Enhancing CNS Drug Delivery By Manipulating The Blood-Brain Barrier

通过操纵血脑屏障增强中枢神经系统药物输送

• 批准号: 8384079
• 负责人: Wen Li
• 金额: $ 7.98万
• 依托单位: VIRGINIA POLYTECHNIC INST AND ST UNIV
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8384079
• 关键词: Affect; Aftercare; Alzheimer' s Disease; Biochemical; Biological Assay; Blood - brain barrier anatomy; Brain; Brain Diseases; Calcium; Cells; Central Nervous System Agents; Central Nervous System Diseases; Characteristics; Chlorpyrifos; Cholinesterase Inhibitors; Confocal Microscopy; Data; Diffusion; Disease; Dose; Down-Regulation; Drug Delivery Systems; Drug usage; Dyes; Electrical Resistance; Enzyme-Linked Immunosorbent Assay; Epilepsy; Evans blue stain; Exposure to; Future; Gene Expression; Genes; Goals; Hour; Imaging Techniques; Immunofluorescence Immunologic; In Vitro; Lead; Magnetic Resonance Imaging; Maintenance; Malignant neoplasm of brain; Mediating; Medicine; Methods; Molecular; Molecular Profiling; Mus; Neuraxis; Neurons; Nutrient; Operative Surgical Procedures; Parkinson Disease; Pathway interactions; Penetration; Permeability; Pharmaceutical Preparations; Pharmacological Treatment; Prodrugs; Proteins; Recombinant Proteins; Recovery; Reporting; Safety; Schizophrenia; Signal Pathway; Signal Transduction; Signaling Protein; Stroke; Structure; Techniques; Therapeutic; Therapeutic Agents; Tight Junctions; Time; Vascular Endothelial Growth Factor Receptor-1; Vascular Endothelial Growth Factors; Water; Western Blotting; base; cytotoxic; design; in vivo; inhibitor/antagonist; nervous system disorder; novel strategies; occludin; prevent; protein expression; protein profiling; protocol development; receptor; research study; small molecule; toxicant

DESCRIPTION (provided by applicant): The blood-brain barrier (BBB) restricts the passive diffusion of many drugs into the brain and constitutes a significant obstacle in the pharmacological treatment of central nervous system (CNS) disorders. Approximately 98% of new small-molecule drugs and almost 100% of large-molecule drugs such as recombinant proteins and gene-based medicines with proven CNS activity have BBB penetration problems. Therefore, our long term goal is to elucidate the regulatory mechanisms associated with the maintenance of the BBB function as a prerequisite to the development of protocols that can be used to facilitate therapeutic drugs entering the CNS for the treatment of neurological disease. We hypothesize that low concentrations of a prototypic BBB disruptor (the lipophilic cholinesterase inhibitor, chlorpyrifos, CPF) facilitate passage of exogenous substances across the BBB by short-term (temporary and reversible) alteration of BBB integrity. The hypothesis is based on our following observations from our preliminary studies: 1). low, nontoxic concentrations of CPF and its metabolites transverse the BBB. 2). CPF and its metabolites contribute to the alteration of BBB integrity and structure, as evidenced by altered tight junction proteins and electric resistance in vitro. 3). CPF disrupts BBB tight junction protein and transien receptor potential channels gene expression in a short-term and reversible way in vitro. Based on these observations, the focus of this R03 proposal is on the molecular and biochemical mechanisms by which the CPF affects the disruption of the BBB in vivo. There are 2 specific aims in the proposal: (1) To determine the time-related effects of low dose CPF treatment on the alteration of BBB-tight junction (BBB-TJ) proteins. (2) To identify the signaling pathways involved in the disruption of BBB-TJ following low dose CPF treatment. The methods to be employed will include three levels of experiments to achieve our goal and to prove our hypothesis. (1) At the functional level, Evan blue dye will be used to evaluate BBB disruption. (2) At the cellular level, Western blot, immunofluorescence assays, ELISA, and enzymatic activities will be used to analyze BBB protein profiles after CPF treatment. (3) At the molecular level, quantitative real-time PCR will be used to verify BBB protein level changes correlating to gene changes. In addition, MR imaging technique will be used to evaluate brain water content after CPF treatment. The data obtained from the proposed studies will significantly advance our understanding of the underlying molecular mechanisms associated with the maintenance of BBB function, and also will be very useful for devising strategies associated with use of low dose, safe lipophilic compounds as an effective pretreatment to allow therapeutic drug delivery to the CNS for treatment of Alzheimer's disease, Parkinson's disease, stroke, schizophrenia, epilepsy and other conditions of the CNS. PUBLIC HEALTH RELEVANCE: The blood-brain barrier functions to keep foreign substances out of the brain. This is beneficial, as passage of nutrients can occur without passage of certain toxicants. This is also detrimental as the barrier prevents useful drugs from entering the brain to treat diseases (e.g. brain cancer, Alzheimer's disease, etc). There is significant value in determining conditions that can transiently and reversibly change the blood-brain barrier, as the information may be used to develop more effective methods for drug delivery to brain for treatment of brain diseases.

