Neuroscience Department, Medical Toxicology Research Division, U.S. Army Medical Research Institute of Chemical Defense, Aberdeen, Proving Ground, MD 21010-5400, USA..
Toxicol Appl Pharmacol. 2023 Apr 1;464:116437. doi: 10.1016/j.taap.2023.116437. Epub 2023 Feb 26.
Recently a novel humanized mouse strain has been successfully generated, in which serum carboxylesterase (CES) knock out (KO) mice (Es1-/-) were further genetically modified by knocking in (KI), or adding, the gene that encodes the human form of acetylcholinesterase (AChE). The resulting human AChE KI and serum CES KO (or KIKO) mouse strain should not only exhibit organophosphorus nerve agent (NA) intoxication in a manner more similar to humans, but also display AChE-specific treatment responses more closely mimicking those of humans to facilitate data translation to pre-clinic trials. In this study, we utilized the KIKO mouse to develop a seizure model for NA medical countermeasure investigation, and then applied it to evaluate the anticonvulsant and neuroprotectant (A/N) efficacy of a specific A adenosine receptor (AAR) agonist, N-bicyclo-(2.2.1)hept-2-yl-5'-chloro-5'-deoxyadenosine (ENBA), which has been shown in a rat seizure model to be a potent A/N compound. Male mice surgically implanted with cortical electroencephalographic (EEG) electrodes a week earlier were pretreated with HI-6 and challenged with various doses (26 to 47 μg/kg, SC) of soman (GD) to determine a minimum effective dose (MED) that induced sustained status epilepticus (SSE) activity in 100% of animals while causing minimum lethality at 24 h. The GD dose selected was then used to investigate the MED doses of ENBA when given either immediately following SSE initiation (similar to wartime military first aid application) or at 15 min after ongoing SSE seizure activity (applicable to civilian chemical attack emergency triage). The selected GD dose of 33 μg/kg (1.4 x LD) generated SSE in 100% of KIKO mice and produced only 30% mortality. ENBA at a dose as little as 10 mg/kg, IP, caused isoelectric EEG activity within minutes after administration in naïve un-exposed KIKO mice. The MED doses of ENBA to terminate GD-induced SSE activity were determined to be 10 and 15 mg/kg when treatment was given at the time of SSE onset and when seizure activity was ongoing for 15 min, respectively. These doses were much lower than in the non-genetically modified rat model, which required an ENBA dose of 60 mg/kg to terminate SSE in 100% GD-exposed rats. At MED doses, all mice survived for 24 h, and no neuropathology was observed when the SSE was stopped. The findings confirmed that ENBA is a potent A/N for both immediate and delayed (i.e., dual purposed) therapy to victims of NA exposure and serves as a promising neuroprotective antidotal and adjunctive medical countermeasure candidate for pre-clinical research and development for human application.
最近成功培育出一种新型的人源化小鼠品系，该品系通过基因敲入（KI）或添加编码人源乙酰胆碱酯酶（AChE）的基因，对血清羧酸酯酶（CES）敲除（KO）小鼠（Es1-/-）进行了进一步的遗传修饰。由此产生的人源 AChE KI 和血清 CES KO（或 KIKO）小鼠品系不仅应该以更类似于人类的方式表现出有机磷神经毒剂（NA）中毒，而且还应该表现出更接近人类的 AChE 特异性治疗反应，以促进数据转化为临床前试验。在这项研究中，我们利用 KIKO 小鼠开发了一种用于 NA 医学对策研究的癫痫发作模型，然后应用该模型评估了一种特定的 A 腺苷受体（AAR）激动剂 N-双环[2.2.1]庚-2-基-5'-氯-5'-脱氧腺苷（ENBA）的抗惊厥和神经保护（A/N）疗效，该激动剂已在大鼠癫痫发作模型中显示出作为一种有效的 A/N 化合物。一周前接受皮层脑电图（EEG）电极手术植入的雄性小鼠先用 HI-6 预处理，然后用各种剂量（26 至 47μg/kg，SC）的梭曼（GD）进行挑战，以确定在 24 小时内引起 100%动物持续性癫痫发作（SSE）活性而导致最小致死性的最小有效剂量（MED）。然后使用选定的 GD 剂量来研究在 SSE 发作开始后立即给予 ENBA 的 MED 剂量（类似于战时军事急救应用）或在持续 SSE 发作活动 15 分钟后给予 ENBA 的 MED 剂量（适用于平民化学攻击紧急分诊）。选定的 GD 剂量 33μg/kg（1.4 x LD）在 100%的 KIKO 小鼠中产生 SSE，仅产生 30%的死亡率。ENBA 的剂量低至 10mg/kg，IP，在未暴露于 GD 的新 KIKO 小鼠中给药后数分钟内即可引起等电 EEG 活动。当在 SSE 发作时给予治疗和当发作活动持续 15 分钟时，ENBA 终止 GD 诱导的 SSE 活性的 MED 剂量分别确定为 10 和 15mg/kg。这些剂量明显低于非基因修饰的大鼠模型，该模型需要 60mg/kg 的 ENBA 剂量才能使 100%GD 暴露的大鼠终止 SSE。在 MED 剂量下，所有小鼠在 24 小时内存活，停止 SSE 时未观察到神经病理学。这些发现证实，ENBA 是人源化动物体内和体外暴露于 NA 后的即刻和延迟（即双重用途）治疗的有效 A/N，是一种有前途的神经保护解毒剂和辅助医学对策候选物，可用于人类应用的临床前研究和开发。
