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 P 450 are responsible for the formation of (R)-8-hydroxywarfarin: a low- affinity enzyme (K-M similar to 1.5 mM), previously identified as P450 1A2; and a high-affinity enzyme (K-M = 330 mu M), now identified as P4 502C19 on the basis of the following evidence. In cross-over inhibitio n studies with P4501A2-depleted human liver microsomes between (R)-war farin and (S)-mephenytoin, reciprocal competitive inhibition was obser ved. Apparent K-M values for (S)-mephenytoin-4'-hydroxylation (52-67 m u M) were similar to the determined K-i values (58-62 mu M) for (S)-me phenytoin inhibition of (R)-8-hydroxywarfarin formation. Similarly, th e apparent K-M for (R)-warfarin 8-hydroxylation in furafylline-pretrea ted microsomes (K-M = 289-395 mu M) was comparable with the K-i values (280-360 mu M) for (R)-warfarin inhibition of (S)-4'-hydroxymephenyto in formation. Inhibition studies with tranylcypromine, a known inhibit or of (S)-mephenytoin hydroxylase activity, acid either substrate in t hree different microsomal preparations yielded nearly identical inhibi tory constants: K-i = 8.7 +/- 1.6 mu M for inhibition of (S)-4'-hydrox ymephenytoin formation and 8.8 +/- 2.5 mu M for inhibition of (R)-8-hy droxywarfarin formation. In addition, fluconazole, a potent inhibitor of (R)-warfarin 8-hydroxylation, K-i = 2 mu M, was found to Inhibit (S )-mephenytoin hydroxylation with an identical K-i (2 mu M). Finally, a strong correlation between (S)-mephenytoin 4-hydroxylation and (R)-wa rfarin 8-hydroxylation activities in furafylline-pretreated microsomes was demonstrated in 14 human liver microsomal preparations (r(2) = 0. 97).