Review Article. Detrimental effect of preservatives in eyedrops: implications for the treatment of glaucoma. Introduction. Christophe Baudouin

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1 Review Article Detrimental effect of preservatives in eyedrops: implications for the treatment of glaucoma Christophe Baudouin Department of Ophthalmology III, Quinze-Vingts National Ophthalmology Hospital, Paris, France ABSTRACT. Antiglaucoma medications are often associated with ocular adverse reactions such as dry eye, and burning or stinging sensations. These undesirable effects may lead to treatment discontinuation and reduced quality of life in patients with glaucoma. Antiglaucoma medications usually contain benzalkonium chloride (BAK) as a preservative. Animal studies, in vitro studies and in vivo experiments have demonstrated various adverse effects of BAK. Clinical studies have also shown an increased incidence of adverse events with BAK and have demonstrated that the withdrawal of preservatives reduces these effects. Collectively, these data suggest that preservative-free antiglaucoma treatments have clinically relevant benefits for patients. Key words: antiglaucoma medications benzalkonium chloride glaucoma preservatives Acta Ophthalmol. 2008: 86: ª 2008 The Author Journal compilation ª 2008 Acta Ophthalmol doi: /j x Introduction Glaucoma is characterized by optic neuropathy, as determined by both structural changes and functional deficits. Glaucoma can be subdivided into primary open-angle glaucoma (POAG), which has an insidious and slow onset, and angle-closure glaucoma (ACG), which is usually more acute (World Health Organization 2007a). Quigley & Broman (2006) estimate that 60.5 million people worldwide will have the disease by 2010 and this number will increase to about 80 million by Approximately 75% of these patients will have POAG. Glaucoma is the second leading cause of blindness in the world and is predicted to account for over 8 million cases of blindness by 2010 rising to over 11 million by 2020 (Quigley & Broman 2006). If the disease is diagnosed at an early stage, medical, surgical or laser treatment can be effective (van der Valk et al. 2005; World Health Organization 2007a). Medical treatment is predominantly used as first-line therapy, and most patients with chronic glaucoma are treated medically (Lindblom 2006). If effective, medical treatment is administered over the longterm, and therefore the majority of patients receive several decades of treatment. Based on data from clinical trials, the tolerability of glaucoma treatments seems satisfactory: few patients are withdrawn from medication as a result of local intolerance or allergy. These results provide reassurance that longterm medical antiglaucoma treatments are not harmful and can be used throughout the patient s lifespan. However, there are several major differences between clinical trials and the real-world progress of antiglaucoma therapy. Clinical trials are usually of short duration (6 months 1 year), and patients usually only receive one test or reference therapy. Patients with known hypersensitivity to the therapy or to the preservative contained within the product, and patients who have active ocular surface diseases such as dry eye, chronic allergy or severe blepharitis are often not included in such trials (Watson & Stjernschantz 1996; Fellman et al. 2002; Manni et al. 2004). In population-based studies, the prevalence of dry eye in elderly patients (aged 65 years) varies between 15% and 34% (Smith et al. 2007). Impaired tear film may therefore interfere with topical treatments in a high proportion of patients, as the ocular surface disease may be encouraged by the drug(s) and or preservatives, and may also reduce the resistance of the cornea and conjunctiva to the presence of toxic or irritant compounds. 716

2 Another major difference with clinical trials is the number of drugs chronically administered to the ocular surface. In clinical practice, patients with glaucoma are treated for a lifetime. Therefore, because of the progressive nature of the disease, a significant majority of patients receive multiple therapies concomitantly. For example, the Ocular Hypertension Treatment Study demonstrated that approximately 40% of patients initially diagnosed with ocular hypertension were using more than one drug after 5 years (Kass et al. 2002), and the Collaborative Initial Glaucoma Treatment Study showed that 75% of patients were using two or more drugs within 2 years of initial glaucoma treatment (Lichter et al. 2001). This cumulative use of polytherapy over time is likely to increase the incidence of adverse events. Therefore, there are likely to be discrepancies between the outcomes observed in clinical trial settings and those seen in longterm treatment, with a higher incidence of side-effects expected in clinical practice. Tolerability issues associated with current antiglaucoma medications Clinical symptoms Ocular surface changes, including burning or stinging sensations, dry eye and tearing, have been