A new safety concern for glaucoma treatment demonstrated by mass spectrometry imaging of benzalkonium chloride distribution in the eye, an experimental study in rabbits.
ABSTRACT: 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.
Project description: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.
Project description:Glaucoma patients often require long-term or even lifelong medical antiglaucomatous treatment. Benzalkonium chloride (BAK) is the most frequently used preservative in medical glaucoma treatment. Laser flare photometry is the noninvasive quantitative measurement of anterior chamber protein level and helps tracking intraocular inflammation. The purpose of our study was to evaluate the ocular aqueous humour flare in glaucoma patients, scheduled for cataract surgery without any other ocular diseases, and the association with pseudoexfoliation (PEX) syndrome, number of medications used, and BAK. A prospective case-control age- and gender-matched study, including open-angle glaucoma patients (>2 years of treatment) with cataract, matched with cataract patients with no other ocular pathology (control group). We found that the aqueous humour flare was higher in the glaucoma group than in the control group. PEX syndrome increased the aqueous humour flare independently from glaucoma diagnosis. The number of used antiglaucomatous medications correlated moderately with the aqueous humour flare. The BAK index showed weak positive correlation with aqueous humour flare. A variety of factors can affect aqueous humour flare increase, including PEX syndrome, medical substance used to treat glaucoma, number of different medications, and presence of BAK. The combination of these factors is of key importance to long-term glaucoma treatment.
Project description:INTRODUCTION:The preservative benzalkonium chloride (BAK) is used to preserve several topical, intraocular pressure (IOP)-lowering glaucoma medications but can cause tolerability concerns that may lead to decreased adherence to treatment and ultimately diminish the effectiveness of IOP control. The study aimed to determine the efficacy and tolerability of BAK-free travoprost preserved with polyquaternium-1 in glaucoma patients switched from BAK-preserved latanoprost or bimatoprost. METHODS:This 12-week, open-label study was conducted in Europe between December 2011 and February 2013. We enrolled adult patients with open-angle glaucoma or ocular hypertension who were receiving BAK-preserved latanoprost 0.005% or bimatoprost 0.01% and, in the opinion of the investigator, would benefit from transition to BAK-free travoprost 0.004% preserved with polyquaternium-1 because of tolerability concerns. Assessments included IOP, proportion of patients with IOP ?18 mmHg, ocular surface status, hyperemia, patient treatment preference, and adherence. Adverse events were recorded throughout the study. RESULTS:Of the 202 patients screened, 187 patients were included in the intent-to-treat population (mean age, 66.6 years; range, 19-90 years). The mean IOP significantly reduced from baseline (17.0 mmHg) to week 6 (mean change, -1.17 mmHg; P<0.001) and week 12 (-1.16 mmHg; P<0.001). At week 12, more patients achieved IOP ?18 mmHg (81.2% versus 73.3% at baseline), and ocular surface disease severity improved from baseline to week 12. Most patients preferred BAK-free travoprost (74.9%) versus their previous medication and were very confident in their adherence (84.1%). Reduced visual acuity and eye pruritus were the most common adverse events (2.5% each). CONCLUSION:BAK-free travoprost 0.004% preserved with polyquaternium-1 was efficacious and well tolerated and may be an advantageous prostaglandin analog option for patients with open-angle glaucoma or ocular hypertension who are intolerant to BAK-preserved latanoprost or bimatoprost.
Project description:The aim of the present study was to examine the effects of switching from Latanoprost ophthalmic solution containing a preservative to preservative-free Tafluprost ophthalmic solution or Tafluprost containing a preservative on ocular surfaces.Forty patients (40 eyes) with glaucoma (mean age: 62.0 ± 10.9 years) using Latanoprost with preservative for six months or longer were assigned either to a Tafluprost-containing-preservative group (20 eyes) or preservative-free-Tafluprost group (20 eyes). The intraocular pressure, corneal epithelial barrier function (fluorescein uptake concentration with fluorophotometer FL-500), superficial punctate keratopathy (AD classification), and tear film breakup time (TBUT) were assessed before switching and at 12 weeks after switching.No significant differences in intraocular pressure were noted after switching in either group. Corneal epithelial barrier function was improved significantly after switching in both the Tafluprost-containing-preservative and the preservative-free-Tafluprost groups. There were no significant differences in AD scores after switching in the Tafluprost-containing-preservative group, but significant improvements were noted in the preservative-free-Tafluprost group. No significant differences in TBUT were noted in the Tafluprost-containing-preservative or preservative-free-Tafluprost groups after switching.After switching from preservative Latanoprost to Tafluprost containing-preservative or preservative-free Tafluprost, corneal epithelial barrier function was improved while the intraocular pressure reduction was retained.
