title: Hydroxywarfarin metabolites potently inhibit CYP2C9 metabolism of S-warfarin.
abstract: Coumadin (R/S-warfarin) anticoagulant therapy poses a risk to over 50 million Americans, in part due to interpersonal variation in drug metabolism. Consequently, it is important to understand how metabolic capacity is influenced among patients. Cytochrome P450s (P450 or CYP for a specific isoform) catalyze the first major step in warfarin metabolism to generate five hydroxywarfarins for each drug enantiomer. These primary metabolites are thought to reach at least 5-fold higher levels in plasma than warfarin. We hypothesized that hydroxywarfarins inhibit the hydroxylation of warfarin by CYP2C9, thereby limiting enzymatic capacity toward S-warfarin. To test this hypothesis, we investigated the ability of all five racemic hydroxywarfarins to block CYP2C9 activity toward S-warfarin using recombinant enzyme and human liver microsomes. We initially screened for the inhibition of CYP2C9 by hydroxywarfarins using a P450-Glo assay to determine IC(50) values for each hydroxywarfarin. Compared to the substrate, CYP2C9 bound its hydroxywarfarin products with less affinity but retained high affinity for 10- and 4'-hydroxywarfarins, products from CYP3A4 reactions. S-Warfarin steady-state inhibition studies with recombinant CYP2C9 and pooled human liver microsomes confirmed that hydroxywarfarin products from CYP reactions possess the capacity to competitively inhibit CYP2C9 with biologically relevant inhibition constants. Inhibition of CYP2C9 by 7-hydroxywarfarin may be significant given its abundance in human plasma, despite its weak affinity for the enzyme. 10-Hydroxywarfarin, which has been reported as the second most abundant plasma metabolite, was the most potent inhibitor of CYP2C9, displaying approximately 3-fold higher affinity than S-warfarin. These results indicate that hydroxywarfarin metabolites produced by CYP2C9 and other CYPs may limit metabolic capacity toward S-warfarin through competitive inhibition. Subsequent processing of hydroxywarfarins to secondary metabolites, such as hydroxywarfarin glucuronides, could suppress product feedback inhibition, and therefore could play an important role in the modulation of metabolic pathways governing warfarin inactivation and elimination.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA.
authors.forename: Drew R
authors.initials: DR
authors.lastname: Jones
authors.forename: So-Young
authors.initials: SY
authors.lastname: Kim
authors.forename: Michael
authors.initials: M
authors.lastname: Guderyon
authors.forename: Chul-Ho
authors.initials: CH
authors.lastname: Yun
authors.forename: Jeffery H
authors.initials: JH
authors.lastname: Moran
authors.forename: Grover P
authors.initials: GP
authors.lastname: Miller
mesh_terms: D000925:Anticoagulants / Q000737:chemistry / Q000378:metabolism* / Q000633:toxicity; D001189:Aryl Hydrocarbon Hydroxylases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D065729:Cytochrome P-450 CYP2C9; D006801:Humans; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology / Q000378:metabolism; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D013237:Stereoisomerism; D014859:Warfarin / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism* / Q000633:toxicity
chemical_list: D000925:Anticoagulants; D011994:Recombinant Proteins; C101436:8-hydroxywarfarin; C059853:4'-hydroxywarfarin; D014859:Warfarin; C065719:10-hydroxywarfarin; C087837:7-hydroxywarfarin; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; C008091:6-hydroxywarfarin