Development of predictive in vitro model of drug absorption across the human BBB

描述(申请人提供)：血脑屏障(BBB)解释了为什么血液中循环的某些物质能够进入大脑，而其他物质则不能。血脑屏障是由看起来像简单的内皮细胞管的大脑毛细血管形成的。实验和临床证据表明，血脑屏障维持神经功能的化学环境，保护大脑免受有害物质的伤害。每年，制药公司花费数百万美元开发和测试使用血脑屏障体外模型的药物，其中许多最终在体内不起作用。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表明，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有时是不完善的。事实上，虽然许多耐药分子在啮齿类动物的大脑中表达，但临床和体外观察表明，人脑似乎要复杂得多。相反，由于疾病相关的多药耐药现象的出现，大多数在体外有效的中枢神经系统药物都未能通过临床试验。我们建议开发和验证一种人性化的、实用的血脑屏障体外模型，以更准确地反映血脑屏障的临床现实。具体地说，我们希望1)将临床相关抗癫痫药物在药物应答患者中的血脑屏障渗透性与体外模型中测量的值进行比较，该模型由来自相同受试者的细胞或其他来源的正常、非癫痫、非耐药人脑的细胞组成。这将通过测量手术中或体外的血清和大脑抗癫痫药物水平来实现；以及2)将在多重耐药患者中测量的相同药物的血脑屏障渗透率值与由相同受试者的细胞组成的体外模型中测量的值进行比较。这将通过测量药物在手术期间对多重耐药人脑和从人脑分离的细胞中的渗透率来实现，以缓解难治性癫痫发作。PAR-07-049希望“加快药物发现的进程”，并“发展允许对化合物进行初步筛选的细胞分析”。我们坚信，提高血脑屏障体外模型的预测价值是朝着这个方向迈出的关键一步。然而，这里提出的实验将对高通量筛选假定的中枢神经系统药物的成功产生深远的影响。公共卫生相关性：三分之一的癫痫患者患有抗药性癫痫，这与死亡风险增加和衰弱的心理社会后果有关。人类癫痫耐药的基础尚不清楚。与癌症耐药的相似之处表明，耐药蛋白可能起到一定作用。我们认为，多药耐药癫痫患者的血管内排列着从大脑中排出药物的分子，从而阻碍了癫痫的成功缓解。如果我们的假设是正确的，我们可能会获得治疗儿童和成人癫痫的新工具。

DESCRIPTION (provided by applicant): The blood-brain barrier (BBB) explains why certain substances circulating in the blood are able to get into the brain and others do not. The BBB is formed by brain capillaries that look like simple tubes of endothelial cells. Experimental and clinical evidence suggests that the BBB maintains the chemical environment for neuronal function and protects the brain from harmful substances. Every year millions of dollars are spent by pharmaceutical companies to develop and test drugs using in vitro models of the BBB, many of which end up not working in vivo. 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 sometimes imperfect. In fact, while a number of drug resistance molecules are expressed in rodent brain, the human brain appears to be much more complicated as demonstrated by both clinical and in vitro observations. Conversely, most of the promising CNS drugs that are effective in vitro have failed clinical trials due to disease- related appearance of the multidrug resistance phenomenon. We propose to develop and validate a humanized, practical in vitro model of the BBB that more accurately reflects the clinical reality of the BBB. In particular, we wish to 1) compare the BBB permeability of clinically relevant antiepileptic drugs in pharmacorespondent patients to values measured in an in vitro model comprised of cells from the same subjects or other sources of normal, non-epileptic, non drug resistant human brain. This will be achieved by measuring serum and brain antiepileptic drug levels intraoperatively or in vitro; and 2) to compare BBB permeability values of the same drugs measured in multiple drug resistant patients to values measured in an in vitro model comprised of cells from the same subjects. This will be performed by measuring the penetration of drugs into multiple drug resistant human brain and across cells isolated from human brain during surgeries to relieve refractory seizures. PAR-07-049 wishes to "accelerate the process of drug discovery" and to "Develop cellular assays that permit the preliminary screening of compounds". We strongly believe that improving the predictive value of in vitro models of the blood- brain barrier is a crucial step in this direction. The experiments proposed herein will however have a profound impact on the success of high-throughput screening of putative CNS drugs. PUBLIC HEALTH RELEVANCE: One third of patients with epilepsy have drug-resistant epilepsy, which is associated with an increased risk of death and debilitating psychosocial consequences. The basis of drug resistance in human epilepsy is not understood. Parallels with resistance in cancer suggest that drug resistance proteins may have a role. We believe that blood vessels of multiple drug resistant epileptics are lined with molecules that extrude drugs from the brain, causing an impediment to successful relief of seizures. If our hypothesis is correct we may acquire new tools to treat epilepsy in children and adults.