Search

Ocular surface disease (OSD) is a complex condition that can cause a range of symptoms (e.g, dryness, irritation, and pain) and can significantly impact the quality of life of affected individuals. Iatrogenic OSD, a common finding in patients with glaucoma who receive chronic therapy with topical ocular antihypertensive drugs containing preservatives such as benzalkonium chloride (BAK), has been linked to damage to the ocular surface barrier, corneal epithelial cells, nerves, conjunctival goblet cells, and trabecular meshwork. Chronic BAK exposure activates inflammatory pathways and worsens symptoms, compromising the success of subsequent filtration surgery in an exposure-dependent manner. In eyes being treated for glaucoma, symptomatic treatment of OSD may provide some relief, but addressing the root cause of the OSD often necessitates reducing or, ideally, eliminating BAK toxicity. Strategies to decrease BAK exposure in patients with glaucoma encompass the use of preservative-free formulations or drugs with alternative and less toxic preservatives such as SofZia®, Polyquad, potassium sorbate, or Purite®. Though the benefits of these alternative preservatives are largely unproven, they might be considered when financial constraints prevent the use of preservative-free versions. For patients receiving multiple topical preserved drugs, the best practice is to switch to nonpreserved equivalents wherever feasible, regardless of OSD severity. Furthermore, nonpharmacological approaches, including laser or incisional procedures, should be considered. This review explores the effects of BAK on the ocular surface and reviews strategies for minimizing or eliminating BAK exposure in patients with glaucoma in order to significantly improve their quality of life and prevent complications associated with chronic exposure to BAK., Competing Interests: Declaration of competing interest MYK: Patent royalties: Alcon, SpyGlass Pharma, and New World Medical. Ownership Interest: New World Medical, SpyGlass Pharma, Aurea Medical Consultant: New World Medical, SpyGlass Pharma. CJR: Avellino, Baxis, Bio-Tissue, Celularity, Dompé, Emmecell, Glaukos, Kala, Oyster Point, Sight Sciences, Tarsus, TearLab/Trukera. EM: Alcon, AbbVie, Bausch + Lomb, DMG, Dompé, GlaxoSmithKline, Kala, Novartis, Sanofi, Santen, Shire, Sifi, Sun Pharmaceuticals, Théa, TRB-Chemedica, Ursapharm Arzneimittel, Visufarma. NMR: Consultant for Alderya, Aerie Pharmaceuticals; Alcon Vision, Alimera Sciences, AbbVie, Bausch + Lomb, Belkin, Dompé, Eyepoint Pharmaceuticals, Glaukos, Imprimis, Iridex, Kala Pharmaceuticals, Lumenis, Novartis, Ocular Science, Ocular Therapeutix, Omeros, Rayner, Reichert, Santen, SpyGlass, Tarsus, TearClear, Théa. AG: Consultant for Novartis, Oyster Point, Dompé, AstraZeneca, Oculis, EyeCool Therapeutics. Grant funding: B&L. CB: Alcon, Horus Pharma, Oculis, Santen, Théa., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction : Preservative-free (PF) medications represent a valuable treatment strategy in the lifelong management of glaucoma. By removing preservative toxicity, PF formulations provide tangible clinical benefits to glaucoma patients worldwide. They improve tolerability and adherence, leading to a positive impact in long-term intraocular pressure (IOP) control. Areas covered : A critical review of the subject is provided, including selected evidence on the safety and tolerability of currently available topical PF formulations. Cumulative evidence confirms that topical PF medications are at least equally efficacious to their preserved equivalents. There is convincing short-term evidence for superior tolerability and safety of PF formulations compared to preserved medications. The long-term benefits and success of PF therapy requires further elucidation. Expert opinion : Successful stepwise administration of medical therapy for glaucoma remains elusive. There is a greater risk for ocular toxicity and therapy failure with preserved topical glaucoma therapy. Currently available and emerging PF therapy options potentially optimize lifelong stepwise glaucoma therapy and may enhance outcome. To avert complications from preservatives leading to poor adherence, ideally, future antiglaucoma therapy should become 100% PF. There are still key aspects of PF therapy that warrant further investigation.

Dry eye disease (DED) is a multifactorial chronic inflammatory disease of the ocular surface characterized by tear film instability, hyperosmolarity, cell damage and inflammation. Hyperosmolarity is strongly established as the core mechanism of the DED. Benzalkonium chloride (BAK) - a quaternary ammonium salt commonly used in eye drops for its microbicidal properties - is well known to favor the onset of DED. Currently, little data are available regarding lipid metabolism alteration in ocular surface epithelial cells in the course of DED. Our aim was to explore the effects of benzalkonium chloride or hyperosmolarity exposure on the human corneal epithelial (HCE) cell lipidome, two different conditions used as in vitro models of DED. For this purpose, we performed a lipidomic analysis using UPLC-HRMS-ESI+/-. Our results demonstrated that BAK or hyperosmolarity induced important modifications in HCE lipidome including major changes in sphingolipids, glycerolipids and glycerophospholipids. For both exposures, an increase in ceramide was especially exhibited. Hyperosmolarity specifically induced triglyceride accumulation resulting in lipid droplet formation. Conversely, BAK induced an increase in lysophospholipids and a decrease in phospholipids. This lipidomic study highlights the lipid changes involved in inflammatory responses following BAK or hyperosmolarity exposures. Thereby, lipid research appears of great interest, as it could lead to the discovery of new biomarkers and therapeutic targets for the diagnosis and treatment of dry eye disease., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Benzalkonium chloride (BAK), a quaternary ammonium compound widely used as disinfecting agent as well as preservative in eye drops is known to induce toxic effects on the ocular surface with inflammation and corneal nerve damage leading to dry eye disease (DED) in the medium-to-long term. The aim of this study was to evaluate in vitro the toxicity of a conditioned medium produced by corneal epithelial cells previously exposed to BAK (BAK-CM) on trigeminal neuronal cells. A human corneal epithelial (HCE) cell line was exposed to 5.10 -3 % BAK (i.e. 0.005% BAK) for 15 min and let recover for 5 h to prepare a BAK-CM. This BAK concentration is the lowest one found in eye drops. After this recovery period, BAK effect on HCE cells displayed cytotoxicity, morphological alteration, apoptosis, oxidative stress, ATP release, CCL2 and IL6 gene induction, as well as an increase in CCL2, IL-6 and MIF release. Next, a mouse trigeminal ganglion primary culture was exposed to the BAK-CM for 2 h, 4 h or 24 h. Whereas BAK-CM did not alter neuronal cell morphology, or induced neuronal cytotoxicity or oxidative stress, BAK-CM induced gene expression of Fos (neuronal activation marker), Atf3 (neuronal injury marker), Ccl2 and Il6 (inflammatory markers). Two and 4 h BAK-CM exposure promoted a neuronal damage (ATF-3, phospho-p38 increases; phospho-Stat3 decreases) while 24 h-BAK-CM exposure initiated a prosurvival pathway activation (phospho-p44/42, phospho-Akt increases; ATF-3, GADD153, active Caspase-3 decreases). In conclusion, this in vitro model, simulating paracrine mechanisms, represents an interesting tool to highlight the indirect toxic effects of BAK or any other xenobiotic on corneal trigeminal neurons and may help to better understand the cellular mechanisms that occur during DED pathophysiology., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Purpose: To validate a new culture model of primary human trabecular meshwork cells (p-hTMCs) using Matrigel®, in order to mimic in vitro 3D-TM organization, and to investigate the proinflammatory effect of benzalkonium chloride (BAK) in 3D p-hTMC cultures., Methods: p-hTMCs, seeded onto Matrigel®-coated inserts were stimulated with BAK (10 -4 %), dexamethasone (DEX) (10 -6 M) or transforming growth factor-beta 2 (TGF-β2) (5ng/ml) for 48h and observed with confocal microscopy. The BAK effect at 10 -4 % or 5.10 -3 % on the gene expressions of interleukin-6 (IL-6), interleukin-8 (IL-8) and matrix metalloproteinase (MMP-9) was investigated using qRT-PCR in 2D and 3D p-hTMC cultures., Results: p-hTMCs seeded in Matrigel® were able to organize themselves in a 3D-spatial conformation in the different conditions tested with cross-linked actin network (CLAN) formation in presence of DEX or TGF-β2 and intercellular space contraction with TGF-β2. IL-6 and IL-8 gene expressions increased in presence of BAK in 2D and in 3D p-hTMC cultures. BAK 10 -4 % only showed a tendency to stimulate MMP-9 expression in p-hTMCs after 24h-recovery., Conclusions: We investigated this new 3D-TM in vitro model in Matrigel ® matrix for pathophysiological and toxicological purposes. It appears as a new promising tool for a better understanding of TM behavior in physiological and stress conditions, as well as toxicological evaluations of antiglaucoma eyedrops and preservatives., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Purpose: Benzalkonium chloride (BAK) is the most commonly used eye drop preservative. Benzalkonium chloride has been associated with toxic effects such as "dry eye" and trabecular meshwork degeneration, but the underlying biochemical mechanism of ocular toxicity by BAK is unclear. In this study, we propose a mechanistic basis for BAK's adverse effects., Method: Mitochondrial O2 consumption rates of human corneal epithelial primary cells (HCEP), osteosarcoma cybrid cells carrying healthy (control) or Leber hereditary optic neuropathy (LHON) mutant mtDNA [11778(G>A)], were measured before and after acute treatment with BAK. Mitochondrial adenosine triphosphate (ATP) synthesis and cell viability were also measured in the BAK-treated control: LHON mutant and human-derived trabecular meshwork cells (HTM3)., Results: Benzalkonium chloride inhibited mitochondrial ATP (IC50, 5.3 μM) and O2 consumption (IC50, 10.9 μM) in a concentration-dependent manner, by directly targeting mitochondrial complex I. At its pharmaceutical concentrations (107-667 μM), BAK inhibited mitochondrial function >90%. In addition, BAK elicited concentration-dependent cytotoxicity to cybrid cells (IC50, 22.8 μM) and induced apoptosis in HTM3 cells at similar concentrations. Furthermore, we show that BAK directly inhibits mitochondrial O2 consumption in HCEP cells (IC50, 3.8 μM) at 50-fold lower concentrations than used in eye drops, and that cells bearing mitochondrial blindness (LHON) mutations are further sensitized to BAK's mitotoxic effect., Conclusions: Benzalkonium chloride inhibits mitochondria of human corneal epithelial cells and cells bearing LHON mutations at pharmacologically relevant concentrations, and we suggest this is the basis of BAK's ocular toxicity. Prescribing BAK-containing eye drops should be avoided in patients with mitochondrial deficiency, including LHON patients, LHON carriers, and possibly primary open-angle glaucoma patients.