描述(申请人提供)：血脑屏障(BBB)限制了许多药物被动扩散到大脑中，并构成了中枢神经系统(CNS)疾病的药物治疗的重大障碍。大约98%的新型小分子药物和几乎100%的大分子药物，如重组蛋白和基因药物，都存在血脑屏障穿透问题。因此，我们的长期目标是阐明与维持血脑屏障功能相关的调节机制，作为开发可用于促进治疗药物进入中枢神经系统治疗神经系统疾病的方案的先决条件。我们假设，低浓度的典型BBB干扰物(亲脂性胆碱酯酶抑制剂，毒死蜱，CPF)通过短期(暂时和可逆)改变BBB完整性，促进外源物质通过BBB。这一假设是基于我们从初步研究中观察到的以下几点：1)。低浓度、无毒的CPF及其代谢产物横跨血脑屏障。2)。CPF及其代谢产物有助于血脑屏障完整性和结构的改变，这是紧密连接改变的证据。 蛋白质和体外电阻。3)。CPF在体外以一种短期和可逆的方式干扰BBB紧密连接蛋白和瞬时受体潜在通道基因的表达。基于这些观察，R03提案的重点是CPF影响体内BBB破坏的分子和生化机制。该提案有两个具体目的：(1)确定低剂量CPF处理对BBB-紧密连接(BBB-TJ)蛋白变化的时间相关效应。(2)探讨小剂量CPF对BBB-TJ功能影响的信号转导途径。所采用的方法将包括三个层次的实验，以实现我们的目标并证明我们的假设。(1)在功能水平上，伊文兰染料将用于评估血脑屏障的破坏。(2)在细胞水平上，采用免疫印迹、免疫荧光、酶联免疫吸附试验和酶活性等方法分析CPF处理后BBB蛋白的表达情况。(3)在分子水平上，实时定量聚合酶链式反应将被用来验证与基因变化相关的BBB蛋白水平的变化。此外，还将使用磁共振成像技术评估CPF治疗后的脑组织水分含量。这些研究的数据将极大地促进我们对维持血脑屏障功能的潜在分子机制的理解，也将非常有助于制定与使用低剂量、安全的亲脂化合物作为有效的预处理以允许治疗性药物输送到中枢神经系统以治疗阿尔茨海默病、帕金森氏病、中风、精神分裂症、癫痫和其他中枢神经系统疾病相关的策略。 与公共健康相关：血脑屏障的功能是将异物挡在大脑之外。这是有益的，因为营养物质可以在不通过某些毒物的情况下通过。这也是有害的，因为屏障阻止有用的药物进入大脑 治疗疾病(如脑癌、阿尔茨海默病等)。确定可以瞬时和可逆地改变血脑屏障的条件具有重大价值，因为这些信息可能被用来开发更有效的药物输送到大脑的方法，用于治疗脑部疾病。