Methylene Blue, A Novel Treatment of H2S Poisoning-induced Brain Injury

 描述(申请人提供)：硫化氢(硫化氢)中毒的流行病学特征在过去几年中发生了变化。尽管硫化氢仍对天然气或石油行业的工人构成持续的化学危险，但硫化氢中毒已成为一种新的自杀方式，在包括美国在内的许多国家，这一事件的发生率以惊人的速度上升。硫化氢毒性是直接抑制神经元线粒体活动和心肺衰竭(呼吸暂停和休克)对氧气输送的后果的综合结果。在低于0.1%的浓度下，硫化氢在几分钟内就会致命；在较低浓度下，硫化氢会导致急性昏迷，导致幸存患者的记忆和认知障碍。我们最近发现，在接触硫化氢后，游离/可溶的硫化氢很快消失，这是中毒后使用金属化合物的一个主要限制，金属化合物是唯一一类被证明有效的解毒剂 在接触硫化氢的过程中。事实上，目前提出的抗硫化氢毒性的Fe3+或Co2+化合物的益处取决于它们捕获血液中游离/可溶性硫化氢的能力，例如 用高铁血红蛋白，或在细胞内，用羟钴胺(Hyco)。然而，这些药物对结合在蛋白质上的H_2S池的作用有限，对H_2S诱导的循环衰竭的缺血后果没有影响。根据我们关于亚甲基蓝在硫化氢中毒中的作用以及亚甲基蓝对缺氧或中毒后脑损伤的显著保护作用的初步数据，我们建议使用亚甲基蓝来治疗硫化氢中毒。这种方法代表了一种新的范式，因为它特异性地中和了硫化氢诱导的细胞和线粒体功能障碍。在这个项目中，我们将确定MB预防硫化氢诱导的脑损伤的短期和长期影响的能力，当在可逆性昏迷的大鼠模型中急性硫化氢中毒后给予MB。我们开发了这个模型来模拟人类最常见的非致命性形式的硫化氢中毒。亚甲蓝在硫化氢诱导昏迷后的短期效应(头12小时)将通过缺氧后损伤的生理和临床参数以及脑缺氧或细胞凋亡的标志物来评估。在另一组大鼠中，将评估非麻醉动物对记忆和认知功能的长期影响，这些动物经过训练，在莫里斯水迷宫中进行测试，在硫化物中毒导致的昏迷发作后，急性或重复给予MB。甲基溴将单独或与Hyco联合使用，以确定这两种解毒剂对抗硫化氢中毒的潜在协同作用。这些研究的结果将导致一种新的策略，使用一种试剂来抵消硫化氢中毒的直接后果。由于MB似乎对其他形式的线粒体中毒也有效，这些结果可能具有超越硫化氢中毒治疗的影响。

 DESCRIPTION (provided by applicant): The epidemiological features of hydrogen sulfide (H2S) intoxication have changed over the last few years. While H2S still poses a persistent risk as a chemical hazard for workers in gas or oil industry, H2S poisoning has become a new method of suicide with an incidence which has increased at an alarming rate in many countries including the US. H2S toxicity results from the combination of a direct inhibition of the mitochondrial activity of neurons and the consequences of the cardio-respiratory failure (apnea and shock) on oxygen delivery. H2S is lethal within minutes at a concentration of less than 0.1 %; at lower concentrations, H2S produces an acute coma leading to memory and cognitive deficits in the surviving patients. Our recent findings of the very fast disappearance of free/soluble H2S following hydrogen sulfide exposure represents a major limitation for using, after the intoxication, metallo-compounds, i.e. the only family of antidotes with a proven efficacy during H2S exposure. Indeed, the benefit of Fe3+or Co2+ compounds currently proposed against H2S toxicity relies on their ability to trap free/soluble H2S in the blood, as for instance with methemoglobin, or inside cells, with Hydroxocobalamin (HyCo). However, these agents have a limited action against the pool of H2S combined on proteins and have no effect against the ischemic consequences of H2S-induced circulatory failure. Based on our preliminary data on the effects of Methylene Blue (MB) during H2S poisoning and the remarkable protective effects of MB on post-anoxic or post-toxic brain injury, we are proposing to use MB for the treatment of H2S poisoning. This approach represents a novel paradigm as it specifically counteracts the cellular and mitochondrial dysfunctions induced by H2S. In this project we will determine the ability of MB to prevent the short and long-term effects of H2S- induced brain injury, when administered after an acute H2S intoxication in a rat model of reversible coma. We have developed this model to mimic the most common type of non-lethal form of H2S poisoning in humans. The short-term effects (first 12 hours) of MB following H2S-induced coma will be evaluated by physiological and clinical parameters of post-anoxic injury and brain markers of hypoxia or apoptosis. In a separate group of rats, the long-term effects on memory and cognitive functions will be evaluated in non-anesthetized animals trained to perform tests in a Morris Water Maze after acute or repetitive administrations of MB following an episode of sulfide poisoning-induced coma. MB will be administered alone or combined with HyCo to determine the potential synergy of these 2 antidotes against H2S poisoning. The results of these studies will lead to a novel strategy using an agent counteracting the direct consequences of H2S poisoning. Since MB appears to be also effective against other forms of mitochondrial poisoning, these results may have implications beyond the treatment of H2S intoxication.