Original article
Variability in the measurement of hERG potassium channel inhibition: Effects of temperature and stimulus pattern

https://doi.org/10.1016/j.vascn.2004.06.003Get rights and content

Abstract

Introduction

In vitro evaluation of drug effects on hERG K+ channels is a valuable tool for identifying potential proarrhythmic side effects in drug safety testing. Patch-clamp recording of hERG K+ current in mammalian cells can accurately evaluate drug effects, but the methodology has not been standardized, and results vary widely. Our objective was to evaluate two potential sources of variability: the temperature at which recordings are performed and the voltage pulse protocol used to activate hERG K+ channels expressed in HEK293 cells.

Methods

A panel of 15 drugs that spanned a broad range of potency for hERG inhibition and pharmacological class was evaluated at both room and near-physiological temperatures using several patch-clamp voltage protocols. Concentration–response analysis was performed with three stimulus protocols: 0.5- and 2-s step pulses, or a step-ramp pattern.

Results

Block by 2 of the 15 drugs tested, d,l-sotalol (antiarrhythmic) and erythromycin (antibiotic), was markedly temperature sensitive. hERG inhibition measured using a 2-s step-pulse protocol underestimated erythromycin potency compared with results obtained with a step-ramp protocol. Using conservative acceptance criteria and the step-ramp protocol, the IC50 values for hERG block differed by less than twofold for 15 drugs.

Discussion

Data obtained at near-physiological temperatures using a step-ramp pattern are highly repeatable and provide a conservative safety evaluation of hERG inhibition.

Introduction

The prolongation of the electrocardiographic QT interval (long QT syndrome, LQTS) has been associated with increased risk of a serious ventricular arrhythmia, torsade de pointes. Several inherited forms of LQTS have been traced to cardiac ion channel mutations, including loss of function mutations affecting hERG (human ether-a-go-go-related gene) potassium channels. Moreover, hERG K+ channel inhibition by both cardiac and noncardiac drugs has been identified as the most common cause of acquired, drug-induced LQTS (Brown & Rampe, 2000, Lacerda et al., 2001, Weirich & Antoni, 1998, Yap & Camm, 1999). hERG K+ channels are responsible for a rapid component (IKr) of the repolarizing currents that terminate the cardiac action potential. Drugs that inhibit hERG have the potential to prolong the cardiac action potential and the QT interval and cause torsade de pointes.

In vitro evaluation of the effects of drugs on hERG K+ channels expressed heterologously in mammalian cells has been recommended as part of the preclinical safety package by the International Conference on Harmonization (ICHS7B Expert Working Group, 2002). Patch-clamp electrophysiological recording of K+ currents in hERG-transfected mammalian cells is considered the most accurate method for evaluating the drug inhibition of functional, voltage-gated K+ channels. However, the methodology for such assays has not been standardized, and wide variability in results has been reported (Witchel, Milnes, Mitcheson, & Hancox, 2002a). Some of the discrepancies in the literature relate to the use of different test systems (e.g., native IKr channel expression in cardiac myocytes, heterologous hERG expression in Xenopus oocyte, and heterologous hERG expression in mammalian cells). Our objective was to test two potential sources of variability within the same test system: the temperature at which the recordings are performed (Crumb, 2000) and the voltage pulse protocol used to activate hERG K+ channels (Rampe, Roy, Dennis, & Brown, 1997) stably expressed in human embryonic kidney cells (HEK293).

A diverse panel of drugs that spanned a broad range of potency for hERG inhibition and pharmacological class was tested at both room and near-physiological temperatures using several voltage protocols. We found that an accurate evaluation of these drugs required the adjustment of the pulse protocol and assessment at near-physiological temperature.

Section snippets

Cell culture

Human embryonic kidney (HEK293) cells were stably transfected with hERG cDNA. Stable transfectants were selected by coexpression with the G418-resistance gene incorporated into the expression plasmid. The selection pressure was maintained by including G418 in the culture medium (Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 supplemented with 10% fetal bovine serum, 100 U/ml penicillin G sodium, 100 μg/ml streptomycin sulfate, and 500 μg/ml G418). Cell culture stock plates (100-mm

hERG assay repeatability

In drug development programs, patch-clamp assays are often used to screen large numbers of compounds for hERG K+ channel inhibition. However, because conventional patch-clamp methods have low throughput, a lack of repeatability over time may compromise the assay. Fig. 1 illustrates repeated measurements of the effects of two well-known hERG blockers that have been used as positive controls for patch-clamp assays of hERG K+ channels stably expressed in HEK293 cells. The effects of 60 nM

Variability of published data

As shown in Fig. 7, published IC50 values obtained either in cloned hERG K+ channels, expressed either in a clonal mammalian cell line or in native IKr in mammalian myocytes, vary greatly. For instance, differences in expression systems (myocytes, Sanguinetti & Jurkiewicz, 1990, vs. transfected cells, Zhou et al., 1998) may explain the reported 50-fold difference in sensitivity to E-4031. However, even when drugs are tested using mammalian heterologous systems, IC50 values obtained in different

Acknowledgements

The authors gratefully acknowledge Karen M. Camarillo, Bryce Waldal, and Jennifer Kozak for their roles in preliminary experiments, William Giroski for technical assistance, and Jennifer Sutkus for secretarial assistance.

References (34)

  • P. Daleau et al.

    Erythromycin blocks the rapid component of the delayed rectifier potassium current and lengthens repolarization of guinea pig ventricular myocytes

    Circulation

    (1995)
  • B. Drolet et al.

    Thioridazine lengthens repolarization of cardiac ventricular myocytes by blocking the delayed rectifier potassium current

    Journal of Pharmacology and Experimental Therapeutics

    (1999)
  • S. Ekins et al.

    Three-dimensional quantitative structure–activity relationship for inhibition of human ether-a-go-go-related gene potassium channel

    Journal of Pharmacology and Experimental Therapeutics

    (2002)
  • E. Hanada et al.

    Inhibitory effect of erythromycin on potassium currents in rat ventricular myocytes in comparison with disopyramide

    Journal of Pharmacy and Pharmacology

    (2003)
  • ICHS7B Expert Working Group. (February, 2002) Safety pharmacology studies for assessing the potential for delayed...
  • M. Ishiguro et al.

    Penetration of macrolides into human polymorphonuclear leucocytes

    Journal of Antimicrobial Chemotherapy

    (1989)
  • M. Khalifa et al.

    Block of potassium currents in guinea pig ventricular myocytes and lengthening of cardiac repolarization in man by the histamine H1 receptor antagonist diphenhydramine

    Journal of Pharmacology and Experimental Therapeutics

    (1999)
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