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

• Published in: European journal of clinical pharmacology Vol. 60; no. 3; pp. 173 - 182
• Main Authors: UFER, Mike, SVENSSON, Jan O, KRAUSZ, Kristopher W, GELBOIN, Harry V, RANE, Anders, TYBRING, Gunnel
• Format: Journal Article
• Language: English
• Published: Heidelberg Springer 01-05-2004; Berlin Springer Nature B.V
• Subjects: Antibodies, Monoclonal - pharmacology; Anticoagulants - pharmacology; Aryl Hydrocarbon Hydroxylases - antagonists & inhibitors; Aryl Hydrocarbon Hydroxylases - chemistry; Aryl Hydrocarbon Hydroxylases - genetics; Aryl Hydrocarbon Hydroxylases - metabolism; Biological and medical sciences; Biotransformation - drug effects; Biotransformation - physiology; Catalysis - drug effects; Cytochrome P-450 CYP2C19; Cytochrome P-450 CYP2C8; Cytochrome P-450 CYP2C9; Cytochrome P-450 CYP3A; Cytochrome P-450 Enzyme System - chemistry; Cytochrome P-450 Enzyme System - genetics; Cytochrome P-450 Enzyme System - metabolism; Drug Therapy, Combination; Enzyme Inhibitors - pharmacology; Forecasting; Gene Expression - drug effects; Gene Expression - genetics; Humans; Hydroxylation; Isomerism; Medical sciences; Medicin och hälsovetenskap; Microsomes, Liver - drug effects; Microsomes, Liver - enzymology; Mixed Function Oxygenases - antagonists & inhibitors; Mixed Function Oxygenases - genetics; Mixed Function Oxygenases - metabolism; Pharmacology. Drug treatments; Phenprocoumon - chemistry; Phenprocoumon - metabolism; Phenprocoumon - pharmacology; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Troleandomycin - pharmacology; Human; Hydroxylation; Enzyme; Isozyme; Cytochrome P450; Anticoagulant; Metabolism; In vitro; Antivitamin K; Cytochrome P450 2C9; Pharmacokinetics; Phenprocoumon; Cytochrome P450 3A4
• ISSN: 0031-6970; 1432-1041
• DOI: 10.1007/s00228-004-0740-5

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.
Relevant catalysts were identified by kinetic, correlation and inhibition experiments using human liver microsomes and recombinant enzymes.
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).
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.