(3): 183-184. 3. Vermani, K. & Garg, S. "The scope and potential of vaginal drug delivery". 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