CHARACTERIZATION OF CYP2C19 AND CYP2C9 FROM HUMAN LIVER - RESPECTIVE ROLES IN MICROSOMAL TOLBUTAMIDE, S-MEPHENYTOIN, AND OMEPRAZOLE HYDROXYLATIONS

Individuals with drug metabolism polymorphisms involving CYP2C enzymes exhibit deficient oxidation of important therapeutic agents, includin g S-mephenytoin, omeprazole, warfarin, tolbutamide, and nonsteroidal a nti-inflammatory drugs. While recombinant CYP2C19 and CYP2C9 proteins expressed in yeast or Escherichia coli have been shown to oxidize thes e agents, the capacity of the corresponding native P450s isolated from human liver to do so is ill defined. To that end, we purified CYP2C19 , CYP2C9, and CYP2C8 from human Liver samples using conventional chrom atographic techniques and examined their capacity to oxidize S-mepheny toin, omeprazole, and tolbutamide. Upon reconstitution, CYP2C19 metabo lized S-mephenytoin and omeprazole at rates that were 11- and 8-fold h igher, respectively, than those of intact liver microsomes, whereas ne ither CYP2C9 nor CYP2C8 displayed appreciable metabolic activity with these substrates. CYP2C19 also proved an efficient catalyst of tolbuta mide metabolism, exhibiting a turnover rate similar to CYP2C9 preparat ions (2.0-6.4 vs 2.4-4.3 nmol hydroxy-tolbutamide formed/min/nmol P450 ). The kinetic parameters of CYP2C19-mediated tolbutamide hydroxylatio n (K-m = 650 mu M, V-max = 3.71 min(-1)) somewhat resembled those of t he CYP2C9-catalyzed reaction (K-m = 178-407 mu M, V-max = 2.95-7.08 mi n(-1)). Polyclonal CYP2C19 antibodies markedly decreased S-mephenytoin 4'-hydroxylation (98% inhibition) and omeprazole 5-hydroxylation (85% inhibition) by human liver microsomes. CYP2C19 antibodies also potent ly inhibited (>90%) microsomal tolbutamide hydroxylation, which was si milar to the inhibition (>85%) observed with antibodies to CYP2C9. Mor eover, excellent correlations were found between immunoreactive CYP2C1 9 content, S-mephenytoin 4'-hydroxylase activity (r = 0.912; P < 0.001 ), and omeprazole 5-hydroxylase activity (r = 0.906; P < 0.001) in liv er samples from 13-17 different subjects. A significant relationship w as Likewise observed between microsomal tolbutamide hydroxylation and CYP2C9 content (r = 0.664; P < 0.02) but not with CYP2C19 content (r = 0.393; P = 0.184). Finally, immunoquantitation revealed that in these human liver samples, expression of CYP2C9 (88.5 +/- 36 nmol/mg) was 5 -fold higher than that of CYP2C19 (17.8 +/- 14 nmol/mg) and nearly 8-f old higher than that of CYP2C8 (11.5 +/- 12 nmol/mg). Our results, lik e those obtained with recombinant CYP2C enzymes, indicate that CYP2C19 is a primary determinant of S-mephenytoin 4'-hydroxylation and low-K- m omeprazole 5-hydroxylation in human liver. Despite its tolbutamide h ydroxylase activity, the low levels of hepatic CYP2C19 expression (rel ative to CYP2C9) may preclude an important role for this enzyme in hep atic tolbutamide metabolism and any polymorphisms thereof. (C) 1998 Ac ademic Press.