INVOLVEMENT OF CYTOCHROME-P450 3A ENZYME FAMILY IN THE MAJOR METABOLIC PATHWAYS OF TOREMIFENE IN HUMAN LIVER-MICROSOMES

The anti-estrogen toremifen-Fc-1157a or 1-[4-[2(N,N-dimethylamino)etho xy]-phenyl]-1-butene is now used for the treatment of breast cancer. T his drug is extensively metabolized by cytochrome P450 dependent hepat ic mixed function oxidase in man, yielding mainly the N-demethyl-(DMTO R), 4-hydroxy-(4OH-TOR) and deamino-hydroxy-(TOR III) toremifene metab olites. The specific forms of cytochrome P450 involved in these oxidat ion reactions were examined in 32 human liver microsomal preparations previously characterized with respect to their contents of several kno wn P450 enzymes. Toremifene was demethylated with an apparent K-m of 1 24 mu M while it was hydroxylated with an apparent K-m of 139 mu M. Th e metabolic rates were 71 +/- 56, 13 +/- 9 and 15 +/- 4 pmol/min/mg mi crosomal protein, respectively, for DMTOR, 4-OH-TOR and TOR III. The N -demethylation activity was strongly correlated with estradiol 2-hydro xylation (r = 0.75), nifedipine oxidation (r = 0.86), tamoxifen N-deme thylation (r = 0.73), testosterone 6 beta-hydroxylation (r = 0.78) and erythromycin N-demethylation (r = 0.84), all these monooxygenase acti vities known to be supported by CYP3A4 isoform. Furthermore, the CYP3A content of liver microsomal samples, measured by western blot analysi s using a monoclonal anti-human CYP3A4 antibody, was strongly correlat ed with DMTOR formation (r = 0.80). Compounds such as cyclosporin, tri acetyl-oleandomycin and testosterone inhibited the N-demethylation of toremifene metabolism at 80, 89 and 56% vs control, respectively, whil e the formation of TOR III was inhibited at 78, 82 and 73% vs control and the 4-hydroxylation pathway was inhibited no more than about 50% v s control. Prior incubation of microsomes with 100 mu M gestodene, kno wn to be a selective mechanism-based inhibitor of CYP3A4 in the presen ce of NADPH, led to 76 +/- 6 and 76 +/- 5% (N = 5 samples) reductions in the N-demethylation and formation of TOR III, respectively. Polyclo nal antibody directed against human CYP3A enzymes inhibited formation of DMTOR and TOR III by 60 and 46%, respectively. The metabolism of to remifene was not activated by cu-naphthoflavone. Finally, the use of y easts genetically engineered for expression of human P4501A1, 1A2, 2C9 and 3A4 allowed us to demonstrate that DMTOR and TOR III formations a re mediated by P4501A and 3A4 enzymes and by contrast these enzymes ar e not involved in the 4-hydroxylation pathway. All these results taken together suggest that the major metabolic pathways of toremifene, nam ely N-demethylation and TOR III formation, are mediated mainly by cyto chrome CYP3A4 enzyme in the human liver microsomes owing to the relati ve hepatic contents in P4501A and 3A.