described after the longterm administration of topical antiglaucoma agents (Pisella et al. 2002). For example, a prospective epidemiological survey was conducted in 2002 in 4107 glaucoma patients to assess the effects of preserved and preservative-free eyedrops on ocular symptoms and conjunctival, corneal and palpebral signs during clinical practice (Pisella et al. 2002). All symptoms were significantly more prevalent in patients using preserved drops compared with those using preservative-free treatment. For example, discomfort upon instillation (43% in the preserved group versus 17% in the non-preserved group), burning or stinging sensations (40% versus 22%, respectively), foreign body sensation (31% versus 14%, respectively), dry eye (23% versus 14%, respectively), Patients with symptoms (%) (A) Patients with symptoms (%) (B) Preservative-containing antiglaucoma medications Preservative-free antiglaucoma medications Pain/ discomfort during instillation Pain/ discomfort during instillation Foreign body sensation Foreign body sensation Stinging/ burning sensation Stinging/ burning sensation tearing (21% versus 14%, respectively) and itchy eyelids (18% versus 10%, respectively) were consistently higher with preserved eyedrops. The prevalence of signs and symptoms was dose-dependent, increasing with the number of preserved eyedrops used. Furthermore, when patients were either switched to preservative-free drugs or given less preservative-containing drugs, all symptoms and signs improved (Pisella et al. 2002). Similar data were obtained when equivalent studies were performed in Italy, Belgium and Portugal (Jaenen et al 2007). Although there were some minor differences between the countries, pooled data from a total of 9658 patients also demonstrated that the incidence of ocular signs and symptoms was significantly (p < ) higher in patients receiving preserved eyedrops, and that the incidence of these signs and symptoms decreased significantly (p < ) by switching to a preservative-free formulation or by reducing the amount of preservative-containing treatment (Figs 1 and 2) (Jaenen et al. 2007). Based on these data, it seems that the use of ophthalmic solutions containing preservatives can cause an increase in ocular surface changes manifesting as adverse events. Impact of symptoms on treatment compliance Discomfort following instillation of eyedrops may lead to patient discontinuation of treatment. In a small study of 83 patients adverse effects was listed as the third most common reason for non-compliance (after forgetting to administer the dose and inappropriately administering the treatment) (Chawla 2007). In a larger survey of patients and 103 ophthalmologists, Zimmerman et al. (2007) showed that approximately half the patients (55.4%) discontinued their index medication within the first 90 days. Prostaglandin-treated patients who identified adverse events as a Dry eyes Dry eyes Tearing Tearing Eyelid itching Preservative-containing antiglaucoma medications Decreased number of preservative-containing antiglaucoma medications Eyelid itching Fig. 1. The incidence of ocular symptoms in (A) patients who switched from preservative-containing to preservative-free antiglaucoma medications and (B) patients who decreased their exposure to preservative-containing antiglaucoma medications; p < for all comparisons. (Data from Jaenen et al. 2007). 717

3 Patients with signs (%) (A) Patients with signs (%) (B) Preservative-containing antiglaucoma medications Preservative-free antiglaucoma medications Anterior Posterior blepharitis blepharitis Anterior Posterior blepharitis blepharitis significant problem were more likely to have poorer adherence to medication (p = 0.04). Almost all physicians (97%) perceived side-effects as a barrier to adherence (Zimmerman et al. 2007). Impact of symptoms on quality of life The side-effects of antiglaucoma treatment can also have an impact on patient quality of life. Nordmann et al. (2003) investigated the quality of life associated with topical glaucoma treatment in 204 randomly selected patients with glaucoma or ocular hypertension. Almost 93% of the patients experienced at least one side-effect. Among these, 25.4% experienced a burning sensation, 20.8% blurred vision and 20.2% tearing. Poor vision-related quality of life was associated with the presence of local side-effects leading to poor patient satisfaction and reduced adherence. Dissatisfied patients also visited their ophthalmologists more frequently (3.50 visits per year) compared with patients who were at least rather satisfied (2.27 visits per year) (Nordmann et al. 2003). Eczema Hyperaemia Follicles Eczema Hyperaemia Follicles FluoresceinSuperficial staining punctate keratitis Preservative-containing antiglaucoma medications Decreased number of preservative-containing antiglaucoma medications Fluorescein Superficial staining punctate keratitis Fig. 2. Incidence of ocular signs in (A) patients who switched from preservative-containing to preservative-free antiglaucoma medications, and (B) patients who decreased their