Françoise Brignole-Baudouin

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

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.

In Vitro Effects of Preservative-Free Tafluprost and Preserved Latanoprost, Travoprost, and Bimatoprost in a Conjunctival Epithelial Cell Line

Purpose: This study compared the toxicity profiles of three antiglaucoma prostaglandin F2α 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.

A multicentre, double-masked, randomized, controlled trial assessing the effect of oral supplementation of omega-3 and omega-6 fatty acids on a conjunctival inflammatory marker in dry eye patients

Purpose:  To determine whether oral supplementation with omega‐3 and omega‐6 fatty acids can reduce conjunctival epithelium expression of the inflammatory marker human leucocyte antigen‐DR (HLA‐DR) in patients with dry eye syndrome (DES).

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

Aims Using an established three-dimensional (3D) toxicological model based on reconstituted human corneal epithelium (HCE), this study investigated the tolerability of four topical intraocular-pressure-lowering agents: the commercial solutions of benzalkonium chloride (BAC)-containing 0.005% latanoprost, 0.004% travoprost, 0.03% bimatoprost containing 0.02%, 0.015% and 0.005% BAC, respectively, and the preservative-free (PF) tafluprost. Solutions of 0.01% and 0.02% BAC alone were also evaluated for comparison. Methods The 3D-HCEs were treated with solutions for 24 h followed or not by a 24 h recovery period. We used a modified MTT (3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) procedure to assess cell viability in the HCE. Frozen sections of HCE were analysed using fluorescence microscopy for the evaluation of apoptosis (terminal deoxynucleotidyl transferase mediated dUTP nick end labelling), inflammation (ICAM-1) and proliferation (Ki67). Corneal epithelial tight junctions (occludin and tight junction protein 1 (zona occludens 1)) were also assessed by en face confocal microscopy in response to the different eye-drops. Results The MTT test revealed that the cytotoxicity of antiglaucoma eye-drops was primarily related to the concentration of their common BAC preservative (0.02% BAC-latanoprost>0.015% BAC-travoprost>0.005% BAC-bimatoprost). PF-tafluprost did not induce any obvious cytotoxicity, showed the least expression of inflammatory or apoptotic markers and revealed preservation of membrane immunostaining of tight junction proteins in comparison with BAC-containing solutions. Conclusion The toxicological model of the 3D reconstructed corneal epithelia model confirmed the ocular surface cytotoxicity of BAC-containing antiglaucomatous solutions. Compared with the formulations containing the toxic preservative BAC, PF-tafluprost was well tolerated without inducing significant corneal epithelium deterioration.

Comparative Anatomy of Laboratory Animal Corneas with a New-Generation High-Resolution In Vivo Confocal Microscope

Purpose: The aim of the current study was to compare the corneas of three commonly used laboratory animals with a new in vivo confocal microscope. Methods: Six eyes of three adult male New Zealand albino rabbits, six eyes of three adult male Lewis rats, and six eyes of three adult male Swiss mice were used in this study. Corneas were analyzed in vivo using the Rostock Cornea Module of the Heidelberg Retina Tomograph (HRT)-II. For all eyes, 20 confocal microscopic images of each layer, that is, the superficial and basal corneal epithelia, the Bowman layer, the anterior and posterior stroma, and the endothelium, were recorded. The images were then analyzed qualitatively and compared among animals. Cellular densities of anterior and posterior stroma keratocytes of rabbits and endothelium density of the three different animals were also measured and compared. Results: The Rostock Cornea Module of the HRT II was successfully used to analyze all corneal layers of these three commonly used laboratory animals. Although the cellular patterns of the corneal layers of these three animals, as observed with in vivo confocal microscopy, were quite similar, some differences were seen in terms of endothelial cell density and stroma appearance. Superficial cells were seen as hyper- and hyporeflective polygonal cells. Basal cells had dark cytoplasm without visible nuclei and were closely organized. A Bowman layer was observed in all three animals as an amorphous tissue containing fine subepithelial nerve plexus. In rabbits, the stroma consisted of an amorphous ground substance with hyper-reflective structures corresponding with keratocyte nuclei. In rats and mice, numerous reflective stellate structures with no clearly visible nuclei were observed within the stroma. Besides endothelial cell density, the endothelium was similar among the three animals and was seen as hyper-reflective cells with dark limits organized in a honeycomb pattern. Conclusions: The Rostock Cornea Module of the HRT II can provide high-resolution images of all corneal layers of rabbits, rats, and mice without sacrificing animals or preparing tissue. This new device may be useful for evaluating the cornea during experimental animal studies.

