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G. Graff, J.–Y. Wei; NSAID–Induced Swelling of Peroxide–Stressed Mitochondria in vitro . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2760.
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Assess the in vitro effect of nepafenac and selected NSAIDs on mitochondria under normal and peroxide–stress conditions.
Mitochondria were prepared from livers of male Sprague Dawley rats according to Broekemeier at al. and were resuspended in ice–cold, iso–osmotic HEPES buffer (3.0 mM), supplemented with mannitol (207 mM), and sucrose (63mM) (pH 7.4). An appropriate aliquot of the mitochondrial preparation was added to 2.95 mL of iso–osmotic, HEPES (3.0 mM) buffer containing sodium succinate (10 mM), and rotenone (1 nmol/mg protein) (pH 7.4). Mitochondrial swelling was monitored at 540 nm with an OLIS/Aminco DW2c dual–beam spectrophotometer. Swelling (positive control) was induced by addition of CaCl2 (60 nmol/mg mitochondrial protein) and tertiary butylhydroperoxide (t–BHP;150 µM).
Exposure of unstressed mitochondria to concentrations of 100 µM of nepafenac, amfenac, bromfenac, ketorolac, ibuprofen and diclofenac in iso–osmotic buffer did not promote swelling as assessed by light scattering. However, when stressed with t–BHP (which by itself does not promote swelling) mitochondria underwent rapid swelling upon addition of i.e., diclofenac. The rate and extent of organelle swelling increased with increasing concentrations of NSAID added. This behavior was qualitatively similar for every NSAID tested, including ibuprofen, amfenac, bromfenac, dicofenac, and ketorolac. The order of potency for induction of half–maximal swelling was bromfenac (EC50 = 9 µM) = amfenac (EC50 = 10 µM) > diclofenac (EC50 = 19 µM) = ibuprofen (EC50 = 22 µM) > ketorolac (EC50 = 529 µM). Notably, nepafenac, the carboxamide derivative and prodrug form of amfenac, did not promote mitochondrial swelling even at the highest concentration tested (i.e., 600 µM).
Classical NSAIDs with free carboxylic acid function were innocuous to unstressed, isolated liver mitochondria, but caused concentration–dependent swelling in the presence of low concentrations of peroxide. Nepafenac, the carboxamide derivative and prodrug form of amfenac, is unique among clinically used ocular NSAIDs in that it had no detectable toxic effects on mitochondria, even when stressed with peroxide.
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