In vitro effects of preservative-free tafluprost and preserved latanoprost, travoprost, and bimatoprost in a conjunctival epithelial cell line.

PURPOSE: This study compared the toxicity profiles of three antiglaucoma prostaglandin F2alpha analogs, latanoprost, travoprost, and bimatoprost which contain benzalkonium chloride (BAK), with tafluprost, a new preservative-free prostaglandin analog. METHODS: IOBA-NHC cells were exposed to BAK-containing prostanoid solutions, their respective BAK concentrations, and preservative-free tafluprost solution for 30 min. Membrane integrity, apoptosis, oxidative stress, and cells morphology were evaluated. RESULTS: Preservative-free tafluprost resulted in significantly higher membrane integrity and lower pro-apoptotic and pro-oxidative effects than preservative-containing prostaglandin analog preparations. CONCLUSIONS: These results suggest that tafluprost, a new preservative-free prostaglandin analog, has very low or no pro-apoptotic, pro-necrotic, or pro-oxidative effects in vitro compared to preservative-containing formulations.

Comparative study of topical anti-allergic eye drops on human conjunctiva-derived cells: responses to histamine and IFN gamma and toxicological profiles.

BACKGROUND: The purpose of the study was to compare toxic effects and responses to histamine and IFN gamma associated with the use of some widely used anti-allergic eye drops commercially available today. METHODS: For dynamic studies, the Wong-Kilbourne cell line was stimulated for 24 h with histamine or IFN gamma in the presence or absence of anti-allergic eye drops. Supernatants of histamine-stimulated cells were evaluated for the production of IL-6 and IL-8 by ELISA, while the expression of ICAM-1 was evaluated by flow cytometry on IFN gamma-stimulated cells. Toxicological assays were performed using cold light cytofluorometry: viability and apoptosis as well as reactive oxygen species (ROS) and O2(.)- production were assessed using neutral red, Hoechst/propidium iodide, H(2)-DCFDA and hydroethidine tests, respectively. RESULTS: Antihistamines reduced IL-6 release and presented dose-dependent inhibitory effects on IL-8 production. None of the eye drops decreased the basal or IFN gamma-stimulated expression of ICAM-1. Conversely, eye drops preserved with benzalkonium chloride (BAC) induced even higher ICAM-1 expression levels on IFN gamma-stimulated cells than did IFN gamma alone, whereas unpreserved drugs had no effect. Toxicological assays confirmed the pivotal role of BAC in proportionally reducing cell viability while increasing apoptosis and oxidative stress. CONCLUSIONS: The ability of topical ocular anti-H(1) drugs to significantly reduce the production of IL-6 and IL-8 argues that they may help treat the inflammatory processes occurring in allergic ocular surface disorders. Nevertheless, preserved ophthalmic formulations may enhance epithelial conjunctival expression of ICAM-1 in the presence of a low inflammatory stimulus, such as IFN gamma, and displayed toxic as well as pro-oxidative effects on these cells. Therefore, BAC used as preservative might in part interfere with the potential anti-inflammatory properties of the active compound by modulating the immuno-inflammatory response of epithelial conjunctival cells.

In vitro comparison of cytoprotective and antioxidative effects of latanoprost, travoprost, and bimatoprost on conjunctiva-derived epithelial cells.

PURPOSE: In a previous study, it was demonstrated that in vitro in a human conjunctiva-derived cell line, latanoprost in its commercial presentation appeared to be less toxic than the benzalkonium chloride (BAC) it contains as a preservative. Through a microplate cytometry technique, the investigation was furthered by study of whether the three commercially available antiglaucoma prostaglandin analogs could protect the same cell line in vitro against BAC toxicity and whether an antioxidative mechanism could be involved in such prostaglandin effects. METHODS: Human conjunctiva-derived epithelial cells from the Chang cell line were exposed to three prostaglandins in their commercial presentation (latanoprost, travoprost, and bimatoprost) and to three concentrations of BAC (0.02%, 0.015%, and 0.005%), corresponding to the concentrations contained in the three prostaglandin eyedrops. Each solution was diluted to 1/10 and was applied for 30 minutes Cellular membrane integrity, cytosolic H2O2, cytosolic O2*- and apoptosis were evaluated using neutral red, H2DCF-DA, hydroethidine, and Yopro-1 probes, respectively. RESULTS: Cellular viability decreased as BAC concentration increased, but it was accompanied by concentration-dependent toxicity. Toxicity of latanoprost and travoprost commercial solutions was statistically significantly lower than their respective BAC concentrations (P < 0.01), whereas bimatoprost induced no significant effects. There was a statistically significant decrease in H2O2 detection with cells exposed to latanoprost (P < 0.01) and travoprost (P < 0.01) and a lower detection of O2*- with cells exposed to latanoprost (P < 0.01) compared with the corresponding BAC concentration alone. The Yopro-1 test showed a BAC-induced apoptotic effect that increased with its concentration. Latanoprost and travoprost produced proapoptotic effects compared with control (P < 0.01), but these were lower than their respective preservative concentrations (statistically significant difference; P < 0.01). CONCLUSIONS: Latanoprost and travoprost were responsible for significant protective effects against BAC toxicity on conjunctiva-derived epithelial cells in vitro, probably related to their antioxidative properties. The low toxicity of the bimatoprost solution did not reveal a possible antioxidative effect. Reduced reactive oxygen species production could be the main mechanism by which prostaglandin analogs protect epithelial cells from the proapoptotic effects of BAC. Further studies will be useful to confirm this hypothesis.

