Abstract

This study represents the first report on the characterization of luciferin-isopropyl acetal (LIPA) as a CYP3A4 substrate in human hepatocytes. LIPA metabolism by human hepatocytes was found to be linear with time up to 120 min and followed Michaelis-Menten kinetics, with apparent K_m value of 15 μM and V_max of 41 pmol/min/million hepatocytes for the hepatocytes used in the study. The nonspecific cytochrome P450 (P450) inhibitor 1-aminobenzotriazole (ABT) and the CYP3A4-selective inhibitor ketoconazole (KTZ) caused concentration-dependent inhibition of LIPA metabolism, with more than 50% inhibition observed at the lowest concentrations evaluated of 7.8 μM (ABT) and 0.78 μM (KTZ), and near 100% inhibition observed at higher concentrations. Substantially lower inhibitory effects were observed for the non-CYP3A4 inhibitors diethyldithiocarbamate, furafylline, omeprazole, orphenadrine, sulfaphenazole, and quinidine. The commonly used organic solvents—acetonitrile, dimethyl sulfoxide (DMSO), and methanol—were found to inhibit LIPA metabolism, with approximately 50% inhibition at concentrations of 5, 1.25, and 5% (by volume), respectively. The comparatively higher inhibitory effects of DMSO relative to that for acetonitrile and methanol on LIPA metabolism were consistent with its known CYP3A4 inhibitory effects reported by others. LIPA metabolism in human hepatocytes was found to be induced by the treatment of human hepatocytes with the prototypical CYP3A4 inducers rifampin, carbamazepine, omeprazole, phenobarbital, and phenytoin but not by the CYP1A2 inducer 3-methylcholanthrene. Although the selectivity toward CYP3A4 needs to be definitively evaluated using cDNA-expressed P450 isoforms, the results suggest that LIPA is a suitable substrate to be used with human hepatocytes for the evaluation of CYP3A4 activities.