Studies on the selective action of cyclophosphamide (NSC-26271): Inactivation of the hydroxylated metabolite by tissue-soluble enzymes.

The hypothesis that selective action of cyclophosphamide, compared to other nitrogen mustards, is due to a balance between enzymatic formation of inactive metabolites and chemical formation of the alkylating product was studied in view of previous observation in our laboratory. Metabolite analysis, inhibition of growth of tumor cells in culture, and kinetic analysis of relevant enzyme activities were used in this investigation. The effect of tissue-soluble enzyme fractions on biochemically prepared aldophosphamide, aldophosphamide analogs, and phosphoramide mustard showed: (a) a range of deactivation abilities with aldophosphamide (liver greater than kidney greater than intestinal mucosa greater than tumor greater than spleen = bovine serum albumin solution); (b) the formation of different amounts of carboxyphosphamide from aldophosphamide; and (c) only comparatively small reductions in the toxicity of phosphoramide mustard and of 4-hydroxy-4methylcyclophosphamide. Correlations were found between NAD+-dependent aldehyde dehydrogenase activity and the deactivation ability of tissue-soluble enzyme fractions. Blockage (by C4 substitution) or inhibition (by disulfiram) of secondary oxidation of aldophosphamide, mediated by aldehyde dehydrogenase, resulted in diminished deactivation ability in vitro and reduced selectivity in vivo.