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Yulia Kaluzhny, Miriam W Kinuthia, Aikaterini M Gremilogianni, Thoa Truong, Patrick Hayden, Mitch Klausner; In Vitro Reconstructed Human Corneal Tissue Model: Applications to Dry Eye and Ophthalmic Drug Delivery. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1963.
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© ARVO (1962-2015); The Authors (2016-present)
The corneal barrier is vitally important for protection from environmental stress but it also presents a major challenge for delivery of ophthalmic drugs. Current methods to investigate pathogenesis of dry eye disease (DED) and permeation of topically applied ophthalmics utilize cell cultures or animals. This study evaluated the utility of an in vitro reconstructed 3-D corneal tissue model for DED and drug permeation.
Reconstructed tissues were characterized by histology, confocal microscopy, barrier function, and expression of genes essential for metabolism, detoxification, and drug transport. Oxidative stress was generated by exposing the tissues to simulated solar UV radiation (60 mJ UVB) or by placing tissues under desiccating stress conditions (DSC, 40% RH, 40°C, and 5% CO2) that stimulate morphological, cellular, and molecular changes relevant to DED. Corneal permeability was evaluated using model compounds with a wide range of hydrophobicity, molecular weight, and excipients, including seven formulations of Latanoprost eye drops.
The tissues express mucin 4 and 16, as well as ALDH3A1 and ALDH1A1, TXNRD1, UGTIA1, P450 cytochromes, efflax (ABC), peptide (SLS15), and uptake (SLS22) transporters. UV exposure resulted in up to 60X increases in cellular ROS accumulation, which droped by 73% 2h post-exposure. 24h incubation at DSC induced up to 5.5X increase in intracellular ROS. Both conditions caused considerable release of IL-1a and IL-8 and gene up-regulation for pro-inflammatory cytokines and enzymes, but didn’t affect tissue viability. Following exposure to UV or DSC, topical application of lubricant gel drops improved tissue morphology and barrier function. The correlation of permeation coefficients to excised animal corneas for model drugs was r2=0.84. Out of seven formulations of eye drops tested, Xalatan had the highest permeation (Papp=8.81) and Poloxamer 407 increased Papp to 6.05 and 6.27 compared to surfactant-free formulations (1.69 to 2.57).
The in vitro reconstructed normal human corneal tissue model structurally and functionally reproduces oxidative stress and DED markers, and its permeability resembles that of the in vivo human cornea. This model is anticipated to be a useful tool to study molecular mechanisms of ocular surface damage, DED, and to evaluate new corneal drug formulations.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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