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ENGLISH SUMMARY: Glaucoma is a leading cause of the global prevalence of irreversible blindness. The pathogenesis of glaucoma is not entirely known, but the major risk factors include advancing age, genetic predisposition, and increased intraocular pressure (IOP). The only evidence‐based treatment is a lowering of IOP through the use of eye drops, laser procedures, or surgical interventions. Although laser treatment is gaining recognition as a first‐choice treatment option, the most common approach for managing glaucoma is IOP‐lowering eye drops. A major challenge in the treatment is the occurrence of adverse events and poor adherence. In this context, the ocular surface is an area of great concern, as most glaucoma patients have dry eye disease (DED), which is largely caused by eye drops. Preservation with benzalkonium chloride (BAK) is a controversial topic due to its potential role as a significant cause of DED. A systematic review and meta‐analyses investigate potential differences in efficacy and safety between BAK‐preserved and BAK‐free anti‐glaucomatous eye drops (I). Many of the included studies report on ocular surface damage caused by the application of BAK‐preserved eye drops. However, the meta‐analyses addressing hyperemia, number of ocular adverse events, and tear break‐up time did not identify any significant differences. The latter is likely due to varying measurement methods, different endpoints, and study durations. It is, therefore, possible that the large variations between the studies conceal differences in the safety profiles. The efficacy meta‐analysis finds that there are no differences in the IOP‐lowering effect between BAK‐preserved and BAK‐free eye drops, indicating that BAK is not necessary for the effectiveness of eye drops. To promote more homogeneous choices of endpoints and methods when evaluating BAK‐preserved and BAK‐free glaucoma treatments, a Delphi consensus statement was performed. In this study, glaucoma experts and ocular surface disease experts reached consensus on the key factors to consider when designing such studies (II). The hope is to have more studies with comparable endpoints that can systematically show the potentially adverse effects of BAK. The preclinical studies in the current Ph.D. research focus on conjunctival goblet cells (GCs). GCs are important for the ocular surface because they release the mucin MUC5AC, which is an essential component of the inner layer of the tear film. BAK preservation may damage the GCs and result in a low GC density, leading to an unstable tear film and DED. The most commonly used IOP‐lowering drugs are prostaglandin analogs (PGAs). Thus, the conducted studies investigate the effect of PGAs preserved in different ways on GCs. BAK‐preserved latanoprost is cytotoxic to primary cultured human conjunctival GCs and results in a scattered expression of MUC5AC, in contrast to negative controls, where MUC5AC is localized around the cell nucleus (III). Preservative‐free (PF) latanoprost is not cytotoxic and does not affect the MUC5AC expression pattern. Furthermore, BAK‐preserved travoprost is found to be cytotoxic in a time‐dependent manner, while Polyquad®‐preserved travoprost does not affect GC survival at any measured time point (IV). Both Polyquad and BAK induce scattered expression of MUC5AC. The cytotoxicity of BAK‐preserved PGA eye drops is higher compared to the safer profile of PF and Polyquad‐preserved PGA eye drops (V). Additionally, PF latanoprost does not increase the release of the inflammatory markers interleukin (IL)‐6 and IL‐8, unlike BAK‐preserved latanoprost. A review highlights the active and inactive components of IOP‐lowering eye drops (VI). Several preclinical and clinical studies have identified adverse effects of BAK. Although other components, such as the active drug and phosphates, can also cause adverse events, the review clearly states that BAK alone is a major source of decreased tolerability. The conclusion of this thesis is that BAK preservation is unnecessary and harmful to the ocular surface. The preclinical studies demonstrate that GCs die when exposed to BAK. Furthermore, they find that BAK induces a pro‐inflammatory response. The review included in the thesis concludes that BAK should be phased out of eye drops for chronic use. Overall, the inclusion of BAK poses a risk of developing DED and poor adherence, which can ultimately lead to disease progression and blindness. [ABSTRACT FROM AUTHOR]

Purpose: To investigate the effect of polyquaternium‐1 (PQ)‐preserved and benzalkonium chloride (BAK)‐preserved travoprost eye drops on viability of primary human conjunctival goblet cell (GC) cultures and on secretion of mucin and cytokines. Furthermore, to evaluate the physicochemical properties of the branded travoprost eye drop Travatan® and available generics. Methods: The effect of travoprost eye drops was evaluated on GC cultures. Cell viability was assessed through lactate dehydrogenase (LDH) and tetrazolium dye (MTT) colorimetric assays. Mucin secretion was evaluated by immunohistochemical staining. Secretion of interleukin (IL)‐6 and IL‐8 was measured using BD Cytometric Bead Arrays. pH, viscosity, droplet mass, osmolality and surface tension were measured for all included eye drops. Results: In the LDH assay, BAK travoprost caused significant GC loss after 2 hrs of incubation compared to the control. PQ travoprost caused no GC loss at any time point. Both PQ‐ and BAK travoprost caused secretion of mucin to the cytoplasma. No difference in IL‐6 and IL‐8 secretion was identified compared to controls. The pH values for the generics were lower (pH 6.0) than the pH value for Travatan (pH 6.7; p < 0.0001). The viscosity was lowest for Travatan, while the mean droplet mass was higher for Travatan (35 mg) than the generics (28–30 mg; p ≤ 0.0318). The osmolality and surface tension did not differ between the eye drops investigated. Conclusion: BAK travoprost caused GC loss, indicating that PQ preservation may be preferable in treatment of glaucoma. Furthermore, physicochemical properties of branded and generic travoprost eye drops can not be assumed to be identical. [ABSTRACT FROM AUTHOR]

