Received for publication June 29, 2007.
Revised October 23, 2007.
Accepted for publication October 23, 2007.

Kinetic identification of membrane transporters that assist P-gp mediated transport of digoxin and loperamide through a confluent monolayer of MDCKII-hMDR1 cells

Abstract

A robust screen for compound interaction with P-gp requires a cell line expressing P-gp and a probe-substrate which is transported solely by P-gp and passive permeability. While this is obvious, 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 hMDR1-MDCKII 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 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 B>A 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 the probe substrate only interacts with P-gp. Attempts to identify the additional digoxin and loperamide transporters using a wide range of substrates/inhibitors of known epithelial transporters (OCT, OAT, OATP, URAT or MRP) failed to inhibit the digoxin or loperamide transport through their basolateral transporter.

Key words: ABC transporters, cellular transport, computer modeling and simulation, inhibition, membrane barriers, membrane permeability, membrane transport, p-glycoprotein, transporters

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