THE IN VITRO METABOLISM OF PROCARBAZINE: MECHANISMS UNDERLYING THE GENERATION OF CYTOTOXIC SPECIES

Procarbazine is an important chemotherapeutic agent used in the treatment of Hodgkins disease and other malignant lymphomas. The mechanisms by which procarbazine produces its cytotoxic effect remain to be completely elucidated, however, it is known that metabolic activation is necessary. This dissertation provides data that characterizes the final steps in the metabolism of procarbazine and identifies the active cytotoxic species responsible for its activity. The metabolism of 1-methyl-2-phenylmethylhydrazine (MBH) by rat liver microsomes was examined and compared to that of procarbazine. These compounds were found to undergo the same metabolic transformations. MBH, therefore, was used as a model compound for the further study of the metabolism of procarbazine. Its metabolites were synthesized and used as substrates and standard references. The compounds synthesized included benzaldehyde methylhydrazone, 1-methyl-2-phenylmethylhydrazine, methyl(phenylmethyl)diazene and a mixture of azoxy isomers, 1-methyl-2-phenylmethyldiazene-1-oxide (methylazoxy) and 2-methyl-1-phenylmethyldiazene-1-oxide (benzylazoxy). The metabolic intermediates were separated and quantified by gas chromatography and gas chromatography-mass spectrometry. Both the methylazoxy and benzylazoxy isomers were identified as metabolic intermediates. These compounds and their deuterium labelled analogs were used as substrates to probe the enzymatic mechanisms involved in the generation of the cytotoxic species. Deuterium isotope effects and product distributions provided evidence for the enzymatic hydroxylation of the azoxy isomers. These highly reactive hydroxylation products degrade to methyl and benzyl diazonium ions, which are believed to be the active alkylating species. The relative cytotoxicity of these alkyldiazonium ions was evaluated by cell culture assays. Benzyl and methyl diazonium ions were generated by the aqueous degradation of benzylnitrosourea and methylnitrosourea, respectively. The methyldiazonium ion proved to be four times more toxic than the benzyl diazonium ion. The results indicate that the cytotoxic activity of procarbazine arises primarily from its metabolic activation via the methylazoxy isomer to form reactive methylating species