Formulation and Characterization of Ciclopirox Olamine Mucoadhesive Effervescent Tablets for Vaginal Delivery

Transcription

1 190 Int J Pharm Sci Nanotech Vol 5; Issue 4 January March 013 International Journal of Pharmaceutical Sciences and Nanotechnology Volume 5 Issue 4 January March 013 Research Paper MS ID: IJPSN-8--1-RACHH Formulation and Characterization of Ciclopirox Olamine Mucoadhesive Effervescent Tablets for Vaginal Delivery M.R. Rachh 1 *, B.S. Barot, P.B. Parejiya, P.K. Shelat and S.S. Deshpande 1 Shankarsinh Vaghela Bapu Institute of Pharmacy, Gandhinagar, Gujarat, India, and K. B. Institute of Pharmaceutical Education and Research, Gandhinagar, Gujarat, India. Received August, 01; accepted September 6, 01 ABSTRACT The study was aimed to design and evaluate a new effervescent mucoadhesive vaginal drug delivery system for ciclopirox olamine (CPO), a broad-spectrum antifungal, antibacterial, and anti-inflammatory agent for effective treatment in vaginal candidiosis. Various polymers like Polyox 303, Carbopol 974P, HPMC K4M, Sodium CMC, HPC, Sodium Alginate, Xanthan Gum, Polycarbophil and Chitosan were used in the formulation of CPO effervescent mucoadhesive vaginal tablet (EMVT) employing direct compression as a method of preparation. The effervescent mixture (citric acid and sodium bicarbonate) was incorporated into the formulations to aid quick wetting and mucoadhesion of tablet followed by drug release modulation. The amount of polymer blends and effervescent mixture was optimized using 3 full factorial design. The swelling, mucoadhesive strength and in-vitro release were studied as dependent responses. The ex-vivo mucoadhesion was determined by modified mucoadhesion assembly. The ex-vivo residence test was carried out by modified USP dissolution test apparatus. In vitro anti-fungal activity of the CPO EMVT was determined in comparison to Candid -V3 tablet. A good sustained effect and a moderate mucoadhesion (0.31N to 0.67N) were obtained with tablets containing HPMC K4M: Polyox 303 (1:1.4) and effervescent mixture (1:3). Ex-vivo mucoadhesion time of all the formulations was in the range of 8 to 4 h. The effervescent CPO tablet showed significantly higher in-vitro antifungal activity as compare to Candid -V3 tablet (p< 0.05). The results of short term stability revealed stable characteristics of optimized formulation. The proposed formulation may provide a potential antifungal activity against Candida albicans. KEYWORDS: Mucoadhesive polymers; vaginal candidiosis; effervescent; C.albicans. Introduction Vaginal candidiasis is a common condition that affects up to 75% of female population, among which 40 50% of patients have frequent occurrence (Ferrer, 000). The prime cause of the infection is Candida albicans. The common treatment is topical therapies which include nystatin, immidazole and hydroxypyridone. ciclopirox olamine (CPO) is a hydroxypyridone analog and antifungal agent developed for the treatment of human mycotic infections. It has proven efficacy against the organisms commonly implicated in vaginal candidiasis; Candida albicans (Carrillo-Munoz et al., 00). Current available topical market vaginal antifungal formulations are aimed towards local delivery (creams, ointments, gels and vaginal tablets). They have some limitations like short residence time at the site of administration, leakage and messiness causing inconvenience to users, leading to poor patient compliance and loss of therapeutic efficacy (Garg et al., 005). Mucoadhesive systems can avoid these problems. Mucoadhesive polymers have capability to adhere to mucous epithelial surface and act as most attractive and promising candidates to be used as drug delivery systems for topical application.(woodley, 001; Sharma et al., 006). For complete and better treatment of vaginal candidiasis, longer residence time and consistent release of therapeutic agent should be targeted. In the present work, an attempt is made to design EMVT of CPO, which targets longer residence time up to 1 hrs due to mucoadhesion and consistent drug release. Sodium bicarbonate and citric acid, added into formulations to facilitate quick wetting even in low moisture content of vagina under normal physiological conditions (Karasulu et al., 00) further this action leads to improve mucoadhesion followed by improved disintegration and modulation of drug release from matrix of CPO EMVT over the desired time period. The study include preliminary screening of different proportions of polymers and effervescent in formulation for mucoadhesion and swelling properties of the tablets to yield consistence drug release. During the study mucoadhesive polymers like Polyox 303, Carbopol 974P, HPMC K4M, Na CMC, HPC, Sodium Alginate, Xanthan Gum, Polycarbophil and Chitosan, were chosen to develop a CPO EMVT. The performances of these 190