exposure to preservative-containing antiglaucoma medications; p < for all comparisons. (Data from Jaenen et al. 2007). Longterm exposure to antiglaucoma medications, and especially to the preservatives they contain, can lead to the condition of dry eye. Preservatives decrease the stability of the tear film (Detry-Morel 2006). Miljanović et al. (2007) studied the impact of dry eye syndrome on quality of life as part of the Women s Health Study and Physicians Health Study (450 women, 240 physicians) and concluded that dry eye syndrome is linked to a measurable adverse impact on quality of life. Effects on surgery outcomes There have been several reports of longterm topical combination therapy causing the failure of glaucoma surgery (Broadway et al. 1994a, 1994b). The effect of different longterm antiglaucoma treatments on the results of glaucoma filtration surgery were studied by Broadway et al. (1994b). A total of 124 patients underwent filtration surgery and were followed for 6 months. Subjects were divided into four groups: patients who received minimal topical therapy (group 1); patients on beta-blockers (group 2); patients receiving betablockers and miotics (group 3), and patients taking beta-blockers, miotics and sympathomimetics (group 4). The surgery success rates in these groups were 90% (group 1), 93% (group 2), 72% (group 3) and 45% (group 4). Surgical outcomes in patients who had received beta-blockers and miotics (group 3) or all three medications (group 4) were significantly less successful than in those treated with minimal topical therapy (p < 0.01 and p < 0.001, respectively). All treatments used in the study contained preservatives. Given the differences in effects on the conjunctiva in the combination therapy arms (groups 3 and 4) compared with those in the single or limited therapy arms (groups 1 and 2), the authors speculated that the degree of exposure to preservatives may also contribute to effects on the conjunctiva (Broadway et al. 1994b) and therefore likely to impact on the success of filtration surgery. Longterm therapy induces several adverse effects such as the inflammation of the conjunctiva with a consequent lower success rate for any filtration surgery. The failure rate of trabeculectomy was also significantly correlated to longterm (> 1 year) topical antiglaucoma therapy (p < 0.001) (Lavin et al. 1990). Topical medications may thus exert adverse effects on the conjunctiva, which result in excessive time taken in postoperative wound healing and further fibrosis (Broadway et al. 1994b). Therefore, the use of topical antiglaucoma drugs can be considered one of the risk factors for failure of filtration surgery, and accurate investigation of drug-induced inflammation of the conjunctiva should be used as a tool to classify patients at high risk of filtration failure (Broadway & Chang 2001). The use of longterm antiglaucoma medications is related to conjunctival foreshortening and shrinkage, which may be associated with an ocular pemphigoid-like condition or evolve into severe scarring conjunctivitis with definitive corneal opacities (Schwab et al. 1992). Other effects related to cataracts The use of antiglaucoma medications has also been associated with the development of cataracts. The Blue 718

4 Mountains Eye Study showed that antiglaucoma therapy can increase the risk of cataract formation (odds ratio [OR] 1.90, 95% confidence interval [CI] ) (Chandrasekaran et al. 2006). Longterm followup in the Ocular Hypertension Treatment Study demonstrated an increased rate of cataract extraction and cataract filtration surgery in patients receiving medication (7.6%) compared with the observation group (5.6%) (hazard ratio 1.56, 95% CI ) (Herman et al. 2006). Furthermore, the use of preserved timolol was associated with a higher incidence of cystoid macular oedema after cataract surgery compared with preservative-free timolol (Miyake et al. 2003). Impact on cost Preservative-containing prostaglandin analogues lower intraocular pressure (IOP) and are widely used to treat POAG. Although these medications are generally well tolerated, their use is associated with topical side-effects (Noecker et al. 2003; Parrish et al. 2003). According to a survey by Stewart et al. (2002a), these side-effects lead to additional telephone calls, more office visits and increased drug discontinuation. This is in line with the findings of Nordmann et al. (2003) and Zimmerman et al. (2007), who found that side-effects caused by antiglaucoma medications containing preservatives can lead to additional costs and resource use. However, to our knowledge, the cost-effectiveness analyses performed to date comparing the various antiglaucoma therapies have not incorporated the cost of managing side-effects into their calculations (Stewart et al. 2002b; Costagliola et al. 2003). In summary, the administration of preserved antiglaucoma medications is associated with significant ocular adverse effects. These effects cause discomfort and pain, lead to the premature discontinuation of treatment, result in a lower