The enantioselective binding of mefloquine enantiomers to P-glycoprotein determined using an immobilized P-glycoprotein liquid chromatographic stationary phase.

Abstract

Mefloquine (MQ), a-2-piperidinyl-2,8-bis(trifluoromethyl)-4-quinolinemethanol (Fig. 1), is an antimalarial agent widely used to treat chloroquine-resistant malaria (1). The agent is administered as a racemic mixture of erythroisomers, (+)-[11R,28S]-MQ {(+)-MQ} and (−)-[11S,28R]-MQ {(−)-MQ}. In humans, there is an enantioselective distribution of MQ with higher plasma and brain concentrations of (−)MQ (2,3). MQ has also been shown to inhibit the activity of the drug efflux transporter P-glycoprotein (Pgp) (4–7). Shao et al. demonstrated that MQ increased the intracellular accumulation of the Pgp substrate daunomycin in the P388/ADR leukemia cell line (4). In addition, when MQ was used concomitantly with the Pgp-substrate vinblastine (VBL), the two agents interacted with each other synergistically in a noncompetitive manner. However, MQ has also been shown to increase the intracellular accumulation of VBL (6,7). In rat brain capillary endothelial GPNT cells, the inhibition of VBL transport was enantioselective with (+)-MQ displaying up to an eightfold greater effect than (−)-MQ (6,7). In Caco-2 human colon carcinoma cells, both (−)-MQ and (+)-MQ significantly increased cellular accumulation of VBL, but the effect was not enantioselective (6,7). The objective of this study was to examine the molecular basis of the observed effect of MQ on VBL transport in light of the observed combined effects of MQ and VBL on daunomycin intracellular accumulation. The experimental approach examined the effect of (+)-MQ and (−)-MQ on the Pgp binding affinities of [H]-VBL and [H]-cyclosporine A ([H]-CsA). The studies were carried out using an immobilized Pgp liquid chromatographic stationary phase (Pgp-SP) (8). The Pgp-SP was constructed using membranes derived from a human cell line transduced with a retroviral vector directing the expression of MDR1 (8). This phase has been used to determine ligand-Pgp binding affinities and to investigate ligand-ligand binding interactions on Pgp (8,9). The experimental approach was competitive binding studies using (+)-MQ and (−)-MQ as the displacers and [H]VBL or [H]-CsA as the ligand. The results demonstrate that the addition of either (+)-MQ or (−)-MQ completely suppressed the binding of [H]-VBL to Pgp, in a manner indicative of an anticooperative allosteric interaction. There was no observed enantioselectivity in this process. However, when [H]-CsA was the marker ligand, (+)-MQ competitively displaced this marker, whereas (−)-MQ had no effect. This indicates that, under the experimental conditions, MQ enantioselectively binds [(+)-MQ > (−)-MQ] at a site at which [H]CsA binds to Pgp. This is the first observation of enantioselective binding to human-derived Pgp.