Development of an In Vitro Release Test (IVRT) for a Vaginal Microbicide Gel

Sensitive and reproducible methods to assess in vitro release rates from semisolid products can provide significant value during drug development. The purpose of the present study was to develop and validate an in vitro release test (IVRT) for a topical gel formulation of an HIV microbicide. The method was developed using a vertical diffusion cell system, commercially available synthetic membranes, and HPLC with UV detection. The IVRT method was robust, reproducible, and sensitive to some of the formulation parameters evaluated. The method was applied to evaluate release rates of the active pharmaceutical ingredient from formulations during stability studies. INTRODUCTION The purpose of a performance test for a dosage form is to predict and monitor the consistency in manufacturing of that dosage form. For topical dosage forms such as gels, creams, and ointments, in vitro release rate testing (IVRT) using the vertical diffusion cell apparatus, or “Franz cell,” has become the most well-accepted performance test for semisolid products (1–5). IVRT can be a useful tool during the development of a topical dosage form. Monitoring the release of a drug from its dosage form during clinical trials can be critical in understanding the efficacy of the formulation in the clinical setting and, in limited cases, has been successful in establishing an in vitro–in vivo correlation for topical products (6). IVRT can be the single test that allows evaluation of drug release from its dosage form following changes to physicochemical properties such as viscosity and particle size, changes in excipients, and changes in manufacturing scale and site. The FDA SUPAC-SS guidance (7) requires IVRT studies following many of these changes to guarantee dosage form performance; thus, IVRT is fast becoming the cornerstone of performance testing for semisolid products. In the present study, we have developed an in vitro release test for a vaginal gel formulation of a non-nucleoside reverse transcriptase inhibitor that is being developed as a vaginal HIV microbicide. IVRT methods should be reproducible and sensitive to small changes in physicochemical properties of the dosage form. The developed test was robust, precise, and discriminatory, and it served well as a performance test for two lead clinical formulations of the active pharmaceutical ingredient (API) during long-term stability studies. MATERIALS AND METHODS IVRT Analysis Typically, six Franz diffusion cells were used for each experiment. Approximately 300–400 mg of the gel samples were applied in the Teflon wafer of the donor chamber and completely occluded by covering the dosage form with an opaque glass disk. The receiving medium was 40:60 ethanol/pH 4 phosphate buffer. A receiving medium with 40% ethanol was selected because of the low aqueous solubility of the API. Nylon membrane was selected as the barrier. The temperature of the diffusion cells was maintained at 37 °C, which is typical for vaginal applications. At 0.5, 1, 2, 4, and 6 h, 200 μL of sample was withdrawn and replenished with fresh, prewarmed media. API concentration in the IVRT samples was determined using the HPLC method described below. The cumulative amount of API released was calculated and plotted against the square root of time to determine the release rate in each diffusion cell. HPLC Analysis API concentration in the IVRT samples was determined by reversed-phase HPLC with UV detection using a Shimadzu liquid chromatography system. Separation was performed on a Thermo Hypersil BDS C18 column, 10 cm × 4.6 mm i.d., 5 μm, maintained at 30 °C. Mobile phase A was 0.5% ammonium acetate buffer, pH 2.3. Mobile phase B was acetonitrile. A thirteen-minute gradient (Table 1) was used to elute the API with a flow rate of 1.0 mL/min and UV detection at 286 nm. Injection volume was 10 μL. Samples were kept at ambient temperature during analysis. RESULTS AND DISCUSSION Release Rate Dependence on Synthetic Membrane Three synthetic membranes, nylon, silastic, and cellulose, were selected to study the in vitro release of the gel formulations. Membrane-binding studies were performed to determine whether the API binds to a specific *Corresponding author. diss-17-04-02.indd 6 11/16/2010 9:14:41 AM dx.doi.org/10.14227/DT170410P6