BAK Concentration Research Articles

Role of Mesenchymal Stem Cell-Derived Conditioned Medium in Modulating the Benzalkonium Chloride-Induced Cytotoxic Effects in Cultured Corneal Epithelial Cells In Vitro

Purpose Benzalkonium chloride (BAK) is a common preservative in ophthalmic formulations that causes cytotoxic damage to the corneal epithelial cells. This study aims to explore the role of mesenchymal stem cell (MSC)-derived conditioned medium in modulating the BAK-induced cytotoxic effects in cultured human corneal epithelial cells (HCECs) as a cell-free therapeutic agent. Methods The in vitro cultured HCECs derived from a HCE cell line were treated with BAK (0.001% and 0.005%, diluted in DMEM/F12, v/v) for 15 min, washed with 1xPBS, and allowed to recover for 24 h in human bone marrow MSC-derived conditioned medium (MSC-CM: undiluted (100%) and diluted (50%, v/v)). On the other hand, HCECs were co-incubated with BAK (0.005%, v/v) and MSC-CM (100% and 50%, v/v) for 24 h. The HCEC-derived conditioned medium (HCE-CM) was used as an optimal control for MSC-CM, whereas HCECs cultured in DMEM/F12 were used as a control. The DMEM/F12 was used as the base medium for the culture of HCECs and preparation of HCE- and MSC-CM. The role of MSC-CM in modulating the metabolic activity, cell death, epithelial repair, and proliferation, in BAK-treated HCECs was evaluated using MTT assay, Propidium iodide staining, scratch assay, and Ki-67 staining, respectively. Results Compared to the control, recovery of BAK-treated (0.001% and 0.005%, for 15 min) HCECs in MSC-CM showed significantly reduced cell death with enhanced metabolic activity, epithelial repair, and proliferation. However, in comparison with HCE-CM, the beneficial effects of MSC-CM were predominantly observed at lower BAK concentration (0.001%, for 15 min). Whereas the co-incubation of BAK (0.005%) and MSC-CM for a longer duration (24 h) was marginally beneficial. Conclusions Our results suggest that the MSC-CM is effective in modulating the BAK-induced cell death, retardation of metabolic activity and proliferation in cultured HCECs, particularly at lower concentration (0.001%) and shorter exposure (15 min) of BAK.

Ocular pharmacokinetics of a new preservative‐free bimatoprost 0.01% ophthalmic gel

Aims/Purpose: The aim of this study was to compare the aqueous humour (AH) and iris‐ciliary body (ICB) concentrations of bimatoprost and its main metabolite bimatoprost free acid after topical instillation in rabbits of a new preservative‐free bimatoprost 0.01% ophthalmic gel (PFB 0.01% gel) versus two commercially available preserved bimatoprost ophthalmic solutions ‐ PB 0.01% (200 ppm benzalkonium chloride [BAK], Lumigan® 0.01%) and PB 0.03% (50 ppm BAK, Lumigan® 0.03%).Methods: Female Dutch Belted rabbits (n = 45) were divided into three treatment groups (30 eyes per group): PFB 0.01% gel, PB 0.01% and PB 0.03% solutions. Animals received a single topical instillation (25 μL) into both eyes. AH and ICB samples were collected at 0.5 h, 1 h, 2 h, 4 h and 8 h post instillation. These samples were analysed by rapid resolution liquid chromatography tandem mass spectrometry for bimatoprost and bimatoprost free acid concentrations.Results: Following a single topical instillation, Tmax of bimatoprost and bimatoprost free acid in both ocular matrices was 1 h across the 3 treatments. Cmax of bimatoprost/bimatoprost free acid was 50.2 ng/mL, 26.3 ng/mL and 59.9 ng/mL of AH respectively following PFB 0.01%, PB 0.01% and PB 0.03% treatments. Maximal concentrations and area under the curve in AH and ICB of bimatoprost/bimatoprost free acid were greater (from 2‐ to 2.6‐fold) with PFB 0.01% gel compared to PB 0.01% and similar (from 0.9‐ to 0.95‐fold) to PB 0.03%.Conclusions: Topical instillation of PFB 0.01% gel produced significantly higher bimatoprost/bimatoprost free acid concentrations in the AH and ICB of rabbits than PB 0.01% (despite a high BAK concentration) and was similar to PB 0.03%. The preservative‐free gel formulation improves corneal penetration and bioavailability of bimatoprost compared to a preserved solution at the same concentration.

