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C. Mehanna, F. Brignole-Baudouin, C. Baudouin; An in vitro Model for Toxicological Comparison of Fixed and Unfixed Combinations: The Case of Brimonidine/Timolol. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3161.
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© ARVO (1962-2015); The Authors (2016-present)
Our objective was to develop an in vitro model to compare cytotoxic effects of fixed combinations (FC) with their corresponding unfixed combinations (UC). We evaluated the effects of brimonidine/timolol FC and compared the solution to its separate components and to the UC.
WKD human conjunctival cells were exposed for 30min to 1/10-diluted solutions of: preserved timolol, brimonidine, brimonidine/timolol-FC (BriT-FC), brimonidine/timolol-UC (BriT-UC), and BAC (0.005%, 0.010% and 0.015%). Cell viability, oxidative stress (O2•-) and apoptosis were evaluated at 30min and 24h using neutral red, hydroethidine and YO-PRO®-1 probes respectively.
BAC reduced cell viability, increased O2•- production and induced apoptosis in a concentration-dependant manner. These effects were increased at 24 hours for high BAC concentrations. FC was significantly less toxic than UC with the three assays (P < 0.0001). There was no significant difference between FC and the corresponding BAC solution whereas BriT-UC reduced O2•- production at 24 hours (P < 0.0001) and had similar viability and apoptotic effects when compared to BAC 0.015%. Timolol/BAC+ was also less toxic than BAC 0.010% at 24 hours by inducing lower superoxide radical production (P < 0.0001). However, difference was not significant for apoptosis. Brimonidine, associated to polyvinyl alcohol, showed better cell viability than control (P = 0.0146) and less apoptosis than BAC 0.005% (P = 0.0181).
Our in vitro model appeared to be adequate for UC-treatment simulation and may have future applications. It showed that timolol induced late cytoprotective effects in toxic environments and proved that FC was less toxic than UC, mainly because of lower BAC concentration. It confirmed the interest of FC for lowering ocular surface side effects in long-term treated glaucoma patients.
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