Human Blood-Brain Barrier Model for CNS Drug Translation

描述（由申请人提供）：本提案涉及主题区域2：将基础科学发现转化为新的和更好的治疗方法。特别是，我们建议开发一种新的人类血脑屏障（BBB）模型，该模型来源于干细胞来源，可用于中枢神经系统（CNS）治疗方法的血脑屏障通透性的高通量评估，这是将新的脑治疗方法转化为临床的关键标准。一个在血脑屏障、人类干细胞和脑肿瘤治疗方面具有专业知识的独特跨学科团队被召集起来，以生成初步的可行性数据，并准备立即部署到拟议的项目中。摘要：数以百万计的美国人患有阿尔茨海默病、帕金森病和脑性艾滋病等神经系统疾病。尽管在小分子药物和生物药物（基因和蛋白质药物）的开发方面取得了重大进展，但很少有新的治疗方法产生。脑药物开发的一个主要障碍是缺乏可靠的药物递送策略，可以通过血液无创地将药物靶向到大脑，这一过程因体内血脑屏障（BBB）的存在而变得复杂。构成血脑屏障（BBB）的内皮具有独特的不渗透性，只允许那些小分子（小于500道尔顿）和亲脂性分子从血液中自由扩散到脑间隙。因此，内皮屏障限制了大多数小分子药物的脑摄取（98%），并且在静脉给药后基本上禁止了蛋白质或基因药物的摄取。在过去的20年里，由于血脑屏障渗透性差，许多有希望的药物在临床试验中失败，这种屏障在一定程度上导致缺乏新的治疗和治愈脑部疾病的方法。事实上，开发神经药物最重要的挑战之一是在全面临床试验之前准确预测血脑屏障的通透性。一种潜在的解决方案是开发基于细胞的体外模型，以模仿在体内观察到的人类血脑屏障特征。这样的模型需要易于操作，可扩展，并适用于药物渗透性筛选，以便对大脑摄取进行先验预测。体外血脑屏障模型通常由生长在可透膜上的单层脑内皮细胞（EC）组成。这些细胞是上下两个充满液体的隔室之间的扩散屏障，上隔室代表血液，下隔室代表大脑。药物可以分别应用于上隔室或下隔室来预测脑流入或流出。不幸的是，尽管体外血脑屏障模型可用于确定候选药物或一组药物的转运特性，但它们很少来自人类。作为一种新颖而及时的解决方案，我们提出了基于人多能干细胞（hPSCs）衍生的内皮细胞建立血脑屏障模型。正如在初步数据中所描述的，我们已经设计了一个强大的方案，用于将人类多能干细胞定向分化为血脑屏障内皮细胞，这些细胞具有发育良好的紧密连接、特征性的转运蛋白表达和对共培养神经细胞提供的线索作出反应的能力。因此，我们已经在培养皿中接近了人类血脑屏障的功能。因此，我们建议进一步验证hpsc衍生的血脑屏障模型，将其转化为多种不同的多能干细胞系，并比较干细胞衍生的血脑屏障内皮细胞与从人脑中收获的血脑屏障内皮细胞的基因表达谱。接下来，我们建议测试一组已知药物分子的渗透性，以证明该模型区分受各种关键血脑屏障运输机制（如被动扩散、主动内流或主动外排）影响的药物的能力。最后，该模型将用于测试一组由约100种化合物组成的脑肿瘤治疗药物，以测试其体外血脑屏障通透性和血脑屏障后的肿瘤抑制功效。综上所述，这些目标有可能通过在开发过程的早期添加人类血脑屏障筛选工具来显著改变中枢神经系统药物的开发方式。通过这种方式，CNS药物从培养皿到临床的翻译可能会被提出的研究大大加强。

DESCRIPTION (provided by applicant): This proposal addresses Thematic Area 2: Translating Basic Science Discoveries into New and Better Treatments. In particular we are proposing the development of a new human blood-brain barrier (BBB) model derived from stem cell sources amenable to high throughput assessment of central nervous system (CNS) therapeutics for their BBB permeability, a key criterion in the translation of new brain therapeutics to the clinic. A unique interdisciplinary team with expertise in blood-brain barrier, human stem cells, and brain tumor therapy was assembled to generate the preliminary feasibility data and is ready for immediate deployment on the proposed project. ABSTRACT: Millions of Americans are afflicted with neurological illnesses such as Alzheimer's disease, Parkinson's disease, and cerebral AIDS. Although significant progress has been made in the development of both small molecule pharmaceuticals and biopharmaceuticals (gene and protein medicines), very few new treatments have resulted. A major hurdle in brain drug development is the lack of robust delivery strategies that can target medicines to the brain non-invasively via the bloodstream, a process that is complicated by the presence of the blood-brain barrier (BBB) in vivo. The endothelium comprising the blood-brain barrier (BBB) is uniquely impermeable and allows only those molecules that are small (less than 500 daltons) and lipophilic to freely diffuse from the bloodstream to the interstices of the brain. Thus the endothelial barrier limits the brain uptake of the majority of small molecule pharmaceuticals (98%) and essentially prohibits the uptake of protein or gene medicines after intravenous administration. Over the past 20 years, many promising drugs have failed in clinical trials as a result of poor BBB permeability and this barrier has in part lead to the lack of new treatments and cures for brain disease. In fact, one of the most significant challenges in