Identification of the Human P450 Isoforms Involved in the Stereoselective Metabolism of Mianserin Enantiomers

Abstract

To identify cytochrome P450 isoform(s) involved in 8-hydroxylation, N-demethylation and N-oxidation of mianserin (US) enantiomers in humans, we studied the metabolism of S(+) and R(-)-MS using human liver microsomes and 6 different recombinant human P450 isoforms (i.e., CYP1A2, 2A6, 2B6, 2D6, 2E1 and 3A4). The rates of 8-hydroxylation and N-oxidation of MS were faster for S(+) than those of R(-)-enantiomer of MS, while that of N-demethylation of MS was faster for R(-) than S(+)-MS in human liver microsomes. When the correlation of the activities of individual forms of P450 with 8-hydroxylation. N-demethylation and N-oxidation of MS at low (5μM) and high (200μM) substrate concentration of MS were evaluated in 10 different human liver microsomes, there were significant correlations between 2-hydroxylation of desipramine and 8-hydroxylation of S(+)-MS (r=0.94, P<.01), between O-deethylation of phenacet i n and N-demethylation of S(+) (r=0.82. P<.05), R(-)-MS (r = 0.71. P<.05) or N-oxidation of S(+)-MS (r=0.95. P<.01) at low concentration of MS. and between 60-hydroxylation of teststerone and 8-hydroxylation, N-demethylation or N-oxidation of S(+)and R(-)-MS (r=0.68 ^ 0.93, P<.05) at high concentration of MS. Quinidine. a selective inhibitor of CYP2D6, inhibited 8-hydroxylation of S(+) and R(-)-MS by approximately 15 and 30%. respectively, while α-naphthoflavone, a selective inhibitor of CYP1A subfamily, inhibited N-demethylation and N-oxidation of S(+) and R(-)-MS by approximately 40 to 60% in human liver microsomes. In addition, troleandamycin, a inhibitor for CYP3A. inhibited 8-hydroxylation, N-demethylation and N-oxidation of S(+) and R(-)-MS by 30 to 80% in human liver microsomes. Among the 6 differnt recombinant human CYP isoforms, CYP2D6. CYP3A4 CYP1A2 and CYP286 catalyzed 8-hydroxylation of both MS enantiomers, while CYP1A2, CYP2B6, CYP2D6 and CYP3A4 showed catalytic activities for N-demethylation of both enantiomers of MS. Catalytic activities for N-oxidation of both enantiomers of MS were detected with CYP3A4 and CYP1A2. The results obtained from human liver microsomes and recombinant P450 isoforms suggested that 8-hydroxylation of MS is catalysed mainly by CYP2D6 and CYP3A4 and at least to some extent by CYP1A2 and CYP286, N-demethylation by CYP1A2, CYP286, CYP2D6 and CYP3A4.and N-oxidation by CYP1A2 and CYP3A4. Therfore, several P450 isoforms appear to be involved in major metabolic pathways of MS enantiomers in human. Since these isoforms have a different stereoselectivity for the metabolism of MS, stereoselective metabolism of MS observed in vivo in human appears to be determined by the sum of stereoselectivities of the different isoforms of P450 responsible for the metabolism of MS enantiomers.