Effect of chlorhexidine, povidone-iodine and betadine antiseptic eye drops on cultured human conjunctival goblet cell survival.

PURPOSE: To compare the effect of the ocular antiseptic treatments 0.05% chlorhexidine, 5% povidone-iodine (PI) and 5% betadine on cell viability and mucin secretion of primary cultured human goblet cells (GCs). METHOD: GC viability was analysed using lactate dehydrogenase (LDH) and tetrazolium dye (MTT) colorimetric assays. Expression of mucin was visualised by immunohistochemical MUC5AC staining. RESULTS: PI and betadine significantly reduced GC survival compared to the control (mean cell survival 23 ± 6% and 23 ± 7%, respectively, p < 0.05), whereas chlorhexidine did not significantly affect GC viability (mean cell survival: 78 ± 17%), as measured by the LDH assay. Similar results were obtained from the MTT assay, where PI and betadine caused a significant loss of GCs (mean cell survival: 26 ± 12% and 26 ± 13%, respectively, p < 0.05). Chlorhexidine did not significantly alter GC survival compared to the control (mean cell survival: 79 ± 8%). PI and betadine caused a dispersion of mucin secretion, which chlorhexidine did not. CONCLUSION: The most used antiseptic treatments, PI and betadine, applied prior to ocular surgery are significantly more cytotoxic to conjunctival GCs than chlorhexidine treatment.

Comparing the effect of benzalkonium chloride-preserved, polyquad-preserved, and preservative-free prostaglandin analogue eye drops on cultured human conjunctival goblet cells

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. Conclusion 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. (AU)

Comparing the effect of benzalkonium chloride-preserved, polyquad-preserved, and preservative-free prostaglandin analogue eye drops on cultured human conjunctival goblet cells.

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. CONCLUSION: 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.

The molecular aspect of anti-glaucomatous eye drops - are we harming our patients?

Glaucoma is one of the leading causes of irreversible blindness. Progression is halted with a reduction in intraocular pressure (IOP), which is most often achieved with eye drops. A major challenge in the topical treatment of glaucoma patients is the many side effects and the resulting reduced adherence. Side effects may of course be due to the molecular properties of the active pharmaceutical ingredients (APIs). There are currently six different APIs available: prostaglandin analogues, ß-adrenergic inhibitors, α-adrenergic agonists, carbonic anhydrase inhibitors, rho-kinase inhibitors and muscarinic 3 agonists. But the additives used in eye drops are also known to cause damage to the ocular surface and to some extent also to the deeper tissues. Said additives are considered inactive molecular components and are added to secure for instance viscosity and pH value, and to prevent contamination. There has been an increasing focus on the harmful effects of preservatives, with the most commonly used preservative benzalkonium chloride (BAK) being particularly controversial. BAK has long been recognized as a toxin that increases the risk of ocular discomfort. This can affect the adherence and ultimately result in lack of disease control. Other issues include the addition of certain buffers, such as phosphates, and varying pH values. This review will address the different molecular components of the IOP-lowering eye drops and what to be aware of when prescribing topical glaucoma treatment.

An Evaluation of the Physicochemical Properties of Preservative-Free 0.005% (w/v) Latanoprost Ophthalmic Solutions, and the Impact on In Vitro Human Conjunctival Goblet Cell Survival.