2 Rachh et al : Formulation and Characterization of Ciclopirox Olamine Mucoadhesive Effervescent Tablets for Vaginal Delivery 1903 mucoadhesive polymers were evaluated by two parameters, the swelling behaviour and the mucoadhesive strength. Anti-fungal activity of the CPO EMVT was determined in comparison to Candid -V3 tablet. Materials and Methods Chemicals and Drugs Ciclopirox olamine was kindly gifted by Cipla Ltd (Mumbai, India). Carbopol 974P was kindly gifted by Corel Pharmaceutical Ltd. (Ahmedabad, India). HPMC K4M, HPC and Sodium CMC were received as gift samples from Torrent Pharmaceuticals Ltd. (Ahmedabad, India). Chitosan, Xanthan Gum and Sodium alginate were obtained as gift sample from Astron Research (Ahmedabad, India). Polycarbophil was gifted from Lubrizol Chemicals Noveon, (Mumbai, India). MCC, Sodium Benzoate, Citric acid and Sodium Bicarbonate were purchased from S.D. Fine Chemicals Ltd. (Mumbai, India). All other ingredients were of laboratory grades. Candid -V3 tablet was purchased from local market. Preliminary Screening of CPO EMVT Different mucoadhesive polymers were screened for formulation of CPO EMVT. The formulations were prepared by direct compression technique. The required quantity of polymers, CPO, effervescent (citric acid and sodium bicarbonate) and the other formulation ingredients were compressed using rotary station tablet machine equipped with round punch of 8 mm (Hardik Engineering, Ahmedabad, India). Swelling study The swelling behaviour of EMVT was described as the water absorbing capacity. Drug-free tablets were weighed individually (W0) and placed separately in % agar gel plates and incubated at 37 ± 1 C. At regular time intervals up to 4 hours, the tablets were removed from the petri dish and wiped with filter paper carefully. The swollen tablet was then reweighed (Wt) and the % swelling were calculated using the following formula: (Kast et al., 00; Karasulu et al., 004) % Swelling = {(Wt W0)/ W0} 100..(1) Where, Wt is the weight of the tablet at time t and W0 is the initial weight of tablet. The swelling was calculated and then plotted as a function of time. The slope of the linear plots was taken as the swelling rate. Optimization using 3 full factorial design A statistical model incorporating interactive and polynomial term was used to evaluate the response: Y = b0 + b1x1+bx + b1x1x + b11x 1 + bx Where, Y is the dependent variables, b0 is the arithmetic mean response of the nine runs, and b1 is the estimated coefficient for the factor X1. The main effects (X1 and X) represent the average result of changing one factor at a time from its low to high value. The interaction terms (X1X) show how the response changes when two factors are simultaneously changed. The polynomial terms (X1 and X ) are included to investigate non-linearity. A 3 full factorial design was adapted to optimize the variables. In this design factors were evaluated, each at 3 levels, and experimental trials were performed at all 9 possible combinations. Polymer ratio (X1) and amount of effervescent mixture (X) were selected as independent variables. Mucoadhesive strength (Y1), % swelling (Y) and t90 (Y3) were selected as dependent variables. The preparation and evaluation method for tablets and amount of CPO was kept constant for all the trials. Criteria for optimized batch Criteria for optimized batch for response are: Y1 (Mucoadhesive Strength): 0.45 to 0.60 N Y (% Swelling): 35 to 49% Y3 (T90): 9 to 10 hrs Physical characterization of CPO EMVT The uniformity of weights of all tablets was determined by using sartorious balance (Model CP- 4 S). The hardness of the tablets was determined by diametral compression using a dial type hardness tester (Model No 1101, Shivani Scientific India). Tablet thickness was measured using vernier calipers (Lachman et al., 1999). Ex-vivo mucoadhesion study Several types of mucosa including rat intestine, pig oral, bovine sublingual, cow vaginal mucosa (Gurny et al., 1984; Gursoy et al., 1989) have been used as model biological tissues for the evaluation of mucoadhesion. A modified self developed force detachment devise was used to measure the minimum detachment force (Figure 1). A piece of rat intestine (.0 cm x 1.0 cm) removed from newly sacrificed rat was adhered to a piece of glass, which was fixed on a plank and the plank was assembled with a little crown block. After hydrating the rat intestine with distilled water, the tablet was brought into contact with the rat intestine by applying little force for minute. After the initial contact, the tablet was encircled by a thread which fastened a light plastic beaker through the crown block. Next, water was dropped into the beaker at a speed of 3.0 ml/min using peristaltic pump until the tablet and rat intestine were pulled apart by the gravity of water. The beaker containing water was weighed and the minimum detachment force was calculated accordingly. The experiments were performed in triplicate and average values with standard deviation (SD) were reported. The Protocol (KBIPER/01/31) was approved by Institutional Animal Ethics Committee (K.B Institute of Pharmaceutical Education and Research) under CPCSEA before carrying out this experiment.

