Functional feature of a novel model of blood brain barrier: studies on permeation of test compounds

Drug delivery to the central nervous system (CNS) is subject to the permeab ility limitations imposed by the blood-brain barrier (BBB). Several systems in vitro have been described to reproduce the physical and biochemical beh avior of intact BBB, most of which lack the feature of the in vivo barrier. We developed a fully formed monolayer of RBE4.B immortalized rat brain mic rovessel endothelial cells (ECs), grown on top of polycarbonate filter inse rts with cortical neuronal cells grown on the outside. Neurons induce ECs t o synthesize and sort occludin to the cell periphery. Occludin localization is regulated by both compositions of the substratum and soluble signals re leased by cortical co-cultured neurons. The observed effects do not require strict physical contact among cells and neurons. To assess the physiologic al function of the barrier we examined the transendothelial transfer of thr ee test compounds: dopamine, L-tryptophan and L-DOPA. Polycarbonate filter inserts, where ECs were co-cultured with neurons, were assumed as open two compartments vertical dynamic models. Permeation studies demonstrated that the ECs/neurons co-cultures possess permeability characteristics approachin g those of a functional BBB: the system behaved as a selective interface th at excludes dopamine permeation, yet permits L-tryptophan and L-DOPA to cro ss. The movement of test compounds from the donor to the acceptor compartme nt was observed at a distinct time from the start of co-culture. Transfer w as determined using standard kinetic equations. Different performance was o bserved after 5 and 7 days of co-culture. After 5 days dopamine, L-tryptoph an and L-DOPA passively permeate through the membrane as indicated by fitti ngs with a first-order kinetic process equation. After 7 days of co-culture , occludin localizes at ECs periphery, dopamine does not cross the barrier to any further extent, while the transfer of L-tryptophan and L-DOPA fits w ell with a saturable Michaelis-Menten kinetic process, thus indicating the involvement of a specific carrier-mediated transport mechanism. Permeation studies confirmed that culture of ECs in the presence of neurons induces th e characteristic permeability limitations of a functional BBB. (C) 2001 Els evier Science B.V. All rights reserved.