In Vitro Human Corneal Epithelial Toxicity of Prostaglandin Analogues Mark McDermott, Fu-Shin X. Yu, Jia Yin, Ashok Kumar, Ke-Ping Xu Mark McDermott, Fu-Shin X. Yu, Jia Yin, Ashok Kumar, Ke-Ping Xu Kresge Eye Institute Wayne State University, Detroit, MI This study was funded by an unrestricted grant from Allergan, Inc. The authors have no financial relationships to disclose.

Abstract  Purpose: To determine the cytotoxicity of prostaglandin analogues (PGAs) on cultured human corneal epithelial cells using ethidium bromide staining.  Methods: Immortalized human corneal epithelial cells were grown in confluence. Cell culture assay performed of commercial formulations of bimatoprost 0.03%, travoprost 0.004%, travoprost 0.004% (BAK-free), latanoprost 0.005%, media and methanol. Cultured microtiter plates (n = 5 per group) were exposed for 25 minutes to agents and controls. Ethidium bromide (EB) staining was performed and fluorescence quantified.  Results: At 25 minutes exposure to agents, the mean EB staining fluorescence measured was 26.8 with bimatoprost, 27.5 with travoprost (BAK-free), 32.7 with travoprost, 60.5 with latanoprost, 12.1 with media, and 51.6 with methanol. At P <.05, using one-way ANOVA, travoprost, travoprost (BAK-free), and bimatoprost all showed significantly less staining florescence than latanoprost.  Conclusion: The EB staining model demonstrates that bimatoprost, travoprost, and travoprost (BAK-free) demonstrate less cellular toxicity than latanoprost or toxic controls. Additional clinical studies will need to be performed to further evaluate the effects of PGAs on the corneal surface.

Introduction  Preservatives are an important component of ophthalmic medications. –Prevent microbial growth and decomposition of active drug  Benzalkonium chloride (BAK) is most commonly used preservative. –Xalatan ® (latanoprost; Pfizer, Inc.; New York, NY) = 0.02% BAK –Travatan ® (travoprost; Alcon Laboratories, Inc.; Fort Worth, TX) = 0.015% BAK –Lumigan ® (bimatoprost; Allergan, Inc.; Irvine, CA) = 0.005% BAK  Higher concentrations of BAK may cause ocular irritation. –Bimatoprost associated with lower corneal toxicity than latanoprost Noecker et al. Cornea. 2004;23:

Introduction  Corneal epithelial cells are a useful in vitro model for evaluating ocular irritancy in humans. 1  Ethidium Bromide (EB) staining in a useful technique for assessing cell toxicity. –Commonly used nucleic acid stain –Yields fluorescent red-orange color after binding to DNA –Degree of staining in cells reflects loss of cell membrane integrity 1. Xu et al. Toxicol Sci. 2000;58:

Purpose  To determine the cytotoxicity of prostaglandin analogues (PGAs) on cultured human corneal epithelial cells using EB staining.

Methods  Immortalized human corneal epithelial cells were grown in confluence.  Cell culture assay performed of commercial formulations of –Lumigan ® (bimatoprost 0.03%) –Travatan ® (travoprost 0.004%) –Travatan Z ® (BAK-free travoprost 0.004%) –Xalatan ® (latanoprost 0.005%) –Media –Methanol  Cultured microtiter plates (n = 5 per group) were exposed for 25 minutes to test agents and controls.  EB staining was performed and fluorescence quantified.

Results  Travatan ® (travoprost), Travatan Z ® (BAK-free travoprost), and Lumigan ® (bimatoprost) all showed significantly less staining florescence than Xalatan ® (latanoprost) (one-way analysis of variance [ANOVA], P <.05) Fluorescence EB Staining in Human Corneal Epithelial Cells Xalatan ® Lumigan ® Travatan ® Travatan Z ® MediaMethanol

Discussion  Results indicate that cellular toxicity in human corneal epithelial cells varies between different PGAs. –May be related to a combination of active ingredient and preservative.  Toxicity and BAK concentrations are higher with Xalatan ® (0.02% BAK) than with Lumigan ® (0.005% BAK), Travatan ® (0.015% BAK) or Travatan Z ® (BAK-free, sofZia TM )

Conclusions  The EB staining model showed that Lumigan ®, Travatan ®, and Travatan Z ® demonstrate less cellular toxicity to human corneal epithelial cells than Xalatan ® or toxic controls.  Additional clinical studies are needed to further evaluate the effects of PGAs on the corneal surface.