COMPETITIVE-INHIBITION OF HUMAN LIVER MICROSOMAL CYTOCHROME-P450 3A-DEPENDENT STEROID 6-BETA-HYDROXYLATION ACTIVITY BY CYCLOPHOSPHAMIDE ANDIFOSFAMIDE IN-VITRO

The prodrugs cyclophosphamide (CP) and ifosfamide (IF) are oxidized by hepatic cytochrome P450 (P450) to the active cytotoxic species, phosp horamide mustard. Acrolein (prop-2-enal) is also formed during CP and IF activation in rat liver and has been associated with P450 destructi on. Analogous inactivation of human liver P450s by CP or IF could lead to pharmacokinetic interactions with coadministered drugs. The presen t study investigated the susceptibilities of human hepatic P450s to in hibition and inactivation by CP and IF in vitro. Unlike the situation in rat liver microsomes, total P450 was not decreased after incubation of CP or IF with NADPH and human fractions. However, CP and IF inhibi ted testosterone 6 beta-hydroxylation mediated by P450s 3A but not P45 0 1A2-dependent 7-ethylresorufin O-deethylation, P450 2C-dependent tol butamide methyl hydroxylation or P450 2E1-mediated N-nitrosodimethylam ine N-demethylation. Kinetic analysis indicated that the drugs were re versible (competitive) inhibitors of testosterone 6 beta-hydroxylation (K-m, 94 +/- 8 mu M) in human liver microsomes (K(l)s, 510 +/- 20 mu M and 490 +/- 40 mu M for CP and IF, respectively). Time-dependent int ensification of the inhibition of the activity by CP or IF did not occ ur; this supports the observation that P450 was refractory to inactiva tion. The rates of acrolein formation from CP and IF in human hepatic microsomes (0.76 +/- 0.23 and 0.19 +/- 0.07 nmol min(-1) mg(-1) of pro tein, respectively) were only 18% and 10% of the rates estimated in fr actions from untreated rat liver (4.20 +/- 0.04 and 1.96 +/- 0.12 nmol min(-1) mg(-1) of protein, respectively). Direct addition of acrolein to human microsomes at a similar concentration to that formed from CP in vitro (similar to 23 mu M) did not result in P450 loss but concent rations similar to those produced from CP in microsomes from phenobarb ital-induced rat liver (similar to 390 mu M) led to the loss of about 35% of total P450. In summary, these findings establish that CP and IF are reversible and preferential inhibitors of human hepatic P450s 3A. The absence of microsomal P450 destruction during NADPH-mediated CP o r IF metabolism seems related to the generation of insufficient quanti ties of acrolein in situ. Possible pharmacokinetic interactions with C P and IF are therefore unlikely to be attributable to P450 inactivatio n by acrolein.