Progress in Retinal and Eye Research - Shroomery · 2013. 7. 12. · The Pharmacopoeia recommends that eye drops must contain an antimicrobial agent (preservative) to avoid or to

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Progress in Retinal and Eye Research 29 (2010) 312e334

Contents lists avai

Progress in Retinal and Eye Research

journal homepage: www.elsevier .com/locate/prer

Preservatives in eyedrops: The good, the bad and the uglyq

Christophe Baudouin a,b,c,d, f,*, Antoine Labbé a,b,c,d, f, Hong Liang a,b,c,d, Aude Pauly b,c,d,Françoise Brignole-Baudouin b,c,d,e

aDepartment of Ophthalmology III, Quinze-Vingts National Ophthalmology Hospital, Paris, Franceb INSERM, U968, Paris, F-75012, FrancecUPMC Univ Paris 06, UMR_S 968, Institut de la Vision, Paris F-75012, FrancedCNRS, UMR_7210, Paris F-75012, FranceeDepartment of Toxicology, Faculty of Biological and Pharmacological Sciences, FrancefAmbroise Paré Hospital, APHP, University of Versailles, France

q Financial disclosure: Christophe Baudouin is consbenefitted from research grants from the same comp* Corresponding author. Quinze-Vingts National Op

E-mail address: [email protected] (C. Bau

1350-9462/$ e see front matter � 2010 Elsevier Ltd.doi:10.1016/j.preteyeres.2010.03.001

a b s t r a c t

There is a large body of evidence from experimental and clinical studies showing that the long-term useof 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 beendescribed in patients receiving antiglaucoma treatments for long periods of time. However, the mech-anisms involved, i.e., allergic, toxic, or inflammatory, as well as the respective roles of the activecompound and the preservative in inducing the toxic and/or proinflammatory effects of ophthalmicsolutions, is still being debated. The most frequently used preservative, benzalkonium chloride (BAK), hasconsistently demonstrated its toxic effects in laboratory, experimental, and clinical studies. As a quater-nary 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, anddamage to deeper ocular tissues. The mechanisms causing these effects have not been fully elucidated,although the involvement of immunoinflammatory reactions with the release of proinflammatorycytokines, apoptosis, oxidative stress, as well as direct interactions with the lipid components of the tearfilm and cell membranes have been well established. Preservative-induced adverse effects are thereforefar from being restricted to only allergic reactions, and side effects are often very difficult to identifybecause they mostly occur in a delayed or poorly specific manner. Care should therefore be taken to avoidthe long-term use of preservatives, otherwise a less toxic alternative to BAK should be developed, as thisweakly allergenic but highly toxic compound exerts dose- and time-dependent effects. On the basis of allthese experimental and clinical reports, it would be advisable to use benzalkonium-free solutionswhenever possible, especially in patients with the greatest exposure to high doses or prolonged treat-ments, in those suffering from preexisting or concomitant ocular surface diseases, and those experi-encing 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.

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Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3132. Preservatives in ophthalmic preparations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .314

2.1. Regulatory aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3142.2. Benzalkonium chloride, the foremost modern preservative . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 314

3. Is BAK an appropriate compound? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .314

ultant for or has received research grants from: Alcon, Allergan, MSD, Pfizer, Santen and Théa. The other authors haveanies.hthalmology Hospital, 28 rue de Charenton, 75012, Paris, france.douin).

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3.1. Efficacy as a preservative . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3143.2. Enhancement of penetration of active compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3153.3. Absence of evidence of better IOP control in most BAK-containing eyedrops . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3153.4. Pharmacokinetic data on BAK . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3153.5. Randomized clinical trials and the real world . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315

4. The ocular surface of glaucomatous patients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3164.1. Allergic reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3164.2. Dry eye symptoms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3164.3. Subconjunctival fibrosis and pseudopemphigoid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3174.4. Increased rate of glaucoma surgery failure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3174.5. Histopathological data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3174.6. Impression cytology specimens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 318

5. Clinical evidence of preservative involvement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3195.1. Prospective studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3195.2. Observational comparative surveys . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3205.3. Switching studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 320

6. In vitro demonstration of BAK toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3216.1. Chemical characteristics of BAK . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3216.2. Models of study in ocular toxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3216.3. Literature data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3226.4. Comparative studies on preservatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3226.5. Effects of BAK on conjunctival epithelial cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3226.6. Three-dimension models of corneal epithelium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323

7. Experimental data in animal models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3247.1. The rabbit as an experimental model: recent improvements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3247.2. BAK toxicity in rat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325

8. Other potential issues with BAK use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3258.1. Dry eye and ocular surface diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3258.2. The crystalline lens: is BAK an inducer of cataract? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3268.3. Trabecular meshwork: raising new hypotheses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3278.4. Retinal involvement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3278.5. Nonocular side effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 328

9. Are there alternatives to BAK? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3289.1. Eliminating the preservative . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3289.2. Is the active compound neutral or does it interact with the preservative? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3289.3. Vehicle/preservative interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3299.4. Non-/less toxic preservatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330

10. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .331References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331

1. Introduction

In the recent past, a large body of evidence from experimentaland clinical studies has accumulated showing that the long-termuse of topical drugs may induce major and frequent ocular surfacechanges. Many clinical manifestations associated with low-gradechronic inflammation have been described in patients receivingantiglaucoma treatments for long periods of time. Glaucomatreatment may cause chronic inflammation or aggravatea concomitant ocular surface disease. In clinical practice, glaucomatreatment often lasts for decades. Patients may present preexistingocular surface impairment, such as dry eye, meibomian glanddysfunction, or chronic allergy, and are very frequently treatedwithtwo or more topical ophthalmic formulations. These findingsexplain that clinical practice apparently often does not fit the safetyprofiles of drugs tested in clinical trials for short periods of time,alone, and only in patients without active ocular surface disorders.

The need for sterility in multidose eye drops requires theinclusion of an antimicrobial preservative in these solutions, mostfrequently the quaternary ammonium benzalkonium chloride(BAK). In some patients e on long-term treatments e allergic orinflammatory reactions are experienced, including redness,stinging, burning, irritation, eye dryness, or less frequently,

conjunctivitis or corneal damage. Many studies indicate a directcorrelation between the presence of preservatives and the symp-toms experienced during antiglaucoma therapy. In a recent large-scale European observational study (Jaenen et al., 2007), involving9658 patients, all recorded symptoms and signs were significantlymore frequent in patients taking preserved medications comparedwith those taking preservative-free formulations. Most effectsobserved in glaucoma patients are thereforemore likely to be due tothe preservative than the active ingredients, since toxicity of thepreservatives has beenwell documented experimentally, as has theharmlessness of most active compounds when unpreserved. BAKtoxicity for eye structures has been reported since the 1940s (Swan,1944) and many studies in experimental or cell models haveconsistently and reliably shown its toxic effects (Baudouin, 2008).Conversely, BAK is a hapten causing relatively few short-termallergic reactions andappears in clinical trials as aneffective and safeagent, especially when compared to mercury derivatives or chlo-rhexidine, formerly used in ophthalmic preparations. Nevertheless,given that the main characteristic of a toxic compound is that itexerts time- and dose-dependent effects, these effects are mostlikely to occur late, with poor specificity in its manifestations, whendelayed, mild, or interacting with other ocular disorders. Moreover,in most cases, the severity of glaucoma as a sight-threatening

Fig. 1. Chemical formula of Benzalkonium chloride (BAK).

C. Baudouin et al. / Progress in Retinal and Eye Research 29 (2010) 312e334314

disease makes dry eye sensation or chronic ocular irritationa comparatively very mild caveat regarding the benefit of glaucomatreatment. However, ocular surface manifestations deeply impactthe quality of life andmay influence patient compliance, in additionto a variety of long-term consequences for the eye. This review isaimed at reviewing the long-term ocular surface side effects oftopical treatments, focusing on BAK toxicity and its potentialharmfulness in ophthalmic preparations.

2. Preservatives in ophthalmic preparations

2.1. Regulatory aspects

The Pharmacopoeia recommends that eye drops must containan antimicrobial agent (preservative) to avoid or to limit microbialproliferation after opening the bottle, which could induce a risk ofpotentially severe eye infection as well as the alteration of theformulation. Eye drops are contaminated essentially by the handswhen handling the bottle or by contact of the tip touching theeyelids, lashes, conjunctiva, or tears. There is also a risk of cross-transmission when the same eye drops are shared by severalpatients, especially in a hospital environment or within the samefamily. Moreover, this type of incident in the 1960s in a Birminghamhospital triggered the British authorities to require the industry todevelop the first unit-doses and to limit the duration of use afteropening (cited in Chibret, 1997). As disposable single-dose units areless cost-effective than multidose bottles, the greatest progress hasbeen made in the field of preservative development since the1960s. However, the regulations requiring addition of an antimi-crobial agent in any multidose ophthalmic preparation appearedonly in the 1970s. Preservatives used in ophthalmic preparationsbelong to a variety of chemical families, including mercury deriv-atives, alcohols, parabens, EDTA, and chlorhexidine, but quaternaryammonium compounds, due to their low allergenic effects andapparently good safety profiles, have progressively become themajor preservative of today’s Pharmacopoeia. The US Pharmaco-peia bases the efficacy of preservatives on the Preservative Effec-tiveness Test (PET) that consists of inoculation with 1x106 colonyforming units (cfu)/mL at Day 0 with various organisms, namelybacteria (Staphylococcus aureus, Pseudomonas aeruginosa, Escher-ichia coli) and fungi (Aspergillus niger and Candida albicans). Theregulatory requirements are: 1.0elog reduction by day 7, 3.0-logreduction by day 14, no increase in survivors at Day 14, no increasein survivors from Days 14e28, and no increase in survivors for thefungi from Day 0 to Day 28 (Rosenthal et al., 2006). Indeed, sucheffectiveness on microorganisms through cytotoxic effects cannotbe achieved without a minimal amount of toxicity to tissues wherepreservatives are applied. Authorities more and more concerned bysuch toxicological issues nowadays tend to favor the developmentby the industrials of new preservatives of preservative-free alter-natives. In a recent statement, the EMEA (European MedicinesAgency), thus addressed the interest of avoiding preservatives in“patients who do not tolerate eyedrops with preservatives” andthose with long term treatment, of using “concentration at theminimum level consistent with satisfactory antimicrobial functionin each individual preparation”, of promoting “new ophthalmicpreparations without any mercury-containing preservatives”,although did not give a general recommendation not to usepreservatives in eye drops (EMEA statement, 2009).

2.2. Benzalkonium chloride, the foremost modern preservative

Today, the most commonly used preservative in ophthalmicpreparations is benzalkonium chloride. BAK is a nitrogenous cationicsurface-acting agent belonging to the quaternary ammonium group.

It has three main categories of use: as a biocide, a cationic surfactant,and a phase transfer agent in the chemical industry. Quaternaryammoniums are bipolar compounds, which are highly hydrosolubleand have surfactant properties. They act mainly via their detergentproperties,which vary in strength anddissolve the bacterialwalls andmembranes, and destroy the semipermeable cytoplasmic layer. Theirbactericidal activity is rapid and is greatest at 37 �C in an alkalinemedium. The spectrum of activity is mainly focused on Gramþ bac-teria (Staphylococcus) even at very low concentrations. Activityagainst Gram� bacteria (P. aeruginosa) is increased when it iscombined with EDTA 0.1%. The quaternary ammoniums are alsoexcellent fungicides and are particularly active against C. albicans andAspergillus fumigatus. Finally, these compounds are potent spermi-cides, and as well as being used in ophthalmic preparations, they areused in a wide range of commonly used products (soaps, cosmetics,cleaning products, disinfectants, etc.).

BAK is a mixture of alkylbenzyldimethylammonium chloride,whose general formula (Fig. 1) is: C6H5CH2N(CH3)2RCl, R repre-senting the alkyl radicals from C8 to C18. BAK-C12 is called ben-zododecinium chloride, BAK-C14, myristalkonium chloride, andBAK-C16, cetalkonium chloride. It is commonly used at concen-trations ranging between 0.004 and 0.025%. Several investigationsusing animal models suggested the existence of links between BAKand cytotoxic effects on several components of the eye. The keystudies are outlined below.

3. Is BAK an appropriate compound?

3.1. Efficacy as a preservative

BAK was mainly used for its apparently good safety/efficacyprofile. This compound is weakly allergenic and has a high rate ofantimicrobial properties. In a study illustrating the potent antimi-crobial properties of BAK, Charnock showed that among fivepreservatives, benzalkonium chloride/EDTA, parabens, chlor-obutanol, silver chloride complex, and the Purite-stabilized oxy-chloro complex, only BAK/EDTA satisfied the major criteria forantimicrobial preservation for all the test microorganisms. More-over, it was the only one tested to inhibit S. aureus (Charnock,2006). Indeed, BAK efficiently destroys the cell membranes ofmicroorganisms and seems to have a good safety profile, eventhough it is almost impossible for a chemical to discriminatemembranes of pathogens from those of normal eye cells.

