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.

在一项近期研究中，我们发现肌萎缩侧索硬化症（amyotrophic lateral sclerosis, ALS）患者的脑脊液（cerebrospinal fluid, 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%。我们同时发现，抗氧化剂白藜芦醇可对抗上述损伤效应；而令人意外的是，利鲁唑（riluzole）竟会拮抗其保护作用。
本研究将前述实验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）及锂剂（lithium）——的相互作用研究。我们获得如下结果：（1）利鲁唑单独作用时，在3~30 µM浓度下可产生神经毒性效应，其引发的细胞损伤程度与30 µM谷氨酸以及10%稀释度的ALS/CSF所致损伤相当；（2）美金刚（0.1~30 µM）、米诺环素（0.03~1 µM）及锂剂（1~80 µg/ml）可对ALS/CSF诱导的神经毒性产生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下述观点：当患者脑内达到利鲁唑的常规血药浓度时，该药物本身的神经毒性效应可能会掩盖临床试验中正在测试的新型化合物的潜在神经保护作用。
因此，基于本研究结果，未来针对ALS患者开展新型神经保护剂临床试验时，纳入一组未接受利鲁唑治疗的患者队列或为必要之举。