Objective: To analyze the effect of preserved antiglaucoma eye drops on the expression of extracellular matrix (ECM) metalloproteinase inducer (EMMPRIN) in conjunctival epithelial cells., Methods: A total of 18 patients treated for primary open-angle glaucoma with benzalkonium chloride (BAK) preserved eye drops and eight age-matched controls were included in this study. Glaucoma patients were divided into two groups according to their daily exposure to BAK: high-exposure (HE) group and low-exposure (LE) group. HLA-DR and EMMPRIN were quantified on conjunctival impression cytology specimens using flow cytometry. In parallel, IOBA-NHC conjunctival epithelial cells were exposed to different BAK concentrations, in the presence or absence of cyclosporine A (CsA), and their total and surface expressions of EMMPRIN were assessed by flow cytometry and results are given in relative fluorescence intensities (RFIs)., Results: Compared to the control group (1.71 ± 0.39 RFI), EMMPRIN was significantly increased in the HE (4.19 ± 1.50 RFI, p < 0.001) and LE groups (2.55 ± 0.40 RFI, p = 0.029). Similar increase was observed in HLA-DR expression in the HE (4.58 ± 1.38 RFI, p < 0.001) and LE groups (2.52 ± 0.47 RFI, p = 0.046) as compared to control subjects (1.75 ± 0.27 RFI). Across all subjects enrolled in the study, there was a significant correlation between HLA-DR and EMMPRIN (R(2) = 0.875, p < 0.0001). IOBA-NHC cells exposed to BAK presented a significant increase in EMMPRIN, which was proportional to the concentration of BAK. The surface expression of EMMPRIN was inhibited by CsA., Conclusions: The increased expression of EMMPRIN in patients topically treated with multiple antiglaucoma BAK-preserved eye drops suggests a matrix metalloproteinase-related modification of conjunctival ECM remodeling. In vitro results suggest that CsA has the potential to limit BAK effects on EMMPRIN.

Benzalkonium (BAK) chloride is the most commonly used preservative in eye drops. It is generally composed of benzyldimethyldodecylammonium C12 and benzyldimethyltetradecylammonium C14 and is supposed to increase penetration of active compounds. However, numerous studies have reported its toxic effect to ocular surface especially in long-term treatments like against glaucoma, a sight-threatening disease. Albino rabbits were treated with a hyperosmolar solution and a high concentration of BAK solution for 1 month. Enucleated eyes were cryo-sectioned and analysed by mass spectrometry. Mass spectrometry imaging using time-of-flight secondary ion mass spectrometry (TOF-SIMS) has been used to characterize the spatial distribution and to determine the relative quantity of BAK at the surface of rabbit eye sections. Liquid chromatography coupled with mass spectrometry (LC-MS) using a hybrid linear ion trap-Orbitrap® mass spectrometer was used to obtain relative quantification of BAK at the sample surface. TOF-SIMS images of BAK ions indicated a distribution at the ocular surface and in deeper structures. Didecyldimethylammonium (DDMAC), which is used in hospitals as a substitute for BAK, was also detected and showed an accumulation around the eyes. After extraction with acetonitrile and chromatographic separation using a Gemini C18 column and an original elution gradient, the relative quantities of BAK and DDMAC present in the whole eye section surface were determined. This LC-MS method was validated in terms of limits of quantification, linearity, repeatability and reproducibility and its feasibility was evaluated in surgically obtained human samples. Specimens of iris, lens capsule or trabecular meshwork were found with significant levels of BAK and DDMAC, thus confirming the penetration of BAK in deep ocular structures, with potential deleterious effects induced by this cytotoxic compound. The analytical method developed here could therefore be of primary interest in the field of pharmaco-toxicology in order to localise, identify and quantify drugs or xenobiotic compounds present at biological sample surfaces.

Purpose: To compare in vitro, on the human reconstituted corneal epithelial SkinEthics model, and in vivo, using an acute rabbit toxicological model, the effects of a benzalkonium chloride (BAK)-preserved solution of latanoprost and a preservative-free (PF) latanoprost solution., Methods: In vitro, the three-dimensional (3D) reconstituted human corneal epithelia (HCE) were treated with PBS, BAK-latanoprost, PF-latanoprost, or 0.02% BAK for 24 hours followed or not followed by a 24 hour post incubation recovery period. Cellular viability was evaluated using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test at 24 hours and the apoptotic cells were counted using TUNEL labeling on frozen sections at 24 hours and 24 hours plus 24 hours. In vivo, rabbits received 50 μL of the same solutions, which were applied at 5 minute intervals a total of 15 times. Ocular surface toxicity was investigated using slit lamp biomicroscopy examination, conjunctival impression cytology (CIC), and corneal in vivo confocal microscopy (IVCM). Standard immunohistology also assessed inflammatory CD45-positive cells., Results: In vitro, BAK-latanoprost and 0.02% BAK induced significant apoptosis in the apical layers that correlated with the significant decrease of cell viability as assessed by the MTT test. PF-latanoprost slightly decreased cell viability and few apoptotic cells were found in the superficial layers, without reaching statistical significance compared with PBS. In vivo, clinical observation and IVCM images showed the lowest ocular surface toxicity with PBS and PF-latanoprost, while BAK-latanoprost and BAK induced abnormal corneoconjunctival aspects. PF-latanoprost showed the lowest CIC score, close to the PBS score and induced fewer CD45-positive cells in both the limbus and the conjunctiva compared with BAK and latanoprost, as assessed by immunohistology., Conclusions: We confirm that rabbit corneoconjunctival surfaces presented better tolerance when treated with PF-latanoprost compared with the standard BAK-latanoprost preparation or the BAK solution.

