Previous kinetic studies have identified a high-affinity (S)-warfarin 7-hydroxylase present in human liver microsomes which appears to be responsible for the termination of warfarin's biological activity. Inhibition of the formation of (S)-7-hydroxywarfarin, the inactive, major metabolite of racemic warfarin in humans, is known to be the cause of several of the drug interactions experienced clinically upon coadministration of warfarin with other therapeutic agents. In order to identify the specific form(s) of human liver cytochrome P-450 involved in this particular toxicity, we have determined the metabolic profiles of 11 human cytochrome P-450 forms expressed in HepG2 cells toward both (R)- and (S)-warfarin. Of the 11 forms examined only 2C9 displayed the regioselectivity and stereoselectivity appropriate for the high-affinity human liver microsomal (S)-7-hydroxylase. We further compared Michaelis-Menten and sulfaphenazole inhibition constants for (S)-warfarin 7-hydroxylation catalyzed by cDNA-expressed 2C9 and by human liver microsomes. Similar kinetic constants were obtained for each enzyme source. It is concluded that 2C9 is likely to be a principal form of human liver P-450 which modulates the in vivo anticoagulant activity of the drug. It is further concluded that those drug interactions with warfarin that arise as a result of decreased clearance of the biologically more potent S-enantiomer may have as their common basis the inhibition of P-450 2C9.

Amiodarone decreased the total body clearance of both (R)- and (S)-warfarin in normal subjects but did not change volumes of distribution. Warfarin excretion products were quantified and clearance and formation clearance values calculated. Amiodarone and metabolites inhibited the reduction of (R)-warfarin to (R,S)-warfarin alcohol-1 and the oxidation of both (R)- and (S)-warfarin to phenolic metabolites. Inhibition of warfarin hydroxylation by amiodarone in human liver microsomes was compared with the in vivo results. In agreement, the in vitro data indicates that amiodarone is a general inhibitor of the cytochrome P450 catalyzed oxidation of both enantiomers of warfarin, but the metabolism of (S)-warfarin is more strongly inhibited than that of (R)-warfarin. These data suggest that the enhanced anticoagulant effect observed when amiodarone and warfarin are coadministered is attributable to inhibition of P4502C9, the isozyme of P-450 primarily responsible for the conversion of (S)-warfarin to its major metabolite, (S)-7-hydroxywarfarin.

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