Conversely, nonpreserved eyedrops could enhance the risk ofcontamination. Preservative-free artificial tears in reclosablecontainers were shown to be at risk of contamination after multipleusesover 10 h (Kimet al., 2008). The riskwashigher inolder patientsand with inappropriate finger manipulation, which may be thehallmark of patients with chronic eye diseases, namely glaucoma ordry eye disease. However, it should be noted that even in the worstconditions of repeatedhandling of unpreserved eyedrops, only 2% ofbottles appeared to be contaminated. Moreover, in a multiple-usesetting, bottles preserved with BAK achieved a 34.8% rate ofcontamination after 15 days, thus demonstrating the very relative


security offered by the preservative (Tasli and Cosar, 2001). More-over, some strains of P. aeruginosa have been found to be resistant toBAK and can be grown in concentrated BAK solutions. The acquiredresistance could be eliminated by the presence of EDTA in thesolution, which may also act as an enhancer of corneal permeation(Pawar and Majumdar, 2006), but whose toxicity, especially whenassociated with BAK, has not been fully addressed.

3.2. Enhancement of penetration of active compounds

One major argument that is proposed to maintain the use ofquaternary ammoniums in eyedrop formulations is that BAKreportedly enhances the penetration of the drug into the anteriorchamber, through disruption of the hydrophobic barrier of thecorneal epithelium. This property may result in a better concen-tration of the active compound and better stimulation/inhibition ofthe target receptors, so that better efficacy should be expected. Theprostaglandin analog tafluprost was therefore tested in a pharma-cokinetic study in rabbits, but it showed no difference in terms ofdrug concentrations in the aqueous humor between the preservedand unpreserved formulations (Pellinen and Lokkila, 2009).Conversely, topical cyclosporine A penetration into the cornea wasfound to be enhanced by a vehicle containing BAK, although almostno differences were observed in the iris/ciliary body (Yenice et al.,2008). BAK, especially when added to other enhancers such aschitosan or EDTA, was also shown to increase transcornealpermeation of acyclovir (Majumdar et al., 2008) and various anti-biotics (Rathore and Majumdar, 2006).

Although major pharmacokinetic differences most likely existbetween the large variety of currently available compounds andformulations (hydrophilic vs. lipophilic drugs, prodrugs vs. activecompounds, solutions vs. suspension or emulsions, etc.), thepharmacokinetic characteristics of an eyedrop do not necessarilycorrelate its effectiveness, e.g., intraocular pressure (IOP) reduction.Indeed, besides the fact that enhancing penetration of a drugthroughmajor histological changes in the corneal barrier is actuallya toxicity-related side effect (de Jong et al., 1994), several studieshave addressed the question in terms of efficacy, showing theoverall equivalence between preserved and unpreserved anti-glaucomatous formulations at least for the tested compounds.

3.3. Absence of evidence of better IOP control in most BAK-containing eyedrops

The question of a possible difference in IOP reduction betweenBAK-containing and unpreserved antiglaucomatous drugs or BAK-free formulations was first addressed with beta-blockers, a familyof hydrophilic compounds most susceptible to be influenced bya decreased penetration of the active compound into the anteriorchamber. Although theoretically beta-blocker penetration shouldbe reduced in the absence of BAK, this was not demonstrated fora topical betaxolol suspension (Denis et al., 1993) or carteolol:a comparative study of 2% carteolol with and without preservativealso confirmed similar IOP reduction by the two formulations(Baudouin and de Lunardo, 1998). More recently, a study comparednonpreserved versus preserved timolol gel 0.1% and demonstratedthat both formulations resulted in a non-significantly differentreduction of IOP (Easty et al., 2006).

Concerning prostaglandin analogs, which are lipophiliccompounds, similar results have been published. Two recent clin-ical trials compared the pharmacokinetic and pharmacodynamicprofiles between preserved and preservative-free formulations ofthe prostaglandin analog tafluprost. There were no statisticallysignificant differences between formulations with regards topharmacokinetic parameters and systemic bioavailability (Uusitalo

et al., 2008). There were no differences in IOP between preservedand preservative-free formulations in the two studies. The overallIOP reduction difference between preservative-free and preservedformulations was within �0.46-mmHg (Hamacher et al., 2008).

In addition, several studies compared Travoprost with andwithout BAK, using a multidose preparation preserved withSofzia�, an ionic buffered preservative system. The two showed nosignificant differences in terms of IOP control: in the first one(Lewis et al., 2007), at all time points the differences in IOPsmeasured during a 3-month study remained within a range of �0.8mmHg. The second one also showed no difference between bothformulations even 60 h after the last administration of the pros-taglandin analog (Gross et al., 2008). Likewise, the fixed combina-tion of timolol and dorzolamide (Cosopt�, Merck, Sharpe & Dohme,NY) showed the equivalent efficacy on IOP control with preservedand preservative-free preparations (Laibovitz et al., 1999).

3.4. Pharmacokinetic data on BAK

Conversely, does BAK enter the eye or infiltrate ocular struc-tures? This question is important regarding a potentially harmfulcompound that may cause very low toxic changes in the short termbut is often used for the rest of the patient’s life and may accu-mulate in ocular tissues. To our knowledge, only one study hasreported BAK pharmacokinetic data after instillation. BAK isretained in tissues and can be found 168 h after a single 30-ml dropof 0.01% BAK in rabbits. The half-life of BAK from corneal andconjunctival tissues in the epithelium is 20 h and the half-life inconjunctival deeper structures is 11 h (Champeau and Edelhauser,1986). This is important to consider because tear washout mayconsiderably reduce tear concentration of toxics, including BAK(Friedlaender et al., 2006), whereas deeper structures may beimpregnated, especially after repeated use of several preservative-containing eyedrops over the long term.

3.5. Randomized clinical trials and the real world

Medical treatment is predominantly used as first-line therapyand the large majority of patients with chronic glaucoma aremedically treated. Thereforemedical treatment is administeredoverthe long term and most of patients receive several decades oftreatment, if not formost of their lifetime, as even those undergoingsurgery often require further adjunctive medical therapy. Eyedropswere developed before being approved after thorough methodo-logical evaluations using randomized clinical trials (RCTs). Based ondata from RCTs, the tolerance of glaucoma treatments seems satis-factory, with only a few patients withdrawn due to local intoleranceor allergy. Although prolonged clinical trials are seldom, medicalantiglaucoma treatments therefore appear not harmful even if theywill most likely be used for much longer periods of time (Alm et al.,1997; Cohen et al., 2004; Fellman et al., 2002).

Although RCTs usually demonstrated good safety profiles fornewly developed eyedrops, it should be recognized that there areseveral major differences between clinical trials and real-life use ofantiglaucoma therapy. First, clinical trials are usually of shortduration (6 months to rarely more than 1 year). The time-depen-dent effects of toxic compounds may require years or decades todevelop at a clinically identifiable level and may therefore belargely underestimated in shorter duration RCTs. Secondly, mostoften patients only receive one test or reference therapy andpossible interactions or additive toxic effects of various compoundsare not addressed. In addition, for ethical reasons patients withknown hypersensitivity to the active molecule or the preservative,and patients who have active concomitant diseases, in particularthose with severe dry eye, blepharitis or chronic allergy, are not


included in such trials, as these pathologies may strongly interferewith ocular tolerance to eyedrops, thus creating a selection bias,potentially underestimating the real-life situation. However, theprevalence of dry eyes in patients over 65 varies between 15 and34% (Dry Eye WorkShop, 2007b). Therefore, an ocular surfacedisease may be enhanced by the drug(s) and/or preservatives, andconversely may reduce the resistance of the cornea and conjunctivato the toxic or irritant compounds.

Finally, in clinical practice, glaucoma very often evolves andrequires additional treatments over time, a large proportion ofpatients thus receiving multiple therapies concomitantly. TheOcular Hypertension Treatment Study demonstrated that approx-imately 40% of patients initially diagnosed with ocular hyperten-sion were using two drugs after 5 years, 9% three or more, witheven higher rates in African-American patients (Kass et al., 2002).Multitherapy is likely to increase the incidence of adverse events,by additivity of side effects of each individual component, orpossible between-drug interactions. This is particularly relevant forthe preservative that may accumulate and reach high tissue levelswhen instilled four or five times a day. Indeed, Osborne et al.showed that the allergic effects of brimonidine might increase thepropensity for multiple allergic reactions to subsequently usedpreparations (Osborne et al., 2005). This illustrates the role ofbetween-drug interactions, both concomitantly and consecutively,and the likelihood of discrepancies between the clinical trial settingand clinical practice, when patients experiencing concomitant orpreexisting ocular surface involvement are treated with severaldrugs for decades. This may indicate that the real world clearlytrumps the reassuring results of RCTs and their low numbers ofpatients who are withdrawn from or intolerant to treatment. Thismust be taken into consideration and emphasizes the discrepancybetween two major principles: evidence-based medicine and theprecautionary principle. The former bases any assumption on RCTs,whereas, according to the latter, any doubtful compound, even iftoxicity occurs only in a minority of patients, should be eliminatedor alternatives developed.

4. The ocular surface of glaucomatous patients

4.1. Allergic reactions

The symptoms of conjunctival allergy (congestion, tearing,photophobia, burning and stinging sensations) induced by theinstillation of preserved eyedrops are various. Simple conjunctivalcongestion or papillary conjunctivitis may be observed with or

Fig. 2. Example of allergic reaction to BAK, through a type IV hypersensitivitymechanism.

without eczema, the worst reaction being patent giant papillaryconjunctivitis. In certain cases, a severe type IV allergic reactionmay develop. Allergic manifestations are often spectacular (Fig. 2),occurring a few days after treatment initiation, and recover rapidlywhen treatment is stopped. In clinical trials, the overall rate ofwithdrawal is relatively low, a few percent of the patients recruited.In a meta-analysis of 28 randomized clinical trials, the overall rateof withdrawal was ranging from 0 to 16%, the majority of RCThaving a rate <8% (van der Valk et al., 2005). However, delayedallergic reactions may occur, often mimicking blepharitis with low-grade inflammation (Fig. 3). In this condition, the relationshipbetween the eyedrops and ocular inflammation is often difficult toassess, especially when the treatment is mandatory for a severesight-threatening condition.

4.2. Dry eye symptoms

In contrast with data obtained from prospective clinical trials,the real world shows a much larger proportion of patients sufferingfrom symptoms and signs related to the ocular surface, as assessedby several observational surveys. Some of them included sucha high number of patients that their clinical value cannot be denied.An epidemiological survey was conducted in 2002 in 4107 glau-coma patients to assess the rate of ocular symptoms and conjunc-tival, corneal, and palpebral signs in a routine clinical practice(Pisella et al., 2002). Prevalence of symptoms was found to be veryhigh in patients using preserved drops, with 43% discomfort uponinstillation, 40% burning or stinging sensation, 31% foreign bodysensation, 23% dry eyes, 21% tearing, and 18% itchy eyelids.Frequency of signs and symptoms increased with the number ofpreserved eye drops used and was significantly lower for all criteriain a group of patients treatedwith unpreserved betablockers. Theseresults were found to be quite similar in several other Europeancountries when the same study was conducted in Italy, Belgium,and Portugal. Pooled data from 9,658 patients demonstrated thatthe incidence of ocular symptoms ranged between 30 and 50% ofpatients (Fig. 4) (Jaenen et al., 2007). Likewise, a total of 20,506patients from 900 centers across Germany were included in anobservational study on dry eye prevalence in glaucoma. The first 30consecutive glaucoma patients at each center were recruited.According to the registry data, more women develop dry eye andglaucoma than men (56.9 vs. 45.7%). Dry eye occurred more

Fig. 3. Example of mild, chronic allergic blepharitis. The relationship to BAK wasconfirmed by complete recovery after switching to a preservative-free topical treat-ment. The onset of blepharitis may be progressive and delayed after starting treatment.

Fig. 4. Frequency of ocular symptoms (stinging or burning, dry eye sensation andtearing) and clinical signs (anterior blepharitis, conjunctival follicles and superficialpunctate keratitis) with preserved and preservative-free glaucoma medications(*, P< 0.0001) (adapted from Jaenen et al., 2007).


frequently in more severe glaucoma cases, when three or moreantiglaucoma drugs were used, and increased with the duration ofglaucoma disease (Erb et al., 2008).

Similarly, in the United States, a cross-sectional study evaluatedthe impact of ocular surface in glaucoma management in 101patients with open-angle glaucoma or ocular hypertension. Usingthe Ocular Surface Disease Index for measuring symptoms of dryeye, 59% of patients reported symptoms in at least one eye, quali-fied as severe in 27% of patients. Schirmer testing showed 61% ofpatients with decreased tear production in at least one eye, 35%being severely affected. Corneal and conjunctival lissamine greenstaining showed positive results in 22% of cases. Tear break-up timewas decreased in 78% of patients (65% qualified as severe) (Leunget al., 2008).

More recently, a new observational survey confirmed the highprevalence of dry eye in glaucoma patients with a clear relationshipwith the number of eyedrops: 39% and 43% of dry eye with two andthree drugs, respectively, whereas dry eye was only found in 11% ofpatients receivingonlyoneeyedrop. Basedona scoreof ocular surfacesymptoms, severe dry eyewas found in 8.7% and 15% of patientswithtwo and three eyedrops, respectively (Rossi et al., 2009).