3 1904 Int J Pharm Sci Nanotech Vol 5; Issue 4 January March 013 Fig. 1 A modified self developed force detachment devise. Ex-vivo mucoadhesion time The ex-vivo residence time was studied using a locally modified USP paddle apparatus (Dissolution test apparatus type-i). Simulated vaginal fluid (ph 4.0) was used as dissolution medium. The medium temperature was maintained at 37±0.5 0 C. A segment of rat intestine,.5 cm long, was glued to the surface of a glass slab, vertically attached to the paddle. The mucoadhesive tablet was hydrated from one surface using 15 ml simulated vaginal fluid and then the hydrated surface was brought into contact with the mucosal membrane. The glass slide was vertically fixed to the paddle and then allowed rotating at 50 rpm. The time required for complete detachment of the tablet from the mucosal surface was recorded (Nafee et al., 004) (n=3). Preparation of simulated vaginal fluid The simulated vaginal fluid (ph 4.0) was prepared as described by Owen and Katz (OwenKatz, 1999) NaCl (3.51 g), KOH (1.40 g), Ca(OH) (0. g), bovine serum albumin (0.018 g), lactic acid ( g), acetic acid (1 g), glycerol (0.16 g), urea (0.4 g), and glucose (5 g) were dissolved in 1 L of deionized water, followed by adjustment to ph 4.0 with HCl. In-vitro CPO release study The release rate of CPO EMVT (n=3) was determined using basket method (USP dissolution testing apparatus I) using 500ml of simulated vaginal fluid ph 4.0 as a dissolution medium. The EMVT was placed in a settling basket to prevent the tablet from floating (WangTang, 008). The rate of stirring was 50 rpm and the medium temperature was maintained at 37 ± 0.5 ºC. A sample (10 ml) of the solution was withdrawn from the dissolution apparatus hourly and the samples were replaced with fresh dissolution medium. The samples were filtered through a filter and diluted to a suitable concentration with simulated vaginal fluid ph 4.0. Absorbance of these solutions was measured at 30 nm using a Shimadzu UV-0 UV-visible double beam spectrophotometer. Cumulative percentage of drug release was calculated using the equation obtained from a standard curve. Statistical analysis Tests for significant differences between means were performed by Student s t-test or one-way ANOVA using the software Sigma Stat version 10. Differences were considered significant at P < 0.05 level. In- vitro antifungal study In-vitro antifungal study was performed against Candida albicans in Sabouraud s agar medium by the cup plate method (Alam et al., 007). The cups cut in the inoculated solidified media were filled with different formulations using sterilized syringes. The marketed tablet (Candid -V3) was crushed into a powder and dissolved in ml of sterilized water was applied using sterilized syringe. The developed CPO EMVT was swelled in ml of sterile water applied into the cups. The covered petri plates were incubated at C in the BOD incubator (Electroquip Pvt. Ltd, India) for 48 hours. The zone of inhibition was measured at the end of 48 hours. Drug release kinetics study To investigate the kinetics of drug release from CPO EMVT, the data of in vitro drug release of optimized batch were fitted to different models. FORTRAN language based program was used for zero order, first order, Higuchi, Hixson-Crowell, Korsmeyer-Peppas, and Weibull models.(korsmeyer et al., 1983) Appropriate drug release kinetic model was selected based on least SSR, least Fisher s ratio (F) and maximum R. Short term stability study CPO EMVTs were subjected to short term stability study in aluminium pack as aluminium strip is considered the best protecting packaging material but in the present study simulation was made using aluminium foil pouch. As the dosage form is formulated for mucoadhesive vaginal drug delivery, no change should occur in its mucoadhesive strength and drug dissolution

