An In Vitro Assay for Screening Chemicals Interacting with the Blood-Brain Barrie

描述(由申请人提供)： 我们申请资助的目的是建立血脑屏障的体外模型，并利用分离的脑微血管检测通透性。与其他器官的毛细血管不同，大脑毛细血管建立了一个屏障，在很大程度上不能渗透到极地和较大的化学物质中，这是由于紧密的连接、没有隔板和胞饮作用。虽然大多数化学物质通过血脑屏障的通透性与其油/水分配系数直接相关，但由于多药物转运体的表达，许多脂溢性化学物质无法进入大脑。相反，由于营养转运蛋白的表达，预计渗透性较差的极地营养物质表现出更高的吸收能力。许多对大脑有潜在毒性的化学物质无法通过血脑屏障。此外，许多治疗精神疾病和神经疾病的候选药物由于血脑屏障而失败。因此，需要一种研究血脑屏障的模型来实现新的范式，在这种范式中，毒性测试将通过体外和计算机方法完成。此外，还需要一个模型来帮助化学家设计神经营养药物。目前的细胞培养模型无法显示体内发现的紧密性和许多转运蛋白。相比之下，分离的脑微血管保留了所有的转运蛋白并显示出一种紧密的屏障，在过去的40年里一直被用于血脑屏障通透性的研究。使用分离的脑微血管的主要障碍是制备过程繁琐和寿命短。一种商业来源的脑微血管将克服这些障碍。我们研究的总体目标是开发冷冻保存的BM作为血脑屏障的模型，并优化试剂和检测方法，以测量通透性和转运，这些将由我们公司进行或出售给其他公司。为了实现这一目标，在具体目标1中，将标准化制备、冷冻保存可存活的牛脑微血管的条件，并确保出售的不同批次微血管的一致性。脑微血管的功能将通过进行氨基酸和糖运输、多药物转运体和生存能力的测定来评估。Z因子接近1.0的分析表明最佳制备条件。在具体目标2中，将开发通过测量非特异性转运来检查血脑屏障的化学毒性的方法。敏感度和特异度将通过计算接收器操作曲线来评估。在第二阶段的赠款申请中，将开发检测方法来测量多个药物外排泵以及腔和腔转运体。通过建立评估血脑屏障的商业试剂来源，制药公司和化学测试实验室将拥有一个现成的模型，以筛选测试化学品的渗透性和毒性。考虑到血脑屏障在神经功能中的重要性，这些产品可能会有广泛的分销和商业成功。 公共卫生相关性： 缺乏有效的模型来衡量药物和化学品对血脑屏障(BBB)的影响，一直是评估化学品对大脑的毒性以及成功开发治疗脑部疾病的药物的障碍。所提出的血脑屏障模型、牛的分离脑微血管(BM)和血脑屏障通透性的测定，在神经毒性测试和药物开发领域具有巨大的潜在影响。这里的创新之处在于，采用了一个成功的研究模型--新鲜分离的BM，并将其转化为冷冻保存的、适销对路的产品，并将其应用于满足以前未满足的评估血脑屏障通透性需求的分析。

DESCRIPTION (provided by applicant): The objective of our grant application is to develop an in vitro model of the blood-brain barrier and assays to examine permeability by using isolated brain microvessels. Unlike capillaries in other organs, brain capillaries establish a barrier that is largely impermeable to polar and larger chemicals, which are due to tight junctions, the absence of both fenestrae and pinocytosis. Although, the permeability of most chemicals to cross the blood-brain barrier is directly associated to its' oil/water partition coefficient, many lipophillic chemicals fail to enter the brain because of the expression of the multidrug transporters. In contrast, polar nutrients that would be predicted to be less permeable display higher uptake because of the expression of nutrient transporters. Many chemicals that are potentially toxic to the brain fail to cross the blood-brain barrier. Additionally, many drug candidates for treating mental illnesses and neurological diseases fail because of the blood-brain barrier. Consequently, a model to study the blood-brain barrier is needed for the new paradigm in which testing chemicals for toxicity will be accomplished with in vitro and in silico methods. Additionally, a model is needed to aid chemists in their attempts to design neurotrophic drugs. Current cell culture models fail to display the tightness and the many transporters found in vivo. In contrast, isolated brain microvessels retain all of the transporters and display a tight barrier, and have been used in the past 40 years in research studies on blood-brain barrier permeability. The major obstacles in using isolated brain microvessels have been the laborious procedure for their preparation and their short life span. A commercial source of brain microvessels will overcome these obstacles. The overall objective of our study is to develop cryopreserved BM as a model of the blood-brain barrier and optimize reagents and assays to measure permeability and transport that will either be conducted by our company or sold to others. To accomplish the objective, in Specific Aim 1 conditions will be standardized for preparing, cryopreserving viable bovine brain microvessels and assure consistency in different batches of microvessels sold. The functionality of the brain microvessels will be assessed by conducting assays to measure amino acid and sugar transport, multidrug transporters, and viability. Assays achieving Z-factors closest to 1.0 will indicate the best preparation conditions. In Specific Aim 2, assays will be developed to examine chemical toxicity of the blood-brain barrier by measuring non specific transport. Sensitivity and specificity will be assessed by computing receiver operating curves. In the phase 2 grant application, assays will be developed to measure multi drug efflux pumps and the luminal and abluminal transporters. By establishing a commercial source of reagents for assessing the blood-brain barrier, pharmaceutical companies and chemical testing laboratories will have a readily available model to screen test chemicals for permeability and toxicity. When considering the importance of the blood-brain barrier in neurological functions, these products will potentially have wide distribution and commercial success. PUBLIC HEALTH RELEVANCE: The lack of an effective model for measuring the effects of drugs and chemicals on the blood-brain barrier (BBB) has been an impediment to evaluating chemicals for toxicity to the brain as well as to the successful development of drugs to treat diseases of the brain18,29. The proposed model of the blood-brain barrier, isolated brain microvessels (BM) from cows, and assay for blood-brain barrier permeability, has the potential for enormous impact in the field of neurotoxicity testing and drug development. The innovation here is in taking a successful research model, freshly isolated BM, and converting it into a cryopreserved, marketable product as well as translating its use into assays that address the previously unmet need for evaluating BBB permeability.