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

 描述（由申请人提供）：硫化氢（H2S）中毒的流行病学特征在过去几年中发生了变化。虽然H2S仍然对天然气或石油工业的工人构成持续的化学危害，但H2S中毒已成为一种新的自杀方法，在包括美国在内的许多国家，其发病率以惊人的速度增长。H2S毒性是由神经元线粒体活性的直接抑制和心脏呼吸衰竭（呼吸暂停和休克）对氧气输送的后果的组合引起的。H2S在浓度低于0.1%时在数分钟内是致命的;在较低浓度下，H2S产生急性昏迷，导致存活患者的记忆和认知缺陷。我们最近发现，在硫化氢暴露后，游离/可溶性H2S非常快速地消失，这代表了中毒后使用金属化合物的主要限制，金属化合物即唯一具有已证实功效的解毒剂家族 在H2S暴露期间。实际上，目前提出的Fe 3+或Co2+化合物对抗H2S毒性的益处依赖于它们捕获血液中游离/可溶H2S的能力，例如 高铁血红蛋白，或细胞内，羟钴胺（HyCo）。然而，这些药物对结合在蛋白质上的H2S池的作用有限，并且对H2S诱导的循环衰竭的缺血性后果没有作用。根据我们对亚甲基蓝（MB）在H2S中毒过程中的作用的初步数据，以及MB对缺氧后或中毒后脑损伤的显著保护作用，我们建议使用MB治疗H2S中毒。这种方法代表了一种新的范例，因为它特异性地抵消了H2S诱导的细胞和线粒体功能障碍。在本项目中，我们将确定MB在可逆性昏迷大鼠模型中急性H2S中毒后给药时预防H2S诱导的脑损伤的短期和长期影响的能力。我们已经开发了这个模型来模拟人类中最常见的非致命形式的H2S中毒。将通过缺氧后损伤的生理和临床参数以及缺氧或细胞凋亡的脑标志物来评价H2S诱导昏迷后MB的短期效应（前12小时）。在另一组大鼠中，在硫化物中毒诱导的昏迷发作后急性或重复给予MB后，将在接受训练以在Morris水迷宫中进行试验的非麻醉动物中评价对记忆和认知功能的长期影响。MB将单独给药或与HyCo联合给药，以确定这2种解毒剂对H2S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，使用代理抵消H2S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出H2S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.