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 P450 (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 Km1 and Km2 of 144.5±34.9 and 10.0±6.49 µM, 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 ~2/3 and ~1/3 using mAb2C8–9-18–19 and TAO, respectively, but (R)-6- and 7-hydroxylation by ~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 (Km <5 µM), whereas CYP3A4 operated with low affinity (Km >100 µM). 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. [ABSTRACT FROM AUTHOR]