In vitro effects of aceclofenac and its metabolites on the production by chondrocytes of inflammatory mediators

Objectives: To investigate the mechanisms of action underlying the anti-inf lammatory action of aceclofenac in vivo, we studied in vitro the effect of aceclofenac and its main metabolite, 4'-hydroxyaceclofenac, in comparison w ith diclofenac, another metabolite, on cyclooxygenases activity as well as interleukin-1 beta, -6 and -8, nitric oxide, and prostaglandin E-2 producti on by human osteoarthritic and normal articular chondrocytes. Methods: Enzymatically isolated human chondrocytes were cultured for 72 It in the absence or presence of interteukin-1 beta (IL-1 beta) or lipopolysac harride (LPS) and with or without increased amounts (1 to 30 muM) of aceclo fenac or metabolites. The production of different cytokines was measured by Enzyme Amplified Sensitivity Immunoassays (EASIA). Prostaglandin E-2 was q uantified by a specific radioimmunoassay. Nitrate and nitrate concentration s in the culture supernatants were determined by spectrophotometric method based upon the Griess reaction. Cyclooxygenase-2, inducible NO synthase and IL-1 beta gene expression were quantified by reverse transcription of mRNA followed by real time and quantitative polymerase chain reaction. Finally, cyclooxygenase inhibitory potency of the drugs was also tested in both a c ell-free system using purified ovine cyclooxygenase-1 and -2 (COX-1 and COX -2) and at a cellular level using human whole blood assay.