Investigation of the major human hepatic cytochrome P450 involved in 4-hydroxylation and N-dechloroethylation of trofosfamide

Trofosfamide and its congeners ifosfamide and cyclophosphamide are cell-cyc le-nonspecific alkylating agents that undergo bioactivation catalyzed by li ver cytochrome P450 (CYP) enzymes. Two NADPH-dependent metabolic routes for the anticancer drug trofosfamide, i.e., 4-hydroxylation and N-dechloroethy lation, were studied in human liver microsomes and in seven recombinant hum an CYP isoforms (i.e., CYP1A1, 1A2, 2A6, 2B6, 2D6, 2E1, and 3A4-OR) to iden tify the CYP enzymes involved. Recombinant human CYP3A4 and CYP2B6 exhibite d catalytic activity with respect to both pathways of trofosfamide. Enzyme kinetic analyses revealed the dominant role of human CYP3A4 in 4-hydroxylat ion and N-dechloroethylation of trofosfamide. This was confirmed by the obs ervation that only the CYP3A4 contents of five samples of human liver micro somes correlated with both pathways of trofosfamide. Furthermore, ketoconaz ole, a selective inhibitor of CYP3A4, substantially inhibited microsomal tr ofosfamide 4-hydroxylation and N-dechloroethylation (50% inhibitory concent ration < 1 mu M for both reactions). The present study indicates that human liver microsomal CYP3A4 preferentially catalyzes the two NADPH-dependent m etabolic routes of trofosfamide, which emphasizes the necessity for awarene ss of potential interactions with any coadministered drugs that are CYP3A4 substrates.