4 Rachh et al : Formulation and Characterization of Ciclopirox Olamine Mucoadhesive Effervescent Tablets for Vaginal Delivery 1905 profile. The effect of aging was studied for optimized formulation. The storage condition was optimized at 40 ± 0.5 C and 75 ± 5 % RH for 3 months in stability chambers (Prashanth et al., 007). The samples were taken out at 30, 60 and 90 days and evaluated physical appearance, hardness, friability, in vitro drug release study, drug content, mucoadhesive strength and swelling. Results and Discussion Preliminary Screening Swelling and mucoadhesion study Swelling is important for the assessment of adhesion. To develop maximum adhesion strength an optimum water concentration was needed for polymer particles. It was observed that the order of swelling rate was Carbopol 974P > HPMC K4M > Polycarbophil > HPC > Polyox 303 > Sodium Alginate = Sod CMC > Chitoson >Xanthan Gum in drug free formulations. Also it was observed that order of mucoadhesion for different polymers was Polyox 303 > HPMC K4M > Carbopol 974P > Polycarbophil > HPC > Chitoson > Sodium CMC > Xanthan Gum > Sodium Alginat. Based on swelling behaviour and mucoadhesive strength further Polyox 303, HPMC K4M and Carbopol 974P were chosen alone and in combination (Table 1). The chosen polymers were further evaluated for swelling and mucoadhesive strength with/without effervescent. According to the comparison of the corresponding swelling profiles of formulations with and without effervescent, it could be seen that the effervescent resulted in a marked increase in swelling rate. Furthermore, most tablets with 30 mg effervescent mixture citric acid and sodium bicarbonate in the ratio (1:3) showed a higher swelling capacity than tablets without effervescent (Table ). The increment in swelling could be explained by the good disintegration effect of effervescent, which increased tablet volume and construct porous channels on surface and inside of tablets. The porous channels increased the area of contacting between polymer particles and water so that the polymers could be hydrated more easily. The presence of CPO in the formulation with effervescent decreased mucoadhesive strength compared to drug free formulations with effervescent. These findings could be correlated to solubility of CPO, as CPO is poorly water soluble molecule which hinders the water imbibition by polymers. Though, HPMC K4M performed better swelling property than Carbopol 974P and Polyox 303, it failed to provide satisfactory mucoadhesion compared to Carbopol 974P and Polyox 303. Further different polymers combinations were tried, to achieve the desired swelling and mucoadhesive strength of formulations. Among three combinations of polymers Carbopol 974P + Polyox 303, HPMC K4M + Polyox 303 and Carbopol 974P + HPMC K4M, it was observed that HPMC K4M + Polyox 303 exhibited optimum swelling and mucoadhesion. Thus, (HPMC + Polyox) was considered to incorporate for the development of CPO EMVT. TABLE 1 Preliminary screening formulation composition of drug free tablets. Batch Code Polymers Polymer weight (mg) MCC (mg) Sod. Benzoate (mg) % Swelling Mucoadhesive Strength (N) P1 Carbopol 974P ± ± P Chitosan ± ± 0.03 P3 Sod CMC ± ± P4 HPMC K4M ± ± P5 HPC ± ± 0.00 P6 Sod Alginate ± ± P7 Xanthan gum ± ± P8 Polycarbophil ± ± P9 Polyox ± ± TABLE Preliminary screening of drug free tablet formulation with and without effervescent mixture. Batch Code Polymer Polymer (Wt) (mg) MCC (mg) Effer Mix (mg) Sod Benz (mg) % Swelling Mucoadhesive Strength (N) E1 Polyox ± ± E HPMC K4M ± ± 0.09 E3 Carbopol 974P ± ± 0.83 E4 Polyox Carbopol 974P ± ± 0.34 E5 Polyox HPMCK4M ± ± 0.31 E6 Carbopol 974P + HPMCK4M ± ± 0.69 E7 Polyox ± ± 0.10 E8 HPMC K4M ± ± 0.75 E9 Carbopol 974P ± ± 0.53 E10 Polyox Carbopol 974P ± ± 0.40 E11 Polyox HPMCK4M ± ± 0.5 E1 Carbopol 974P + HPMCK4M ± ± 0.91

5 1906 Int J Pharm Sci Nanotech Vol 5; Issue 4 January March 013 Optimization using 3 full factorial design The number of experiments required for these studies are dependent on the number of independent variables selected. The response (Yi) is measured for each trial. In order to investigate factors systematically, a factorial design was employed in the present investigation. On the basis of the preliminary trials a 3 full factorial design was employed to study the effect of independent variables i.e. polymer ratio (X1) [HPMC K4M: Polyox 303] and amount of effervescent (X) [Citric acid: Sodium bicarbonate] on dependent variables mucoadhesive strength (Y1), % swelling (Y) and t90 (Y3). The full factorial