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（由申请人提供）：血脑屏障（BBB）限制了许多药物在大脑中的被动扩散，并构成了中枢神经系统（CNS）疾病的药理学治疗中的重要障碍。大约98％的新小分子药物和几乎100％的大分子药物（例如重组蛋白和基于基因的CNS活性）具有BBB渗透问题。因此，我们的长期目标是阐明与维持BBB相关的调节机制，作为开发方案的开发的先决条件，可用于促进进入中枢神经系统治疗神经系统疾病的治疗药物。我们假设低浓度的原型BBB破坏者（亲脂性胆碱酯酶抑制剂，毒死rif虫，CPF）促进了外源物质通过短期（临时和可逆的）BBB完整性的变化来传递BBB。该假设基于我们的初步研究的以下观察结果：1）。 CPF及其代谢物的低，无毒浓度横向BBB。 2）。 CPF及其代谢产物有助于改变BBB的完整性和结构，这是通过改变的紧密连接所证明的 体外蛋白质和电阻。 3）。 CPF以短期和可逆的方式破坏BBB紧密连接蛋白和跨性别受体电势通道基因表达。基于这些观察结果，该R03提案的重点是CPF影响体内BBB破坏的分子和生化机制。该提案中有2个特定的目的：（1）确定低剂量CPF处理对BBB紧密连接（BBB-TJ）蛋白改变的时间相关的影响。 （2）确定低剂量CPF处理后涉及BBB-TJ涉及的信号传导途径。要使用的方法将包括三个级别的实验，以实现我们的目标并证明我们的假设。 （1）在功能水平上，Evan Blue Dye将用于评估BBB的破坏。 （2）在细胞水平，蛋白质印迹，免疫荧光测定，ELISA和酶促活性将用于分析CPF处理后的BBB蛋白谱。 （3）在分子水平上，定量实时PCR将用于验证与基因变化相关的BBB蛋白水平变化。此外，MR成像技术将用于评估CPF治疗后脑含水量。从拟议的研究中获得的数据将显着提高我们对与维持BBB功能相关的潜在分子机制的理解，并且对于设计与使用低剂量，安全的亲脂性化合物相关的策略也将非常有用中枢神经系统的条件。 公共卫生相关性：使外来物质远离大脑的血脑屏障功能。这是有益的，因为在没有某些有毒物质的情况下可以通过营养的流逝。这也有害，因为屏障阻止有用的药物进入大脑 治疗疾病（例如脑癌，阿尔茨海默氏病等）。确定可以瞬时和可逆地改变血脑屏障的疾病具有显着价值，因为该信息可用于开发更有效的药物以治疗脑部疾病的脑部药物。