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.

A model for the regulation of apoptosis intrinsic pathway: The potential role of the transcriptional regulator E2F in the point of no return

Apoptosis has been extensively characterized by both experimental approaches and model simulations. However, it is still not fully understood how the regulation occurs, especially in the intrinsic pathway, which can be activated by a great variety of signals. In addition, the conditions in which a point of no return could be reached remain elusive. In this work, we use differential equations models to approach these issues. Our starting point was the model for caspase activation of Legewie et al. (Legewie S, et al., PLoS Computational Biology 2006, 2(9): e120), which exhibits irreversible bistability. We added an activation module to this model, with the main events related to mitochondrial outer membrane permeabilization, which includes cytochrome C release by the mitochondria and its effects on caspase activation and respiratory chain disruption. This “Extended Legewie Model” (ELM) uses BAK as the apoptotic stimulus and active caspase 3 as a measure of apoptosis activation. Unexpectedly, in the extended model, BAK cannot trigger apoptosis activation using physiologically sound initial values of the variables, due to limitations in apoptosome concentration increase. Therefore, the next step was to find a regulatory mechanism, allowing apoptosis activation in the ELM, starting from physiological initial concentrations. For this aim, we performed a sensitivity analysis on the 61 parameters of the system, finding that those producing the most relevant changes in the qualitative behaviour were the rates of synthesis of caspase 3, caspase 9 and XIAP. Based on these results, the transcription factor E2F was included in the ELM because it directly regulates the rate of synthesis of caspase 3 and 9. Depending on the concentration of E2F, the ELM shows different qualitative behaviours. On one hand, for low E2F apoptosis is impossible and for high E2F apoptosis is inevitable. Therefore, if E2F is sufficiently increased, the point of no return is crossed. On the other hand, for intermediate values of E2F there is a bistable region where the fate of the system also depends on the concentration of BAK and other signalling species.

Safety and Efficacy of Benzalkonium Chloride-optimized Tafluprost in Japanese Glaucoma Patients With Existing Superficial Punctate Keratitis

To evaluate the safety and efficacy of benzalkonium chloride (BAK)-optimized tafluprost (with a BAK concentration reduced from 0.01% to 0.001%) in glaucoma patients with existing superficial punctate keratitis (SPK). A prospective, multicenter, open-label study was designed to compare BAK-optimized tafluprost administered over 12 weeks relative to other preserved prostaglandin analogs previously administered in Japanese glaucoma patients. Thirty patients with SPK graded at <6 points by area density (AD) scoring in 1 eye were recruited. The primary outcome measure was change in AD score at 12 weeks after the switch in treatment compared with that at baseline. Secondary outcome measures included changes in tear film breakup time (TBUT), hyperemia score, and intraocular pressure (IOP). Four patients were excluded from analysis because of treatment discontinuation. Mean AD score±SD decreased significantly from 3.4±0.9 to 1.8±1.8 after the switch (P<0.0001). Mean TBUT increased significantly from 6.3±3.3 to 8.0±4.2 seconds (P<0.01). Mean hyperemia score remained unchanged, whereas mean IOP decreased significantly from 15.6±2.6 to 14.4±2.0 mm Hg (P<0.01). For patients previously treated with BAK-preserved latanoprost (n=17) or bimatoprost (n=2), mean AD score decreased significantly from 3.4±0.9 to 1.8±1.8 (P<0.01) and mean TBUT increased significantly from 6.4±3.6 to 8.2±4.3 seconds (P<0.01); no such changes were apparent for patients previously treated with sofZia-preserved travoprost (n=7). BAK-optimized tafluprost is a treatment option to improve the condition of the ocular surface and to maintain IOP control in glaucoma patients with existing SPK who have been previously treated with other BAK-preserved prostaglandin analogs.