quality of life, and impact the outcome of eventual glaucoma surgery. Furthermore, although no formal cost-effectiveness analyses are available, we would speculate that these ocular effects are also likely to be associated with an overall increased cost of therapy. Toxicity or allergy? The adverse events observed with the use of antiglaucoma medications may result from either an allergic or a toxic reaction. Although allergy can occur in a small proportion of patients, toxicity is probably the predominant cause. The side-effects may be caused by either the active compound of the antiglaucoma medication, or, as is more likely, the preservatives included within the formulation. Toxicity of preservatives: animal models The most common preservative in antiglaucoma medications and other topical ophthalmic preparations is benzalkonium chloride (BAK). This is a quaternary ammonium compound composed of a mixture of alkylbenzyldimethylammonium chloride homologues with n-c 12 H 25, n-c 14 H 29 and n-c 16 H 33 comprising a major portion of the alkyl groups present (United States Pharmacopeia National Formulary [USP NF] 2005). It is commonly used at concentrations of 0.004)0.025%. Several investigations using animal models have suggested the existence of links between BAK and cytotoxic effects on several components of the eye. The key studies are outlined here and a full list of references is provided in Table 1. Tear film The tear film is important because it functions as both a lubricating and protective layer. The cornea holds the tear film on its surface, probably with the aid of mucin-producing goblet cells throughout the conjunctiva (Lemp et al. 1970). Over 30 years ago, Wilson et al. (1975) demonstrated that 0.01% BAK hastened the drying of the precorneal tear film in rabbits. The compound shortened the length of time taken for dry spots to appear on the corneal surface by a factor of about four (Wilson et al. 1975). More recently, Pisella et al. (2000) found that albino rabbits given a preserved beta-blocker (Timoptol Ò 0.25% and 0.50%, preserved with 0.01% BAK; MSD-Chibret, Paris, France) displayed a significantly greater reduction in tear film break-up time compared with those given a non-preserved beta-blocker (Timabak Ò 0.25% and 0.50%; Laboratoires The a SA, Paris, France). Through the loss of the protective layer, an impaired tear film will cause dry eye symptoms and corneal damage, and also convey cytotoxic inflammatory mediators throughout the ocular surface (Baudouin 2001). Conjunctiva and cornea Using a rabbit model, Furrer et al. (2001) demonstrated that 28 days of treatment with beta-blockers preserved in BAK 0.01% or benzododecinium bromide 0.012% resulted in microlesions covering nearly 9% of the corneal surface. Administration of solutions containing BAK 0.01% has been linked to the infiltration of immunocompetent cells into the limbus and bulbar conjunctiva in rats (Becquet et al. 1998). The inflammatory reaction was associated with severe damage to the cornea and conjunctiva, including epithelial alterations and keratinization. Several other animal studies have shown that preservatives are linked to the onset of chronic fibrosis in the conjunctiva (Mietz et al. 1994, 1997; Noecker et al. 2004). Noecker et al. (2004) investigated the extent of epithelial and corneal damage associated with Purite Ò (Allergan, Inc, Irvine, California, USA), a stabilized oxychloro complex, and with topical antiglaucoma medications preserved with BAK. Fifteen New Zealand white rabbits were randomized to six treatment groups: artificial tears (Refresh Tears Ò, carboxymethyl cellulose 0.5% twice daily; Allergan, Inc.); brimonidine Purite Ò 0.15% twice daily; bimatoprost 0.03% once daily; dorzolamide 2% twice daily; timolol maleate 0.5% twice daily, or latanoprost 0.005% once daily, for 30 days. The bimatoprost, dorzolamide, timolol and latanoprost solutions contained BAK at concentrations of 0.005%, 0.008%, 0.001% and 0.02%, respectively. The dorzolamide, timolol and latanoprost groups experienced significantly more changes in both the cornea and the conjunctiva than did the groups with artificial tears (containing Purite Ò ) and brimonidine Purite Ò (p = 0.001). 719

5 Table 1. Summary of studies investigating the effects of different antiglaucoma therapies on the various components of the eye. Tear film Conjunctiva Cornea Trabecular cells Animal studies Wilson et al Young et al Burnstein 1980 Chou et al Pisella et al. 2000, 2002 Mietz et al. 1994, 1997 Dormans & van Logten 1982 Baudouin et al Becquet et al Lo pez Bernal & Ubels 1991 Baudouin et al Ichijima et al Noecker et al Imayasu et al Ito et al Becquet et al Baudouin et al Furrer et al. 1999, 2001 Noecker et al Human cell lines (in vitro) Takahashi 1982 Samples et al Samples et al De Saint Jean et al. 1999, 2004 Saarinen- Savolainen et al. Hamard et al. 2002, Debbasch et al. Ito et al a, 2001b Pisella et al Yee et al Guenoun et al. 2005a, 2005b Human cells (ex vivo) Broadway et al. 1994a Baudouin et al Baudouin 1996 Baudouin et al. 1994, 1999, 2004 