Polymorphism in stereoselective hydroxylations of mephenytoin and hexobarbital by Japanese liver samples in relation to cytochrome P-450 human-2 (IIC9)

Abstract

1. Stereoselective 4′-hydroxylations of R-(—)-mephenytoin and S-(+)-mephenytoin were determined in liver microsomes of 19 Japanese subjects.

2. The content of P-450 human-2 assessed by Western blots correlated with micro-somal S-(+)-mephenytoin 4′-hydroxylation. Antibody raised against P-450 human-2 effectively inhibited microsomal S-(+)-mephenytoin 4′-hydroxylation, but was less efficient for inhibition of R-(—)-mephenytoin 4′-hydroxylation in extensive metabolizers, and 4′-hydroxylation of both mephenytoin enantiomers in poor metabolizers.

3. Similar results were observed on the stereoselective hydroxylations of R-(+)- and S-(+)-hexobarbital. Clear correlations were observed for the content of P-450 human-2 and microsomal R-(—)-hexobarbital 3′α-hydroxylation and S-(+)-hexobarbital 3′β-hydroxylation.

4. Moreover, yeast microsomes expressing P-450 human-2 cDNA showed high stereoselectivities for hydroxylations of mephenytoin and hexobarbital similar to those observed in human liver.

5. Two other cytochromes P-450(IIC 9/10) expressed in yeast, whose cDNA were synthesized by site-directed mutagenesis from human-2 cDNA, showed no stereo-selectivity for the hydroxylations of mephenytoin and hexobarbital, in spite of the modification of only two amino acid substitutions or deletions in the whole sequence.

6. Only a cytochrome derived from P-450 human cDNA corresponding to P-450 human-2 was expressed in human livers, the two cytochromes of the three related IIC9/10 forms were not expressed.

7. These findings indicate that P-450 human-2 is the major cytochrome P-450 responsible for the polymorphisms in stereoselective hydroxylations of mephenytoin and hexobarbital.