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）是最常见的原发性脑肿瘤，也是最恶性的星形细胞瘤（WHO分级IV级）。我们已经证明，GBM肿瘤细胞及其血管内皮都表达细胞表面受体组织因子（TF），这是血栓形成的关键引发剂，而正常大脑的脉管系统则不表达。在这里，我们探索了与GBM血管中TF上调相关的机制，并假设结合到TF载体的细胞毒性药物将特异性靶向GBM的血管，而不是非肿瘤性脑。因此，本提案的目的是开发一种新的GBM治疗方法，其中表达tf的血管系统是特异性靶向的。我们开发的细胞毒性药物是EF24，一种合成姜黄素类似物，它将与酶无活性凝血因子VIIa （fVIIa）结合，这是一种对TF具有高度特异性的高亲和力配体。我们假设这种药物偶联物（EF24-FFRck-fVIIa）将与GBM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，并引发细胞毒性反应。针对VECs的血脑屏障（BBB）的破坏以及GBM的组织破坏性也应该允许药物偶联物直接与表达TF的肿瘤细胞结合。因此，这种治疗方法极有可能同时具有抗血管生成和直接抗肿瘤的作用。本研究将探讨恶性胶质瘤诱导血管内皮细胞表达TF的机制。我们将在体外确定PTEN缺失和缺氧是否会导致胶质瘤释放诱导内皮TF表达的因子，并确定这些机制是否与人类脑肿瘤标本相关。目的II将建立药物偶联物和药物在胶质瘤异种移植物及其相关血管和邻近正常大脑中的分布。使用cy5.5标记的EF24-FFRck- fVIIa进行近红外光学成像，确定药物偶联物在体外和体内的分布。利用生物素化EF24结合链霉亲和素组织化学来测定药物的分布，将更精确地定位药物在胶质瘤异种移植组织切片中的位置。目的III将在小鼠异种移植模型中确定药物偶联物对恶性胶质瘤的疗效。我们将在该模型中检查EF24-FFRck-fVIIa对生存的影响，并检查药物偶联治疗的生物学相关性，包括肿瘤生长、血管生成和血脑屏障的破坏。

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.