hERG Assay Research Articles

Electrophysiological effects of brompheniramine on cardiac ion channels and action potential

Some antihistamines (mainly terfenadine and astemizole) have been demonstrated to cause QT interval prolongation and, in some cases, torsade-de-pointes. We investigated the cardiac electrophysiological effects of brompheniramine, a conventional antihistamine. Brompheniramine was reported to prolong QT interval in isolated hearts. To evaluate the electrophysiological effects of brompheniramine, we used whole-cell patch clamp techniques in human ether-a-go-go related gene (hERG)-stably transfected CHO cells, the SCN5A sodium channel transiently transfected CHO cells, and rat myocytes and conventional microelectrode recording techniques in isolated guinea pig papillary muscles. As for the I hERG, the IC 50 value of brompheniramine was found to be 0.90 ± 0.14 μM with a Hill coefficient ( n H) of 1.75 ± 0.42. Action potential duration at 90% repolarization (APD 90) was slightly prolonged by brompheniramine at 10 and 100 μM, but APD 50 was shortened by 100 μM. Moreover, despite the potent hERG current block, reductions of the V max and total amplitude of action potential were observed at high concentrations of brompheniramine. The change in action potential parameters and poor correlations between hERG and APD assay indicated additional effects of brompheniramine on non-hERG channels. In agreement with this hypothesis, the inhibition of I Na (IC 50 values: 21.26 ± 2.52 μM) and I Ca (IC 50 values: 16.12 ± 9.43 μM) by brompheniramine was observed. The results of this study suggest that brompheniramine may possess classes III, Ib and IV properties, especially at high concentrations and that additional studies on non-hERG channels will be necessary to elucidate the complex electrophysiological effects of brompheniramine on the heart.

Optimisation and validation of a medium-throughput electrophysiology-based hERG assay using IonWorks™ HT

Introduction Regulatory and competitive pressure to reduce the QT interval prolongation risk of potential new drugs has led to focus on methods to test for inhibition of the human ether-a-go-go-related gene (hERG)-encoded K + channel, the primary molecular target underlying this safety issue. Here we describe the validation of a method that combines medium-throughput with direct assessment of channel function. Methods The electrophysiological and pharmacological properties of hERG were compared using two methods: conventional, low-throughput electrophysiology and planar-array-based, medium-throughput electrophysiology (IonWorks™ HT). A pharmacological comparison was also made between IonWorks™ HT and an indirect assay (Rb + efflux). Results Basic electrophysiological properties of hERG were similar whether recorded conventionally (HEK cells) or using IonWorks™ HT (CHO cells): for example, tail current V ½ − 12.1 ± 5.0 mV (32) for conventional and − 9.5 ± 6.0 mV (46) for IonWorks™ HT (mean ± S.D. ( n)). A key finding was that as the number of cells per well was increased in IonWorks™ HT, the potency reported for a given compound decreased. Using the lowest possible cell concentration (250,000 cells/ml) and 89 compounds spanning a broad potency range, the pIC 50 values from IonWorks™ HT (CHO-hERG) were found to correlate well with those obtained using conventional methodology (HEK-hERG)( r = 0.90; p < 0.001). Further validation using CHO-hERG cells with both methods confirmed the correlation ( r = 0.94; p < 0.001). In contrast, a comparison of IonWorks™ HT and Rb + efflux data with 649 compounds using CHO-hERG cells showed that the indirect assay consistently reported compounds as being, on average, 6-fold less potent, though the differences varied depending on chemical series. Discussion The main finding of this work is that providing a relatively low cell concentration is used in IonWorks™ HT, the potency information generated correlates well with that determined using conventional electrophysiology. The effect on potency of increasing cell concentration may relate to a reduced free concentration of test compound owing to partitioning into cell membranes. In summary, the IonWorks™ HT hERG assay can generate pIC 50 values based on a direct assessment of channel function in a timeframe short enough to influence chemical design.