Purpose: Long-term antiglaucomatous drug administration may cause irritation, dry eye, allergy, subconjunctival fibrosis, or increased risk of glaucoma surgery failure, potentially due to the preservative benzalkonium chloride (BAK), whose toxic, proinflammatory, and detergent effects have extensively been shown experimentally. We hypothesize that BAK also influences trabecular meshwork (TM) degeneration., Methods: Trabecular specimens were examined using immunohistology and reverse transcriptase-polymerase chain reaction. A trabecular cell line was stimulated by BAK and examined for apoptosis, oxidative stress, fractalkine and SDF-1 expression, and modulation of their receptors. An experimental model was developed with BAK subconjunctival injections to induce TM degeneration. Mass spectrometry (MS) imaging assessed BAK penetration after repeated instillations in rabbit eyes., Results: Trabecular specimens showed extremely low densities of trabecular cells and presence of cells expressing fractalkine and fractalkine receptor and their respective mRNAs. Benzalkonium in vitro induced apoptosis, oxidative stress, and fractalkine expression and inhibited the protective chemokine SDF-1 and Bcl2, also inducing a sustained intraocular pressure (IOP) increase, with dramatic apoptosis of trabecular cells and reduction of aqueous outflow. MS imaging showed that BAK could access the TM at measurable levels after repeated instillations., Conclusion: BAK enhances all characteristics of TM degeneration typical of glaucoma-trabecular apoptosis, oxidative stress, induction of inflammatory chemokines-and causes degeneration in acute experimental conditions, potentially mimicking long-term accumulation. BAK was also shown to access the TM after repeated instillations. These findings support the hypothesis that antiglaucoma medications, through toxicity of their preservative, may cause further long-term trabecular degeneration and therefore enhance outflow resistance, reducing the impact of IOP-lowering agents.

Purpose: Cationic emulsions (CEs), developed as vehicles for lipophilic drugs, have been shown to be safe and effective for the treatment of dry eye. The aim of this study was to investigate the effects of a preservative-free latanoprost 0.005% CE (latanoprost-CE) in in vitro and in vivo models of corneal wound healing., Methods: An in vitro wound was made by scraping through a confluent layer of human corneal epithelial cells. Cytotoxicity, cell migration, and proliferation were analyzed after an exposure to phosphate-buffered saline, CE, latanoprost-CE, 0.02% benzalkonium chloride (0.02%BAK), and Xalatan (latanoprost). In vivo, the recovery and integrity of corneal wound healing were assessed in rat eyes instilled twice a day for 5 days with the above treatments after deepithelialization of the superior cornea., Results: In vitro wound distances decreased at 2 and 24 hours for human corneal epithelial cells exposed to CE, latanoprost-CE, and phosphate-buffered saline, whereas they progressively increased for 0.02%BAK-treated and latanoprost-treated cells. The greater wound closure was associated with a higher number of Ki67-positive cells. In CE- and latanoprost-CE-treated rats, reepithelialization of the cornea was enhanced, restoring normal appearance and function. In contrast, 0.02%BAK or latanoprost delayed corneal healing, induced inflammation, and decreased MUC5-AC expression., Conclusions: Both models effectively evaluated the cytotoxicity and dynamic recovery of corneal wound healing, and their correlation supports the potential of the in vitro model as a reliable alternative to in vivo ocular toxicity tests. Both models demonstrated that in the face of corneal injury, CEs favored corneal healing, whereas BAK was deleterious.

The study used a validated acute in vivo model to compare a new formulation of travoprost 0.004% ophthalmic solution(travoprost PQ), preserved with polyquaternium-1 (PQ), with commercially available formulations of benzalkonium-chloride(BAK)-preserved travoprost 0.004% ophthalmic solution(travoprost BAK) and BAK-preserved latanoprost 0.005%ophthalmic solution (latanoprost BAK). Adult male New Zealand albino rabbits (n = 36) were randomly divided into 6 groups. Phosphate-buffered saline (PBS), 0.001% PQ, 0.015% BAK, travoprost PQ, travoprost BAK or latanoprost BAK were applied onto rabbit eyes as 1 drop, for 15 times at 5-min intervals.The ocular surface reactions were investigated at hour 4 and day 1 using slitlamp examination; in vivo confocal microscopy (IVCM) for cornea, limbus and conjunctiva/conjunctiva-associated lymphoid tissue, conjunctival impression cytology and standard immunohistology in cryosections for detecting CD45+ infiltrating cells and MUC-5AC-labeled cells. PBS, PQ and travoprost PQ did not induce obvious irritation by clinical observation, changes in microstructures of the whole ocular surface as measured by IVCM analysis,inflammatory infiltration or cell damage as measured by impression cytology, altered levels of goblet cell counts or numerous CD45+ cells in the cornea. In contrast, all BAK-containing products induced diffuse conjunctival hyperemia and chemosis, abnormal changes in the ocular surface microstructure,significant total ocular surface toxicity scores,damaged epithelial cells, inflammatory cell infiltration and decreased goblet cell density. Travoprost PQ did not elicitocular surface toxicity when administered to rabbit eyes.These results suggest a greater safety advantage for the ocular surface of patients receiving chronic glaucoma treatment with PQ-preserved drugs.

Conjunctiva-associated lymphoid tissue (CALT) is closely associated with ocular surface immunity. This study investigated the effects of antiglaucoma prostaglandin analogs with or without benzalkonium chloride (BAK) preservative on organized CALT using an acute toxic model. A total of 48 albino rabbits were used and seven groups of treatments were constituted. Solutions (50 µl) of PBS, 0.02%BAK, (0.02%BAK+)latanoprost, (0.015%BAK+)travoprost, (0.005%BAK+)bimatoprost, (BAK-free)travoprost preserved with the SofZia® system or (BAK-free)tafluprost were instilled 15 times at 5-min intervals in both eyes. CALT changes were analyzed using in vivo confocal microscopy (IVCM), immunohistology in cryosections for detecting MUC-5AC+ mucocytes and CD45+ hematopoietic cells. Antiglaucoma eye drops stimulated inflammatory cell infiltration in the CALT, and seemed to be primarily related to the concentration of their BAK content. The CALT reaction after instillation of BAK-containing eye drops was characterized by inflammatory cell infiltration in the dome and intrafollicular layers and by cell circulation inside the lymph vessels. CD45 was strongly expressed in the CALT after instillation of all BAK-containing solutions at 4 h and decreased at 24 h. The number of MUC-5AC+ mucocytes around the CALT structure decreased dramatically after instillation of BAK-containing solutions. This study showed for the first time the in vivo aspect of rabbit CALT after toxic stimuli, confirming the concentration-dependent toxic effects of BAK. IVCM-CALT analysis could be a pertinent tool in the future for understanding the immunotoxicologic challenges in the ocular surface and would provide useful criteria for evaluating newly developed eye drops.

Purpose: Benzalkonium chloride (BAK), the most commonly used preservative in eye drops, is known to induce ocular irritation symptoms and dry eye in long-term treated patients and animal models. As tear film hyperosmolarity is diagnostic of some types of dry eye disease, we determined in vitro on conjunctival epithelial cells the cytoxicity of BAK in hyperosmolar conditions through cell viability, apoptosis, and oxidative stress assays., Methods: The Wong Kilbourne derivative of Chang conjunctival epithelial cells were cultured for 24 h or 48 h either in NaCl-induced hyperosmolar conditions (400-425-500 mOsM), in low concentrations of BAK (10(-4)%, 3.10(-4)%, and 5.10(-4)%), or in combination of both. We investigated cell viability through lysosomal integrity evaluation, cell death (cell membrane permeability and chromatin condensation), and oxidative stress (reactive oxygen species, superoxide anion) using spectrofluorimetry. Immunohistochemistry was performed for cytoskeleton shrinkage (phalloidin staining), mitochondrial permeability transition pore (cytochrome c release), the apoptosis effector active caspase-3, and the caspase-independent apoptosis factor AIF. We also observed early effects induced by the experimental conditions on the conjunctival cell layers using phase contrast imaging of live cells., Results: As compared to standard culture solutions, hyperosmolar stress potentiated BAK cytotoxicity on conjunctival cells through the induction of oxidative stress; reduction of cell viability; cell membrane permeability increase; cell shrinkage with cell blebbing, as shown in phase contrast imaging of live cells; and chromatin condensation. Like BAK, but to a much lesser extent, hyperosmolarity increased cell death in a concentration-dependent manner through a caspase-dependent apoptosis characterized by a release of cytochrome c in the cytoplasm from mitochondria and the activation of caspase-3. Moreover, the caspase-independent apoptosis factor AIF was found translocated from mitochondria to the nucleus in both conditions., Conclusions: This study showed increased cytotoxic effects of BAK in hyperosmotic conditions, with characteristic cell death processes, namely caspase-dependent and independent apoptosis and oxidative stress. As BAK is known to disrupt tear film, which could promote evaporative dry eye and tear hyperosmolarity, BAK could promote the conditions enhancing its own cytotoxicity. This in vitro hyperosmolarity model thus highlights the risk of inducing a vicious cycle and the importance of avoiding BAK in patients with dry eye conditions.

