Introduction. A common microbial problem in the vulvovaginal tract is infection with Candida albicans. Approximately 75% of women will have a vaginal infection with a Candida strain during their life and about 40 to 50% of them will suffer a second one, and a small percentage will show a chronic course. Our aim of this study is to generate a novel drug delivery system for the application of Fluconazole, a synthetic azole, and widely used antifungal agent for the treatment of Candidiasis. The goal of this formulation and development project is to evaluate various formulations for the preparation of a safe, effective and stable gel containing Fluconazole as a model drug.

During the last decade, there has been an increasing desire among scientists, manufacturers, and consumers to use alternative in-vitro models as a humane approach to reduce or replace the use of laboratory animals. This should minimize expensive, and sometimes controversial, animal studies for irritation, penetration and toxicity testing. In-vitro methods include a wide range of techniques that measure biological activity of different cell cultures by monitoring specific endpoints. One important technique is cell culture. Human equivalent cultures were investigated as possible pre-clinical irritation screens to aid safety assessments for chemicals and product formulations. An advantage of human cultures is that they are relevant to the organ and species of concern, and as such, are mechanistically sound alternatives to traditional rabbit vaginal irritation tests.

A tissue culture based model for the vaginal epithelium (EpiVaginal™) has been developed by MatTek Corporation. The EpiVaginal culture model is a cell culture system made of normal, human ectocervico-vaginal (ECV) epithelial cells that were induced to form a three-dimensional tissue using specially formulated serum free medium.

Purpose. The goal of our research on formulation is to evaluate various ingredients with varying concentrations for the preparation of a safe, effective and stable gel containing Fluconazole and evaluate its in-vitro performance, its stability and also evaluate the use of reconstituted human vaginal epithelium EpiVaginal™ as a model for drug irritation, toxicity and penetration for an antifungal gel containing Fluconazole.

Methods. The gel forming agents Carbopol 974 P NF, HPMC a white chemical (hydroxyl propyl methyl cellulose) and a combination of both Carbopol 971P NF polymer and Noveoon AA1 USP a polycarbophil were investigated because of their satisfactory profile for low irritation and toxicity. Gels with 1% w/w Fluconazole were formulated and the release of the drug from these formulations has been compared using both Franz diffusion cell and EpiVaginal™ tissue cultures. The skin permeation studies were conducted using tissue cultures. Hanks Buffer pH 7.4 was used as a receptor medium. 100 mg of test product were deposited onto the surface of the tissues. The cultures are incubated at 37°C for a specific time. A plot of the amount of Fluconazole permeated μg/cm ² versus time (hr) was used to calculate the flux.

Based on validated protocols the EpiVaginal model will allow us to screen and measure with high precision, the irritation of the developed gel formulation. The in-vitro endpoints have previously been shown to be relevant to human irritation in-vivo, including MTT-metabolism that is an indicator of culture viability, and the release of inflammatory mediators or cytokines (multiple end point analysis) using the ELISA technique.

Results. In the in-vitro permeation tests using cellulose acetate membrane mounted onto a Franz cell both CA 974 and HPMC gels had significantly lower flux when compared to CA 971. Significantly lower fluxes were measured (P < 0.05) when gels were formulated with HA as compared to the same formulation without HA. The effect of HA in the formulations was found to be concentration dependent.

When utilizing reconstituted human vaginal epithelium only the formulations with METHOCEL yielded the least flux, thus yielding a lower rate of permeation as compared to the carbopol containing formulations. There is a statistically significant difference between the mean flux of the METHOCEL gels as compared to the Carbopol gels (P < 0.05).

Conclusion. Stable gel formulations were formulated. The use of a recently developed normal (non-cancerous), human cell-derived, tissue can be investigated in this study as a model of drug delivery.