title: Validation of an immortalized human (hBMEC) in vitro blood-brain barrier model.
abstract: We recently established and optimized an immortalized human in vitro blood-brain barrier (BBB) model based on the hBMEC cell line. In the present work, we validated this mono-culture 24-well model with a representative series of drug substances which are known to cross or not to cross the BBB. For each individual compound, a quantitative UHPLC-MS/MS method in Ringer HEPES buffer was developed and validated according to current regulatory guidelines, with respect to selectivity, precision, and reliability. Various biological and analytical challenges were met during method validation, highlighting the importance of careful method development. The positive controls antipyrine, caffeine, diazepam, and propranolol showed mean endothelial permeability coefficients (P e) in the range of 17-70 × 10(-6) cm/s, indicating moderate to high BBB permeability when compared to the barrier integrity marker sodium fluorescein (mean P e 3-5 × 10(-6) cm/s). The negative controls atenolol, cimetidine, and vinblastine showed mean P e values < 10 × 10(-6) cm/s, suggesting low permeability. In silico calculations were in agreement with in vitro data. With the exception of quinidine (P-glycoprotein inhibitor and substrate), BBB permeability of all control compounds was correctly predicted by this new, easy, and fast to set up human in vitro BBB model. Addition of retinoic acid and puromycin did not increase transendothelial electrical resistance (TEER) values of the BBB model.
authors.affiliation: Pharmaceutical Biology, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, 4056, Basel, Switzerland.
authors.forename: Daniela Elisabeth
authors.initials: DE
authors.lastname: Eigenmann
authors.affiliation: Pharmaceutical Biology, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, 4056, Basel, Switzerland.
authors.forename: Evelyn Andrea
authors.initials: EA
authors.lastname: Jähne
authors.affiliation: Molecular Modeling, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, 4056, Basel, Switzerland.
authors.forename: Martin
authors.initials: M
authors.lastname: Smieško
authors.affiliation: Pharmaceutical Biology, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, 4056, Basel, Switzerland.
authors.forename: Matthias
authors.initials: M
authors.lastname: Hamburger
authors.affiliation: Pharmaceutical Biology, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, 4056, Basel, Switzerland. mouhssin.oufir@unibas.ch.
authors.forename: Mouhssin
authors.initials: M
authors.lastname: Oufir
mesh_terms: D001812:Blood-Brain Barrier / Q000378:metabolism*; D001921:Brain / Q000098:blood supply; D002199:Capillary Permeability; D018929:Cell Culture Techniques; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D003198:Computer Simulation; D042783:Endothelial Cells / Q000378:metabolism*; D006801:Humans; D008954:Models, Biological; D010599:Pharmacokinetics; D053719:Tandem Mass Spectrometry
keywords: FDA/EMA; In vitro human blood-brain barrier (BBB) model; Method validation; Permeability coefficient; UHPLC-MS/MS; hBMEC cell line