The last two studies concluded that ocular surface symptomshada substantial impact on the patients’ quality of life, as is well knownin dry eye disease irrespective of the cause (Dry Eye WorkShop,2007b). The impact on compliance is very likely, although moredifficult to assess. In an observational study conducted in 204patients, the high rate of symptoms, with 25.4% experiencingburning sensation, 20.8% blurred vision, and 20.2% tearing, led topoor patient satisfaction and reduced adherence. Dissatisfiedpatients also visited their ophthalmologists more frequently(Nordmann et al., 2003). In a survey performed in the US between2001 and 2004, based on the refilling rate of initial therapy in 300patients, adverse effects were found the second most commonreasons noted by physicians for switching medications after lack ofefficacy (19% vs. 43%, respectively) (Zimmerman et al., 2009).

4.3. Subconjunctival fibrosis and pseudopemphigoid

The development of subconjunctival fibrosis has been reportedin patients treated with antiglaucomamedications for a long period

of time, most likely resulting from an increase in fibroblast densityin the subepithelial substantia propria, related to an increase ininflammatory cells (Baudouin et al., 1999; Broadway et al., 1994a;Sherwood et al., 1989). The use of long-term antiglaucoma medi-cations has been shown to cause conjunctival foreshortening andshrinkage, which may be associated with an ocular pemphigoid-like condition or evolve into severe scarring conjunctivitis withdefinitive corneal opacities (Schwab et al., 1992). In a series of 145patients presenting a pseudopemphigoid, Thorne et al. showed thatexposure to antiglaucoma eyedrops was the primary cause ofpseudopemphigoid. Almost all the cases reported (97.4%) involvedan association of antiglaucoma medications (Thorne et al., 2004).

4.4. Increased rate of glaucoma surgery failure

There have been several reports of long-term topical combina-tion therapy as a significant risk factor for glaucoma surgery failure(Lavin et al., 1990; Broadway et al., 1994b). The success rate oftrabeculectomy was thus found to be negatively influenced bylong-term topical antiglaucoma therapy (Lavin et al., 1990).Broadway and colleagues further studied the effect of differentlong-term antiglaucoma treatments on the results of glaucomafiltration surgery and found a significant relationship between therisk of failure of filtering surgery and the number of drugs used andduration of treatment (Broadway et al., 1994b). The failure ofglaucoma surgery in most cases originates from a fibroblasticconjunctival response at the bleb level in the early postoperativemonths. An excessive wound healing process characterized byinflammation and fibroblast proliferation and extracellular matrixdeposition results in blockade of aqueous outflow in the subcon-junctival space. Increased postoperative fibrosis may be enhancedin populations of patients or conditions known to be at higher riskof surgical failure, but several reports have pointed out the roleplayed by preoperative inflammation of the conjunctiva caused byantiglaucomatous drugs used over the long term (Broadway andChang, 2001).

Long-term therapy induces an inflammation of the conjunctivawith consequently an increased postoperative fibrosis. Therefore,the use of topical antiglaucoma drugs can be considered as animportant risk factor for failure of filtration surgery and wheneverpossible, improvement of the ocular surface and reduction ofinflammation prior to surgery should be considered (Broadway andChang, 2001). Although these results were collected at a timewhennew powerful drugs had not yet been developed, more than 50% ofglaucomatous patients require two or more drugs and are stilloperated on after prolonged medical treatment with multipledrugs. Filtration surgery success rates do not appear to be improvedin the recent years (Gedde et al., 2009).

4.5. Histopathological data

As clinical data and observations often lack specificity,numerous reports have investigated the ocular surface of glau-comatous patients by means of histopathological techniques. Theyclearly demonstrated that, even without evident symptoms orclinical manifestations, inflammation is abnormally observed in theconjunctival epithelium and substantia propria. In most cases,conjunctival biopsies have been taken at the time of glaucomasurgery and examined using immunohistological techniques.Infiltration of immunocompetent cells in the conjunctiva hastherefore been consistently reported. Sherwood et al. comparedtwo groups of patients, one with primary glaucoma surgery, theother one after long-term eyedrop therapy (Sherwood et al., 1989).A significant increase in the number of macrophages, lymphocytes,mast cells, and fibroblasts in the conjunctiva and the Tenon capsule

Fig. 5. Immunohistochemistry in a conjunctival impression cytology showing ina multitreated glaucoma patient a major dendritiform cell infiltration (vimentin greenimmunostaining, propidium iodide red staining of nuclei, confocal microscopy; scalebar: 50 mm)).


and a significant decrease in the number of epithelial goblet cellswere observed in the latter group. In another study, conducted on124 conjunctival biopsy specimens from patients undergoingfiltration surgery, Broadway et al. found that administration oftopical medication, irrespective of type, for 3 years or more induceda significant degree of subclinical inflammation in the conjunctiva(Broadway et al., 1994a). Associations of two or more medicationsthus induced a significant decrease in goblet cells, an increase inpale cells, macrophages, and lymphocytes within the epithelium,and an increase in fibroblasts, macrophages, mast cells, andlymphocytes in the substantia propria. In addition, administrationof one topical medication for more than 3 years was found to beassociated with similar inflammatory and fibroblast infiltration.These data were directly correlated to risk of surgical failure(Broadway et al., 1994b). Similarly, in medium- and long-termtherapy patients, Nuzzi et al. confirmed significant increases in thethickness and number of epithelial cell layers, in the fibroblastdensity in both subepithelial and deep connective tissue, anda more compact connective tissue, richer in collagen fibersarranged in whirls, with some inflammatory elements andincreased expression of inflammatory markers (HLA-DR, CD1a,CD4, CD8, IL-2, and C3b) (Nuzzi et al., 1995).

Our group also conducted an immunohistological study inconjunctival biopsies and trabecular meshwork specimens frompatients undergoing glaucoma surgery after various durations andnumber of medical treatments. Immunohistochemical analysesrevealed that samples from patients who were receiving treat-ments had significantly greater expression of fibroblastic andinflammatory markers compared with untreated participants.Furthermore, patients who received multiple therapies had greaterexpression of markers compared with those who were on mono-therapy (Baudouin et al., 1999). Apoptotic rates in the conjunctivalepithelium were also found to be increased in glaucoma patientstreated chronically compared to normal eyes, independent of drugfamily, which could again raise the possible if not likely role playedby the preservative in epithelial toxicity (Dogan et al., 2004).Conversely, the most common sign related to prostaglandinanalogs, namely hyperemia, was histologically confirmed asresulting from vascular congestion (Leal et al., 2004) and was notassociated with inflammatory changes when examining specimensreceiving bimatoprost eye drops, a preparation that frequentlycauses hyperemia but contains low doses of BAK, i.e., 0.005%.However, little information is available on the conjunctival changesinvolved in patients receiving monotherapy of prostaglandinanalogs. In conjunctival specimens from 20 patients treated witheither latanoprost or timolol, both containing BAK as preservative,latanoprost-treated conjunctival specimens showed a decreasedstromal collagen density and a less pronounced inflammatoryinfiltration than those receiving timolol, with an upregulation ofMMP-1 and MMP-3 in latanoprost-treated eyes (Terai et al., 2009).

4.6. Impression cytology specimens

Developed at the end of the 1970s, impression cytology is awell-known, easily repeated technique for collecting conjunctivalepithelial cells in a noninvasive, rapid, and almost painless way forbiological analyses of ocular surface disorders. Using conjunctivalimpression, the most superficial cells of the conjunctival epitheliumare collected, namely epithelial cells, mucin-secreting goblet cells,and inflammatory cells infiltrating the conjunctiva (Fig. 5) (Brignole-Baudouin et al., 2004). Classical techniques of conjunctival impres-sion cytology allow calculation of goblet cell density and the stagingof squamous metaplasia, especially in dry eye syndrome and ocularsurface diseases. In glaucoma, several reports showed consistentocular surface changes as assessed by impression cytology. In 72

patients, Brandt et al. showed statistically significant degrees ofmetaplasia in patients treated over the long-term and associatedwith the number of medications (Brandt et al., 1991). Clinicalimpairment of the tear film and a rapid decrease of goblet celldensity were also demonstrated after starting treatment withpreserved timolol (Herreras et al., 1992). Schirmer’s test and break-up time were altered compared to the basal control already signifi-cantly in the first month of treatment (P< 0.01 and P< 0.001,respectively). Similarly, impression cytology showed a progressivedecrease in goblet cell density also significant at the first month(P< 0.001).

More recently, impression cytology specimens also showedocular surface changes in glaucoma patients. Cytology scores weresignificantly higher in the medication group than the untreatedcontrol group. The mean scores of the control, timolol, latanoprost,dorzolamide, timololþ latanoprost, and timololþ dorzolamidegroups were 0.20, 1.62, 2.00, 1.75, 2.13, and 2.44, respectively, usingthe Nelson’s scoring system ranging from 0 to 3 (Nelson et al.,1983). Among the medication groups, cytology scores were signif-icantly lower in the monotherapy groups than the fixed-combina-tion therapy groups (Hong et al., 2006).

In addition, immunocytological techniques have been developedto better assess inflammatory changes in ocular surface diseases.Our group was able to quantify HLA-DR expression in impressioncytologyspecimens.HLA-DRantigens arenormallynotexpressedbyepithelial cells, unless in inflammatory processes. ThereforeHLA-DRhas become a very reliable marker for demonstrating that epithelialcells may be involved in inflammatory ocular surface diseases andfor quantifying the level of inflammation, showing special useful-ness in disorders that are clinically notor veryweakly inflammatory,such as dry eye and glaucoma. In the first immunocytological studyperformed in glaucoma, conjunctival inflammatory antigens wereinvestigated in impression cytologyspecimens frompatientswithorwithout glaucoma treatment, including 107 eyes from 55 patientswith primary open-angle glaucoma. None of the untreated glau-coma eyes or controls showed reactivity for either monoclonalantibody,HLA-DRorCD23, a lowaffinity receptor for IgE. In contrast,


HLA-DR expression by conjunctival cells was found in 43 of 88treated eyes (mean percentage of reactive cells, 70%� 28%) andpositive staining for the receptor to IgE in 26of 68 eyes (52%� 28%ofconjunctival cells). The results were not related to a specific treat-ment or combination of antiglaucoma drugs. However, the propor-tion of positive specimens (3/14 for both antigens) in the groupreceiving chlorhexidine-containing eye drops was significantlylower than that found in the patients who had been in repetitivecontact with antiglaucomatous treatments and their commonpreservative, BAK (Baudouin et al., 1994).

Our group further developed the technique of flow cytometry inimpression cytology and demonstrated it to be a reliable methodfor assessing inflammatory changes in ocular surface cells. It isminimally invasive and can be routinely performed for investi-gating ocular surface disorders and the effects of topical drugs onthe eye surface, since the technique of cell collection is the same asthat used in standard impression cytology. We showed that glau-coma patients, even though clinically asymptomatic, exhibitsignificant overexpression of HLA-DR class II antigens (Fig. 6),ICAM-1, or interleukins IL-6, IL-8, and IL-10 in the epithelium(Baudouin et al., 2004, 2005, 2008; Pisella et al., 2004). Preserveddrugs and multiple treatments reliably showed higher levels ofinflammatory markers or cytokines. Interestingly, in an ex vivo andin vitro study investigating latanoprost eye drops, the only prosta-glandin available at that time, we found that pro-inflammatory andpro-apoptotic effects were lower than expected, regarding theconcentration of benzalkonium contained in the commercialpreparation, and these effects were at even lower levels than thosefound in a preserved beta-blocker group (Pisella et al., 2004). Wealso found a relatively well-preserved goblet cell density, lowerthan with unpreserved beta-blocker but significantly less impairedthan in the preserved beta-blocker-treated eyes. Another groupsimilarly investigated HLA-DR in glaucomatous patients and theexpression of trefoil factor family (TFF)1, MUC5AC, and HLA-DR,which were significantly higher in patients than in controls(Souchier et al., 2006). However, most interestingly, a higherMUC5AC expression and a lower HLA-DR expressionwere observedin patients with further successful glaucoma surgeries than infailures. Surface marker expression could therefore becomea predictive factor of successful glaucoma surgery.

In a more recent study also conducted in impression cytologyusing a similar technique, we further investigated markers associ-ated with the T helper (Th) 1/Th2 profiles of inflammation invarious ocular surface diseases, using the expression of CCR5 and

Fig. 6. Comparative expression of HLA DR class II antigens using flow cytometry inimpression cytology specimens in normal subjects, and glaucomatous patients treatedwith an unpreserved or preserved beta-blocker, or receiving multiple therapy (adaptedfrom Baudouin et al., 2004).

