6-hydroxytaxol and acetonitrile

The effect of common organic solvents on the activities of various human cytochromes P450 has been reported. However, very little is known about their influence on CYP2B6 and CYP2C8 enzymes. The purpose of this study was to investigate the effect of solvents on the kinetics of representative CYP2B6 (bupropion hydroxylase) and CYP2C8 (paclitaxel hydroxylase) reactions in human liver microsomes. Methanol, ethanol, dimethyl sulfoxide (DMSO), and acetonitrile were studied at increasing volumes (v/v). Acetonitrile, DMSO, and ethanol were shown to increase the Km and decrease the intrinsic clearance (CLint) of CYP2B6-mediated bupropion hydroxylation in a concentration-dependent manner. These solvents did not noticeably alter the Vmax at concentrations of < or =1% (v/v). Unlike the other solvents studied, the effect of methanol (< or =0.5%, v/v) on CYP2B6 kinetics was negligible. Both DMSO and ethanol increased the Km and decreased the CL(int) of CYP2C8-mediated paclitaxel hydroxylation in a concentration-dependent manner. Acetonitrile had minimal influence on CYP2C8 enzyme kinetics at concentrations of < or =1% (v/v). Methanol decreased the Km of paclitaxel at low concentrations followed by an increase at concentrations of > or =2% (v/v). This differential influence on Km resulted in an increased CLint at low concentrations followed by a decrease at high concentrations. The studied solvents had minimal influence on Vmax of paclitaxel. Collectively, DMSO and ethanol were not suitable for characterizing CYP2B6- and CYP2C8-mediated reactions because they showed concentration-dependent inhibition. Methanol and acetonitrile at concentrations of < or =0.5% and < or =1% (v/v) appeared to be suitable for the measurement of CYP2B6- and CYP2C8-mediated activities, respectively.

Topics: Acetonitriles; Aryl Hydrocarbon Hydroxylases; Bupropion; Cytochrome P-450 CYP2B6; Cytochrome P-450 CYP2C8; Cytochrome P-450 Enzyme System; Dimethyl Sulfoxide; Ethanol; Humans; Hydroxylation; Kinetics; Methanol; Microsomes, Liver; Paclitaxel; Solvents; Taxoids

A reversed-phase high-performance liquid chromatographic (HPLC) method has been validated for the quantitative determination of the three major paclitaxel metabolites (6 alpha-hydroxypaclitaxel, 3'-p-hydroxypaclitaxel, 6 alpha,3'-p-dihydroxypaclitaxel) in human plasma. The HPLC system consists of an APEX-octyl analytical column and acetonitrile-methanol-0.02 M ammonium acetate buffer pH 5 (AMW; 4:1:5, v/v/v) as the mobile phase. Detection is performed by UV absorbance measurement at 227 nm. The sample pretreatment of the plasma samples involves solid-phase extraction (SPE) on Cyano Bond Elut columns. The concentrations of the metabolic products could be determined by using the paclitaxel standard curve with a correction factor of 1.14 for 6 alpha,3'-p-dihydroxypaclitaxel. The recoveries of paclitaxel and the metabolites 6 alpha,3'-p-dihydroxypaclitaxel, 3'-p-hydroxypaclitaxel and 6 alpha-hydroxypaclitaxel in human plasma were 89, 78, 91 and 89%, respectively. The accuracy of the assay for the determination of paclitaxel and its metabolites varied between 95 and 97%, at a 50 ng/ml analyte concentration. The lower limit of quantitation was 10 ng/ml for both the parent drug and its metabolites.

Topics: Acetates; Acetonitriles; Antineoplastic Agents, Phytogenic; Buffers; Chromatography, High Pressure Liquid; Humans; Hydrogen-Ion Concentration; Methanol; Paclitaxel; Spectrophotometry, Ultraviolet; Taxoids