Novel primary epithelial cell toxicity assay using porcine corneal explants.

The purpose of this study was to develop a novel primary epithelial cell toxicity assay using porcine corneal explant and evaluate the assay using benzalkonium chloride (BAK). Circular corneal explants were trephined from the peripheral cornea of porcine eyes using 2-mm biopsy punches, and placed on 6-well culture dishes with a culture medium. After incubation for 12 hours, 50 μL of BAK at 0.00001%, 0.0001%, 0.001%, or 0.01% was applied to each well for 2 minutes. After washing, explants were cultured for another 24 hours, then epithelial outgrowth was photographed and measured. Corneal immunohistochemical characteristics were evaluated by cytokeratin (CK) 3, CK12, and ZO-1. Cell toxicity was evaluated by WST-8 assay, Ki-67 staining, and TUNEL assay. Epithelial cells migrated outward concentrically from the corneal explant as time elapsed and were positive for CK3, CK12, and ZO-1. The outgrowth rate decreased significantly with 0.0001%, 0.001%, and 0.01% BAK compared with the phosphate-buffered saline (PBS) control (P < 0.01), and the decrease was BAK concentration dependent. Numbers of viable cells and Ki-67-positive cells also decreased significantly with 0.01% BAK compared with the PBS control (P < 0.05). TUNEL-positive cells were present in the epithelial outgrowth. Moreover, TUNEL-positive cell density tended to increase with 0.01% BAK compared with the PBS control (P = 0.14) and 0.00001% BAK (P = 0.081). Our novel toxicity assay using porcine corneal explant is simple and reflects the effect of single and short-duration instillation of eye drops. The method is useful for evaluation of corneal toxicity at low concentrations of BAK.

Reduction of cytotoxicity of benzalkonium chloride and octenidine by Brilliant Blue G.

The irritative effects of preservatives found in ophthalmologic solution, or of antiseptics used for skin disinfection is a consistent problem for the patients. The reduction of the toxic effects of these compounds is desired. Brilliant Blue G (BBG) has shown to meet the expected effect in presence of benzalkonium chloride (BAK), a well known preservative in ophthalmic solutions, and octenidine dihydrochloride (Oct), used as antiseptic in skin and wound disinfection. BBG shows a significant protective effect on human corneal epithelial (HCE) cells against BAK and Oct toxicity, increasing the cell survival up to 51 % at the highest BAK or Oct concentration tested, which is 0.01 %, both at 30 min incubation. Although BBG is described as a P2x7 receptor antagonist, other selective P2x7 receptor antagonists, OxATP (adenosine 5’-triphosphate-2’,3’-dialdehyde) and DPPH (N’-(3,5-dichloropyridin-4-yl)-3-phenylpropanehydrazide), did not reduce the cytotoxicity of neither BAK nor Oct. Therefore we assume that the protective effect of BBG is not due to its action on the P2x7 receptor. Brilliant Blue R (BBR), a dye similar to BBG, was also tested for protective effect on BAK and Oct toxicity. In presence of BAK no significant protective effect was observed. Instead, with Oct a comparable protective effect was seen with that of BBG. To assure that the bacteriostatic effect is not affected by the combinations of BAK/BBG, Oct/BBG and Oct/BBR, bacterial growth inhibition was analyzed on different Gram-negative and Gram-positive bacteria. All combinations of BAK or Oct with BBG hinder growth of Gram-positive bacteria. The combinations of 0.001 % Oct and BBR above 0.025 % do not hinder the growth of B. subtilis. For Gram-negative bacteria, BBG and BBR reduce, but do not abolish, the antimicrobial effect of BAK nor of Oct. In conclusion, the addition of BBG at bacterial inhibitory concentrations is suggested in the ready-to-use ophthalmic preparations and antiseptic solutions.

