CYP2D6 Catalyzes Tamoxifen 4-Hydroxylation in Human Liver¹

The major metabolites of tamoxifen (tam) formed by animal and human liver microsomes are mono-N-demethylated tam, 4-hydroxy-tam (4-OH-tam), and tam-N-oxide. The N-desmethylated-tam and 4-OH-tam are formed by P450s, whereas the N-oxide is primarily formed by flavin-containing monooxygenase. Because 4-OH-tam is a highly potent antiestrogen (and possibly is the active anticancer tam metabolite) and is on the path of formation of the reactive intermediate that binds covalently to proteins and DNA, it was of importance to identify the P450(s) catalyzing its formation. In the current study, three different preparations of expressed human P450s in Escherichia coli, lymphoblastoma cells, and insect cell line and livers from several human donors were used to identify the P450 isoform catalyzing the 4-hydroxylation (preliminary results were reported by Dehal et al., Eleventh International Symposium on Microsomes and Drug Oxidations, p. 71. Los Angeles, 1996). Tam metabolism was examined with human CYP2C8, 2C9, 2C18, 2C19, and 2D6 expressed in E. coli. These P450s were reconstituted with P450 reductase and lipid and were incubated with 50 µm [³H]tam and NADPH at 37°C for 60 min. Essentially all of the recombinant P450s catalyzed the N-demethylation to various degrees; however, only 2D6 yielded detectable levels of 4-OH-tam. The inclusion of cytochrome b₅ in the reconstituted system of 2D6 and 2C9 did not significantly affect the rate of 4-hydroxylation, indicating that b₅ is not essential for this activity. Tam metabolism by CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2D6, 2E1, and 3A4, expressed in lymphoblastoma cells, revealed that only 2D6 significantly catalyzed the 4-hydroxylation. Tam metabolism by CYP2D6 coexpressed with P450 reductase in a baculovirus infected insect cell line (“supersomes”) exhibited marked tam 4-hydroxy-lation. In an experiment with human liver microsomes, the inclusion of quinidine, a specific 2D6 inhibitor, resulted in approximately 50% inhibition of tam 4-hydroxylation without affecting N-demethylation. Polyclonal antibodies raised against 2D6 moderately inhibited (approximately 30%) the 4-hydroxylation in human liver microsomes. These results demonstrate a significant contribution by CYP2D6 to the catalysis of tam-4-hydroxylation by human liver.