Non-enzymatic activation of procarbazine to active cytotoxic species

Abstract

The cellular cytotoxicity of procarbazine is thought to result from bioactivation of the parent compound through reactive intermediates to an ultimate alkylating species. Procarbazine is converted initially to azoprocarbazine, which is then N-oxidized through a cytochrome P-450-mediated process to a mixture of the positional isomers, benzylazoxyprocarbazine and methylazoxyprocarbazine. In order to define the bioactivation events that lead to the cytotoxic species, the in vitro cytotoxicities of the purified azoxy isomers as well as of the parent compound, procarbazine, were evaluated with the human leukemia cell line, CCRF-CEM. The methylazoxy isomer was found to be the most active species. Procarbazine inhibited the growth of CCRF-CEM cells but at a concentration much higher than that required for the methylazoxy isomer. Since procarbazine must be metabolized to form the cytotoxic species, we sought to determine if the active metabolite, methylazoxyprocarbazine, was being formed in the incubations. Solutions of procarbazine incubated with and without cells at 37 degrees C were analyzed by combined liquid chromatography-mass spectrometry with a thermospray interface. The azoxy metabolites of procarbazine appeared rapidly in cellular incubations and in the aqueous solutions without cells. More of the methylazoxy isomer was formed initially, but by 72 hr the benzylazoxy isomer was the predominant species. Thus, in these studies it appears that procarbazine was benzylazoxy isomer was the predominant species. Thus, in these studies it appears that procarbazine was non-enzymatically oxidized to the two azoxyprocarbazine isomers and that the methylazoxy compound was the most cytotoxic to CCRF-CEM cells.