An Electrically Tight In Vitro Blood–Brain Barrier Model Displays Net Brain-to-Blood Efflux of Substrates for the ABC Transporters, P-gp, Bcrp and Mrp-1

• Published in: The AAPS journal Vol. 16; no. 5; pp. 1046 - 1055
• Main Authors: Helms, Hans Christian, Hersom, Maria, Kuhlmann, Louise Borella, Badolo, Lasina, Nielsen, Carsten Uhd, Brodin, Birger
• Format: Journal Article
• Language: English
• Published: Boston Springer US 01.09.2014
• Subjects: Animals; Astrocytes - metabolism; ATP Binding Cassette Transporter, Sub-Family B - metabolism; ATP Binding Cassette Transporter, Sub-Family G, Member 2; ATP-Binding Cassette Transporters - metabolism; Biochemistry; Biomedical and Life Sciences; Biomedicine; Biotechnology; Blood-Brain Barrier - cytology; Blood-Brain Barrier - metabolism; Cattle; Cell Communication; Cell Polarity; Cells, Cultured; Coculture Techniques; Digoxin - metabolism; Electric Conductivity; Endothelial Cells - metabolism; Estrone - analogs & derivatives; Estrone - metabolism; Etoposide - metabolism; Kinetics; Mannitol - metabolism; Multidrug Resistance-Associated Proteins - metabolism; Permeability; Pharmacology/Toxicology; Pharmacy; Phenotype; Rats; Research Article; polarized small molecule transport; p-glycoprotein; blood–brain barrier; breast cancer resistance protein; multidrug resistance-associated protein
• ISSN: 1550-7416; 1550-7416
• DOI: 10.1208/s12248-014-9628-1

Efflux transporters of the ATP-binding cassette superfamily including breast cancer resistance protein (Bcrp/ Abcg2 ), P-glycoprotein (P-gp/ Abcb1 ) and multidrug resistance-associated proteins (Mrp’s/Abcc’s) are expressed in the blood–brain barrier (BBB). The aim of this study was to investigate if a bovine endothelial/rat astrocyte in vitro BBB co-culture model displayed polarized transport of known efflux transporter substrates. The co-culture model displayed low mannitol permeabilities of 0.95 ± 0.1 · 10 −6  cm·s −1 and high transendothelial electrical resistances of 1,177 ± 101 Ω·cm 2 . Bidirectional transport studies with 3 H-digoxin, 3 H-estrone-3-sulphate and 3 H-etoposide revealed polarized transport favouring the brain-to-blood direction for all substrates. Steady state efflux ratios of 2.5 ± 0.2 for digoxin, 4.4 ± 0.5 for estrone-3-sulphate and 2.4 ± 0.1 for etoposide were observed. These were reduced to 1.1 ± 0.08, 1.4 ± 0.2 and 1.5 ± 0.1, by addition of verapamil (digoxin), Ko143 (estrone-3-sulphate) or zosuquidar + reversan (etoposide), respectively. Brain-to-blood permeability of all substrates was investigated in the presence of the efflux transporter inhibitors verapamil, Ko143, zosuquidar, reversan and MK 571 alone or in combinations. Digoxin was mainly transported via P-gp, estrone-3-sulphate via Bcrp and Mrp’s and etoposide via P-gp and Mrp’s. The expression of P-gp, Bcrp and Mrp-1 was confirmed using immunocytochemistry. The findings indicate that P-gp, Bcrp and at least one isoform of Mrp are functionally expressed in our bovine/rat co-culture model and that the model is suitable for investigations of small molecule transport.