Oxidative stress in corneal injuries of different origin: Utilization of 3D human corneal epithelial tissue model.

The purpose of the current work was to utilize a three dimensional (3D) corneal epithelial tissue model to study dry eye disease and oxidative stress-related corneal epithelial injuries for the advancement of ocular therapeutics. Air-liquid interface cultures of normal human corneal epithelial cells were used to produce 3D corneal epithelial tissues appropriate for physiologically relevant exposure to environmental factors. Oxidative stress was generated by exposing the tissues to non-toxic doses of ultraviolet radiation (UV), hydrogen peroxide, vesicating agent nitrogen mustard, or desiccating conditions that stimulated morphological, cellular, and molecular changes relevant to dry eye disease. Corneal specific responses, including barrier function, tissue viability, reactive oxygen species (ROS) accumulation, lipid peroxidation, cytokine release, histology, and gene expression were evaluated. 3D corneal epithelial tissue model structurally and functionally reproduced key features of molecular responses of various types of oxidative stress-induced ocular damage. The most pronounced effects for different treatments were: UV irradiation - intracellular ROS accumulation; hydrogen peroxide exposure - barrier impairment and IL-8 release; nitrogen mustard exposure - lipid peroxidation and IL-8 release; desiccating conditions - tissue thinning, a decline in mucin expression, increased lipid peroxidation and IL-8 release. Utilizing a PCR gene array, we compared the effects of corneal epithelial damage on the expression of 84 oxidative stress-responsive genes and found specific molecular responses for each type of damage. The topical application of lubricant eye drops improved tissue morphology while decreasing lipid peroxidation and IL-8 release from tissues incubated at desiccating conditions. This model is anticipated to be a valuable tool to study molecular mechanisms of corneal epithelial damage and aid in the development of therapies against dry eye disease, oxidative stress- and vesicant-induced ocular injuries.

New Human Organotypic Corneal Tissue Model for Ophthalmic Drug Delivery Studies.

Purpose: The purpose of the current work was to develop a physiologically relevant, in vitro human three-dimensional (3D) corneal epithelial tissue model for use in ophthalmic drug development. Methods: Normal human corneal epithelial cells were cultured at the air-liquid interface to produce the 3D corneal tissue model. Corneal barrier was determined by measuring transepithelial electrical resistance (TEER). Quantitative PCR arrays were utilized to investigate expression of 84 phase I/II metabolizing enzymes and 84 drug transporter genes. Permeability was evaluated using model compounds with a wide range of hydrophobicity, molecular weight, and excipients. Finally, different formulations of latanoprost and bimatoprost were administered and drug absorption and tissue viability and integrity were investigated. Results: Histologic assessment and TEER of the corneal tissue model revealed tissue structure, thickness, and barrier formation (1000 ± 146 Ω·cm2) comparable to native human corneal epithelium. The 3D corneal tissue expressed tight junctions, mucins, and key corneal epithelial detoxification enzymes. Drug-metabolizing enzyme and transporter gene expression in 3D corneal tissue and excised human corneal epithelium were highly correlated (r2 = 0.87). Coefficients of permeation for model drugs in the tissue model and excised rabbit corneas also showed a high correlation (r2 = 0.94). As expected, latanoprost and bimatoprost free acids had much lower permeability (Papp = 1.2 × 10-6 and 1.9 × 10-6) than the corresponding prodrugs (Papp = 2.5 × 10-5 and 5.6 × 10-5), respectively. The presence of 0.02% benzalkonium chloride in ophthalmic formulations significantly affected tissue barrier and viability. Conclusions: The newly developed 3D corneal tissue model appears to be very useful for evaluation of corneal drug permeability and safety during ophthalmic drug development.

The EpiOcular Eye Irritation Test (EIT) for hazard identification and labelling of eye irritating chemicals: protocol optimisation for solid materials and the results after extended shipment.

The 7th Amendment to the EU Cosmetics Directive and the EU REACH Regulation have reinforced the need for in vitro ocular test methods. Validated in vitro ocular toxicity tests that can predict the human response to chemicals, cosmetics and other consumer products are required for the safety assessment of materials that intentionally, or inadvertently, come into contact with the eye. The EpiOcular Eye Irritation Test (EIT), which uses the normal human cell-based EpiOcular™ tissue model, was developed to address this need. The EpiOcular-EIT is able to discriminate, with high sensitivity and accuracy, between ocular irritant/corrosive materials and those that require no labelling. Although the original EpiOcular-EIT protocol was successfully pre-validated in an international, multicentre study sponsored by COLIPA (the predecessor to Cosmetics Europe), data from two larger studies (the EURL ECVAM-COLIPA validation study and an independent in-house validation at BASF SE) resulted in a sensitivity for the protocol for solids that was below the acceptance criteria set by the Validation Management Group (VMG) for eye irritation, and indicated the need for improvement of the assay's sensitivity for solids. By increasing the exposure time for solid materials from 90 minutes to 6 hours, the optimised EpiOcular-EIT protocol achieved 100% sensitivity, 68.4% specificity and 84.6% accuracy, thereby meeting all the acceptance criteria set by the VMG. In addition, to satisfy the needs of Japan and the Pacific region, the EpiOcular-EIT method was evaluated for its performance after extended shipment and storage of the tissues (4-5 days), and it was confirmed that the assay performs with similar levels of sensitivity, specificity and reproducibility in these circumstances.