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.

National Institute of Health (NIH)
National Eye Institute (NEI)
Research Project (R01)
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Anterior Eye Disease Study Section (AED)
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Mckie, George Ann
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Columbia University (N.Y.)
Schools of Medicine
New York
United States
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Paik, David C; Trokel, Stephen L; Suh, Leejee H (2018) Just What Do We Know About Corneal Collagen Turnover? Cornea 37:e49-e50
Zyablitskaya, Mariya; Munteanu, E Laura; Nagasaki, Takayuki et al. (2018) Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking (TXL) for Myopia. J Vis Exp :
Rapuano, Patrick B; Scanameo, Alexandra H; Amponin, Daeryl E et al. (2018) Antimicrobial Studies Using the Therapeutic Tissue Cross-Linking Agent, Sodium Hydroxymethylglycinate: Implication for Treating Infectious Keratitis. Invest Ophthalmol Vis Sci 59:332-337
Zyablitskaya, Mariya; Takaoka, Anna; Munteanu, Emilia L et al. (2017) Evaluation of Therapeutic Tissue Crosslinking (TXL) for Myopia Using Second Harmonic Generation Signal Microscopy in Rabbit Sclera. Invest Ophthalmol Vis Sci 58:21-29
Takaoka, Anna; Babar, Natasha; Hogan, Julia et al. (2016) An Evaluation of Lysyl Oxidase-Derived Cross-Linking in Keratoconus by Liquid Chromatography/Mass Spectrometry. Invest Ophthalmol Vis Sci 57:126-36
Kim, Su-Young; Babar, Natasha; Munteanu, Emilia Laura et al. (2016) Evaluating the Toxicity/Fixation Balance for Corneal Cross-Linking With Sodium Hydroxymethylglycinate (SMG) and Riboflavin-UVA (CXL) in an Ex Vivo Rabbit Model Using Confocal Laser Scanning Fluorescence Microscopy. Cornea 35:550-6
Babar, Natasha; Kim, MiJung; Cao, Kerry et al. (2015) Cosmetic preservatives as therapeutic corneal and scleral tissue cross-linking agents. Invest Ophthalmol Vis Sci 56:1274-82
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
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
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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