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V. R. Sharma, R. Z. Migon, S. Mansoor, A. U. Sapkal, N. Gupta, C. A. Ramirez, M. C. Kennney, B. D. Kuppermann; In vitro Safety of Diclofenac Sodium on Human Retinal Pigment Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4747.
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© ARVO (1962-2015); The Authors (2016-present)
To evaluate the in vitro safety of diclofenac sodium ophthalmic solution (DS) on human retinal pigment epithelial cells (ARPE-19).
ARPE-19 cells were treated with 0.1% DS at 0.025 mg/ml (1X clinical dose), 0.05 mg/ml (2X), 0.1mg/ml (4X), 0.0125 mg/ml (X/2), or 0.00625 mg/ml (X/4). Clinical dose was defined as the concentration of drug in 0.1ml of 0.1% DS solution injected into the 4ml human vitreous assuming equal distribution throughout the vitreous. Cells were plated in 6 well plates for the cell viability (CV) assay (5x105 cells/well) and 24-well plates for JC-1 assay (1.2x105 cells/well) and were incubated at 37º C until confluent. Before drug exposure, the cells were incubated for 24 hours in 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 and JC-1 to measure changes in mitochondrial membrane potential (ΔΨM).
ARPE-19 cells treated with 4X, 2X, 1X ,X/2 and X/4 DS showed mean CV of 14.95±2.70% (p<0.001), 26.65±1.53% (p<0.001), 67.30±2.75% (p<0.001) and 90.68± 2.36 (p<0.05) respectively, compared to untreated ARPE-19 controls (95.63±1.98%). Mean CV of ARPE-19 cells treated with X/4 was not significantly reduced: 93.20±1.43% (p>0.05). The ΔΨM/JC 1 activity was decreased (p<0.001) in ARPE-19 cells treated with all DS concentrations: 3.30±0.23, 3.26±0.15, 3.93±0.26, 4.67±0.79, 6.62±0.28 for 4X, 2X, X, X/2 and X/4 respectively compared to untreated ARPE-19 controls (11.03±0.24).
Diclofenac Sodium at concentrations X/2 clinical dose or higher decreases ARPE-19 cell viability in vitro. All measured DS concentrations (X/4 or greater) significantly decrease JC1 activity/mitochondrial membrane potential.
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