Turbo Eye Drops to Treat Ocular Toxicity and Blindness from Sulfur Mustard

摘要 硫芥子气（SM）是一种起泡剂和战剂，自第一次世界大战以来已被用于许多战争; 最近在叙利亚。SM在接触后迅速穿透眼睛，并通过损伤角膜组织导致失明- 组织和功能。临床上，患者表现出被称为芥子气角膜病变的病理学， 严重眼部炎症，复发性上皮糜烂，上皮-基质分离，角膜缘干细胞 缺乏、角膜溃疡、混浊和新生血管形成。MGK病理生理学是双相的，包括急性 和延迟发病，涉及多种机制。我们开发了一种新型的，多模式的，非甾体局部 滴眼液，Turbo Eye Drop（TED），含有4种FDA批准的仿制药， 作用，并在环境温度下稳定。我们的初步研究发现，局部TED有效地治疗急性和 在兔体内和离体人角膜中的迟发型MGK，没有显著的副作用。我们的中央 假设是局部TED通过抑制SM诱导的早期MGK来治疗体内急性和延迟发作的MGK。 炎症反应，细胞外基质降解，以及过度促纤维化和促纤维化的产生。 无明显副作用的血管生成因子。该项目测试了两个新的假设，以建立一个 有效和安全的局部治疗急性和迟发型MGK在体内，使用四个特定的目标：目的-1 通过测试增加频率和持续时间的假设， TED的应用将有效地治疗急性MGK和失明，而没有明显的副作用。Aim-2建立 通过检验低TED局部给药持续时间较长的假设， 持续时间将有效地治愈延迟发作的MGK，而不会在家兔中出现问题。Aim-3揭示了 TED在体内和体外缓解急性和延迟发作的MGK。Aim-4保护知识产权， 制定监管策略，并将TED局部滴眼液作为SM诱导的 对人类应用的眼损伤。这将使用已建立的SM-蒸汽家兔体内完成 和人角膜器官培养离体模型，GMP级TED滴眼液，以及活兔中的监测眼 以时间依赖的方式与临床眼科检查和诊断成像。的表征 TED用于减轻MGK的机制将使用安乐死后收集的角膜组织进行研究， 测量角膜上皮基底膜、上皮基质组织和胶原纤维的完整性 使用qPCR、ELISA、免疫荧光、H&E和透射电子显微镜进行排列 利用我们公布的方法。该项目的成功完成将导致 急性和迟发性MGK的有效和安全的治疗方法，以及减少眼部损伤的医学对策 在人类中意外或故意使用SM导致的闭塞，因此将产生非常高的影响 在现场和公共安全。

ABSTRACT Sulfur mustard gas (SM), a vesicating and warfare agent, has been used in many wars since World War I; most recently in Syria. SM rapidly penetrates the eye on contact and causes blindness by injuring corneal tissue- organization and function. Clinically, patients show a pathology termed as Mustard Gas Keratopathy that involves severe ocular inflammation, recurrent epithelial-erosions, epithelial-stromal separation limbal stem cell deficiency, corneal ulceration, haze and neovascularization. MGK pathophysiology is biphasic including acute and delayed-onset, and involves multiple mechanisms. We developed a novel, multimodal, non-steroidal topical ophthalmic drops, Turbo Eye Drop (TED), containing 4 FDA-approved generic drugs with differing mode of action, and stable at ambient temperature. Our pilot studies found that topical TED efficaciously treats acute and delayed-onset MGK in rabbits in vivo and human cornea ex vivo without significant side effects. Our central hypothesis is that topical TED treats acute and delayed-onset MGK in vivo by curbing SM-induced early inflammatory responses, extracellular matrix degradation, and production of excessive pro-fibrotic and pro- angiogenic factors without significant side effects. This project tests two novel hypotheses to establish an efficacious and safe topical therapy for acute and delayed-onset MGK in vivo, using four specific aims: Aim-1 defines TED treatment for acute MGK in vivo by testing the hypothesis that increasing frequency and duration of TED application will potently treat acute MGK and blindness without significant side effects. Aim-2 establishes TED treatment for delayed-onset MGK in vivo by testing the hypothesis that low TED topical dosing for longer duration will effectively cure delayed-onset MGK without issues in rabbits. Aim-3 uncovers mechanisms used by TED in mitigating acute and delayed-onset MGK in vivo and in vitro. Aim-4 secures intellectual property rights, develops regulatory strategies, and advances TED topical ophthalmic drops as an antidote for SM-induced ocular injury towards human application. This will be accomplished using an established SM-vapor rabbit in vivo and human cornea organ culture ex vivo models, GMP-grade TED eye drops, and monitoring eyes in live rabbits in a time-dependent manner with clinical eye exams and diagnostic imaging. The characterization of mechanisms used by TED in mitigating MGK will be studied using corneal tissues collected after euthanasia by measuring integrity of corneal epithelial basement membrane, epithelial-stromal organization, and collagen fibril arrangement using qPCR, ELISAs, immunofluorescence, H&E, and transmission electron microscopy techniques utilizing our published methods. Successful completion of the project will lead to the development of an effective and safe therapy for acute and delayed MGK and medical countermeasure to minimize ocular obliteration caused by the accidental or intentional use of SM in humans, and therefore will have very high impact in field and public safety.