Developing a New Antioxidant Activator to Intervene in Lung Inflammatory Diseases

描述（由申请人提供）：与肺组织中的氧化应激相关的肺部炎症性疾病代表了美国的主要健康问题。这些疾病包括过敏性哮喘、慢性阻塞性肺病、病毒和细菌感染以及相关的脓毒症诱导的急性肺损伤。有证据表明，组织中的抗氧化防御在减少肺部炎症中起着关键作用，并且这些保护途径的增强可以导致组织损伤和发病率的减少。新出现的证据已经确定了转录因子Nrf 2在响应氧化剂相关刺激诱导组织保护性抗氧化酶的全球网络中的核心作用。许多注意力都集中在刺激Nrf 2激活的方法上，用于预防性和干预性治疗。我们的初步研究表明，天然油的成分诱导Nrf 2的显着激活，并随后刺激其下游抗氧化基因靶点。第一阶段研究的目的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开发的潜力。第一阶段的具体目标是：（1）确定先前测试的油混合物的组分油，其中存在主要的Nrf 2刺激活性。这将通过使用Nrf 2报告基因构建体稳定转染的细胞，然后验证野生型细胞中的抗氧化剂基因和蛋白质表达来实现。(2)测定活性油的药代动力学和Nrf 2-选择性。将检查抗氧化剂和细胞毒性标记物基因相对于已知Nrf 2化学活化剂的表达的时间过程和刺激功效。将通过核蛋白积累和DNA结合（EMSA）以及Nrf 2-/-细胞中活性的缺乏来评估Nrf 2信号传导。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急性肺损伤小鼠模型中，评估活性成分在减少炎症和组织损伤标志物方面的有效性。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学相关的基因和蛋白质的表达。这些研究将使用研究者目前使用的细胞培养和动物模型系统和方法进行。第一阶段的活动预计将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I期和II期研究和开发的结果将是鉴定出一种分子，该分子作为商业产品将是有吸引力的，可以作为治疗剂开发。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。已知肺组织中合成的抗氧化剂可提供保护以对抗与许多这些疾病相关的病理过程。该项目的目标是分离和测试在精油中发现的一种药剂的有效性，该药剂已被证明可以激活肺中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.