Developing a New Antioxidant Activator to Intervene in Lung Inflammatory Diseases

描述(申请人提供)：与肺组织中的氧化应激有关的肺部炎症性疾病在美国是一个主要的健康问题。这些疾病包括过敏性哮喘、慢性阻塞性肺疾病、病毒和细菌感染，以及相关的脓毒症引起的急性肺损伤。有证据表明，组织中的抗氧化防御在减轻肺部炎症方面起着关键作用，加强这些保护途径可以减少组织损伤和发病率。新出现的证据表明，转录因子Nrf2在组织保护性抗氧化酶全球网络的诱导中发挥核心作用，以响应氧化剂相关的刺激。人们非常关注通过何种手段刺激NRF2的激活，以进行预防性和干预性治疗。我们的初步研究表明，天然油的一种成分诱导Nrf2显着激活，并随后刺激其下游抗氧化基因靶标。第一阶段研究的目标是测试含有活性成分的油的有效性和安全性，以确定其作为治疗剂在第二阶段研发中继续发展的潜力。第一阶段的具体目标是：(1)确定主要刺激NRF2活性所在的先前测试的油混合物的组成油。这将通过稳定地转染Nrf2报告结构的细胞来实现，然后验证野生型细胞中的抗氧化基因和蛋白质的表达。(2)测定活性油的药代动力学和NRF2选择性。将检测与已知Nrf2化学激活剂相关的抗氧化剂和细胞毒性标记基因的表达时程和刺激效果。Nrf2信号将通过核蛋白积累和DNA结合(EMSA)以及Nrf2-/-细胞中活性的缺失来评估。微阵列分析将识别同时激活的通路的存在。(3)在过敏性哮喘和内毒素诱导的小鼠急性肺损伤模型上，评价其降低炎症和组织损伤标志物的作用。标记物将包括组织病理学测量以及与这些模型的氧化剂相关炎症病理相关的基因和蛋白质的表达。这些研究将使用研究人员目前使用的细胞培养和动物模型系统和方法进行。第一阶段的活动有望为进一步分离和开发天然油中的活性成分提供科学依据。预计第一阶段和第二阶段研究和开发的结果将是确定一种分子，该分子将作为一种准备作为治疗剂开发的商业产品而具有吸引力。公共卫生相关性：超过3500万美国人患有急性和慢性肺部疾病，其病理与炎症有关。众所周知，在肺组织中合成的抗氧化剂可以提供保护，防止与许多此类疾病相关的病理过程。该项目的目标是分离和测试一种在精油中发现的制剂的有效性，该制剂已被证明可以激活肺内保护性抗氧化剂的广泛合成。

DESCRIPTION (provided by applicant): Pulmonary inflammatory diseases linked to oxidant stress in lung tissues represent a major health concern in the U.S. These diseases include allergic asthma, chronic obstructive pulmonary disease, viral and bacterial infections, and related sepsis-induced acute lung injury. Evidence indicates that antioxidant defenses in the tissues play a pivotal role in reducing pulmonary inflammation and that enhancement of these protective pathways can lead to a reduction of tissue damage and morbidity. Emerging evidence has identified a central role for the transcription factor, Nrf2, in orchestrating the induction of a global network of tissue-protective antioxidant enzymes in response to oxidant-related stimuli. Much attention is being focused on means by which to stimulate activation of Nrf2 for both preventive and interventive therapies. Our preliminary studies have demonstrated that a constituent of a natural oil induces marked activation of Nrf2 and subsequent stimulation of its down-stream antioxidant gene targets. The Objectives of the Phase I research are to test the effectiveness and safety of the oil containing the active constituent in order to determine its potential for continued development in Phase II R&D as a therapeutic agent. Phase I Specific Aims are to: (1) Identify the component oil of the previously-tested oil mixture in which the primary Nrf2-stimulating activity resides. This will be achieved using cells stably transfected with an Nrf2 reporter construct followed by verification of antioxidant gene and protein expression in wild-type cells. (2) Determine the pharmacokinetics and Nrf2-selectivity of the active oil. The time-course of expression and efficacy of stimulation of antioxidant and cytotoxicity marker genes relative to known Nrf2 chemical activators will be examined. Nrf2 signaling will be assessed by nuclear protein accumulation and DNA binding (EMSA) and the absence of activity in Nrf2 -/- cells. Microarray analysis will identify the presence of concurrently-activated pathways. (3) Assess the effectiveness of the active constituent in reducing markers of inflammation and tissue damage in mouse models of allergic asthma and endotoxin- induced acute lung injury. Markers will include histopathologic measures and the expression of genes and proteins associated with the oxidant-related inflammatory pathology of these models. These studies will be carried out using cell culture and animal model systems and methodologies currently in use by the investigators. The activities of Phase I are expected to provide the scientific basis for further isolation and development of the active constituent present in the natural oil. It is anticipated that the outcome of Phase I and II research and development will be the identification of a molecule that will be attractive as a commercial product ready for development as a therapeutic agent. PUBLIC HEALTH RELEVANCE: Over 35 million Americans live with acute and chronic lung diseases whose pathology is linked to inflammation. Antioxidants synthesized in lung tissues are known to offer protection against pathologic processes associated with many of these diseases. The goal of this project is to isolate and test the effectiveness of an agent found in an essential oil that has been demonstrated to activate broad synthesis of protective antioxidants in the lung.