Abstract
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.
Footnotes
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.107.017301.
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ABBREVIATIONS: P-gp, the P-glycoprotein product of the hMDR1 gene; MDCKII-hMDR1, Madin-Darby canine kidney II cell line that overexpresses human multidrug resistance 1; bcrp/BRCP, breast cancer resistance protein; GF120918, 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; DMEM, Dulbecco's modified Eagle's medium; A>B (or B> transport across the confluent cell monolayer when the donor chamber is apical (or basolateral) and the receiver chamber is basolateral (or apical); TM, transport medium; BT, basolateral transporter; AT, apical transporter; RMS, root-mean-square; OAT, organic anion transporters; OATP, organic ion-transporting polypeptide; OCT, organic cation transporters; MRP, multidrug resistance-associated protein.
- Received June 29, 2007.
- Accepted October 23, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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