Purpose: To compare, in vitro, the cytotoxicity profile of a new formulation of travoprost 0.004%/timolol 0.5% fixed combination ophthalmic solution preserved with polyquaternium-1 0.001% (travoprost/timolol PQ) instead of benzalkonium chloride (BAK) with (1) commercially available travoprost 0.004%/timolol 0.5% fixed combination ophthalmic solution (travoprost/timolol BAK), (2) commercially available latanoprost 0.005%/timolol 0.5% fixed combination ophthalmic solution (latanoprost/timolol BAK), and (3) their associated BAK concentrations., Methods: Compounds tested on Wong-Kilbourne-derived human conjunctival epithelial cells: (1) phosphate-buffered saline, (2) polyquaternium-1 0.001% (Polyquad(®), PQ), (3) travoprost/timolol PQ, (4) travoprost/timolol BAK with 0.015% BAK (DuoTrav(®)), (5) BAK 0.015%, (6) latanoprost/timolol BAK with 0.020% BAK (Xalacom(®)), and (7) BAK 0.020%. Toxicological assays were used to assess cell viability [neutral red (NR), Alamar blue (AB)], apoptosis (YO-PRO-1, Hoechst 33342), and oxidative stress (H(2)DCF-DA, hydroethidine). The apoptosis and oxidative stress assays were each reported according to cell viability as observed with NR and AB (totaling 10 analyses per treatment)., Results: The NR and AB assays demonstrated that cells incubated with travoprost/timolol PQ had significantly better viability than cells incubated with latanoprost/timolol BAK, travoprost/timolol BAK, BAK 0.015%, and BAK 0.020% (P<0.0001 for all). As assessed with YO-PRO-1 and Hoechst 33342 relative to cell viability determined with NR or AB, travoprost/timolol PQ produced significantly less apoptosis than travoprost/timolol BAK and latanoprost/timolol BAK and their respective BAK concentrations alone (P<0.0001 for all). Also, travoprost/timolol BAK induced less apoptosis than latanoprost/timolol BAK (P<0.0001). As assessed with H(2)DCF-DA as a ratio to NR or AB, all of the compounds without BAK (phosphate-buffered saline, PQ 0.001%, and travoprost/timolol PQ) and travoprost/timolol BAK produced significantly less reactive oxygen species than latanoprost/timolol BAK (P<0.0001 for all). As assessed with hydroethidine as a ratio to NR or AB, travoprost/timolol PQ produced significantly fewer superoxide anions than latanoprost/timolol BAK (P<0.0001). In contrast, release of superoxide anions (hydroethidine method) after incubation with travoprost/timolol BAK was not significantly different from incubation with latanoprost/timolol BAK or travoprost/timolol PQ., Conclusion: Travoprost/timolol PQ may be better for ocular surface health than either BAK preserved formulations of latanoprost/timolol or travoprost/timolol.

The side effects of glaucoma hypotensive treatments usually remain moderate and limited to local side effects - conjunctival hyperemia, itching, discomfort after instillation - but are very often a leading source of poor compliance to treatment and thereby may decrease its efficacy. Moreover, these symptoms usually reflect progressive and irreversible major ocular surface changes. These ocular surface changes induced by glaucoma eyedrops may include subconjunctival fibrosis, increasing the risk of failure of a further glaucoma surgery. All the components of the hypotensive eye drops, including the active ingredient, the preservatives, and the excipients, may be involved in the occurrence of these side effects. It is therefore important to identify the agents involved and the mechanisms of these side effects, in order to choose a treatment minimizing their risk and the discomfort felt by patients, and therefore increasing the likelihood of good compliance. When available, preservative-free solutions should be considered., (Copyright © 2011 Elsevier Masson SAS. All rights reserved.)

Purpose: To compare the effects of benzalkonium chloride (BAC)-preserved and unpreserved antiallergic eye drops on the human 3D-reconstituted corneal epithelial model (3D-HCE)., Methods: 3D-HCE were treated for 24 h followed or not by a 24 h post-incubation recovery period (24 h+24 h) with phosphate-buffered saline (PBS), 0.01% BAC, unpreserved formulations of ketotifen, N Acetyl-Aspartyl Glutamic Acid (NAAGA), cromoglycate, or BAC-preserved commercial formulations of ketotifen, olopatadine, epinastine, and levocabastine. The 3D-HCE viability was evaluated using the 3-(4,5-Dimethylthiazol-2-yl) -2,5-Diphenyltetrazolium Bromide (MTT) test at 24 h and 24 h+24 h. At 24 h, the numbers of Cluster of Differentiation 54 (CD54)- and Ki67-immunopositive cells as well as the number of apoptotic deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells were evaluated on 3D-HCE frozen sections. The expression of the tight junction-associated protein occludin was also assessed using fluorescence confocal microscopy on flat-mounted 3D-HCE epithelia., Results: The MTT and the TUNEL tests revealed a significant decrease of cell viability and an increased apoptosis in the superficial layers of the 3D-HCE only when treated with BAC-containing formulations and in a BAC concentration-dependent manner. The expression of CD54 and Ki67 in the basal layers was also increased in this group. A concentration-dependent disorganization of occludin distribution in the epithelium treated with BAC-containing solutions was also observed. The unpreserved formulations induced effects comparable to the control., Conclusions: BAC-preserved solutions decreased cell viability and induced apoptosis in a concentration-dependent manner. Moreover, they induced CD54 expression, proliferation in the basal layers, and changes in the distribution of occludin, which is consistent with a disorganization of the tight-junctions and suggests the loss of the epithelial barrier function. On the contrary, the unpreserved solutions did not impair cell structures and viability, suggesting a better tolerance for the ocular surface. As allergic patients often exhibit impaired and inflammatory ocular surface, BAC-free compounds should be the first choice when treating allergic conjunctivitis.

Preservatives are present in numerous multidose eyedrops and provide the sterility of the solution against bacteria and fungi. However, numerous studies have shown their toxicity for the ocular surface, particularly in long-term treatments. The most widely used preservative in eyedrops is benzalkonium chloride. This quaternary ammonium acts as a detergent, antiseptic, disinfectant, fungicide, bactericide, and spermicide. Its use on the ocular surface therefore has significant consequences. Indeed, the preservatives are pro-apoptotic, pro-inflammatory and they cause the dissolution of the lachrymal film. The prolonged administration of one or several eye drops containing preservatives induces changes in the superficial structures (conjunctiva, cornea) as well as in deeper structures (trabecula, lens). The least severe symptoms are irritation and discomfort, including sensation of a foreign body, itching, or burning sensations. However, more severe side effects have been described, such as chronic inflammation of variable intensity or the progressive development of fibrosis with higher risk of failure after glaucoma filtering surgery. Ideally, preservative-free eyedrops should be recommended, or at least a reduction of the number of instilled preserved eyedrops should be considered. All these strategies could increase patient comfort, quality of life, and compliance, with better outcome at the time of filtering surgery., (Copyright © 2010 Elsevier Masson SAS. All rights reserved.)

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

Purpose: To investigate the effects of benzalkonium chloride (BAK) on the human reconstituted corneal epithelial model (HCE) and to optimize the operating potential of this model in the field of ophthalmic toxicology., Methods: The HCEs were treated with 0.001% to 0.5% BAK for 24 hours followed or not by a 24-hour postincubation period. To complete the histologic analysis, the authors designed a new MTT procedure to assess cellular viability. Frozen sections were analyzed by using fluorescence confocal microscopy for the presence of TUNEL, activated caspase-3, Ki67, ICAM-1, HLA-DR, E-cadherin, and occludin. Occludin gene expression was also investigated by using quantitative RT-PCR., Results: The MTT test revealed a dose-dependent response of BAK with significant toxic effects for concentrations as low as 0.005%. Increasing BAK concentrations induced an increased number of apoptotic cells, found from the superficial to the deeper layers, with the activation of caspase-3 at 0.01% and 0.02% concentrations. The number of Ki67- and ICAM-1-positive cells increased with 0.01% BAK and with 0.001% to 0.01% BAK, respectively. BAK induced the dose-dependent disappearance of occludin in the superficial layers while increasing its gene expression up to the 0.02% BAK concentration., Conclusions: Fluorescence techniques conjugated with confocal microscopy on 3D-reconstructed corneal epithelia were well suited for the investigation of toxicological markers such as cell junction alteration, apoptosis, cell activation, and proliferation and gave relevant results compared with the known human data. They complement the new sensitive MTT test and improve the operating potential of this new, valuable 3D model in ophthalmic toxicology.

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.

