An Enzyme-Based Antidote for Acute Nicotine Toxicity

项目摘要/摘要。电子烟作为传统香烟的替代品越来越受欢迎， 美国的销售额从2012年的2.83亿美元增加到2018年的25亿美元。全球电子烟市场 预计到2024年将达到242亿美元。电子烟解决方案，也被称为电子烟液体，与 诱人的味道，如烟草、薄荷、水果、糖果和甜点。液体尼古丁浓度在这些地区各不相同 根据FDA的一项研究，产品的浓度从0到100毫克/毫升。尽管尼古丁在成人中的毒性是罕见的，但 估计致死剂量在60到500毫克(0.8-6.7毫克/公斤)之间，加味的电子液体正在被越来越多地摄入 在口头上，使儿童面临接触高浓度尼古丁的风险。的确，尼古丁对儿童的毒性 5岁以下儿童只需摄入一茶匙液态尼古丁即可。不可预见的事情 在儿童尼古丁中毒方面，电子烟的后果提出了一个尚未得到满足的需要 抵消这一弱势群体中可能出现的有害和潜在的致命后果。它是 我们计划在这项提案中讨论的儿科人群，是意外摄入液体的高危人群 电子烟中的尼古丁是存在的。尽管与尼古丁有关的中毒报告有所增加，但没有 急性尼古丁中毒的治疗。目前尼古丁中毒的治疗方案有支持性的 护理，使用活性木炭，使用机械通风进行呼吸支持。一种改变的替代方法 尼古丁中毒的毒性可以通过简单地隔离药物来实现。尼古丁的抗体有 作为阻断这种药物的药理作用的一种手段。到目前为止，吸烟疫苗 停用疫苗在临床前动物模型中显示了希望；然而，在临床研究中，这些疫苗失败了。 以衡量干预组和安慰剂组在戒烟方面的显著差异。因此， 一种抗体减弱急性剂量尼古丁的可能性是值得怀疑的。现在需要的是一个足够的 药代动力学(PK)生物，不仅能隔离尼古丁，还能促进其新陈代谢。 我们设想一种能够分解尼古丁而不是简单地隔离药物的生物将具有 有可能治疗急性尼古丁中毒。手头的提案详细说明了一种细菌菌株恶臭假单胞菌， 它已经进化成使用尼古丁作为其唯一的碳和氮来源。从这种细菌菌株中，我们将 研究一种一流的酶，一种名为NicA2的尼古丁氧化还原酶，作为治疗尼古丁中毒的手段。 我们对这种酶的初步表征表明，它可能是改变尼古丁的一个很好的候选者。 下毒了。然而，这种酶逆转尼古丁中毒的成功演示需要 几项实验工作包括：(1)评估NicA2减轻急性尼古丁的效果 啮齿动物模型的毒性，包括体积描记、血/脑分布和致死率。(2)定向进化 通过基因重组和随机诱变来提高其催化能力。

Project Summary/Abstract. Electronic cigarettes are gaining popularity as alternative to traditional cigarettes, with sales increasing from $283 million in 2012 to $2.5 billion in 2018 in the US. The global e-cigarette market is estimated to reach $24.2 billion by 2024. E-cigarette solutions, also known as e-liquids, are highly variable with enticing flavors, such as tobacco, menthol, fruit, candy, and dessert. Liquid nicotine concentrations vary in these products from 0 to 100 mg/ml according to an FDA study. Although nicotine toxicity in adults is rare with an estimated lethal dose between 60 and 500 mg (0.8-6.7 mg/kg), flavored e-liquids are increasingly being ingested orally, putting children at risk for exposure to high concentrations of nicotine. Indeed, nicotine toxicity in children under 5 years of age can occur with consumption of as little as a teaspoon of liquid nicotine. The unforeseen consequences of e-cigarettes, with respect to nicotine poisoning in children, presents an unmet need to counteract the harmful and potentially fatal outcomes that may occur among this vulnerable population. It is the pediatric population we plan to address in this proposal, as a high potential of accidental ingestion of liquid nicotine from e-cigarettes exists. Despite the increase in nicotine-related poisonings reported, there is no treatment for acute nicotine toxicity. Current treatment regimens for nicotine poisoning range from supportive care, to activated charcoal, to respiratory support with mechanical ventilation. An alternative means of altering the toxicity of nicotine poisoning could come via simple sequestering of the drug. Antibodies to nicotine have been prepared as a means to block the pharmacological effects of this drug. To date, vaccines for smoking cessation have shown promise in preclinical animal models; however, in clinical studies, these vaccines failed to measure significant differences in smoking abstinence between the intervention and placebo groups. Thus, the likelihood of an antibody attenuating an acute dose of nicotine is doubtful. What is needed is a sufficient pharmacokinetic (PK) biologic with the capacity to not just sequester nicotine but also increase its metabolism. We envision a biologic able to catabolize nicotine rather than simply sequestering the drug would have the potential to treat acute nicotine poisoning. The proposal at hand details a bacterial strain, Pseudomonas putida, which has evolved to use nicotine as its sole source of carbon and nitrogen. From this bacterial strain, we will examine a first-in-class enzyme, a nicotine oxidoreductase termed NicA2, as a means to treat nicotine poisoning. Our initial characterization of the enzyme indicates that it could be an excellent candidate for altering nicotine poisoning. However, the successful demonstration of this enzyme reversing nicotine poisoning will require several experimental undertakings including: (1) Evaluating the efficacy of NicA2 to attenuate acute nicotine toxicity in rodent models including plethysmography, blood/brain distribution and lethality. (2) Directed evolution of NicA2 to increase its catalytic capacity through gene recombination and random mutagenesis.