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...
Multidrug resistance-associated protein 1 (MRP1) was originally shown to confer resistance of human tumor cells to a broad range of natural product anticancer drugs. MRP1 has also been shown to mediate efflux transport of glutathione and glucuronide conjugates of drugs and endogenous substrates. An ortholog of MRP1 in the mouse has been cloned and characterized. Significant functional differences between murine and human MRP1 have been noted. Since drug disposition and pharmacology studies often are conducted in rats, there is a need to clone and characterize the rat ortholog of MRP1. We isolated a rat MRP1 (rMRP1) cDNA from rat brain astrocytes, characterized its coding sequences, and verified the transport activity of the protein expressed in MRP1 cDNA-transfected Madin-Darby canine kidney (MDCK) cells. Our results showed that rMRP1 has a coding sequence of 4599 bp, which predicts a polypeptide of 1533 amino acids with an apparent molecular weight of 190 kd by Western immunoblot analysis. rMRP1-transfected MDCK cells are capable of efflux transport of a fluorescent MRP1 marker-calcein-that is inhibitable by known MRP1 inhibitors, indomethacin, and MK571. Sequence analysis indicates that rMRP1 is more closely related to mouse MRP1 than human MRP1.
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