New human organotypic corneal tissue model for ophthalmic drug delivery, dry eye, and wound healing studies

Abstract

Purpose : The purpose of the current work was to develop a physiologically-relevant, in vitro human 3D corneal epithelial tissue model (EpiCorneal) for ophthalmic drug development applications.

Methods : Normal human corneal epithelial cells were cultured at the air-liquid interface to produce the 3D corneal tissue model. Oxidative stress was generated by exposing the tissues to non-toxic doses of UV radiation, hydrogen peroxide, or by desiccating stress conditions that stimulate morphological, cellular, and molecular changes relevant to dry eye disease (DED). Corneal wounds were introduced by mechanical abrasion or alkali application. EpiCorneal permeability was evaluated using model compounds with a wide range of hydrophobicity, molecular weight, and excipients, including latanoprost family eye drops. qPCR arrays were utilized to investigate expression of 84 Phase I/II metabolizing enzymes and 84 drug transporter genes in the in vitro model and in isolated human corneal epithelium.

Results : EpiCorneal histology, expression of tight junctions, mucins, and tight barrier formation (1000±250Ω●cm²) are comparable to in vivo human corneal epithelium. Oxidative stress conditions resulted in significant accumulation of intracellular ROS and 8-Isoprostane, release of IL-8 and gene upregulation of pro-inflammatory cytokines and enzymes. Upregulation of Cyclin D1 expression was observed after 24h post-abrasion and the presence of EGFR inhibitor prevented wound healing. Drug metabolizing enzyme and transporter gene expression in EpiCorneal and excised human corneal epithelial were highly correlated (r²=0.87). Coefficients of permeation for model drugs in EpiCorneal and excised animal corneas also showed a high correlation (r²=0.84). As expected, Latanoprost and Bimatoprost free acids had much lower permeability (Papp=1.2x10^-6 and 1.9x10^-6) than corresponding prodrugs (Papp=2.5x10^-5 and 5.6x10^-5). The presence of 0.02%Benzalkonium chloride in ophthalmic formulations significantly affected tissue barrier and viability.

Conclusions : The newly developed EpiCorneal tissue model structurally and functionally reproduces DED, oxidative stress, wound healing markers, and its permeability resembles that of the in vivo human cornea. This model will be useful for evaluation of corneal drug permeability and safety during ophthalmic drug development and to study molecular mechanisms of ocular surface damage.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.