Hypoxia-Activated O6-Benzylguanine Prodrugs

描述（由申请人提供）：将DNA鸟嘌呤的O-6位氯乙基化的烷基化剂前药包括Cloretazine，一种目前处于III期临床试验的药物; BCNU，一种FDA批准的临床使用的亚硝基脲;和KS 119 W，一种在实体瘤的缺氧细胞中选择性活化的水溶性磺酰肼前药。这种DNA损伤易于被O 6-烷基鸟嘌呤-DNA烷基转移酶（ACT）修复，ACT是一种将烷基从鸟嘌呤的O-6位置转移到ACT分子的蛋白质。这种作用代表了肿瘤和宿主组织对Cloretazine，BCNU和KS 119 W耐药的主要机制。已知最有效的ACT抑制剂之一是O 6-苄基鸟嘌呤（O 6-BG），其与ACT反应在蛋白质的活性位点形成S-苄基半胱氨酸。因此，O 6-BG消耗ACT并增加肿瘤和宿主细胞对Cloretazine，BCNU和KS 119 W的敏感性。在患者中，全身性O 6-BG的无毒剂量已被证明可消耗肿瘤的ACT含量。然而，这种作用也使宿主组织对联合使用的BCNU敏感，由于骨髓抑制，需要将该药剂的剂量减少80%，导致BCNU的无效水平。本申请的具体目的是（a）设计和合成通过实体瘤的缺氧细胞中的还原酶选择性活化的O 6-BG前药，从而选择性地消耗AGT的肿瘤，同时保留氧合的正常组织;（B）评价在体外用O 6-BG前药预处理缺氧和氧合的肿瘤细胞，然后用氯雷他嗪、BCNU和KS 119 W预处理;（c）在体内评价这些组合对含有高水平AGT活性的各种移植的人肿瘤的作用;和（d）合成的前药的作用机制的药理学和生物化学研究。主要总体目标是选择用于临床开发的O 6-BG前药与BCNU、Cloretazine和KS 119 W联合使用。所选的O 6-BG前药应选择性地消耗实体瘤中的AGT，从而允许以接近全治疗剂量的氯乙基化剂顺序组合使用，而不会增加骨髓抑制或对其他正常组织的毒性。预期由于组成性高水平的AGT而对Cloretazine、KS 119 W和BCNU的细胞毒性作用具有抗性的实体瘤将通过用O 6-BG前药预处理而选择性地耗尽抗性诱导蛋白，导致氯乙基化剂抗性肿瘤转化为药物敏感性肿瘤，从而增加可受益于Cloretazine、KS 119 W、和BCNU。

DESCRIPTION (provided by applicant): 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.