Increased Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) Expression in the Conjunctival Epithelium Exposed to Antiglaucoma Treatments

Objective: To analyze the effect of preserved antiglaucoma eye drops on the expression of extracellular matrix (ECM) metalloproteinase inducer (EMMPRIN) in conjunctival epithelial cells.Methods: A total of 18 patients treated for primary open-angle glaucoma with benzalkonium chloride (BAK) preserved eye drops and eight age-matched controls were included in this study. Glaucoma patients were divided into two groups according to their daily exposure to BAK: high-exposure (HE) group and low-exposure (LE) group. HLA-DR and EMMPRIN were quantified on conjunctival impression cytology specimens using flow cytometry. In parallel, IOBA-NHC conjunctival epithelial cells were exposed to different BAK concentrations, in the presence or absence of cyclosporine A (CsA), and their total and surface expressions of EMMPRIN were assessed by flow cytometry and results are given in relative fluorescence intensities (RFIs).Results: Compared to the control group (1.71 ± 0.39 RFI), EMMPRIN was significantly increased in the HE (4.19 ± 1.50 RFI, p < 0.001) and LE groups (2.55 ± 0.40 RFI, p = 0.029). Similar increase was observed in HLA-DR expression in the HE (4.58 ± 1.38 RFI, p < 0.001) and LE groups (2.52 ± 0.47 RFI, p = 0.046) as compared to control subjects (1.75 ± 0.27 RFI). Across all subjects enrolled in the study, there was a significant correlation between HLA-DR and EMMPRIN (R2 = 0.875, p < 0.0001). IOBA-NHC cells exposed to BAK presented a significant increase in EMMPRIN, which was proportional to the concentration of BAK. The surface expression of EMMPRIN was inhibited by CsA.Conclusions: The increased expression of EMMPRIN in patients topically treated with multiple antiglaucoma BAK-preserved eye drops suggests a matrix metalloproteinase-related modification of conjunctival ECM remodeling. In vitro results suggest that CsA has the potential to limit BAK effects on EMMPRIN.

Effects of Benzalkonium Chloride on THP-1 Differentiated Macrophages In Vitro

PurposeTo characterize the effects of benzalkonium chloride (BAK) in THP-1 differentiated cells in vitro.MethodsMacrophages were obtained after differentiation of THP-1 cells, a human monocytic leukemia cell line. Macrophages were exposed for 24 h to 33 nM (10−5%) benzalkonium chloride (BAK), 10 nM dinitrochlorobenzene (DNCB), 100 ng/mL lipopolysaccharide (LPS), 5 ng/mL tumor necrosis factor alpha (TNF-α) or phosphate buffered saline (PBS) as controls. The expression of CD11b, CD11c, CD33 and CD54 was evaluated using immunohistochemistry and flow cytometry (FCM). Phagocytosis function was analyzed using carboxylate-modified fluorescent microspheres and quantified by FCM. Migration was evaluated in cocultures with conjunctival epithelial cells. Cytokine production was detected and quantified in culture supernatants using a human cytokine array.ResultsStimulation of THP-1-derived macrophages with a low concentration of BAK increased CD11b and CD11c expression and decreased CD33. Macrophages exposed to BAK, LPS and TNF-α had increased phagocytosis. In contrast to LPS, BAK and TNF-α increased macrophage migration. Cytokines in supernatants of macrophages exposed to BAK revealed an increased release of CCL1, CCL4/MIP-1β, TNF-α, soluble CD54/ICAM-1 and IL-1β.Conclusion In vitro, BAK has a direct stimulating effect on macrophages, increasing phagocytosis, cytokine release, migration and expression of CD11b and CD11c. Long-term exposure to low concentrations of BAK should be considered as a stimulating factor responsible for inflammation through macrophage activation.

