Inhibition of hERG K+ currents by antimalarial drugs in stably transfected HEK293 cells

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Abstract

Several antimalarial drugs are known to produce a QT interval prolongation via a blockade of the rapidly activating delayed rectifier K+ current (IKr), encoded by the human-ether-a-go-go-related gene (hERG). We investigated the influence of lumefantrine and its major metabolite desbutyl-lumefantrine, as well as halofantrine, chloroquine, and mefloquine, on wild type hERG K+ channels in stably transfected human embryonic kidney cells (HEK293) using the whole cell patch-clamp technique. All of the tested antimalarial drugs inhibited the hERG K+ channels in a concentration- and time-dependent manner. Only halofantrine blocked hERG tail currents voltage-dependently. The ranking of the half-maximal inhibitory concentrations (IC50) of the antimalarials was: halofantrine (0.04 μM)<chloroquine (2.5 μM)<mefloquine (2.6 μM)<desbutyl-lumefantrine (5.5 μM)<lumefantrine (8.1 μM). Lumefantrine and desbutyl-lumefantrine showed a slower inhibition of IKr than the other tested antimalarials. In conclusion, lumefantrine and desbutyl-lumefantrine inhibited significantly the hERG tail current with a higher IC50-value than mefloquine, chloroquine and halofantrine. This, together with the calculated cardiac safety indices, suggests that lumefantrine and desbutyl-lumefantrine have a weaker proarrhythmic potential than their comparator compounds.

Introduction

Malaria is one of the most widespread reasons for disease and death in tropical and subtropical areas of the world. Unfortunately, there has been a growing body of evidence that certain antimalarial drugs induce cardiotoxicity with respect to QT prolongation which may lead to life-threatening cardiac arrhythmias like Torsade de Pointes Nosten and Price, 1995, Touze et al., 2002. For example, the widely used antimalarial drug halofantrine has been associated with a QT interval prolongation in both clinical trials Nosten et al., 1993, Wesche et al., 2000, Abernethy et al., 2001 and in in vitro assays (Tie et al., 2000). Although the structural similarity between lumefantrine and the aryl-amino alcohol group of antimalarials (in particular halofantrine) has raised concerns over cardiotoxicity, there has been no evidence of QT interval prolongation and Torsade de Pointes in humans so far (Bakshi et al., 2000).

The QT interval of the electrocardiogram is the time period needed for depolarization and repolarization of ventricular myocytes and its prolongation is mainly caused by delayed repolarization. Human cardiac ventricular myocytes are mainly repolarized by the activity of outward K+ currents. One of the major currents is the delayed rectifier K+ current IK, which consists of a rapidly and a slowly activating component (IKr and IKs; Sanguinetti and Jurkiewicz, 1990). The human ether-a-go-go-related gene (hERG) encodes the α-subunit of the voltage gated K+ channels underlying the native current IKr Sanguinetti et al., 1995, Trudeau et al., 1995. When transfected into heterologous systems such as human embryonic kidney cells (HEK293), hERG expresses a K+ channel with properties similar to IKr (Zhou et al., 1998). Inhibition of the hERG current (IhERG) can lead to a prolongation of the action potential duration and consequently of the QT interval of the electrocardiogram, which may, under certain circumstances, lead to the polymorphic ventricular tachyarrhythmia, Torsade de Pointes (Keating and Sanguinetti, 2001). A wide range of pharmacological agents of different therapeutic classes is known to induce QT prolongation by blocking IhERG, including type III antiarrhythmics Sanguinetti and Jurkiewicz, 1990, Spector et al., 1996, antihistaminics Roy et al., 1996, Zhou et al., 1999 and antipsychotics (Rampe et al., 1998, Drolet et al., 1999a, Drolet et al., 1999b, Kang et al., 2000; review of: Buckley and Sanders, 2000). Although there is no clear relationship between the potency of a compound to block IhERG and the likelihood to prolong QT and to induce Torsade de Pointes, it is generally accepted to consider a high potency (low IC50-value) for IhERG inhibition as a risk factor for fatal arrhythmias related to QT interval prolongation, especially if the free therapeutic plasma concentration of a drug is near or even below the IC50-value for IhERG inhibition (Webster et al., 2002). Hence, to compare the proarrhythmic potential of drugs it is reasonable to calculate cardiac safety indices by dividing the hERG current inhibiting potency (e.g. IC50-value) by the respective therapeutic free plasma concentration Crump and Cavero, 1999, Cavero et al., 2000. Antimalarial drugs represent a chemically diverse group of compounds. Although these drugs are associated with a higher risk for QT prolongation, the literature is poor regarding their respective effects on cardiac ion currents in relation to their chemical structure and no clear structure activity relationships related to a risk of IhERG inhibition have yet been elucidated.

The purpose of the present study was to assess the proarrhythmic potential and the structure–activity relationships with regard to IhERG inhibition of the antimalarial drugs lumefantrine, desbutyl-lumefantrine in comparison with halofantrine, chloroquine and mefloquine. Electrophysiological experiments on HEK293 cells stably expressing the hERG channel were conducted using the patch-clamp technique.

Section snippets

Cell culture

HERG.T.HEK (HEK293 cells stably transfected with HERG cDNA) were obtained from the University of Wisconsin (Zhou et al., 1998). The cells were continuously maintained in and passaged using Minimum Essential Medium (MEM; Gibco-BRL, UK) supplemented with 10% foetal bovine serum (Gibco-BRL, UK), 1% non-essential amino acids (lot no. 1085497, Gibco-BRL, UK) and 0.4 mg/ml geneticin (Gibco-BRL, UK). For studies, the cells were plated onto sterile glass coverslips in 35 mm2 dishes (containing 3 ml

Results

Fig. 1 shows the chemical structures of five antimalarial drugs, which were electrophysiologically tested for their ability to inhibit IhERG. In these experiments, a 4.8-s depolarization to +20 mV from a holding potential of−80 mV was followed by 5-s repolarization to −50 mV to produce large, slowly deactivating tail currents characteristic of hERG K+ channels Sanguinetti et al., 1995, Roy et al., 1996. The currents in the presence of each concentration of the respective drug were corrected for

Discussion

This is the first report of a direct blockade of the hERG channel activity by desbutyl-lumefantrine and of the comparison of its hERG inhibitory potency with four other antimalarials, among them its parent compound lumefantrine (also known as benflumetol).

All agents tested (lumefantrine, desbutyl-lumefantrine, halofantrine, chloroquine and mefloquine) inhibited IhERG of stably transfected HEK293 cells in a concentration-and time-dependent manner. Our results reveal halofantrine to be the most

Acknowledgments

The authors wish to thank the staff of Quintiles Limited (Edinburgh, UK), especially Kirsty McGullogh for major experimental contributions.

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