Metabolism of cyclophosphamide by rat hepatic microsomes.

cellular enzymes, presumably phosphatases and phosphoramidases. Selective toxicity was to be achieved because the concentrations of these enzymes was believed to be much higher in tumor cells as compared to normal cells (24). Cyclophosphamide itself exerts minimal alkylating activity and does not exert a cytostatic effect in vitro, but it does exert its biological effect following in vivo activation to metabolites capable of alkylation, indicating that the enzymes responsible for its activation are not present in the tumor tissue itself 18) revealed that cyclophosphamide activation takes place in the liver and that various other organs, prostate acid phosphatase, renal alkaline phosphatase, and spleen phosphoramidase are not capable of activating cyclophosphamide. In contrast, 1 laboratory has reported that activation of cyclophosphamide takes place not only in liver, but also in bone marrow, kidney, several experimental tumors, and various solid human carcinomas (29). Subcellular distribution studies showed that hepatic activation takes place in the microsomes and that NADPH and oxygen are required for this purpose (8, 12). In vivo studies revealed that phenobarbital stimulates and SKF 525A,2 2 ,4-dichloro-6-phenylphenoxy ethyl-diethylamine, and chloramphenicol inhibit the metabolism of cyclophosphamide to its active alkylating form that in vitro hepatic microsomal metabolism of cyclophosphamide was inhibited by (a) carbon monoxide, (b) the interruption of the microsomal electron transport chain by cytochrome c or p-hydroxymercuribenzoate, and (c) several well-known substrates for hepatic microsomal enzymes, including hexobarbital , p-nitroanisole , testosterone, and cortisol (1 1, 12). Furthermore, hepatic microsomes obtained from male rats metabolized cyclophosphamide at a much faster rate than did similar preparations from female rats, and enzymatic activity was markedly increased by phenobarbital pretreatment (I 1, 12). These findings all strongly suggest that the hepatic microsomal enzyme system(s) which oxidatively metabolize many drugs as well as endogenous steroids and which have come to be known collectively as the hepatic microsomal mixed-function oxidases (21†" 23, 33, 34) also metabolize and thus activate cyclophosphamide. The investigations reported in the current manuscript were undertaken in an attempt to provide rpore information regarding the characteristics of cyclophosphamide metabolism 2 The abbreviation used is: SKF 525A, 2-diethylan-iinoethyl 2,2-diphenylvalerate; norHN2, bis(2-chloroethyl)amine hydrochloride. SUMMARY The conversion of cyclophosphamide to its alkylating metabolite(s) by microsomes obtained from male rat liver was characterized. Under the conditions used, the reaction was found (a) to be temperature dependent; (b) to be enzyme concentration dependent; (c) to proceed in a linear fashion for 20 mm; (d) to …