Effect of Low Concentrations of Benzalkonium Chloride on Acanthamoebal Survival and Its Potential Impact on Empirical Therapy of Infectious Keratitis

The significant antiacanthamoebal effect of benzalkonium chloride, at or below concentrations used for preservation of common ophthalmic preparations, should be understood both when choosing empiric antibiotic therapy for infectious keratitis and when assessing the persistent rise in Acanthamoeba cases in the United States since 2003. To characterize the antiacanthamoebal efficacy of low concentrations of benzalkonium chloride (BAK) for drug preservation and therapeutic effect against Acanthamoeba. Experimental study with a review of the literature. Laboratory. A concentration of 10(4) trophozoites of 3 well-characterized clinical strains of Acanthamoeba were exposed at 0.5, 2.0, 3.5, 5.0, and 6.5 hours to BAK (0.001%, 0.002%, and 0.003%), moxifloxacin hydrochloride (0.5%), and moxifloxacin (0.5%) + BAK (0.001% and 0.003%) with hydrogen peroxide (3%) and amoeba saline controls. Amoeba survival was calculated using the most probable number method recorded as log kill values. The relationship of BAK concentration and exposure time as well as the relative effect of BAK and moxifloxacin on acanthamoebal survival were analyzed. Amoebicidal activity of BAK is both time dependent and concentration dependent in pooled and strain-stratified analyses (P < .001). Moxifloxacin demonstrated no significant independent inhibitory effect or additive effect to BAK efficacy on acanthamoebal survival. The profound antiacanthamoebal effect of BAK, 0.003%, was similar to that of hydrogen peroxide for certain strains. Low concentrations of BAK, previously demonstrated to concentrate and persist in ocular surface epithelium, exhibit significant antiacanthamoebal activity in vitro at or below concentrations found in commercially available ophthalmic anti-infectives. The unexplained persistence of the Acanthamoeba keratitis outbreak in the United States, clusters abroad, and clinical studies reporting resolution or modification of Acanthamoeba keratitis without specific antiacanthamoebal therapy suggests that other contributing factors should be considered, including changes in the formulations used for empirical therapy of presumed infectious keratitis occurring in the same period.

Cell viability score (CVS) as a good indicator of critical concentration of benzalkonium chloride for toxicity in cultured ocular surface cell lines

Cytotoxicity of benzalkonium chloride (BAK) is a major factor affecting drug cytotoxicity. This study aimed to determine the critical concentration of BAK for cultured ocular cells, using SIRC (rabbit corneal epithelium), BCE C/D-1b (bovine corneal epithelial cells), RC-1 (rabbit corneal epithelium), and Chang (human conjunctival cells). Cell viability was determined following the exposure of cells to 11 concentrations of BAK for 10, 30, or 60min using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and neutral red assays, and the cell viability score (CVS) was used to evaluate comprehensively the toxicity of BAK. The CVS system consists of two values. The CVS50 was determined by the number of measurements for viability ⩾50% of control. The CVS40/80 was calculated as follows: CVS40/80=(number of measurements for viability values >80%)−(number of measurements for viability values <40%). Both %CVS50 and %CVS40/80 decreased with concentrations of BAK. When BAK concentrations were 0.01% or higher, %CVS50 and %CVS40/80 became 0 and less than −90, respectively. Meanwhile, when BAK concentrations were 0.001% or lower, %CVS50 became 100. In the case of %CVS40/80, when the BAK concentrations were 0.002% or lower, the values reached 75 or more, and when 0.0005% or lower, the %CVS40/80 value reached 100. Accordingly, BAK induced very low cytotoxicity in the cultured ocular cell lines at concentrations of 0.002% or lower. The concentration-dependency confirmed that the CVS score is useful for expressing drug cytotoxicity in a simple and comprehensive manner.

