Toxicological evaluation of preservative-containing and preservative-free topical prostaglandin analogues on a three-dimensional-reconstituted corneal epithelium system

Author(s): Hong Liang ¹ ^, ² ^, ³ ^, ⁴ ^, ⁵ , Aude Pauly ² ^, ³ ^, ⁴ ^, ⁵ , Luisa Riancho ² ^, ³ ^, ⁴ ^, ⁵ , Christophe Baudouin ¹ ^, ² ^, ³ ^, ⁴ ^, ⁵ ^, ⁶ , Françoise Brignole-Baudouin ² ^, ³ ^, ⁴ ^, ⁵ ^, ⁷ ^,

Publication date (Electronic): 22 March 2011

Journal: The British Journal of Ophthalmology

Publisher: BMJ Group

Keywords: Cornea, immunology, inflammation, drugs, ocular surface

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Abstract

Aims

Using an established three-dimensional (3D) toxicological model based on reconstituted human corneal epithelium (HCE), this study investigated the tolerability of four topical intraocular-pressure-lowering agents: the commercial solutions of benzalkonium chloride (BAC)-containing 0.005% latanoprost, 0.004% travoprost, 0.03% bimatoprost containing 0.02%, 0.015% and 0.005% BAC, respectively, and the preservative-free (PF) tafluprost. Solutions of 0.01% and 0.02% BAC alone were also evaluated for comparison.

Methods

The 3D-HCEs were treated with solutions for 24 h followed or not by a 24 h recovery period. We used a modified MTT (3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) procedure to assess cell viability in the HCE. Frozen sections of HCE were analysed using fluorescence microscopy for the evaluation of apoptosis (terminal deoxynucleotidyl transferase mediated dUTP nick end labelling), inflammation (ICAM-1) and proliferation (Ki67). Corneal epithelial tight junctions (occludin and tight junction protein 1 (zona occludens 1)) were also assessed by en face confocal microscopy in response to the different eye-drops.

Results

The MTT test revealed that the cytotoxicity of antiglaucoma eye-drops was primarily related to the concentration of their common BAC preservative (0.02% BAC-latanoprost>0.015% BAC-travoprost>0.005% BAC-bimatoprost). PF-tafluprost did not induce any obvious cytotoxicity, showed the least expression of inflammatory or apoptotic markers and revealed preservation of membrane immunostaining of tight junction proteins in comparison with BAC-containing solutions.

Conclusion

The toxicological model of the 3D reconstructed corneal epithelia model confirmed the ocular surface cytotoxicity of BAC-containing antiglaucomatous solutions. Compared with the formulations containing the toxic preservative BAC, PF-tafluprost was well tolerated without inducing significant corneal epithelium deterioration.

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Most cited references 23

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Preservatives in eyedrops: the good, the bad and the ugly.

Christophe Baudouin, Antoine Labbe, Hong Liang … (2010)

There is a large body of evidence from experimental and clinical studies showing that the long-term use of topical drugs may induce ocular surface changes, causing ocular discomfort, tear film instability, conjunctival inflammation, subconjunctival fibrosis, epithelial apoptosis, corneal surface impairment, and the potential risk of failure for further glaucoma surgery. Subclinical inflammation has also been described in patients receiving antiglaucoma treatments for long periods of time. However, the mechanisms involved, i.e., allergic, toxic, or inflammatory, as well as the respective roles of the active compound and the preservative in inducing the toxic and/or proinflammatory effects of ophthalmic solutions, is still being debated. The most frequently used preservative, benzalkonium chloride (BAK), has consistently demonstrated its toxic effects in laboratory, experimental, and clinical studies. As a quaternary ammonium, this compound has been shown to cause tear film instability, loss of goblet cells, conjunctival squamous metaplasia and apoptosis, disruption of the corneal epithelium barrier, and damage to deeper ocular tissues. The mechanisms causing these effects have not been fully elucidated, although the involvement of immunoinflammatory reactions with the release of proinflammatory cytokines, apoptosis, oxidative stress, as well as direct interactions with the lipid components of the tear film and cell membranes have been well established. Preservative-induced adverse effects are therefore far from being restricted to only allergic reactions, and side effects are often very difficult to identify because they mostly occur in a delayed or poorly specific manner. Care should therefore be taken to avoid the long-term use of preservatives, otherwise a less toxic alternative to BAK should be developed, as this weakly allergenic but highly toxic compound exerts dose- and time-dependent effects. On the basis of all these experimental and clinical reports, it would be advisable to use benzalkonium-free solutions whenever possible, especially in patients with the greatest exposure to high doses or prolonged treatments, in those suffering from preexisting or concomitant ocular surface diseases, and those experiencing side effects related to the ocular surface. Indeed, mild symptoms should not be underestimated, neglected, or denied, because they may very well be the apparent manifestations of more severe, potentially threatening subclinical reactions that may later cause major concerns. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

