title: The antineoplastic efficacy of the prodrug Cloretazine is produced by the synergistic interaction of carbamoylating and alkylating products of its activation.
abstract: 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.
authors.affiliation: Department of Pharmacology and Developmental Therapeutics Program, Cancer Center, Yale University School of Medicine, New Haven, CT 06520, USA.
authors.forename: Raymond P
authors.initials: RP
authors.lastname: Baumann
authors.forename: Helen A
authors.initials: HA
authors.lastname: Seow
authors.forename: Krishnamurthy
authors.initials: K
authors.lastname: Shyam
authors.forename: Philip G
authors.initials: PG
authors.lastname: Penketh
authors.forename: Alan C
authors.initials: AC
authors.lastname: Sartorelli
mesh_terms: D000818:Animals; D018906:Antineoplastic Agents, Alkylating / Q000493:pharmacokinetics / Q000494:pharmacology; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D001711:Biotransformation; D016466:CHO Cells; D002460:Cell Line; D002478:Cells, Cultured; D006224:Cricetinae; D004357:Drug Synergism; D006147:Guanine / Q000031:analogs & derivatives / Q000494:pharmacology; D018922:HL-60 Cells; D006801:Humans; D006834:Hydrazines / Q000493:pharmacokinetics / Q000494:pharmacology*; D017953:Isocyanates / Q000493:pharmacokinetics / Q000494:pharmacology; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D011355:Prodrugs / Q000493:pharmacokinetics / Q000494:pharmacology*; D013449:Sulfonamides / Q000493:pharmacokinetics / Q000494:pharmacology*; D014162:Transfection
chemical_list: D018906:Antineoplastic Agents, Alkylating; D006834:Hydrazines; D017953:Isocyanates; D011355:Prodrugs; D013449:Sulfonamides; C064976:O(6)-benzylguanine; C483604:laromustine; D006147:Guanine; C008461:methyl isocyanate; D019853:O(6)-Methylguanine-DNA Methyltransferase