developing neuropharmaceuticals is the accurate prediction of BBB permeability prior to full-blown clinical trials. One potential solution is the development of cell-based in vitro models that can mimic the human BBB characteristics observed in vivo. Such models would need to be facile, scaleable and amenable to drug permeability screening for a priori prediction of brain uptake. An in vitro BBB model typically consists of a monolayer of brain endothelial cells (EC) that is grown on a permeable membrane. These cells serve as a diffusion barrier between upper and lower liquid filled compartments, the upper compartment representing the bloodstream and the lower compartment the brain. Drugs can be applied to either the upper or lower compartment to predict brain influx or efflux, respectively. Unfortunately, while in vitro BBB models can be useful in determining the transport characteristics of a candidate drug or panel of drugs, they are rarely of human origin. As a novel and timely solution, we propose creating a blood-brain barrier model based on endothelial cells derived from human pluripotent stem cells (hPSCs). As described in the preliminary data, we have devised a robust protocol for directed differentiation of human pluripotent stem cells to blood-brain barrier endothelial cells having well developed tight junctions, characteristic transporter expression, and capability to respond to cues provided by co-cultured neural cells. As such, we have approximated human BBB function in the culture dish. Thus, we propose further validation of the hPSC-derived BBB model by translating it to a variety of different pluripotent stem cell lines, and comparing the gene expression profile of stem cell-derived BBB endothelial cells to BBB endothelial cells harvested from human brain. Next, we propose to test the permeability of a panel of known drug molecules to demonstrate the model's capability of differentiating drugs that are subject to various key BBB transport mechanisms such as passive diffusion, active influx, or active efflux. Finally, the model will be used to test a panel of brain tumor therapeutics, comprised of ~100 compounds, for their human BBB permeability and post-BBB efficacy in tumor repression in vitro. Taken together, these aims have the potential to significantly alter the way CNS drugs are developed by adding a human BBB screening tool early in the development process. In this way, translation of CNS drugs from the Petri dish to the clinic may be substantially enhanced by the proposed research. PUBLIC HEALTH RELEVANCE: Developing a human BBB model with predictive capability would impact the drug development process for CNS diseases that afflict millions of people worldwide. Moreover the capability to produce large amounts of human brain endothelial cells along with NPC-derived astrocytes and neurons on demand would allow researchers and drug developers alike a new and unique resource for modeling the human BBB to pursue drug development, drug targeting, stem cell therapy, and many other endeavors that are currently intractable in humans. Finally, a detailed understanding of EGFR inhibitor permeability and efficacy using the human BBB model along with brain tumor isolates could have substantial impact on the translation of this class of medicines to the clinic.