Purpose: To investigate whether contact lenses used after surgery for congenital cataracts act as a depot for dexamethasone, which would allow the prescribed amount of drops to be reduced, and to examine whether the preservative benzalkonium chloride accumulates in the contact lens matrix, which would suggest a need for more frequent replacements. Methods: Contact lenses (n = 10) worn by infants treated with dexamethasone eye drops after congenital cataract surgery were analysed with scanning electron microscopy, UV‐vis, 1H‐NMR and LDI‐MS for chemical deposits and for changes on the contact lens surface. Unused lenses (n = 5) and lenses (n = 4) from patients with no eye drop treatment were analysed as reference. Results: The treated contact lenses displayed ruptured surfaces in comparison with unused and reference lenses. Dexamethasone and BAK were not detected in any of the lenses. A polyethylene oxide component was found in the treated lenses, likely originating from the dexamethasone eye drops or the contact lens solution. Conclusion: Dexamethasone and BAK do not accumulate in the contact lenses, and a depot effect of any clinical significance is unlikely. Therefore, the number of drops given after surgery should remain the same regardless of whether the child has contact lenses. The ruptured surface may both decrease the child's comfort and increase the risk of microbial adhesion, and so it is recommended that contact lenses should be replaced once a month throughout the course of anti‐inflammatory eye drop treatment after surgery for congenital cataract. [ABSTRACT FROM AUTHOR]

Benzalkonium chloride (BAK) is a common preservative found in glaucoma medications used to increase shelf‐life, prevent bacterial contamination, and enhance ocular penetration. Its detergent properties kill microorganisms by disrupting cell membranes and causing cell lysis. Unfortunately BAK also has potentially harmful cytotoxic effects, particularly for the ocular surface, with evidence of BAK‐induced apoptosis of conjunctival and corneal epithelial cells, decreased density of conjunctival goblet cells, delayed corneal wound healing and reduced density of corneal nerved. It may also affect deeper ocular structures including the lens and trabecular meshwork, with BAK shown to cause apoptosis of human trabecular meshwork cells in culture. Clinical consequences may include increased risk of ocular surface disease, reduced adherence, and with long‐term use, reduced success of glaucoma surgery. [ABSTRACT FROM AUTHOR]

Ocular surface disease (OSD) is a major and often unrecognized issue in patients with glaucoma. The prevalence of ODS symptoms in glaucoma patients is between 48% and 59%. The prevalence of ODS signs in glaucoma patients stretches from 22% for corneal fluorescein staining until 78% for tear film BUT. Possible risk factors for OSD in glaucoma include female gender, higher age, pre‐existing DED, glaucoma itself, glaucoma type, glaucoma duration, number of glaucoma medications and frequency of instillation, active ingredient of glaucoma drug, preservatives esp. Benzalkonium chloride in glaucoma drug, and glaucoma filtration surgery. [ABSTRACT FROM AUTHOR]

Aims/Purpose: To investigate the effect of benzalkonium chloride (BAK)‐preserved latanoprost and bimatoprost, polyquad (PQ)‐preserved travoprost, and preservative‐free (PF) latanoprost and tafluprost, all prostaglandin analogues (PGAs), on human conjunctival goblet cell (GC) survival. Furthermore, to investigate the effect of BAK‐preserved and PF latanoprost on the cytokine secretion from GC. Methods: Primary human conjunctival GCs were cultivated from donor tissue. Lactate dehydrogenase (LDH) and tetrazolium dye colorimetric (MTT) assays were used for the assessment of GC survival. A cytometric bead array was employed for measuring secretion of interleukin (IL)‐6 and IL‐8 from GC. Results: BAK‐preserved latanoprost and bimatoprost reduced cell survival by 28% (p = 0.0133) and 20% (p = 0.0208), respectively, in the LDH assay compared to a negative control. BAK‐preserved latanoprost reduced cell proliferation by 54% (p = 0.003), BAK‐preserved bimatoprost by 45% (p = 0.006), PQ‐preserved travoprost by 16% (p = 0.0041), and PF latanoprost by 19% (p = 0.0001), in the MTT assay compared to a negative control. Only PF tafluprost did not affect the GCs in either assay. BAK‐preserved latanoprost caused an increase in the secretion of pro‐inflammatory IL‐6 and IL‐8 (p = 0.0001 and p = 0.0019, respectively) compared to a negative control, which PF latanoprost did not. Conclusions: BAK‐preserved PGA eye drops were more cytotoxic to GCs than PQ‐preserved and PF PGA eye drops. BAK‐preserved latanoprost induced an inflammatory response in GC. Treatment with PF and PQ‐preserved PGA eye drops could mean better tolerability and adherence in glaucoma patients compared to treatment with BAK‐preserved PGA eye drops. [ABSTRACT FROM AUTHOR]

