Received for publication April 13, 2005.
Revised June 8, 2005.
Accepted for publication June 13, 2005.

CYP2C9 Genotype-Dependent Effects on In Vitro Drug-Drug Interactions: Switching of Benzbromarone Effect from Inhibition to Activation in the CYP2C9.3 Variant

Abstract

The CYP2C9.3 variant exhibits marked decreases in substrate turnover compared to the wild type enzyme but little is known regarding the effect this variant form may have on the occurrence of drug-drug interactions. To examine this possibility, the effect of the potent CYP2C9 inhibitor benzbromarone was studied with regard to CYP2C9.1- and CYP2C9.3-mediated flurbiprofen metabolism to evaluate whether the variant enzyme exhibits differential inhibition kinetics. While benzbromarone inhibited CYP2C9.1 activity as expected, CYP2C9.3-mediated flurbiprofen 4'-hydroxylation was activated in the presence of benzbromarone. T₁ relaxation studies revealed little change in distances of flurbiprofen protons from the heme-iron of either CYP2C9.1 or CYP2C9.3 in the presence of benzbromarone as compared to flurbiprofen alone. Spectral binding studies were also performed to investigate whether benzbromarone affected substrate binding, with addition of benzbromarone having little effect on flurbiprofen binding affinity in both CYP2C9.1 and CYP2C9.3. Docking studies with the 2C9.1 structure crystallized with a closed active site identified multiple, but overlapping sub-sites with sufficient space for benzbromarone binding in the enzyme when flurbiprofen was positioned nearest to the heme. If the closed conformation of 2C9.3 is structurally similar to 2C9.1, as expected for the conservative I359L mutation, then the dynamics of benzbromarone binding may account for the switching of drug interaction effects. In conclusion, the I359L amino acid substitution found in CYP2C9.3 not only reduces metabolism compared to CYP2C9.1, but can also dramatically alter inhibitor effects suggesting that differential degrees of drug inhibition interactions may occur in individuals with this variant form of CYP2C9.

Key words: Cytochrome P450, Genetics, Structure/function/mechanism

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