INTRODUCTION:

Vaginal administration of drugs is used for the treatment of female specific conditions such as vaginitis, bacterial vaginosis and candidiasis. In addition, vaginal formulations have a great potential for systemic absorption of drugs because of large surface area, rich blood supply and permeability to a wide range of compounds. The rate and extension of drug absorption after intravaginal administration may vary depending on formulation factors, vaginal physiology, age of the patient and menstrual cycle. The main advantages of vaginal drug delivery over conventional drug delivery are the ability to by-pass first pass metabolism, ease of administration and high permeability of low molecular weight compounds¹. Vaginal candidiasis is a common condition and up to 75% of all women have atleast one episode of this infection during their life time. About 40-50% of them will suffer a second one and some will show a chronic course^{2, 3}. Candida albicans is the most important cause of vaginal candidiasis accounting for over 80% of the infection. Most patients with candida vaginitis respond to topical treatment with nystatin or imidazole.

Ketoconazole (KTZ) is an imidazole derivative antifungal agent developed for the treatment of human mycotic infections and plays an essential role in the antifungal chemotherapy⁴. It is a weak base with limited water solubility⁵. The molecular formula of ketoconazole is C₂₆H₂₈Cl₂N₄O_4;its molecular weight is 531.4. Ketoconazole is practically insoluble in water; sparingly soluble in alcohol; freely soluble in dichloromethane. Half-life is 2 hours and melting point is 148-152⁰C. The present study was aimed to formulate and evaluate ketoconazole vaginal films for effective treatment of vaginal candidiasis.

MATERIALS AND METHODS:

Materials

Ketoconazole was obtained from Yarrow Chem Products Mumbai, India. Hydroxyl propyl methyl cellulose (HPMC K-100) and Sodium carboxy methyl cellulose (SCMC), PEG-400, Tween-80 were obtained from Loba Chemie, Mumbai, India. All other materials used in the current study were of analytical grade.

Formulations of ketoconazole vaginal films:

Bioadhesive vaginal films of ketoconazole were formulated by solvent casting technique. HPMC K-100 and Sodium CMC were the polymers used along with PEG-400 and Tween-80 as plasticizer and dispersing agent. The compositions of formulations were shown in the table 1. Required quantity of HPMC K-100 and Sodium CMC were taken in separate beakers containing distilled water and stirred continuously. Both the polymeric solutions were mixed together and drug was dispersed into the polymeric solution. Tween-80 and PEG-400 was added and made up the volume with distilled water. The films were casted by transferring the solution to petri plate and kept for drying in oven at 45⁰C for 24–48 hours. The formed films were wrapped in an aluminum foil, stored at room temperature until further use. Ketoconazole was added in the formulation to get a constant amount of 10mg/cm².

Preparation of Simulated Vaginal Fluid:

Simulated Vaginal fluid (SVF) was prepared from 3.51 g/L NaCl, 1.40 g/L KOH,0.222 g/L Ca(OH)₂, 0.018 g/L bovine serum albumin, 2 g/L lactic acid, 1 g/L acetic acid, 0.16 g/L glycerol, 0.4 g/L urea and 5 g/L glucose. The pH of the mixture was adjusted to 4.5 using 0.1M HCl⁶.

Evaluation of ketoconazole vaginal films:

Film weight and thickness: Five films of every composition were taken and weighed individually on digital balance and average weights were calculated. Thickness of each film was measured at five different locations (centre and four peripheral locations) using a micrometer screw gauge and a mean value of five measurements was used as the film thickness.

Folding endurance: Five 2x2 cm square shaped films of each formulation were cut by using a sharp scissors. Folding endurance was determined by repeatedly folding a strip of the film at the same place by 90⁰ and 180⁰ at the rate of 30-35 folds/min till it broke. The number of times, the film could be folded at the same place without breaking gave the value of folding endurance.

Drug excipient compatibility studies: Compatibility studies of Ketoconazole, HPMC K-100 and Sodium CMC and the physical mixture of Ketoconazole and bioadhesive polymers were carried out using Fourier Transform Infrared Spectrophotometer (Shimadzu FT-IR 8400-S) in the range of 400-4000cm^-1 by KBr disc method.

Swelling studies: The films were weighed individually (W₁) and placed separately in a petri plate containing 5ml of simulated vaginal fluid. At regular intervals films were removed from the petri plate and wiped with tissue paper. The films were then reweighed (W₂) and % swelling index was calculated using the formula.

