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.

National Institute of Health (NIH)
National Eye Institute (NEI)
Exploratory/Developmental Grants (R21)
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Anterior Eye Disease Study Section (AED)
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Shen, Grace L
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Columbia University (N.Y.)
Schools of Medicine
New York
United States
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Li, Xia; Li, Yongjun; Kim, Mijung et al. (2014) Aliphatic ?-nitroalcohols for therapeutic corneoscleral cross-linking: chemical stability studies using 1H-NMR spectroscopy. Photochem Photobiol 90:338-43
Kim, MiJung; Takaoka, Anna; Hoang, Quan V et al. (2014) Pharmacologic alternatives to riboflavin photochemical corneal cross-linking: a comparison study of cell toxicity thresholds. Invest Ophthalmol Vis Sci 55:3247-57
Wen, Quan; Trokel, Stephen L; Kim, MiJung et al. (2013) Aliphatic ýý-nitroalcohols for therapeutic corneoscleral cross-linking: corneal permeability considerations. Cornea 32:179-84
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Solomon, Marissa R; O'Connor, Naphtali A; Paik, David C et al. (2010) Nitroalcohol Induced Hydrogel Formation in Amine-Functionalized Polymers. J Appl Polym Sci 117:1193-1196
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