Formation of a dihydroxy metabolite of phenytoin in human liver microsomes/cytosol: Roles of cytochromes P4502C9, 2C19, and 3A4

Formation of four oxidative metabolites from the anticonvulsant drug phenyt oin (DPH) catalyzed by human liver microsomal cytochrome P450 (P450) enzyme s was determined simultaneously. Under the conditions in which linearity fo r formation of 4'-hydroxylated DPH (4'-HPPH; main metabolite) was observed, human liver cytosol increased microsome-mediated DPH oxidation. 3',4'-Dihy droxylated product (3',4'-diHPPH) formation was 10 to 40% of total DPH oxid ation in the presence of liver cytosol. 3'-Hydroxy DPH formation was cataly zed by only one of the human liver microsomal samples examined and 3',4'-di hydrodiol formation could not be detected in all samples. In the presence o f liver cytosol, 3',4'-diHPPH formation activity from 100 mu M 4'-HPPH was correlated with testosterone 6 beta-hydroxylation activity and CYP3A4 conte nt. However, 3',4'-diHPPH formation using 1 or 10 mu M 4'-HPPH as a substra te was not correlated with contents of any P450s or marker activities. Of 1 0 cDNA-expressed human P450 enzymes examined, CYP2C19, CYP2C9, and CYP3A4 c atalyzed 3',4'-diHPPH formation from the primary hydroxylated metabolites ( 3'-hydroxy-DPH and 4'-HPPH). Fluvoxamine and anti-CYP2C antibody inhibited 3',4'-diHPPH formation from 10 mu M 4'-HPPH in a human liver sample that co ntained relatively high levels of CYP2C, whereas ketoconazole and anti-CYP3 A antibody showed inhibitory effects on the activities in liver microsomal samples in which CYP3A4 levels were relatively high. These results suggest that CYP2C9, CYP2C19, and CYP3A4 all have catalytic activities in 3',4'-diH PPH formation from primary hydroxylated metabolites in human liver and that the hepatic contents of these three P450 forms determine which P450 enzyme s play major roles of DPH oxidation in individual humans.