Determining P-glycoprotein-drug interactions: evaluation of reconstituted P-glycoprotein in a liposomal system and LLC-MDR1 polarized cell monolayers

Abstract

Introduction: P-Glycoprotein (ABCB1, MDR1) is a multidrug efflux pump that is a member of the ATP-binding cassette (ABC) superfamily. Many drugs in common clinical use are either substrates or inhibitors of this transporter. Quantitative details of P-glycoprotein inhibition by pharmaceutical agents are essential for assessment of their pharmacokinetic behavior and prevention of negative patient reactions. Cell-based systems have been widely used for determination of drug interactions with P-glycoprotein, but they suffer from several disadvantages, and results are often widely variable between laboratories. We aimed to demonstrate that a novel liposomal system employing contemporary biochemical methodologies could measure the ability of clinically used drugs to inhibit the P-glycoprotein pump. To accomplish this we compared results with those of cell-based approaches.

Methods: Purified transport-competent hamster Abcb1a P-glycoprotein was reconstituted into a unilamellar liposomal system, Fluorosome-trans-pgp, whose aqueous interior contains fluorescent drug sensors. This provides a well-defined system for measuring P-glycoprotein transport inhibition by test drugs in real time using rapid fluorescence-based technology.

Results: Inhibition of ATP-driven transport by Fluorosome-trans-pgp employed a panel of 46 representative drugs. Resulting IC50 values correlated well (r2=0.80) with Kd values for drug binding to purified P-glycoprotein. They also showed a similar trend to transport inhibition data obtained using LLC-MDR1 cell monolayers. Fluorosome-trans-pgp IC50 values were in agreement with published results of digoxin drug-drug interaction studies in humans.

Discussion: This novel approach using a liposomal system and fluorescence-based technology is shown to be suitable to study whether marketed drugs and drug candidates are P-glycoprotein inhibitors. The assay is rapid, allowing a 7-point IC50 determination in <6 min, and requires minimal quantities of test drug. The method is amenable to robotics and offers a cost advantage relative to conventional cell-based assays. The well-defined nature of this assay also obviates many of the inherent complications and ambiguities of cell-based systems.

Fig. 1

Fl-t-pgp principles of operation. Fl-t-pgp consists of purified Pgp reconstituted into the bilayers of unilamellar liposomes that contain drug-binding fluorescent sensor molecules in their aqueous interior. Prior to the actual assay, the Pgp substrate S-HR is introduced into a preparation of Fl-t-pgp and passively diffuses through the membrane to reach a state of equilibrium. Upon entering the aqueous interior of the Fl-t-pgp particle, the substrate rapidly binds to the fluorescent sensor, resulting in a new and stable fluorescence baseline (not shown). 1. To conduct a transport assay, “Fl-t-pgp reagent” in the wells of a microplate is placed in a fluorescence plate reader, and measurement is initiated. 2. ATP is injected. 3. Substrate is actively pumped by Pgp against a concentration gradient into the interior of the Fl-t-pgp particle, and the influxed substrate is rapidly bound by the encapsulated fluorescent sensor. 4. A linear, time-dependent alteration of the fluorescence intensity is observed.

Fig. 2

Screen shot of Pgp activity assay using Fl-t-pgp reagent in the presence of three concentrations of cyclosporin A. Samples (100 µL each) in a BMG NOVOStar injecting fluorescence plate reader were scanned in well mode for 20 s to establish a baseline. At 20 s, 5 µL of ATP stock solution was rapidly injected into each well (final concentration 2 mM), and fluorescence increase was monitored over 30 s, beginning 10 s post-injection. (The sharp drop in fluorescence observed upon ATP injection is an instrumental artifact.) Onboard software calculated the slope of fluorescence at each concentration of cyclosporin A, and % of Pgp inhibition at each concentration of cyclosporin A is the ratio of that slope to the control slope.

Fig. 3

Representative IC₅₀ plots derived from Fl-t-pgp assays. Individual points represent the activity of Pgp in the presence and absence of test compound, normalized to a value of 1 for controls. IC₅₀ values were calculated by fitting the relative activity at each test compound concentration to a monophasic decay. The IC₅₀ value is the test compound concentration causing 50% inhibition of Pgp transport activity.

Fig. 4

Plot (log-log) of the IC₅₀ values for Pgp transport inhibition obtained with Fl-t-pgp and K_d values for Pgp binding for 33 test compounds. IC₅₀ values for transport inhibition were determined using the Fl-t-pgp assay, and K_d values were determined by fluorescence quenching of purified Pgp in 2 mM CHAPS.

Fig. 5

Representative IC₅₀ plots derived from net transport measurements of [³H]digoxin transport in LLC-MDR1 cell monolayers. Individual points represent the inhibition of Pgp-mediated digoxin transport at varying concentrations of test compounds. IC₅₀ values were calculated by non-linear regression analysis as described in Materials and methods.

Fig. 6

Plot (log-log) of the IC₅₀ values for transport inhibition determined using LLC-MDR1 cell monolayers and IC₅₀ values for transport inhibition determined using the Fl-t-pgp assay for 16 test compounds.

Fig. 7

(a) Plot of the ratio of AUC for digoxin in the presence and absence of drug (inhibitor) vs. the ratio of peak plasma concentration of drug (inhibitor) and the IC₅₀ for Fl-t-pgp. (b) Plot of the ratio of AUC for digoxin in the presence and absence of drug (inhibitor) vs. the ratio of gastrointestinal concentration of drug (inhibitor) and the IC₅₀ for Fl-t-pgp. (c) Plot of the ratio of C_max for digoxin in the presence and absence of drug (inhibitor) vs. the ratio of peak plasma concentration of drug (inhibitor) and the IC₅₀ for Fl-t-pgp. (d) Plot of the ratio of C_max for digoxin in the presence and absence of drug (inhibitor) vs. the ratio of gastrointestinal concentration of drug (inhibitor) and the IC₅₀ for Fl-t-pgp. In vivo data are taken from Fenner et al. (Fenner et al., 2009).