Pisella et al Aritu rk et al Nuzzi et al Sherwood et al Albietz & Bruce 2001 Dogan et al Hong et al Clinical studies Wilson et al Schwab et al Lemp & Zimmerman 1988 Herreras et al Levrat et al de Jong et al Kuppens et al Jaenen et al Levrat et al Yalvaç et al Albietz & Eleftheriadis et al Bruce 2001 Nuzzi et al Kozobolis et al Baudouin & Jaenen et al de Lunardo 1998 Kozobolis et al Manni et al Treatment with glaucoma medications that contained higher levels of BAK resulted in greater damage to the cornea and conjunctiva compared with treatment with preparations preserved with lower concentrations of BAK. Bimatoprost, containing the lowest concentration of BAK, was associated with significantly less corneal and conjunctival damage (Noecker et al. 2004). Other eye structures In addition to damaging the cornea and conjunctiva, preservatives may also cause severe lesions in the retina. In pigmented rabbits, subconjunctival injection of timolol 0.5% or befunolol 1% preserved with BAK caused retinal lesions, retinal detachment, loss of visual acuity and atrophy of the pigmented epithelium of the retina and choroids (Chou et al. 1985). Toxicity of preservatives: human cell lines Many studies have also demonstrated the effects of BAK on human tissuederived cells cultured in vitro. The key studies are outlined here and a full list of references is provided in Table 1. Conjunctival cells De Saint Jean et al. (1999) studied the effect of different concentrations of BAK ( %) on a continuous human conjunctival cell line: the Wong Kilbourne derivative of Chang conjunctiva. Cells were treated for 10 mins and were assessed before treatment and at 3, 24, 48 and 72 hours after treatment. Benzalkonium chloride at concentrations of 0.1% and 0.05% caused immediate cell lysis. Exposure to 0.01% BAK was associated with cell death within 24 hours, and doses of % induced apoptotic cell death at 24)72 hours in a dose-dependent manner. These findings suggest that BAK at concentrations as low as % causes cell death, and that cell destruction is BAK concentrationdependent (De Saint Jean et al. 1999). Pisella et al. (2004) compared the toxicities of 0.005% latanoprost preserved with 0.02% BAK, 0.5% timolol preserved with 0.02% BAK, unpreserved 0.5% timolol and 0.02% BAK alone on the Wong Kilbournederived human conjunctival cell line. Cells were treated for 15 mins and subsequently left to recover for 0, 4 and 24 hours in a normal medium. Both latanoprost and timolol were associated with toxic proapoptotic effects on conjunctival cells, whereas no toxic effect was observed with unpreserved timolol. Both medications were less toxic than BAK alone. These findings suggest that the preservative itself causes the damage, and that preservative alone is even more harmful than preservatives within medications (Pisella et al. 2004). Trabecular and corneal cells Benzalkonium chloride at a concentration of 0.002% inhibits the growth of human trabecular cells and corneal cells in vitro (Samples et al. 1989). Studies performed on human immortalized trabecular cells have shown that even a short exposure (15 mins) 720

6 to BAK % induced apoptosis. The fact that betaxolol preserved with % BAK showed a moderate proapoptotic effect, whereas unpreserved betaxolol did not display any apoptosis, further supports the BAK-dependence of cellular apoptosis (Hamard et al. 2002). These results confirm that BAK alone can trigger apoptosis in more than 95% of healthy cells and glaucoma donorderived trabecular cell lines (Hamard et al. 2003). Lens cells Goto et al. (2003) investigated the effects of latanoprost, timolol maleate and BAK on the secretion of chemical mediators of stress and wound healing in a human lens epithelial cell line. The results showed that BAK alone caused the most damage by strongly stimulating the expression of soluble chemical mediators of stress (prostaglandin E2, interleukin-1a and interleukin 6) in lens epithelial cells. These effects were also observed with BAK-containing latanoprost or timolol formulations, but to a lesser extent than with BAK alone (Goto et al. 2003). In summary, evidence from several animal studies and human cell line experiments supports the hypothesis that antiglaucoma therapies containing the preservative BAK are associated with various adverse reactions on surface and deep ocular tissues. Toxicity of preservatives: evidence from clinical studies Evidence from animal studies and human tissue cell culture experiments, which suggests that preservatives can cause detrimental effects on the superficial ocular tissues, is supported by evidence from clinical studies (Wilson et al. 1975; Lemp & Zimmerman 1988; Sherwood et al. 1989; Herreras et al. 1992; Baudouin et al. 1994; Broadway et al. 1994a; de Jong et al. 1994; Kuppens et al. 1995; Yalvac et al. 1995; A- ritu rk et al. 1996; Baudouin 1996; Baudouin & de Lunardo 1998; Nuzzi et al. 1998; Levrat et al. 1999; Albietz & Bruce 2001; Eleftheriadis et al. 2002; Dogan et al. 2004; Pisella et al. 2004; Kozobolis et al. 2005; Manni et al. 2005; Hong et al. 2006). The