Enhancing CNS Drug Delivery By Manipulating The Blood-Brain Barrier

描述（由申请人提供）：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）疾病药物治疗的重大障碍。大约98%的新的小分子药物和几乎100%的大分子药物，如重组蛋白和基因为基础的药物与证明的中枢神经系统活性有血脑屏障渗透问题。因此，我们的长期目标是阐明与维持BBB功能相关的调节机制，作为开发可用于促进治疗药物进入CNS以治疗神经系统疾病的方案的先决条件。我们假设，低浓度的原型血脑屏障干扰剂（亲脂性胆碱酯酶抑制剂，毒死蜱，CPF）促进通过血脑屏障的完整性的短期（临时和可逆的）改变的外源性物质通过血脑屏障。该假设基于我们初步研究的以下观察结果：1）。低、无毒浓度的CPF及其代谢物穿过血脑屏障。2）。CPF及其代谢产物导致BBB完整性和结构的改变，如改变的紧密连接所证明的 蛋白质和体外电阻。3）。CPF在体外以短期可逆的方式干扰血脑屏障紧密连接蛋白和瞬时受体电位通道基因表达。基于这些观察结果，R 03提案的重点是CPF影响体内BBB破坏的分子和生化机制。本研究的主要目的有两个：（1）研究低剂量CPF处理对BBB-紧密连接（BBB-TJ）蛋白的时间效应。(2)确定低剂量CPF处理后BBB-TJ破坏所涉及的信号通路。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，将使用伊文思蓝染料评价BBB破坏。(2)在细胞水平上，将使用Western印迹、免疫荧光测定、ELISA和酶活性来分析CPF处理后的BBB蛋白质谱。(3)在分子水平上，将使用定量实时PCR来验证与基因变化相关的BBB蛋白水平变化。此外，将使用MR成像技术来评估CPF治疗后的脑水含量。从所提出的研究中获得的数据将显著推进我们对与维持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作为有效的预处理以允许治疗药物递送至CNS以治疗阿尔茨海默病、帕金森病、中风、精神分裂症、癫痫和其他CNS疾病。

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.