The initial metabolic products of cyclophosphamide (4-hydroxy-cyclophosphamide and aldophosphamide) were prepared biologically in unpurified form. Their toxicity to tumor cells were tested by bioassay techniques and in cell culture, and the deactivation abilities of various tissue-soluble fractions were quantitated. Liver and kidney cytosol effectively deactivated the primary metabolites, whereas cytosols from gastrointestinal tract mucosa, Walker ascites tumor, and spleen were less efficient. When [14C]cyclophosphamide was activated and incubated with liver cytosol, 34% of all radioactivity was identified as carboxyphosphamide, by mass spectrometry of the methyl ester.

Measurement of alcohol dehydrogenase (EC and aldehyde dehydrogenase (EC activities by reduced nicotinamide adenine dinucleotide production revealed a qualitative correspondence between aldehyde dehydrogenase activity and deactivation ability. Unpurified aldophosphamide and the analogs prepared from 6-methyl- and 5,5-dimethylcyclophosphamides were substrates for nicotinamide adenine dinucleotide-requiring enzymes, whereas incubation of 4-hydroxy-4-methylcyclophosphamide in an unfractionated incubation mixture with liver soluble enzymes did not cause reduced nicotinamide adenine dinucleotide production.

Activated 4-methylcyclophosphamide was deactivated by liver cytosol to the same extent as phosphoramide mustard (dose reduction factors, 2.2 and 2.7, respectively); that for liver cytosol and activated cyclophosphamide was 49.1.

It was concluded that the selective action of cyclophosphamide when compared to other nitrogen mustards is largely dependent on the balance between the enzymatic production of nontoxic metabolites (principally carboxyphosphamide) and chemical decomposition of aldophosphamide to the ultimate alkylating agent (phosphoramide mustard).


This investigation was supported by a grant to the Chester Beatty Research Institute from the Medical Research Council (Grant G973/787/K).

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