Targeted Delivery of a Novel Synthetic Curcumin Analog, EF24 to Glioblastoma and

描述(申请人提供)：胶质母细胞瘤(GBM)是最常见的原发脑肿瘤，也是最恶性的星形细胞瘤(WHO IV级)。我们已经证明，GBM肿瘤细胞及其血管内皮细胞都表达细胞表面受体组织因子(Tf)，这是血栓形成的关键启动者，而正常脑血管系统不表达。在这里，我们探索了与基底膜血管系统中转铁蛋白上调有关的机制，并假设结合到载体上的针对转铁蛋白的细胞毒剂将特异性地靶向基底膜血管，而不是非肿瘤性脑。因此，本提案的目的是开发一种新的治疗GBM的方法，其中表达TF的血管系统是特异性靶向的。我们开发的细胞毒剂是EF24，一种合成的姜黄素类似物，它将与酶失活的凝血因子VIIa(FVIIa)相连，FVIIa是TF的高亲和力配体，具有极高的特异性。我们推测这种药物结合物(EF24-FFRck-FVIIa)将与基底膜内血管内皮细胞(VECs)上的Tf结合，通过配体-受体介导的内吞作用进入靶细胞，并引发细胞毒反应。由于靶向血管内皮细胞和GBM的组织破坏性导致的血脑屏障(BBB)的破坏，也应该允许药物结合物直接与表达TF的肿瘤细胞结合。因此，这种治疗方法很有可能同时具有抗血管生成和直接抗肿瘤的作用。具体目的I将确定恶性胶质瘤通过血管内皮细胞诱导TF表达的机制。我们将在体外确定PTEN缺失和低氧是否导致胶质瘤释放诱导内皮因子表达的因子，并将确定这些机制是否在人脑肿瘤标本中具有相关性。目的建立药物结合物和药物在人脑胶质瘤移植瘤、相关血管和邻近正常脑组织中的分布。通过使用Cy5.5标记的EF24-FFRck-FVIIa，近红外光学成像将被用来确定药物结合物在体内外的分布。使用生物素标记的EF24结合链霉亲和素组织化学的药物分布将被用来在胶质瘤异种移植组织切片中更准确地定位药物。目的III将在小鼠异种移植模型中确定该药物结合物对恶性胶质瘤的疗效。我们将在该模型中检测EF24-FFRCK-FVIIa对存活率的影响，并检测药物结合治疗的生物相关性，包括肿瘤生长、血管生成和破坏血脑屏障。 公共卫生相关性：胶质母细胞瘤(GBM)是最常见的原发脑瘤，在美国每年有8700例新发病例。这些肿瘤普遍是致命的，按照目前的治疗方法，患者的平均生存时间只有60周。这项建议采用了一种高度创新的方法，专门针对基底膜的异常血管，以减缓其生长。虽然这项建议专门针对GBM中的异常血管，但该方法也可能应用于其他血管生长异常的疾病，如糖尿病视网膜病变、黄斑变性、子宫内膜异位症、克罗恩病、牛皮癣和其他癌症。

DESCRIPTION (provided by applicant): Glioblastoma (GBM) is the most common primary brain tumor and the most malignant form of astrocytoma (WHO grade IV). We have demonstrated that both GBM tumor cells and its vascular endothelium express the cell surface receptor tissue factor (TF), a critical initiator of thrombosis, while the vasculature of the normal brain does not. Here we explore mechanisms related to the upregulation of TF in the vasculature of GBM and hypothesize that a cytotoxic agent conjugated to a carrier directed at TF will specifically target blood vessels of GBM, but not non-neoplastic brain. Thus, the objective of this proposal is to develop a novel therapeutic approach for GBM, in which the TF-expressing vasculature is specifically targeted. The cytotoxic agent we have developed is EF24, a synthetic curcumin analog, which will be linked to enzymatically inactive coagulation factor VIIa (fVIIa), the high affinity ligand for TF that has exquisite specificity. We hypothesize that this drug conjugate (EF24-FFRck-fVIIa) will bind to TF on vascular endothelial cells (VECs) within the GBM, enter target cells by ligand-receptor mediated endocytosis, and elicit a cytotoxic response. The disruption of the blood brain barrier (BBB) due to targeting the VECs and to the tissue-destructive nature of GBM should also permit binding of the drug-conjugate directly to TF expressing neoplastic cells. Thus, this therapeutic approach has a high likelihood of having both an anti-angiogenic and direct anti-tumor effect. Specific Aim I will determine the mechanisms by which malignant gliomas induce the expression of TF by vascular endothelial cells. We will establish whether PTEN loss and hypoxia lead to release of factors by gliomas that induce endothelial TF expression in vitro and will determine if these mechanisms have correlates in human brain tumor specimens. Aim II will establish the distribution of the drug-conjugate and the drug in glioma xenografts, their associated vasculature and the adjacent normal brain. Near infrared optical imaging will be used to determine the distribution of the drug-conjugate in vitro and in vivo using Cy5.5-labeled EF24-FFRck- fVIIa. The distribution of the drug using biotinylated EF24 combined with streptavidin histochemistry will be used to more precisely localize the drug in glioma xenografts tissue sections. Aim III will determine the efficacy of the drug-conjugate against malignant gliomas in a mouse xenograft model. We will examine the effects of the EF24-FFRck-fVIIa on survival in this model and examine the biologic correlates of drug-conjugate treatment, including tumor growth, angiogenesis and disruption of the BBB. PUBLIC HEALTH RELEVANCE: Glioblastoma (GBM) is the most common primary brain tumor, with 8700 new cases per year in the United States. These tumors are universally fatal and the average length of patient survival is only 60 weeks with current therapies. This proposal uses a highly innovative approach to specifically target the abnormal blood vessels of GBM in order to slow their growth. Although this proposal specifically addresses the abnormal vessels in GBM, the approach may find application in other diseases with abnormal blood vessel growth, such as diabetic retinopathy, macular degeneration, endometriosis, Crohn's disease, psoriasis, and other cancers.