Abstract

Cytochrome P450 (P450) fluorometric high-throughput inhibition assays have been widely used for drug-drug interaction screening particularly at the preclinical drug discovery stages. Many fluorometric substrates have been investigated for their selectivity, but most are found to be catalyzed by multiple P450 isozymes, limiting their utility. In this study, 3-O-methylfluorescein (OMF) was examined as a selective fluorescence substrate for CYP2C19 in human liver microsomes (HLMs). The kinetic studies of OMF O-demethylation in HLMs using a liquid chromatography/mass spectrometry method exhibited two-enzyme kinetics with apparent K_m and V_max values of 1.14 ± 0.90 μM and 11.3 ± 4.6 pmol/mg/min, respectively, for the high affinity component(s) and 57.0 ± 6.4 μM and 258 ± 6 pmol/mg/min, respectively, for the low affinity component(s). Studies utilizing cDNA-expressed individual P450 isoforms and P450-selective chemical inhibitors showed that OMF O-demethylation to fluorescein was selective for CYP2C19 at substrate concentrations ≤1 μM. At substrate concentrations ≥10 μM, other P450 isozymes were found to catalyze OMF O-demethylation. In HLMs, analysis of the two-enzyme kinetics in the presence of P450 isozyme-selective chemical inhibitors (ticlopidine for CYP2C19, sulfaphenazole for CYP2C9, and furafylline for CYP1A2) indicated that CYP2C19 was the high affinity component and CYP2C9 was the low affinity component. Based on these findings, a fluorometric assay was developed using 1 μM OMF and 2 μM sulfaphenazole for probing CYP2C19-mediated inhibition in HLMs. The IC₅₀ data of 13 substrates obtained from the fluorometric assay developed in this study correlated well with that reported in the literature using nonfluorescence assays.