design layout, coded values for polymer ratio(x1) and amount of effervescent mixture(x), and composition of factorial batches V1 to V9 are shown in (Table 3). TABLE 3 Full factorial design layout. Batch code X1 Variable level in coded form V V -1 0 V3-1 1 V4 0-1 V5 0 0 V6 0 1 V7 1-1 V8 1 0 V9 1 1 Check point (V10) Coded value Polymer ratio HPMC K4M: Polyox303 X1 X Amount of effervescent Citric acid and Sod. bicarbonate (1:3) X -1 1: : : Check point 1: Physicochemical characterization of factorial design batches Based on preliminary studies [ HPMC K4M + Polyox 303] mucoadhesive polymers were used in different proportions along with different portions of effervescent mixture [Citric acid + Sodium bicarbonate] for preparation CPO factorial design batches (Batch V1 to V9) (Table 5). The formulated tablets were then evaluated. Tablets had an average weight ± 4.88 mg, 3.11 ± mm thickness and 8 ± 0.1 mm Diameter. The hardness of all the tablets was in the range of 4.7 ± 0.45 (Table 5). Drug content for CPO was TABLE 4 Composition of 3 full factorial design batches. carried out by measuring the absorbance of samples at 30 nm using Shimadzu UV-0 UV/Visible double beam spectrophotometer and comparing the content from a calibration curve prepared with standard CPO in the same medium. The drug content of factorial batches was in the range of 98.5 to 99.93%. Swelling study of factorial design batches The swelling index of factorial batches showed linear relationship with concentration of Polyox 303, as the amount of Polyox was increased in the formulations V1 to V9 the swelling index was also increased from 1 % to 49 % (Table 4). Ex-vivo mucoadhesion study In general, the swelling state of polymer contributes to its mucoadhesive behaviour. It was observed that the swelling rate was increased with increasing amount of effervescent; however, the effervescent led to a significant drop in adhesive strength. The influences of effervescent on swelling and mucoadhesion were opposite, mainly due to the tiny bubbles created by effervescent. These tiny bubbles depressed the mucosapolymer interaction, resulting in a decrease in the mucoadhesive strength (Bottenberg et al., 1991). The minimum adhesion strength (0.31N) was observed in formulation V3, which could be due to the lower ratio of HPMC K4M:Polyox 303 and the higher content of effervescent mixture. On the contrary, with an increase in HPMC K4M:Polyox 303 ratio and decrease in effervescent, the maximum adhesion strength (0.67N) was obtained for formulation V8 (Table 5). Ex-vivo mucoadhesion time The time for the tablet to detach from the rat intestine was recorded as the mucoadhesion time. The increase in concentration of polymer blend in series from formulation V1 to V9, showed a gradual increase in mucoadhesion time. The factorial batches V1 to V3 showed mucoadhesion time 8-10 hours, batches V4 to V6 showed 1-15 hours and batches V7 to V9 showed 4 hours. In- vitro drug release study of factorial design batches The release rate of CPO EMVT was described as a function of time as shown in (Figure ). In all the formulations, the burst release of CPO was observed within first hrs, and then gradually increased up to 8-1 hrs. More drug release could be seen as decreasing HPMC K4M and Polyox 303 and increasing amount of effervescent mixture. Ingredients Quantity (mg) V1 V V3 V4 V5 V6 V7 V8 V9 Ciclopirox Olamine HPMC K4M Polyox MCC Citric acid Sod. Bicarbonate Sod Benzoate Total Wt

6 Rachh et al : Formulation and Characterization of Ciclopirox Olamine Mucoadhesive Effervescent Tablets for Vaginal Delivery 1907 TABLE 5 Physicochemical characterizations of factorial batches. Batch Code Mucoadhesive strength(n) % Swelling t90 (hrs.) Hardness kg/cm Thickness mm(n=3) Weight mg(n=3) V ± ± ± ± ± 5.50 V 0.39 ± ± ± ± ± 5.0 V ± ± ± ± ± 7.81 V ± ± ± ± ±.11 V ± ± ± ± ± 5.86 V ± ± ± ± ±.65 V ± ± ± ± ± 4.51 V ± ± ± ± ± 6.03 V ± ± ± ± ± 4.58 V ± ± ± ± ± 3.85 Fig. In vitro drug release of factorial batches (V1-V9). Full factorial design batches The mucoadhesive strength, % swelling and t90 for the nine batches showed wide variation. The results clearly indicate that all the dependent variables are strongly dependent on the selected