Determination of Surface pH: The Surface pH of the prepared Ketoconazole vaginal film was determined to evaluate the possible irritation effects on the mucosa. The patch was left to swell in 10ml of SVF in small beakers and the pH was measured at regular time intervals 1, 2, 3 and 4 h by placing the electrode in contact with the surface of the film.

Drug content: The prepared films were cut in to 2 x 2 cm² and taken into a 100ml volumetric flask and dissolved in 100ml of SVF. Suitable dilutions were made and absorbance was measured at 225 nm, using UV- Visible spectrophotometer (Shimadzu -1700, Japan) against SVF as blank.

Tensile strength and percentage elongation of patches: Films were evaluated using fabricated instrument. Films of dimension 50x10 mm and free from air bubbles or physical imperfections, were held between two clamps which are supported on metal base. One of the clamps is fixed and other one is movable which is tied to the weighing pan. During measurement, the films were pulled by the movable clamp with the addition of weights. The strength and elongation were measured when the films broke and tensile strength was calculated using the following formula.

Ex – vivo mucoadhesive strength: Mucoadhesive strength of the film was determined by using modified balance method. Fresh sheep vaginal mucosa was obtained from local slaughter house and suitable dimension of the mucosa was fixed to the apparatus using cyanoacrylate adhesive. One end of the film is adhered on to the mucosa with little amount of simulated vaginal fluid there by creating adhesive bond between the film and the membrane. The other end of the film is connected to the pan for the addition of weights. The weights were added slowly to the pan until the film gets detached from the mucosal surface, from which mucoadhesive strength of the film in grams was determined.

Ex – vivo mucoadhesion time: A segment of sheep vaginal mucosa was glued to the inner surface of 50ml beaker. The mucoadhesive film was hydrated from one surface using simulated vaginal fluid and then the hydrated surface was brought into contact with the mucosal membrane. The beaker is filled with 30ml of simulated vaginal fluid and stirred at 50 rpm using magnetic stirrer. The time required for complete detachment of the film from the mucosal surface is recorded⁷.

In – vitro drug release studies: The film measuring 2 × 2 cm² were kept in a basket mesh. This assembly was made to dip into beaker containing 100ml of simulated vaginal fluid. The contents in the assembly was maintained at 37±2°C and stirred on a magnetic stirrer at 50 rpm. Samples are withdrawn at regular intervals and replaced with the same volume of SVF in order to maintain sink conditions. The resulting solution was diluted suitably and absorbance was measured at 225nm using UV – Visible spectrophotometer (Shimadzu -1700, Japan) against VSF as blank.

Kinetic modeling of drug release mechanism: The dissolution data of all formulations were fitted to zero-order, first-order, Hixson-Crowell, Higuchi and Korsemeyer and Peppas models to predict the drug release mechanism.

In–vitro permeation studies: In–vitro permeation studies were carried out using Keischery – Chein cell. Suitable dimension of sheep vaginal mucosa was glued on to the receptor compartment containing SVF. The film measuring 2 × 2 cm² were placed on to the mucosal surface between the donor and receptor compartment. Temperature was maintained at 37±2⁰C with constant stirring at 50 rpm. Samples are withdrawn at regular intervals and replaced with the same volume of SVF in order to maintain sink conditions. Suitable dilutions were made and absorbance of the resulting solution was measured at 225 nm using UV – Visible spectrophotometer (Shimadzu -1700, Japan) against SVF as blank.

RESULTS AND DISCUSSION:

The polymers, Sodium CMC and HPMC K-100, were selected owing to their excellent bioadhesive strength, release rate controlling ability, non-toxicity, non-irritancy and stability at vaginal pH⁸. Successful use of the polymer combination of SCMC and HPMC K-100 is known to provide the formulation with controlled drug release along with desired mucoadhesive properties⁹. Variations in the weight and thickness were studied. This variation was due to the concentration of polymers and volume of the solvent used. So decrease in the polymer concentration and volume of solvent decreases weight and thickness of the film. Drug content in the film of all batches found to be uniform, considering the fact that drug is dispersed uniformly throughout the film. Folding endurance evaluates the ability of the film to withstand repeated folding, bending and measuring quality of the film. The folding endurance was found to be increasing with the increase in polymer concentrations. The prepared bioadhesive films showed acidic pH indicating that none of the formulation will cause irritation, since the vaginal pH varied from 4.5–5. The results of drug content, surface pH and physical evaluation parameters are shown below in the Table – 2.