PURPOSE: To examine the physicochemical properties of five preservative-free (PF) 0.005% latanoprost ophthalmic products; Monoprost®, Latanest®, Gaap Ofteno®, Xalmono®, and Xaloptic® Free. Furthermore, the study investigated the mucin production and cell survival of primary cultured human conjunctival goblet cells when treated with PF eye drops. METHOD: The pH value, osmolality, and surface tension were examined. Cell survival was analyzed using lactate dehydrogenase and tetrazolium dye colorimetric assays. Mucin production was analyzed with immunohistochemical staining. RESULTS: Monoprost® (pH value 6.84 ± 0.032) had a pH value closest to the pH value of tear fluid (pH value 7.4-7.6), whereas Gaap Ofteno® (pH value 6.34 ± 0.004) and Latanest® (pH value 6.33 ± 0.003) had the lowest pH values. Gaap Ofteno® (325.9 ± 2.9 mosmol/kg) showed iso-osmolar probabilities, whereas the other products were hypo-osmolar. Gaap Ofteno® (60.31 ± 0.35 mN/m) had a higher surface tension compared to the tear fluid (40 to 46 mN/m), as described in the literature. No significant differences in goblet cell survival or mucin release were observed between the treatments and control. CONCLUSION: Significant differences in pH value, osmolality, and surface tension were observed. However, this did not affect the viability of the goblet cells or the release of mucin. Clinical studies are required to evaluate the long-term effects of use on efficacy and safety.

Generic benzalkonium chloride-preserved travoprost eye drops are not identical to the branded polyquarternium-1-preserved travoprost eye drop: Effect on cultured human conjunctival goblet cells and their physicochemical properties.

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.

Adverse Effects and Safety in Glaucoma Patients: Agreement on Clinical Trial Outcomes for Reports on Eye Drops (ASGARD)-A Delphi Consensus Statement.

PURPOSE: The purpose of this study is to establish consensus among experts on outcomes and methods to be used in clinical trials to assess adverse effects of anti-glaucomatous eye drops. DESIGN: Modified Delphi method. METHODS: Clinical experts from Europe, North America, South America, the Middle East, and Asia were invited to participate in 2 sequential web-based surveys administered from June 27 to August 29, 2021. A total of 91 clinical experts were invited to participate. Of these, 71 (78%) experts from 23 different countries accepted the invitation and answered the first questionnaire. The importance of items was ranked using a 10-point scale (1 as not important, 10 as very important). RESULTS: A total of 84 items were rated in round one by 71 participants. Of these, 68 (81%) reached consensus. In round 2, 19 items, including 3 additional items, were rated by 53 (75%) participants. Consensus was reached in 98% of investigated items. Eight outcomes were agreed as important to assess when conducting future trials: ocular surface, dryness, epithelial damage, local adverse effects related to eye drops as reported by patients, periocular surroundings and eyelids, quality of life questionnaires, hyperemia, visual acuity, tear film, and anterior chamber inflammation. CONCLUSION: We propose a consensus-based series of outcomes and assessment methods to be used in clinical trials assessing adverse effects of antiglaucomatous eye drops. This, we hope, will improve the comparability of results from future trials and thus facilitate meta-analyses and progress in this field.

Benzalkonium Chloride-Preserved Anti-Glaucomatous Eye Drops and Their Effect on Human Conjunctival Goblet Cells in vitro.

INTRODUCTION: Most intraocular pressure (IOP)-lowering eye drops are preserved with benzalkonium chloride (BAK). This can increase side effects and decrease adherence. Particularly, damage to the mucin-producing conjunctival goblet cells may be an issue due to instability of the tear film. We aimed to investigate the effect of IOP-lowering eye drops preserved with BAK on cultured human conjunctival goblet cells. METHODS: Eye drops Brimonidine Tartrate Teva (BT) with 0.005% BAK, Dorzolamide Stada (DS) with 0.0075% BAK, Optimol® (OP) with 0.01% BAK, and Latanoprost Teva (LT) with 0.02% BAK were included. Human primary cultured goblet cell survival was evaluated using a lactate dehydrogenase assay on human goblet cells after treatment for 30 min and 6 h with the different anti-glaucoma drug formulations. RESULTS: All eye drops examined, except BT, reduced goblet cell survival. The impact of eye drops on goblet cell viability was correlated with the time of exposure as well as to the concentration of BAK. After 30 min of exposure, cell viability was 93% for BT (0.005% BAK; p = 0.93), 71% for DS (0.0075% BAK; p = 0.067), 70% for OP (0.01% BAK; p = 0.054), and 69% for LT (0.02% BAK; p = 0.022), and exposure for 6 h reduced cell survival to 74% for BT (p = 0.217), 52% for DS (p = 0.011), 34% for OP (p = 0.017), and 31% for LT (p = 0.0007). CONCLUSION: LT, OP, and DS reduced human goblet cell survival in a time-dependent manner. BT did not affect goblet cell survival. Cell survival was correlated with the BAK concentration in the eye drops making 0.02% BAK-preserved LT most toxic and 0.005% BAK-preserved BT least toxic. Based on the present study, decreasing BAK in eye drops for chronic use seems important to reduce damage to the goblet cells. However, future studies are needed to further explore this finding.

