Optimizing NGF for Topical Treatment of Glaucoma

摘要 青光眼是不可逆性视力丧失的主要原因，其特征是进行性退化。 视网膜神经节细胞(RGC)及其视神经轴突。虽然年龄是一个关键的危险因素，但眼内高度升高 压力(IOP)是唯一可改变的危险因素，局部降眼压药物是一线治疗方法。 然而，在服用这些药物的患者中，有一半的患者RGC退化和视力丧失仍在继续。到那时 当检测到特征性视野缺陷时，30%-50%的视网膜节细胞已经丢失。因此，在那里 迫切需要开发与降低眼压无关的新疗法，以保护RGC免受 在疾病中受到挑战的视网膜节细胞的退化和增强功能。 神经生长因子(NGF)与TrkA结合可减轻反应性星形细胞增多症并促进RGC 生存和繁殖。相反，与p75NTR的结合导致星形胶质细胞和RGC的凋亡。血清 与健康对照组相比，早、中度青光眼患者的NGF水平降低。野生型 人NGF(OxervateTM，Cenegermim，Dompe)于2018年被FDA批准为治疗眼药水 神经性角膜炎。在最近的一项1b期临床试验中，已经表明局部使用西尼格明是 对开角型青光眼患者安全可耐受。然而，野生型NGF治疗未能提供 P75NTR活性上调拮抗临床前青光眼模型的神经保护作用。 此外，使用大肠杆菌表达系统生产Cenegermim的效率低下会损害蛋白质 稳定性和质量，而慢性治疗的成本高得令人望而却步。 在第一阶段的研究中，我们通过设计和验证人类神经生长因子成功地达到了所有里程碑 变种人，HC201。突变体通过增强TrkA受体的活性来保持蛋白质的稳定性和表达 但取消了p75NTR的结合和信号转导。在巩膜外静脉灼烧法诱导的大鼠青光眼模型中， 用HC201强力保护RGC进行局部治疗。相反，野生型NGF并不有效。HC201 在糖尿病角膜溃疡和干眼病模型上也观察到了疗效。与此同时，我们已经开发出 一种在哺乳动物细胞中生产HC201的高成本效益和可扩展的方法。在第二阶段SBIR中 应用后，我们将继续验证诱导微球的疗效并阐明其作用机制。 大鼠和松鼠猴青光眼模型的建立。不同方法提高眼压的临床研究 两个物种将显著提高转化为人类临床试验的成功。拥有一位经验丰富的 作为药物开发团队的一员，我们还将启动必要的关键活动，使IND备案成为可能。 具体目标包括：1)确定使用HC201的局部治疗是否保持视网膜完整性和 微珠阻塞大鼠和松鼠青光眼模型与安慰剂对照的功能比较 2)从100L工程运行中生产出cGMP级HC201；3)评价其非临床安全性 重复剂量HC201。 长期目标是单独或与目前的降压药联合开发HC201，以 保护甚至恢复青光眼患者的视力。

ABSTRACT Glaucoma is a leading cause of irreversible vision loss, which is characterized by progressive degeneration of retinal ganglion cells (RGC) and their optic nerve axons. While age is a key risk factor, elevated intraocular pressure (IOP) is the only modifiable risk factor, with topical IOP-lowering drugs as the first line treatment. However, RGC degeneration and vision loss continues in half the patients taking these medications. By the time when characteristic visual field defects are detected, 30-50% of the RGCs have already been lost. Thus, there is an urgent need to develop novel therapies, independent of IOP reduction, which protect RGCs from degeneration and boost the function of RGCs challenged in the disease. The binding of nerve growth factor (NGF) to TrkA attenuates reactive astrocytosis and promotes RGC survival and proliferation. In contrast, the binding to p75NTR leads to astrocytosis and RGC apoptosis. Serum levels of NGF are reduced in early and moderate glaucoma patients compared to healthy controls. Wildtype human NGF (OxervateTM, Cenegermim, Dompe) was approved by FDA in 2018 as eye drops to treat neurotrophic keratitis. In a recent Phase 1b clinical trial, it has been shown that topical use of Cenegermim was safe and tolerable in open-angle glaucoma patients. However, wildtype NGF treatment failed to provide neuroprotection in preclinical models of established glaucoma, antagonized by up-regulated p75NTR activity. Moreover, inefficient manufacturing of Cenegermim using an E coli expression system compromises protein stability and quality at a prohibitive cost for chronic treatment. In the Phase 1 study, we successfully reached all the milestones by designing and validating a human NGF mutein, HC201. The mutein preserves protein stability and expression with enhanced TrkA receptor activities but abrogated p75NTR binding and signaling. In rat models of glaucoma induced by episcleral vein cauterization, topical treatment with HC201 robustly protected RGCs. In contrast, wildtype NGF was not effective. HC201 efficacy was also observed in diabetic corneal ulcer and dry eye disease models. Meanwhile, we have developed a highly cost-effective and scalable process to produce HC201 in mammalian cells. In the Phase II SBIR application, we will continue to validate the efficacy and elucidate mechanism of action in the inducible microbead occlusion model of glaucoma in rats and squirrel monkeys. The use of different approaches to elevate IOP in two species will significantly improve the success for translation to human clinical trials. With a highly experienced drug development team, we also will initiate critical activities necessary to enable IND filing. Specific Aims include: 1) Determine whether topical treatment with HC201 preserves retinal integrity and function compared to the placebo control using the microbead occlusion model of glaucoma in rats and squirrel monkeys; 2) Manufacture cGMP grade HC201 from 100L engineering run; 3) Evaluate nonclinical safety of repeat dose HC201. The long-term goal is to develop HC201, alone or in combination with current hypotensive medication, to preserve and even restore vision in glaucoma patients.