Supplementary Material for: The Neuroprotection Exerted by Memantine, Minocycline and Lithium, against Neurotoxicity of CSF from Patients with Amyotrophic Lateral Sclerosis, Is Antagonized by Riluzole

In a recent study we found that cerebrospinal fluids (CSFs) from amyotrophic lateral sclerosis (ALS) patients caused 20-30% loss of cell viability in primary cultures of rat embryo motor cortex neurons. We also found that the antioxidant resveratrol protected against such damaging effects and that, surprisingly, riluzole antagonized its protecting effects. Here we have extended this study to the interactions of riluzole with 3 other recognized neuroprotective agents, namely memantine, minocycline and lithium. We found: (1) by itself riluzole exerted neurotoxic effects at concentrations of 3-30 µM; this cell damage was similar to that elicited by 30 µM glutamate and a 10% dilution of ALS/CSF; (2) memantine (0.1-30 µM), minocycline (0.03-1 µM) and lithium (1-80 µg/ml) afforded 10-30% protection against ALS/CSF-elicited neurotoxicity, and (3) at 1-10 µM, riluzole antagonized the protection afforded by the 3 agents. These results strongly support the view that at the riluzole concentrations reached in the brain of patients, the neurotoxic effects of this drug could be masking the potential neuroprotective actions of new compounds being tested in clinical trials. Therefore, in the light of the present results, the inclusion of a group of patients free of riluzole treatment may be mandatory in future clinical trials performed in ALS patients with novel neuroprotective compounds.

在近期一项研究中，我们发现肌萎缩侧索硬化症 (amyotrophic lateral sclerosis, ALS) 患者的脑脊液 (cerebrospinal fluids, CSF) 可导致大鼠胚胎运动皮层神经元原代培养物的细胞活力下降20%~30%。
我们还发现抗氧化剂白藜芦醇 (resveratrol) 可对抗此种损伤效应，且令人意外的是，利鲁唑 (riluzole) 反而会拮抗其保护作用。
本研究将此前的工作拓展至利鲁唑与另外三种公认的神经保护剂——美金刚 (memantine)、米诺环素 (minocycline) 与锂 (lithium)——的相互作用研究。
我们得到如下发现：(1) 利鲁唑单独使用时，在3~30 µM浓度下会产生神经毒性作用；此种细胞损伤程度与30 µM谷氨酸 (glutamate) 以及10%稀释度的ALS脑脊液所引发的损伤相当；(2) 美金刚 (0.1~30 µM)、米诺环素 (0.03~1 µM) 与锂 (1~80 µg/ml) 可对ALS脑脊液诱导的神经毒性提供10%~30%的保护作用；(3) 在1~10 µM浓度下，利鲁唑会拮抗上述三种药物的保护效应。
上述结果有力支持了这一观点：当利鲁唑浓度达到患者脑内的实际水平时，该药物自身的神经毒性作用可能会掩盖新型神经保护化合物在临床试验中展现出的潜在神经保护功效。
因此，结合本研究结果，未来针对ALS患者开展新型神经保护化合物临床试验时，纳入一组未接受利鲁唑治疗的患者队列或许是必要的。