independent variables as they show a wide variation among the nine batches (V1 to V9). Statistical analysis of factorial design batches The statistical analysis of the factorial design batches was performed by multiple linear regression analysis carried out in Design Expert software (Design- Expert State-Ease, Inc.). The data clearly indicate that the values of mucoadhesive strength, % swelling and t90, are strongly dependent on the independent variables. The fitted equations (full and reduced) relating the responses mucoadhesive strength, % swelling and t90 to the transformed factors are shown in (Table 6).The polynomial equations can be used to draw conclusions after considering the magnitude of coefficient and mathematical sign it carries (i.e. positive or negative). Table 7 shows the results of the analysis of variance (ANOVA), which was performed to identify insignificant factors. (MendenhallSincich, 1993) High value of correlation coefficient for mucoadhesive strength, % swelling, and t90 indicates good fit i.e., good agreement between dependent and independent variables. The equations may be used to obtain estimates of the responses as small error of variance was noticed in the replicates. The significant test for regression coefficients was performed by applying student F test. A coefficient is significant if the calculated F value is greater than the critical value of F. Full and reduced model for mucoadhesive strength The significant level of coefficient b, b1 and b11 was found to be having p > 0.05 hence it were omitted from full model to generate reduced model. The results of statistical analysis are shown in (Table 8). The coefficients b1 and b were found to be significant at p < 0.05; hence they were retained in reduced model. The results of multiple linear regression analysis (reduced model) revealed that, on increasing the concentration of polymer, the mucoadhesive strength was increased; the coefficients b1 bear positive sign. When high

7 1908 Int J Pharm Sci Nanotech Vol 5; Issue 4 January March 013 concentration of polymer was used, higher adhesion was expected. While in case of amount of effervescent, opposite relation was observed for mucoadhesive strength. It is obvious that the presence of low amount of effervescent adhesion is facilitated. The fitted equations for full and reduced model relating the response are given below. TABLE 6 Summary of results of regression analysis. Coefficients for mucoadhesive strength Model b0 b1 b b1 b11 b R FM RM Coefficients for % swelling (4hrs.) b0 b1 b b1 b11 b R FM RM Coefficients for t90 b0 b1 b b1 b11 b R FM RM FM indicates full model and RM indicates reduced model TABLE 7 Calculation for testing the model in portion (ANOVA). For mucoadhesive strength Regression DF SS MS F R FM Fcal=176.0 RM Ftab=4.7*10-6 Error DF=(1,5) FM RM For % swelling Regression DF SS MS F R FM Fcal=585 RM Ftab=1.3*10-7 Error DF=(1,5) FM RM For t90 Regression DF SS MS F R FM Fcal=54.7 RM Ftab=1.8*10-7 Error DF=(1,5) FM RM TABLE 8 Model summary statistics of the responses. Factor Y1 Y Y3 Factor effect p-value Factor effect p-value Factor effect p-value X < < < X < < X1X X X Refined regression equations of the fitted model Y1 = Y = X X Y = Y = X X Y3 = 0 Y = X1 1.08X

8 Rachh et al : Formulation and Characterization of Ciclopirox Olamine Mucoadhesive Effervescent Tablets for Vaginal Delivery 1909 Full model (mucoadhesive strength) Y = X X + 0X1X 0.019X X Reduced model (mucoadhesive strength) Y = X X Full and reduced model for % swelling The equation of % swelling follows linear model, and all its coefficients have p value more than Hence, % swelling was not altered by the interaction terms of polymer concentration ratio and effervescent mixture ratio. Percentage swelling was influenced greatly by polymer concentration and moderately by effervescent mixture ratio. Full model (% Swelling) Y = X X Full and reduced model for t90 The equation of t90 follows linear model, and all its coefficients have p value more than Hence, t90 was not altered by the interaction terms of polymer concentration ratio and effervescent mixture ratio. The magnitude of t90 was increased with increase in polymer concentration ratio but effervescent