A Novel Treatment for Keratoconus and Keratectasias using Nitro Technology

描述（申请人提供）：在圆锥角膜和lasik术后角膜扩张的治疗上取得了重大突破。德国Wollensak， Spoerl和Seiler小组最近的工作表明，通过核黄素和紫外线（UVR）的应用交联角膜胶原蛋白可以限制圆锥角膜患者的进行性视力丧失。尽管取得了这些成功，但紫外线照射疗法也带来了风险，特别是与紫外线照射有关的风险。因此，FDA最近才批准在美国进行临床试验。本应用的主旨是利用新技术开发一种角膜胶原交联的替代方法。我们最近的研究表明，通过与亚硝酸盐和相关药物的反应，胶原交联可以诱导与UVR治疗相适应的角膜变化。此外，已发表的毒性/诱变性研究表明，这些药物具有良好的安全性。因此，硝基技术可以作为UVR治疗的“眼药水替代品”。与目前的UVR治疗相比，潜在的优势包括不需要紫外线照射，能够治疗薄角膜，患者不适较少，易于自我给药，剂量反应效应，以及能够治疗周围角膜。该项目旨在为将该技术快速转化为治疗方法奠定基础。具体来说，我们将：1。在模拟人类角膜的条件下（即pH值7.4和34℃），确定一种使用硝基技术进行角膜交联的有效方法。新鲜的兔和人眼库角膜将在各种条件下孵育，包括1。亚硝酸盐与催化剂和2。替代亚硝酸盐相关剂。热收缩温度测定将用于确定交联效果。将使用双轴材料试验机研究引起的刚度变化。特异性目标1旨在确定一种或多种“候选眼药水”，然后用于兔体内实验。2. 验证角膜细胞能够耐受通过硝基技术进行的角膜交联的假设。兔内皮细胞、角化细胞和上皮细胞的原代培养将在体外培养。然后将细胞暴露于被发现对交联有效的硝基化合物中。使用标准的细胞凋亡和坏死试验评估细胞毒性。本研究结果将确定硝基化合物对角膜细胞的体外毒性水平。具体目标3。通过硝基技术验证角膜交联对活体眼睛的有效性和安全性。将33只年轻成年雌性新西兰白兔每天（1-6周）在右眼涂抹局部硝基溶液，另一只眼作为对照。动物将接受3种非侵入性仪器的连续检查：角膜地形仪、体内共聚焦显微镜和眼压计。死后，生物力学强度测试，组织学和扫描电镜将进行。这一目标的结果将确定该技术的体内有效性和安全性，并将决定人体I期试验的可行性。本研究旨在开发一种简单的、新的治疗角膜不稳定疾病的方法，包括圆锥角膜和术后角膜扩张。后一种情况是毁灭性的，长期（5-10年）并发症后LASIK手术。它们以前是无法预测的，目前的流行病学比例是未知的。

DESCRIPTION (provided by applicant): A major breakthrough in the treatment of keratoconus and post-LASIK keratectasias has been realized. Recent work by the German group of Wollensak, Spoerl, and Seiler has shown that cross-linking corneal collagen through application of riboflavin and ultraviolet light (UVR) can limit progressive vision loss in keratoconus patients. Despite these successes, the UVR therapy poses attendant risks, particularly related to ultraviolet irradiation. As such, FDA approval for clinical trials in the US has only recently been granted. The thrust of this application is to develop an alternative method of corneal collagen cross-linking using novel technology. Our recent studies indicate that collagen cross-linking through reactions with nitrite and related agents can induce corneal changes commensurate with UVR therapy. In addition, published toxicity/mutagenicity studies suggest a good safety profile for these agents. Thus, nitro technology could be used as an "eye drop alternative" to UVR treatment. Potential advantages over current UVR therapy include the omission of ultraviolet light exposure, the ability to treat thin corneas, less patient discomfort, ease of self- administration, a dose-response effect, and the ability to treat the peripheral cornea. This project is designed to lay groundwork for rapid translation of this technology into a treatment. Specifically, we will: 1. Determine an effective way to cross-link the cornea using nitro technology under conditions simulating the human cornea (i.e. pH 7.4 and 34oC). Fresh rabbit and human eye bank corneas will be incubated under a variety of conditions that include 1. nitrite with catalysts and 2. alternative nitrite related agents. The assay of thermal shrinkage temperature will be used to determine cross-linking efficacy. Stiffness changes induced will be studied using a biaxial material tester. Specific aim 1 is designed to identify one or more "candidate eye drops" that will then be used for in vivo rabbit experiments. 2. Test the hypothesis that corneal cross-linking through nitro technology is tolerated by corneal cells. Primary cultures of rabbit endothelial cells, keratocytes, and epithelial cells will be grown in vitro. The cells will then be exposed to nitro compounds found to be efficacious for cross-linking. Cytotoxicity will be evaluated using standard apoptosis and necrosis assays. The results from this aim will establish the in vitro toxicity level of nitro compounds to corneal cells. Specific aim 3. Test the hypothesis that corneal cross-linking through nitro technology has efficacy and is safe for the living eye. 33 young adult female New Zealand white rabbits will undergo daily application (for 1-6wks) of the topical nitro solution to the right eye with the fellow eye serving as a control. The animals will undergo serial examinations using 3 non-invasive instruments, a corneal topographer, in vivo confocal microscope, and applanation tonometer. Post-mortem, biomechanical strength testing, histology, and SEM will be performed. The results of this aim will establish the in vivo efficacy and safety of this technology and will dictate the feasibility of a human phase I trial. PUBLIC HEALTH RELEVANCE This research aims to develop a simple, new treatment for diseases of corneal destabilization which include keratoconus and post-surgical keratectasias. The latter conditions are devastating, long-term (5-10 years) complications following LASIK procedures. They were previously unpredicted and are currently of unknown epidemiologic proportion.