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0026-895X/05/6803-644 –651$20.00 MOLECULAR PHARMACOLOGY Copyright © 2005 The American Society for Pharmacology and Experimental Therapeutics Mol Pharmacol 68:644–651, 2005

Vol. 68, No. 3 13763/3048546 Printed in U.S.A.

CYP2C9 Genotype-Dependent Effects on in Vitro Drug-Drug Interactions: Switching of Benzbromarone Effect from Inhibition to Activation in the CYP2C9.3 Variant
Matthew A.Hummel, Charles W. Locuson, Peter M. Gannett, Dan A. Rock, Carrie M. Mosher, Allan E. Rettie, and Timothy S. Tracy
Department of Experimental and Clinical Pharmacology, University of Minnesota, Minneapolis, Minnesota (M.A.H., C.W.L., T.S.T.); Department of Basic Pharmaceutical Sciences, West Virginia University, Morgantown, West Virginia (P.M.G.); Global Pharmacokinetics, Dynamics, and Metabolism,Pfizer, Inc., St. Louis, Missouri (D.A.R.); and Department of Medicinal Chemistry, University of Washington, Seattle, Washington (C.M.M., A.E.R.)
Received April 13, 2005; accepted June 13, 2005

ABSTRACT The CYP2C9.3 variant exhibits marked decreases in substrate turnover compared with the wild-type enzyme, but little is known regarding the effect this variant form may have on the occurrence ofdrug-drug interactions. To examine this possibility, the effect of the potent CYP2C9 inhibitor, benzbromarone, was studied with regard to CYP2C9.1- and CYP2C9.3mediated flurbiprofen metabolism to evaluate whether the variant enzyme exhibits differential inhibition kinetics. Although benzbromarone inhibited CYP2C9.1 activity as expected, CYP2C9.3-mediated flurbiprofen 4 -hydroxylation was activatedin 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 compared with flurbiprofen alone. Spectral binding studies were also performed to investigate whether

benzbromarone affected substrate binding, with the addition of benzbromarone having littleeffect 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 subsites with sufficient space for benzbromarone binding in the enzyme when flurbiprofen was positioned closest to the heme. If the closed conformation of 2C9.3 is structurally similar to 2C9.1, as expected for theconservative 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 with CYP2C9.1 but can also dramatically alter inhibitor effects, suggesting that differential degrees of drug inhibition interactions may occur in individuals with thisvariant form of CYP2C9.

Prediction of drug interactions involving the cytochrome P450 (P450) enzyme system requires an accurate estimation of the inhibition kinetics. New compounds are routinely tested in an in vitro system using liver microsomes or purified enzymes against P450 isoform-specific probe substrates to gauge which and to what extent a particular P450 isoform is inhibited. Oneprobe substrate is often selected for each P450 isoform. However, it is known that the CYP2C9 and CYP3A4 isoforms exhibit atypical kinetic profiles, presumably because of the binding of more than one molecule within
This study was supported in part by National Institutes of Health grants GM069753 and GM063215 (T.S.T) and GM032165 (A.E.R.), the University of Minnesota School of Medicine NationalScience Foundation grant BIR-961477, and the Minnesota Medical Foundation. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.105.013763.

its active site (Shou et al., 1994, 1999, 2001; Hutzler et al., 2001, 2003; Galetin et al., 2002; Nakamura et al., 2002). The crystal structure of CYP2C9 has been solved and has facilitated...