Comprehensive evaluation of tamoxifen sequential biotransformation by the human cytochrome P450 system in vitro: prominent roles for CYP3A and CYP2D6.

We performed comprehensive kinetic, inhibition, and correlation analyses in human liver microsomes and experiments in expressed human cytochromes P450 (P450s) to identify primary and secondary metabolic routes of tamoxifen (TAM) and the P450s catalyzing these reactions at therapeutically relevant concentrations. N-Desmethyl-TAM formation catalyzed by CYP3A4/5 was quantitatively the major primary metabolite of TAM; 4-hydroxy-TAM formation catalyzed by CYP2D6 (and other P450s) represents a minor route. Other minor primary metabolites include alpha -, 3-, and 4'-hydroxyTAM and one unidentified metabolite (M-I) and were primarily catalyzed by CYP3A4, CYP3A5, CYP2B6/2C19, and CYP3A4, respectively. TAM secondary metabolism was examined using N-desmethyl- and 4-hydroxy-TAM as intermediate substrates. N-Desmethyl-TAM was predominantly biotransformed to alpha-hydroxy N-desmethyl-, N-didesmethyl-, and 4-hydroxy N-desmethyl-TAM (endoxifen), whereas 4-hydroxy-TAM was converted to 3,4-dihydroxyTAM and endoxifen. Except for the biotransformation of N-desmethyl-TAM to endoxifen, which was exclusively catalyzed by CYP2D6, all other routes of N-desmethyl- and 4-hydroxy-TAM biotransformation were catalyzed predominantly by the CYP3A subfamily. TAM and its primary metabolites undergo extensive oxidation, principally by CYP3A and CYP2D6 to metabolites that exhibit a range of pharmacological effects. Variable activity of these P450s, brought about by genetic polymorphisms and drug interactions, may alter the balance of TAM effects in vivo.

Triethylenethiophosphoramide is a specific inhibitor of cytochrome P450 2B6: implications for cyclophosphamide metabolism.

Cytochrome P450 2B6 is a genetically polymorphic enzyme that is important in the metabolism of a number of clinically used drugs. This enzyme is not as well studied as other cytochrome P450 (P450) isoforms because of the lack of specific antibodies, probe drugs, and inhibitors. Although recent progress has been made toward specific antibodies and probe drugs, a specific enzyme inhibitor is still lacking. Studies suggest that CYP2B6 plays an important role in the 4-hydroxylation of cyclophosphamide and that this reaction can be inhibited by triethylenethiophosphoramide (thioTEPA). We therefore wished to test the hypothesis that thioTEPA is an inhibitor of CYP2B6. Using human liver microsomes (HLMs) and recombinant P450 enzymes, we demonstrated that thioTEPA is a potent and specific inhibitor of CYP2B6. Enzyme activity was reduced 78.1 +/- 0.2% by 50 microM thioTEPA when CYP2B6 activity was measured by following the metabolism of 200 microM S-mephenytoin to nirvanol. thioTEPA did not significantly inhibit (<20% at 100 microM) the other isoforms tested (CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4). thioTEPA seems to be a potent noncompetitive inhibitor of CYP2B6, with K(i) values of 4.8 +/- 0.3 and 6.2 +/- 0.7 microM for HLMs and recombinant CYP2B6, respectively, values that are within the plasma concentration range of thioTEPA at therapeutic doses (1.1-18.6 microM). We conclude that thioTEPA is a potent and specific inhibitor of CYP2B6 and that this is the likely mechanism by which thioTEPA inhibits the activation of cyclophosphamide. Furthermore, thioTEPA may prove to be a valuable new tool for the study of this important drug-metabolizing enzyme.