Preclinical Evaluation of a Novel Artificial Tear (AT) for Dry Eye

Abstract

Abstract: : Purpose: Using a risk analysis approach, data obtained during screening analyses were used to develop a unique battery of in vitro and in vivo tests to evaluate the ocular safety of a novel AT solution. Methods: Cytotoxicity was evaluated in an exaggerated neutral red release (NRR) assay using MDCK cells, an agar overlay assay, and by imaging of corneal epithelial (CE) cells using Hoffman modulation contrast optics. Corneal permeability to carboxyfluoroscein (CF) was determined ex vivo in a modified corneal perfusion system following epithelial exposure to AT, or in vivo by fluorophotometry in anesthetized rabbits after 3 min exposure in a conjunctival cup model or QID dosing for 5 days with AT, buffered saline or BAC–containing solutions. Protection of corneas of anesthetized rabbits from desiccation was measured by methylene blue uptake, and promotion of recovery of corneas damaged with 0.01% BAC was measured by CF uptake. Ocular toxicity potential was evaluated by biomicroscopy and/or SEM in rabbits after normal use 4–8 times/day for up to 3 months. Exaggerated use, or misuse, was evaluated in rabbits dosed 12 times/day, or during soft contact lens (SCL) wear and following microkeratotomy. Results: The AT solution passed the agar overlay assay but significantly reduced cell viability in the NRR assay after 20–60 min exposure compared to BAC–containing solutions. Exposure of CE cells to AT for 20–60 min caused vacuolization and loss of cell junctions. The effect of AT on ex vivo corneal permeability to CF was equivalent to that of buffered saline and significantly lower than the effect of BAC–containing solutions. The effect of AT on in vivo CF permeability was not different from control and was lower than a BAC–containing product. The AT completely protected the cornea during a 2 hr desiccation compared to untreated controls, and promoted significant recovery of the epithelial barrier of BAC–damaged corneas. SEM after 7 days of normal use showed normal cell junctions and morphology. Biomicroscopy showed no ocular effects of AT during 3 months of normal use, short–term exaggerated use, or misuse. Conclusion: In vitro assays showed a higher cytotoxic potential of the novel AT than marketed solutions, while extensive ex vivo and in vivo tests selected through risk analysis showed the AT to be equivalent or better than marketed solutions. In vitro assays are valuable screening tools, but the data must be evaluated with caution since they are not directly predictive of the in vivo safety of ophthalmic solutions.

Keywords: ocular irritancy/toxicity testing • cornea: tears/tear film/dry eye • cornea: epithelium