Project description:Latanoprost is a common glaucoma medication. Here, we study longitudinal effects of sustained latanoprost treatment on intraocular pressure (IOP) in C57BL/6J mice, as well as two potential side-effects, changes in iris pigmentation and central corneal thickness (CCT). Male C57BL/6J mice were treated daily for 16 weeks with latanoprost. Control mice were treated on the same schedule with the preservative used with latanoprost, benzalkonium chloride (BAK), or handled, without ocular treatments. IOP and CCT were studied at pre-treatment, 2 "early" time points, and 2 "late" time points; slit-lamp analysis performed at a late time point; and expression of corneal and iridial candidate genes analyzed at the end of the experiment. Latanoprost lowered IOP short, but not long-term. Sustained application of BAK consistently resulted in significant corneal thinning, whereas sustained treatment with latanoprost resulted in smaller and less consistent changes. Neither treatment affected iris pigmentation, corneal matrix metalloprotease expression or iridial pigment-related genes expression. In summary, latanoprost initially lowered IOP in C57BL/6J mice, but became less effective with sustained treatment, likely due to physiological adaptation. These results identify a new resource for studying changes in responsiveness associated with long-term treatment with latanoprost and highlight detrimental effects of commonly used preservative BAK.
Project description:PURPOSE:To investigate the usefulness of meibomian gland (MG) dropout rate in the evaluation of MG morphological change associated with the use of prostaglandin for glaucoma treatment through the association between MG and the ocular surface parameters and medication duration and presence of preservative. METHODS:This cross-sectional study was conducted on 88 eyes of 88 patients who were diagnosed with glaucoma and used only Tafluprost as treatment. The patients were divided into four "user" groups: 1) 23 patients used preservative-free (PF) Tafluprost for 6 months; 2) 21 patients used preservative-containing (PC) Tafluprost for 6 months; 3) 23 patients used PF-Tafluprost for 24 months; 4) 21 patients used PC-Tafluprost for 24 months. Ocular surface parameters and the MG condition, including MG dropout rate and meiboscale, were evaluated. Multiple regression was used to identify associations. RESULTS:There were significant differences in age (p = 0.003), tear breakup time (p = 0.016), lid margin abnormality (p = 0.016), expressibility (p = 0.039), meiboscale (p<0.001), and MG dropout rate (p<0.001) among the 4 groups. MG dropout rate and meiboscale showed significant differences in all post hoc analyses, except for the comparison between the PF-Tafluprost and PC-Tafluprost 6-month user groups. Medication duration, preservative status, and meiboscale were significantly correlated with MG dropout rate (p<0.001, p = 0.024, p<0.001, respectively). In the 6-month user group, preservative status significantly correlated with MG dropout rate (p = 0.015). However, in the 24-month user group, meiboscale was the only parameter significantly associated with MG dropout rate (p<0.001). CONCLUSION:MG dropout rate in patients using Tafluprost showed a significant correlation with medication duration and preservative status. This result indicates MG dropout rate reflects MG morphologic change associated with prostaglandin.
Project description:OBJECTIVES:Bimatoprost-timolol (bimatoprost 0.03%-timolol 0.5% fixed-dose combination [FDC]) and tafluprost-timolol (tafluprost 0.0015%-timolol 0.5% FDC) eye drops are currently the only topical intraocular pressure (IOP)-reducing therapies available as preservative-free (PF) prostaglandin and timolol FDC. The aim of this study was to investigate changes to ocular signs and symptoms when patients with ocular hypertension (OH) or open-angle glaucoma (OAG) switched from PF or benzalkonium chloride (BAK)-preserved bimatoprost-timolol to PF tafluprost-timolol eye drops. DESIGN:This was a 12-week, open-label, phase IV study. SETTING:Sixteen centres in Finland, Germany, Italy and the UK. PARTICIPANTS:Patients with OH or OAG (IOP on medication ?21?mm Hg), treated with PF or BAK-preserved bimatoprost-timolol for ?4 weeks before screening, and presenting with conjunctival hyperaemia and ?1?ocular symptom. INTERVENTIONS:Patients were switched to PF tafluprost-timolol once daily in the treated eye(s). PRIMARY AND SECONDARY OUTCOME MEASURES:The primary endpoints were change from screening to week 12 in conjunctival hyperaemia and worst ocular symptom. The secondary outcome measures were changes from screening in ocular signs (other than conjunctival hyperaemia) and symptoms at week 12. RESULTS:Of 123 enrolled patients, 121 were included in the intention-to-treat dataset, of which all were Caucasian and 54.5% were female; 76 patients used BAK-preserved bimatoprost-timolol and 45 used PF drops. Conjunctival hyperaemia and severity of worst ocular symptom following switch to PF tafluprost-timolol significantly reduced from screening to week 12 in all patients (p<0.001). The percentage of patients with ocular signs and symptoms was significantly reduced at week 12 compared with screening (p<0.001). IOP was not affected by the change of treatment. CONCLUSIONS:Switching from BAK-preserved or PF bimatoprost-timolol to tafluprost-timolol reduced both signs and symptoms of ocular surface disease with no clinically relevant effect on IOP. TRIAL REGISTRATION NUMBER:EudraCT2014-005273-37; Results.