Aims/Purpose: To investigate the cell survival of human conjunctival goblet cells (CGs) when incubated with preserved and preservative‐free (PF) artificial tears (AT) used against dry eye disease (DED). The following ATs were tested: 0.011% benzalkonium chloride (BAK)‐preserved Viskøse® Øjendråber, 0.006% BAK‐preserved Oftagel®, 0.005% BAK‐preserved Oculac®, 0.001% PolyQuad™‐preserved Systane® Ultra, 0.01% Cetrimide‐preserved Artelac®, and PF ATs: Systane® Ultra, Oculac®, Oftagel®, and Thealoz® Duo. Methods: Primary cultures of human conjunctival CGs were cultivated from donor conjunctiva. After incubation with the above‐mentioned ATs for 2 h, CG survival was analysed using a lactate dehydrogenase assay and calculated to a negative control. Results: AT preserved with 0.011% BAK reduced GC survival by 28% compared to negative control (p = 0.001). The remaining ATs did not significantly alter the cell survival. Conclusions: BAK‐preserved ATs cause GC death in a concentration‐dependent manner. Reduction of the GC density may contribute to development of DED. PF‐ATs and PolyQuad‐ and Cetrimide‐preserved ATs may thus be a better choice for treating DED than BAK preserved ATs, as the latter may add to the ocular surface disease. [ABSTRACT FROM AUTHOR]

Purpose: The majority of dry eye disease (DED) patients suffer from the tear film lipid layer (TFLL) instability issues associated with increased evaporation of water from the ocular surface. Therefore, we developed a liposomal formulation based on selected TFLL lipid classes that would restore the tear film stability and retard the evaporation of the aqueous tear fluid. Methods: The liposomal formulation was prepared by the thin film hydration method and subsequently characterized intermittently by standard techniques for at least 6 months. The mitochondrial metabolic activity (MTT) assay with human corneal epithelial (HCE) cells was utilized to assess the formulation toxicity in vitro. The evaporation resistance properties of the formulation were determined in vitro by the Langmuir trough methods, while a DED cellular model engendered by exposing the HCE cells to benzalkonium chloride (BAC) enabled additional assessment of the in vitro efficacy. In vivo safety was evaluated in rats and rabbits in one‐week studies. The conditions of the animal eyes were assessed by a non‐invasive biomicroscope, an optical coherence tomography, and histological staining of the dissected eyes. Results: The lipid‐based formulation with favourable pharmaceutical and functional properties (e.g., the evaporation resistance, and the promotion of HCE cell recovery after damage) was developed successfully, while also the physical stability of the formulation was found promising. No evidence of toxicity was observed during in vitro and in vivo experiments. Conclusions: The results suggest that the developed formulation is well‐tolerated and possesses properties and a safety profile compatible with ocular administration. Utilized in vitro efficacy models imply that the developed formulation is a promising candidate towards more efficient DED treatment. In vivo efficacy remains to be clarified in future studies. [ABSTRACT FROM AUTHOR]

Aims/Purpose: To investigate the use of artificial tears (AT), identify which eye diseases are most prevalent among the users, determine which products are preferred by the population, and analyse their preservative content. Methods: Data originate from the FOREVER questionnaire. Respondents are customers from Synoptik A/S aged from 18 years and of both genders, and have given written consent for the use of their data. All 48 AT in the questionnaire appear in The Nordic Guidelines Dry Eye Disease 2022. T‐test and chi2 were performed to examine significant differences in the data. Results: 41.723 participants had answered the questionnaire, 15.613 men and 26.110 women, with a mean age of 55.4 (15.0) years. 10.1% reported having an eye disease diagnosed by a doctor, the most prevalent being cataract, then glaucoma, vitreous detachment and age‐related macular degeneration (AMD). 5129 (12.3%) used AT. This subpopulation consisted of 1268 men and 3861 women with a mean age of 61.2 (14.8) years. 21.8% had an eye disease diagnosed by a doctor. The most prevalent eye disease remained cataract, then glaucoma, vitreous detachment and AMD. Thealoz Duo (preservative free (PF)) was the most used brand, then Systane Ultra (PF or PolyQuad 0.001%) and Viscous Eye Drops (benzalkonium chloride (BAK) 0.011%). It was possible to identify 2560 respondents using a PF product and 874 using a product with a preservative. The mean age was significantly higher among those using AT, compared with those who did not use AT (54.6 ± 14.8 years) with p < 2.2·10−16. Also, a significant distribution between eye disease and the use of AT was found (p < 2.2·10−16). Conclusions: Around 12% of the population used AT. Their mean age was significantly higher than those not using AT (61.2 years compared to 54.6 with p < 2.2·10−16) and the prevalence of eye disease was also significantly higher (21.8% compared to 10.1%, p < 2.2·10−16), but the four most common eye diseases was similar in both groups. [ABSTRACT FROM AUTHOR]