A New Safety Concern for Glaucoma Treatment Demonstrated by Mass Spectrometry Imaging of Benzalkonium Chloride Distribution in the Eye, an Experimental Study in Rabbits

We investigated in a rabbit model, the eye distribution of topically instilled benzalkonium_(BAK) chloride a commonly used preservative in eye drops using mass spectrometry imaging. Three groups of three New Zealand rabbits each were used: a control one without instillation, one receiving 0.01%BAK twice a day for 5 months and one with 0.2%BAK one drop a day for 1 month. After sacrifice, eyes were embedded and frozen in tragacanth gum. Serial cryosections were alternately deposited on glass slides for histological (hematoxylin-eosin staining) and immunohistological controls (CD45, RLA-DR and vimentin for inflammatory cell infiltration as well as vimentin for Müller glial cell activation) and ITO or stainless steel plates for MSI experiments using Matrix-assisted laser desorption ionization time-of-flight. The MSI results were confirmed by a round-robin study on several adjacent sections conducted in two different laboratories using different sample preparation methods, mass spectrometers and data analysis softwares. BAK was shown to penetrate healthy eyes even after a short duration and was not only detected on the ocular surface structures, but also in deeper tissues, especially in sensitive areas involved in glaucoma pathophysiology, such as the trabecular meshwork and the optic nerve areas, as confirmed by images with histological stainings. CD45-, RLA-DR- and vimentin-positive cells increased in treated eyes. Vimentin was found only in the inner layer of retina in normal eyes and increased in all retinal layers in treated eyes, confirming an activation response to a cell stress. This ocular toxicological study confirms the presence of BAK preservative in ocular surface structures as well as in deeper structures involved in glaucoma disease. The inflammatory cell infiltration and Müller glial cell activation confirmed the deleterious effect of BAK. Although these results were obtained in animals, they highlight the importance of the safety-first principle for the treatment of glaucoma patients.

Polyquad-preserved travoprost/timolol, benzalkonium chloride (BAK)-preserved travoprost/timolol, and latanoprost/timolol in fixed combinations: a rabbit ocular surface study.

IntroductionThe aim of this study was to use a validated acute rabbit model to test the toxicity of a novel formulation of fixed-combination travoprost 0.004%/timolol 0.5% ophthalmic solution, which contains the antimicrobial preservative polyquaternium-1 (PQ), compared with the commercial formulation of fixed combinations travoprost 0.004%/timolol 0.5% ophthalmic solution and latanoprost 0.005%/timolol 0.5% ophthalmic solution, which both contain the preservative benzalkonium chloride (BAK).MethodsAdult male New Zealand albino rabbits (n=24) were randomly divided into four groups. Phosphatebuffered saline (PBS), travoprost/timolol PQ, travoprost/timolol BAK, or latanoprost/timolol BAK were instilled onto rabbit eyes one drop, 15 times at 5 minute intervals. The ocular surface reactions were investigated at hour 4 and day 1 using slit lamp examination; in-vivo confocal microscopy (IVCM) for cornea, limbus, and conjunctiva-associated lymphoid tissue (CALT); conjunctival impression cytology; and standard immunohistology in cryosections for detecting CD45+ infiltrating cells and MUC-5AC-labeled cells.ResultsTravoprost/timolol PQ was better tolerated than travoprost/timolol BAK or latanoprost/timolol BAK. This improved tolerance was evident via clinical observation under slit lamp, IVCM in different layers of the cornea and conjunctiva, conjunctival impression cytology of superficial epithelium aspects, and immunohistochemistry for inflammatory infiltration of CD45+ cells in the cornea and goblet cell distribution. Travoprost/timolol PQ was similar to PBS in regards to in-vivo findings, the Draize test for ocular irritation, and epithelial and limbal aspects as evaluated with IVCM. Treatment with either travoprost/timolol PQ or PBS produced no obvious inflammatory infiltration inside and outside the CALT follicles, yielded similar IVCM toxicity scores and CD45+ cell counts, and eyes treated with either solution had normal goblet cells.ConclusionThe fixed combination of travoprost/timolol with 0.001% PQ had decreased ocular surface toxicity relative to the BAK-containing solutions. The potential benefit to the human ocular surface with oncedaily dosing needs to be evaluated clinically.

Reduced in vivo ocular surface toxicity with polyquad-preserved travoprost versus benzalkonium-preserved travoprost or latanoprost ophthalmic solutions.

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 elicit ocular 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.

In vitro and in vivo comparative toxicological study of a new preservative-free latanoprost formulation.

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.

Conjunctiva-associated lymphoid tissue (CALT) reactions to antiglaucoma prostaglandins with or without BAK-preservative in rabbit acute toxicity study.

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-freetravoprost preserved with the SofZia® system or BAK-freetafluprost 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.