Use of New Bacterial Enzymes to Improve Nitro-Prodrug Cancer Therapy

说明书(申请人提供)：还原前体药物在天然状态下是无害的，但在还原时既能杀死生长中的肿瘤细胞，也能杀死不生长的肿瘤细胞。癌症治疗需要选择性地杀死所有的癌细胞，副作用最小，但这很少实现，因为大多数药物不能到达实体肿瘤中的所有细胞(存在于90%的人类癌症中)，而且没有选择性，也可以被正常的人类细胞激活。因此，需要一种专门针对肿瘤的药物/催化剂体系，并允许可视化肿瘤对药物渗透的障碍。这项建议涉及新发现的硝基前药[6-chloro-9-nitro-5-oxo-5H-benzo[a]phenoxazine(CNOB)]和由PI发现和进化的一种进化的高活性硝基还原酶。CNOB是一种有效的药物，其独特的性质是其激活的产物是荧光的，并且可以在活体动物身上看到。还提出了将药物/催化剂输送到肿瘤和清除肿瘤屏障的新方法。我们将追求四个目标。前两项涉及体外研究：针对不同癌症的有效性的普遍性，以及代谢物的鉴定。AIM 3涉及药物/催化剂对肿瘤的特定递送。针对CD44受体在几乎所有癌症中过度表达的透明质酸标记纳米脂质体将被用于输送药物、催化剂或两者兼而有之。然而，由于一些正常细胞也可以表达CD44，因此可能会发生有害的非靶向传递。因此，还将采用另一种方法，针对通常在癌细胞中过度表达的两种受体。虽然正常细胞可能表达其中一种受体，但它们极不可能同时表达两种受体。在这种方法中，CNOB将由CD44靶向脂质体递送，并由细胞因子诱导的针对肿瘤特异性NKG2D受体的杀伤细胞作为催化剂。这样，正常细胞可能会接受这种疗法的一种或另一种成分(药物或催化剂)，但不会同时接受两者，从而逃脱伤害。对于给药方法，将检查药物的药代动力学。AIM 4将解剖肿瘤内的药物和活化产物REACH，并将利用最先进的显微和活体成像技术(共焦、IVIS、活体内成像、肿瘤切片的放射自显影)和染色(差异荧光标记、血管染色、缺氧和坏死区域)来检测阻碍穿透的肿瘤区域。清除这些屏障的方法是，例如，使用胶原酶溶解凝块，恢复肿瘤中的正常血管系统。治疗老鼠癌症的有效方法将会产生，为这种令人兴奋的新癌症疗法的人类试验铺平道路。与公共卫生的相关性。抗癌药物通常不能令人满意，原因有两个：它们也会伤害非癌细胞；它们不能杀死肿瘤内的所有细胞。正在研究的药物对正常细胞无害，但会杀死癌细胞，因为一种特殊开发的酶将只对癌细胞产生特异性作用。这种药物也可以在体内看到；这将允许检测肿瘤内的渗透屏障并消除它们。

DESCRIPTION (provided by applicant): Reductive prodrugs are harmless in their native state but kill both growing and non-growing tumor cells upon reduction. Cancer cure requires selective killing of all cancerous cell with minimal side effects, but this is rarely achieved because most drugs cannot reach all the cells within solid tumors (present in >90% human cancers), and are not selective, being activated also by normal human cells. Thus, a drug/catalyst regime is needed that specifically targets tumors and permits visualization of tumor barriers to drug penetration. This proposal is concerned with a newly discovered nitro-prodrug [6-chloro-9-nitro-5-oxo-5H-benzo[a]phenoxazine (CNOB)] and an evolved high-activity nitroreductase discovered and evolved by the PI. CNOB is an effective drug with the unique quality that its activated product is fluorescent and can be visualized in living animals. Novel methods of drug/catalyst delivery to tumors and removal of tumor barriers are also proposed. Four aims will be pursued. The first two involve in vitro studies: the generality of effectiveness against different cancers, and identification of metabolites. Aim 3 deals with specific delivery of the drug/catalyst to tumors. Hyaluronan-labeled nanoliposomes that target the CD44 receptor over-expressed in nearly all cancers will be used to deliver the drug, the catalyst, or both. However, since some normal cells could also express CD44, harmful non-target delivery could occur. Another approach will therefore also be employed that targets two receptors typically overexpressed in cancer cells. While normal cells may express one of these receptors, they would be highly unlikely to express both. In this approach, CNOB will be delivered by CD44-targeting liposomes, and the catalyst by the cytokine induced killer cells that target the tumor-specific NKG2D receptors. This way the normal cells might receive one or the other component of this therapy (the drug or the catalyst) but not both, thus escaping harm. The pharmacokinetics of the drug will be examined for the delivery methods. Aim 4 will dissect the drug and activated product reach within the tumors and will utilize state of the art microscopic and in vivo imaging technology (confocal, IVIS, intravital, autoradiography of tumor sections) and staining (differential fluorescent labels, staining of blood vessels, regions of hypoxia and necrosis) to detect the tumor regions hindering penetration. Removal of these barriers will be attempted by, for example, use of collagenases to dissolve clots, and restoration of normal vasculature in the tumor. Effective means for treating cancer in mice will result, paving the way for human trials of this exciting new cancer therapy. Relevance to public health. Cancer drugs are often unsatisfactory for two reasons: they also harm the non-cancerous cells; and they fail to kill all the cells within a tumor. The drug under study is harmless to normal cells, and will kill cancer cells because a specially developed enzyme will be delivered specifically only to them. This drug can also be seen inside the body; this will allow detection of penetration barriers within the tumors and their elimination.