Aim: To compare the conjunctival and corneal reactions of commercially available solution of latanoprost (Xalatan) and preservative-free (PF) tafluprost in rabbits., Methods: The rabbits received 50 microl of phosphate-buffered saline (PBS), PF-tafluprost 0.0015%, latanoprost 0.005% or benzalkonium chloride (BAK) 0.02%; all solutions were applied at 5 min intervals for a total of 15 times. The ocular surface toxicity was investigated using slit-lamp biomicroscopy examination, flow cytometry (FCM) and on imprints for CD45 and tumour necrosis factor-receptor 1 (TNFR1) conjunctival impression cytology (CIC) and corneal in vivo confocal microscopy (IVCM). Standard immunohistology also assessed inflammatory/apoptotic cells., Results: Clinical observation and IVCM images showed the highest ocular surface toxicity with latanoprost and BAK, while PF-tafluprost and PBS eyes presented almost normal corneoconjunctival aspects. FCM showed a higher expression of CD45+ and TNFR1+ in latanoprost- or BAK-instilled groups, compared with PF-tafluprost and PBS groups. Latanoprost induced fewer positive cells for inflammatory marker expressions in CIC specimens compared with BAK-alone, both of which were higher than with PF-tafluprost or PBS. Immunohistology showed the same tendency of toxic ranking., Conclusion: The authors confirm that rabbit corneoconjunctival surfaces presented a better tolerance when treated with PF-tafluprost compared with commercially available latanoprost or BAK solution.

Purpose: This study compared the toxicity profiles of three antiglaucoma prostaglandin F2alpha analogs, latanoprost, travoprost, and bimatoprost which contain benzalkonium chloride (BAK), with tafluprost, a new preservative-free prostaglandin analog., Methods: IOBA-NHC cells were exposed to BAK-containing prostanoid solutions, their respective BAK concentrations, and preservative-free tafluprost solution for 30 min. Membrane integrity, apoptosis, oxidative stress, and cells morphology were evaluated., Results: Preservative-free tafluprost resulted in significantly higher membrane integrity and lower pro-apoptotic and pro-oxidative effects than preservative-containing prostaglandin analog preparations., Conclusions: These results suggest that tafluprost, a new preservative-free prostaglandin analog, has very low or no pro-apoptotic, pro-necrotic, or pro-oxidative effects in vitro compared to preservative-containing formulations.

Purpose: To assess the usefulness of noninvasive combined technologies used to observe ocular surface changes in toxicology studies., Methods: Benzalkonium chloride (BAC) at 0.01%, 0.1%, 0.25%, and 0.5% was applied to rat corneas for 11 days. The eye was evaluated macroscopically from day (D)0 to D52. The cornea was examined with the slit lamp, a fluorescein test was performed, and a confocal microscope was used in vivo to calculate corneal thickness, score corneal epithelial and endothelial defects, and quantify corneal stromal inflammation and neovascularization. Conjunctival impression and brush cytology specimens were taken for labeling with MUC-5AC antibodies and sub-G1 peak analysis by flow cytometry, respectively. Histologic analyses were performed on D11., Results: Although macroscopic and slit lamp examinations revealed signs of ocular irritation in the 0.25% and 0.5% BAC-treated eyes only, in vivo confocal microscopy revealed epithelial defects in the 0.01% and 0.1% BAC-treated corneas, and sub-G1 peak analyses showed increased apoptosis for all the BAC concentrations on D8 and D11. BAC at 0.25% and 0.5% induced increased corneal thickness, loss of goblet cells, reversible corneal inflammation, and persistent neovascularization., Conclusions: Sub-G1 peak analysis of conjunctival brushings, in conjunction with in vivo confocal microscopy of the cornea and immunolabeling of conjunctival imprints, constitutes a noninvasive, reliable, and sensitive tool to evaluate toxic drug-induced ocular surface damage in rats, in addition to standard clinical assessments and at a wide range of concentrations, including the lowest ones. This study is consistent with the international strategy aimed at reducing the use of animals and refining animal toxicologic models.

Purpose: With use of the Wong-Kilbourne derivative Chang conjunctival cell line, this study compared in vitro the ocular toxicity of three topical intraocular pressure (IOP)-lowering agents: travoprost 0.004% containing 0.015% benzalkonium chloride (BAK), travoprost Z 0.004%, a new formulation without BAK, and latanoprost 0.005% containing 0.02% BAK., Methods: Neutral red, Alamar blue, YOPRO-1, and annexin V/7-AAD assays were used to evaluate the effects of the IOP-lowering agents and BAK on cellular viability, membrane integrity, and apoptosis in the conjunctival cell line using microtitration fluorometric analysis and flow cytometry. All assessments were performed in a masked manner., Results: Assessment of cell viability and membrane integrity revealed a significant effect by latanoprost with BAK or BAK alone but no effect by travoprost Z without BAK or buffer alone (P < 0.0001). Latanoprost with BAK, travoprost with BAK, and BAK alone were cytotoxic in Chang conjunctival cells, whereas no cytotoxicity was observed in cells exposed to travoprost Z without BAK or in cells treated with buffer (P < 0.0001). No increase in apoptosis or necrosis was observed in cells treated with control or travoprost Z without BAK compared with BAK, travoprost with BAK, and latanoprost with BAK (P < 0.0001)., Conclusions: Latanoprost with BAK, travoprost with BAK, and BAK alone have significant cytotoxic effects on human conjunctiva-derived cells and are associated with apoptosis. These effects likely result from BAK used as a preservative. IOP-lowering agents with alternative preservatives instead of BAK will most likely have fewer ocular surface adverse effects than agents containing BAK.

Purpose: To compare the prevalence of side effects between eyedrops with or without preservatives, in terms of subjective symptoms and objective signs in patients with open-angle glaucoma., Methods: In a multicenter cross-sectional epidemiologic survey in four European countries, ophthalmologists in private practice enrolled 9658 nonconsecutive patients using preservative (P) or preservative-free (PF) beta-blocking eyedrops between June 1997 and December 2003. Subjective symptoms, conjunctival and palpebral signs, and superficial punctate keratitis were explored before and after a change in therapy. For statistical analysis, a Chi-square test was used to calculate the differences in the prevalence of symptoms and signs with or without preservatives., Results: A total of 74% of the patients used P, 12% PF, 10% a P-PF combination, and in 4% the type of medication was unknown. Each recorded symptom and all the palpebral, conjunctival, and corneal signs were significantly more frequent (p<0.0001) in the P-group than in the PF-group, such as pain or discomfort during instillation (48 vs 19%), foreign body sensation (42 vs 15%), stinging or burning (48 vs 20%), and dry eye sensation (35 vs 16%). A total of 68% of the patients had a sec-ond visit performed, of whom 63% (6083) had been evaluated on treatment difference. A significant decrease (p<0.0001) of all ocular symptoms and signs was observed in patients in whom the preserved eyedrops were diminished in number or altered into preservative free drops., Conclusions: Compared to preserved eyedrops, preservative free eyedrops are significantly less associated with ocular symptoms and signs of irritation.

Background: The purpose of the study was to compare toxic effects and responses to histamine and IFN gamma associated with the use of some widely used anti-allergic eye drops commercially available today., Methods: For dynamic studies, the Wong-Kilbourne cell line was stimulated for 24 h with histamine or IFN gamma in the presence or absence of anti-allergic eye drops. Supernatants of histamine-stimulated cells were evaluated for the production of IL-6 and IL-8 by ELISA, while the expression of ICAM-1 was evaluated by flow cytometry on IFN gamma-stimulated cells. Toxicological assays were performed using cold light cytofluorometry: viability and apoptosis as well as reactive oxygen species (ROS) and O2(.)- production were assessed using neutral red, Hoechst/propidium iodide, H(2)-DCFDA and hydroethidine tests, respectively., Results: Antihistamines reduced IL-6 release and presented dose-dependent inhibitory effects on IL-8 production. None of the eye drops decreased the basal or IFN gamma-stimulated expression of ICAM-1. Conversely, eye drops preserved with benzalkonium chloride (BAC) induced even higher ICAM-1 expression levels on IFN gamma-stimulated cells than did IFN gamma alone, whereas unpreserved drugs had no effect. Toxicological assays confirmed the pivotal role of BAC in proportionally reducing cell viability while increasing apoptosis and oxidative stress., Conclusions: The ability of topical ocular anti-H(1) drugs to significantly reduce the production of IL-6 and IL-8 argues that they may help treat the inflammatory processes occurring in allergic ocular surface disorders. Nevertheless, preserved ophthalmic formulations may enhance epithelial conjunctival expression of ICAM-1 in the presence of a low inflammatory stimulus, such as IFN gamma, and displayed toxic as well as pro-oxidative effects on these cells. Therefore, BAC used as preservative might in part interfere with the potential anti-inflammatory properties of the active compound by modulating the immuno-inflammatory response of epithelial conjunctival cells.

