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N. S. Patel, R. Migon, S. Mansoor, M. C. Kenney, B. D. Kuppermann; In vitro Safety of Bromfenac on Human Retinal Pigment Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4740.
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© ARVO (1962-2015); The Authors (2016-present)
To evaluate the in vitro safety of bromfenac ophthalmic solution (BOS) on human retinal pigment epithelial cells (ARPE-19).
ARPE-19 cells were treated with 0.09% BOS at 22.5mcg/ml (1X clinical dose), 45.0mcg/ml (2X), 90.0mcg/ml (4X), 11.25mcg/ml (X/2) or 5.625mcg/ml (X/4). Clinical dose was defined as the concentration of drug in 0.1ml of 0.09% BOS injected into the 4ml human vitreous assuming equal drug distribution throughout the vitreous. Cells were incubated at 37º C until confluent. Before drug exposure, the cells were incubated for 24 hours in fetal bovine serum free medium to make them relatively non-proliferating. After 24 hours of drug exposure, the following assays were performed: trypan blue dye exclusion to measure cell viability (CV), JC-1 to measure changes in mitochondrial membrane potential (ΔΨM), caspase-3/7 as an indicator of apoptotic activity and the 2’, 7’-dichlorodihydrofluorescin diacetate (DCFH-DA) assay to measure accumulation of reactive oxygen species (ROS).
ARPE-19 cells treated with 4X and 2X BOS showed mean CV of 16.95±3.04% (p<0.001), and 28.45±2.33% (p0.05). The ΔΨM was decreased and the caspase 3 /7 activity increased in ARPE-19 cells treated with all BOS concentrations when compared to untreated ARPE 19 controls (15.15±1.05). The ΔΨM of ARPE-19 cells were 2.74±0.36 (p<0.001), 2.35±0.33 (p<0.001), 2.63±0.20 (p<0.001), 5.30±0.18 (p<0.001) and 5.22±0.15 (p<0.001) for 4X, 2X, X, X/2 and X/4 concentrations respectively. The ROS activity was significantly increased for all BOS concentrations when compared to untreated control. Caspase 3/7 activity was significantly increased for all BOS concentrations.
Bromfenac ophthalmic solution at concentrations 2X clinical dose or higher decreases retinal cell viability in vitro and all BOS concentrations significantly induce JC1, ROS, and caspase 3/7 activity.
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