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).


CONCLUSIONS
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.
Ufer
Mike
M
Department of Laboratory Medicine, Division of Clinical Pharmacology, Karolinska Institutet, Huddinge University Hospital, Stockholm, Sweden. mikeufer@gmx.de
Svensson
Jan O
JO
Krausz
Kristopher W
KW
Gelboin
Harry V
HV
Rane
Anders
A
Tybring
Gunnel
G
D000911:Antibodies, Monoclonal / Q000494:pharmacology; D000925:Anticoagulants / Q000494:pharmacology; D001189:Aryl Hydrocarbon Hydroxylases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001711:Biotransformation / Q000187:drug effects / Q000502:physiology; D002384:Catalysis / Q000187:drug effects; D065731:Cytochrome P-450 CYP2C19; D065727:Cytochrome P-450 CYP2C8; D065729:Cytochrome P-450 CYP2C9; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004359:Drug Therapy, Combination; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005544:Forecasting; D015870:Gene Expression / Q000187:drug effects / Q000235:genetics; D006801:Humans; D006900:Hydroxylation; D007536:Isomerism; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D010644:Phenprocoumon / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014217:Troleandomycin / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D000925:Anticoagulants; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D014217:Troleandomycin; D006899:Mixed Function Oxygenases; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; C082185:CYP2C18 protein, human; C045793:CYP2C19 protein, human; C450259:CYP2C8 protein, human; C104464:CYP3A protein, human; D065731:Cytochrome P-450 CYP2C19; D065727:Cytochrome P-450 CYP2C8; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D010644:Phenprocoumon