Abstract

Reliable human in vitro blood-brain barrier (BBB) models suitable for high-throughput screening are needed in early CNS drug discovery and development programs for assessing the ability of promising bioactive compounds to cross the BBB. A human brain microvascular endothelial cell line hBMEC was recently established and validated as an immortalized monoculture model of the human blood-brain barrier (BBB) applicable in 24-well plate format.
For further validation of the model we assessed, by means of quantitative targeted absolute proteomics (QTAP), the protein expression levels in hBMEC cells of ten selected transporters, for comparison after normalization with BBB models involving primary isolated human brain microvessels, human umbilical vein endothelial cells (HUVEC), and immortalized human brain microvascular endothelial cell line (hCMEC/D3). These transporters were further confirmed by real-time PCR (RT-PCR) and Western blot analysis.
Among ten selected target proteins, only two transporters (hMRP1 and hMCT1) were present at quantifiable levels in the plasma membrane fraction of hBMEC cells using the two approaches (QTAP and RT-PCR). After normalization based on Na+/K+ ATPase expression for the QTAP approach, the protein expression levels for the transporter proteins hMRP1 and hMCT1 in the immortalized cell lines (hBMEC and hCMEC/D3) were within the same range. The quantitative expression levels of transporters (QTAP) showed the limitations of the hBMEC cell line as a BBB functional model since the two key transporters (multidrug resistant protein 1 hMDR1 and breast cancer resistant protein hBCRP), recommended in the FDA/EMA regulatory “drug-drug interactions” (DDI) guidances, are not quantifiable but only detectable. In addition, both ABCB1 and hBCRP were confirmed as not detectable by RT-PCR and Western-blot analysis.