Hypoxia-Activated O6-Benzylguanine Prodrugs

说明(申请人提供)：可使DNA鸟嘌呤的O-6位氯乙基化的烷化剂前药包括目前处于第三阶段临床试验的Cloretazine；FDA批准用于临床的亚硝脲BCNU；以及KS119W，一种在实体肿瘤的缺氧细胞中选择性激活的水溶性磺酰肼前药。这种DNA损伤很容易被O6-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位转移到ACT分子的蛋白质。这一作用代表了肿瘤和宿主组织对Cloretazine、BCNU和KS119W耐药的主要机制。已知的最有效的ACT抑制剂之一是O6-苄基鸟嘌呤(06-BG)，它与ACT反应在蛋白质的活性部位形成S-苄基半胱氨酸。因此，O6-BG耗尽了ACT，增加了肿瘤细胞和宿主细胞对Cloretazine、BCNU和KS119W的敏感性。患者服用无毒剂量的全身性O6-BG可耗尽肿瘤的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，这种药物的剂量必须减少80%，导致BCNU的无效水平。本应用的具体目的在于：(A)设计和合成通过降低实体瘤缺氧细胞中的酶活性而选择性激活的O6-BG前体药物，从而选择性地消耗AGT肿瘤细胞，同时保留含氧的正常组织；(B)体外评估O6-BG前体药物预处理缺氧和氧化肿瘤细胞的效果，然后再用Cloretazine、BCNU和KS119W进行预处理；(C)在体内评价这些组合对多种含有高AGT活性的人体移植肿瘤的抑制作用；(D)对合成前体药物的作用机制进行药理学和生物化学研究。主要的总体目标是选择一种用于临床开发的O6-BG前体药物，与BCNU、Cloretazine和KS119W联合使用。所选的O6-BG前药应该选择性地消耗实体肿瘤中的AGT，从而允许在接近全部治疗剂量的情况下连续联合使用氯乙基化试剂，而不会增加骨髓抑制或对其他正常组织的毒性。预期的是，由于AGT的结构性高水平而对CloRetazine、KS119W和BCNU的细胞毒作用具有抵抗力的实体肿瘤，将通过O6-BG前药的预处理选择性地耗尽耐药诱导蛋白，导致氯乙化剂耐药的肿瘤向药物敏感的肿瘤转化，从而增加可能受益于Cloretazine、KS119W和BCNU的恶性肿瘤的谱。

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.