Project description:Müller cells create the external limiting membrane (ELM) by forming junctions with photoreceptor cells. This study evaluated the relationship between focal photoreceptors and RPE loss in geographic atrophy (GA) and Müller cell extension into the subretinal space.Human donor eyes with no retinal disease or geographic atrophy (GA) were fixed and the eye cups imaged. The retinal posterior pole was stained for glial fibrillary acidic protein (GFAP; astrocytes and activated Müller cells) and vimentin (Müller cells) while the submacular choroids were labeled with Ulex Europaeus Agglutinin lectin (blood vessels). Choroids and retinas were imaged using a Zeiss 710 confocal microscope. Additional eyes were cryopreserved or processed for transmission electron microscopy (TEM) to better visualize the Müller cells.Vimentin staining of aged control retinas (n = 4) revealed a panretinal cobblestone-like ELM. While this pattern was also observed in the GA retinas (n = 7), each also had a distinct area in which vimentin+ and vimentin+/GFAP+ processes created a subretinal membrane. Subretinal glial membranes closely matched areas of RPE atrophy in the gross photos. Choroidal vascular loss was also evident in these atrophic areas. Smaller glial projections were noted, which correlated with drusen in gross photos. The presence of glia in the subretinal space was confirmed by TEM and cross cross-section immunohistochemistry.In eyes with GA, subretinal Müller cell membranes present in areas of RPE atrophy may be a Müller cell attempt to replace the ELM. These membranes could interfere with treatments such as stem cell therapy.
Project description:Background:Benzalkonium chloride (BAK), commonly used in glaucoma treatment, is an eye drop preservative with dose-dependent toxicity. Previous studies have observed the multi-functional benefits of angiogenin (ANG) against glaucoma. In our study, we evaluated ANG's cytoprotective effect on the trabecular meshwork (TM) damage induced by BAK. Additionally, we developed a plant-derived ANG fusion protein and evaluated its effect on TM structure and function. Methods:We synthesized plant-derived ANG (ANG-FcK) by fuzing immunoglobulin G's Fc region and KDEL to conventional recombinant human ANG (Rh-ANG) purified from transgenic tobacco plants. We established a mouse model using BAK to look for degenerative changes in the TM, and to evaluate the protective effects of ANG-FcK and Rh-ANG. Intraocular pressure (IOP) was measured for 4 weeks and ultrastructural changes, deposition of fluorescent microbeads, type I and IV collagen, fibronectin, laminin and ?-SMA expression were analyzed after the mice were euthanized. Results:TM structural and functional degeneration were induced by 0.1% BAK instillation in mice. ANG co-treatment preserved TM outflow function, which we measured using IOP and a microbead tracer. ANG prevented phenotypic and ultrastructure changes, and that protective effect might be related to the anti-fibrosis mechanism. We observed a similar cytoprotective effect in the BAK-induced degenerative TM mouse model, suggesting that plant-derived ANG-FcK could be a promising glaucoma treatment.
Project description:Purpose: Adenoviral conjunctivitis is the most common cause of conjunctivitis worldwide with no approved antiviral treatment. Benzalkonium chloride (BAK) is a common preservative in ophthalmic medications and is the active ingredient in some skin disinfectants and hand sanitizers. BAK is known to be effective in killing bacteria and enveloped viruses; however, its activity against ocular types of nonenveloped adenoviruses (Ads) is unknown. The goal was to determine whether BAK is an effective antiviral agent against common human ocular types of adenovirus in vitro. Methods: The direct inactivating activity of BAK was determined by incubating several human adenovirus types with BAK concentrations of 0.001%, 0.003%, 0.005%, 0.01%, 0.1%, and 0% for 1?h at 33°C. Resulting adenovirus titers were determined after treatment. Decreases in titers of ?3 Log10 were considered virucidal, while decreases in titers of <1 Log10 were considered ineffective. Results: BAK 0.1% was virucidal for Ad3, Ad5, Ad7a, Ad19/64, and Ad37, while it reduced titers >1 Log10, but <3 Log10 for Ad4 and Ad8. Decreases in titers >1 Log10 were demonstrated for BAK 0.003%, 0.005%, and 0.01% for Ad5 only. Decreases in titers for the other adenovirus types for those concentrations were ?0.53 Log10. 0.001% BAK produced minimal decreases in titers for all types. Conclusions: BAK, at 0.01% or less was not consistently effective as an antiviral against adenovirus, but higher concentrations, such as 0.1%, should be further investigated as a possible topical treatment for adenoviral ocular infections, providing ocular toxicity is not an issue.