The role of CYP2C enzymes in human drug metabolism

The CYP2C enzymes of the cytochrome P450 system are important drug metabolizing enzymes in humans. Variations in substrate specificity and expression among these enzymes contribute to interindividual variability in drug metabolism associated with the CYP2C family of enzymes. Genetic polymorphisms play a role in this variation. The S-mephenytoin drug metabolism polymorphism has been well characterized, yet 20% of poor metabolizers in caucasian populations can not be attributed to known alleles of CYP2C19. We have identified a variant of CYP2C19, containing a histidine to isoleucine substitution at codon 99, that displays reduced ability to 4{dollar}spprime{dollar}-hydroxylate S-mephenytoin. Kinetic analysis of S-mephenytoin metabolism by mutant CYP2C19-Ile{dollar}sp{lcub}99{rcub}{dollar} exhibited a 6.5 fold increase in K{dollar}sb{lcub}rm M{rcub}{dollar} and a 3 fold decrease in V{dollar}sb{lcub}rm MAX{rcub}{dollar} compared to wild type CYP2C19-His{dollar}sp{lcub}99{rcub}{dollar} indicating decreased catalytic efficiency by the mutant. While this allele may explain a poor metabolizer phenotype, screening of DNA samples isolated from 85 different individuals revealed only the wild type CYP2C19-His{dollar}sp{lcub}99{rcub},{dollar} samples isolated from 85 different individuals revealed only the wild type CYP2C19-His{dollar}sp{lcub}99{rcub},{dollar} suggesting that the CYP2C19-Ile{dollar}sp{lcub}99{rcub}{dollar} allele possessing the S-mephenytoin poor metabolizer phenotype was rare.; The metabolic capacity of CYP2C enzymes isolated from human liver microsomes toward the substrates S-mephenytoin, omeprazole and tolbutamide was also evaluated. CUP2C19 conventionally purified from human liver microsomes metabolized S-mephenytoin and omeprazole at rates 11- and 8-fold greater than the microsomes from which the enzyme was purified while neither CYP2C8 nor CYP2C9 catalyzed these reactions. CYP2C19 was also found to metabolize tolbutamide at rates similar to CYP2C9. These findings were consistent with results obtained using recombinant P450 enzymes. Immunoblot analysis showed that polyclonal antibodies raised against a single purified CYP2C enzyme displayed cross reactivity with the other CYP2C members. The polyclonal antibodies also potently inhibited S-mephenytoin (98% inhibition), tolbutamide ({dollar}>{dollar}90% inhibition) and omeprazole (85% inhibition) metabolism by human liver microsomes. Strong correlations between immunoreactive CYP2C19 and either S-mephenytoin (r = 0.912) or omeprazole (r = 0.906) hydroxylation rates were observed. Correlations between tolbutamide hydroxylation and CYP2C9 (r = 0.664) were significant, but not between CYP2C9 and S-mephenytoin (r = 0.393). Immunoquantitation showed 7.7 fold greater expression of CYP2C9 compared to CYP2C19 in a panel of human liver microsomes. These data confirmed that CYP2C19 was the major S-mephenytoin and omeprazole hydroxylase, and suggested that CYP2C19 contributed to in vivo tolbutamide hydroxylation but its role was minimal due to relatively low expression of CYP2C19.; In order to render monospecificity to anti-2C antibodies, recombinant CYP2C19 was linked to an affinity resin. Immunoblot analysis showed that the back-adsorption with expressed CYP2C19 produced an anti-CYP2C9 antibody that reacted only with CYP2C9. The monospecific anti-CYP2C9 IgG was capable of inhibiting CYP2C9 mediated diclofenac metabolism in human liver microsomes or reconstituted systems but was devoid of inhibitory capacity toward CYP2C19 or microsome mediated S-mephenytoin hydroxylation, a reaction catalyzed by both CYP2C 9 and CYP2C19 by 78% in reactions catalyzed by microsomes containing CYP2C19 and 92% when microsomes with undetectable levels of CYP2C19 were used. These results suggest that CYP2C19 contributed approximately 17-25% toward microsomeal tolbutamide metabolism. While this contribution is limited, it might be of consequence when factors such as polymorphic CYP2C9 express...