Cloretazine {1,2-bis(methylsulfonyl)-1-[(2-chloroethyl)-2-(methylamino)carbonyl]
hydrazine;
VNP40101M; 101M} is a sulfonylhydrazine
prodrug that possesses broad spectrum antitumor efficacy against transplanted murine and human
tumor models and has shown activity in clinical trials against relapsed or refractory
acute myeloid leukemia. Base catalyzed activation of this
prodrug generates two different reactive intermediates: chloroethylating species that covalently interact with
DNA at the O6-position of
guanine residues that progress to a G-C interstrand cross-link, and a carbamoylating agent,
methyl isocyanate. Previous findings from this laboratory have provided initial evidence that
methyl isocyanate can contribute to the efficacy of
Cloretazine by enhancing the cytotoxicity of the generated chloroethylating species. This action may be due in part to inhibition of the DNA repair
protein O6-alkylguanine-DNA
alkyltransferase (AGT); however, activity in cells devoid of AGT indicates that other actions are involved in the synergistic cytotoxicity. Herein we demonstrate that
O6-benzylguanine can also produce synergistic cell kill with the alkylating component of
Cloretazine but differs from
methyl isocyanate in that the enhancement occurs in AGT-containing cells, but not in cells devoid of AGT.
Methyl isocyanate generated by the decomposition of 1,2-bis(methylsulfonyl)-1-[methylaminocarbonyl]
hydrazine also acts to enhance the activity of a variety of
DNA cross-linking agents, while only producing additive cytotoxicity with methylating agents. Flow cytometric studies using
annexin as a marker for apoptosis indicate that in Chinese hamster ovary cells and in human
leukemia cells
Cloretazine-induced apoptosis is primarily caused by the generated
methyl isocyanate. Comet assays designed to detect
DNA cross-links in intact cells indicate that the chloroethylating species generated by the activation of
Cloretazine produce
DNA cross-links, with the co-generated
methyl isocyanate increasing the degree of cross-linking produced by the reactive chloroethylating species. These findings provide further evidence that the
methyl isocyanate produced by the activation of
Cloretazine can be a major contributor to the cytotoxicity produced by this
antineoplastic agent.