Oxidative Metabolism of Cyclophosphamide: Identification of the Hepatic Monooxygenase Catalysts of Drug Activation1

Cytochrome P-450-catalyzed activation of Cyclophosphamide to alkylating metabolites was studied in isolated rat liver microsomes and puri fied, reconstituted P-450 enzyme systems in order to identify the major enzymatic catalysts of drug activation in both uninduced and drug-induced liver tissue. P-450 form PB-4 (P-450 gene UBI) activated Cyclophospha mide with high efficiency [\ „¿ ,», (app) = 18.2 nmol metabolite/min/nmol P-450; A,.,(app) = 0.16 HIM]via the formation of 4-hydroxycyclophosphamide, which was quantitatively trapped as a bisulfite adduct then characterized following its conversion to cyano derivatives. Antibodies to P-450 PB-4 inhibited Cyclophosphamide activation catalyzed by phenobarbital-induced adult male rat liver microsomes (specific activity, 5.4 nmol metabolite/min/mg liver microsomes) in a selective and near quan titative (>80%) fashion; little or no inhibition was obtained using anti bodies inhibitory towards six other rat hepatic P-450 forms. Cyclophos phamide activation catalyzed by uninduced adult male rat liver micro somes (specific activity, 0.68 nmol/min/mg), although not inhibited by anti-P-450 PB-4 antibodies, was partially inhibited (~60%) by antibodies to P-450 I'll I (gene IIC6) and more completely inhibited (>95%) by antibodies reactive with both P-450 PB-1 and P-450 2c (gene IU II)Consistent with these observations, P-450 PB-1 and P-450 2c both activated Cyclophosphamide at moderate rates in reconstituted systems (turnover, 1.6-2.7 nmol metabolite/min/nmol P-450), while seven other purified hepatic P-450 forms exhibited significantly lower activities (turn over £0.5nmol metabolite/min/nmol P-450). Further studies revealed that the changes in liver microsomal Cyclophosphamide activation rates with age and sex and in response to in vivo administration of cisplatin primarily reflect changes in the levels of P-450 forms PB-1 and 2c. These studies establish that P-450 forms PB-1, 2c, and PB-4 are the major catalysts of Cyclophosphamide activation in rat hepatic tissue and that the modulation of microsomal Cyclophosphamide activation with devel opment and in response to drug exposure largely reflects alterations in the levels of these three hepatic P-450 enzymes.