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G. Cattaneo, B. De Servi, M. Meloni; In Vitro Assessment Of Long Term Eye Compatibility Of Preserved Or Preservative Free Timolol Eye Drops And Eye Gel Formulations.. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3195. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
to assess eye irritation potential and long term compatibility of four marketed ophthalmological timolol preparations by using a biologically relevant and sensitive 3D model of human corneal epithelium (HCE) and Multiple Endpoint Analysis (MEA)
Protocol for MEA was structured on 4 time points: 24h, 24h followed by product wash and by a post incubation period, and repeated application (twice daily) for a continuous exposure of 72h followed by a 72h recovery. After application of Timolabak®(preservative free eye drops),Timoptol®(BAK preserved eye drops) , Nyogel®(BAK preserved eye gel),Timogel®(preservative free eye gel) and BAK 0.01% as positive control, the following parameters were quantified:Cellular viability, Histological analysis (H&E staining),IL-1α and IL-8 passive release by ELISA and the OCCLUDIN gene expression by quantitative real time-PCR
Cell viability measured at basal level of HCE model by MTT test method were reduced under the 50%cut-off value after acute exposure (24h and 24h +24h) by BAK 0.01%, Timoptol, and Nyogel. After repeated application (72h and 72h+72h) Timolabak and Timogel never reduced under 50 % viability after all the exposures. Morphological results were consistent with the viability data, Timolabak and Timogel didn't significantly modify HCE morphology after different exposures. Release of the pro-inflammatory cytokine IL-1 α has been quantified higher than the negative control (>20pg/ml) for the positive control (BAK 0.01% ) Nyogel and Timoptol treatments and it was not different after treatment with Timolabak and Timogel. A prediction model based on occludin mRNA allowed to classify the products as No irritant (Timolabak), borderline between Non irritants and Extremely mild ( Nyogel and Timogel), Slightly mild (Timoptol and BAK 0.01%).
By introducing a molecular endpoint such as the mRNA expression of occludin into the MEA, we added an early, sensitive and quantitative biomarker able to reveal significant effects often neglected as the early and superficial signs of toxicity.
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