Validation of a P-Glycoprotein (P-gp) Humanized Mouse Model by Integrating Selective Absolute Quantification of Human MDR1, Mouse Mdr1a and Mdr1b Protein Expressions with In Vivo Functional Analysis for Blood-Brain Barrier Transport.
It is essential to establish a useful validation method for newly generated humanized mouse models. The novel approach of combining our established species-specific protein quantification method combined with in vivo functional studies is evaluated to validate a humanized mouse model of P-gp/MDR1 efflux transporter. The P-gp substrates digoxin, verapamil and docetaxel were administered to male FVB Mdr1a/1b(+/+) (FVB WT), FVB Mdr1a/1b(-/-) (Mdr1a/1b(-/-)), C57BL/6 Mdr1a/1b(+/+) (C57BL/6 WT) and humanized C57BL (hMDR1) mice. Brain-to-plasma total concentration ratios (Kp) were measured. Quantitative targeted absolute proteomic (QTAP) analysis was used to selectively quantify the protein expression levels of hMDR1, Mdr1a and Mdr1b in the isolated brain capillaries. The protein expressions of other transporters, receptors and claudin-5 were also quantified. The Kp for digoxin, verapamil, and docetaxel were 20, 30 and 4 times higher in the Mdr1a/1b(-/-) mice than in the FVB WT controls, as expected. The Kp for digoxin, verapamil and docetaxel were 2, 16 and 2-times higher in the hMDR1 compared to the C57BL/6 WT mice. The hMDR1 mice had 63- and 9.1-fold lower expressions of the hMDR1 and Mdr1a proteins than the corresponding expression of Mdr1a in C57BL/6 WT mice, respectively. The protein expression levels of other molecules were almost consistent between C57BL/6 WT and hMDR1 mice. The P-gp function at the BBB in the hMDR1 mice was smaller than that in WT mice due to lower protein expression levels of hMDR1 and Mdr1a. The combination of QTAP and in vivo functional analyses was successfully applied to validate the humanized animal model and evaluates its suitability for further studies.
Department of Pharmaceutical Biosciences, Uppsala University, Uppsala, Sweden.
Muhammad Waqas
MW
Sadiq
Division of Membrane Transport and Drug Targeting, Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aoba, Aramaki, Aoba-ku, Sendai, Japan.
Yasuo
Y
Uchida
Division of Membrane Transport and Drug Targeting, Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aoba, Aramaki, Aoba-ku, Sendai, Japan.
Yutaro
Y
Hoshi
Division of Membrane Transport and Drug Targeting, Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aoba, Aramaki, Aoba-ku, Sendai, Japan.
Masanori
M
Tachikawa
Division of Membrane Transport and Drug Targeting, Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aoba, Aramaki, Aoba-ku, Sendai, Japan.
Tetsuya
T
Terasaki
Department of Pharmaceutical Biosciences, Uppsala University, Uppsala, Sweden.
Margareta
M
Hammarlund-Udenaes
D018435:ATP Binding Cassette Transporter, Subfamily B / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D001812:Blood-Brain Barrier / Q000187:drug effects / Q000378:metabolism*; D001921:Brain / Q000098:blood supply / Q000187:drug effects; D002196:Capillaries / Q000187:drug effects / Q000378:metabolism; D016572:Cyclosporine / Q000494:pharmacology; D004077:Digoxin / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D008810:Mice, Inbred C57BL; D023421:Models, Animal; D010098:Oxycodone / Q000378:metabolism; D040901:Proteomics; D000097202:ATP-Binding Cassette Sub-Family B Member 4
D018435:ATP Binding Cassette Transporter, Subfamily B; D016572:Cyclosporine; D004077:Digoxin; D008565:Membrane Proteins; D010098:Oxycodone; D000097202:ATP-Binding Cassette Sub-Family B Member 4; C095122:multidrug resistance protein 3