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Prevalence of ocular surface disease in glaucoma patients.

Eamon Leung, Felipe Medeiros, Robert N. Weinreb (2008)

To examine the prevalence of ocular surface disease (OSD) in glaucoma patients. This was a cross-sectional study. One hundred and one patients, 18 years of age or older, with open-angle glaucoma or ocular hypertension were consecutively recruited for the study. Patients with a history of use of cyclosporine, steroids, topical ocular nonsteroidal anti-inflammatory drugs, or punctal plugs within the last 3 months were excluded. Each patient completed an Ocular Surface Disease Index questionnaire and underwent evaluation by Schirmer test, corneal and conjunctival lissamine green staining, and tear break-up time. Using Ocular Surface Disease Index for measuring symptoms of dry eye, 60 (59%) patients reported symptoms in at least 1 eye. Severe symptoms were reported by 27 (27%) patients. Schirmer testing showed 62 (61%) patients with decrease in tear production in at least 1 eye. Severe tear deficiency was presented in 35 (35%) patients. Corneal and conjunctival lissamine green staining showed positive results in 22 (22%) patients. None had severe staining. Tear break-up time showed abnormal tear quality in 79 (78%) patients and severe decrease in tear quality was found in at least 1 eye in 66 (65%) patients. Multivariate logistic regression models were used to investigate the association between the number of benzalkonium chloride (BAK)-containing eyedrops and results on the clinical tests of OSD. After adjustment for age and sex, each additional BAK-containing eyedrop was associated with an approximately 2 times higher odds of showing abnormal results on the lissamine green staining test (odds ratio=2.03; 95% confidence interval: 1.06 to 3.89; P=0.034). A large proportion of patients with open-angle glaucoma or ocular hypertension had signs and/or symptoms of OSD in at least 1 eye. The coexistence of OSD and the use of BAK-containing medications may impact vision-related quality of life in this patient population.

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Conjunctival proinflammatory and proapoptotic effects of latanoprost and preserved and unpreserved timolol: an ex vivo and in vitro study.

C Debbasch, Patrice Rat, Pierre-Jean Pisella … (2004)

