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

Abstract.

Objectives: To investigate the mechanisms of action underlying the anti-inflammatory action of aceclofenac in vivo, we studied in vitro the effect of aceclofenac and its main metabolite, 4′-hydroxyaceclofenac, in comparison with diclofenac, another metabolite, on cyclooxygenases activity as well as interleukin-1β, -6 and -8, nitric oxide, and prostaglandin E₂ production by human osteoarthritic and normal articular chondrocytes. Methods: Enzymatically isolated human chondrocytes were cultured for 72 h in the absence or presence of interleukin-1β (IL-1β) or lipopolysacharride (LPS) and with or without increased amounts (1 to 30 μM) of aceclofenac or metabolites. The production of different cytokines was measured by Enzyme Amplified Sensitivity Immunoassays (EASIA). Prostaglandin E₂ was quantified by a specific radioimmunoassay. Nitrite and nitrate concentrations in the culture supernatants were determined by spectrophotometric method based upon the Griess reaction. Cyclooxygenase-2, inducible NO synthase and IL-1β 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 cell-free system using purified ovine cyclooxygenase-1 and -2 (COX-1 and COX-2) and at a cellular level using human whole blood assay. Results: We have demonstrated that aceclofenac, 4′-hydroxyaceclofenac and diclofenac significantly decreased interleukin-6 production at concentrations ranged among 1 to 30 μM and fully blocked prostaglandin E2 synthesis by IL-1β- or LPS-stimulated human chondrocytes. Aceclofenac and diclofenac had no effect on interleukin-8 production while 4′-hydroxyaceclofenac slightly decreased this parameter at the highest dose (30 μM). Aceclofenac was without effect on IL-1β- or LPS-stimulated nitric oxide production. At 30 μM, 4′-hydroxyaceclofenac inhibited both IL-1β or LPS-stimulated nitric oxide production while diclofenac inhibited only the LPS-stimulated production. Finally, at 30 μM, the three drugs significantly decreased IL-1β mRNA. In the whole blood test, aceclofenac and 4′-hydroxyaceclofenac weakly inhibited COX-1 with IC₅₀ values superior to 100 μM, but decreased by 50% COX-2 activity at the concentration of 0.77 and 36 μM, respectively. Diclofenac strongly inhibited both COX-1 and COX-2 with IC₅₀ values of 0.6 and 0.04 μM, respectively. On the other hand, aceclofenac and diclofenac weakly inhibited purified ovine cyclooxygenases with IC₅₀ values superior to 100 μM, whereas 4′-hydroxyaceclofenac was without effect. Conclusions: These results suggest that aceclofenac actions are multifactorial and that metabolites could contribute to its anti-inflammatory actions.