mixture ratio had inhibitory effect on it. These findings may be due to increased disintegration and dissolution of the drug due to effervescent action. Full model (t90) Y = X1 1.08X The response surface plot For drawing the conclusions, response surface plot was used. Figure 4 to Figure 6 shows the plot of polymer ratio (X1) and amount of effervescent (X) versus mucoadhesive strength, % swelling and t90 respectively. The plots were drawn using Design Expert software (Design- Expert State-Ease,Inc.). The plots demonstrate the effect of X1 and X for mucoadhesive strength, % swelling and t90. It was arbitrarily decided to select a batch of tablet that would give moderate mucoadhesive strength and drug release in a control manner. The final selection was done after considering some aspects such as drug release profile, ex-vivo retention time and t90. A checkpoint batch V10 was prepared at level (X1 = -0.3 and X = 0.7) from the reduced model. It is expected that the value of mucoadhesive strength of the checkpoint batch should be 0.47 N; the value of % swelling should be 34.9, and the value of t90 of the checkpoint batch should be 7.4 hrs. The practical output of check point batch for desired responses showed values; 0.53N mucoadhesive strength, 37 % swelling and 8.1 hrs for t90. These findings revealed insignificance difference between theoretical prediction and practical output. Thus, it was concluded that the statistical model is mathematically valid. The optimized batch was evolved from the overlay region of surface plots (X1=0.08 and X= -0.18) to achieve desired criteria (Figure 7). The optimized batch V11 exhibited satisfactory mucoadhesion strength (0.56 N), % Swelling (36%) and t90 (9.5hrs). These results were further compared with theoretical values of mucoadhesion strength (0.57N), % swelling (35.41%) and t90 (9.46hrs) and no significance difference was observed. Fig. 3 In vitro drug release study of optimized CPO EMVT (Batch V11).

9 1910 Int J Pharm Sci Nanotech Vol 5; Issue 4 January March 013 Fig. 4 Response surface plot for mucoadhesive strength. Fig. 5 Response surface plot for % swelling. Fig. 6 Response surface plot for t90%.

10 Rachh et al : Formulation and Characterization of Ciclopirox Olamine Mucoadhesive Effervescent Tablets for Vaginal Delivery 1911 Fig. 7 Overlay plot. Ex-vivo mucoadhesion time The increase in concentration of polymer ratio in series from formulations V1 to V9, showed a gradual rise in mucoadhesion time. The values of mucoadhesion time for batches V1 to V3, batches V4 to V6 and batches V7 to V9 were 10 hours, 15 hours and 4 hours respectively. Anti fungal study Mucoadhesive polymers of the tablet had prolonged drug release and provided better contact with the wells cut in the plate. The zone of inhibition was measured at the end of 48 hours. The CPO EMVT showed significantly higher in-vitro antifungal activity as compare to Candid -V3 tablet (p<0.05). The results of antifungal studies are reported in (Figure 8). Drug release kinetics In vitro dissolution data of the optimized formulation V11 were fitted to various mathematical models (zero order, first order, Higuchi, Hixson-Crowell, Korsmeyer- Peppas, and Weibull) in order to describe the kinetics of drug release. Drug release from optimized formulation (V11) fitted well into Zero order kinetics with least sum of square of residuals (SSR =17.0), Fischer s ratio (F =1.70) and maximum R value (Table 9). The value of drug release exponent (n=0.893) indicates Case II transport as a mechanism of drug transport from CPO EMVT. Short term stability study of optimized batch The optimized formulations subjected to short term stability study and were evaluated for physical appearance, hardness, friability, swellings, mucoadhesion, in vitro drug release study and drug content. There was no change in physical appearance of batch V11. There was insignificant change in drug release profile before and after stability study period (f value =79.3±0.63). Table 10 depicts the results of various evaluation parameters of batch V11 before and after stability study which were found to be in official limits. Fig. 8 Comparative anti fungal activity of optimized formulation.