Cell Viability Score as an Integrated Indicator for Cytotoxicity of Benzalkonium Chloride-Containing Antiglaucoma Eyedrops

We evaluated the in vitro cytotoxicity of benzalkonium chloride (BAK)-containing antiglaucoma eyedrops. We prepared cell cultures of SIRC, BCE C/D-1b, RC-1, and Chang conjunctiva. The viability of cell cultures was determined using the MTT and neutral red assays. The cell viability score (CVS) was used to compare the toxicity of test solutions. %CVS50 and %CVS40/80 of each eyedrop solution were 71 and 26 for Lumigan(®) (0.002% bimatoprost with 0.005% BAK), 100 and 99 for Tapros(®) (0.0015% tafluprost, a new formula from 2010 with 0.001% BAK), 39 and -29 for 2% Trusopt(®) (2% dorzolamide with 0.0075% BAK), 28 and -43 for Xalacom(®) (latanoprost/0.5% timolol with 0.02% BAK), 88 and 66 for DuoTrav(®) (travoprost/0.5% timolol with no BAK), 36 and -35 for Cosopt(®) (2% dorzolamide/0.5% timolol with 0.0075% BAK) and 53 and -1 for Combigan(®) (0.15% brimonidin/0.5% timolol with 0.005% BAK). Only Xalacom(®) and Tapros(®) did not show an apparent decrease in %CVS as compared to the corresponding concentration of BAK. In conclusion, the cytotoxicity of tested eyedrops was dependent on BAK. Only the eyedrops containing latanoprost or tafluprost showed a reduction in the cytotoxicity of BAK.

Effects of benzalkonium chloride on antigen presenting cells in vitro

AbstractPurpose 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.

Effects of benzalkonium chloride- and polyquad-preserved combination glaucoma medications on cultured human ocular surface cells

The aim of this study is to investigate potential adverse effects of fixed combination glaucoma medications preserved with either benzalkonium chloride (BAK) or Polyquad® (PQ; Alcon Research Ltd., Fort Worth, TX, USA) on cultured ocular epithelial cells. Confluent cultures of human cornea and conjunctival cell lines were exposed for 25 minutes to different glaucoma medications as well as a range of concentrations of BAK (0.001%-0.050%). Balanced salt solution was used as the "live" control and a solution containing 70% methanol and 0.2% saponin was used as a "dead" control. The number of dead and live cells were determined via ethidium homodimer (Eth-1) and calcein acetoxymethyl ester (AM) fluorescence, respectively. The toxicity of the prostaglandin analog with beta-blocker timolol fixed-combination formulations preserved with BAK was different from that observed in the respective BAK concentrations. Travoprost plus timolol fixed combination with BAK performed better than its respective BAK concentration alone, while the latanoprost plus timolol fixed combination performed worse than its respective BAK concentration. Travoprost plus timolol fixed combination preserved with PQ had greater corneal and conjunctival cell survival than either the travoprost plus timolol fixed combination preserved with BAK or the latanoprost plus timolol fixed combination. Ocular surface side effects have previously been demonstrated with chronic, long-term exposure to intraocular-pressure-lowering medications containing the common preservative BAK. BAK alone has significant in-vitro cytotoxicity to cultured ocular epithelial cells. Substitution of BAK with PQ resulted in significantly higher percentages of live conjunctival and corneal cells. Further studies are needed to understand the clinical implications of these findings.

In Vitro Assessment of the Cytotoxicity of Anti-allergic Eye Drops Using 5 Cultured Corneal and Conjunctival Cell Lines

The aim of this study was to evaluate the cytotoxicity of anti-allergic eye drops for human corneal endothelial cells (HCEC) and commercially available ocular surface cells. A primary HCEC culture was derived from human eye bank specimens. SIRC (rabbit corneal epithelium), BCE C/D-1b (bovine corneal epithelial cells), RC-1 (rabbit corneal epithelium), and Chang (human conjunctival cells) were obtained commercially. The WST-1 assay was used to measure HCEC viability, and the viability of other cells was measured using the MTT assay. Cells were treated with 7 commercially available anti-allergic eye drops for 48 h and cell viability was measured and calculated as a percentage of control. The degree of toxicity for each eye-drop solution was based on the cell viability score (CVS). HCECs treated with a 1000-fold dilution of the eye-drop solution had a viability score of 67% for Rizaben and ≥80% for the other solutions with Zepelin being the least toxic. Cell viability in response to eye-drop solutions preserved with benzalkonium chloride (BAK) was dependent on the concentration of the drug solution and exposure time. Treatment of ocular surface cells with a 20-fold dilution of the eye-drop solution resulted in the following order of cell viability as determined by their CVS: Zepelin > Ketas = Zaditen ≥ Tramelas PF = Patanol ≥ Rizaben ≥ Livostin. This order was similar to that observed for HCECs, and cell viability was found to be concentration-dependent. Based on the penetration of the drug into eye tissues, HCECs are only likely to be pharmaceutically damaging in rare cases. Epithelial cell viability depends primarily on the concentration of BAK rather than on the action of the active component in the eye-drop solution. CVS values were useful for comparison of toxicity.