CCR4, respectively, two markers already demonstrated to reflectthe two T helper pathways (Baudouin et al., 2005). In this study,vernal keratoconjunctivitis (VKC) was characterized by a highCCR4/low CCR5 profile, whereas keratoconjunctivitis sicca (KCS)had a low CCR4/high CCR5 profile, consistent with what wasexpected to be related to Th2 and Th1 involvements, respectively.Interestingly, we also studied a group of glaucoma patients treatedover the long term and found that their conjunctiva expressed highlevels of both CCR4 and CCR5, suggesting the simultaneousinvolvement of Th1 and Th2 pathways under the stimulation oftopical drugs. To our knowledge, no other research had focused onthe Th1/Th2 profile in glaucoma patients, and little informationwasknown on the conjunctival effects of the currently available pros-taglandin analogs. We therefore undertook a complementary studyto improve our knowledge on the inflammatory pathways involvedin glaucoma patients treated over the long term (i.e., Th1, Th2, orboth) and to look for possible differences in the inflammatoryprofiles of the three commercially available prostaglandin analogs,namely latanoprost, travoprost, and bimatoprost. Our results firstconfirmed that the conjunctiva of glaucomatous patients treatedover the long term was highly inflammatory, especially in patientsreceiving more than one eye drop (Baudouin et al., 2008). More-over, glaucoma treatments were confirmed to activate not onlyCCR4, therefore most likely associated with the Th2 pathway sug-gesting allergic reactions, but also CCR5, themarker associatedwithTh1. This was found at a high level in patients receiving multipletreatments. Consequently, the Th1 and Th2 pathways seemed to besimultaneously implicated in this iatrogenic ocular pathology,resembling what has been found with other techniques in complexocular surface diseases such as atopic keratoconjunctivitis (Metzet al., 1997). This illustrates the complexity of the mechanismsoccurring in the ocular surface in glaucoma, possibly involvingallergic or toxic reactions, direct stimulation of inflammatory cells,impairment of the lacrimal film, or destruction of epithelial cellsand their further stimulation. Although not significant, a tendencytoward slight differences between the three prostaglandins wasfound with HLA-DR expression, parallel to the concentrations ofBAK contained in the eye drops, namely the higher the concen-tration, the higher the HLA-DR expression. No apparent relation-ship could be established with the known frequency of hyperemia,which therefore seems to correspond to a different mechanism,likely not caused by inflammatory processes as found in conjunc-tival biopsies (Leal et al., 2004). Interestingly, the inflammationinduced by prostaglandin analogs, as assessed by HLA-DR expres-sion in impression cytology, was also found by another group, withno significant differences between the three compounds currentlyavailable on the market, but very early after starting the treatmentbecause increased expressions were found at 1 month (RodriguesMde et al., 2009).

5. Clinical evidence of preservative involvement

5.1. Prospective studies

Only very few prospective studies have addressed the questionof the deleterious role of the preservative, in part because of thecurrent lack of preservative-free compounds and to a large extentbecause a normal ocular surface will experience weak toxic effectsafter a short duration of treatment, especially with a monotherapy,that is at a low BAK exposure rate. Nevertheless, in healthyvolunteers Ishibashi et al. demonstrated that preserved timololcaused significantly higher tear film instability and disruption ofcorneal barrier function than preservative-free timolol (Ishibashiet al., 2003). Our group found very similar results also in healthyvolunteers when comparing preservative-free and BAK-containing


carteolol (Baudouin and de Lunardo, 1998). The tear break-up time(BUT) was significantly lower in volunteers receiving BAK. Whenconsidering that both studies were conducted in young subjectswith a fully normal ocular surface, these results may help betterunderstand the high rate of dry eye symptoms and signs in glau-comatous patients who accumulate a long duration of treatment,a higher number of medications, and an increased risk of impairedocular surface. Indeed, one study was conducted in healthyvolunteers prospectively evaluating various concentrations of BAKin tear substitutes given eight times a day for 7 days. Even a lowconcentration of BAK induced goblet cell loss and increased thecytoplasmic/nucleus ratio, two characteristics of dry eye disease(Rolando et al., 1991). A subsequent study prospectively assigned132 subjects to one or two BAK-containing eye drops, BAK twicedaily, or no treatment. All groups receiving BAK showed signifi-cantly decreased Schirmer test values compared with subjects notreceiving therapy (Nuzzi et al., 1998).

Another more recent study used impression cytology and thetechnique of in vivo confocal microscopy (IVCM) to prospectivelyinvestigate in 27 eyes of 27 patients the effects of preserved orunpreserved levobunolol in the conjunctival epithelium. Patientswere naive of previous antiglaucoma treatment. IVCM andimpression cytology were performed before and after 6 months oftherapy and showed significant differences from baseline in bothgroups and between the two groups. The IVCM analysis showed61% and 17% of goblet cell density reduction from baseline,respectively, in groups 1 (preserved levobunolol) and 2 (unpre-served levobunolol) (p< 0.001). Similarly, using the Nelson’s score(Nelson et al., 1983), the grading of impression cytology parameterswas significantly higher in group 1 than in group 2. Moreover, theseconjunctival epithelial changes were observed only after 6 monthsof therapy suggesting that preservatives exert their toxic effects ina rather short period of time at least at a subclinical level(Ciancaglini et al., 2008).

5.2. Observational comparative surveys

Several studies have in recent years compared the ocular surfaceparameters in patients receiving preserved and unpreservedeyedrops. The ocular surface of 20 patients with open-angle glau-coma receiving 0.5% timolol (with 0.01% BAK) or bitherapy withpreserved 0.5% timolol plus 1% dipivefrin were evaluated andcompared with those of 20 healthy control participants receivingplacebo. The Schirmer test and tear film break-up time values weresignificantly lower in the two treatment groups when comparedwith placebo (Yalvac et al., 1995). To evaluate the long-term effectsof preservative-free and preservative-containing antiglaucoma eyedrops on the ocular surface, a total of 84 patients were evaluatedusing the sophisticated method of IVCM with respect to theirtreatment. Significant differences were found between groups ontopical BAK-containing hypotensive therapy, namely preservedbeta-blocker, preserved prostaglandin, fixed or unfixed combina-tions of both drugs, and a preservative-free beta-blocker group. Inparticular, the density of superficial epithelial cells and the numberof sub-basal nerves were reduced in the preservative-containinggroups. In contrast, the density of basal epithelial cells, stromalkeratocyte activation, and bead-like nerve shapeing were higher inthe glaucomatous preservative therapy groups than the control andpreservative-free groups (Martone et al., 2009). Moreover thisstudy pointed out a decreased corneal sensitivity, based on esthe-siometry, in all preserved groups (ranging from 39.1 in the unfixedcombination group to 49.4 in the fixed combination group)compared to control or unpreserved eyedrops (58.2�1.7 and55.8� 4.3, respectively, P< 0.05). No differences were foundbetween preservative-containing groups. This study focused on

corneal nerves and structural but also functional changes, witha decreased sensitivity, are quite consistent with the knowndenervation effects of BAK in other systems like the gastrointestinalmyenteric plexus or the urinary tract (Rolle et al., 2003). Thisproperty of BAK could thus participate to the overall good comfortof patients receiving BAK-containing eyedrops, but if at least in partdue to corneal hypoesthesia, this would raise new concerns abouta falsely apparent good tolerance.

At a larger scale, in the above-mentioned epidemiologicalsurvey conducted in 2002 in 4107 glaucoma patients to assess theeffects of preserved and preservative-free eye drops on ocularsymptoms (Pisella et al., 2002), all were significantly more preva-lent (about twice as much) in patients using preserved dropscompared with those on preservative-free treatment. Likewise, thesimilarly conducted European survey also demonstrated that theincidence of ocular signs and symptoms was higher in patientsreceiving preserved eye drops (Fig. 4) (Jaenen et al., 2007).

5.3. Switching studies

Cross-sectional study observations have been confirmed byswitching studies. In patients receiving preserved eyedrops andexperiencing symptoms and signs evocative of poor tolerance, suchas allergy, blepharitis, or dry eye surface, changing to preservative-free eyedrops reliably leads to rapid improvement. In the Pisellastudy, conducted in private practices on patients treated for chronicopen-angle glaucoma, ophthalmologists prescribed preservative-free eyedrops to 349 patients presenting with signs and symptomsof ocular impairment and previously treated by preservedeyedrops. After 4 months, a sizeable and significant decrease(p< 0.001) of all functional signs and symptoms was observed. Aless marked improvement was also obtained in approximately 50patients who had reduced the number of preserved eye dropsolutions used with respect to the first visit. No change wasobserved in patients having continued their previous treatment(Pisella et al., 2002). Similarly, in the larger European survey theincidence of signs and symptoms decreased significantly byswitching to a preservative-free formulation or by reducing theamount of preservative-containing treatment (Jaenen et al., 2007).

Bron and colleagues report similar results in another multi-center, prospective, open-label study conducted on 435 patientswith open-angle glaucoma or ocular hypertension, by replacinga preserved solution by a preservative-free solution of the samefamily (timolol). After 3 months, changing to preservative-free eyedrops led to a notable improvement in local tolerance, whilemaintaining a good pressure balance. On instillation, irritation, dryeye, or foreign body sensations and blurred vision or stuck eyelidswere diminished. Between instillations, dry eye and foreign bodysensations were reduced by half (15.4% vs. 8.0%). The percentage ofconjunctival congestion dropped from 24.4% to 14.6%. Folli-culopapillary and superficial punctuate keratitis rates were alsoreduced by half (Bron et al., 2003).

The improvements described above are related to a significantdecrease in the cytologic and inflammatory processes of theconjunctiva and the cornea (Fig. 7). In a single blind study, Cam-pagna et al. analyzed the cytologic impression of 20 glaucomapatients before and after 3 months using preservative-free timololin place of preserved timolol. Switching to a preservative-freesolution significantly increased mucus cells and significantlyimproved conjunctival epithelial cell impairment (rose Bengalstaining). The subjective symptoms (stinging, foreign body sensa-tion) present on enrollment also diminished. These improvementswere significant beginning at the second month of treatment.Intraocular pressure control was also maintained during thetreatment change (Campagna et al., 1997). Likewise, in another

Fig. 7. A. Example of toxic reaction resulting from long term use of multiple treatment for glaucoma: the limbus is distended, hyperemic, with a pseudogerontoxon resulting frominflammatory infiltrates. B. A few weeks after surgery and treatment removal. The ocular surface rapidly recovered.


study of 21 patients treated with preserved timolol only 2 weeks oftreatment with preservative-free timolol resulted in partialnormalization of corneal permeability as measured by fluoropho-tometry (increase of þ27%, p¼ 0.025). In parallel, the improvementor disappearance of symptoms was obtained in eight out of tenpatients complaining of a burning or dry eye sensation and efficacywas not lost after removal of BAK (de Jong et al., 1994). Veryrecently, a new open-label multicenter switching study conductedin 158 patients treated with preserved latanoprost and experi-encing signs and symptoms of intolerance showed a markedimprovement when switching to preservative-free tafluprost(Uusitalo et al., in press). All symptoms and signs were significantlydecreased, as well as tear break-up time increased, whereas IOPcontrol remained unchanged after switching the prostaglandinanalog. Such improvements are therefore susceptible to improvequality of life, which is known to be deeply impaired in glaucom-atous patients by ocular surface-related side effects (Nordmannet al., 2003), and most likely compliance (Zimmerman et al.,2009), although these issues have not been specifically addressedin the latter studies.

6. In vitro demonstration of BAK toxicity

6.1. Chemical characteristics of BAK

Benzalkonium chloride (BAK) is a bactericidal cationic ten-sioactive compound used as preservative in various medical prep-arations in concentrations ranging from 0.004% to 0.025% in eyedrops. BAK is a mixture of alkyl benzyl dimethyl ammoniumchlorides with several analogs varying in the length of the aliphaticalkyl chain. In commercial preparations, the aliphatic alkyl chainspossess lengths of 12, 14 and 16 carbon atoms (Gardner and Girard,2000). At low concentrations, BAK in aqueous solutions is positivelyloaded, with amphipathic conformation. These cations associateand form micelles when the concentration of BAK reaches thecritical micellar concentration (CMC). Micelles are roughly spher-ical or globular aggregates characterized by a hydrophobic core anda hydrophilic outer surface. The CMC of BAK depends on the rela-tive composition of C12-, C14-, and C16-alkyl chains. As a conse-quence of micelle formation, the physical and biological propertiesof the solution may change abruptly, which was considereda possible cause of the abrupt termination of BAK activity at highconcentrations. CMC has been reported to occur over a concentra-tion of 0.02%. The effects of BAK micelles on ocular cells has notbeen fully addressed and seem to vary according to the systemtested in vitro (Deutschle et al., 2006). However, even thoughtoxicity may be less than expected in cell lines at high

concentration, probably due to micelle formation, in vivo highconcentrations on BAK cause major cell loss and tissue alterations(Pauly et al., 2008).

The biological activity of BAK is based on its interaction withproteins, lipids, and G proteins in biological membranes (Patarcaet al., 2000). As a microbicidal agent, it is therefore highly toxic,in a time- and dose-dependent manner, widely and reliablydemonstrated in a large variety of systems and organs, demon-strating not only cell toxicity but also genotoxicity andmutagenesispotential (Deutschle et al., 2006).

