Determination of CYP2C9-Catalyzed Diclofenac 4-Hydroxylation by High-Performance Liquid Chromatography

CYP2C9 is a major CYP2C enzyme expressed in human liver (1 –3 ). Recent experiments with primary cultures of human hepatocytes have suggested that CYP2C9 protein expression can be increased in cells treated with a P450 inducer such as phenobarbital, dexamethasone, or rlfampin (rifampicin) (3 ). This is consistent with the clmical finding that rlfampin increases the total body clearance of tolbutamide (4 ), which is metabolized almost entirely by hepatic CYP2C9 (5 ). Other substrates for CYP2C9 include phenytom (5 , 6 ), warfarin (7 ) and diclofenac (8 ). Sulfaphenazole is a CYP2C9-selective chemical mhlbitor (9 –12 ) and inhibition experiments with this sulfur-containing compound have suggested that human liver microsomal diclofenac 4′-hydroxylatlon IS selectively catalyzed by CYP2C9 (8 ). This chapter describes a high-performance liquid chromatographic (HPLC) assay for the determination of diclofenac 4′-hydroxylase activity. Methods for other CYP2C9 assays such as tolbutamide methylhydroxylase (13 ) and (S)-warfarin 7-hydroxylase (14 , 15 ) can be found in the cited references.