Therapeutic Corneal Cross-linking Using Aliphatic Beta-Nitroalcohols

描述(申请人提供)：圆锥角膜和LASIK术后角膜扩张症的治疗已经实现了重大突破。德国Wollensak、Spoerl和Seler小组最近的研究表明，通过应用核黄素和紫外线(UVAR)使角膜胶原交联可以限制圆锥角膜患者的进行性视力损失。尽管取得了这些成功，但UVAR疗法带来了随之而来的风险，特别是与紫外线照射有关的风险，薄角膜是被禁止的，需要痛苦的上皮切除。一种更好的方法或许是可能的。这项提议的主旨是利用新技术开发一种替代角膜胶原交联剂的方法。特别是，我们已经确定了一类化合物(即脂肪族？-硝基醇=BNAs)，它们在生理pH和温度条件下似乎是安全和有效的组织交联剂。与目前的UVAR疗法相比，UVAR疗法的潜在优势包括避免紫外线照射，治疗薄角膜的能力，不进行上皮清创，减少患者不适和感染机会，剂量-反应效应，以及治疗周边角膜的能力。该项目旨在为将这项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。机械测定将导致确定有效的催化剂，这将通过生化收缩温度分析和机械故障测试来确认。AIM 1旨在鉴定一种或多种“候选滴眼液制剂”，然后将其用于在体兔体内实验。在目标2中，将使用角膜上皮细胞、角膜基质细胞和内皮细胞的原代培养来确定BNAs(包括高阶BNAs=HONAs)和催化剂的毒性阈值。这一目标的结果将确定硝基化合物对角膜细胞的毒性水平。在目标3中，将使用Franz扩散池和体外兔角膜来测定BNA经角膜的渗透性。此外，还将研究优化渗透性(如丙泊卡因和苯扎氯铵等增强剂)和传递(Merocel海绵、水凝胶隐形眼镜和胶原膜等设备)的方法。在目标4中，将进行活兔的局部交联性研究。所使用的给药方法和制剂将根据从AIMS 1、2和3获得的信息确定。将进行实时体内疗效和安全性评估(使用体内共焦显微镜、超声测厚、角膜地形图和压平眼压计)，然后进行死后机械故障测试、收缩温度分析、组织学和透射电子显微镜。这一目标的结果将确定这项技术的体内有效性和安全性，并将决定人类I期试验的可行性。 与公共健康相关：这项研究旨在开发一种安全有效的方法来增加活体组织的机械强度(治疗交联物)，并可在许多医学领域中应用。特别是，我们正在利用这项技术开发一种简单的、新的治疗角膜不稳定疾病的方法，包括圆锥角膜和手术后角膜扩张。后一种情况可能是众所周知的LASIK手术(激光辅助原位角膜磨镶术)后的破坏性、长期(长达10年或更长时间)的并发症，目前流行病学比例尚不清楚。

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. PUBLIC HEALTH RELEVANCE: This research aims to develop a safe and effective way to increase the mechanical strength of tissues (therapeutic cross-linking) in a living human being and could have applications in many fields of medicine. In particular, we are using this technology to develop a simple, new treatment for diseases of corneal destabilization which includes keratoconus and post-surgical keratectasia. The latter condition can be a devastating, long-term (up to 10 years or more) complication following the well-known LASIK procedure (Laser-Assisted In situ Keratomileusis) and is currently of unknown epidemiologic proportion.