ABSTRACT: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 PB-MEC 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 r 2 of 0.60.Removal of the three system L substrates increased the r 2 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.One major hurdle of successful central nervous system drug delivery is to penetrate the blood-brain barrier (BBB) to reach the therapeutic targets. The BBB is a continuous layer of endothelial cells that are connected to each other through tight junctions. In contrast to the endothelial cells of peripheral blood vessels, the brain microvessel endothelial cells are characterized by unique intercellular tight junctions, the absence of fenestrations, and minimal pinocytic activity. The BBB represents a physiological barrier that efficiently restricts free paracellular passage of most substances from the blood to the brain extracellular environment. Furthermore, the brain microvessel endothelial cells possess a variety of metabolic enzyme systems, which further limit the brain entry of compounds (Pardridge, 1983). Finally, a complex of membrane-bound transport systems, including active efflux transporters, such as P-glycoprotein (Pgp) (Schinkel et al., 1996;Miller et al., 2000) and multidrug resistanceassociated protein (MRP) Zhang et al., 2000), and active uptake transporters, such as the system L amino acid transporter (LAT) (Pardridge, 1983;Smith, 1991), further regulates brain penetration.Pharmaceutical companies have been actively pursuing various methods to accurately predict the brain penetrati...
