Human Blood-Brain Barrier Model for CNS Drug Translation

描述（由申请人提供）：本提案涉及主题领域2：将基础科学发现转化为新的更好的治疗方法。特别是，我们建议开发一种新的人血脑屏障（BBB）模型，该模型来源于干细胞来源，适合于高通量评估中枢神经系统（CNS）治疗剂的BBB渗透性，这是将新的脑治疗剂转化为临床的关键标准。一个独特的跨学科团队在血脑屏障，人类干细胞和脑肿瘤治疗方面的专业知识被组装起来，以产生初步的可行性数据，并准备立即部署在拟议的项目。摘要：数以百万计的美国人患有神经系统疾病，如阿尔茨海默病、帕金森病和脑艾滋病。虽然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。脑药物开发的一个主要障碍是缺乏强大的递送策略，可以通过血流非侵入性地将药物靶向大脑，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）的内皮是独特的不可渗透的，并且仅允许那些小分子（小于500道尔顿）和亲脂性的分子从血流自由扩散到脑的间隙。因此，内皮屏障限制了大多数小分子药物（98%）的脑摄取，并基本上阻止了静脉给药后蛋白质或基因药物的摄取。在过去的20年里，许多有前途的药物在临床试验中失败，这是由于BBB渗透性差，这种屏障在一定程度上导致了缺乏新的治疗方法和治疗脑部疾病的方法。事实上，开发神经药物的最大挑战之一是在全面临床试验之前准确预测BBB渗透性。一种潜在的解决方案是开发基于细胞的体外模型，其可以模拟在体内观察到的人BBB特征。这种模型需要是简单的，可缩放的，并服从于药物渗透性筛选的大脑摄取的先验预测。体外BBB模型通常由在可渗透膜上生长的单层脑内皮细胞（EC）组成。这些细胞充当填充液体的上隔室和下隔室之间的扩散屏障，上隔室代表血流，下隔室代表脑。药物可以应用于上室或下室，以分别预测脑流入或流出。不幸的是，虽然体外血脑屏障模型可用于确定候选药物或药物组的转运特征，但它们很少是人类来源的。作为一种新颖而及时的解决方案，我们提出建立一个基于人多能干细胞（hPSC）衍生的内皮细胞的血脑屏障模型。如在初步数据中所述，我们已经设计了一种稳健的方案，用于将人多能干细胞定向分化为血脑屏障内皮细胞，所述血脑屏障内皮细胞具有发育良好的紧密连接、特征性转运蛋白表达和对共培养的神经细胞提供的线索作出响应的能力。因此，我们在培养皿中近似了人BBB功能。因此，我们建议通过将hPSC衍生的BBB模型转化为各种不同的多能干细胞系，并将干细胞衍生的BBB内皮细胞的基因表达谱与从人脑收获的BBB内皮细胞进行比较，来进一步验证hPSC衍生的BBB模型。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BBB转运机制（如被动扩散、主动流入或主动流出）影响的药物的能力。最后，该模型将用于测试由约100种化合物组成的一组脑肿瘤治疗剂的体外肿瘤抑制中的人BBB渗透性和BBB后功效。总之，这些目标有可能通过在开发过程的早期添加人类BBB筛选工具来显著改变CNS药物的开发方式。以这种方式，CNS药物从培养皿到临床的翻译可以通过所提出的研究得到显著增强。 公共卫生相关性：开发具有预测能力的人类BBB模型将影响CNS疾病的药物开发过程，这些疾病困扰着全球数百万人。此外，根据需要产生大量人脑内皮细胞沿着NPC衍生的星形胶质细胞和神经元的能力将允许研究人员和药物开发者等获得用于模拟人BBB的新的和独特的资源，以追求药物开发、药物靶向、干细胞治疗和目前在人类中难以解决的许多其他努力。最后，使用人血脑屏障模型沿着脑肿瘤分离株对EGFR抑制剂渗透性和疗效的详细了解可能对此类药物向临床的转化产生重大影响。

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.