To compare the toxicity of latanoprost and preserved and unpreserved timolol on conjunctival cells. Expression of inflammatory markers and MUC5AC-related mucin production were evaluated by impression cytology in a case-control ex vivo study. The proapoptotic effect of the same drugs was also evaluated in vitro in a conjunctival cell line and compared with that of benzalkonium chloride (BAC). Impression cytology (IC) specimens were obtained from a series of normal subjects and from patients with glaucoma treated for at least 1 year with latanoprost eye drops or preserved or unpreserved timolol. All groups were comparable in age and duration of treatment. Expression of HLA-DR, intercellular adhesion molecule (ICAM)-1, and mucin was evaluated in a masked manner by flow cytometry. For the in vitro study, a human conjunctiva-derived cell line was treated with 0.02% BAC-containing latanoprost or timolol, unpreserved timolol, or 0.02% BAC alone for 15 minutes, followed or not by 4 or 24 hours of cell recovery in normal medium. Cell viability and chromatin condensation were evaluated using microplate cold light cytofluorometry with the neutral red and the Hoechst 33342 tests, respectively. The Hoechst-neutral red ratio was defined for the apoptosis assay, and cytoskeleton changes were assessed by confocal microscopy. No difference was found between normal eyes and those receiving unpreserved timolol. Preserved latanoprost and timolol significantly increased the inflammatory marker expression and decreased MUC5AC expression, but to a significantly higher extent in the preserved timolol group compared with latanoprost. In vitro, 0.02% BAC-containing timolol and latanoprost triggered conjunctival cell apoptosis-however, to a significantly lesser extent than did 0.02% BAC alone. Unpreserved timolol did not cause any cell toxicity. These ex vivo and in vitro studies demonstrate that BAC-containing latanoprost and timolol exhibit higher proinflammatory and proapoptotic effects on conjunctival cells than does unpreserved timolol. Latanoprost caused less toxicity, however, than preserved timolol, and both drugs were less toxic than BAC alone. These results suggest a potential protective effect of the prostaglandin analogue and to a lesser extent of timolol against the toxicity of BAC in conjunctival cells.

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Author and article information

Journal

Journal ID (nlm-ta): Br J Ophthalmol

Journal ID (publisher-id): bjo

Journal ID (hwp): bjophthalmol

Title: The British Journal of Ophthalmology

Publisher: BMJ Group (BMA House, Tavistock Square, London, WC1H 9JR )

ISSN (Print): 0007-1161

ISSN (Electronic): 1468-2079

Publication date (Electronic): 22 March 2011

Publication date (Print): June 2011

Publication date PMC-release: 22 March 2011

Volume: 95

Issue: 6

Pages: 869-875

Affiliations

[1 ]Department of Ophthalmology III, Quinze-Vingts National Ophthalmology Hospital, Paris, France

[2 ]Institut National de la Santé et de la Recherche Médicale, U968, Paris, France

[3 ]Université Pierre et Marie Curie University Paris, France

[4 ]Unité Mixte de Recherche en Santé 968, Institut de la Vision, Paris, France

[5 ]Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7210, Paris, France

[6 ]Ambroise Paré Hospital, APHP, University of Versailles Saint-Quentin-en-Yvelines, Versailles, France

[7 ]Department of Toxicology, Faculty of Biological and Pharmacological Sciences, Paris, France

Author notes

Correspondence to Dr Françoise Brignole-Baudouin, UMR_S968, INSERM, Institut de la Vision, 17 Rue Moreau, Paris 75012, France; frbaudouin@ 123456aol.com

Article

Publisher ID: bjophthalmol189449

DOI: 10.1136/bjo.2010.189449

PMC ID: 3099447

PubMed ID: 21429894

SO-VID: bb1e7bba-845d-4c47-81a5-b51f7ed5bb8b

Copyright © © 2011, Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

License:

This is an open-access article distributed under the terms of the Creative Commons Attribution Non-commercial License, which permits use, distribution, and reproduction in any medium, provided the original work is properly cited, the use is non commercial and is otherwise in compliance with the license. See: http://creativecommons.org/licenses/by-nc/2.0/ and http://creativecommons.org/licenses/by-nc/2.0/legalcode.

Toxicological evaluation of preservative-containing and preservative-free topical prostaglandin analogues on a three-dimensional-reconstituted corneal epithelium system

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Abstract

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Results

Conclusion

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Karger: Ophthalmology

Most cited references 23

Preservatives in eyedrops: the good, the bad and the ugly.

Prevalence of ocular surface disease in glaucoma patients.

Conjunctival proinflammatory and proapoptotic effects of latanoprost and preserved and unpreserved timolol: an ex vivo and in vitro study.

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