In vivo comparative study of ocular vasodilation, a relative indicator of hyperemia, in guinea pigs following treatment with bimatoprost ophthalmic solutions 0.01% and 0.03%

The objective of this in vivo study was to compare the incidence of vasodilation in guinea pigs following topical administration of bimatoprost ophthalmic solutions 0.01% and 0.03%. The study comprised 20 guinea pigs assigned to 2 treatment groups (10 per treatment group) to receive either bimatoprost 0.01% or bimatoprost 0.03%. Animals were hand-held under 2.75 x magnification to score ocular vasodilation (a measure of hyperemia), using a scoring system developed at Alcon Research, Ltd. Following baseline ocular scoring, each animal received a 30 muL dose to the left eye of either bimatoprost 0.01% (3 mug) or bimatoprost 0.03% (9 mug). Vasodilation was again scored at 1, 2, 3, 4, 5 and 6 hours after dosing. Incidence of vasodilation was calculated as the percent of total eyes in each 2-hour time interval with scores >/=2. The incidence of vasodilation was higher in the bimatoprost 0.01% treatment group (range, 45.0% to 60.0%) than the bimatoprost 0.03% treatment group (range, 30.0% to 52.2%) at all post-dosing time points. The 2 bimatoprost formulations elicited ocular vasodilation of long duration (>6 hours) in the guinea pig model, with the bimatoprost 0.01% treatment group showing a higher incidence of ocular vasodilation than the bimatoprost 0.03% treatment group. Further clinical studies would be needed to determine whether the higher incidence of vasodilation may also be attributed to the increased BAK concentration in the bimatoprost 0.01% formulation.

Impact of topical bimatoprost 0.01% and bimatoprost 0.03% on conjunctival irritation in rabbits

The purpose of this study was to examine and compare the conjunctival irritation (congestion, swelling, and discharge) of topical bimatoprost ophthalmic solution 0.01% and bimatoprost ophthalmic solution 0.03% in rabbits. Six healthy New Zealand White rabbits were treated with either bimatoprost 0.01% or bimatoprost 0.03% (3 animals/group). One dose (2 drops/dose) of study medication was administered to the right eye of each animal every 30 minutes for 4.5 hours. Approximately 1 hour after the last dose, conjunctival irritation was assessed using a slit-lamp biomicroscope to individually evaluate conjunctival congestion, swelling, and discharge. The mean conjunctival congestion, swelling and discharge scores for bimatoprost 0.03% were 1.67, 0.33 and 0.33, respectively, and for bimatoprost 0.01% were 2.00, 0.33 and 1.33, respectively. Despite the lower drug concentration of the 0.01% formulation, bimatoprost 0.01% does not reduce conjunctival irritation, including conjunctival congestion, swelling, and discharge, in rabbits compared to bimatoprost 0.03%. Further studies would be needed to determine whether the increase in the mean conjunctival congestion and discharge scores may be attributed to the increased BAK concentration in the bimatoprost 0.01% formulation.

