Abstract

The roles of individual liver cytochrome P450 (P450) enzymes in N-dechloroethylation leading to deactivation and neurotoxification of the isomeric alkylating agent prodrugs ifosfamide (IF) and cyclophosphamide (CPA) were investigated using an in vitro rat liver model. Rats were pretreated with a panel of drugs, including phenobarbital (a strong inducer of liver P450 2B1/2B2) and dexamethasone (a strong inducer of P450 3A enzymes), to examine the effects of these P450-inducing agents on IF and CPA N-dechloroethylation catalyzed by rat hepatic microsomes. The P450 3A-specific inhibitor troleandomycin and inhibitory monoclonal antibodies reactive with P450 2B and 2C enzymes were used to identify the individual P450 subfamilies involved in microsomal N-dechloroethylation of IF and CPA. It was found that dexamethasone pretreatment preferentially elevated microsomal CPA N-dechloroethylation activity (12-fold increase) and that P450 3A enzymes catalyzed up to > 95% of this reaction in both uninduced and drug-induced liver. In contrast, IF N-dechloroethylation activity was stimulated (approximately 8-fold increase) in liver microsomes by phenobarbital pretreatment, and P450 2B1/2B2 were responsible for the majority of this activity. In addition, P450 2C11 catalyzed approximately 50% of IF N-dechloroethylation in uninduced male rat liver microsomes. inducers of P450 1A and 4A enzymes had no effect on N-dechloroethylation of IF or CPA. These P450 enzyme patterns for the N-dechloroethylation reaction are distinct from those previously determined for IF and CPA activation via 4-hydroxylation. In accord with this observation, the balance between oxazaphosphorine activation (4-hydroxylation pathway) and deactivation/neurotoxication (N-dechloroethylation pathway) could be modulated by P450 form-selective inducers and inhibitors. Thus, dexamethasone pretreatment substantially decreased the extent of IF N-dechloroethylation, from 47% to 24% of total metabolism, whereas it increased CPA N-dechloroethylation from 29% to 84% of total metabolism. Moreover, troleandomycin selectively inhibited CPA N-dechloroethylation, thereby increasing net metabolism of the drug via the therapeutically productive 4-hydroxylation pathway. Oxazaphosphorine activation and deactivation/neurotoxication are thus catalyzed by distinct subsets of liver P450 enzymes, in a manner that may allow for improvements in therapeutic indices for this class of drugs by using P450 form-selective modulators.