CYP2Cl g-Mediated (S)-Mephenytoin 4-Hydroxylation Assayed by High-Performance Liquid Chromatography with Radiometric Detection

CYP2C 19 is one of at least three CYP2C enzymes expressed in human hver (1 , 2 ), although the abundance of this P450 is generally less than that of etther CYP2C8 or CYP2C9 (3 ). Considerable interindividual differences occur in hepatic CYP2C19 content (1 , 2 ), primarily owmg to a genetic polymorphism in this enzyme (4). Approximately 15-20% of Orientals and 3% of Caucasians exhibit the CYP2C19 poor-metabolizer phenotype, which is associated with at least two mutant alleles, designated CYP2C 19ml and CYP2C 19m2 (4 ). Studies with cDNA-expressed CYP2C 19 have identified several drugs as substrates for this polymorphically expressed P450, including omeprazole (5 , 6 ), diazepam (7 ), cyclophosphamlde (3 ), and lfosfamide (3 ). However, the precise role of human hepatic CYP2C19 in drug biotransformation is poorly understood because mhibitory monospecific antiCYP2C19 antibodies are not available and a CYP2C19-specific chemical inhibitor has yet to be identified. Recent correlational analyses have led to the conclusion that CYP2C19 is a major P450 responsible for (S)-mephenytoin 4’-hydroxylation activity in human liver microsomes (1 , 2 ). This is supported by the finding that cDNA-expressed CYP2C19 has a 50-to lOO-fold greater turnover number than CYP2C8, CYP2C9, or CYP2C 18 for (S)-mephenytoin 4−hydroxylatlon (2 ). As a result, (S)-mephenytom 4’-hydroxylase activity is frequently used as a catalytic monitor for human hepatic CYP2C19. In addmon, (S)-mephenytom 4’-hydroxylation is a useful indicator of the catalytic activity of cDNA-expressed CYP2C 19 (2 ). Varrous analytical methods have been developed to measure the enzymatic formation of 4’-hydroxymephenytoin, including isocratrc, reversed-phase hrgh performance hqutd chromatography (8 ). This chapter describes a method for the determination of (S)-mephenytoin 4’-hydroxylation by gradient, reversedphase high performance llqurd chromatography using ¹⁴ C-labeled substrate and radiometrrc detection.