Impact of benzalkonium chloride-preserved and preservative-free latanoprost eye drops on cultured human conjunctival goblet cells upon acute exposure and differences in physicochemical properties of the eye drops.

OBJECTIVE: To investigate the short-term impact on human conjunctival goblet cell (GC) survival and mucin release of acute exposure to 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 AND ANALYSIS: Primary GC cultures were established from human conjunctival donor tissue. The impact of eye drops on GC survival was assessed using a lactate dehydrogenase assay. 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 min (min), the GC survival was maintained compared with control (p=0.9941), while all BAK-LT eye drops reduced survival with approximately 30% (p<0.02). 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 value 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 drops. The mean drop mass was 26-31 mg for the BAK-LT eye drops and 30 mg for PF-LT. The surface tension was lower for all BAK-LT eye drops (31.1-32.1 mN/m) compared with PF-LT (42 mN/m). CONCLUSION: PF-LT compared with various branded and generic LT preparations containing BAK are less cytotoxic when applied to cultured GCs.

Efficacy and safety evaluation of benzalkonium chloride preserved eye-drops compared with alternatively preserved and preservative-free eye-drops in the treatment of glaucoma: a systematic review and meta-analysis.

BACKGROUND/AIMS: This systematic review compared the efficacy and safety of benzalkonium chloride (BAK)-preserved eye-drops with alternatively preserved (AP) and preservative-free (PF) eye-drops. METHODS: PubMed, EMBASE and MEDLINE were searched for randomised controlled trials in June and October 2019. Study selection, data extraction and risk of bias assessment were made by two independent reviewers using the Cochrane Handbook. Studies on prostaglandin analogue or beta-blocker eye-drops and patients with glaucoma or ocular hypertension were included. Primary outcome was change in intraocular pressure (IOP). Secondary outcomes were safety measures as assessed in original study. RESULTS: Of 433 articles screened, 16 studies were included. IOP meta-analysis was conducted on 13 studies (4201 patients) ranging from 15 days to 6 months. No significant differences between BAK versus PF and AP were identified (95% CI -0.00 to 0.30 mm Hg, p=0.05). Meta-analyses revealed no differences between BAK versus AP and PF with regards to conjunctival hyperaemia (risk ratio (RR) 1.05, 95% CI 0.91 to 1.22, 3800 patients, 9 studies), ocular hyperaemia (RR 1.31, 95% CI 0.96 to 1.78, 2268 patients, 5 studies), total ocular adverse events (RR 1.03, 95% CI 0.88 to 1.20, 1906 patients, 5 studies) or tear break-up time (mean difference 0.89, 95% CI -0.03 to 1.81, 130 patients, 3 studies). Diverse reporting on safety measures made comparison challenging. Risk of bias was assessed as high or unclear in many relevant domains, suggesting potential selective reporting or under-reporting. CONCLUSION: No clinically significant differences on efficacy or safety could be determined between BAK versus AP and PF. However, there were substantial uncertainties on safety.PROSPERO registration numberCRD42019139692.

Predicting the effects of supplemental EPA and DHA on the omega-3 index.

