CYP2C19 participates in tolbutamide hydroxylation by human liver microsomes

Tolbutamide is a sulfonylurea-type oral hypoglycemic agent whose action is terminated by hydroxylation of the tolylsulfonyl methyl moiety catalyzed by cytochrome P-450 (CYP) enzymes of the human CYP2C subfamily. Although most studies have implicated CYP2C9 as the exclusive catalyst of hepatic tolbut amide hydroxylation in humans, there is evidence that other CYP2C enzymes ( e.g., CYP2C19) may also participate. To that end, we used an immunochemical approach to assess the role of individual CYP2Cs in microsomal tolbutamide metabolism. Polyclonal antibodies were raised to CYP2C9 purified from huma n liver, and were then back-adsorbed against recombinant CYP2C19 coupled to a solid-phase support. Western blotting revealed that the absorbed antihum an CYP2C9 preparation reacted with only recombinant CYP2C9 and the correspo nding native protein in hepatic microsomes, and no longer recognized CYP2C1 9 and CYP2C8. Monospecific anti-CYP2C9 not only retained the ability to inh ibit CYP2C9-catalyzed reactions, as evidenced by its marked (90%) inhibitio n of diclofenac 4'-hydroxylation by purified CYP2C9 and by human liver micr osomes, but also exhibited metabolic specificity, as indicated by its negli gible (< 15%) inhibitory effect on S-mephenytoin 4'-hydroxylation by purifi ed CYP2C19 or hepatic microsomes containing CYP2C19. Monospecific anti-CYP2 C9 was also found to inhibit rates of tolbutamide hydroxylation by 93 +/- 4 and 78 +/- 6% in CYP2C19-deficient and CYP2C19-containing human liver micr osomes, respectively. Taken together, our results indicate that both CYP2C9 and CYP2C19 are involved in tolbutamide hydroxylation by human liver micro somes, and that CYP2C19 underlies at least 14 to 22% of tolbutamide metabol ism. Although expression of CYP2C19 in human liver is less than that of CYP 2C9, it may play an important role in tolbutamide disposition in subjects e xpressing either high levels of CYP2C19 or a catalytically deficient CYP2C9 enzyme.