Zhang Yan, Li Cheryl S W, Ye Yuyang, Johnson Kjell, Poe Julie, Johnson Shannon, Bobrowski Walter, Garrido Rosario, Madhu Cherukury
Department of Pharmacokinetics, Dynamics & Metabolism, Global Research & Development, Pfizer Inc, Ann Arbor, MI 48105, USA.
Drug Metab Dispos. 2006 Nov;34(11):1935-43. doi: 10.1124/dmd.105.006437. Epub 2006 Aug 8.
The objective of the study was to establish primary cultured porcine brain microvessel endothelial cells (PBMECs) as an in vitro model to predict the blood-brain barrier (BBB) permeability in vivo. The intercellular tight junction formation of PBMECs was examined by electron microscopy and measured by transendothelial electrical resistance (TEER). The mRNA expression of several BBB transporters in PBMECs was determined by reverse transcriptionpolymerase chain reaction analysis. The in vitro permeability of 16 structurally diverse compounds, representing a range of passive diffusion and transporter-mediated mechanisms of brain penetration, was determined in PBMECs. Except for the perfusion flow rate marker diazepam, the BBB permeability of these compounds was determined either in our laboratory or as reported in literature using in situ brain perfusion technique in rats. Results in the present study showed that PBMECs had a high endothelium homogeneity, an mRNA expression of several BBB transporters, and high TEER values. Culturing with rat astrocyte-conditioned medium increased the TEER of PBMECs, but had no effect on the permeability of sucrose, a paracellular diffusion marker. The PBMEC permeability of lipophilic compounds measured under stirred conditions was greatly increased compared with that measured under unstirred conditions. The PBMEC permeability of the 15 test compounds, determined under the optimized study conditions, correlated with the in situ BBB permeability with an r2 of 0.60. Removal of the three system L substrates increased the r2 to 0.89. In conclusion, the present PBMEC model may be used to predict or rank the in vivo BBB permeability of new chemical entities in a drug discovery setting.
本研究的目的是建立原代培养的猪脑微血管内皮细胞（PBMECs）作为体外模型，以预测体内血脑屏障（BBB）的通透性。通过电子显微镜检查PBMECs的细胞间紧密连接形成，并通过跨内皮电阻（TEER）进行测量。通过逆转录聚合酶链反应分析确定PBMECs中几种BBB转运蛋白的mRNA表达。在PBMECs中测定了16种结构多样的化合物的体外通透性，这些化合物代表了一系列被动扩散和转运蛋白介导的脑渗透机制。除灌注流速标记物地西泮外，这些化合物的BBB通透性在我们实验室测定，或如文献报道使用大鼠原位脑灌注技术测定。本研究结果表明，PBMECs具有高度的内皮细胞同质性、几种BBB转运蛋白的mRNA表达以及高TEER值。用大鼠星形胶质细胞条件培养基培养可提高PBMECs的TEER，但对细胞旁扩散标记物蔗糖的通透性没有影响。在搅拌条件下测量的亲脂性化合物的PBMEC通透性与在非搅拌条件下测量的相比大大增加。在优化的研究条件下测定的15种测试化合物的PBMEC通透性与原位BBB通透性相关，r2为0.60。去除三种系统L底物后，r2提高到0.89。总之，目前的PBMEC模型可用于在药物发现环境中预测或排列新化学实体的体内BBB通透性。
