An Enzyme-Based Antidote for Acute Nicotine Toxicity

一种基于酶的急性尼古丁中毒解毒剂

• 批准号: 10790758
• 负责人: Kim Janda
• 金额: $ 27.15万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-30 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10790758
• 关键词: 5 year old; Accident and Emergency department; Accidents; Acetylcholine; Activated Charcoal; Acute; Address; Adrenergic Antagonists; Adult; Affinity; Agonist; Animal Model; Antibodies; Antidotes; Attenuated; Autonomic ganglion; Benzodiazepines; Binding; Biodistribution; Biological; Biological Assay; Blood; Blood Pressure; Bradycardia; Brain; Brain region; Buffers; Candy; Carbon; Cessation of life; Child; Childhood; Cholinergic Receptors; Chronic; Clinical Research; Clinical Trials; Coma; Complex; Consumption; Directed Molecular Evolution; Dose; Drug Kinetics; Dyspnea; Electronic cigarette; Engineering; Enzymes; Evolution; Exposure to; Failure; Fatal Outcome; Fruit; Gastric Lavage; Gene Library; Generations; Genes; Genetic Recombination; Goals; Half-Life; Hand; Heart Rate; Hour; Hypotension; Immunologics; In Situ; Information Systems; Ingestion; Intervention; Libraries; Marketing; Measures; Mechanical ventilation; Menthol; Metabolism; Modeling; Monoclonal Antibodies; Muscle; Mutagenesis; Neuromuscular Junction; Neurons; Nicotine; Nicotinic Receptors; Nitrogen; Oral Ingestion; Overdose; Oxidoreductase; Patients; Peripheral; Pharmaceutical Preparations; Pharmacodynamics; Plethysmography; Poison; Poisoning; Population; Positioning Attribute; Pseudomonas putida; Publishing; Reporting; Respiratory Failure; Risk; Rodent; Rodent Model; Sales; Seizures; Serum; Signs and Symptoms; Site; Societies; Source; Structure; Supportive care; System; Therapeutic; Tobacco; Toxic effect; Treatment Protocols; Uncertainty; Vaccines; Variant; Vulnerable Populations; aspirate; cardiovascular collapse; cholinergic; combustible cigarette; efficacy evaluation; electronic cigarette use; electronic liquid; experimental study; improved; liquid nicotine; mouse model; mutant; nerve agent; neural; nicotine cessation; nicotine exposure; nicotine oxidase; nicotine use; nicotine vaccine; organophosphate poisoning; pharmacologic; placebo group; pre-clinical; receptor; respiratory; response; smoking abstinence; smoking cessation; therapeutic enzyme; thermostability; tobacco products

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.

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 亿美元增至 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（也称为电子液体）变化很大， 诱人的口味，如烟草、薄荷醇、水果、糖果和甜点。液体尼古丁浓度各不相同 根据 FDA 研究，产品浓度为 0 至 100 mg/ml。尽管尼古丁对成人的毒性很少见 估计致死剂量在 60 至 500 毫克（0.8-6.7 毫克/千克）之间，调味电子烟液的摄入量越来越多 口服，使儿童面临接触高浓度尼古丁的风险。事实上，尼古丁对儿童的毒性 5岁以下的儿童只要摄入一茶匙液体尼古丁就可能发生这种情况。不可预见的 电子烟对儿童尼古丁中毒的后果提出了一个未得到满足的需求 抵消这一弱势群体中可能发生的有害和潜在致命的后果。它是 我们计划在本提案中解决儿科人群的问题，因为意外摄入液体的可能性很高 电子烟中存在尼古丁。尽管尼古丁相关中毒的报道有所增加，但尚无 治疗急性尼古丁中毒。目前尼古丁中毒的治疗方案包括支持性治疗 护理、活性炭、机械通气呼吸支持。另一种改变方法 尼古丁中毒的毒性可能是通过简单地隔离药物来实现的。尼古丁抗体有 已被制备作为阻断该药物的药理作用的手段。迄今为止，针对吸烟的疫苗 戒烟在临床前动物模型中显示出希望；然而，在临床研究中，这些疫苗失败了 测量干预组和安慰剂组之间戒烟的显着差异。因此， 抗体减弱急性剂量尼古丁的可能性值得怀疑。需要的是足够的 药代动力学 (PK) 生物制剂不仅能够隔离尼古丁，还能够增加尼古丁的代谢。 我们设想一种能够分解代谢尼古丁而不是简单地隔离药物的生物制剂 具有治疗急性尼古丁中毒的潜力。手头的提案详细介绍了一种细菌菌株，恶臭假单胞菌， 它已经发展到使用尼古丁作为其唯一的碳和氮来源。从这种菌株中，我们将 检查一种一流的酶，一种称为 NicA2 的尼古丁氧化还原酶，作为治疗尼古丁中毒的方法。 我们对该酶的初步表征表明它可能是改变尼古丁的绝佳候选者 中毒。然而，成功证明这种酶逆转尼古丁中毒需要 多项实验工作包括： (1) 评估 NicA2 减弱急性尼古丁的功效 啮齿动物模型中的毒性，包括体积描记法、血液/大脑分布和致死率。 (2)定向进化 NicA2 通过基因重组和随机突变来提高其催化能力。