Received for publication February 25, 2009.
Revised May 15, 2009.
Accepted for publication May 18, 2009.

Evaluation of Luciferin-IPA as a CYP3A4 Substrate for Human Hepatocytes: Effects of Organic Solvents, P450 Inhibitors, and P450 Inducers

Abstract

This study represents the first report on the characterization of luciferin-IPA (LIPA) as a CYP3A4 substrate in human hepatocytes. LIPA metabolism by human hepatocytes was found to be linear with time up to 120 minutes and followed Michaelis-Menten kinetics, with apparent Km value of 15 uM and Vmax of 41 pmoles/min/million hepatocytes for the hepatocytes used in the study. The nonspecific P450 inhibitor 1-aminobenzotriazole (ABT) and the CYP3A4-selective inhibitor ketoconazole (KTZ) caused concentration-dependent inhibition of LIPA metabolism, with over 50% inhibition observed at the lowest concentrations evaluated of 7.8 uM (ABT) and 0.78 uM (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 was 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. While the selectivity towards CYP3A4 needs to be definitively evaluated using cDNA-expressed CYP isoforms , the results suggest that LIPA is a suitable substrate to be used with human hepatocytes for the evaluation of CYP3A4 activities.

Key words: CYP induction, CYP inhibition, CYP3A, cytochrome P450, drug-drug interactions, enzyme induction, enzyme inhibitors, hepatocytes, high throughput screening, human CYP enzymes