Purpose: The aim of the study was to evaluate the presence of alterations in the tear film osmolarity and in other parameters that can define the OSD in POAG/OH patients treated with different topical therapies in order to better define the role of osmolarimetry in the clinical management of patients in a real life setting. Methods: This is a cross‐sectional, observational study. All patients with POAG/OH treated with topical medications and 20 subjects age and sex matched as a control group, were consecutively and prospectively enrolled. The following exams were performed: tear film osmolarity assessed using Tearlab®, tear break‐up time (TBUT), corneal staining score (Oxford scale), ocular symptom assessment using Ocular Surface Disease Index (OSDI) questionnaire. These data were compared between POAG/OH patients and control group and correlations were analysed between tear film osmolarity and other DED parameters. Results: 50 POAG/OH patients (25 M/25F; mean age 69 ± 17y) and 20 controls, matched for age and sex, were enrolled. The mean treatment duration was 2.5 ± 1.2 years. Benzalkonium chloride (BAK) was used in 92% of cases. The mean osmolarity value (mOsms/L) in POAG/OH patients was 306 ± 12 versus 296 ± 12 in control group (p < 0.05). TBUT (sec) was 5 ± 2 versus 9 ± 2 (p < 0.05). OSDI ranged from 0 to 96 and the mean value was 18 ± 19 in the study group vs 16 ± 14 in controls (NS). No significant difference was found in corneal staining between the two groups. In POAG/OH eyes tear film osmolarity values were ≤308 in 17/50 (34%) patients and in control group were 4/20 (20%). TBUT<8 s in 41/50(82%) vs 5/20 (25%). OSDI was <12 in 50% of cases vs 55% of controls, ranging from 13 to 32 in 36% versus 30% and from 33 to 100 in 14% versus 20%; corneal staining >1 in 12% versus 10%. Tear osmolarity was significantly correlated to TBUT (r = 0.11; p = 0.05). Conclusions: The differences between OSM and TBUT are statistically significant between the two groups. OSDI score difference is not significant, but the self‐administered questionnaire has inherent limitations. Tear film osmolarimetry has the advantage of providing a quantitative, operator‐independent indication of possible ocular surface dysfunction and it may be useful in the real life practice of a glaucoma unit in identifying the most vulnerable patients who might benefit from a switch to BAK‐free preparations. [ABSTRACT FROM AUTHOR]

Purpose: To evaluate the protective effect of different concentrations of sodium hyaluronate (SH) on the Benzalkonium chloride (BAK)‐induced toxicity using an in vitro model. Methods: The NAV14 cell line (SV40‐immortalized murine conjunctival epithelium) was used. Cell monolayers were exposed to different combinations of BAK (0.001%; 0.005%; 0.01%) and SH (0.2%; 0.3%; 0.4%) for 15 minutes; then cells were washed, and fresh culture media was added. Cell viability was evaluated after 2 h by resazurin reduction and lactate dehydrogenase (LDH) release. Also, cell migration and proliferation over 24 hours were determined by the scratch wound‐healing assay. Data were analysed by two‐way ANOVA and are shown as mean ± SD of two independent experiment with 4–6 replicates each. Results: BAK induced a concentration‐dependent decrease on cell viability (BAK 0.001%: 91 ± 14%, BAK 0.005%: 45 ± 9% and BAK 0.01%: 22 ± 10% of control cells, p < 0.001) and an increase in LDH release (no BAK:0.22 ± 0.02, BAK 0.001%: 0.31 ± 0.02, BAK 0.005%: 1.14 ± 0.05 and BAK 0.01%: 1.21 ± 0.05, p < 0.001). Conversely, SH neutralized these effects also in a concentration‐dependent manner (p < 0.001). In the presence of SH 0.4% (highest effect), cell viability was BAK 0.001%: 104 ± 22%, BAK 0.005%: 109 ± 9% and BAK 0.01%: 75 ± 13% of control cells (p < 0.001 for BAK 0.005–0.01%) while LDH release was no BAK: 0.24 ± 0.03, BAK 0.001%: 0.26 ± 0.01, BAK 0.005%: 0.37 ± 0.02 and BAK 0.01%: 0.49 ± 0.22, (vs. no SH: p < 0.001 for BAK 0.005–0.01%). BAK also reduced wound closure in vitro (after 24 h, no BAK: 76 ± 14%, BAK 0.001%: 42 ± 12%, BAK 0.005%: 17 ± 16% and BAK 0.01%: 0.0% wound closure, p < 0.001 for all BAK). Conversely, SH neutralized this effect in a concentration‐dependent fashion (p < 0.001). In the presence of SH 0.4% (highest effect), wound closure at 24 h was: no BAK: 81 ± 15%, BAK 0.001%: 58 ± 6%, BAK 0.005%: 63 ± 10%, BAK 0.01%: 60 ± 8% (vs no SH: p < 0.001 for BAK 0.005–0.01%). Conclusions: Sodium hyaluronate neutralized BAK toxicity on conjunctival epithelial cells in a concentration‐dependent manner. SH 0.4% was even protective at the highest preservative concentrations. These findings support the use of SH to mitigate BAK toxicity in long‐term antiglaucomatous treatment, although more studies are needed. [ABSTRACT FROM AUTHOR]

