Review of Fluconazole Properties and Analytical Methods for Its Determination

The therapeutic potential of UK-49,858, a difluorophenyl bis-triazole derivative, has been assessed by evaluating its activity against systemic infections with Candida albicans in normal mice and rats and in mice with impaired defence mechanisms, against vaginal infections with C. albicans in mice, and against dermal infections with Trichophyton mentagrophytes in guinea pigs. Orally administered ketoconazole was used as a comparative agent throughout, and parenterally administered amphotericin B was included in the study of C. albicans systemic infection in normal mice. The activity of UK-49,858 given orally to mice or rats infected systemically with C. albicans was far superior to that of ketoconazole. In addition, UK-49,858 showed activity comparable to that of amphotericin B when given parenterally, although the latter gave more prolonged protection. UK-49,858 was also effective orally in curing experimental candidal vaginitis in mice and trichophytosis in guinea pigs, against which it was approximately 10 times more active than ketoconazole. These data suggest that UK-49,858 may be of value in the treatment of both C. albicans and dermatophyte fungal infections in man.

High-performance liquid chromatographic technique has been developed for the determination or some azolcsantifungals namely, clotrimazole (CZ), ketoconazole (KZ) and fluconazole (FZ), in pure forms and in pharmaceutical formulations. The proposed HPLC-method can be successfully applied as a stability indicating method for the determination of CZ in presence of its acid degradation products; viz (2-chlorophenyl)-diphenyl methanol and imidazole. The analyzed drugs were separated on a reversed-phase column [Bondapak C18 (10 microm, 25 cm x 4.6 mm, i.d.)] using a mobile phase containing acetonitrilc+25 mM trishydroxymeihyl aminomethane in phosphate butter (pH 7) = 55:45 (v/v), with UV-detection at 260 nm. The differences in the retention times (tR) of the three azoles permit their use as internal standard for each other. In addition, a coupled TLC-densitometric method has been also applied as a stability indicating method to separate and quantify CZ alone or in presence of byproducts impurities and/or its acid degradation products. The TLC-fractionation was performed on a precoated silica gel F254 plates using a solvent system consisting of chloroform+acetone+ammonia (25%) (7:1:0.1, by volumes), CZ was well separated from its acid degradation products and quantified by densitometric scanning at 260 nm.

The current study focused on the development of an automated IC50 cocktail assay in a miniaturized 384 well assay format. This was developed in combination with a significantly shorter high pressure liquid chromatography (HPLC) separation and liquid chromatography-mass spectrometry (LC-MS/MS) run-time; than those currently reported in the literature. The 384-well assay used human liver microsomes in conjunction with a cocktail of probe substrates metabolized by the five major CYPs (tacrine for CYP1A2, diclofenac for CYP2C9, (S)-mephenytoin for CYP2C19, dextromethorphan for CYP2D6 and midazolam for CYP3A4). To validate the usefulness of the automated and analytical methodologies, IC50 determinations were performed for a series of test compounds known to exhibit inhibition across these five major P450s. Eight compounds (sertraline, disulfuram, ticlopidine fluconazole, fluvoxamine, ketoconazole, miconazole, paroxetine, flunitrazepam) were studied as part of a cocktail assay, and against each CYPs individually. The data showed that the IC50s generated with cocktail incubations did not differ to a great extent from those obtained in the single probe experiments and hence unlikely to significantly influence the predicted clinical DDI risk. In addition the present method offered a significant advantage over some of the existing cocktail analytical methodology in that separation can be achieved with run times as short as 1 min without compromising data integrity. Although numerous studies have been reported to measure CYP inhibition in a cocktail format the need to support growing discovery libraries not only relies on higher throughput assays but quicker analytical run times. The current study reports a miniaturized high-throughput cocktail IC50 assay, in conjunction with a robust, rapid resolution LC-MS/MS end-point offered increased sample throughput without compromising analytical sensitivity or analyte resolution.