QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) All Versions of this Article: dmd.106.010249v1 34/11/1903    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kumar, V. Articles by Wahlstrom, J. L. Search for Related Content PubMed PubMed Citation Articles by Kumar, V. Articles by Wahlstrom, J. L.

Enzyme Source Effects on CYP2C9 Kinetics and Inhibition

Department of Experimental and Clinical Pharmacology, University of Minnesota, Minneapolis, Minnesota (V.K., T.S.T.); and Global Pharmacokinetics, Dynamics and Metabolism, Pfizer Global Research & Development, St. Louis Laboratories, Pfizer Inc., St. Louis, Missouri (D.A.R., C.J.W., J.L.W.)

When choosing a recombinant cytochrome P450 (P450) enzyme system for in vitro studies, it is critical to understand the strengths, limitations, and applicability of the enzyme system to the study design. Although literature kinetic data may be available to assist in enzyme system selection, comparison of data from separate laboratories is often confounded by differences in experimental conditions and bioanalytical techniques. We measured the Michaelis-Menten kinetic parameters for four CYP2C9 substrates (diclofenac, (S)-warfarin, tolbutamide, and (S)-flurbiprofen) using four recombinant CYP2C9 enzyme systems (Supersomes, Baculosomes, RECO system, and in-house purified, reconstituted enzyme) to determine whether the enzyme systems exhibited kinetic differences in metabolic product formation rates under uniform experimental conditions. The purified, reconstituted enzyme systems exhibited higher K_m values, reduced substrate affinity, and lower calculated intrinsic clearance values compared with baculovirus microsomal preparations. Six- to 25-fold differences in predicted intrinsic clearance values were calculated for each substrate depending on the enzyme system-substrate combination. Results suggest that P450 reductase interactions with the CYP2C9 protein and varying ratios of CYP2C9/P450 reductase in the enzyme preparations may play a role in these observed differences. In addition, when (S)-flurbiprofen was used as a substrate probe to determine CYP2C9 inhibition with a set of 12 inhibitors, decreased inhibition potency was observed across 11 of those inhibitors in the RECO purified, reconstituted enzyme compared with the Supersomes baculovirus microsomal preparation and pooled human liver microsomes. Considering these differences, consistent use of an enzyme source is an important component in producing comparable and reproducible kinetics and inhibition data with CYP2C9.

This article has been cited by other articles:

				A. Ghosal, R. Ramanathan, Y. Yuan, N. Hapangama, S. K. Chowdhury, N. S. Kishnani, and K. B. Alton Identification of Human Liver Cytochrome P450 Enzymes Involved in Biotransformation of Vicriviroc, a CCR5 Receptor Antagonist Drug Metab. Dispos., December 1, 2007; 35(12): 2186 - 2195. [Abstract] [Full Text] [PDF]