Hypoxia-Activated O6-Benzylguanine Prodrugs

说明(申请人提供)：使DNA鸟嘌呤O-6位氯乙基化的烷化剂前药包括由我们实验室设计合成的正处于晚期临床试验的onrigin(氯氮肼)；FDA批准的临床使用的亚硝脲和甲基化药物卡莫司汀(BCNU)和替莫唑胺(TMZ)；以及可在实体肿瘤的缺氧细胞中选择性激活的磺酰肼前药KS119。DNA损伤是由胆碱化试剂引起的，DNA鸟嘌呤的O6位发生烷基化，导致G-C交联键，容易被O6-烷基鸟嘌呤-DNA烷基转移酶(AGT)修复，AGT是一种将烷基和甲基从鸟嘌呤的O-6位转移到AGT分子的蛋白质。这一作用代表了肿瘤和宿主组织对onrigin、KS119、BCNU和TMZ耐药的主要机制。在癌症患者中，全身剂量的O6-BG被证明是无毒的，可以耗尽肿瘤的AGT含量；这种作用也会耗尽正常组织中的AGT，使两种肿瘤宿主组织对联合使用的BCNU敏感。由于骨髓抑制，AGT的耗竭需要减少80%的BCNU剂量，导致这种亚硝脲的抗肿瘤水平无效。本申请的具体目的是：(A)设计和合成可处方的O6-BG前药，该前药含有通过还原实体瘤缺氧细胞中的酶选择性/优先激活的对-硝基苄基羰基触发器/连接子，从而选择性地耗尽AGT的肿瘤，同时保留有氧的正常组织；(B)评价O6-BG前药体外对缺氧和充氧肿瘤细胞的预处理，随后是onrigin、KS119、BCNU和TMZ；(C)体内评估这些药物与合成的AGT抑制剂的组合对含有不同水平AGT的各种人和小鼠移植肿瘤的作用机理和(D)合成前药的作用机制的生物化学研究预期，由于固有的高AGT水平而对O6-DNA鸟嘌呤靶向剂的细胞毒作用产生抗药性的实体肿瘤，将通过O6-BG前体药物的预处理选择性/优先地耗尽耐药诱导蛋白AGT，导致具有氯乙基化/甲基化试剂抗性的肿瘤转化为对药物敏感的肿瘤，从而增加可能受益于onrigin、KS119、BCNU和TMZ的恶性肿瘤的谱。 与公共卫生相关：缺氧肿瘤组织中AGT抑制前体药物的选择性激活增强了烷基化和甲基化药物的抗肿瘤潜力，这些药物使DNA鸟嘌呤的O6位发生氯乙基化和甲基化，并逆转了这类药物(即针对DNA鸟嘌呤的O6位的烷基化药物)的主要耐药机制。新的AGT抑制药的设计、合成和表征与已知的特定DNA O6-鸟嘌呤靶向药物的持续开发相结合，很可能会为各种目前无反应的肿瘤产生独特的新疗法。

DESCRIPTION (provided by applicant): Alkylating agent prodrugs that chloroethylate the O-6 position of DNA guanine include onrigin (cloretazine), an agent designed and synthesized in our laboratory, currently in late stage clinical trial; carmustine (BCNU) and temozolomide (TMZ), an FDA approved clinically used nitrosourea and methylating agent, respectively, and KS119, a sulfonylhydrazine prodrug selectively activated in hypoxic cells of solid tumors will be employed. The DNA lesion, produced by the cholorethylating agents, an alkylation of the O6-position of DNA guanine which leads to a G-C crosslink, is susceptible to repair by O6-alkylguanine-DNA alkyltransferase (AGT), a protein that transfers alkyl and methyl groups from the O-6 position of guanine to the AGT molecule. This action represents the primary mechanism of tumor and host tissue resistance to onrigin, KS119, BCNU, and TMZ. Non-toxic doses of systemic O6-BG have been shown in cancer patients to deplete the AGT content of tumors; this action also depletes AGT in normal tissue, sensitizing both tumor host tissues to BCNU used in combination. The depletion of AGT necessitates an 80% decrease in dosage of BCNU because of myelosuppression, leading to an ineffective antineoplastic level of this nitrosourea. The Specific Aims of this application are (a) the design and synthesis of formulatable O6-BG prodrugs containing a p-nitrobenzylcarbonyl trigger/linker activated selectively/preferentially by reducing enzymes in oxygen-deficient cells of solid tumors, thereby selectively depleting tumors of AGT while sparing oxygenated normal tissue; (b) evaluation of pretreatment of hypoxic and oxygenated tumor cells with O6-BG prodrugs in vitro followed by onrigin, KS119, BCNU, and TMZ; (c) evaluation in vivo of combinations of these agents with synthesized AGT inhibitors against a variety of transplanted human and murine tumors containing varied levels of AGT and (d) biochemical studies of the mechanism of action of the synthesized prodrugs The expectation is that solid tumors, resistant to the cytotoxic actions of O6-DNA guanine targeting agents because of constitutively high levels of AGT, will be selectively/preferentially depleted of the resistance inducing protein AGT by pretreatment with the O6-BG prodrug, leading to the conversion of chloroethylating/methylating agent resistant neoplasms to drug sensitive ones, thereby increasing the spectrum of malignancies that may derive benefit from onrigin, KS119, BCNU and TMZ. PUBLIC HEALTH RELEVANCE: The selective activation of AGT inhibitory prodrugs in oxygen-deficient tumor tissue enhance the antitumor potential of alkylating and methylating drugs that chloroethylate and methylate the O6-position of DNA guanine, as well as reverse the primary mechanism of resistance of this class of agents (i.e., alkylating agents targeting the O6-position of DNA guanine). The design, synthesis and characterization of new AGT inhibitory agents in concert with the continued development of specific known DNA O6-guanine targeting agents to use in combination may well prove to yield unique new treatments for a variety of currently non-responsive tumors.