Maffei Facino R, Carini M, Aldini G, Calloni M T
Istituto Chimico Farmaceutico Tossicologico, Milan, Italy.
Drugs Exp Clin Res. 1997;23(5-6):157-65.
The protective effect of hydroxynimesulide, the main metabolite of the nonsteroidal antiinflammatory drug nimesulide, on red blood cells (RBCs, 0.2%; 3.5 x 10(7) cell/ml) hemolysis induced by cumene hydroperoxide (CuOOH; 50 microM) 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 micron (% hemolysis 85.2 +/- 3.4%) and increases at the higher concentrations (63.5 +/- 3.9% at 5 microM; 43.5 +/- 6.3% at 10 microM; and, 14.5 +/- 4.3% at 20 microM). In addition, in the samples protected with 10 microM and 20 microM, there is a significant delay (30 and 60 min) in the onset of the hemolytic response. Inhibition of hemolysis is the result of protection of RBC membrane integrity, both on lipid (cis-Parinaric acid fluorescence quenching was delayed by 53 +/- 10 sec vs. the controls at 1 micron, by 115 +/- 15 sec at 5 microM, with a lag phase of 240 +/C- 18 sec at 10 microM) 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 microM) 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.
通过比浊法和形态学分析，评估了非甾体抗炎药尼美舒利的主要代谢产物羟基尼美舒利对氢过氧化异丙苯（CuOOH；50微摩尔）诱导的红细胞（RBCs，0.2%；3.5×10⁷个细胞/毫升）溶血的保护作用。羟基尼美舒利以剂量依赖性方式抑制CuOOH诱导的溶血：在孵育150分钟后计算保护作用（对照组溶血100%），在1微摩尔时开始有保护作用（溶血率85.2±3.4%），且在较高浓度时增加（5微摩尔时为63.5±3.9%；10微摩尔时为43.5±6.3%；20微摩尔时为14.5±4.3%）。此外，在用10微摩尔和20微摩尔保护的样品中，溶血反应的起始有显著延迟（分别为30分钟和60分钟）。溶血的抑制是保护红细胞膜完整性的结果，包括脂质（顺式紫黄质酸荧光猝灭在1微摩尔时比对照组延迟53±10秒，在5微摩尔时延迟115±15秒，在10微摩尔时有240±18秒的延迟期）和蛋白质成分，这通过SDS-PAGE电泳确定。在溶血实验中，羟基尼美舒利的功效与α-生育酚相当，并观察到羟基尼美舒利和α-生育酚（均为10微摩尔）之间存在协同相互作用。这些结果表明，羟基尼美舒利通过直接淬灭血红蛋白/过氧化物相互作用产生的活性氧来保护红细胞膜。该代谢产物直接清除自由基干预的证据来自HPLC研究，其表明羟基尼美舒利随时间消耗，并伴随形成两种主要反应（加成/氧化）产物。
