Functional and targeted proteomics characterization of a human primary endothelial cell model of the blood-brain barrier (BBB) for drug permeability studies

The blood-brain barrier (BBB) protects the brain from toxins but hinders the penetration of neurotherapeutic drugs. Therefore, the blood-to-brain permeability of chemotherapeutics must be carefully evaluated. Here, we aimed to establish a workflow to generate primary cultures of human brain microvascular endothelial cells (BMVECs) to study drug brain permeability and bioavailability. Furthermore, we characterized and validated this BBB model in terms of quantitative expression of junction and drug-transport proteins, and drug permeability. We isolated brain microvessels (MVs) and cultured BMVECs from glioma patient biopsies. Then, we employed targeted LC-MS proteomics for absolute protein quantification and immunostaining to characterize protein localization and radiolabeled drugs to predict drug behavior at the Human BBB. The abundance levels of ABC transporters, junction proteins, and cell markers in the cultured BMVECs were similar to the MVs and correctly localized to the cell membrane. Permeability values (entrance and exit) and efflux ratios tested in vitro using the primary BMVECs were within the expected in vivo values. They correctly reflected the transport mechanism for 20 drugs (carbamazepine, diazepam, imipramine, ketoprofen, paracetamol, propranolol, sulfasalazine, terbutaline, warfarin, cimetidine, ciprofloxacin, digoxin, indinavir, methotrexate, ofloxacin, azidothymidine (AZT), indomethacin, verapamil, quinidine, and prazosin). We established a human primary in vitro model suitable for studying blood-to-brain drug permeability with a characterized quantitative abundance of transport and junction proteins, and drug permeability profiles, mimicking the human BBB. Our results indicate that this approach could be employed to generate patient-specific BMVEC cultures to evaluate BBB drug permeability and develop personalized therapeutic strategies.