BACKGROUND: Supplemental long-chain omega-3 (n-3) fatty acids (EPA and DHA) raise erythrocyte EPA + DHA [omega-3 index (O3I)] concentrations, but the magnitude or variability of this effect is unclear. OBJECTIVE: The purpose of this study was to model the effects of supplemental EPA + DHA on the O3I. METHODS: Deidentified data from 1422 individuals from 14 published n-3 intervention trials were included. Variables considered included dose, baseline O3I, sex, age, weight, height, chemical form [ethyl ester (EE) compared with triglyceride (TG)], and duration of treatment. The O3I was measured by the same method in all included studies. Variables were selected by stepwise regression using the Bayesian information criterion. RESULTS: Individuals supplemented with EPA + DHA (n = 846) took a mean ± SD of 1983 ± 1297 mg/d, and the placebo controls (n = 576) took none. The mean duration of supplementation was 13.6 ± 6.0 wk. The O3I increased from 4.9% ± 1.7% to 8.1% ± 2.7% in the supplemented individuals ( P < 0.0001). The final model included dose, baseline O3I, and chemical formulation type (EE or TG), and these explained 62% of the variance in response (P < 0.0001). The model predicted that the final O3I (and 95% CI) for a population like this, with a baseline concentration of 4.9%, given 850 mg/d of EPA + DHA EE would be ∼6.5% (95% CI: 6.3%, 6.7%). Gram for gram, TG-based supplements increased the O3I by about 1 percentage point more than EE products. CONCLUSIONS: Of the factors tested, only baseline O3I, dose, and chemical formulation were significant predictors of O3I response to supplementation. The model developed here can be used by researchers to help estimate the O3I response to a given EPA + DHA dose and chemical form.

n-3 PUFA esterified to glycerol or as ethyl esters reduce non-fasting plasma triacylglycerol in subjects with hypertriglyceridemia: a randomized trial.

To date, treatment of hypertriglyceridemia with long-chain n-3 polyunsaturated fatty acids (n-3 PUFA) has been investigated solely in fasting and postprandial subjects. However, non-fasting triacylglycerols are more strongly associated with risk of cardiovascular disease. The objective of this study was to investigate the effect of long-chain n-3 PUFA on non-fasting triacylglycerol levels and to compare the effects of n-3 PUFA formulated as acylglycerol (AG-PUFA) or ethyl esters (EE-PUFA). The study was a double-blinded randomized placebo-controlled interventional trial, and included 120 subjects with non-fasting plasma triacylglycerol levels of 1.7-5.65 mmol/L (150-500 mg/dL). The participants received approximately 3 g/day of AG-PUFA, EE-PUFA, or placebo for a period of eight weeks. The levels of non-fasting plasma triacylglycerols decreased 28% in the AG-PUFA group and 22% in the EE-PUFA group (P < 0.001 vs. placebo), with no significant difference between the two groups. The triacylglycerol lowering effect was evident after four weeks, and was inversely correlated with the omega-3 index (EPA + DHA content in erythrocyte membranes). The omega-3 index increased 63.2% in the AG-PUFA group and 58.5% in the EE-PUFA group (P < 0.001). Overall, the heart rate in the AG-PUFA group decreased by three beats per minute (P = 0.045). High-density lipoprotein (HDL) cholesterol increased in the AG-PUFA group (P < 0.001). Neither total nor non-HDL cholesterol changed in any group. Lipoprotein-associated phospholipase A2 (LpPLA2) decreased in the EE-PUFA group (P = 0.001). No serious adverse events were observed. Supplementation with long-chain n-3 PUFA lowered non-fasting triacylglycerol levels, suggestive of a reduction in cardiovascular risk. Regardless of the different effects on heart rate, HDL, and LpPLA2 that were observed, compared to placebo, AG-PUFA, and EE-PUFA are equally effective in reducing non-fasting triacylglycerol levels.