In Vivo Corneal Epithelial Permeability following Treatment with Prostaglandin Analoges with or without Benzalkonium Chloride

The effects of two intraocular pressure (IOP)-lowering agents, latanoprost with 0.02% benzylalkonium chloride (BAK) and travoprost Z without BAK, on corneal epithelial permeability, assessed by carboxyfluorescein uptake, were compared in a rabbit cornea model (n = 12). Loss of epithelial tight junctions was measured by ruthenium red uptake with transmission electron microscopy. There was a significant difference between corneal epithelium permeability of rabbits exposed to either travoprost Z or latanoprost preserved with 0.02% BAK (P < 0.0001). There was no difference between untreated controls and those receiving travoprost Z (P = 0.224). Epithelial permeability was significantly increased in the corneas of rabbits exposed to latanoprost in the 3-min drop test (2.16 ± 1.094 nm/sec) and the 3-min exposure (16.69 ± 3.15 nm/sec), compared with control or travoprost (P = 0.002), likely due to the presence of a 0.02% concentration of BAK in the latanoprost solutions. There was no detectable loss of epithelial tight junctions in corneas from the control or travoprost Z groups, compared with significant loss in the latanoprost group. Agents such as travoprost Z with an alternative preservative decreased the epithelial toxicity associated with the long-term use of IOP-lowering agents preserved with BAK.

The dilution of benzalkonium chloride (BAK) in the tear Film

The claim that benzalkonium chloride (BAK) synergistically enhances the antibiotic efficacy of gatifloxacin ophthalmic solution 0.3% (preserved with 0.005% BAK [50 microg/mL), Zymar; Allergan Inc., Irvine, Calif, USA) has been the subject of several studies. The purpose of this prospective clinical study was to test the hypothesis that BAK would be significantly diluted shortly after topical ocular administration and would thereafter have little or no effect on the enhancement of the antibiotic efficacy of commercial gatifloxacin on the ocular surface. This hypothesis was tested by investigators who measured the concentration of tear film BAK at successive time points after topical administration of commercial gatifloxacin. After subjects (N=10) received 5 separate instillations of a single 35-microL drop of gatifloxacin 0.3% ophthalmic solution in each eye, tear samples were collected at 30 sec, 1 min, 3 min, 5 min, and 20 min, with the use of graduated 5-microL glass microcapillaries. A validated high-performance liquid chromatography method was used to measure the concentration of BAK in each tear sample. The results showed rapid BAK dilution to 6.4 microg/mL, 3.2 microg/mL, 1.4 microg/mL, below the detection limit, and below the detection limit at 30 sec, 1 min, 3 min, 5 min, and 20 min after instillation of a single 35-microL drop of gatifloxacin. Because such rapid dilution reduces the concentration of BAK to near zero in minutes and does not allow the time (1 h) required for effective bacterial kill power, BAK is not expected to have a clinically significant effect on enhancement of the antimicrobial efficacy of gatifloxacin on the human ocular surface.

Assessment of corneal epithelial integrity after acute exposure to ocular hypotensive agents preserved with and without benzalkonium chloride

The corneal toxicity of 2 intraocular pressure-lowering agents was compared in a rabbit cornea model with New Zealand White rabbits. Corneal epithelial morphology and cell size were assessed by in vivo confocal microscopy. Baseline microscopic examinations were performed on 1 eye of each animal. Two weeks later, the eyes were bathed for 3 min in travoprost 0.004% preserved without benzalkonium chloride (BAK( or latanoprost 0.005% preserved with 0.02% BAK; the eyes were then rinsed with balanced salt solution, and the corneas were again examined by confocal microscopy (n=4/group). A second group of animals was exposed to the medications through a dosing regimen of 1 drop/min (lpar3 drops total) (n=4/group). In eyes treated with travoprost without BAK (3-min bath), superficial epithelial cells were similar to baseline, as indicated by their visible cell borders and bright nuclei. In contrast, the surface cells in eyes treated with latanoprost were significantly smaller and brighter and had less distinct borders. Surface cell size was significantly smaller as compared with baseline size and as compared with rabbits treated with travoprost without BAK for 3 min. Similar effects on corneal epithelial cell morphology were observed with the 1-drop/min dosing regimen. In this rabbit model, travoprost 0.004% preserved without BAK did not cause corneal epithelial toxicity; latanoprost 0.005% induced superficial cell loss, most likely caused by the presence of a relatively high concentration of BAK (0.02%).