Received for publication December 26, 2006.
Revised February 26, 2007.
Accepted for publication February 27, 2007.

EVALUATION OF 3-O-METHYLFLUORESCEIN AS A SELECTIVE FLUOROMETRIC SUBSTRATE FOR CYP2C19 IN HUMAN LIVER MICROSOMES

Abstract

Cytochrome P450 (CYP) fluorometric high throughput inhibition assays have been widely used for drug-drug interaction (DDI) screening particularly at the pre-clinical drug discovery stages. Many fluorometric substrates have been investigated for their selectivity, but most are found to be catalyzed by multiple CYP isozymes, limiting their utility. In this study 3-O-methylfluorescein (OMF) was examined as a selective fluorescence substrate for CYP2C19 in human liver microsomes (HLM). The kinetic studies of OMF O-demethylation in HLM using a LC/MS 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 CYP isoforms and CYP-selective chemical inhibitors demonstrated that OMF O-demethylation to fluorescein (FL) was selective for CYP2C19 at substrate concentrations 1 µM. At substrate concentrations 10 µM, other CYP isozymes were found to catalyze OMF O-demethylation. In HLM, analysis of the two-enzyme kinetics in the presence of CYP isozyme-selective chemical inhibitors (ticlopidine for CYP2C19, sulfaphenazole for CYP2C9, and furafylline for CYP1A2) indicated that CYP2C19 was the high affinity component and CYP2C9 the low affinity component. On the basis of these findings, a fluorometric assay was developed using 1 µM OMF and 2 µM sulfaphenazole for probing CYP2C19-mediated inhibition in HLM. The IC₅₀ data of thirteen substrates obtained from the fluorometric assay developed in this study correlated well with that reported in the literature using non-fluorescence assays.

Key words: CYP inhibition, drug discovery, drug-drug interactions, enzyme kinetics, high throughput screening, human CYP enzymes, liver microsomes