Purpose: The aim of this study was to compare, in vivo on a rat model, two different preservatives- benzalkonium chloride (BAC) and polyquaternium-1 (PQ-1)-using new experimental approaches., Methods: Thirty (30) eyes of 15 male Lewis rats were used in this study. Rats were randomly divided into five groups instilled twice a day for 11 days with eye drops containing different concentrations of preservatives, 0.1% BAC, 0.5% BAC, 0.1% PQ-1, 0.5% PQ-1, and balanced salt solution (BSS) as a control. The ocular surface toxicity of these two preservatives was investigated using new in vivo experimental approaches. Slit-lamp examination, the fluorescein test, the red phenol test, impression cytology, and in vivo corneal confocal microscopy were used to evaluate the rat ocular surface after preservative instillation. Histology sections and immunohistochemistry were also examined to confirm these results., Results: Compared to PQ-1, BAC consistently and dramatically altered the corneoconjunctival surface as evaluated by slit-lamp examination, the fluorescein test, impression cytology, in vivo confocal microscopy, and histology. The 0.5% BAC solution also significantly decreased tear production compared to the control. Although 0.5% PQ-1 significantly decreased goblet cell density in comparison to the control, and some abnormalities were observed with in vivo confocal microscopy, no statistically significant differences were observed between these two groups using the tear production test, slit-lamp and fluorescein evaluation, or histology., Conclusion: Using an acute rat model of ocular toxicity by comparing preservatives at high concentrations, we demonstrated in vivo that high doses of PQ-1 were much less toxic than BAC. In vivo confocal microscopy and impression cytology are new promising experimental approaches to studying the rat corneoconjunctival surface, particularly in the field of ocular surface toxicity.

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

Background: The mechanisms of trabecular cell loss in glaucoma patients are poorly understood. In order to determine whether drug-induced apoptosis could be one of the mechanisms by which trabecular cells die in glaucoma, we evaluated the effect of benzalkonium-preserved (BAC+) or preservative-free (BAC-) antiglaucoma medications on apoptotic marker expression by cultured human trabecular meshwork (HTM) cells., Methods: Normal and glaucomatous trabecular cell lines were treated for 15 min with antiglaucoma drugs (1/100 and 1/10 dilutions): timolol BAC+ or BAC-, betaxolol BAC+ or BAC-, latanoprost BAC+ or pure BAC. Apo2.7 expression, annexin V binding and DNA content were evaluated by flow cytometry and confocal microscopy., Results: Results obtained in the two cell lines were similar for all tested drugs and criteria. In a 1/100 dilution, unpreserved beta-blockers had no apoptotic effect, preserved beta-blockers and latanoprost significantly increased Apo2.7 expression only, while BAC significantly increased all three apoptotic markers. When tested in a 1/10 dilution, all drugs except unpreserved timolol triggered a 2- to 3.5-fold increase in apoptotic features, whereas up to 95% of the cells underwent apoptosis upon treatment with BAC (representing a 9-fold increase over the background level)., Conclusion: At concentrations higher than those supposed to be found in the aqueous humor after instillation (1/100 dilution), unpreserved beta-blocker exhibited no proapoptotic activity on HTM cells in vitro. Benzalkonium-containing beta-blockers and prostaglandin analogue triggered mild expression of one out of three apoptotic markers, while the pro-apoptotic effect observed with BAC appeared to be largely hindered by active compounds in the preserved eyedrops.

Purpose: Antiglaucoma drugs have been associated with conjunctival and trabecular inflammatory cell infiltrates. However, the underlying mechanisms are still poorly understood. The aim of this study was to assess the effects of antiglaucoma medications on the complement system, an early mediator of the inflammatory response., Methods: Human serum was first treated with a classical or alternative pathway activator (aggregated human IgG or zymosan, respectively) in the presence or the absence of preservative-free or benzalkonium (BAK)-preserved antiglaucoma drugs. CH50 assay was then performed to assess the functional activity of residual complement in treated serum., Results: In the absence of complement activator, the antiglaucoma drugs tested in this study were all devoid of intrinsic complement-activating potency. Preserved and preservative-free carteolol as well as preserved latanoprost did not worsen or prevent complement activation induced by zymosan or aggregated IgG. Unexpectedly, both preserved and unpreserved timolol and betaxolol significantly counteracted the effects of complement activators. Timolol prevented activation triggered by both IgG and zymosan to the same extent (24% to 29%), despite the presence of BAK in the preserved formulation. Betaxolol was twice as effective at preventing the effect of IgG (34% to 37%) than that of zymosan (14%), regardless of the presence of BAK. However, BAK itself strongly aggravated complement activation by both activators., Conclusions: Carteolol, timolol, betaxolol and latanoprost did not activate complement system. On the contrary, the beta-blockers timolol and betaxolol exerted an anti-inflammatory effect by preventing complement activation. The deleterious effect of benzalkonium seems to have been neutralized within the preserved eyedrops through a mechanism that remains to be elucidated. Our study suggests that inflammatory signs in glaucoma patients should not be attributed to complement activation by antiglaucoma drugs.

Purpose: To compare the effect of timolol with or without preservatives on the conjunctival epithelium of glaucoma patients., Methods: A retrospective study using impression cytology (IC) was conducted on patients treated with 0.5% timolol with or without 0.01% benzalkonium chloride (BAC). Fifteen eyes from 15 patients treated with timolol, BAC+ and 17 eyes from 17 patients treated with timolol, BAC- were included in two groups comparable for age and duration of treatment lasting at least 1 year. Specimens were analyzed by flow cytometry for inflammatory profile (using antibodies directed against HLA DR and ICAM-1) and mucin detection (anti-M1/MUC5AC antibody)., Results: IC analyses showed a significant increase in the expression of the two inflammatory markers, HLA DR and ICAM-1 in the timolol, BAC+ group and also a significant decrease in goblet cell density in the same group as compared to the timolol, BAC- group., Conclusion: The use of long-term preserved beta-blocker in glaucoma patients is associated with a direct subclinical epithelial toxicity in the conjunctiva, as already demonstrated by previous experimental studies.

Purpose: Trabecular meshwork, which is involved in aqueous outflow resistance, is deeply modified in glaucoma patients, with a decrease in the trabecular cell number. Trabecular toxicity of antiglaucoma medications cannot be excluded. On a human cultured trabecular cell line, we investigated the potential proapoptotic effect of a beta-blocker with or without preservative, benzalkonium chloride (0.01% BAC), by flow cytometry and confocal microscopy., Material: and, Methods: A human immortalized trabecular cell line (HTM-5) obtained from a normal donor was cultured under normal conditions. Preserved 0.25% betaxolol suspension (betaxolol BAC +), unpreserved 0.25% betaxolol suspension, and 0.01% BAC were respectively added to the culture medium in a 1/10 or 1/100 dilution for 15 minutes. After a 24-hour recovery period in normal culture conditions, cell size and the expression of an apoptotic marker, Apo 2.7, were evaluated by flow cytometry and confocal microscopy. Untreated trabecular cells were used as control cells., Results: Preserved and unpreserved betaxolol in a 1/10 dilution induced a significant decrease in trabecular cell size compared to controls. However, this cell size decrease was less pronounced than that induced by BAC at the same dilution. Similar results were obtained with betaxolol and BAC in a 1/100 dilution. Trabecular cell Apo 2.7 expression was significantly increased after treatment with betaxolol BAC + and BAC- in a 1/10 dilution compared to controls (36.8%, 28.1%, and 15.4%, respectively p<0.005). However, this proapoptotic activity was much less pronounced than that induced by BAC- at the same dilution (96.9%, p<10(-4)). Unpreserved betaxolol in a 1/100 dilution had no apoptotic activity on trabecular cells. Trabecular cell Apo 2.7 expression slightly increased with betaxolol BAC + at a 1/100 dilution (24.9%, p=0.04), while it was greatly increased with BAC at the same dilution (39.9%; p<10(-4))., Conclusion: In our model, unpreserved betaxolol at a low concentration displayed no proapoptotic activity on trabecular cells. On the other hand, preserved betaxolol displayed a moderate proapoptotic activity by triggering cell death of around 25% of cells. Trabecular cell toxicity appeared to be mainly due to the preservative benzalkonium chloride (BAC). Taken together, our results demonstrated that the strong apoptotic activity of BAC was greatly reduced within the preserved eye drops, probably through the interaction of BAC with the active compound.