. Purpose: To assess the effect of SofZia-preserved travoprost on ocular surface conditions in comparison with benzalkonium chloride (BAK)-preserved latanoprost. Methods: A prospective randomized multicentre single-masked comparative study. Patients with open-angle glaucoma or ocular hypertension who had been treated with BAK-preserved latanoprost 0.005% (Xalatan®) monotherapy for at least 3 months. Patients were enrolled at 23 facilities. Patients were randomly divided into the X-X group, continuous use of Xalatan®, or the X-T group, switching from Xalatan® to SofZia-preserved travoprost 0.004% (TravatanZ®), and followed for 3 months. The superficial punctate keratopathy (SPK), conjunctival epitheliopathy, hyperaemia, tear break-up time (TBUT) and intraocular pressure (IOP) were examined for each patient in a masked manner. Changes in the frequency of keratoconjunctival epitheliopathy were evaluated 3 months after study initiation. Intra- and intergroup comparisons of changes in SPK, conjunctival epitheliopathy, hyperaemia, TBUT and IOP were also carried out. Results: Two hundred twenty patients participated and 215 completed the 3-month study. The frequency of keratoconjunctival epitheliopathy significantly decreased in the X-T group (p = 0.036) and the intergroup difference was also significant (p = 0.001). SPK scores and TBUT were significantly improved in the X-T group (p = 0.034, 0.049), also with significant intergroup differences in the cornea excluding the inferior area and TBUT. There were no significant intergroup differences in changes of the hyperaemia scores and the IOP reduction. Conclusion: Switching to SofZia-preserved travoprost after BAK-preserved latanoprost resulted in a lower incidence of keratoconjunctival epitheliopathy, especially in the cornea, with no clinically relevant changes in hyperaemia and IOP. [ABSTRACT FROM AUTHOR]

. Purpose: The deleterious effects of benzalkonium chloride (BAK) on the ocular surface are well known. However, few clinical data are available to prove a toxic effect at the level of the anterior chamber. The laser flare meter is a reliable tool to detect low levels of inflammation in the anterior chamber. We wanted to know whether instillation of BAK-preserved timolol in one eye would result in higher laser flare values than the instillation of preservative-free timolol in the fellow eye. Methods: Randomized prospective, single-masked clinical trial. Twenty-eight untreated patients with ocular hypertension were recruited. After obtaining baseline flare values, we randomly assigned one eye to BAK-preserved timolol and the fellow eye to preservative-free timolol. After 1 month, flare measurements were repeated. Results: There was a significant increase in the flare values in the two treatment regimens, but the increase in the BAK-treated eyes was higher than in the preservative-free treated eyes, and this difference in increase was statistically significant. Conclusion: This is the first study to show that short-term BAK administration produces inflammation in the anterior segment of previously untreated patients whose blood-aqueous barrier was not affected by recent intraocular surgery. [ABSTRACT FROM AUTHOR]

Purpose: To evaluate the cytotoxicity of a new Latanoprost Benzalkonium chloride (BAK)‐free nanoemulsion and to compare it with a Latanoprost solution containing BAK. Methods: Normal Human Conjunctival epithelial cells (IOBA‐NHC) were incubated for 15, 30, and 60 minutes with 1:6 dilutions of either latanoprost solution containing BAK (LSc), (latanoprost 0.005%, BAK 0.02%, Xalatan, Pfizer) or latanoprost nanoemulsion (LNe), (latanoprost 0.005%, potassium sorbate 0.18%; Louten Emulsion, POEN). Cytotoxicity was determined by MTT assay. Cell death was measured by flow cytometry using annexin V‐FITC and propidium iodide. Results: Cytotoxicity evaluation: the values of cell viability and proliferation obtained from cells exposed to LNe were between 80 and 90% relative to control group, whereas values obtained from cells exposed to LSc were around 30% relative to control group at all study times (p < 0.05 at 15 and 30 minutes; p < 0.01 at 60 minutes). Cell death evaluation: the percentage of viability was significantly lower in cells exposed to LSc compared with those incubated with LNe at all study times (p < 0.05 at 15 and 30 minutes; p < 0.01 at 60 minutes). Viability of cells exposed to LNe was higher than 90% at all study times, whereas there was a time‐dependent decrease in cells exposed to Lsc. The percentage of viable cells in the LNe group was comparable to the viability of control group (93.1%) at all study times. No statistical differences between groups reached in early apoptotic cells. A significant increase of late apoptosis and necrosis cells were observed at all study times for those incubated with Lsc (p < 0.05; p < 0.01 at 30 and 60 minutes). Conclusions: The new latanoprost nanoemulsion is significantly less cytotoxic on human conjunctival cells than LSc. It suggests that the new formulation could be gentler on the eye surface than currently available BAK preserved latanoprost solutions, with levels of cytotoxicity equivalent to control. [ABSTRACT FROM AUTHOR]

