Mechanism and Countermeasure of Carfentanil-induced Respiratory Disorder and Death

项目摘要 阿片类药物在2020年在美国涉及75，673例过量死亡，其中芬太尼是一种合成阿片类药物， 最致命的原因是诱发抑郁症。卡芬太尼（CFN）是另一种作用于阿片类药物的合成阿片类药物 μ受体（MORs）的效力是芬太尼的100倍。虽然不打算用于治疗用途， 雾化CFN已被用作致命剂（生物武器），以引起呼吸障碍（RD）和死亡 在人类身上。纳洛酮增加早期报告中过量阿片类药物的存活率;然而， 在合成阿片类药物时代后，由于临床环境中不成功的复苏尝试而增加。它也没有 逆转雾化CFN诱导的动物RD/死亡。迄今为止， 急性CFN切除引起RD/死亡的对策尚不清楚。 通过静脉（IV）灌注过量芬太尼会产生RD，包括渐进性抑郁（dVE） 然后在死前呼吸暂停/喘气。RD被认为是由于抑制颈动脉体O2-受体 和中央CO2化学感受器，特别是在后斜方核和面旁核（RTN/PFN）中的化学感受器， 前Bötzinger复合体（PBC，呼吸节律发生器）中的呼吸神经元。有意思的是，我们 最近报道，静脉推注芬太尼引起的立即呼吸暂停仅由 支气管肺泡C纤维（PCF）在麻醉大鼠。我们的初步研究进一步表明， 与喉闭合、胸部呼气肌收缩和吸气肌沉默有关 (i.e.,阻塞性和中枢性呼吸暂停伴胸壁僵硬），如果过量，这是致命的。此外，雾化 芬太尼（100 mg/m3，持续10 min）最初触发类似的呼吸暂停，随后发生dVE和喘息，导致死亡 麻醉大鼠暴露后约10 min。因此，在本提案中，我们将描述心肺功能 （呼吸肌）对清醒大鼠急性CFN暴露的反应，并确定 外周和中央MORs（Aim 1）。随后，我们将机械地证明CFN暴露 刺激PCF触发初始呼吸暂停，然后抑制颈动脉体、RTN/PFN CO2敏感性和PBC 呼吸神经元通过作用于局部MORs产生dVE和喘息（呼吸暂停）（目的2）。多沙普仑是一种 CX 717对颈动脉体有较强的呼吸兴奋作用，而CX 717能增强呼吸频率和呼吸强度， 通过作用于PBC驱动，加强中枢CO2化学感受。两种药物都能逆转 阿片类药物在动物和人类中诱导的RD。这沿着迷走神经感觉神经元中的莫尔内化， 通过皮吗啡预处理（外周作用莫尔）阻断静脉注射芬太尼诱导的呼吸暂停 激动剂，IV），使我们能够确定皮吗啡，多沙普仑和CX 717的鸡尾酒预处理是否 是针对CBN诱导的RD/死亡的对策（目的3）。 多学科方法（电生理学，免疫组织化学和药理学）在全身和 将采用细胞水平来阐明急性CFN阻断诱导的RD/死亡的潜在机制 并评估鸡尾酒预处理的功效。预期的数据将首次机械地 揭示了PCF、颈动脉体、RTN/PFN和PBC在产生CFN诱导的RD中的关键作用， 为预防/减少RD/死亡提供了一种新的潜在对策。

PROJECT SUMMARY Opioids were involved in 75,673 overdose deaths in 2020 in the U.S, in which fentanyl, a synthetic opioid, is the deadliest due to induction of ventilatory depression. Carfentanil (CFN) is another synthetic opioid acting on opioid mu-receptors (MORs) with a potency 100 times that of fentanyl. Though no intending for therapeutic use, aerosolized CFN has been applied as a lethal agent (bioweapon) to cause respiratory disorder (RD) and death in humans. Naloxone increases survival rate of overdose opioids in early reports; however, deaths have been increasing after synthetic opioid era due to unsuccessful resuscitation attempts in clinical settings. It also fails to reverse aerosolized CFN-induced RD/death in animals. To date, the characteristics, mechanisms, and effective countermeasure of acute CFN exposure-induced RD/death are still unclear. Overdose fentanyl via intravenous (IV) perfusion produces RD including gradual ventilatory depression (dVE) and then apneas/gasping before death. The RD is believed to result from inhibiting the carotid body O2-receptor and central CO2-chemoreceptors, especially those in the retrotrapezoid and parafacial nucleus (RTN/PFN) and the respiratory neurons in the pre-Bötzinger complex (PBC, the respiratory rhythm-generator). Interestingly, we recently reported that IV bolus injection of fentanyl induced an immediate apnea solely mediated by bronchopulmonary C-fibers (PCFs) in anesthetized rats. Our pilot study further shows that the apnea is associated with closure of the larynx, constriction of chest expiratory muscles, and silence of inspiratory muscles (i.e., obstructive and central apnea with chest wall rigidity), which is lethal if overdosed. Moreover, aerosolized fentanyl (100 mg/m3 for 10 min) initially triggers the similar apnea followed by dVE and gasping, leading to death ~10 min post exposure in anesthetized rats. In this proposal, we, therefore, will characterize the cardiorespiratory (respiratory muscles’) responses to acute CFN exposure in awake rats and determine the causal roles of peripheral and central MORs (Aim 1). Subsequently, we will mechanistically demonstrate that CFN exposure stimulates PCFs to trigger the initial apneas and then inhibits the carotid body, RTN/PFN CO2-sensitive and PBC respiratory neurons to produce dVE and gasping (apneas) by acting on local MORs (Aim 2). Doxapram is a potent respiratory stimulant to the carotid body, while CX717 can enhance respiratory frequency and respiratory drive via acting on the PBC and strengthen central CO2-chemoreception. Both agents are capable of reversing opioid-induced RD in animals and humans. This, along with MOR internalization in vagal sensory neurons and blockade of IV injection of fentanyl-induced apnea by dermorphin pretreatment (a peripherally acting MOR agonist, IV) in our pilot study, allows us to define if a cocktail pretreatment of dermorphin, doxapram, and CX717 is a countermeasure for CFN-induced RD/death (Aim 3). A multidisciplinary approach (electrophysiology, immunohistochemistry, and pharmacology) at systemic and cellular levels will be employed to elucidate the mechanisms underlying acute CFN exposure-induced RD/death and evaluate the efficacy of the cocktail pretreatment. The expected data will, for the first time, mechanistically reveal the key roles of PCFs, the carotid body, RTN/PFN and PBC in generating the CFN-induced RD and provide a novel and potential countermeasure to prevent/diminish the RD/death.