Swelling study: Polymer swelling permits the mechanical entanglement by exposing the bioadhesive site for hydrogen bonding and/or electrostatic interaction between the polymer and mucin network of mucus¹⁰. The capability of polymer to swell governs the release rate of incorporated drug and also bioadhesiveness of the formulation¹¹. The films were rapidly swelled within 30-45 min and thereafter gradually reach a plateau. The high initial uptake of water was due to the faster hydration rate of HPMC¹². The swelling rate for formulations F1–F6 was varied from 30–90 min whereas for the formulations F7–F12 varied from 30–180 min, there by confirming that as the concentration of polymers is less, swelling is maximum. The effect of various compositions on the swelling index of ketoconazole films are shown in Figure 1.

Figure 1. Swelling index of ketoconazole vaginal films.

Tensile strength and percentage elongation studies: Tensile strength is defined as the maximum stress sustained by the material. Tensile strength varied according to the composition of the formulation as shown in figure 2. The films should withstand mechanical damage during production and handling. As the concentration of polymer increased, tensile strength also increased. Percentage elongation is useful for studying flexibility of the film. The films possess both flexibility and strength for desired effectiveness. Films showed increased percentage elongation with the increase in the polymer concentration which is shown in figure 3.

Table 1. Composition of ketoconazole films containing different polymers

Formulations	Ingredients
Formulations	HPMC(mg)	SCMC(mg)	PEG-400(ml)	Tween-80(ml)	Distilled water(ml)
F1	250	250	0.5	0.3	30
F2	300	250	0.5	0.3	30
F3	300	300	0.5	0.3	30
F4	350	300	0.5	0.5	30
F5	350	350	0.5	0.3	30
F6	400	350	0.5	0.3	30
F7	400	500	0.5	0.5	50
F8	500	400	0.5	0.5	50
F9	500	500	0.5	0.5	50
F10	600	500	0.5	0.5	50
F11	600	600	0.5	0.5	50
F12	700	600	0.5	0.5	50

Table 2. Evaluation of vaginal films

Formulations	Average weight(mg)	Thickness(mm)	Drug content (%)	Folding endurance	Surface P^H
F1	24±3.80	0.21±0.015	98.11±0.94	182±2.775	4.48±0.102
F2	36±8.94	0.28±0.022	99.46±0.41	176±1.924	4.55±0.035
F3	35±10.0	0.29±0.020	99.10±0.98	192±2.702	4.58±0.047
F4	36±8.94	0.29±0.025	96.01±1.25	202±2.408	4.63±0.034
F5	38±8.36	0.33±0.034	98.50±0.70	212±3.962	4.57±0.062
F6	40±7.90	0.34±0.020	98.90±0.85	203±2.702	4.74±0.050
F7	44±4.18	0.32±0.024	99.68±0.62	290±2.236	4.69±0.086
F8	47±5.47	0.33±0.016	99.03±0.55	264±4.123	4.72±0.063
F9	48±4.47	0.36±0.029	99.50±0.81	325±2.864	4.75±0.049
F10	49±2.22	0.35±0.025	99.59±0.52	352±2.588	4.83±0.054
F11	47±2.73	0.36±0.015	99.86±0.39	325±3.899	4.81±0.055
F12	47±4.47	0.39±0.019	99.78±0.34	365±2.964	4.92±0.050

Data are depicted as the mean±SD (n=5)

Table – 3. Drug release kinetics of ketoconazole vaginal films

FORMULATIONS	Drug release kinetics
FORMULATIONS	R	model	K
F1	0.9371	Zero order	0.6813
F2	0.9551	Zero order	0.5586
F3	0.9158	Zero order	0.5519
F4	0.9600	Zero order	0.5310
F5	0.9801	Peppas	0.2232
F6	0.9614	Peppas	0.3613
F7	0.9784	Zero order	21.922
F8	0.9929	Zero order	20.164
F9	0.9960	Zero order	20.2385
F10	0.9885	Zero order	15.1172
F11	0.9824	Zero order	12.6942
F12	0.9843	peppas	10.5929

