A novel Stat3 inhibitor for treating retinal inflammation and neovascularization

描述（由申请人提供）：本提案的目的是检查CLT-005（一种新型Stat 3小分子抑制剂）治疗糖尿病视网膜病变视网膜炎性和血管渗漏的疗效。已知视网膜血管形成由血管内皮生长因子（VEGF）蛋白介导，并且目前FDA批准的AMD治疗是该蛋白及其信号传导能力的抑制剂。这些药物是治疗AMD的一大进步，但它们1）未能解决疾病的炎症性质; 2）对所有AMD患者无效; 3）需要每月注射到眼睛中以递送药物; 4）只能在已经发生视力丧失时使用。尽管这些疗法在临床上显示出前景，但靶向新途径的新疗法对于更好的疾病管理和组合治疗选择是非常理想的。同行评审文献中的几条证据表明Stat 3密切参与视网膜炎症和新生血管形成：1）在人类中，在患有糖尿病或一些其他形式的视网膜病变的患者的玻璃体中观察到瘦素水平显著升高。2)瘦素是一种强促血管生成因子，其启动导致Stat 3（pStat 3）磷酸化/活化的活化级联。3)pStat 3是一种转录因子，可促进多种炎症和新生血管基因（如VEGF）的表达。4)在急性视网膜新生血管形成的动物模型中，仅在视网膜的新生血管区域中观察到pStat 3。5)炎性细胞因子IL-6还引起视网膜中pStat 3的磷酸化，其引起脉络膜新生血管形成，并且阻断该途径抑制新生血管事件。由于pStat 3可以快速改变细胞基因表达，并且似乎是这些途径的效应分子，我们开发了一种小分子（CLT-005），其特异性抑制Stat 3的磷酸化和随后的二聚化。在我们的初步数据中，我们已经表明CLT-005：1）预测结合Stat 3的磷酸化位点，从而阻断该转录因子的二聚化和活化; 2）选择性抑制内皮细胞增殖，而对周细胞活力没有任何强烈影响; 3）降低糖尿病大鼠模型中视网膜ICAM-1和VEGF水平。基于这些有希望的数据，我们建议进一步评估CLT-005在糖尿病和视网膜病啮齿动物模型中减少视网膜炎症和血管通透性的功效。在这个I期计划中，我们将玻璃体内给予糖尿病大鼠眼睛不同浓度的CLT-005，并定量促炎细胞因子IL-6、TNF-1和MCP-1的水平。我们还将评估和量化这些视网膜中的白细胞停滞作为炎症的早期标志物。此外，我们还将量化CLT-005在减少糖尿病大鼠眼睛和呈现急性视网膜新生血管形成的氧诱导视网膜病大鼠模型中的血管渗漏中的功效。如果这个I期项目证明了疗效，这些实验将为II期研究奠定坚实的基础，以进一步证实CLT-005作为视网膜炎症和黄斑水肿治疗的前景。公共卫生相关性：视网膜的新生血管疾病，包括糖尿病视网膜病变（DR）和视网膜相关性黄斑变性（AMD），是全世界50岁以上患者失明的主要原因。在几种体外和体内模型中，Stat 3的激活已被证明在引发视网膜中的炎症和新生血管事件中起主要作用。我们最近开发了一种新型小分子（CLT-005），其抑制Stat 3的这种激活，因此作为治疗DR和AMD的理想治疗剂呈现。该提案的目的是证明CLT-005在糖尿病和视网膜病啮齿动物模型中预防视网膜炎症和血管渗漏的功效。

DESCRIPTION (provided by applicant): The objective of this proposal is to examine the efficacy of CLT-005, a novel small molecule inhibitor of Stat3, for treating inflammatory and vascular leakage of the retina of diabetic retinopathy. Retinal blood vessel formation is known to be mediated by the vascular endothelial growth factor (VEGF) protein, and current FDA approved treatments for AMD are inhibitors of this protein and its ability to signal. These drugs are a great advancement in the treatment of AMD, but they 1) fail to address the inflammatory nature of the disease; 2) are not effective in all AMD patients; 3) require monthly injections into the eye to deliver the drug; 4) can only be used once visual loss has already occurred. Although these therapies have shown promise in the clinic, new treatments that target novel pathways are highly desirable for better disease management and combinatorial therapeutic options. Several lines of evidence in the peer reviewed literature suggest the intimate involvement of Stat3 in retinal inflammation and neovascularization: 1) In humans, significantly elevated levels of Leptin are observed in the vitreous of patients with diabetes or some other form of retinopathy. 2) Leptin is a strong pro- angiogenic factor that initiates an activation cascade resulting in the phosphorylation/activation of Stat3 (pStat3). 3) pStat3 is a transcription factor that promotes expression of several inflammatory and neovascular genes, such as VEGF. 4) In animal models of acute retinal neovascularization, pStat3 is observed solely in the neovascular areas of the retina. 5) The inflammatory cytokine IL-6 also causes phosphorylation of pStat3 in the retina which causes choroidal neovascularization, and blockade of this pathway inhibits neovascular events. As pStat3 can rapidly change cellular gene expression and appears to be the effector molecule of these pathways, we developed a small molecule (CLT-005) that specifically inhibits phosphorylation and subsequent dimerization of Stat3. In our preliminary data, we have shown the CLT-005: 1) is predicted to bind to the phosphorylation site of Stat3, thus blocking dimerization and activation of this transcription factor; 2) selectively inhibits endothelial cell proliferation without any strong effect on pericyte cell viability; 3) reduces retinal ICAM-1 and VEGF levels in a rat model of diabetes. Based on these promising data, we propose to further evaluate the efficacy of CLT-005 in reducing retinal inflammation and vascular permeability in rodent models of diabetes and retinopathy. In this Phase I proposal, we will intravitreally administer varying concentrations of CLT-005 to the diabetic rat eyes and quantify the levels of the pro-inflammatory cytokines IL-6, TNF-1, and MCP-1. We will also assess and quantify leukostasis in these retinas as an early marker of inflammation. In addition, we will also quantify the efficacy of CLT-005 in reducing vascular leakage in the diabetic rat eye and in the rat model of oxygen-induced retinopathy which presents with acute retinal neovascularization. If this Phase I project proves the efficacy, these experiments will lay a solid ground for Phase II studies to further substantiate the promise of CLT-005 as a treatment for retinal inflammatory and macular edema. PUBLIC HEALTH RELEVANCE: Neovascular diseases of the retina, including Diabetic Retinopathy (DR) and Age-related Macular Degeneration (AMD), are the leading cause of blindness worldwide in patients over the age of 50. In several in vitro and in vivo models, activation of Stat3 has been shown to play a major role in initiating inflammatory and neovascular events in the retina. We have recently developed a novel small molecule (CLT-005) that inhibits this activation of Stat3, and thus presents as an ideal therapeutic for treating DR and AMD. The goal of this proposal is to prove the efficacy of CLT-005 is preventing retinal inflammation and vascular leakage in rodent models of diabetes and retinopathy.