6.2. Models of study in ocular toxicology

One useful alternative to animal models relies on cell cultures.Of course one major limitation is the monocharacteristic of the cellmonolayer that cannot mimic a more complex structure likea tissue. In particular, conjunctiva, the most reactive tissue of theocular structure from an immunological point of view, with thecomplex association of epithelial cells, mucin-secreting goblet cells,dendritic cells, and intraepithelial lymphocytes, cannot be fullystudied on the basis of merely epithelial cells. Nevertheless, despitethis drawback, cell cultures have beenwidely used for investigatingsome pathophysiological aspects of human conjunctiva, especiallyfor toxicological purposes. Particularly, cell lines permit easy andquick cell development and have the advantage over primaryculture of being independent from conjunctival biopsy availability.Currently, theWong-Kilbourne derivative of the Chang conjunctivalcell line has been widely used for toxicological and functional invitro studies on ocular surface diseases. This cell line has beenimmortalized from normal human conjunctival epithelial cells, butpresents some characteristics distinguishing it from normal tissueand primary cultures, particularly in their response to inflamma-tory cytokines such as TNFa and INFg (De Saint Jean et al., 2004).Indeed, like other cell lines, it has acquired some differences fromnormal conjunctiva in its phenotypic characteristics. However, themost common reserve to this cell line is the reported contamina-tion with HeLa cells (Lavappa, 1978). Recently, Diebold et al. char-acterized the IOBA-NHC spontaneously immortalized cell line,which showed no other cell type contamination but some pheno-typic differences compared to the normal conjunctival epithelium(Diebold et al., 2003). Our group evaluated the relevance of theIOBA-NHC cell line in toxicological research studies, determiningwhether these cell lines would be fully comparable and suitable fortoxicological in vitro studies (Brasnu et al., 2008b). As expected, BAKshowed similar toxicity profiles in the two cell lines. We thusconfirmed the validity of using the Chang cell line for toxicologicalin vitro studies. However, this line seems to have a higher sensitivity


against BAK despite their reported contamination with HeLa cells.In addition, in this study, we showed a dose-dependent toxicity ofBAK on IOBA-NHC cells, validating this cell line for toxicological invitro studies, particularly for the comparison of the apoptotic oroxidative effects of different ophthalmic medications. Nevertheless,as Chang and IOBA-NHC cell lines are in vitro models, the resultsobtained with each cell line cannot be fully extrapolated to in vivoconditions. In vivo, cells are protected with the action of the eyelids,the preocular mucin layer and glycocalyx, and there is a permanentrenewal of ocular surface epithelia. Furthermore, tissues have highregeneration and defense capacities and conjunctival epitheliumhas a stratified structure that enhances protection of the ocularsurface against preservative toxicity.

6.3. Literature data

As a quaternary ammonium, the preservative BAK may act atdifferent levels of the cellmachinery and has been shown to interactwith cell membranes and cell receptors, especially those involved incell death. In Chang conjunctival cells and IOBA-NHC cells, Buronet al. demonstrated both similarities and differences between BAKtoxicity and ultraviolet-induced stress (Buron et al., 2006). Bothagents induced cytochrome c release from the mitochondria, cas-pase activation, and nuclear chromatin condensation, suggestingcaspase-dependent apoptosis, prevented by stable expression ofBcl-2 protein and only partially prevented by baculovirus p35 cas-pase inhibitors, which more efficiently delayed the UV irradiation-induced than the BAK-induced nuclear chromatin condensation.Therefore BAK also induced caspase-independent cell death andautophagic features, likely to play a protective or at least adaptiverole. Whereas both agents induced a redistribution of Fas in plasmamembrane rafts and the Fas-ligand-independent formation ofa death-inducing complex leading to caspase-8 activation, BAKspecifically activated a caspase-independent pathway by inducingthe mitochondrial release of apoptosis-inducing factor (AIF). BAK-treated cells contained autophagosomes/autolysosomes, a charac-teristic feature of autophagy, and siRNA-mediated downregulationof the beclin-1 gene, whose product is crucial for autophagy,increased BAK toxicity (Buron et al., 2006). In addition, a significantmutagenic effect of BAKhas been found at environmentally relevantconcentrations andeven thoughnoevidenceof genotoxicity-relatedocular side effects has been reported to date, this effect should beconsidered an additional potentially harmful effect in patientsreceiving BAK over the long term (Ferk et al., 2007).

6.4. Comparative studies on preservatives

Very few comparative toxicological studies have been con-ducted, either in vitro or in vivo. As we developed efficient in vitrotesting models in conjunctival cell lines using cold light cyto-fluorimetry and flow cytometry, dealing with apoptosis, cellviability, cell cycle, and oxidative stress, we conducted a compara-tive assessment of various preservatives used in ophthalmic prep-arations (Debbasch et al., 2001b). BAK in three differenthydrocarbon chain lengths, benzododecinium bromide (BOB),cetrimide (CET), phenylmercuric nitrate, thimerosal, methyl para-hydroxybenzoate, chlorobutanol, and EDTA were submitted totoxicological screening. Consistent toxicity was reliably found withall quaternary ammonium compounds, namely BAK, BOB, and CET,at a threshold of toxicity as low as 0.005%. Nonquaternary ammo-nium preservatives caused oxidative stress but to a significantlylesser extent and induced a lower cell death rate.

More recently, Epstein et al. also investigated various preser-vative compounds in immortalized human conjunctival andcorneal epithelial cells. BAK, methyl paraben (MP), sodium

perborate (SP), chlorobutanol (Cbl), and stabilized thimerosal (Thi),and EDTA with negative and positive controls were tested. Theauthors confirmed that all preservatives caused significantconjunctival and corneal cell toxicity, depending upon concentra-tion. BAK exhibited more toxicity than Cbl, MP, and SP, Thi beingmore toxic in this model than quaternary ammoniums and EDTAand exerting minimal toxicity (Epstein et al., 2009).

6.5. Effects of BAK on conjunctival epithelial cells

Chang human conjunctival-derived cells have been widely usedto demonstrate and study the toxic effects of BAK. Our groupdeveloped and used new cytotoxicity tests using cytofluorimetricmicrotitration and flow cytometry. The techniques of MiFALC(Microtitration Fluorimetric Assays on Live Cells) allow tests on livecells, consistent with the recommendations of the EuropeanCommittee for the Validation of Alternative Methods. We thereforeused a series of innovative tests and methods for multiple evalua-tions in ocular toxicology by investigating objective biologicalcriteria such as cellular viability, chromatin condensation,production of reactive oxygen species, and transmembrane mito-chondrial potential. Therefore, many antiglaucoma drugs and theirpreservatives have been studied. All preservatives, even at very lowconcentrations, appeared cytotoxic for ocular cells and responsiblefor apoptosis and free radical production. Conversely, most activecompounds did not induce any apoptotic mechanism; nor did theyshow interesting antioxidative and antiapoptotic properties(Brasnu et al., 2008a; De Saint Jean et al., 1999, 2000; Debbaschet al., 2001a,b, 2002; Guenoun et al., 2005a,b; Labbe et al., 2006b;Pisella et al., 2004).

From all these in vitro studies, it appears that BAK inducesa significant, concentration-dependent decrease in cellular viability,whereas unpreserved preparations of timolol, carteolol, and taflu-prost did not show any toxicity. Preparations containing thepreservative induced chromatin condensation associated with analteration of mitochondrial activity and a decrease of glutathione,suggesting an apoptotic phenomenon. ROS production was alsofound with preserved formulations at significantly higher levelsthan those observed with unpreserved drugs. Furthermore, wedeveloped an in vitro proinflammatory model using the stimulationof intercellular adhesionmolecule (ICAM)-1using interferon (IFN)-gin order to address the possible mechanisms leading to ICAM-1 orHLA-DR overexpression in glaucomatous patients treated over thelong term. In contrast with proapoptotic cell death-promotingeffects, eye drops preservedwith BAKdid not cause direct ICAM-1 orHLA-DR expression, but they did induce even higher ICAM-1expression levels on IFNg-stimulated cells than did IFNg alone,whereas unpreserved drugs had no effect. This would demonstrateBAK is a costimulatory factor with inflammatory cytokinesenhancing inflammatory cascade activation (Pauly et al., 2007a).

Indeed, the proapoptotic effects were seen at very lowconcentrations of BAK with a threshold of toxicity found at about0.005%. Interestingly, a reliable observation found in our successivestudies was the fact that commercially available prostaglandinanalogs often induced a lower toxicity to conjunctival cells than thesolution of BAK alone at the same concentration (Guenoun et al.,2005a,b; Pisella et al., 2004). A chemical combination reducingthe amount of toxic free BAK could not be excluded. However, asthe reduction of effects observed in vitro was mainly effective onfree radical production, a positive antioxidative effect of the pros-taglandin analog was thus hypothesized that could in part coun-teract the deleterious oxidative stress caused by the preservative(Fig. 8) (Guenoun et al., 2005a). This was confirmed both using thecommercial formulations of travoprost and latanoprost and whentesting nonpreserved latanoprost in a dose-dependent manner

Fig. 8. O2� detection using hydroethidine test after 30 minutes of treatment with BAC

0.02%, BAC 0.015%, BAC 0.005%, latanoprost, travoprost, or bimatoprost. Each solutionwas diluted to 1/10. BAC concentrations are matched with those of the three prosta-glandin solutions, respectively. Means� SE. White star, statistically significant differ-ence compared with its respective BAC concentration (P< 0.01). Black star, statisticallysignificant compared with control (P< 0.01). From Guenoun et al., 2005a. In vitrocomparison of cytoprotective and antioxidative effects of latanoprost, travoprost, andbimatoprost on conjunctiva-derived epithelial cells. Invest. Ophthalmol. Vis. Sci. 46,4594e4599. (Permission from IOVS asked, answer currently in progress).


(Guenoun et al., 2005a), whereas bimatoprost seemed not toexhibit antioxidative properties, at least in the experimentalconditions we tested. This hypothesis is therefore in line with theresults of clinical trials and postmarketing observations that alsoargue in favor of relatively good tolerance of such compoundsdespite a rather high amount of BAK instilled over the long term, atleast in patients receiving only one drug.

6.6. Three-dimension models of corneal epithelium

In vitro experiments with immortalized cells do not really reflectthe conditions of a mucosa in vivo, as isolated cells may be moresensitive than a tissue. On the other hand, a tissue exposed tovarious stresses may involve all cell components, which maychange the response of complex tissues, such as the conjunctiva,which associates epithelial, inflammatory, and mucus cells. Suchcomplex interactions may thus stimulate compensation modeswhose result may be at the ocular surface levels a mixture of anincreased cell death rate, tear film impairment, inflammatorystimulation and increased epithelial turnover. Therefore, the searchfor more appropriate models than cell monolayers has led tocomplex three-dimensional (3D) epithelial systems that are usefulfor toxicological purposes.

The reconstructed 3D model of human corneal epithelial cells(HCEs), supplied by SkinEthic� Laboratories, was found to resemblethe corneal epithelium of the human eye in morphology andthickness (Nguyen et al., 2003). It was proposed as a useful alter-native model to the classical Draize test (Doucet et al., 1998, 2006)for the assessment of eye irritation potential of chemicals andcosmetic products. However, as the standard procedures are basedon the MTT (3-(4, 5-dimethylthiazol-2-y1) 2,5-diphenyl tetrazo-lium bromide succinate)-based colorimetric assay for toxicologicalassessments and because the MTT solution is usually placed underthe construct, it could penetrate only two or three layers from thebasal side, yielding results that may not correlate with histo-morphological findings in the superficial layers. This proceduremay therefore underestimate any mild toxic effect occurring at theapical surface of the constructs. Our group (Pauly et al., 2009)adapted the MTT technique to overcome this drawback andincrease sensitivity, making this procedure better suited to the

prediction of low- to very-low-irritant potential, especially whenthe products are used repeatedly over long periods of time.

In our study, HCEs were treated with concentrations of BAKranging from 0.001% to 0.5% for 6 to 24 h followed or not by a 24-hpostincubation period. Frozen sections were analyzed using fluo-rescence confocal microscopy for the presence of TUNEL, activatedcaspase-3, Ki67, ICAM-1, HLA-DR, E-cadherin, and occludin.

As expected and similar to what was previously observed inmonolayer cell models, the MTT test revealed a dose-dependentresponse of BAK with significant toxic effects for concentrations aslow as 0.005%. Increasing BAK concentrations induced an increasednumber of apoptotic cells found from the superficial to the deeperlayers (Fig. 9). Interestingly, the most superficial cell layer, i.e., thelayer most exposed to the toxic effect, showed large TUNEL posi-tivity, quite consistent with apoptotic and cell death featuresobserved in Chang or IOBA cells. However, the deepest layers,namely those receiving much lower doses of toxic compounds,showed the activation of caspase-3, consistent with the early stageof apoptosis and demonstrating the range of effect that a toxicsubstance will cause in a multilayered epithelium. Indeed, it isnoteworthy that BAK at low concentrations (0.001e0.01%) seemedto elicit a defense response by increasing expressions of Ki67, a cellmarker of proliferation, and ICAM-1, an adhesion molecule relatedto inflammatory responses and cell recruitment, as well as byincreasing occludin mRNA expression, most likely to compensatethe tight junction disruption due to BAK. BAK thus induced thedose-dependent disappearance of occludin, suggesting thedisruption of the epithelial tight junctions in the superficial cells.These results are consistent with the known enhancer properties ofBAK used to increase corneal permeation of active compounds.

