Identification of cytochromes P450 2C9 and 3A4 as the major catalysts of phenprocoumon hydroxylation in vitro.

OBJECTIVE: This in-vitro study aimed at an identification of cytochrome P(450) (CYP) enzymes catalysing the (S)- and (R)-hydroxylation of the widely used anticoagulant phenprocoumon (PPC) to its major, inactive metabolites. METHODS: Relevant catalysts were identified by kinetic, correlation and inhibition experiments using human liver microsomes and recombinant enzymes. RESULTS: Kinetics revealed (S)-7-hydroxylation as quantitatively most important. Biphasic Eadie-Hofstee plots indicated more than one catalyst for the 4'-, 6- and 7-hydroxylation of both enantiomers with mean K(m1) and K(m2) of 144.5+/-34.9 and 10.0+/-6.49 microM, respectively. PPC hydroxylation rates were significantly correlated with CYP2C9 and CYP3A4 activity and expression analysing 11 different CYP-specific probes. Complete inhibition of PPC hydroxylation was achieved by combined addition of the CYP3A4-specific inhibitor triacetyloleandomycin (TAO) and a monoclonal, inhibitory antibody (mAb) directed against CYP2C8, 9, 18 and 19, except for the (R)-4'-hydroxylation that was, however, inhibited by ~80% using TAO alone. (S)-PPC hydroxylation was reduced by approximately 2/3 and approximately 1/3 using mAb2C8-9-18-19 and TAO, respectively, but (R)-6- and 7-hydroxylation by approximately 50% each. Experiments with mAbs directed against single CYP2C enzymes clearly indicated CYP2C9 as a major catalyst of the 6- and 7-hydroxylation for both enantiomers. However, CYP2C8 was equally important regarding the (S)-4'-hydroxylation. Recombinant CYP2C8 and CYP2C9 were high-affinity catalysts (K(m) <5 microM), whereas CYP3A4 operated with low affinity (K(m) >100 microM). CONCLUSION: CYP2C9 and CYP3A4 are major catalysts of (S)- and (R)-PPC hydroxylation, while CYP2C8 partly catalysed the (S)-4'-hydroxylation. Increased vigilance is warranted when PPC treatment is combined with substrates, inhibitors, or inducers of these enzymes.