Territrems B and C metabolism in human liver microsomes: Major role of CYP3A4 and CYP3A5

Human liver microsomes, supersomes from baculovirus-transformed insect cells expressing different human CYP450 isoforms, and control and CYP3A4 cDNA-transfected V79 Chinese hamster cells were tested for their ability to metabolize territrem B (TRB) and territrem C (TRC). Two TRB metabolites, designated MB2 and MB4, and one TRC metabolite, designated MC, were formed by all of these preparations. Of the nine supersomes from baculovirus-transformed insect cells expressing different human CYP450 isoforms (1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, and 3A5), only those expressing CYP3A4 or CYP3A5 metabolized TRB and TRC. MB2, MB4, and MC were formed by CYP3A4 cDNA-transfected V79MZ Chinese hamster cells, but not by non-transfected cells. In order to investigate which CYP450 isoforms were responsible for MB2, MB4 and MC formation in human liver microsomal preparations, six isoform-specific chemical inhibitors (furafylline, sulfaphenazole, omeprazole, quinidine, ketoconazole, and diethyldithiocarbamate) and antibodies against CYP3A4 were used. MB2, MB4, and MC formation was markedly inhibited by ketoconazole, but less affected by quinidine and sulfaphenazole. Anti-CYP3A4 antibody markedly inhibited MB2, MB4, and MC formation and also 6β-hydroxytestosterone formation from testosterone. The CYP3A-dependent reaction of testosterone 6β-hydroxylation showed a high correlation with 4β-C hydroxylation of TRB (r2 = 0.97, P < 0.0001), O-demethylation of TRB (r2 = 0.95, P < 0.0001), and 4β-C hydroxylation of TRC (r2 = 0.99, P < 0.0001). Immunoblotting and RT-PCR showed that CYP3A4 and CYP3A5 were expressed in all four human liver microsomal preparations tested (HLM1-HLM4). The amount of MB2, MB4, and MC formed using different HLM preparations was related to the 6β-testosterone hydroxylase activity of the preparations. However, the extent of MB2, MB4, and MC formation was not related to the age or gender of the person from whom the microsomal sample was prepared. It was therefore suggest that CYP3A4 and CYP3A5 are the major enzymes responsible for TRB and TRC metabolism by human liver microsomes.