Aim: To determine the incidence of ocular toxicity of preservatives with glaucoma medications., Methods: A prospective epidemiological survey was carried out in 1999 by 249 ophthalmologists on 4107 patients. Ocular symptoms, conjunctiva, cornea, and eyelids were assessed. A chi(2) test was used for differences between preserved eye drops (P) and preservative free eye drops (PF)., Results: 84% patients used P, 13% received PF, and 3% a combination of P and PF eye drops. All symptoms were more prevalent with P than with PF drops (p<0.001): discomfort upon instillation (43% versus 17%), and symptoms between instillations such as burning-stinging (40% versus 22%), foreign body sensation (31% versus 14%), dry eye sensation (23% versus 14%), tearing (21% versus 14%), and eyelid itching (18% versus 10%). An increased incidence (>2 times) of ocular signs was seen with P eye drops. The prevalence of signs and symptoms was dose dependent, increasing with the number of P drops. A reduction in the symptoms and signs was observed when patients changed from P to PF eye drops (p<0.001)., Conclusions: Symptoms and signs are less prevalent when PF drops are used. Moreover, most of the adverse reactions induced by P glaucoma medication are reversible after removing preservatives.

Purpose: Quaternary ammonium ions have been demonstrated to induce apoptosis correlated with superoxide anion production in vitro. The purpose of this study was to further investigate the mechanisms of benzalkonium chloride (BAC), unpreserved and preserved beta-blocker eye-drops-induced programmed cell death, with special attention to the roles of mitochondrial transmembrane potential and intracellular reduced glutathione., Methods: Chang conjunctival cells were incubated with different concentrations of unpreserved or preserved timolol (0.1%, 0.25%, and 0.4%), or carteolol (1% and 2%), or BAC (0.0001% to 0.01%) for 15 minutes, or for 15 minutes with a 24-hour recovery period in normal medium. Cellular viability (neutral red test), mitochondrial activity (rhodamine 123 test), intracellular reduced glutathione (monochlorobimane test), DNA condensation (Hoechst 33342 test), and reactive oxygen species (ROS) production (dichlorofluorescein diacetate and hydroethidine tests) were evaluated using microplate cold-light cytofluorometry., Results: A significant, concentration-dependent decrease in cellular viability was found with preserved beta-blockers and with BAC alone, whereas unpreserved preparations did not show any toxicity. Only preserved beta-blockers induced chromatin condensation associated with an alteration of mitochondrial activity and a decrease of glutathione, suggesting an apoptotic phenomenon. BAC increased glutathione after 15 minutes, whereas a decrease was observed after a recovery period. ROS production was found with preserved formulations at significantly higher levels than those observed with unpreserved drugs., Conclusions: This in vitro study demonstrates that oxidative stress, evidenced by enhanced ROS production and mitochondrial injury rather than by cellular glutathione depletion, is a mechanism involved in apoptosis induced by preservative-containing eye-drops.

Purpose: To investigate some of the toxicity mechanisms of 10 preservatives currently used in ophthalmic solutions in vitro., Methods: A continuous human conjunctival cell line was treated with different concentrations of various preservatives for 15 minutes and for 15 minutes followed by 24 hours of cell recovery: three benzalkonium chlorides (BACs) with different hydrocarbon chain length, benzododecinium bromide (BOB), cetrimide (Cet), phenylmercuric nitrate (PM), thimerosal (thi), methyl parahydroxybenzoate (MPHB), chlorobutanol (clb), and EDTA. An inhibition study was then conducted using a 1-hour vitamin E pretreatment followed by a 15-minute BAC treatment. Membrane integrity was assessed using a neutral red test and chromatin condensation with a Hoechst 33342 test. Reactive oxygen species were measured using dichlorofluorescein diacetate test for H2O2 production and hydroethidine test for O2.- production. These tests were performed using microplate cold light cytofluorometry. Cell size and DNA content were also analyzed using flow cytometry. Confocal microscopy was used to explore morphologic changes., Results: A significant decrease of membrane integrity with chromatin condensation was observed with all the quaternary ammoniums tested at concentrations of 0.005% and higher. The effect was amplified after 24 hours of cell recovery. The other preservatives tested did not decrease membrane integrity. H2O2 production was observed with all the preservatives, whereas O2.- production was significantly higher with the quaternary ammoniums at 0.005% and 0.01%, compared with the other preservatives. Flow cytometry results confirmed the cytotoxicity observed with cold light cytofluorometry., Conclusions: The quaternary ammoniums tested (BAC, BOB, and Cet) were the most cytotoxic preservatives in the current model. An apoptotic mechanism appeared to be present at low concentrations of quaternary ammoniums, whereas a necrotic process appeared at higher concentrations. Superoxide anions may play an important role in tissue damage induced by preservatives in ocular surface disorders.

Purpose: To compare the toxicity of a short-time application of timolol with benzalkonium chloride (timolol-BAC+) and unpreserved timolol (timolol-BAC-) in an in vitro model of human conjunctival cells., Methods: Chang's conjunctival cell line (ATCC CCL 20.2) was treated for 15min. with 0.1%, 0.25% or 0.4% timolol-BAC(+) or BAC(-) and then examined immediately or 24h later. Cell viability, chromatin condensation, mitochondrial mass and activity, free radicals production were studied by microplate cold light cytometry. Moreover, relative cell number was evaluated by crystal violet colorimetric test. In addition, cell size and the expression of an apoptotic marker Apo2.7 were studied by flow cytometry., Results: Timolol-BAC(+) induced a rapid decrease in cell viability ranging from 40% immediately after treatment to 85% 24h later. A small, significantly less important decrease in cell viability was also observed with all tested concentrations of timolol-BAC(-). 24h after treatment with 0.25% timolol-BAC(+), the relative cell number was reduced by 55% whereas it did not vary after 0.25% timolol -BAC(-) treatment. Only timolol-BAC(+) induced chromatin condensation, decrease in mitochondrial membrane potential and cell size reduction. Moreover, cells treated with timolol-BAC(+) overexpressed the apoptotic marker Apo2.7. Also reactive oxygen species (ROS) production was significantly more important after cell exposure to timolol-BAC(+)., Conclusion: In our model of conjunctival cells in vitro, timolol-BAC(+) induced irreversible cytotoxic damage with some characteristics of apoptosis. The active compound of timolol-BAC(-) could be responsible for ROS production and for cell viability variations. Oxidative stress could also play a role in timolol-BAC(+)-induced toxicity. In vitro toxic effects of antiglaucoma drugs could, in part, explain some ocular surface disorders in long-term treated patients.

Purpose: To compare the toxicity of a short-time application of timolol with benzalkonium chloride (timolol-BAC+) and unpreserved timolol (timolol-BAC-) in a human conjunctival cell line., Methods: Chang's conjunctival cell line (ATCC CCL 20.2) was treated for 15min. with 0.1%, 0.25% or 0.4% timolol-BAC(+) or BAC(-) and then examined immediately or 24h later. Cell viability, chromatin condensation and free radicals production were studied by microplate cold light cytometry. Moreover, relative cell number was evaluated by crystal violet colorimetric test. The comparison was done with an oxidative stress model of cells treated with 0.001-0.000001% hydrogen peroxide (H(2)O(2)). In addition, cell size and the expression of an apoptotic marker Apo2.7 were evaluated by flow cytometry., Results: Timolol-BAC(+) induced a rapid decrease in cell viability ranging from 40% immediately after treatment to 85% 24h later. A small initial decrease in cell viability was also observed with all tested concentrations of timolol-BAC(-) but, 24h later, cell viability either tended to remain constant or cells completely recovered. Cell viability fell down after 24h exposure to 0.001% H(2)O( 2) whereas it was not modified at lower concentrations. 24h after treatment with 0.25% timolol-BAC(+), the relative cell number was reduced by 55% whereas it did not vary after 0.25% timolol-BAC(-) treatment. Only timolol-BAC(+) induced chromatin condensation and cell size reduction. Moreover, cells treated with timolol-BAC(+) overexpressed the apoptotic marker Apo2.7. Both timolol-BAC(+) and BAC(-) induced reactive oxygen species (ROS) production which was significantly more important when 0.25% or 0.4% timolol-BAC(+) were applied. Only 0.001% and 0.0001% H(2)O(2) generated a significant free radicals production., Conclusion: In our model of conjunctival cells in vitro timolol-BAC(+) induced irreversible cytotoxic damage with some characteristics of apoptosis. The active compound of timolol-BAC(-) could be responsible for reactive oxygen species production and for cell viability variations. The role of oxidative stress in timolol-BAC(+)-induced toxicity seems not to be predominant. in vitro toxic effects of antiglaucoma drugs could, in part, explain some ocular surface disorders in long-term treated patients.