key studies are outlined here and a full list of references is provided in Table 1. It should be noted that at least one study in the literature showed no significant difference in the histologic parameters between 18 patients receiving at least 12 months of antiglaucoma treatments, when compared with 18 age matched controls (Banu 1995). Tear film Ocular surfaces of 20 patients with POAG receiving 0.5% timolol (0.01% BAK) or bi-therapy with 0.5% timolol plus 1% dipivefrin (0.04% BAK) were evaluated and compared with those of 20 healthy control participants receiving placebo (Kuppens et al. 1995). Schirmer s test values were significantly lower in the two treatment groups when compared with placebo (means ± 1.58 mm, 8.20 ± 1.55 mm and ± 2.21 mm, respectively; both p < 0.001), and tear film break-up times were significantly abnormal in both treatment groups (p < 0.001). This study suggests that the toxic effects on the tear film increase with the number of antiglaucoma therapies containing BAK administered (Yalvaç et al. 1995). A subsequent study corroborated the link between longterm use of antiglaucoma treatment drops containing preservatives and tear film modifications. A total of 132 subjects were assigned to one of the following groups: 29 patients with POAG were given timolol 0.5% twice daily (group A); 23 POAG patients received timolol 0.5% twice daily plus pilocarpine 2% three times daily (group B); 20 healthy participants were given BAK twice daily (BAK group), and 60 healthy recipients did not receive any therapy (group C). Schirmer s test resulted in average readings of ± 5.12 mm for group A, 9.08 ± 3.20 mm for group B, ± 3.40 mm for the BAK group, and ± 6.17 mm for group C. The differences between participants receiving treatment containing BAK or BAK alone compared with those not receiving therapy were significant (p < 0.05) (Nuzzi et al. 1998). Conjunctiva and cornea Longterm use of preservatives leads to corneal damage. The effect of topical timolol with and without BAK on the epithelial permeability and autofluorescence of the cornea has been investigated in patients with POAG or ocular hypertension. The corneas of 21 patients were examined during treatment with timolol preserved with BAK at concentrations of 0.25% or 0.5%. After 2 weeks, patients were switched to treatment with timolol without BAK. Corneal epithelial permeability decreased significantly (mean decrease per patient 27%; p = 0.025), whereas corneal autofluorescence increased significantly (mean increase per patient 6%; p = 0.003). These results suggested an improvement in corneal epithelial function following the withdrawal of BAK (de Jong et al. 1994). In line with these data, the results from the large survey showed that signs of damage to the conjunctiva, cornea and eyelids significantly decreased when patients were switched from preserved to preservative-free medication, or even when the number of eyedrops containing BAK was decreased. This effect demonstrated the dose-dependency of BAK-induced manifestations (Jaenen et al. 2007). Furthermore, numerous reports have shown that even without evident symptoms or clinical manifestations, inflammation is abnormally observed in the conjunctival epithelium. Infiltration of immunocompetent cells into the outer layers of the epithelium has therefore been reported (Sherwood et al. 1989; Liesegang 1998). All immuno-inflammatory markers and mediators of the conjunctival epithelium of patients with glaucoma were found to be significantly increased compared with healthy controls (Baudouin et al. 2004, 2007; Pisella et al. 2004). The intensity of this inflammatory reaction seems to be related to the number of antiglaucoma medications used, and the duration of treatment (Aritu rk et al. 1996). The human leucocyte antigen DR (HLA- DR) and the intercellular adhesion molecule-1 (ICAM-1) were both significantly higher in patients treated with preserved drugs compared with those treated with preservative-free therapies (Baudouin et al. 2004; Pisella et al. 2004). Patients who received preservative-free treatments showed mild or no inflammatory response (Fig. 3) (Baudouin et al. 2004; Pisella et al. 2004). Furthermore, the two chemokine 721

7 Fluorescence levels (ABC x 1000) receptors CCR4 and CCR5 were also found to be over-expressed in glaucoma patients receiving longterm treatment (Costagliola et al. 2001), suggesting that the chronic use of antiglaucoma topical drugs induces a complex network of inflammatory reactions. The conjunctival cytology specimens of patients on longterm antiglaucoma medication also have distinct characteristics, including epithelial keratinization, squamous metaplasia and a reduction in the number of goblet cells (Table 2) (Costagliola et al. 2001). Histomorphological changes to the epithelium can appear as early as 2 weeks after the start of antiglaucoma treatment (Lewis et al. 2007). Table 2. Histopathological changes linked with preserved antiglaucoma medications. Reproduced with permission from Liesegang, Cornea 1998;17: Histopathological changes Reduction