The BAK-induced overexpression of ICAM-1 on 3D-HCE suggeststhat it can contribute to corneal epithelial cell injury by aiding theattachment of inflammatory cells that express the receptor forICAM-1 and the beta2 integrins (CD11a,b,c/CD18). Overexpressionof ICAM-1 by conjunctival cells was found in humans treated overthe long term (Pisella et al., 2004). However, in this in vitro model,HLA-DR was not found, unlike in human eyes. This may beexplained by the lack of inflammatory cells and mediators in the invitro corneal model, and possibly structural and functional differ-ences between the cornea and the conjunctiva. Nevertheless, theseresults illustrate the tissue-level response to a toxic, most likely onan inflammatory mode in response to the cell-level death mode.Taken together, these results appear important to better compre-hend the effects of low doses of a toxic compound on a morecomplex structure than single cells. This would fill the gap betweenthe cell death-related observations in in vitro models and theinflammatory responses observed in humans or animal models.

However, conflicting results were also reported, minimizing theimpact of preservative on ocular tissues. In contrast with our resultsand all the literature data on BAK toxicity, Khoh-Reiter and Jessen,in a study conducted by the Pfizer laboratory, using the samemodelof 3D corneal epithelium, did not find any loss of viability whentesting preserved latanoprost or BAK after a 10- to 60-min contactduration (Khoh-Reiter and Jessen, 2009). This raises severalimportant issues such as the choice of the model and the mostappropriate duration of contact to best fit real-world and clinicalconditions. Actually, a normal tissue in a young patient, submittedto low concentrations of a toxic agent once a day, will unlikelyundergo major changes, unless treated for a very long period oftime. Indeed, individual sensitivity of the tissue and accumulationof drugs will directly influence the outcome of the ocular surfaceover the long term. The question of the actual duration of BAKexposure to corneal cells, and subsequently the accuracy of themodel, is important to consider. Khoh-Reiter and Jessen stated thatBAK would only be in contact with the eye structures for few

Fig. 9. Immunolocalization of occludin (cell junctions), ICAM-1 (Intercellular adhesion molecule), TUNEL (late apoptosis), activated caspase-3 (early apoptosis), and Ki67 (cellproliferation) in a 3D model of corneal epithelium (Skinethics�), in control (left) or after 24 hours of contact with BAK 0.01% (right). Immunostainings appear in green. The nuclei arestained in blue with DAPI (raws 3 and 4) or in red with propidium iodide (raws 1,2 and 5). BAK was administered on the top of the epithelium for 24 hours. Results clearly showa disruption of tight junctions in the most superficial layers, namely the most exposed to BAK, overexpression of ICAM-1, late apoptosis of the most superficial layer, early apoptosisin deeper layers and a compensatory multiplication of epithelial cells corresponding to an increased turnover of the corneal epithelium (Scale bar: 150 mm).


seconds after instillation. This assumption seemed in accordancewith a previous study investigating the concentration of BAK in thetears after instillation and showing rapid dilution (Friedlaenderet al., 2006). However, very few data exist on ocular pharmacoki-netics of BAK after instillation. Champeau and Edelhauser evaluatedthe presence of BAK in tissues, thus evaluating not dilution butimpregnation of ocular surface tissues by the chemical compound.They found measurable concentrations in the conjunctiva andcornea up to 7 days after a single instillation in a normal rabbit eye(Champeau and Edelhauser, 1986). As BAK is usually considereda potent and useful enhancer of drug penetration into deep ocularstructures, all experimental models should be considered in light ofcontact time, depending not on tear concentrations of BAK butinstead on much more prolonged contact times, as most likelyoccurs with a compound that disrupts cell membranes, increasescorneal epithelium permeability, and is repeatedly used overextremely long periods of time.

7. Experimental data in animal models

Chemicals, cosmetics, and pharmaceuticals have to be assessedfor their irritancy potential and risk to the human eye. However, theonly method accepted worldwide by regulatory authorities for theassessment of acute eye irritation potential is the Draize rabbit test(Draize et al., 1944), which has been criticized by animal welfare

advocates and whose relevance, validity, and precision have beenchallenged because of the variability and low predictiveness of thehuman response (Curren and Harbell, 2002;Wilhelmus, 2001). Thistest is mainly based on scoring observed macroscopic changes inthe rabbit cornea, conjunctiva, and iris, and various scoring systemsare currently accepted (Maximum Average Score MAS, ModifiedMaximum Average Score MMAS, Globally Harmonized System,etc.). The rabbit model showed weaknesses for assessing nonsevereirritants and low toxics. There has therefore been an attempt toimprove the sensitivity and relevance of rabbit eye models intoxicology. One major technique, in addition to standard clinicalassessment and postmortem histology, is the development ofcorneal confocal microscopy (IVCM), an in vivo nontraumatictechnique for investigating the ocular surface, first developed inhumans but suitable for animal models.

7.1. The rabbit as an experimental model: recent improvements

The first toxicological study was conducted by Ichijima et al.,who developed an acute stress mode suitable for mimicking theeffects of long-term use of low toxicity compounds over a shortperiod of time. Furthermore, the authors used IVCM to improve thedetermination of ocular surface changes following drug adminis-tration. The effects of BAK on the living rabbit cornea were studiedby in vivo Tandem scanning confocal microscopy (TSCM) and


confirmed by conventional scanning electron microscopy (SEM).Two drops of saline or phosphate-buffered saline (PBS) containingBAK in concentrations of 0.02, 0.01, or 0.005%were applied to rabbiteyes 15 times at 5-min intervals. Immediately after application of0.02 and 0.01% BAK, no normal corneal superficial epithelial cellscould be imaged by in vivo TSCM. Application of as little as 0.005%BAK caused the superficial epithelial cells to swell and desquamate.The observed desquamation of corneal superficial epithelial cellsincreased with higher BAK concentrations applied to the eye. Onehour after final drug application, inflammatory cells appeared onthe surface of the cornea treated with 0.02% BAK. These findingswere correlated with SEM observations, whereas nonpreservedcontrol solutions showed no toxicity (Ichijima et al., 1992).

Our group also used the rabbit eye for toxicological purposes,developing the most recent technical improvement of IVCM toenhance ocular surface investigations at a histological-like level ofresolution. The recently developed Rostock Corneal Module of theHeidelberg Retina Tomograph (HRT/RCM, Heidelberg Engineering,Heidelberg, Germany) now allows resolutions close to 1 mm andfacilitates examination of the whole ocular surface. Major appli-cations have been developed in human diseases (Labbe et al., 2005,2006a, 2009) and the device has been adapted to animal use (Labbeet al., 2006b; Liang et al., 2008a,b). In addition to toxicity-relatedchanges such as corneal epithelium alterations, IVCM was able toshow inflammatory infiltrates in the peripheral cornea, the limbus,and the conjunctiva, features previously only accessible to post-mortem histology (Fig. 10). Furthermore, for the first time we wereable to identify in vivo the conjunctiva-associated lymphoid tissue(CALT) and its major inflammatory infiltration after an acute chal-lenge with BAK or BAK-containing eyedrops (Liang et al., 2009).Until now, no study has been carried out in vivo or even using exvivo techniques focusing on the CALT reactions after instillations oftoxic eye drops. In our toxicological model, the inflammatory celltrafficking in CALT seemed to be primarily related to the concen-tration of BAK because major infiltration could be observed, ina dose-dependent manner with regard to BAK concentration, andnonpreserved control eyedrops or active compounds did not causeany change. These immunoinflammatory changes in CALT, as wellas in a more diffuse way in the whole conjunctiva, may participatein the strong inflammatory and apoptotic reactions observed afterapplications of BAK-containing eye drops, thus demonstrating thetwo pathways induced by BAK, namely cell death and inflamma-tion. This approach in ocular toxicology therefore allows betterinvestigations of low-toxicity compounds and refined assessmentof active compounds or possible preservative alternatives to BAKuse in this acute stress model.

Other tools for a better analysis of toxicity in rabbit eyes mayalso include impression cytology specimens (Liang et al., 2008a,b),another technique that allows repeated examinations beforeanimal sacrifice, thus reducing the number of animals used forexperimental purposes, in accordance with the current require-ments for animal use (the Three R rule).

Nevertheless, histology, immunohistology, and electronmicroscopy have also widely been used for toxicological purposesand several reports have clearly confirmed the quite consistenttoxicity of BAK, for any model, duration of application, and possibleassociation with an active drug. For example, Noecker et al. foundless damage and lower inflammatory scores after a 30-day appli-cation of bimatoprost, the prostaglandin analog with the lowestBAK concentration, containing 0.005% BAK, compared to timolol orlatanoprost eyedrops (0.01% and 0.02%, respectively), thus con-firming the reliable dose-dependent effects of BAK (Noecker et al.,2004). Consistent with this report, Kahook and Noecker countedgoblet cell density after 30 days of treatment in rabbit eyes andfound significantly lower density in the BAK-containing latanoprost

group than in the preservative-free artificial tear group (2.21�0.40vs. 7.03�1.33; p¼ 0.0001) (Kahook and Noecker, 2008). Aftera treatment of only one drop a day in healthy animals and a rathershort duration, these findings must be understood in light ofdramatic tear film changes and dry eye prevalence in glaucomapatients treated over the long term. They are quite consistent withhuman findings of reduced goblet cell density in treated patients(Herreras et al., 1992; Pisella et al., 2004).

7.2. BAK toxicity in rat

The rat is a useful model that is more suitable for handling andhousing than the rabbit. It has already been used to demonstrateBAK toxicity, confirming that even in healthy animals a rather shortduration of contact may exert significant histological changes thatare observable in BAK-containing solutions but not with timololalone (Baudouin et al., 1999). However, a high variability of ocularbehavior in rat models may render this animal poorly reliable andless often used than the rabbit for toxicological purposes. IVCM hastherefore improved the usefulness of rats and the accuracy of ocularexaminations in addition to biomicroscopy and postmortemhistological analyses (Labbe et al., 2006b). We performed a series oftoxicological assessments of BAK in the rat eye after repeatedinstillations and developed IVCM techniques in rats in order toimprove in vivo investigations. The effects of BAK were first eval-uated using the Draize-derived criteria and irritation was onlyevidenced after instillation of highly concentrated BAK solutions, i.e., 0.5% and 0.25%. Then we characterized the effects of BAK usingthe HRT-II confocal microscope and the results showed a dose-response relationship for BAK-induced scores in rat cornea. Thehighest 0.25% and 0.5% concentrations caused epithelial denuda-tion as well as major damage in the deep structures such as stromalinflammation and neovascularization as well as loss of endothelialvisibility and fibrosis. The HRT-derived scores calculated from ourdata were therefore very high. We evidenced incomplete cornealrecovery even long after toxic removal, as assessed by a decreasedbut still high HRT score due to anisocytosis, abnormal epithelialreflectivity and shape patterns, and persistent stromal neo-vascularization. In contrast with clinical data, the lower 0.01% and0.1% BAK concentrations were characterized by a significantlyabnormal total HRT score, resulting from damage mainly restrictedto the epithelium that extended from cell border loss (0.01%) toepithelial erosion exposing the underlying, smaller wing cells(0.1%). The lowest 0.01% BAK concentration was a good example ofa slight damage inducer, with changes restricted to the mostsuperficial cell layers of the cornea and only identified because themost sensitive evaluation criteria were used (Pauly et al., 2007b).This detection of slight corneal changes must be seriously consid-ered, given that it was obtained for a much shorter treatmentduration than used in common treatments in humans. We there-fore developed a scoring system based on IVCM findings in order tostandardize toxicological changes and allow various laboratoriesworldwide to better evaluate low toxicity compounds using a singletechnique (Pauly et al., 2008).

8. Other potential issues with BAK use

8.1. Dry eye and ocular surface diseases

BAK is a well-known irritant in dermatological and allergologicalinvestigations, but it is rarely recognized as the main allergenresponsible for contact dermatitis (Uter et al., 2008). However, bytrulyallergenicmechanismsorasa cofactorandan irritant compoundit may cause or enhance various clinical manifestations at the ocularsurface level, such as allergic or nonallergenic blepharitis,meibomian

Fig. 10. In vivo confocal microscopy (IVCM) images of rabbit cornea superficialepithelium (column 1) and CALT structure (column 2), 4 hrs after 15 instillations ofvarious antiglaucoma prostaglandin analogs. After instillations of PBS (A), BAK-freeTafluprost (B), or BAK-free travoprost (Travatan Z�, C), rabbits presented a normalcorneal epithelium with a regular polygonal mosaic appearance and brightly reflectivenuclei. They did not induce any obvious changes in CALT structure. However, the threeBAK-containing solutions, 0.02%BAKþlatanoprost (D), 0.015%BAKþtravoprost (E), andto a lesser extent 0.005%BAKþbimatoprost (F) induced partial desquamation ofepithelial cells, with some epithelial cells showing an irregular aspect with hyper-reflectivity patterns or a loss of cell borders (column 1). They also induced substantial


gland dysfunction, chronic conjunctival inflammation, or tear filminstability. Through its toxic and proinflammatory effects, BAK maytherefore cause or aggravate dry eye disease and allergic conjuncti-vitis. The tear film is a fundamental protective layer for the ocularsurface through its lubricating,mechanical, trophic, andantimicrobialproperties.Goblet cellsproducesolublemucinsandparticipate in tearfilm stability. These cells are extremely sensitive to toxic andinflammatory stresses. They have been shown in humans to bedecreased indensity after short exposure to BAK (Rolandoet al.,1991)or BAK-containing timolol (Herreras et al.,1992). Long-termexposureto preserved antiglaucoma drugs also caused decreased goblet celldensity (Pisella et al., 2004). In animalmodels,Wilson and colleaguesdemonstrated that 0.01% BAK caused precorneal tear film disruptionin rabbits (Wilson et al., 1975). More recently, an experimental studyconducted in albino rabbits showed significantly greater reduction inthe tear film break-up time in animals receiving a preserved beta-blocker compared with those given a nonpreserved beta-blocker(Pisella et al., 2000). At the biological level, MUC1 and MUC16 werefound to be reduced after exposure to BAK in human corneal-limbalepithelial cells and human corneal epithelium. Transmission electronmicroscopy of the anterior corneal surface revealed fixation of themucus layer after exposure to 0.01% BAK for 5 or 15 min, whereasmoreprolongedexposure (60 min) to0.01%BAKdestroyed themucuslayer (Chung et al., 2006).