Ex–vivo mucoadhesive strength and time: The work of adhesion and the force required to produce joint failure (the maximum detachment force) have been used to assess the strength of the adhesive joint. It has been proposed that work of adhesion is the best method for quantifying mucoadhesion. It has been proposed that mucoadhesion occurs by a process of wetting and then interpenetration of the mucoadhesive polymer with the mucus gel. The initial interaction between the polymer and the biological surface is through electrostatic interaction followed by mechanical interlocking of the polymer chains and therefore, the surface charge density of polymers is important for electrostatic behavior during the adhesion process^13,14. SCMC can increase surface charge density and the carboxylic group can form hydrogen bonds with tissue¹⁵. HPMC is the long chained, non-ionic polymer and the mucoadhesive property could be due to the formation of physical or hydrogen bonding with the mucus components. HPMC can relieve the dryness and irritation even in the case of reduced mucus secretions¹⁶. SCMC and HPMC show faster hydration rate and thereby swelling which helps in the interpenetration of mucus and polymer resulting in bioadhesion. The prepared bioadhesive films showed maximum force of detachment with the increase in the polymer concentration as shown in the figure 4. Ex–vivo mucoadhesion time varied according to the polymer concentration. Formulations, F1- F6 showed mucoadhesion time varying from 8 – 12 h where as the formulations, F7 - F12 showed mucoadhesive time from 8 – 20 h, revealing the fact that as the polymer concentration increased, mucoadhesion time increased.

Figure 2. Tensile strength of polymeric films. Each data point represent the mean±S.D (n=3)

Figure 3. Percentage elongation of polymeric films. Each data point represents the mean ± S.D (n=3).

Figure 4. Force of adhesion of polymeric films. Each data point represents the mean ± S.D (n=3)

Figure 5. IR spectra of pure Ketoconazole

Figure 6. IR Spectra of HPMC

Figure 7. IR Spectra of SCMC

Figure 8. IR Spectra of Ketoconazole + HPMC

Figure 9. IR Spectra of Ketoconazole + SCMC

Figure 10. In–vitro permeation profile of ketoconazole vaginal films.

Figure 11. Drug release profile of vaginal films (F1 – F6)

Figure 12. Drug release profile of vaginal films (F7 – F12)

Drug – excipients compatibility: Drug – excipient compatibility studies are done to evaluate interactions between the drug and polymer. The IR spectra of pure drug, polymers like HPMC, SCMC and physical mixture of drug and polymers in the figure 5 - 9. The IR spectra of pure drug has shown characteristic peaks at 1903cm^-1 indicating C=N stretching, 1108cm^-1indicating C-O stretching, 3039cm^-1 indicating C-H stretching aromatic, and 730cm^-1 indicating C-Cl are the major peaks of the drug. The IR spectra of physical mixture also showed the characteristic peaks of pure drug indicating that there were no interaction between the drug and the polymers and significantly can be used for the studies.

In–vitro permeation studies: The permeation studies were performed in Keischery–Chein cell using sheep vaginal mucosa and the medium used is simulated vaginal fluid to mimic the conditions of the vagina. Figure 10, shows the permeation profile of ketoconazole vaginal film. It was observed that, as the concentration of polymers increased, the ketoconazole permeation rate from the film got decreased.

In–vitro dissolution studies: The dissolution studies were performed in simulated vaginal fluid to mimic the conditions of vaginal secretions. Figure 11 and 12 shows the drug release profile of ketoconazole from the film. In all the formulations burst release was observed, formulations F1 – F6 showed burst release with in 30 min, where as the in the formulations F7 – F12 burst release was seen after 1 hour. The burst release is seen because of HPMC, formulations F1 – F6 showed maximum release of drug up to 150 min where as formulations F7 – F12 showed release up to 7 hours. It was observed that as the polymer concentration increases, the ketoconazole release rate from films decreases. The drug release kinetics of all the formulations with their R and k values is shown in the table - 3.

CONCLUSION:

In the present study, ketoconazole vaginal films have been developed. The results of the study revealed that with the developed formulations, the permeation, mucoadhesion and the drug release properties of vaginal films can be modified by changing the polymer concentration. Ex–vivo mucoadhesion studies shows that prolong retention of the film on the mucosal surface. Surface P^H studies show the P^H of the films is acidic in simulated vaginal fluid indicating no irritancy effects. Tensile strength studies indicates that as the polymer concentration increases tensile strength also increases, films showed good force of adhesion with sheep vaginal mucosa. The overall results obtained during studies suggest that bioadhesive vaginal film showed optimum physico–mechanical, pharmaceutical and biological properties, making the films effective for vaginal candidiasis.

ACKNOWLEDGEMENT:

The authors are thankful to Gokula Education Foundation for providing necessary facilities to carry out the research work.

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Received on 07.12.2011 Modified on 14.01.2012

Research J. Pharm. and Tech. 5(3): March 2012; Page 376-382