A HESI consortium approach to assess the human predictive value of non-clinical repolarization assays.

Drug-induced ventricular arrhythmia and Torsades de Pointes remain a serious public health issues in bringing safe new pharmaceuticals to the market place. Under the auspices of the International Life Science Institute (ILSI)-Health and Environmental Sciences Institute (HESI), a consortium involving representatives from pharmaceutical companies, regulatory agencies and opinion leaders from the scientific and medical research communities has been initiated. The objectives are (1) to assess the concordance between signals in non-clinical repolarization assays and clinical QT interval prolongation; (2) to investigate the mechanisms for any discrepancy identified between non-clinical and clinical results and to determine viable and successful alternative approaches to identify these compounds; and (3) to assess the proarrhythmic potential of such compounds. At present, the consortium is conducting a retrospective analysis of non-clinical and clinical data from both FDA and contributing companies' databases and supplementing with a literature review. The overall objectives of these initial efforts are to establish a quantitative integrated risk assessment for each compound; to define criteria for concordance and apply them to the database in order to identify non-concordant compounds.

The hERG channel and risk of drug-acquired cardiac arrhythmia: an overview.

This review summarizes current knowledge of the cardiac rapidly activating delayed rectifier potassium current (I(Kr)), and its connection to drug-acquired QT prolongation and the associated risk of ventricular arrhythmia and fibrillation. The molecular characterization of hERG as the structural correlate of I(Kr) and the link between inherited long QT and the KCNH2 gene (hERG), have facilitated mechanistic studies of drug-acquired QT prolongation. The development of high throughput assays to evaluate drug effects on hERG has provided an avenue to determine structure activity relations (SAR) within chemical series. More than 10 years of collective data and structural considerations support the notion that hERG is an unusually promiscuous target among potassium channels, but that defining SAR within a chemical series is a viable strategy to reduce or eliminate hERG activity. Despite a critical need to minimize drug effects on hERG, one should always keep in mind that hERG is not the only structural correlate of QT prolongation, and that QT prolongation is a sub-optimal biomarker for ventricular arrhythmia and fibrillation.

Improved throughput of PatchXpress hERG assay using intracellular potassium fluoride.

Blockade of the human ether-a-go-go-related gene (hERG) potassium channel, with a consequent possibility of QT prolongation and increased susceptibility to a characteristic polymorphic ventricular arrhythmia, torsade de pointes, is an important cause of withdrawal of drugs from the market. In the aftermath of recent drug withdrawals, regulatory agencies now require in vitro hERG screening of all pharmaceutical compounds that are targeted for human use. To minimize the potential for failure in later-stage drug development, many pharmaceutical and biotechnology companies have begun to use automated patch clamp systems with higher throughput than conventional manual patch-clamp techniques to conduct routine functional hERG screening during drug discovery and early development. We have optimized an automated patch-clamp hERG screening method for the PatchXpress 7000A system (Molecular Devices, Sunnyvale, CA) using potassium fluoride (KF) in the internal recording solution. In this study we show that (1) the biophysical and pharmacological properties of hERG current recorded with KF are similar to those with standard potassium chloride solutions, (2) use of KF significantly improves the success rate of hERG screening using PatchXpress without compromising data quality, and (3) utilization of KF can significantly increase the throughput of hERG screening with PatchXpress.

Application of PatchXpress planar patch clamp technology to the screening of new drug candidates for cardiac KCNQ1/KCNE1 (I Ks) activity.

A cardiac safety concern for QT prolongation and potential for pro-arrhythmia exists due to inhibition of the cardiac slowly activating delayed rectifier potassium current, I(Ks). Selective inhibitors of I Ks have been shown to prolong the QT interval in animal models. On the other hand, I Ks has been considered as a target for anti-arrhythmic therapy due to certain biophysical and pharmacological properties and its expression pattern in the heart. Consequently, we have developed a method utilizing a human embryonic kidney (HEK)-293 cell line expressing KCNQ1/KCNE1 (genes that encode for the I Ks channel) as a model for screening of new compounds for I Ks activity. This study was designed (1) to establish and optimize the experimental conditions for measurement of I Ks using PatchXpress() 7000A (Molecular Devices Corporation, Sunnyvale, CA) and (2) to test the effects of I Ks inhibitors and compare the 50% inhibitory concentration (IC50) values determined with PatchXpress versus conventional patch clamp in order to validate the PatchXpress approach for higher-throughput I Ks screening. Biophysical properties of HEK/I Ks recorded with PatchXpress were similar to those recorded with conventional patch-clamp and reported in the literature. The IC50 values for I Ks block determined with PatchXpress correlated well with conventional patch-clamp values from HEK-293 cells as well as from native cardiac myocytes for the majority of compounds tested. Electrophysiological recording of I Ks expressed in HEK-293 cells with the PatchXpress is of acceptable quality for screening purposes. This approach can be utilized for functional prescreening of development compounds for I Ks inhibition either for optimizing lead anti-arrhythmic or other therapeutic candidates or to exclude compounds with the potential to prolong QT.

Flunarizine is a highly potent inhibitor of cardiac hERG potassium current.

Flunarizine has been widely used for the management of a variety of disorders such as peripheral vascular diseases, migraine, and epilepsy. The majority of its beneficial effects have been attributed to its ability to inhibit voltage-gated Ca2+ channels in the low micromolar range, albeit non-selectively, as flunarizine has been shown to inhibit a variety of ion channels. We examined the effects of flunarizine on potassium currents through cardiac channels encoded by the human ether-a-go-go related gene (hERG) stably expressed in CHO cells. In this study, we have characterized the effect of flunarizine on biophysical properties of hERG potassium currents with standard whole-cell voltage-clamp techniques. Notably, flunarizine is a highly potent inhibitor of hERG current with an IC50 value of 5.7 nM. The effect of flunarizine on hERG potassium current is concentration and time dependent, and displays voltage dependence over the voltage range between -40 and 0 mV. At concentrations near or above the IC50, flunarizine causes a negative shift in the voltage dependence of hERG current activation and accelerates tail current deactivation. Flunarizine preferentially blocks the activated state of the channel and displays weak frequency dependence of inhibition. Flunarizine also inhibits KCNQ1/KCNE1 channel current with an IC50 of 0.76 microM.

Variability in the measurement of hERG potassium channel inhibition: effects of temperature and stimulus pattern.

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 IC(50) 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.