Quantitative Targeted Absolute Proteomics for Better Characterization of an In Vitro Human Blood-Brain Barrier Model Derived from Hematopoietic Stem Cells.

We previously developed an in vitro model of the human blood-brain barrier (BBB) based on the use of endothelial cells derived from CD34+-hematopoietic stem cells and cultured with brain pericytes. The purpose of the present study was to provide information on the protein expression levels of the transporters, receptors, tight junction/adherence junction molecules, and transporter-associated molecules of human brain-like endothelial cells (hBLECs). The absolute protein expression levels were determined by liquid chromatography-mass spectrometry-based quantitative targeted absolute proteomics and compared with those from human brain microvessels (hBMVs). The protein levels of CD144, CD147, MRP4, Annexin A6 and caveolin-1 showed more than 3-fold abundance in hBLECs, those of MCT1, Connexin 43, TfR1, and claudin-5 showed less than 3-fold differences, and the protein levels of other drug efflux transporters and nutrient transporters were less represented in hBLECs than in hBMVs. It is noteworthy that BCRP was more expressed than MDR1 in hBLECs, as this was the case for hBMVs. These results suggest that transports mediated by MCT1, TfR1, and claudin-5-related tight junction function reflect the in vivo BBB situation. The present study provided a better characterization of hBLECs and clarified the equivalence of the transport characteristics between in vitro BBB models and in vivo BBB models using LC-MS/MS-based protein quantification.

Developmental changes in transporter and receptor protein expression levels at the rat blood-brain barrier based on quantitative targeted absolute proteomics.

The blood-brain barrier (BBB) transport systems regulate the supply of nutrients, amino acids, vitamins, and hormones to the developing brain, as well as blocking the entry of xenobiotics and drugs. The purpose of this study was to clarify the developmental changes in the absolute protein expression levels of BBB transport-related proteins in developing rat brain capillaries, using quantitative targeted absolute proteomics (QTAP). The changing patterns of ATP-binding cassette (ABC) and solute carrier (SLC) transporters, receptors, and tight junction/adherence junction-related proteins were classified into 4 types: uphill (continuously increasing expression from postnatal day (P) 1 to P56), bell-shape/inverted bell-shape (increased/decreased expression from P1 to P14 followed by decreased/increased expression from P21 to P56), downhill (continuously decreasing expression from P1 to P56), and constant (no significant difference from P1 to P56). Proteins showing uphill-type expression included P-glycoprotein/Mdr1a/Abcb1, Mrp4/Abcc4, Bcrp/Abcg2, Glut1/Slc2a1, Oatp1c1/Slco1c1, FcRn, 4F2hc/Slc3a2, claudin-5, caveolin-1, Cd29/integrin ß1. Those showing bell-shape/inverted bell-shape expression included Mct1/Slc16a1, Oat3/Slc22a8, Tfr1, Lrp1, and CD147. On the other hand, Cat1/Slc7a1 and Cd54/Icam-1 showed downhill expression, and Insr showed constant expression. These results suggest that the protein expression levels of transporters and receptors at the BBB change in various ways to meet the changing requirements of the developing brain.

Increased Expression of Renal Drug Transporters in a Mouse Model of Familial Alzheimer's Disease.

It is well established that the expression and function of drug transporters at the blood-brain barrier are altered in Alzheimer's disease (AD). However, we recently demonstrated in a mouse model of AD that the expression of key drug transporters and metabolizing enzymes was modified in peripheral organs, such as the small intestine and liver, suggesting that systemic drug absorption may be altered in AD. The purpose of this study was to determine whether the expression of drug transporters in the kidneys differed between 8- to 9-month-old wild-type mice and APPswe/PSEN1dE9 (APP/PS1) transgenic mice, a mouse model of familial AD, using a quantitative targeted absolute proteomics approach. The protein expression of the drug transporters-multidrug resistance-associated protein 2, organic anion transporter 3, and organic cation transporter 2-was upregulated 1.6-, 1.3-, and 1.4-fold, respectively, in kidneys from APP/PS1 mice relative to wild-type mice. These results suggest that in addition to modified oral absorption of certain drugs, it is possible that the renal excretion of drugs that are multidrug resistance-associated protein 2, organic anion transporter 3, and organic cation transporter 2 substrates could be altered in AD. These changes could affect the interpretation of studies conducted during drug development using this mouse model of AD and potentially impact dosage regimens of such drugs prescribed in this patient population.

Altered Expression of Small Intestinal Drug Transporters and Hepatic Metabolic Enzymes in a Mouse Model of Familial Alzheimer's Disease.

Drug transporter expression and function at the blood-brain barrier is altered in Alzheimer's disease (AD). However, the impact of AD on the expression of transporters and metabolizing enzymes in peripheral tissues has received little attention. The current study evaluated the expression of drug transporters and metabolizing enzymes in the small intestine and liver from 8- to 9-month-old female wild-type (WT) and APPswe/PSEN 1dE9 (APP/PS1) transgenic mice, a widely used AD model, using a quantitative targeted absolute proteomics (QTAP) approach. Furthermore, the general morphological appearance of the liver was assessed by immunohistochemistry, and lipid content was visualized using Oil Red O staining. The small intestines of APP/PS1 mice exhibited a significant 2.3-fold increase in multidrug resistance-associated protein 2 (Mrp2), a 1.9-fold decrease in monocarboxylate transporter 1 (Mct1), and a 3.6-fold increase in UDP-glucuronosyltransferase (Ugt) 2b5 relative to those from WT mice based on QTAP analysis. While the liver from APP/PS1 mice exhibited no changes in drug transporter expression, there was a 1.3-fold elevation in cytochrome P450 (Cyp) 51a1 and a 1.2-fold reduction in Cyp2c29 protein expression, and this was associated with morphological alterations including accumulation of hepatocyte lipids. These studies are the first to demonstrate that the protein expression of transporters and metabolizing enzymes important in oral drug absorption are modified in a mouse model of familial AD, which may lead to altered disposition of some orally administered drugs in AD.