1. 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.

2. 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.

3. 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. 1 1. Noecker et al. Cornea. 2004;23:490-496.

4. 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;58:306-314.

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

6. 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.

7. 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). 0 10 20 30 40 50 60 70 Fluorescence EB Staining in Human Corneal Epithelial Cells Xalatan ® Lumigan ® Travatan ® Travatan Z ® MediaMethanol

8. 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 )

9. 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.