of goblet cells Epithelial keratinization Squamous metaplasia Loss of microvilli Increased number of desmosomes Bullous dystrophy of the epithelium Increased number of subepithelial fibroblasts Subepithelial fibrosis Reduction of intravascular spaces Increased number of subepithelial lymphocytes and plasma cells Thickening of basal membrane Basement membrane staining for immunoglobulin * Normal Timolol without BAK Latanoprost Timolol with BAK HLA-DR ICAM-1 (CD 54) Fig. 3. Inflammatory markers with latanoprost, preserved and unpreserved timolol and normal controls. * p = 0.01 compared with normal controls; p compared with the other three groups. ABC = antibody-binding capacity; BAK= benzalkonium chloride; HLA- DR = human leucocyte antigen DR; ICAM-1 = intercellular adhesion molecule-1. (Reproduced with permission from Pisella et al. Investigative Ophthalmology & Visual Science, 2004; 45: (copyright of the Association for Research in Vision and Ophthalmology). Development of subconjunctival fibrosis without clinical signs of intolerance has frequently been documented in patients who have taken antiglaucoma medication for long periods of time (Baudouin et al. 1994). The fibrosis is believed to result from an increase in the fibroblast density in the subepithelial substantia propria, linked with an increase in inflammatory cells (Sherwood et al. 1989; Baudouin et al. 1999). This fibrosis, together with inflammatory infiltrates and cytokine release, plays a role in postoperative fibrotic scarring reaction and therefore contributes to surgical failure (Broadway et al. 1994b). Immunohistochemical analyses of conjunctival and trabecular surgical specimens from patients treated with antiglaucoma eyedrops revealed that samples from patients who were receiving treatment had significantly greater expression of fibroblastic and inflammatory markers compared with samples from non-treated participants. Furthermore, patients who received multiple therapies had a greater expression of markers compared with those on monotherapy (Baudouin et al. 1999). Effects of preservatives on safety: evidence from clinical trials Most studies imply a direct correlation between the presence of preservatives and the symptoms experienced during antiglaucoma therapy (Levrat et al. 1999; Pisella et al. 2002; Jaenen et al. 2007). In a recent study involving 9658 patients (Jaenen et al. 2007), all recorded symptoms and signs were significantly more frequent in patients taking preserved medications compared with those taking preservativefree formulations (p < for all). For example, foreign body sensation was experienced by 42% of the preservative recipients compared with 15% of those taking non-preserved medication, and stinging or burning was noted in 48% versus 20% of recipients, respectively, and dry eye sensation in 35% versus 16%, respectively. Jaenen et al. (2007) concluded that preservative-free eyedrops are significantly less likely to be associated with ocular symptoms and signs of irritation. These findings were similar to those seen in observational studies reported by Pisella et al. (2002) and Levrat et al. (1999). A significantly higher proportion of patients receiving preserved therapies experienced discomfort or pain during therapy compared with those treated with preservativefree formulations (58% versus 30%; p < 0.001) (Levrat et al. 1999). All signs and symptoms were significantly more prevalent in patients using preserved drops compared with those using preservative-free drops (p < 0.001) (Figs 1 and 2) (Jaenen et al. 2007). Furthermore, the effect was dose-dependent (Pisella et al. 2002). Preliminary short-term studies with preserved and preservative-free tafluprost, a novel prostaglandin analogue in development, have shown similar safety results (Hamacher et al. 2007; Uusitalo et al. 2007). In one of these studies, ocular hyperaemia was classified as predominantly moderate with preserved tafluprost and predominantly mild with preservative-free tafluprost (Uusitalo et al. 2007). Some studies have even demonstrated a direct correlation between the degree of damage to eye structures and clinical symptoms. Indirectly, Nuzzi et al. (1998) showed that the treatment groups associated with the greatest reductions in tear film (timolol plus pilocarpine and BAK) also displayed the highest frequencies of subjective symptoms. In more direct analyses, patients complaints have been correlated with objective signs of conjunctival damage (e.g. conjunctival 722