In addition, as a tensioactive compound, BAK is also a detergentfor the lipid layer of the tear film, causing evaporation of theaqueous tear film. The result of both mechanisms, namely mucusand lipid-layer alterations, is a globally impaired tear filmwith tearinstability and excessive evaporation, hallmarks of dry eye disease(Dry Eye WorkShop, 2007a). For example, in humans, a decreasedbreak-up time was found after a short duration of treatment withBAK-containing carteolol in healthy volunteers (Baudouin and deLunardo, 1998) and increased corneal epithelial permeability ofdry eye patients was shown in dry eye with additional impairmentwhen using artificial tears containing BAK compared to non-preserved eyedrops (Gobbels and Spitznas, 1989, 1992).

Through the loss of its protective properties, an impaired tearfilm will cause dry eye symptoms and corneal damage and alsoconvey cytotoxic inflammatory mediators throughout the ocularsurface. Tear film alterations may even stimulate a series of bio-logical changes in the ocular surface leading to subsequentneurogenic inflammation and further impairment of the tear film,actually creating a vicious circle (Baudouin, 2007; Dry EyeWorkShop, 2007a).

8.2. The crystalline lens: is BAK an inducer of cataract?

The use of antiglaucoma medications has also been associatedwith the development of cataract. In the Ocular HypertensionTreatment Study (OHTS) an increased rate of cataract extractionand cataract/filtration surgery was observed in patients receivingmedication (7.6%) compared with the non-treated control group(5.6%), with an odds ratio of 1.56 (Herman et al., 2006). The BlueMountains Eye Study also showed that antiglaucoma therapyseems to increase the risk of cataract formation (odds ratio of 1.90)(Chandrasekaran et al., 2006). These observations raise thehypothesis that antiglaucoma treatment may increase the lens

inflammatory cell infiltration in the CALT structure, in the follicle area but at an evenhigher level in the parafollicle areas, consisting of lymphocyte-like and dendritiformcells. Quantification of epithelial changes and cell infiltrates clearly showed a dose-dependent effect of BAK (0.02%BAKþlatanoprost> 0.015%BAKþtravoprost> 0.005%BAKþbimatoprost). All images: 400mm X 400mm; numbers on the figures show depthfrom the surface.


opacity rate and/or accelerate cataract formation (Brandt, 2003). Noevidence of a specific role for one or another compound could befound, which again argues for a possible interaction with thecommon toxic compound found in eye drops, namely the preser-vative that accumulates in the eyes of patients treated with severaldrugs over the long term. Indeed, Goto and colleagues investigatedthe effects of latanoprost, timolol maleate, and BAK in a human lensepithelial cell line. The results showed that BAK alone, and toa much lesser extent latanoprost or timolol, strongly stimulated theexpression by lens epithelial cells of soluble mediators involved ininflammatory and/or apoptotic processes, i.e., prostaglandin E2,interleukin-1a, and interleukin-6 (Goto et al., 2003). These medi-ators could therefore favor or stimulate lens opacification.

8.3. Trabecular meshwork: raising new hypotheses

The glaucomatous trabecular meshwork is characterized bya dramatic trabecular cell loss, increased trabecular extracellularmatrix, and accelerated senescence, resulting in increased outflowresistance. The mechanisms of trabecular cell loss in glaucomapatients are still poorly understood. A possible role for oxidativestress has been proposed and since BAK could stimulate oxidativestress, cell death, and fibronectin mRNA in an in vitro model oftrabecular cells, the involvement of BAK in trabecular meshworkdegeneration was therefore hypothesized (Yu et al., 2008). Like-wise, Samples and colleagues demonstrated that BAK causeda significant inhibition of the growth of trabecular meshwork cellsat extremely low concentrations (Samples et al., 1989). To deter-mine whether drug-induced apoptosis could be one of the mech-anisms by which trabecular cells die in glaucoma, we evaluated theeffects of BAK-containing or BAK-free antiglaucomamedications oncell cytoskeleton and apoptotic marker expression in two culturedhuman trabecular meshwork cell lines (Fig. 11). In a 1/100 dilution,i.e., an extremely low concentration, unpreserved beta-blockershad no apoptotic effect, preserved beta-blockers and latanoprostsignificantly increased expression of only Apo2.7, an early apoptosismarker, while BAK significantly increased all the apoptotic markersinvestigated. When tested in a 1/10 dilution, all drugs exceptunpreserved timolol triggered a 2- to 3.5-fold increase in apoptoticpatterns, whereas up to 95% of the cells underwent apoptosis upontreatment with BAK, representing a nine fold increase over thebackground level (Hamard et al., 2003). The most toxic concen-trations seemed slightly higher than those assumed to be found inthe aqueous humor after instillation (supposedly corresponding toa 1/100 dilution). However, repeated instillation may cause further

Fig. 11. Cultured trabecular cell line immunostained with alexa-phalloidin (green staining ofollowing 15 minutes of exposure with 0.01% BAK; A. Normal control cells; B. Total disorgan25 mm (Adapted from Hamard et al., 2003).

involvement of trabecular cells submitted to long-term treatmentsand the impact of toxics on diseased trabecular tissues have notbeen evaluated. Moreover, BAK was shown to accumulate inconjunctival space since only a single drop of BAK could causemeasurable amounts in the conjunctiva up to 7 days after instilla-tion (Champeau and Edelhauser, 1986). No data are available for thetrabecular meshwork, but it is most likely that BAK accumulates indeep tissues after prolonged administration and the aqueoushumor route is probably not the only one to cause contact of BAKwith trabecular tissues. In addition, the impact of inflammatory andtoxic reactions, resulting from chronic treatments with BAK-con-taining eyedrops over the long term, remains to elucidate. From allthe above-mentioned studies and surveys, in humans, in experi-mental models, and in vitro, it can be assessedwhether conjunctivalepithelial cells are involved as targets and/or stimulators, in majorinflammatory changes such as overexpression and/or synthesis ofclass II antigens, adhesion molecules, chemokines, chemokinereceptors, interleukins, or cell death markers and mediators.Additionally, the substantia propria of the conjunctiva is infiltratedby inflammatory cells (Baudouin et al., 1999; Broadway et al.,1994a) and at least in the rabbit eye where it can be assessed invivo, CALT follicles are stimulated by repeated instillations of BAK-containing eyedrops. Therefore, the impact of high rates of celldeath and inflammatory signals on deeper but still very closestructures such as the trabecular meshwork cannot be excludedand this hypothesis deserves further attention.

This would indeed raise the new and ominous hypothesis thatBAK present in antiglaucomatous treatments could stimulatetrabecular senescence, resulting in further damage to the trabeculawith a possible impact on outflow and consequently IOP, at thesame time as drugs are precisely aimed at decreasing IOP.

8.4. Retinal involvement

Several antiglaucoma eye drops have been reported to causecystoid macular edema following cataract surgery. Although pros-taglandin analogs used as antiglaucoma treatment would be moreprone to cause this complication, the association with othercompounds such as timolol raised the hypothesis of a role played bythe common preservative BAK. Therefore, after a series of investi-gations on the possible causes of macular edema following cataractextraction and the role of antiglaucoma drugs and their preserva-tive, a clinical trial was conducted by Miyake et al., comparing theimpact of preserved timolol with preservative-free timolol. Theauthors found significantly greater aqueous flare and a higher

f actin) and propidium iodide (red staining of nuclei). Degeneration of trabecular cellsization of cell cytoskeleton; C. No effect of unpreserved timolol. The scale bars indicate


incidence of angiographic cystoid macular edema after cataractsurgery in the preservative-containing group. Based on the findingsof these studies, which indicate that the preservative causesincreased synthesis of proinflammatory mediators and intensifiedpostoperative inflammation, the term pseudophakic preservativemaculopathy was proposed (Miyake et al., 2003).

8.5. Nonocular side effects

As the volume of the preocular tear film is much lower than thevolume of one instilled drop, the excess volume may reach thenasopharyngeal mucosa, resulting in a significant systemicabsorption, specially when the patient instills more than one drop,which is quite common, either due to misusing eye drops or simplybecause about 50% of patients are treated with two or more anti-glaucomatous drugs, in both eyes. In such cases, the effects ofsystemic absorption or even of BAK in the bronchial tract arecertainly underestimated. In the late 1980s, a series of studiesdemonstrated that BAK was a potent bronchoconstrictor agonistwhen inhaled by patients with asthma in concentrations similar tothose in which it was present in commonly used bronchodilatorsolutions (Beasley et al., 2001). BAKwas only eight times less potentas a bronchoconstrictor agonist than histamine and it was shown tocause bronchoconstriction through a combination of mast cellactivation and stimulation of neural pathways. It has been shownthat the inclusion of BAK not only reduces the overall bronchodi-lator efficacy of the product, but also causes marked bronchocon-striction in a significant proportion of patients with asthma,whereas preservative-free bronchodilators did not show theseparadoxical effects. Therefore Beasley and colleagues stated:“When considered together with previous studies, these findingsindicate the urgent requirement for the worldwide withdrawal ofBAK from bronchodilator nebulizer solutions” (Beasley et al., 2001).The impact of BAK contained in eye drops on respiratory mucosaemust still be evaluated, but in light of pneumological assessmentson BAK, it could be hypothesized that repeated instillations of BAK,alone or in interaction with the active compound, e.g., beta-blockers or prostaglandin analogs, could play a role in the respi-ratory side effects experienced by some glaucomatous patients.

9. Are there alternatives to BAK?

9.1. Eliminating the preservative

Considerable efforts have been made in the recent past by thepharmaceutical industry to develop new antiglaucomatouscompounds that would bring about efficacy, safety, and compli-ance. As BAK toxicity is mainly dose-dependent, reducing thenumber of instillations has improved ocular tolerance. Long-actingpreparations of timolol and prostaglandins are given once a day,mathematically reducing the amount of BAK in contact with the eyeby 50%. The same has occurred for various fixed combinations oftimolol and prostaglandins, carbonic anhydrase inhibitors, andbrimonidine, which are commercially available in many countries.Little information is available on the safety and tolerance of fixedcombinations compared to their unfixed counterpart. As expected,it appears that the fixed combinations have a better safety profilethan the unfixed association (Hommer, 2007), but all the studieswere conducted in selected patients for a short duration and maynot reflect the actual effects of reducing preservatives. We con-ducted a preliminary in vitro toxicological study aimed atcomparing associations of eye drops, which raises technical issuesand explains the scarcity of such evaluations, confirming the lowerproapoptotic and oxidative effects of fixed combinations of timolol/

brimonidine and timolol/bimatoprost compared to their unfixedcounterparts (Mehana, submitted for publication).

Nevertheless, the only way to totally eliminate BAK-related sideeffects, especially in the most sensitive patients, would obviouslybe to remove BAK from eye drops; however, this raises industrialand regulatory concerns. Single-dose units are the most frequentlyused preservative-free preparations and depending on the country,some compounds are available, mainly beta-blockers, mostlybecause they came first to generics and could easily be developed.However, several drawbacks have been pointed out regardingsingle-dose units, such as higher cost and difficult handling, espe-cially in elderly patients. Multidose bottles have therefore beendeveloped, either by allowing preservative filtration through andadsorption on a porous membrane or by using a valve system thathinders penetration of bacteria into the bottle. The ABAK� (Labo-ratoires Théa, France) and COMOD� (Ursapharm, Germany)systems have thus been patented and commercialized with variousbeta-blockers such as timolol, carteolol, and nonantiglaucomatouscompounds (Meloni et al., 2009; Pisella et al., 2000; Teping andWiedemann, 1994). Clinically, all switch studies have confirmedthat removal of BAK substantially benefitted the patient’s ocularsurface (de Jong et al., 1994; Jaenen et al., 2007; Kuppens et al.,1995; Pisella et al., 2002).

More recently, an unpreserved fixed combination of timolol anddorzolamide (Merck, Sharpe & Dohme, NY) and a prostaglandinanalog, tafluprost (Santen, Japan), have been developed andapproved in several countries.