Optical coherence tomography in the acute and chronic phases of Vogt-Koyanagi-Harada disease.

PURPOSE: To assess the potential of optical coherence tomography (OCT) in the diagnosis and monitoring of serous retinal detachment in Vogt-Koyanagi-Harada (VKH) disease and to describe OCT characteristics of subretinal sequelae of the disease. METHODS: Six patients in the acute phase of VKH disease with serous retinal detachment were followed in our department from July 2001 to December 2003 using slit-lamp biomicroscopy, OCT, and fluorescein angiography. RESULTS: OCT was effective in objectively quantifying the amount of serous retinal detachment present and then in following the resolution of subretinal fluid accumulation. Subretinal pigmented lesions on angiography corresponded with retinal pigment epithelium hypertrophy and fibrosis on OCT. CONCLUSION: A beneficial effect of treatment was observed within days, paralleling the improvement in visual acuity. Retinal pigment epithelium hypertrophy and fibrosis in the chronic phase of the disease were analyzed with OCT for the first time.

In vitro study of inflammatory potential and toxicity profile of latanoprost, travoprost, and bimatoprost in conjunctiva-derived epithelial cells.

PURPOSE: Conjunctiva-derived epithelial cells were used to investigate, in vitro, the expression of various inflammation-associated markers known to be overexpressed in patients with glaucoma after contact with the three major commercially available eye drops containing prostaglandin analogues. The impact on cellular viability and apoptosis in the same cell line was evaluated, to address the possible proinflammatory and/or toxic origin of the most frequent clinical impairments induced by prostanoids (i.e., conjunctival hyperemia). METHODS: Conjunctiva-derived cells were treated in vitro with the commercial solutions of latanoprost, travoprost, bimatoprost, prostaglandin (PG)F2alpha, tumor necrosis factor (TNF)-alpha, and different concentrations of benzalkonium chloride (BAC). Expressions of three inflammation- and immune-related markers, intercellular adhesion molecule (ICAM)-1, platelet-endothelial cell adhesion molecule (PECAM)-1 and HLA DR, were evaluated with flow cytometry after 24 to 72 hours of contact at low, subtoxic concentrations. Toxicological tests were also performed with cold-light cytofluorometry, in which cellular viability and apoptosis were evaluated with the neutral red and Hoechst/propidium iodide tests, respectively. RESULTS: TNFalpha induced or stimulated expression of the three inflammatory markers, whereas the PGF2alpha, latanoprost, travoprost, and bimatoprost solutions did not induce an increase in these markers and even produced a marked reduction of ICAM-1 and PECAM-1 expression in those solutions most concentrated in BAC, thus suggesting a toxic phenomenon in cellular membranes induced by the preservative rather than the medication itself. Cytotoxic assays confirmed this hypothesis and showed significant toxicity with prostaglandin analogues after prolonged contact, proportional to the concentration of BAC in the solution and similar to that of the corresponding concentration of BAC alone, bimatoprost having both the least concentration of BAC and the least cytotoxic in these experimental conditions. CONCLUSIONS: The comparison of latanoprost, travoprost, and bimatoprost, in their commercial formulations, showed that none of them appeared to induce direct stimulation of the inflammatory pathways involving adhesion molecules or class II antigens, although these markers have been found ex vivo in conjunctival specimens from patients treated with prostaglandins. In fact, their toxicity was mild and seemed to be primarily related to the concentration of BAC, their common preservative, which may be the major factor responsible for long-term ocular surface reactions in patients receiving topical prostaglandins, but most likely is not a factor in early and transient conjunctival hyperemia.