8 redness) and corneal damage (superficial punctuate keratitis) (Levrat et al. 1999; Jaenen et al. 2007). Furthermore, switching from a preservative-containing to a preservative-free formulation leads to a reduction of complaints regarding burning or dry eye sensation, reduces all signs of structural eye damage and objective clinical symptoms, and improves tolerance (Levrat et al. 1999; Albietz & Bruce 2001; Pisella et al. 2002; Dogan et al. 2004; Jaenen et al. 2007). Preserved versus preservative-free therapies: pharmacokinetics, pharmacodynamics and efficacy parameters Two recent clinical trials comparing the respective pharmacokinetic and pharmacodynamic profiles of preserved and preservative-free tafluprost demonstrated no significant differences in these parameters between the two formulations (Hamacher et al. 2007; Uusitalo et al. 2007). There were no significant differences between formulations with regard to pharmacokinetics parameters of AUC 0-last, C max and t max, and systemic bioavailability was comparable between groups (Hamacher et al. 2007). There is no difference in efficacy between preserved and preservativefree formulations (Liesegang 1998; Albietz & Bruce 2001; Hamard et al. 2003). In a trial using tafluprost, the estimated overall treatment difference (preservative-free versus preserved) in the reduction of IOP was 0.01 mmhg (95% CI ) 0.46 to 0.49; p = 0.96) (Hamacher et al. 2007). Results from a recent study demonstrated total equivalence in IOP control between two formulations of travoprost, one containing BAK and the other using an ionic buffered preservative system (Lewis et al. 2007). Easty et al. (2006) tested nonpreserved T-Gel 0.1% versus preserved T-Gel 0.1% and demonstrated that both formulations resulted in a mean IOP reduction of 24%. A comparative study of 2% carteolol with and without preservative confirmed similar efficacies between the two formulations and comparable reductions in IOP (Baudouin & de Lunardo 1998). Conclusions Animal studies and in vitro studies have demonstrated that preservatives such as BAK cause harmful effects to several eye structures, including the tear film, cornea, conjunctiva and trabecular cells. The detrimental effects of BAK are also recognized by the regulatory authorities. For example, the European summaries of product information for both bimatoprost and travoprost, both of which contain BAK, include the following text: Benzalkonium chloride has been reported to cause punctate keratopathy and or toxic ulcerative keratopathy, may cause eye irritation and is known to discolour soft contact lenses. Close monitoring is required with frequent or prolonged use of Lumigan Ò Travatan Ò in dry eye patients, or in conditions where the cornea is compromised. Benzalkonium chloride may cause eye irritation (Alcon, Inc. 2004; Allergan, Inc. 2006). The same warning has been issued for Xalatan Ò (Pfizer, Inc. 2006). Furthermore, data from clinical trials suggest that these adverse effects are correlated to the increase in clinical symptoms in patients treated with preservative-containing antiglaucoma medications. Reducing exposure to preservatives may reduce adverse events, which could lead to better tolerability, fewer incidents of treatment discontinuation and higher levels of adherence in patients treated with antiglaucoma medications. This in turn would improve outcomes for these patients, in terms of both glaucoma management and quality of life, which may contribute to reducing the costs of longterm glaucoma complications. It is most likely that the outcomes of glaucoma surgery would improve in line with a decrease in inflammation of the conjunctiva at the time of surgery. Moreover, although no studies on this topic have been published to date, a significant number of surgical procedures are performed in patients with poor tolerance to the drugs they require to control IOP, and when the maximally tolerable medical treatment is insufficient to stabilize glaucoma. Extending the limits of tolerance ) by improving the tolerability of medications ) should become one of the key objectives of glaucoma therapy. Overall, the provision of preservative-free formulations would offer clinically relevant benefits to patients who are highly sensitive to preservatives because of pre-existing or concomitant ocular surface diseases, as well as to patients who use combinations of two or more drugs, those at risk of undergoing surgery, and all patients who will need treatment over several decades. Preservative-free formulations should therefore become the gold standard for glaucoma therapy in the near future. Acknowledgements The author received editorial and writing support in the preparation of this manuscript, funded by Santen Oy, Tampere, Finland. The author is fully responsible for the content of this paper. The author is a consultant for and has received research grants from Alcon, Inc., Allergan, Inc., Pfizer, Inc., Santen Oy and Laboratoires The a SA. References Albietz JM & Bruce AS (2001): The conjunctival epithelium in dry eye subtypes: effect of preserved and non-preserved topical treatments. Curr Eye Res 22: Alcon, Inc. (2004): Travatan Summary of Product Information. alcon.com/alcon-products/pharmaceutical.asp. [Accessed 27 September 2007.] Allergan, Inc. (2006): Lumigan Product Information. [Accessed 27 September 2007.] Aritu rk N, Oge I, Baris S, Erkan D, Su lu Y & Koc F (1996): The effects of antiglaucomatous agents on conjunctiva used for various durations. Int Ophthalmol 20: Baudouin C (1996): Side-effects of antiglaucomatous drugs on the ocular surface. 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