4--hydroxywarfarin and 8-hydroxywarfarin

Coumadin (R/S-warfarin) is a highly efficacious and widely used anticoagulant; however, its highly variable metabolism remains an important contributor to uncertainties in therapeutic responses. Pharmacogenetic studies report conflicting findings on the clinical relevance of CYP2C19. A resolution to this controversy is impeded by a lack of de tailon the potential role of CYP2C19 in warfarin metabolism. Consequently, we assessed the efficiency of CYP2C19 metabolism of R- and S-warfarin and explored possible contributions in the liver using in vitro methods. Recombinant CYP2C19 metabolized R- and S-warfarin mainly to 6-, 7-, and 8-hydroxywarfarin, while 4'-hydroxywarfarin was a minormetabolite. Over all R-warfarin metabolism was slightly more efficient than that for S-warfarin. Metabolic pathways thatproduce R-6-, 7-, and 8-hydroxywarfarin in human liver microsomal reactions correlated strongly with CYP2C19 Smephenytoinhydroxylase activity. Similarly, CYP1A2 activity toward phenacetin correlated with formation of R-6 and 7-hydroxywarfarin such that R-8-hydroxywarfarin seems unique to CYP2C19 and possibly a biomarker. In following, CYP2C19 likely impacts R-warfarin metabolism and patient response to therapy. Intriguingly, CYP2C19 may contributeto S-warfarin metabolism in patients, especially when CYP2C9 activity is compromised due to drug interactions orgenetic polymorphisms.

Topics: Anticoagulants; Aryl Hydrocarbon Hydroxylases; Cytochrome P-450 CYP2C19; Female; Humans; Male; Microsomes, Liver; Pharmacogenetics; Stereoisomerism; Warfarin

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.

Topics: Anticoagulants; Aryl Hydrocarbon Hydroxylases; Cytochrome P-450 CYP2C9; Humans; Kinetics; Microsomes, Liver; Recombinant Proteins; Stereoisomerism; Warfarin