Transcript In Vitro Human Corneal Epithelial Toxicity of Prostaglandin

In Vitro Human Corneal Epithelial
Toxicity of Prostaglandin Analogues
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.1
1. Noecker et al. Cornea. 2004;23:490-496.
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.
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
EB Staining in Human Corneal Epithelial Cells
70
60
Fluorescence
50
40
30
20
10
0
Xalatan®
Lumigan®
Travatan® Travatan Z®
Media
Methanol
 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).
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, sofZiaTM)
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.