Effects of RGTA OTR4131 on Ocular Surface: in vivo Evaluation on a Rabbit Corneal Wound Healing Model and in vitro Toxicological Studies

Abstract

Abstract: : Purpose: Regenerating Agent, RGTA, is an engineered biopolymer mimicking heparan sulfates as a protector and stabilizer of the actions of heparin–binding growth factors and is known to stimulate wound healing in different in vivo systems. In this study, we analyzed its effects both in vivo and in vitro in order to assess its potential interest in ocular surface diseases. Methods: 1/ In vivo, we investigated the wound healing activity of a single application of RGTA 1h after an alkali burn using NaOH in 1 eye of 20 rabbits. During 7d, we scored the clinical signs and quantified the corneal opacity and the deepithelialization. Histology was performed at day 7. 2/ In vitro, we studied, on human conjunctival epithelial cells and rabbit corneal fibroblasts, the effects of RGTA on cell proliferation, apoptosis using flow cytometry and oxidative stress (OS) with a previously validated technique of cold light cytometry on live cells. We looked also after its eventual protective effect in an in vitro model of Benzalkonium Chloride (BAC)–induced cell toxicity. Results: In vivo, RGTA was efficient in enhancing reepithelialization, reducing clinical signs, corneal opacity and improving histological patterns. In vitro, it did not induce any alteration of cell viability nor apoptosis on both cell lines. On corneal fibroblasts but not on epithelial cells, it reduced cell proliferation in a dose–dependent manner. Used in combination with a toxic dose of BAC, it showed a protective effect on conjunctival epithelial cells. RGTA did not induce by itself OS, but could protect conjunctival cells from OS induced by BAC through a reduction of H₂O₂ production and glutathione uptake. Conclusions:RGTA is a promising drug in controlling ocular surface inflammation and promoting corneal wound healing, and is not toxic both in vivo and in vitro. Its antioxidative and antifibroblast proliferation effects also suggest that it could be interesting for reducing uncontrolled corneal scarring. These properties may deserve further interest for future therapeutic applications in patients suffering of various ocular surface diseases.

Keywords: ocular irritancy/toxicity testing • wound healing • antioxidants