METABOLISM OF THE ANTIMAMMARY CANCER ANTIESTROGENIC AGENT TAMOXIFEN .1. CYTOCHROME P-450-CATALYZED N-DEMETHYLATION AND 4-HYDROXYLATION

Previous studies suggested that the therapeutic effect of the antimamm ary cancer agent tamoxifen might be related to its metabolism. This st udy examined the cytochrome P-450 enzymes in rat and human liver catal yzing the metabolism of tamoxifen. Incubations of tamoxifen with rat l iver microsomes yielded three major polar metabolites identified as th e N-oxide, N-desmethyl, and 4-hydroxy derivatives. N-Oxide formation w as catalyzed by the flavin-containing monooxygenase (see part II). Car bon monoxide, SKF-525A, metyrapone, and benzylimidazole strongly inhib ited N-demethylation and 4-hydroxylation, indicating the participation of P-450 monooxygenase in these reactions. Antibodies to NADPH-P450 r eductase inhibited N-demethylation and 4-hydroxylation. Comparison of the metabolism of tamoxifen in untreated male and female rats demonstr ated some sexual dimorphism. N-Demethylation was higher in the male ra t and 4-hydroxylation was higher in the female. Treatment of rats with phenobarbital (PB), pregnenolone-16alpha-carbonitrile (PCN), and meth ylcholanthrene ( MC) enhanced N-demethylation, demonstrating the poten tial participation of multiple P-450s in N-demethylation. Evidence str ongly indicates that CYP3A enzyme(s) catalyzes N-demethylation in live r microsomes of PB- and PCN-treated rats (PB and PCN microsomes, respe ctively): i) N-demethylation was inhibited by cortisol and erythromyci n (alternate substrates) and a time-dependent inhibition was observed with troleandomycin (TAO) in vitro; ii) treatment of female rats with TAO, followed by dissociation of the microsomal TAO-P-450 complex, ele vated N-demethylation; iii) treatment of PCN-induced female rats with chloramphenicol inhibited N-demethylation; and iv) polyclonal antibodi es (PAbs) to CYP3A1 inhibited N-demethylation in PCN- and PB-treated f emale rats. Although we were unable to reconstitute the N-demethylatio n activity with purified CYP3A1, which is difficult to reconstitute, c ollectively the evidence demonstrated that CYP3A enzymes catalyze N-de methylation in PB and PCN microsomes. By contrast, antibodies against CYP2B1/B2 did not inhibit N-demethylation and reconstituted 2B1 did no t catalyze N-demethylation of tamoxifen, indicating that 2B1 was not i nvolved. The increase in N-demethylation by MC treatment appears to be due to elevation of CYP1A1/1A2 (P-450c/d). Alternate substrates of CY P1A1/1A2 inhibited N-demethylation and reconstituted rat CYP 1A1-catal yzed N-demethylation. Surprisingly, monoclonal antibodies (MAbs) again st CYP1A1/1A2 only partially inhibited, and PAbs against CYP1A1 did no t inhibit N-demethylation in MC microsomes, indicating that in MC micr osomes, 1A1 does not contribute significantly to that reaction. MAb an ti-CYP2C11/2C6 (P-450h/k) inhibited N-demethylation in PB, PCN, and co ntrol male rat liver microsomes, suggesting that CYP2C11 and/or CYP2C6 catalyze this reaction to some extent. Human liver microsomes formed tamoxifen metabolites at a much lower rate than rats. Inhibitors of P- 450 diminished the formation of N-desmethyl and 4-hydroxy metabolites in human liver microsomes. Cortisol and erythromycin inhibited N-demet hylation, but not 4-hydroxylation in human microsomes. Based on these findings, we concluded that tamoxifen N-demethylation is catalyzed in the rat by CYP1A, CYP2C, and CYP3A enzymes and in the human by CYP3A e nzyme(s). By contrast, 4-hydroxylation appears to be catalyzed by cons titutive P-450s: i) inducers of P-450 did not enhance that reaction; i i) neither alternate substrates of induced P-450s nor antibodies to th ese P-450s inhibited that reaction; and iii) neither reconstituted CYP 2B1 nor CYP1A1 catalyzed 4-hydroxylation.