P47 A novel hydrogen sulfide-releasing NMDA receptor antagonist prevents ischemic neuronal death

Background Hydrogen sulfide (H2S) is an endogenously produced gaseous signaling molecule. A number of studies examined therapeutic potential of H2S-donating compounds and H2S gas itself in a variety of animal models of human disease and injury. In the majority of experimental studies, sodium sulfide (Na2S) and sodium hydrosulfide (NaHS) have been used as H2S donor compounds. However, because the short half-lives of these sulfide salts in biological fluid, plasma sulfide levels rapidly increase after bolus administration of Na2S or NaHS and then return to baseline instantaneously. To sustain “physiological” levels of sulfide in circulation after bolus administration, many slow-releasing H2S donor compounds, including 4-(3-thioxo-3H-1,2-dithiol-4-yl)-benzoic acid (ACS48), have been developed. While it has been reported that low and physiological levels of H2S protect neurons, H2S also exhibits neurotoxicity especially at high concentrations. Some investigators have suggested that H2S-induced neurotoxicity may be mediated via enhancement of N-methyl-D-aspartate receptor (NMDAR) activity, because toxicity of H2S was abolished by NMDAR antagonist in vitro and in vivo. Based on these observations, we hypothesized that a hybrid NMDAR antagonist that is capable of slowly releasing H2S in circulation is more effective in protecting neurons than H2S donor compounds alone. Method To characterize the neuroprotective effects of combination of exogenous H2S and NMDAR antagonism, we synthesized a novel H2S-releasing NMDAR antagonist N-((1r,3R,5S,7r)-3,5-dimethyladamantan-1-yl)-4-(3-thioxo-3H-1,2-dithiol- 4-yl)-benzamide (sulfur-memantine) by chemically combining a slow releasing H2S donor ACS48 with a NMDAR antagonist memantine. We examined protective effects of sulfur-memantine in cultured cells at baseline and after oxygen glucose deprivation (OGD) using a panel of cytotoxicity assays. Neuroprotective effects of sulfur-memantine were validated in a mice model of cerebral ischemia and reperfusion injury. Result While sulfur-memantine and ACS48 increased sulfide levels similarly in cell culture medium, sulfur-memantine augmented intracellular sulfide concentration in human neuroblastoma cells (SH-SY5Y) ten-fold higher than did ACS48 ( Fig. 1 ). Incubation with sulfur-memantine after reoxygenation following OGD protected SH-SY5Y and murine primary cortical neurons more markedly than did ACS48 or memantine. Although Na2S and ACS48 exhibited dose-dependent cytotoxicity to primary cortical neurons measured by lactate dehydrogenase release, memantine and sulfur-memantine showed no cytotoxicity up to 50 μM. Glutamate-induced intracellular calcium accumulation in primary cortical neurons were aggravated by Na2S or ACS48, but suppressed by memantine and sulfur-memantine. Sulfur-memantine prevented glutamate-induced glutathione depletion in SH-SY5Y more markedly than did Na2S or ACS48. Sulfur-memantine prevented caspase-3 activation in SH-SY5Y subjected to OGD. The anti-apoptotic effects of sulfur-memantine were associated with maintenance of extracellular-signal regulated kinase 1/2 (ERK) phosphorylation but not with changes in Akt phosphorylation. Administration of sulfur-memantine 1 min after reperfusion following 45 min of bilateral carotid occlusion reduced cerebral infarct volume and improved survival and neurological function of mice compared to Na2S, ACS48, or memantine up to 60 days after the ischemic insult ( Fig. 2 ). Conclusion These results suggest that a novel H2S-releasing NMDAR antagonist derivative sulfur-memantine prevents cerebral ischemia–reperfusion injury. Hybrid H2S-releasing NMDAR antagonists may provide a novel therapeutic strategy for ischemic brain injury and other forms for neuredegeneration.