Purpose: To investigate the impact on human conjunctival goblet cell (GC) survival and mucin release for benzalkonium chloride (BAK) preserved and preservative‐free (PF) 0.005% (w/v) latanoprost (LT) eye drops, and to compare the eye drops' physicochemical properties. Methods: GC cultures were established from human conjunctival donor tissue. The impact of eye drops on GC survival was assessed using lactate dehydrogenase (LDH) and tetrazolium dye (MTT) colorimetric assays. Mucin release was evaluated through mucin‐specific immunostaining. pH‐value, osmolality, drop mass and surface tension for all LT eye drops were measured. Results: After application with PF‐LT for 30 minutes (min), the GC survival was maintained (p = 0.7043), while all BAK‐LT eye drops reduced survival to approximately 70% (p<0.0001). Following application with PF‐LT for 30 min, mucin was found around the GC nucleus, as seen in the vehicle control, indicating no secretion. In contrast, BAK‐LT caused diffuse staining of mucin, similar to the secretagogue histamine, indicating stimulation of secretion. The pH of the BAK‐LT and PF‐LT eye drops were 6.0‐6.9 and 6.8, respectively. The osmolality was 258‐288 mOsm/kg for the BAK‐LT eye drops and 276 for PF‐LT eye drop. The mean drop mass was 26‐31 mg for the BAK‐LT eye drops and 30mg for PF‐LT. The surface tension was lower for all BAK‐LT eye drops (31.1‐32.1 mN/m) compared to PF‐LT (42 mN/m). Conclusions: PF‐LT compared to various branded and generic LT preparations containing BAK should be used to improve goblet cell tolerability and ocular surface health. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Purpose: Prostanoid F2α (PF2α) analogues are commonly used as first-line treatment of glaucoma. Tafluprost is a newly synthesized PF2α derivative and represents the first PF2α analogue with a fully preservative-free formulation. Methods: A randomized, investigator-masked, single-centre, crossover phase I study evaluated the pharmacokinetics, efficacy and safety profiles of preserved and preservative-free tafluprost 0.0015% eyedrops in healthy volunteers. Both formulations were administered once/day for 8 days each. Plasma concentrations and, consequently, area under the curve (AUC0–last), maximum concentration ( Cmax) and time to maximum concentration ( tmax) were determined for tafluprost acid, the biologically active metabolite. Intraocular pressure, adverse events, and ocular and systemic safety parameters were analysed. Results: There were no statistically significant differences in pharmacokinetic parameters between preserved and preservative-free formulations after either single (day 1) or repeated (day 8) dosing. The mean (± standard deviation) results for preserved and preservative-free formulations on day 8 were, respectively: AUC0–last 581.1 ± 529.9 pg/min/ml versus 431.9 ± 457.8 pg/min/ml (p = 0.462); Cmax 31.4 ± 19.5 pg/ml versus 26.6 ± 18.0 pg/ml (p = 0.294), and median (range) tmax 10 (5–15) for both. Generally, plasma concentrations of tafluprost acid were low at all time-points and were cleared rapidly from the circulatory system. There were no unexpected safety findings. The incidence of ocular hyperaemia was similar in both formulations and was of predominantly moderate severity with preserved tafluprost and mild severity with preservative-free tafluprost. Conclusions: Preservative-free tafluprost appeared to have similar pharmacokinetic properties to the preserved formulation and was generally well tolerated. [ABSTRACT FROM AUTHOR]

Purpose: Tafluprost is a new prostaglandin F2α (PGF2α) derivative in development for the treatment of glaucoma. Tafluprost is the first PGF2α analogue with a preservative-free formulation. Methods: This randomized, investigator-masked, multicentre, crossover phase III study evaluated the pharmacodynamics and safety of preserved and preservative-free tafluprost 0.0015% eyedrops administered for 4 weeks in 43 patients with open-angle glaucoma or ocular hypertension. The primary variable was change from baseline in overall diurnal intraocular pressure (IOP) at 4 weeks. Adverse events and other safety parameters were also analysed. Results: Decreased IOP was clearly observed with both formulations at week 1 and was sustained until week 4. The overall treatment difference (preservative-free versus preserved formulations) at week 4 was 0.01 mmHg (95% confidence interval − 0.46 to 0.49; p = 0.96). There were no unexpected safety-related findings. Both formulations were well tolerated and most adverse events were ocular and mild in severity. Conclusions: The reduction in IOP achieved by preservative-free tafluprost is equivalent to that obtained with the preserved formulation. The preservative-free formulation was generally well tolerated. [ABSTRACT FROM AUTHOR]

Purpose: To investigate goblet cell (GC) survival after treatment with benzalkonium chloride (BAC)‐preserved anti‐glaucomatous eye drops. Conjunctival GCs produce mucin which stabilizes the tear film and protects against dry eyes. Eyedrop‐induced GC death may cause ocular surface disorders. Method: Latanoprost Teva (LT; prostaglandin analogue), Optimol® (OP; beta‐blocker), Dorzolamid Stada (DS; carbonic anhydrase inhibitor) and Brimondine tartrat Teva (BT; alfa2‐agonist) BAC‐preserved eye drops were used for treatment of ex vivo cultivated human GCs. The cell survival was evaluated by lactate dehydrogenase assay on human GCs after 30 min and 6 hr exposure to the different treatments and compared to the untreated control group. pH‐value and osmolality were also measured. Results: After 30 min of treatment, the human GC survival was 69.05% for LT, 69.78% for OP, 71.12% for DS and 93.42% for BT. The survival was significantly reduced only for the treatment with LT compared to the controls (p = 0.044). After 6 hr of exposure, GC survival was 31.08% for LT, 33.81 % for OP, 51.64 % for DS and 74.49 % for BT. The survival was significantly reduced for LT, OP, and DS compared to controls (p = 0.002, p = 0.004, p = 0.022). DS had the lowest pH (5.58), while OP the highest (6.84). LT had the lowest osmolality (267.6 mOsm/kg), while BT had the highest (293.2). There was no apparent correlation between GC survival, pH and osmolality. However, there seemed to be a connection between GC survival and BAC concentration. LT caused the lowest survival with a BAC concentration of 0.02%, while OP had a concentration of 0.01%, DS of 0.0075% and BT of 0.005%. Conclusion: Latanoprost Teva was the most cytotoxic, causing significant GC death after 30 min and 6 hr of treatment. OP and DS caused significant GC death only after 6 hr of exposure. BT did not affect the GC survival. The cytotoxicity appeared to correlate with the BAC concentration, but not pH and osmolality. [ABSTRACT FROM AUTHOR]

