Kinetic identification of membrane transporters that assist P-glycoprotein-mediated transport of digoxin and loperamide through a confluent monolayer of MDCKII-hMDR1 cells.
A robust screen for compound interaction with P-glycoprotein (P-gp) has some obvious requirements, such as a cell line expressing P-gp and a probe substrate that is transported solely by P-gp and passive permeability. It is actually difficult to prove that a particular probe substrate interacts only with P-gp in the chosen cell line. Using a confluent monolayer of MDCKII-hMDR1 cells, we have determined the elementary rate constants for the P-gp efflux of amprenavir, digoxin, loperamide, and quinidine. For amprenavir and quinidine, transport was fitted with just P-gp and passive permeability. For digoxin and loperamide, fitting required a basolateral transporter (p < 0.01), which was inhibited by the P-gp inhibitor N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918). This means that when digoxin is used as a probe substrate and a compound is shown to inhibit digoxin flux, it could be that the inhibition occurs at the basolateral transporter rather than at P-gp. Digoxin basolateral>apical efflux also required an apical importer (p < 0.05). We propose that amprenavir and quinidine are robust probe substrates for assessing P-gp interactions using the MDCKII-hMDR1 confluent cell monolayer. Usage of another cell line, e.g., LLC-hMDR1 or Caco-2, would require the same kinetic validation to ensure that the probe substrate interacts only with P-gp. Attempts to identify the additional digoxin and loperamide transporters using a wide range of substrates/inhibitors of known epithelial transporters (organic cation transporters, organic anion transporters, organic ion-transporting polypeptide, uric acid transporter, or multidrug resistance-associated protein) failed to inhibit the digoxin or loperamide transport through their basolateral transporter.
Acharya
Poulomi
P
Department of Bioscience & Biotechnology, Drexel University, 32nd and Chestnut Sts., Philadelphia, PA 19104, USA.
O'Connor
Michael P
MP
Polli
Joseph W
JW
Ayrton
Andrew
A
Ellens
Harma
H
Bentz
Joe
J
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism*; D000818:Animals; D002219:Carbamates / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004077:Digoxin / Q000378:metabolism*; D004285:Dogs; D005663:Furans; D007700:Kinetics; D008139:Loperamide / Q000378:metabolism*; D011802:Quinidine / Q000378:metabolism; D013449:Sulfonamides / Q000378:metabolism
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D002219:Carbamates; D005663:Furans; D013449:Sulfonamides; C095108:amprenavir; D008139:Loperamide; D004077:Digoxin; D011802:Quinidine