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开发一种血脑屏障的体外模型，并通过使用分离的脑微血管检测渗透性。与其他器官中的毛细血管不同，脑毛细血管建立了一种屏障，这种屏障对极性和较大的化学物质基本上是不可渗透的，这是由于紧密连接，没有窗孔和胞饮作用。虽然大多数化学物质穿过血脑屏障的渗透性与其油/水分配系数直接相关，但许多亲脂性化学物质由于多药物转运蛋白的表达而不能进入脑。相反，由于营养转运蛋白的表达，预计渗透性较低的极性营养素显示出较高的吸收。许多对大脑有潜在毒性的化学物质无法穿过血脑屏障。此外，许多治疗精神疾病和神经系统疾病的候选药物由于血脑屏障而失败。因此，需要一个模型来研究血脑屏障的新范式，其中测试化学品的毒性将在体外和计算机方法完成。此外，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未能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，并显示出紧密的屏障，在过去的40年中已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其制备过程费力且寿命短。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，并优化试剂和测定方法，以测量渗透性和运输，这些方法将由我们公司进行或出售给他人。为了实现这一目标，在特定目标1中，将对制备、冷冻保存活牛脑微血管的条件进行标准化，并确保不同批次销售的微血管的一致性。将通过测定氨基酸和糖转运、多药转运蛋白和活力来评估脑微血管的功能。达到Z因子最接近1.0的测定将指示最佳制备条件。在特定目标2中，将开发试验，通过测量非特异性转运来检查血脑屏障的化学毒性。通过计算受试者工作曲线评估灵敏度和特异性。在II期资助申请中，将开发用于测量多药外排泵以及管腔和近管腔转运蛋白的检测方法。通过建立用于评估血脑屏障的试剂的商业来源，制药公司和化学测试实验室将有一个现成的模型来筛选测试化学品的渗透性和毒性。当考虑到血脑屏障在神经功能中的重要性时，这些产品将有可能获得广泛的销售和商业成功。 公共卫生相关性： 缺乏有效的模型来测量药物和化学品对血脑屏障（BBB）的影响，这一直是评估化学品对大脑毒性的障碍，也是成功开发治疗大脑疾病的药物的障碍18，29。提出的血脑屏障模型，从牛分离的脑微血管（BM），血脑屏障通透性测定，有可能在神经毒性测试和药物开发领域产生巨大的影响。这里的创新在于采用成功的研究模型，新鲜分离的BM，并将其转化为冷冻保存的可销售的产品，并将其用于解决先前未满足的评估BBB渗透性的需求的测定。

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.