Enzyme source effects on CYP2C9 kinetics and inhibition.
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.
Kumar
Vikas
V
Department of Experimental and Clinical Pharmacology, University of Minnesota, Minneapolis, MN, USA.
Rock
Dan A
DA
Warren
Chad J
CJ
Tracy
Timothy S
TS
Wahlstrom
Jan L
JL
D001189:Aryl Hydrocarbon Hydroxylases / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D016367:Baculoviridae / Q000201:enzymology / Q000235:genetics; D065729:Cytochrome P-450 CYP2C9; D003579:Cytochrome Reductases / Q000737:chemistry; D015786:Cytochromes b5 / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology; D011485:Protein Binding; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D015786:Cytochromes b5; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; D003579:Cytochrome Reductases