Therapeutic Corneal Cross-linking Using Aliphatic Beta-Nitroalcohols

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（UVAR）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UVAR治疗仍存在风险，特别是与紫外线照射有关，禁止在薄角膜中使用，并且需要痛苦的上皮切除。或许有更好的办法。本提案的主旨是开发一种使用新技术的角膜胶原交联的替代方法。特别是，我们已经确定了一类化合物(即脂肪族？-硝基醇= BNAs)在生理pH值和温度条件下作为组织交联剂似乎是安全有效的。与目前的UVAR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，不需要对上皮进行清创，从而减少患者的不适和感染的机会，有剂量反应效应，并且能够治疗周围角膜。该项目旨在为将该技术快速转化为治疗方法奠定基础。在目标1中，将使用分析仪器确定BNA交联反应所涉及的化学机制。机械测定将导致有效催化剂的鉴定，这将通过生化收缩温度分析和机械失效测试来证实。目的1旨在确定一种或多种“候选滴眼液制剂”，然后用于兔体内实验。在目标2中，将使用角膜上皮细胞、角质细胞和内皮细胞的原代培养物来确定BNAs（包括高阶BNAs = HONAs）和催化剂的毒性阈值。本目的结果将确定硝基化合物对角膜细胞的毒性水平。在目的3中，使用Franz扩散细胞和离体兔角膜来测定BNA经角膜通透性。此外，还将研究优化渗透性（增强剂，如丙帕卡因和苯扎氯铵）和递送（如merocel海绵、水凝胶隐形眼镜和胶原蛋白保护罩）的方法。在目标4中，将对活兔子进行局部交联。所使用的递送方法和药剂将由从目标1、2和3中获得的信息决定。将进行实时体内疗效和安全性评估（使用体内共聚焦显微镜、超声测厚仪、角膜地形图和压平眼压计），随后进行死后机械失效测试、收缩温度分析、组织学和透射电镜。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。

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 (UVAR) can limit progressive vision loss in keratoconus patients. Despite these successes, the UVAR therapy poses attendant risks, particularly related to ultraviolet irradiation, is prohibited in thin corneas, and requires painful epithelial removal. A better approach may be possible. The thrust of this proposal is to develop an alternative method of corneal collagen cross- linking using novel technology. In particular, we have identified a class of compounds (i.e. aliphatic ?-nitro alcohols = BNAs) that appear to be safe and effective as tissue cross-linking agents under conditions of physiologic pH and temperature. Potential advantages over current UVAR therapy include the omission of ultraviolet light exposure, the ability to treat thin corneas, no epithelial debridement resulting in less patient discomfort and a lower chance of infection, 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. In aim 1, the chemical mechanisms involved in BNA cross-linking reactions will be determined using analytical instrumentation. Mechanistic determinations will lead to the identification of effective catalysts which will be confirmed using biochemical shrinkage temperature analysis and mechanical failure testing. Aim 1 is designed to identify one or more "candidate eye drop preparations" that will then be used for in vivo rabbit experiments. In aim 2, primary cultures of corneal epithelial cells, keratocytes, and endothelial cells will be used to determine the toxic thresholds for BNAs (including higher order BNAs = HONAs) and catalysts. The results from this aim will establish the toxicity level of nitro compounds to corneal cells. In aim 3, BNA transcorneal permeability using a Franz diffusion cell and ex vivo rabbit corneas will be determined. In addition, ways of optimizing permeability (enhancing agents such as proparacaine and benzalkonium chloride) and delivery (devices such as merocel sponges, hydrogel contact lenses, and collagen shields) will be studied. In aim 4, topical cross-linking of live rabbits will be undertaken. The delivery method and agents used will be determined by the information gained from aims 1, 2, and 3. Real time in vivo efficacy and safety evaluations (using in vivo confocal microscopy, ultrasound pachymetry, corneal topography, and applanation tonometry) will be performed, followed by post-mortem mechanical failure testing, shrinkage temperature analysis, histology, and TEM. 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.