OF TAMOXIFEN BY RECOMBINANT HUMAN CYTOCHROME P 450 ENZYMES : FORMATION OF THE 4-HYDROXY , 4-HYDROXY AND N-DESMETHYL METABOLITES AND ISOMERIZATION OF trans-4-HYDROXYTAMOXIFEN

The cytochrome P450 (P450)-mediated biotransformation of tamoxifen is important in determining both the clearance of the drug and its conversion to the active metabolite, trans-4-hydroxytamoxifen. Biotransformation by P450 forms expressed extrahepatically, such as in the breast and endometrium, may be particularly important in determining tissue-specific effects of tamoxifen. Moreover, tamoxifen may serve as a useful probe drug to examine the regioselectivity of different forms. Tamoxifen metabolism was investigated in vitro using recombinant human P450s. Forms CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, 3A5, and 3A7 were coexpressed in Escherichia coli with recombinant human NADPHcytochrome P450 reductase. Bacterial membranes were harvested and incubated with tamoxifen or trans-4-hydroxytamoxifen under conditions supporting P450-mediated catalysis. CYP2D6 was the major catalyst of 4-hydroxylation at low tamoxifen concentrations (170 20 pmol/40 min/0.2 nmol P450 using 18 M tamoxifen), but CYP2B6 showed significant activity at high substrate concentrations (28.1 0.8 and 3.1 0.5 nmol/120 min/0.2 nmol P450 for CYP2D6 and CYP2B6, respectively, using 250 M tamoxifen). These two forms also catalyzed 4 -hydroxylation (13.0 1.9 and 1.4 0.1 nmol/120 min/0.2 nmol P450, respectively, for CYP2B6 and CYP2D6 at 250 M tamoxifen; 0.51 0.08 pmol/40 min/0.2 nmol P450 for CYP2B6 at 18 M tamoxifen). Tamoxifen N-demethylation was mediated by CYP2D6, 1A1, 1A2, and 3A4, at low substrate concentrations, with contributions by CYP1B1, 2C9, 2C19 and 3A5 at high concentrations. CYP1B1 was the principal catalyst of 4-hydroxytamoxifen trans-cis isomerization but CYP2B6 and CYP2C19 also contributed. The nonsteroidal antiestrogen tamoxifen is currently the most widely used chemotherapeutic agent for the adjuvant treatment of breast cancer. It may act, at least in part, as a prodrug, by conversion to the primary metabolite, trans-4-hydroxytamoxifen, which has enhanced binding affinity and potency compared with the parent compound (Katzenellenbogen et al., 1984). Tamoxifen has also been shown to halve the risk of breast cancer in women with high susceptibility to the disease (Fisher et al., 1998). However, its prophylactic benefit is compromised by a small but statistically significant increase in the risk of developing endometrial cancer (International Agency for Research on Cancer, 1996). The P450-mediated biotransformation of tamoxifen is important in determining both the clearance of the drug and its conversion to the active metabolite, trans-4-hydroxytamoxifen. Flavin-containing monooxygenases appear to be responsible for N-oxide formation. Tamoxifen has been shown to be metabolized by human P450s to N-desmethyl, 4-hydroxy, and -hydroxy metabolites. Previous studies have pointed to CYP3A4 being the major catalyst of N-demethylation (Jacolot et al., 1991; Crewe et al., 1997) with potential contributions by CYP1A1 and 1A2 (Simon et al., 1993). Studies in one of our laboratories (Crewe et al., 1997) and others (Dehal and Kupfer, 1997) have suggested that CYP2D6 plays a predominant role in the 4-hydroxylation of tamoxifen, with contributions from CYP2C9 and 3A4. In addition, secondary metabolites resulting from further metabolism of these derivatives may be formed by phase I processes. P450catalyzed isomerization of trans-4-hydroxytamoxifen has been reported (Williams et al., 1994), resulting in the conversion of a potent antiestrogen (trans-4-hydroxytamoxifen) into a weak estrogen agonist (cis-4-hydroxytamoxifen). Clinical resistance to tamoxifen therapy has been associated with decreased tumor concentrations of tamoxifen and an increase in the cis/trans ratio of 4-hydroxytamoxifen (Osborne et al., 1992). Biotransformation of tamoxifen by P450 forms expressed extrahepatically, such as in the breast and endometrium, may be particularly important in determining tissue-specific effects of the drug, with respect to both the removal of parent drug and the generation of therapeutic or toxic metabolites. The objectives of the current study were to determine which human P450 forms might participate in the extrahepatic metabolism of tamoxifen to its N-desmethyl, 4-hydroxy, and 4 -hydroxy metabolites and in the isomerization of trans-4-hydroxytamoxifen (Fig. 1). In Funding for this study was provided by the Kathleen Cuningham Foundation for Breast Cancer Research and the Australian Cancer Fund. 1 Abbreviations used are: P450, cytochrome P450 (heme-thiolate protein P450); HPLC, high-performance liquid chromatography; hNPR, human NADPHcytochrome P450 reductase. Address correspondence to: Dr. E. M. J. Gillam, Department of Physiology and Pharmacology, School of Biomedical Sciences, The University of Queensland, St. Lucia, Queensland, Australia 4072. E-mail: e.gillam@uq.edu. au 0090-9556/02/3008-869–874$7.00 DRUG METABOLISM AND DISPOSITION Vol. 30, No. 8 Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics 661/995627 DMD 30:869–874, 2002 Printed in U.S.A. 869 at A PE T Jornals on Sptem er 8, 2017 dm d.aspurnals. org D ow nladed from