Formation of (R)-8-hydroxywarfarin in human liver microsomes. A new metabolic marker for the (S)-mephenytoin hydroxylase, P4502C19.
Kinetic studies demonstrate that two forms of human liver cytochrome P450 are responsible for the formation of (R)-8-hydroxywarfarin: a low-affinity enzyme (KM approximately 1.5 mM), previously identified as P4501A2; and a high-affinity enzyme (KM = 330 microM), now identified as P4502C19 on the basis of the following evidence. In crossover inhibition studies with P4501A2-depleted human liver microsomes between (R)-warfarin and (S)-mephenytoin, reciprocal competitive inhibition was observed. Apparent KM values for (S)-mephenytoin-4'-hydroxylation (52-67 microM) were similar to the determined Ki values (58-62 microM) for (S)-mephenytoin inhibition of (R)-8-hydroxywarfarin formation. Similarly, the apparent KM for (R)-warfarin 8-hydroxylation in furafylline-pretreated microsomes (KM = 289-395 microM) was comparable with the Ki values (280-360 microM) for (R)-warfarin inhibition of (S)-4'-hydroxymephenytoin formation. Inhibition studies with tranylcypromine, a known inhibitor of (S)-mephenytoin hydroxylase activity, and either substrate in three different microsomal preparations yielded nearly identical inhibitory constants: Ki = 8.7 +/- 1.6 microM for inhibition of (S)-4'-hydroxymephenytoin formation and 8.8 +/- 2.5 microM for inhibition of (R)-8-hydroxywarfarin formation. In addition, fluconazole, a potent inhibitor of (R)-warfarin 8-hydroxylation, Ki = 2 microM, was found to inhibit (S)-mephenytoin hydroxylation with an identical Ki (2 microM). Finally, a strong correlation between (S)-mephenytoin 4-hydroxylation and (R)-warfarin 8-hydroxylation activities in furafylline-pretreated microsomes was demonstrated in 14 human liver microsomal preparations (r2 = 0.97).
Wienkers
L C
LC
Department of Medicinal Chemistry, University of Washington, Seattle 98195, USA.
Wurden
C J
CJ
Storch
E
E
Kunze
K L
KL
Rettie
A E
AE
Trager
W F
WF
D001189:Aryl Hydrocarbon Hydroxylases; D015415:Biomarkers; D065731:Cytochrome P-450 CYP2C19; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015725:Fluconazole / Q000494:pharmacology; D006801:Humans; D006900:Hydroxylation; D007700:Kinetics; D008617:Mephenytoin / Q000031:analogs & derivatives / Q000494:pharmacology; D008862:Microsomes, Liver / Q000201:enzymology / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D008996:Monoamine Oxidase Inhibitors / Q000494:pharmacology; D013806:Theophylline / Q000031:analogs & derivatives / Q000494:pharmacology; D014191:Tranylcypromine / Q000494:pharmacology; D014859:Warfarin / Q000031:analogs & derivatives* / Q000378:metabolism*
D015415:Biomarkers; D004791:Enzyme Inhibitors; D008996:Monoamine Oxidase Inhibitors; C101436:8-hydroxywarfarin; D014191:Tranylcypromine; D014859:Warfarin; C025687:4-hydroxymephenytoin; D015725:Fluconazole; D003577:Cytochrome P-450 Enzyme System; D013806:Theophylline; C050131:furafylline; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; C045793:CYP2C19 protein, human; D065731:Cytochrome P-450 CYP2C19; D008617:Mephenytoin