Purpose We present a new Label free quantitative imaging of small molecules in order to investigate their roles in ophthalmic disorders (toxicity, inflammation) Methods Quantitative Mass Spectrometry imaging has been developed and applied to ophthalmology to assess the distribution and quantification of Benzalkonium chloride (BAK) compound in specific areas of the eye after instillation. This method has been compared to gold standard technique as liquid chromatography ‐ Mass spectrometry to validate the data. Results The distribution of two BAK compounds (BAK C12 and BAK C14) were investigated in small specific histological regions of the eye (such as iridocorneal angle or sclera, choroid, retina regions) in order to estimate efficiency of action or adverse effects of the treatment. High spatial resolution images were performed at cells level (30 µm). Molecular distribution was also correlated to tissue histology using H&E staining. Then, our methodology of quantification by MSI was applied. Conclusion MSI offers new insight in ocular therapeutic/pharmaceutical research, especially for high‐precision distribution and quantification studies [ABSTRACT FROM AUTHOR]

Purpose To elucidate the effect of Benzalkonium chloride (BAK) on the intraocular pressure (IOP) lowering efficacy of a local ROCK‐inhibitor AMA0076 (Amakem NV) in the rabbit eye. Methods Topical administration of AMA0076 (0.1%, 0.3% and 0.5%) was tested in a normotensive New Zealand White rabbit model (5 rabbits/group). The IOP lowering efficacy of the compound was determined with or without the addition of 0.01% BAK. The contralateral eye was used as a control and was treated with vehicle (H2O PEG) in all groups. IOP was measured at baseline and at 1, 2, 3, 4, 5, 6, 7 and 8h post administration. Data at individual time points were analyzed using mixed model analysis for repeated measures. P < 0.05 was considered to be statistically significant. Results Single topical administration of AMA0076 significantly lowered IOP in a concentration dependent manner compared to the control eye (p<0.05). Placebo/vehicle administration did not induce significant changes in IOP. The maximal IOP lowering effect of AMA0076 0.1%, 0.3% and 0.5% containing 0.01% BAK was 38%, 45% and 53%, which was significantly stronger compared to their BAK‐free equivalents: 21%, 28% and 37% (p=0.005, p=<0.001; p=0.008 respectively). Conclusion The local ROCK inhibitor, AMA0076 was significantly more effective in lowering IOP in the presence of 0.01% BAK. [ABSTRACT FROM AUTHOR]

Purpose Friction is an inherent potential source of wounding for ocular surface tissues during the blink and bulbus movements and is identified as a major contributor to ocular surface disease of the dry eye type. The expression of lubricin, a boundary lubricant in articulating joints that protects against frictional forces, cell adhesion and protein deposition, was investigated at the ocular surface. Methods Human corneal, conjunctival and control cartilage tissues were fixed with 4% paraformaldehyde, embedded in paraffin, sectioned serially, exposed to antigen retrieval buffers, and stained for lubricin by immunohistochemistry (IHC) with a rabbit polyclonal anti‐human lubricin antibody. Several negative and positive controls were performed. Respective fresh tissues were analysed by reverse transcriptase polymerase chain reaction (RT‐PCR) in order to verify the presence of the mRNA. Results The mRNA for lubricin was found in human corneal and conjunctival cell lines and in complete ocular surface tissues similar to cartilage. The mRNA is translated and expressed into lubricin protein and protein staining was observed along the complete corneal and conjunctival epithelial surface, most strongly expressed in the corneal epithelium. The specificity of staining was verified in several positive and negative controls including use of irrelevant antibodies and preincubation of the anti‐lubricin antibody with a pure peptide, which had served to generate the antibody. Conclusion These findings verify that lubricin is synthesized and expressed by epithelial cells of the human cornea and conjunctiva and conceivably serves protective functions against frictional forces at the human ocular surface similar to the joint. [ABSTRACT FROM AUTHOR]

Purpose To characterize the phenotype, function and cytokine production of antigen presenting cells (APC) when exposed to the most common preservatives in eye drops, benzalkonium chloride (BAK). Methods APC were obtained from a human leukemia cell line THP‐1. APC were exposed to 4 concentrations of BAK (10‐5%, 5.10‐5%, 10‐4% and 5.10‐4%) and PBS during 24 hours. Cellular toxicity was evaluated with an annexin V‐PE/7‐AAD double‐staining flow cytometry analysis. Phenotype modification was evaluated by flow cytometry through a panel of cluster of differentiation: CD11b, CD11c, CD33, CD45, CD54 and CD86. Phagocytosis function was analyzed using carboxylate‐modified fluorescent microspheres and quantified by flow cytometry. The cytokine production of APC exposed to BAK was measured in supernatants by a human cytokine array. Results BAK had almost no cellular toxicity at concentrations below 5.10‐5%. A dose‐dependent cellular toxicity of BAK was observed from 5.10‐5% to 5.10‐4%. At low concentrations, BAK modified the phenotype of APC with an increased expression of CD11b, CD11c, CD54 and CD86, and a decrease of CD33 and CD45. APC phagocytosis function was also increased when exposed to low concentration of BAK. Cytokines in supernatants of APC exposed to 10‐5% BAK during 24 hours revealed decreased levels of IL‐1β and CXCL10, and increased levels of CD40L, CXCL11, G‐CSF, S‐TREM‐1, IL‐17E, IL‐6, IL‐23, IL‐27 and IFN‐γ. Conclusion The interaction between APC and epithelial conjunctival cells are involved in iatrogenic ocular surface diseases. Low concentrations of BAK could have a stimulating effect on APC, modifying phenotype, function and cytokine production. [ABSTRACT FROM AUTHOR]

