Alkylating agent prodrugs that chloroethylate the O-6 position of DNA guanine include Cloretazine, an agent currently in Phase III clinical trial; BCNU, an FDA approved clinically used nitrosourea; and KS119W, a water-soluble sulfonyl hydrazine prodrug selectively activated in hypoxic cells of solid tumors. This DNA lesion is susceptible to repair by O6-alkylguanine-DNA alkyltransferase (ACT), a protein that transfers alkyl groups from the O-6 position of guanine to the ACT molecule. This action represents the primary mechanism of tumor and host tissue resistance to Cloretazine, BCNU, and KS119W. One of the most potent known inhibitors of ACT is O6-benzylguanine (06-BG), which reacts with ACT to form S- benzylcysteine in the active site of the protein. As a result, O6-BG depletes ACT and increases the sensitivity of tumor and host cells to Cloretazine, BCNU and KS119W. Non-toxic doses of systemic O6-BG have been shown in patients to deplete the ACT content of tumors. This action, however, also sensitizes host tissues to BCNU used in combination, necessitating an 80% decrease in the dosage of this agent because of myelosuppression, leading to an ineffective level of BCNU.
The Specific Aims of this application are the (a) design and synthesis of O6-BG prodrugs activated selectively by reducing enzymes in hypoxic cells of solid tumors, thereby selectively depleting tumors of AGT while sparing oxygenated normal tissues; (b) evaluation of pretreatment of hypoxic and oxygenated tumor cells with O6-BG prodrugs in vitro followed by Cloretazine, BCNU, and KS119W; (c) evaluation in vivo of these combinations against a variety of transplanted human tumors containing high levels of AGT activity; and (d) pharmacological and biochemical studies of the mechanism of action of synthesized prodrugs. The primary overall objective is the selection of an O6-BG prodrug for clinical development to use in combination with BCNU, Cloretazine and KS119W. The O6-BG prodrug selected should deplete AGT selectively in solid tumors, thereby permitting use in sequential combination at close to full therapeutic dosage of a chloroethylating agent without increased myelosuppression or toxicities to other normal tissues. The expectation is that solid tumors, resistant to the cytotoxic actions of Cloretazine, KS119W and BCNU because of constitutively high levels of AGT, will be selectively depleted of the resistance inducing protein by pretreatment with the O6-BG prodrug, resulting in conversion of chloroethylating agent resistant neoplasms to drug sensitive ones, thereby increasing the spectrum of malignancies that may benefit from Cloretazine, KS119W, and BCNU. ? ? ? ?
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