Hydroxynimesulide, the main metabolite of nimesulide, prevents hydroperoxide/hemoglobin-induced hemolysis of rat erythrocytes

The protective effect of hydroxynimesulide, the main metabolite of the nonsteroidal antiinflammatory drug nimesulide, on red blood cell (RBCs, 0.2%; 3.5x10(7) cells/ml) hemolysis induced by cumene hydroperoxide (CuOOH; 50 mu M) was evaluated by turbidimetric and morphological analyses. Hydroxynimesulide inhibits the CuOOH-induced hemolysis in a dose dependent fashion: the protective effect, calculated after 150 min incubation (100% hemolysis in the controls), starts at 1 mu M (% hemolysis 85.2 +/- 3.4%) and increases at the higher concentrations (63.5 +/- 3.9% at 5 mu M; 43.5 +/- 6.3% at 10 mu M; and, 14.5 +/- 4.3% at 20 mu M). In addition, in the samples protected with 10 mu M and 20 mu M, there is a significant delay (30 and 60 min) in the onset of the hemolytic response. Inhibition of hemolysis is the result of a protection of RBC membrane integrity both on lipid (cis-Parinaric acid fluorescence quenching was delayed by 53 +/- 10 sec vs. the controls at 1 mu M, by 115 +/- 15 sec at 5 mu M, with a lag phase of 240 +/- 18 sec at 10 mu M) and protein constituents, as determined by SDS-PAGE electrophoresis. In hemolysis experiments, the efficacy of hydroxynimesulide is comparable to that of alpha-tocopherol and a cooperative interaction between hydroxynimesulide and alpha-tocopherol (both at 10 mu M) has been observed These results indicate that hydroxynimesulide protects RBC membranes by directly quenching reactive oxygen species generated by hemoglobin/peroxide interaction. Evidence for a direct radical scavenging intervention of the metabolite comes from HPLC studies, which demonstrate a time-dependent consumption of hydroxynimesulide, with the concomitant formation of two main reaction (addition/oxidation) products.