Metabolism of (-)-fenchone by CYP2A6 and CYP2B6 in human liver microsomes

The in vitro metabolism of (-)-fenchone was examined in human liver microsomes and recombinant enzymes. The biotransformation of (-)-fenchone was investigated by gas chromatography-mass spectrometry. (-)-Fenchone was found to be oxidized to 6-exo-hydroxyfenchone, 6-endo-hydroxyfenchone and 10-hydroxyfenchone by human liver microsomal P450 enzymes. The formation of metabolites was determined by the relative abundance of mass fragments and retention times on gas chromatography (GC). CYP2A6 and CYP2B6 were major enzymes involved in the hydroxylation of (-)-fenchone by human liver microsomes, based on the following lines of evidence. First, of 11 recombinant human P450 enzymes tested, CYP2A6 and CYP2B6 catalysed the oxidation of (-)-fenchone. Second, oxidation of (-)-fenchone was inhibited by thioTEPA and (+)-menthofuran. Finally, there was a good correlation between CYP2A6, CYP2B6 contents and (-)-fenchone hydroxylation activities in liver microsomes of 11 human samples. CYP2A6 may be more important than CYP2B6 in human liver microsomes. Kinetic analysis showed that the V-max/K-m values for (-)fenchone 6-endo-, 6-exo- and 10-hydroxylation catalysed by liver microsomes of human sample HG-03 were 24.3, 44.0 and 1.3nM(-1) min(-1), respectively. Human recombinant CYP2A6 and CYP2B6 catalysed (-)-fenchone 6-exo-hydroxylation with V-max values of 2.7 and 12.9 nmol min(-1) nmol(-1) P450 and apparent K-m values of 0.18 and 0.15 mM and (-)-fenchone 6-endo-hydroxylation with Vmax values of 1.26 and 5.33 nmol min(-1) nmol(-1) P450 with apparent K. values of 0.29 and 0.26 mM. (-)-Fenchone 10-hydroxylation was catalysed by CYP2B6 with K-m and V-max values of 0.2mM and 10.66 nmol min(-1) nmol(-1) P450, respectively.