Purpose Ocular Surface Disease (OSD), recognized in 50% of glaucoma patients, can decrease anti‐glaucoma therapy compliance. Benzalkonium chloride (BAK) has been shown to induce OSD. Catioprost® is an unpreserved latanoprost 0.005% cationic emulsion. The design of a study comparing the efficacy and safety of Catioprost® versus a BAK‐free prostaglandin agonist, Travatan Z® (travoprost 0.004%) in patients with glaucoma and OSD is described. Methods Patients with elevated IOP at baseline requiring treatment with ocular anti‐hypertensive therapy were enrolled in this multicenter, phase II, investigator‐masked, randomized study. Following a washout period, patients demonstrating OSD symptoms and signs (corneal fluorescein staining, CFS, score≥1 on the modified Oxford scale and a tear film break up time, TFBUT, ≤10) in at least one eye at the baseline visit were randomized to either Catioprost® or Travatan Z® QD with follow‐up study evaluations at month 1 and 3. Results 105 patients were randomized. Change in diurnal IOP, a change in the global symptoms (ocular discomfort, burning, dryness, grittiness, stinging) and objective signs (CFS, TFBUT, conjunctival hyperemia) of OSD at month 1 and 3 will be presented. Safety outcomes will also be reported. Conclusion Preservative free prostaglandin agonists reduce the risk of ocular toxicity associated with BAK. However, their effect on restoring ocular surface damage is unclear. In this study, the efficacy and safety of an unpreserved latanoprost 0.005% cationic emulsion relative to BAK‐free travoprost 0.004% solution on reducing IOP and improving the signs and symptoms of OSD will be revealed. Commercial interest [ABSTRACT FROM AUTHOR]

Lowering intraocular pressure is currently the only proven approach to preserve visual function in glaucoma. Hereby medical therapy is the initial treatment option. According to the guidelines of the European Glaucoma Society monotherapy is the first step in medical therapy. Prostaglandin analogues (PG) are approved for first line therapy for several years and they are the most frequently used IOP lowering agents now, because of their strong IOP lowering effect and once daily application. All PG that have been on the market are containing Benzalkonium chloride (BAK) as preservatives. These preservatives may cause tolerability problems and inflammation. Dry eyes and ocular surface disorders (OSD) have been limiting the use of PG with BAK. The introduction of Tafluprost without BAK is a significant addition in our medical options for glaucoma treatment. In clinical use we can consider Tafluprost sine as a useful option in patients with dry eye, ocular surface disorder, allergy to BAK or intolerability. The IOP lowering effect is comparable to the most frequently used PG Latanoprost. The application is once daily as well. Because of the high rate of dry eye patients and OSD patients Tafluprost sine is a very useful option. If these patients need more than one medication the combination of Cosopt sine and Tafluprost sine is an option of three IOP lowering molecules with three drops a day in total but unpreserved. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Both glaucoma and ocular surface disease (OSD) are very common conditions in the elderly. In order to provide sufficient intraocular pressure (IOP) reduction and low target pressure, long‐term use of IOP lowering medication as well as optimal adherence to the prescribed medication are essential. Most topical glaucoma medications are preserved with benzalkonium chloride (BAK). Chronic exposition to BAK (as preservative of most preserved eye drops) was repeatedly shown to decrease tear film quality, increase the frequency of topical side effects, and worsen the existing ocular surface disease. In addition, chronic and multiple instillation of BAK in preserved topical IOP lowering medication is a risk for failure of later filtering surgery. Patients suffering from OSD use BAK‐free artificial tears, but the same patients if they also have glaucoma mostly use BAK‐preserved IOP lowering drops. This practice is obviously not optimal. Use of unpreserved glaucoma medications in routine clinical practice is needed for IOP lowering in glaucoma. [ABSTRACT FROM AUTHOR]

Purpose To investigate whether the chemokine CX3CL1 could play a role in the conjunctival inflammatory cell trafficking induced by an exposure to benzalkonium chloride (BAC). Methods Expression of CX3CL1 was assessed in BAC‐exposed human conjunctival cell (HCC)line using flow cytometry and RT‐PCR. In vitro migration of leukocytes to BAC‐stimulated HCCs was assessed by migration assays and immuno flow cytometry with or without inhibition of CX3CR1. In vivo inflammatory cell trafficking was investigated by immunohistochemistry and confocal microscopy in the conjunctiva of wild‐type or CX3CR1‐deficient mice exposed to BAC. Results BAC induced an overexpression of CX3CL1 in HCCs. Leukocyte chemotaxis was observed when HCCs are stimulated with BAC. Human monocytic cells (CD45+,CD14+) and NK cells (CD45+,CD56+) migration was inhibited by blocking the CXCR3 receptor. In a mouse model of BAC‐induced ocular surface toxicity, the inflammatory cell infiltration of the conjunctiva was altered in the CX3CR1‐deficient mice compared to wild‐type. Conclusion The CX3CL1/CX3CR1 axis is involved in the BAC‐induced conjunctival inflammation. Specific blockade of this receptor could regulate the cell trafficking in the ocular surface. [ABSTRACT FROM AUTHOR]