In Vitro Human Corneal Epithelial In Vitro Human Corneal Epithelial Toxicity of Prostaglandin Analogues Toxicity of Prostaglandin Analogues

Mark McDermott, Fu-Shin X. Yu, Jia Yin, Mark McDermott, Fu-Shin X. Yu, Jia Yin, Ashok Kumar, Ashok Kumar, Ke-Ping XuKe-Ping Xu

Kresge Eye InstituteKresge Eye InstituteWayne State University, Detroit, MI Wayne State University, Detroit, MI

This study was funded by an unrestricted grant from Allergan, Inc.This study was funded by an unrestricted grant from Allergan, Inc.The authors have no financial relationships to disclose.The authors have no financial relationships to disclose.

AbstractAbstract

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

Methods:Methods: Immortalized human corneal epithelial cells were grown in Immortalized human corneal epithelial cells were grown in confluence. Cell culture assay performed of commercial formulations of confluence. Cell culture assay performed of commercial formulations of bimatoprost 0.03%, travoprost 0.004%, travoprost 0.004% (BAK-free), bimatoprost 0.03%, travoprost 0.004%, travoprost 0.004% (BAK-free), latanoprost 0.005%, media and methanol. Cultured microtiter plates (n = 5 per latanoprost 0.005%, media and methanol. Cultured microtiter plates (n = 5 per group) were exposed for 25 minutes to agents and controls. Ethidium bromide group) were exposed for 25 minutes to agents and controls. Ethidium bromide (EB) staining was performed and fluorescence quantified.(EB) staining was performed and fluorescence quantified.

Results:Results: At 25 minutes exposure to agents, the mean EB staining At 25 minutes exposure to agents, the mean EB staining fluorescence measured was 26.8 with bimatoprost, 27.5 with travoprostfluorescence 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 (BAK-free), 32.7 with travoprost, 60.5 with latanoprost, 12.1 with media, and 51.6 with methanol. At 51.6 with methanol. At PP < .05, using one-way ANOVA, travoprost, travoprost < .05, using one-way ANOVA, travoprost, travoprost (BAK-free), and bimatoprost all showed significantly less staining florescence (BAK-free), and bimatoprost all showed significantly less staining florescence than latanoprost.than latanoprost.

Conclusion:Conclusion: The EB staining model demonstrates that bimatoprost, The EB staining model demonstrates that bimatoprost, travoprost, and travoprost (BAK-free) demonstrate less cellular toxicity than travoprost, and travoprost (BAK-free) demonstrate less cellular toxicity than latanoprost or toxic controls. Additional clinical studies will need to be latanoprost or toxic controls. Additional clinical studies will need to be performed to further evaluate the effects of PGAs on the corneal surface.performed to further evaluate the effects of PGAs on the corneal surface.

IntroductionIntroduction

Preservatives are an important component of Preservatives are an important component of ophthalmic medications.ophthalmic medications.

– Prevent microbial growth and decomposition of active drugPrevent microbial growth and decomposition of active drug

Benzalkonium chloride (BAK) is most commonly Benzalkonium chloride (BAK) is most commonly used preservative.used preservative.

– XalatanXalatan®® (latanoprost; Pfizer, Inc.; New York, NY) = 0.02% BAK (latanoprost; Pfizer, Inc.; New York, NY) = 0.02% BAK– TravatanTravatan®® (travoprost; Alcon Laboratories, Inc.; Fort Worth, TX) (travoprost; Alcon Laboratories, Inc.; Fort Worth, TX)

= 0.015% BAK= 0.015% BAK– LumiganLumigan®® (bimatoprost; Allergan, Inc.; Irvine, CA) = 0.005% BAK (bimatoprost; Allergan, Inc.; Irvine, CA) = 0.005% BAK

Higher concentrations of BAK may cause ocular Higher concentrations of BAK may cause ocular irritation. irritation.

– Bimatoprost associated with lower corneal toxicity than Bimatoprost associated with lower corneal toxicity than latanoprost.latanoprost.11

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

IntroductionIntroduction

Corneal epithelial cells are a useful in vitro model for Corneal epithelial cells are a useful in vitro model for evaluating ocular irritancy in humans.evaluating ocular irritancy in humans.11

Ethidium Bromide (EB) staining in a useful technique Ethidium Bromide (EB) staining in a useful technique for assessing cell toxicity.for assessing cell toxicity.

– Commonly used nucleic acid stain Commonly used nucleic acid stain

– Yields fluorescent red-orange color after binding to DNAYields fluorescent red-orange color after binding to DNA

– DegreeDegree of staining in cells reflects loss of cell membrane of staining in cells reflects loss of cell membrane integrityintegrity

1. Xu et al. Toxicol Sci. 2000;58:306-314.

PurposePurpose

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

MethodsMethods

Immortalized human corneal epithelial cells were grown in Immortalized human corneal epithelial cells were grown in confluence. confluence.

Cell culture assay performed of commercial formulations of Cell culture assay performed of commercial formulations of

– LumiganLumigan®® (bimatoprost 0.03%) (bimatoprost 0.03%)

– TravatanTravatan®® (travoprost 0.004%) (travoprost 0.004%)

– Travatan ZTravatan Z®® (BAK-free travoprost 0.004%) (BAK-free travoprost 0.004%)

– XalatanXalatan®® (latanoprost 0.005%) (latanoprost 0.005%)

– Media Media

– MethanolMethanol

Cultured microtiter plates (n = 5 per group) were exposed for Cultured microtiter plates (n = 5 per group) were exposed for 25 minutes to test agents and controls.25 minutes to test agents and controls.

EB staining was performed and fluorescence quantified. EB staining was performed and fluorescence quantified.

ResultsResults

TravatanTravatan®® (travoprost), Travatan Z (travoprost), Travatan Z®® (BAK-free travoprost), and (BAK-free travoprost), and LumiganLumigan®® (bimatoprost) all showed significantly less staining (bimatoprost) all showed significantly less staining florescence than Xalatanflorescence than Xalatan®® (latanoprost) (one-way analysis of (latanoprost) (one-way analysis of variance [ANOVA], variance [ANOVA], PP < .05). < .05).

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EB Staining in Human Corneal Epithelial Cells

Xalatan® Lumigan® Travatan® Travatan Z® Media Methanol

DiscussionDiscussion

Results indicate that cellular toxicity in human corneal Results indicate that cellular toxicity in human corneal epithelial cells varies between different PGAs.epithelial cells varies between different PGAs.

– May be related to a combination of active ingredient and May be related to a combination of active ingredient and preservative.preservative. Toxicity and BAK concentrations are higher with XalatanToxicity and BAK concentrations are higher with Xalatan®® (0.02% (0.02%

BAK) than with LumiganBAK) than with Lumigan®® (0.005% BAK), Travatan (0.005% BAK), Travatan®® (0.015% BAK) (0.015% BAK) or Travatan Zor Travatan Z®® (BAK-free, sofZia (BAK-free, sofZiaTMTM))

ConclusionsConclusions

The EB staining model showed that LumiganThe EB staining model showed that Lumigan®®, , TravatanTravatan®®, and Travatan Z, and Travatan Z®® demonstrate less cellular demonstrate less cellular toxicity to human corneal epithelial cells than toxicity to human corneal epithelial cells than XalatanXalatan®® or toxic controls. or toxic controls.

Additional clinical studies are needed to further Additional clinical studies are needed to further evaluate the effects of PGAs on the corneal surface.evaluate the effects of PGAs on the corneal surface.