Side-by-side comparison of blood-brain barrier models

描述(申请人提供)：血脑屏障(BBB)对于复杂的问题至关重要，例如药物输送、涉及BBB功能障碍的慢性神经疾病的发病机制(例如脑瘤、缺血、缺氧、脑水肿、多发性硬化症和脑膜炎)以及与生物防御相关的问题。因为血脑屏障选择性地(通过特定的运输机制)排除了大多数血液传播物质和外来物质进入大脑，保护它免受全身的影响。不幸的是，为保护大脑免受潜在危险物质伤害而开发的生物防御系统也可能在治疗几种中枢神经系统疾病(如药物难治性癫痫或难治性脑瘤)过程中导致多药耐药(MDR)现象。每年，制药公司花费数百万美元开发绕过脑实质屏蔽的替代药物策略，并使用血脑屏障的体内或体外模型研究新的治疗方法，其中许多最终都不起作用。合理的中枢神经系统药物设计不能完全和完全依赖于假定的神经疗法的物理化学性质，因为亲脂性本身不能很好地预测药物渗透到中枢神经系统。抗肿瘤药物、抗病毒药物和抗癫痫药物这三大类中枢神经系统药物尤其如此。在包括啮齿动物在内的小动物身上进行的研究不能直接推论到人类组织。该实验室和其他实验室的初步结果令人信服地证明，使用啮齿动物脑内皮细胞和一般非人类来源的内皮细胞系作为临床药理学模型是有缺陷的。我们建议：1)比较临床相关抗癫痫药物在体内和体外的渗透值。这将通过与对照(即非多药耐药)血脑屏障内皮细胞和神经胶质细胞建立的DIV-BBB相比，通过测量药物对幼稚啮齿动物大脑的渗透率来实现。2)比较直接在耐药患者的血清和脑中测量的抗癫痫药物的渗透值，以及在我们的体外模型中由可比的多药耐药对象的细胞组成的渗透值。3)开发自动配药和采样系统，实现对大量模块的自动控制、数据采集和分析。我们还将评估使用微透析探头进行在线采样的可行性，这将克服在渗透性研究中使用放射性或荧光标记化合物的需要。4)实现跨内皮细胞电阻测量系统的新设计，以便利用不同频率的斜坡来测量血脑屏障的阻抗。我们建议开发和验证一种改进的动态体外血脑屏障(DIV-BBB)模型，该模型再现了体内BBB的功能特征，具有更高的可预测性，并且完全可扩展和定制。因此，它将非常适合广泛的药理学和生理学研究。此外，DIV-BBB的使用还可以扩展到神经科学中的研究应用。DIV-BBB可用于更好地了解脑给药原理，获得BBB解剖学和生理学的相关知识，并研究内皮细胞分化为BBB表型的机制。考虑到这些现实前提，DIV-BBB可用于研究病理生理条件的影响，促进针对大脑的新型中枢神经系统药物和治疗方法的设计，对患者有明显的好处。

DESCRIPTION (provided by applicant): The blood-brain barrier (BBB) is crucial for complex issues such as drug delivery, pathogenesis of chronic neurological diseases involving BBB dysfunction (e.g., brain tumors, ischemia, hypoxia, brain edema, multiple sclerosis, and meningitis) and issues related to bio-defense. Since the BBB selectively (by specific transport mechanisms) excludes most blood-borne substances and xenobiotics from entering the brain, protecting it from systemic influences. Unfortunately, bio-defense systems that developed to protect the brain from potentially dangerous substances may also contribute to the phenomenon known as multiple drug resistance (MDR) during treatment of several CNS disorders, such as drug refractory epilepsy or intractable brain tumors. Every year millions of dollars are spent by pharmaceutical companies to develop alternative pharmaceutical strategies that bypass the shielding of brain parenchyma and to study new therapeutic approaches using in vivo or in vitro models of the BBB, many of which end up not working. Rational CNS drug design cannot entirely and exclusively rely upon the physical-chemical properties of putative neurotherapeutics, since lipophilicity alone is a poor predictor for drug penetration into the CNS. This is particularly true for three large families of CNS drugs, antineoplastics, antivirals and antiepileptics. Studies performed in small animals including rodents cannot be directly extrapolated to human tissue. Preliminary results from this and other laboratories have convincingly demonstrated that use of rodent brain endothelial cells and in general endothelial cell lines from non human sources as models of clinical pharmacology are flawed. We propose to: 1) To compare the permeability values of clinically relevant antiepileptic drugs in vivo versus in vitro. This will be performed by measuring the penetration of drugs into the naive rodent brain compared to DIV-BBB established with control (i.e., non-multiple drug resistant) blood-brain barrier endothelial and glial cells. 2) To compare the permeability value of antiepileptic drugs measured directly in serum and brain of pharmacoresistant patients or in our in vitro model comprising of cells from comparable multiple drug resistant subjects. 3) To develop an automated drug dosing and sampling system to allow for automated control, data collection and analysis on large number of modules at the same time. We will also assess the feasibility of the use of a microdialysis probe for on line sampling which will overcome the need of radio- or fluorescent labeled compounds for permeability studies. 4) To implement a new design of the trans-endothelial electrical resistance measurement system in order to allow for the measurement of the impedance of BBB by the use of a ramp of different frequencies. We propose to develop and validate an improved dynamic in vitro blood-brain barrier (DIV-BBB) model that reproduces the functional characteristics of the BBB in vivo, features higher predictability, and is fully scalable and customizable. As such, it will be perfectly suited for extensive pharmacological and physiological studies. In addition, the use of DIV-BBB can be extended to research applications in Neuroscience. The DIV- BBB can be successfully used to better understand the principles of brain drug delivery, to acquire relevant knowledge of the BBB anatomy and physiology, and to study the mechanism of endothelial cells differentiation into a BBB phenotype. Considering these realistic premises, the DIV-BBB can be used to study the effects of pathophysiological conditions and facilitate the design of novel CNS drugs and therapeutical approaches targeting the brain with clear benefits for the patients.