Thirty-six albino rabbits, randomly divided into six groups, were used to study their ocular tolerance to (a) 0.25 and 0.50% Timoptol preserved with 0.01% benzalkonium chloride, (b) 0.25 and 0.50% Timoptol-LP, a gel-forming solution preserved with 0. 012% benzododecinium bromide, and (c) 0.25 and 0.50% Timabak unpreserved in the ABAK eyedrops dispenser. All eyedrops were applied in the right eye for 60 days. A clinical follow-up with slitlamp examination and break-up time evaluation was performed for 2 months. At the end of the experimentation, the animals were sacrificed and their eyes enucleated for histological analyses of the conjunctiva and cornea. There was no significant difference in the clinical examination between each group, except for the break-up time evaluation between Timoptol and Timabak at each concentration which was better with the unpreserved timolol. Histological results showed a significant difference in the corneal stroma edema between preserved and unpreserved timolol. This study confirms that using unpreserved timolol may be beneficial for the long-term treatment of glaucomatous patients as it increases tear film stability and decreases epithelial permeability and stromal aggression of the cornea., (Copyright 2000 S. Karger AG, Basel.)

Purpose: Ophthalmic preparations can induce conjunctival toxicity, often caused by preservatives. The aim of this study was to evaluate in vitro cytotoxicity of quaternary ammonium., Methods: Cytotoxicity tests were done on a continuous human conjunctival cell line using microplate cold light cytofluorimetry. Membrane integrity (neutral red test), DNA condensation (Hoechst 33342 test) and reactive oxygen species (ROS) production (dichlorofluorescein diacetate and hydroethidine tests) were evaluated on living cells treated with different concentrations of benzalkonium chloride, benzododecinium bromide and cetrimide (0.00001 to 0.01%) after 15 minutes of treatment or 15 minutes and 24 hours of cell recovery., Results: All the compounds tested showed similar in vitro effects. Using the neutral red test, we observed a decrease in membrane integrity even at 0.005% and 0.01% (p < 0.001) and after a short time (15 minutes). A stimulation of ROS production (H2O2 and O2) was observed at 0.00001% and above (p < 0.001), associated with a chromatine condensation due to an apoptotic phenomenon., Conclusion: A necrotic phenomenon is suggested at high concentrations of quaternary ammonium preservatives whereas an apoptotic mechanism exists for lower concentrations. This toxicity observed in vitro can explain some of the ocular surface damage caused by long-term use of preserved eye-drops.

Purpose: To evaluate the clinical tolerance of antiglaucoma eye drops with and without a preservative in routine practice., Methods: A total of 125 ophthalmologists in private practice located throughout France examined altogether 919 glaucomatous patients treated with eye drops containing a preservative or not. For some patients the examination was repeated during a second visit. Functional signs as well as conjunctival and corneal examination results were recorded., Results: The proportion of patients experiencing discomfort or pain during instillation was 58% for eye drops containing a preservative and 30% for eye drops with no preservative (p < 0.001). Moreover, the proportion of patients presenting at least one symptom of eye irritation (sensation of itching or burning, sensation of a foreign body in the eye, and flow of tears) was greater with preservative-containing eye drops (53% vs 34%; p < 0.001). The experience of discomfort during instillation was more often associated with problems later on. The patient's complaints were correlated with objective signs of conjunctival (conjunctival redness, conjunctival follicles), or corneal (superficial punctuate keratitis) damages. A higher proportion of patients treated with eye drops containing a preservative showed at least one conjunctival sign (52% vs 35%; p = 0.001) or superficial punctuate keratitis (12% vs 4%; p = 0.01). In 164 patients, whose treatment was changed from eye drops containing a preservative to eye drops with no preservative and who were examined a second time (mean interval between visits: 3.3 months) the frequency of all symptoms and objective signs fell by a factor of 3 to 4 (p < 0.001)., Conclusion: Patient complaints and objective damage to the ocular surface are more frequent among patients treated with eye drops containing a preservative. Ocular surface damage due to preservatives is partially reversible. Patient complaints should consequently be taken into account and preservative-free eye drops should be prescribed when ever possible.

Purpose: The aim of this study was to investigate the action of benzalkonium chloride (BAC), used as a preservative in most ophthalmic topical solutions, on epithelial conjunctival cells in vitro., Methods: A continuous human conjunctival cell line (Wong-Kilbourne derivative of Chang conjunctiva) was exposed to BAC solutions at various concentrations (0.1%-0.0001%) during a period of 10 minutes. Cells were examined before treatment and 3, 24, 48, and 72 hours later, after reexposure to normal cell culture conditions. Cell number and viability were assessed with crystal violet and 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide colorimetric assays. The expression of the apoptotic marker Apo 2.7, nuclear antigen p53, membrane proteins Fas and Fas ligand, and DNA content was studied by flow cytometry. Morphologic aspects of cell nuclei were analyzed on slides with a nucleic acid-specific dye, 4',6'-diamidino-2-phenylindole dihydrochloride. Cytoskeleton was labeled with a monoclonal anti-pancytokeratin antibody. In addition, apoptosis was measured by DNA electrophoresis assays in agarose gel., Results: Cell exposure to 0.1% and 0.05% BAC induced cell lysis immediately after treatment. All cells (100%) treated with 0.01% BAC died in a delayed manner within 24 hours, with most of the characteristics of apoptosis (chromatin condensation and DNA fragmentation, reduction in cell volume, expression of the apoptotic marker Apo 2.7, and apoptotic changes in DNA content). Aliquots of 0.005%, 0.001%, 0.0005%, and 0.0001% BAC induced growth arrest and apoptotic cell death in a dose-dependent manner between 24 and 72 hours after treatment. The expressions of Fas and p53 did not vary after BAC treatment. Fas ligand was always negative., Conclusions: These results suggest that BAC induces cell growth arrest and death at a concentration as low as 0.0001%. The mode of BAC-induced cell death is dose-dependent. Cells die by necrosis after BAC treatment at high concentrations and by apoptosis if low concentrations of BAC are applied. This new aspect of in vitro toxicity of BAC could in part explain some ocular surface disorders observed in patients undergoing long-term topical treatments with preservative-containing drugs.

Aim: A crossover, randomised double blind study was undertaken in 30 healthy volunteers, in order to compare the tolerance of 2% carteolol with and without preservative in short term use., Methods: Complete ophthalmic examinations were performed before and 30, 60, and 180 minutes after instillation of one drop of the solution, and after 3 days of preservative treatment. After a 5 day washout, the same examinations were done with the second drug., Results: Results showed good general tolerance for both formulations. No significant difference in subjective tolerance, corneal aesthesiometry, punctuate keratitis, Schirmer's test, intraocular pressure (IOP) decrease (about 25% in the two groups at 3 hours, 10% after 3 days of treatment), resting cardiac frequency, or blood pressure was observed. However, break up time was significantly reduced from baseline by preserved carteolol both at 3 hours (10.40 (5.9) seconds to 6.15 (3.9) seconds, p = 0.001) and after 3 days (7.72 (5.5) seconds, p = 0.04). Preservative free carteolol did not significantly change the break up time (baseline 9.08 (5.7) seconds; 3 hours = 7.88 (5.5) seconds, not significant; day 3 = 8.35 (5.8), non-significant)., Conclusions: These results confirm that carteolol is well tolerated, either with or without preservative. The preservative free group showed better stability of the tear film, without loss of effect on IOP. This difference, although mild in the healthy young subjects in the present study could be much more relevant in those patients treated long term, older patients, and/or those suffering from ocular surface disorders. In such instances, preservative free drugs could be of potential benefit to protect the lacrimal fluid integrity and corneoconjunctival surface.

An increasing number of studies, both experimental and epidemiologic, have provided evidence that filtering glaucoma surgery may be less effective than initially described. Of a number of risk factors for failure, duration and number of antiglaucoma drugs prior to surgery seem to play a critical role and highly accumulated antiglaucoma topical treatments significantly reduce success rates. Histopathological studies have confirmed that topically applied drugs may exert toxic effects to the corneoconjunctival surface, and induce chronic infraclinical inflammation, as shown by the presence of immune and inflammatory infiltrates in multitreated eyes. The origin of topical inflammation has not yet been clearly determined, but a common component of ophthalmic drugs, the benzalkonium chloride used as preservative in almost all antiglaucoma preparations, has shown strong evidence of toxicity. A number of questions remain to be investigated, but suppression of preservatives from chronically applied drugs should be a critical issue in the near future.