9.2. Is the active compound neutral or does it interact with thepreservative?

Given that very few compounds have been developed inpreservative-free formulations, most of the information availableon the possible interactions with the active compounds is availablebased on in vitro or experimental studies. Almost all currentlyavailable data have clearly confirmed that beta-blockers have notoxic effects on their own (Baudouin et al., 1999; De Saint Jean et al.,2000; Pisella et al., 2000, 2004). The same absence of toxic effectswas confirmed both in vitro and in an experimental acute challengein rabbit eyes with preservative-free tafluprost, which showed nosignificant clinical, histopathological, apoptotic, or inflammatoryeffect (Brasnu et al., 2008a; Liang et al., 2008a).

However, in addition to the simple neutrality of preservative-free compounds, a possible specific effect of prostaglandin analogshas been proposed in several in vitro assays. Indeed, in a conjunc-tival cell line we found that two preservative-containing prosta-glandins, latanoprost and travoprost, appeared slightly butconsistently less toxic than their BAK counterpart, througha protective mechanism perhaps related to the antioxidant prop-erties of prostaglandins (Guenoun et al., 2005a). These results wereconfirmed by Yu et al. in cultured human trabecular cells byshowing a reduced effect of BAK in the presence of prostaglandinanalogs, through antioxidative effects that would counteract thepro-oxidative and toxic properties of the preservative (Yu et al.,2008).

Another specific positive effect of the prostaglandin agonistcould target goblet cells that are known in other systems to bestimulated by prostaglandin derivatives. In the human eye, inter-esting studies using impression cytology have shown a relativelylower decrease in goblet cell density compared to preservedtimolol, despite a higher concentration of BAK in the prostaglandinsolution (Pisella et al., 2004), or a transient increase in goblet celldensity after a short duration of treatment before goblet celldensity decreased after longer periods of treatment, likely as


a consequence of the delayed cytotoxic effects of the preservativeover the long-term (Moreno et al., 2003).

Therefore, when administering a drug that contains both a toxicand an antitoxic compound, the final result may be very confusing,especially since the prostaglandin in all experiments was not ableto totally counteract the detrimental effects of BAK. This wouldfurther argue in favor of definitively eliminating the toxiccompound in order to better evaluate the other properties of theactive drug besides their IOP-reducing effect.

9.3. Vehicle/preservative interactions

To reduce the impact of the preservative, several attempts havebeen made to provide vehicles that would interact with BAK tolimit its toxic effects. Although no data are clinically available, invitro studies showed that carbomer and hyaluronates, two widelyused compounds in tear substitutes, can decrease BAK toxicitycompared to the solution. In Chang conjunctival cells, Debbaschet al. investigated the cytotoxicity of low concentrations of unpre-served or preserved carbomer 934P (0.03% and 0.3%), unpreservedor preserved hyaluronic acid (0.018% and 0.18%), and BAK (0.0005%and 0.005%) for 15 min or for 15 min with 24 h of cell recovery,using cold light fluorimetry and confocal microscopy (Debbaschet al., 2002). The authors found that cytotoxicity of the two tear

Fig. 12. Immunostaining of inflammatory (CD45, green staining, lines 1 and 2) and apoptoticinstillations of an ophthalmic solution (15 instillations in 90 minutes). A. PBS; B. BAK SolutioEmulsion, at Day 1. Nuclei are stained in red with propidium iodide. Immunohistology cinflammatory cells and TUNELþ apoptotic cells infiltrating the limbus and conjunctival arealess inflammatory/apoptotic cells. Only occasional inflammatory/apoptotic cells were foundthat ocular surface toxicity was reduced by using an emulsion instead of a traditional solut

substitutes was significantly lower than that observed with BAKalone, although the same concentrations of preservative were used.Moreover, these two ophthalmic hydrogels seemed to possessantioxidative properties. Consistent with these results, in a morerecent study, high-molecular-weight hyaluronan was able todecrease the toxic effects of BAK in epithelial cell lines, reducingboth apoptosis and the oxidative effects of BAK, through an inter-action between BAK and the vehicle or by blocking cell deathreceptors at the cell membrane level (Pauloin et al., 2008).

Another approach to reducing BAK cytotoxicity is to combinethe quaternary ammoniums that are highly lipophilic and posi-tively charged in an emulsion of lipids. Recent in vivo studies con-ducted in rabbits in our laboratory have demonstrated the roleplayed by cationic emulsions in reducing ocular cytotoxicityinduced by quaternary ammonium-containing solutions (Lianget al., 2008b). Repeated instillations of a cationic emulsion formu-lation containing 0.02% BAK on the ocular surface of the rabbitinduced fewer changes of the ocular surface microstructures anda lower expression pattern of inflammatory markers than did 0.02%BAK in solution. Likewise, the same model also demonstrated thatcationic emulsions with 0.002% cetalkonium, another quaternaryammonium, were not toxic for the ocular surface and displayedfindings quite similar to those found in the control group (Fig. 12)(Liang et al., 2008b). Moreover, compared with traditional

(TUNEL, green staining, lines 3 and 4) markers in rabbit eye cryosections after repeatedn (Sol); C. BAK Cationic Emulsion (Em); D. Cetalkonium solution (CKC Sol); and E. CKClearly showed that repeated instillations of BAK solution induced numerous CD45þs. Comparatively BAK Em and CKC Sol induced moderate infiltrates, with significantlyafter CKC Em or PBS instillations. These experimental approaches clearly demonstratedion. The scale bars indicate 100 mm (adapted from Liang et al., 2008b).


solutions, the emulsion optimized the ocular surface homeostasisby its oily properties, since the emulsion alone was developed asa tear substitute for dry eye symptoms (Cationorm�, NovagaliPharma SA, France). Since lipid compounds have been proposed astear substitutes with good relief of patient symptoms and signs, thisapproach to improving the vehicle can be proposed for glaucomapatients, especially for those suffering from ocular surface diseasessuch as dry eye or allergy. Indeed, an emulsion of latanoprost wasfound not to be toxic in the acute challenge model in rabbitscompared to BAK-containing prostaglandin analogs (Liang et al.,2009).

9.4. Non-/less toxic preservatives

Extensive research has been conducted to discover and developless toxic preservatives than BAK and quaternary ammoniums.However, since a preservative must be a potent antimicrobial agentwhile not being cytotoxic, only very few agents have been proposedand are commercially available. Noecker and colleagues investi-gated in rabbit eyes the effects of Purite�, a stabilized oxychlorocomplex, compared with topical BAK-containing antiglaucomamedications. After 30 days, both in the cornea and the conjunctiva,BAK-containing dorzolamide, timolol, and latanoprost producedsignificantly more changes than did artificial tears containingPurite� and brimonidine Purite� (p¼ 0.001). In this study, treat-ment with glaucoma medications that contained higher levels ofBAK resulted in greater damage of the cornea and conjunctivacompared with preparations preserved with lower concentrationsof BAK, consistent with all previous studies conducted by our group

Fig. 13. Conjunctival impression cytology specimens of a rat model after application of BSS (polyquaternium-1 (PQ) at day 3 (D3), day 8 (D8) and day 11 (D11). Confocal microscopy imagreen. Impression cytology of control eyes showed no morphological anomaly. Impression cyand size, and irregular epithelial cells. Concerning the 0.25% and 0.5% group, impression cyobserved. In contrast impression cytology specimens of rats instilled with 0.5% PQ showed refrom Labbe et al., 2006b).

(Noecker et al., 2004). Purite� was also shown to be effective inenhancing penetration of the active compound brimonidine intoaqueous humor (Dong et al., 2004), which reduced the brimonidineconcentration from 0.2% to 0.15% without significant loss of IOP-reducing effects. Clinically, the two changes e moving from BAK toPurite� and reducing brimonidine concentration e resulted insignificantly better tolerance, especially in irritated eyes (Mundorfet al., 2003).

Another approach has been developed for avoiding BAK asa preservative in a prostaglandin analog solution, travoprost, whichexists both preserved with BAK and with a new preservativesystem, Sofzia�, composed of boric acid, propylene glycol, sorbitol,and zinc chloride. In randomized clinical trials, BAK-free travoprostreduced IOP similarly to BAK-containing travoprost, with a slightreduction of hyperemia (Lewis et al., 2007), thus further demon-strating that BAK is not absolutely mandatory to obtain appropriatepenetration of the active compound into the anterior chamber. Wecarried out a toxicological study using our previously validatedprotocols in conjunctival cells and confirmed that BAK-free trav-oprost induced significantly less apoptosis and fewer alterations ofcell viability and membrane integrity than did BAK-containinglatanoprost or travoprost (Baudouin et al., 2007). In animal models,a once-daily application in the rabbit eye also caused significantlyless corneal epithelium damage as assessed by transmission elec-tron microscopy and less conjunctival lymphocyte infiltration thanlatanoprost preserved with BAK, with no differences with thecontrol eyes receiving artificial tears (Kahook and Noecker, 2008).The same authors also compared changes in the number of gobletcells after chronic exposure to latanoprost preserved with 0.02%

Balanced Salt Solution), 0.1% benzalkonium chloride (BAC), 0.25% BAC, 0.5% BAC or 0.5%ges after immunostaining (�200); epithelial cell nuclei appear in red and goblet cells intology of rats instilled with 0.1% BAC showed goblet cells that were reduced in numbertology samples showed many irregular and small nuclei, almost no goblet cells weregular epithelial cells with numerous goblet cells. The scale bar indicates 50 mm (adapted


BAK to travoprost preserved with Sofzia� or preservative-freeartificial tears and found a dramatic decrease of goblet cells in theBAK-receiving group (2.21�0.40 per high-power field), contrastingwith the values found in the Sofzia� group, which were not onlynonsignificantly different from those found in the control group(6.02�1.20), but even slightly higher after only 1 month(7.03�1.33, nonsignificant), which may corroborate a possiblestimulatory effect of the prostaglandin analog to goblet cells(Kahook and Noecker, 2008). In an open-label study, 691 patientstreated with latanoprost or bimatoprost switching to BAK-freetravoprost due to tolerability issues demonstrated significantimprovement of ocular surface symptoms and hyperemia, withoutreducing or even improving IOP control (Henry et al., 2008).

A third candidate as a preservative that could be a less toxicalternative to BAK is polyquaternium, a family of polycationicpolymers that are used in the personal care industry. At least 37different polymers exist under the polyquaternium designation butpolyquaternium-1 is commonly used as amultipurpose solution forcontact lens care and showed a good safety and tolerance profilecompared to other multipurpose solutions (Lipener, 2009). Thiscompound is currently developed in eye drops as an alternative toBAK. Our group therefore tested this compound in rats and useda set of toxicological tools including clinical and histopathologicalcriteria, and IVCM. Except at a very high concentration of 0.5%,polyquaternium-1 (Polyquad) did not cause significant changes inthe ocular surface compared to saline, whereas BAK induced majortoxic effects at mild to high concentrations, with a dramatic andextremely rapid destruction of goblet cells (Fig. 13) (Labbe et al.,2006b). Interestingly, it should be noted that Polyquad is used asa disinfectant in multipurpose contact lens solutions and asa preservative in eye drops at a very low concentration, namely0.001%, instead of the standard concentrations of BAK that rangebetween 0.004 and 0.025% in commercially available eye drops.

10. Conclusion

Over more than two decades, a huge number of animal and invitro studies, using a considerable variety of models, cells, andtissues, have demonstrated that BAKmay cause or enhance harmfulconsequences on the eye structures of the anterior segment,including the tear film, cornea, conjunctiva, and even trabecularmeshwork. Despite these consistent and solid data, and warningscoming from observational surveys and individual case series, BAKis still used as the main preservative in eye drops and very fewalternatives have been developed. Paradoxically, whereas BAK usehas dropped off in the therapeutic area of dry eye disease in the lastdecade, BAK issues are still highly underestimated in glaucoma caredespite the well-known but unexpectedly high prevalence of dryeye in the glaucoma population, most likely in relation to theprolonged use of topical medications for very long periods of time.Obviously, glaucoma is a severe, sight-threatening diseasedeserving thorough treatments, but the ocular surface side effectsof antiglaucoma medications should not be neglected because theymay deeply impact patients’ quality of life, compliance, or latersurgical outcome.

Reducing the exposure to preservatives may lessen the adverseevents, which could lead to better tolerability, lower treatmentdiscontinuation, and higher adherence in patients treated withantiglaucomamedications. This in turnwould improveoutcomes forthese patients, both in terms of glaucoma management and theirquality of life,whichmayalso contribute to a lower cost of long-termglaucoma complications. Reversing the limits of tolerance shouldalso become one of the key objectives of glaucoma therapy. There-fore, for patientswhoarehighly sensitive topreservatives becauseofpreexisting or concomitant ocular surface diseases, for patients

receiving combinations of two or more drugs, for those who are atrisk of undergoing surgery, and finally for all patients whowill needtreatments over several decades, preservative-free formulationswould provide clinically relevant benefits and should become a goldstandard in ocular pharmacology in the near future.

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Progress in Retinal and Eye Research - Shroomery · 2013. 7. 12. · The Pharmacopoeia recommends that eye drops must contain an antimicrobial agent (preservative) to avoid or to