11 191 Int J Pharm Sci Nanotech Vol 5; Issue 4 January March 013 TABLE 9 Results of drug release kinetics of batch V11 Parameter Zero order First Order Higuchi model Hixon Crowell Korsmeyer -Peppas Weibull R F SSR Slope Intercept n F is Fisher s ratio and SSR is sum of square of residuals. TABLE 10 Evaluation parameters for optimized batch V11 subjected to stability study. Parameters Time period Initial 1 month months 3 months Drug content (%) ± ± ± ± Hardness ((kg/cm ) 4.76 ± ± ± ± 0.37 Friability 0.60 ± ± ± ± 0.0 T90% (hrs) 9.50 ± ± ± ± Mucoadhesive strength (N) 0.56 ± ± ± ± Swelling (%) 36 ± ± ± ± 0.66 Conclusions The results of this study show that incorporation of effervescent into the mucoadhesive tablets leads to increase in swelling and drug release and conversely decrease mucoadhesion. It was observed that with the developed formulations of CPO, release and mucoadhesion properties of EMVT can be controlled by changing the polymer type, polymer concentration and effervescent content. Ex-vivo retention studies justified the prolong retention of the tablet inside the vaginal tract. Consequently, the mucoadhesive form of the drug would increase the time of contact with the vaginal mucosa and thus its therapeutic effect. In addition, the soft and rubbery nature of mucoadhesive polymers will minimize mechanical and frictional irritation to the surrounding tissue. Finally it is concluded that formulated CPO EMVT is a better alternative compared to the available gels, creams, pessaries and tablets which may increase patient compliance. References Alam MA, Ahmad FJ, Khan ZI, Khar RK and Ali M (007). Development and evaluation of acid-buffering bioadhesive vaginal tablet for mixed vaginal infections. AAPS PharmSciTech 8: E109. Bottenberg P, Cleymaet R, de Muynck C, Remon JP, Coomans D, Michotte Y and Slop D (1991). Development and testing of bioadhesive, fluoride-containing slow-release tablets for oral use. J Pharm Pharmacol 43: Carrillo-Munoz AJ, Brio S, Alonso R, del Valle O, Santos P and Quindos G (00). Ciclopiroxolamine: in vitro antifungal activity against clinical yeast isolates. Int J Antimicrob Agents 0: Ferrer J (000). Vaginal candidosis: epidemiological and etiological factors. Int J Gynaecol Obstet 71 Suppl 1: S1-7. Garg S, Vermani K, Garg A, Anderson RA, Rencher WB and Zaneveld LJ (005). Development and characterization of bioadhesive vaginal films of sodium polystyrene sulfonate (PSS), a novel contraceptive antimicrobial agent. Pharm Res : Gurny R, Meyer JM and Peppas NA (1984). Bioadhesive intraoral release systems: design, testing and analysis. Biomaterials 5: Gursoy A, Sohtorik I, Uyanik N and Peppas NA (1989). Mucoadhesive controlled release systems for vaginal delivery. STP Pharma 5: Karasulu HY, Hilmioglu S, Metin DY and Guneri T (004). Efficacy of a new ketoconazole bioadhesive vaginal tablet on candida albicans. Farmaco 59: Karasulu HY, Taneri F, Sanal E, Guneri T and Ertan G (00). Sustained release bioadhesive effervescent ketoconazole microcapsules tabletted for vaginal delivery. J Microencapsul 19: Kast CE, Valenta C, Leopold M and Bernkop-Schnurch A (00). Design and in vitro evaluation of a novel bioadhesive vaginal drug delivery system for clotrimazole. J Control Release 81: Korsmeyer RW, Gurny R, Doelker E, Buri P and Peppas NA (1983). Mechanisms of solute release from porous hydrophilic polymers. Int J Pharm 15: Lachman L, Lieberman LI and Kanig JL (1999). The theory and practice of industrial pharmacy, 4 th Ed. Mumbai: Varghese Publishing House. Mendenhall W and Sincich T. (1993). Regression analysis. in: A second course in business statistics: regression analysis, 4 th Ed. San Francisco: Prentice Hall College Div. Nafee NA, Ismail FA, Boraie NA and Mortada LM (004). Mucoadhesive delivery systems. I. Evaluation of mucoadhesive polymers for buccal tablet formulation. Drug Dev Ind Pharm 30: Owen DH and Katz DF (1999). A vaginal fluid simulant. Contraception 59: Prashanth B, Swamy P, Areefulla S, Shirsand S and Gandra S (007). Orodispersible tablets of meloxicam using disintegrant blends for improved efficacy. Ind J Pharm Sci 69: Sharma G, Jain S, Tiwary AK and Kaur G (006). Once daily bioadhesive vaginal clotrimazole tablets: design and evaluation. Acta Pharm 56: Wang L and Tang X (008). A novel ketoconazole bioadhesive effervescent tablet for vaginal delivery: design, in vitro and 'in vivo' evaluation. Int J Pharm 350: Woodley J (001). Bioadhesion: new possibilities for drug administration. Clin Pharmacokinet 40: Address correspondence to: Milan R. Rachh, Associate Professor, Department of Pharmaceutics, Shankarsinh Vaghela Bapu Institute of Pharmacy, Gandhinagar, Gujarat, India. Mob: ; milanrachh@yahoo.com