CYP2D6 CATALYZES TAMOXIFEN 4-HYDROXYLATION IN HUMAN LIVER

The major metabolites of tamoxifen (tam) formed by animal and human li ver 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 P4 50s, whereas the N-oxide is primarily formed by flavin-containing mono oxygenase. Because 4-OH-tam is a highly potent antiestrogen (and possi bly is the active anticancer tam metabolite) and is on the path of for mation of the reactive intermediate that binds covalently to proteins and DNA, it was of importance to identify the P450(s) catalyzing its f ormation, In the current study, three different preparations of expres sed human P450s in Eschericia coli, lymphoblastoma cells, and insect c ell line and livers from several human donors were used to identify th e P450 isoform catalyzing the 4-hydroxylation (preliminary results wer e reported by Dehal et al., Eleventh International Symposium on Micros omes and Drug Oxidations, p, 71, Los Angeles, 1996), Tam metabolism wa s examined with human CYP2C8, 2C9, 2C18, 2C19, and 2D6 expressed in E. coli, These P450s were reconstituted with P450 reductase and lipid an d were incubated with 50 mu M [H-3]tam and NADPH at 37 degrees C for 6 0 min. Essentially all of the recombinant P450s catalyzed the N-demeth ylation to various degrees; however, only 2D6 yielded detectable level s of 4-OH-tam. The inclusion of cytochrome b(5) in the reconstituted s ystem of 2D6 and 2C9 did not significantly affect the rate of 4-hydrox ylation, indicating that b(5) is not essential for this activity, Tam metabolism by CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2D6, 2E1, and 3A4, expr essed in lymphoblastoma cells, revealed that only 2D6 significantly ca talyzed the 4-hydroxylation. Tam metabolism by CYP2D6 coexpressed with P450 reductase in a baculovirus infected insect cell line (''supersom es'') exhibited marked tam 4-hydroxylation, In an experiment with huma n liver microsomes, the inclusion of quinidine, a specific 2D6 inhibit or, resulted in approximately 50% inhibition of tam 4-hydroxylation wi thout affecting N-demethylation, Polyclonal antibodies raised against 2D6 moderately inhibited (approximately 30%) the 4-hydroxylation in hu man liver microsomes, These results demonstrate a significant contribu tion by CYP2D6 to the catalysis of tam-4-hydroxylation by human liver.