Utility of hERG Assays as Surrogate Markers of Delayed Cardiac Repolarization and QT Safety

HERG (human-ether-a-go-go-related gene) encodes for a cardiac potassium channel that plays a critical role in defining ventricular repolarization. Noncardiovascular drugs associated with a rare but potentially lethal ventricular arrhythmia (Torsades de Pointes) have been linked to delayed cardiac repolarization and block of hERG current. This brief overview will discuss the role of hERG current in cardiac electrophysiology, its involvement in drug-induced delayed repolarization, and approaches used to define drug effects on hERG current. In addition, examples of hERG blocking drugs acting differently (i.e., overt and covert hERG blockade due to multichannel block) together with the utility and limitations of hERG assays as tools to predict the risk of delayed repolarization and proarrhythmia are discussed.

The Different Effects of Phenothiazines on Cardiac Action Potential Duration

The Different Effects of Phenothiazines on Cardiac Action Potential Duration

Drugs, hERG and sudden death

Early recognition of potential QT/TdP liability is now an essential component of the drug discovery/drug development program. The hERG assay is an indispensable step and a high-quality assay must accompany any investigational new drug (IND) application. While it is the gold standard at present, the hERG assay is too labor-intensive and too low throughput to be used as a screen early in the discovery/development process. A variety of indirect high throughput screens have been used.

The utility of hERG and repolarization assays in evaluating delayed cardiac repolarization: influence of multi-channel block.

Drug-induced delayed cardiac repolarization is a recognized risk factor for proarrhythmia and is associated with block of IKr (the potassium current encoded by the human ether-a- go-go-related gene [hERG]). To evaluate the utility of 2 in vitro assays widely used to assess delayed repolarization, we compared the effects of haloperidol and 9 structurally diverse drugs in a hERG and repolarization (canine Purkinje fiber action potential duration [APD]) assay over wide concentrations. Despite potent hERG current block (IC50 = 0.174 microM), haloperidol elicited a bell-shaped concentration-response relationship for APD prolongation, with lesser prolongation (and reduced plateau height) observed with concentrations eliciting maximal hERG block, consistent with multi-channel block at higher concentrations. Consistent with this hypothesis, APD prolongation with the specific IKr blocker dofetilide was a) reduced by concomitant administration of nifedipine (calcium current block) and b) reversed by lidocaine (late sodium current block). Additional studies demonstrated prominent (>50%) hERG inhibition with most (9/10) drugs despite wide APD changes (158% prolongation - 16% shortening), consistent with multi-channel block. The poor correlation between hERG and repolarization assays suggests that the hERG assay oversimplifies drug effects on the complex repolarization process for drugs demonstrating multi-channel block and that neither assay alone adequately predicts proarrhythmic risk.

Micro-electrode arrays in cardiac safety pharmacology: a novel tool to study QT interval prolongation.

Drug-induced QT interval prolongation is now a major concern in safety pharmacology. Regulatory authorities such as the US FDA and the European Medicines Agency require in vitro testing of all drug candidates against the potential risk for QT interval prolongation prior to clinical trials. Common in vitro methods include organ models (Langendorff heart), conventional electrophysiology on cardiac myocytes, and heterologous expression systems of human ether-a-go-go-related gene (hERG) channels. A novel approach is to study electrophysiological properties of cultured cardiac myocytes by micro-electrode arrays (MEA). This technology utilises multi channel recording from an array of embedded substrate-integrated extracellular electrodes using cardiac tissue from the ventricles of embryonic chickens. The detected field potentials allow a partial reconstruction of the shape and time course of the underlying action potential. In particular, the duration of action potentials of ventricular myocytes is closely related to the QT interval on an ECG. This novel technique was used to study reference substances with a reported QT interval prolonging effect. These substances were E4031, amiodarone, quinidine and sotalol. These substances show a significant prolongation of the field potential. However, verapamil, a typical 'false positive' when using the hERG assay does not cause any field potential prolongation using the MEA assay. Whereas the heterologous hERG assay limits cardiac repolarisation to just one channel, the MEA assay reflects the full range of mechanisms involved in cardiac action potential regulation. In summary, screening compounds in cardiac myocytes with the MEA technology against QT interval prolongation can overcome the problem of a single cell assay to potentially report 'false positives'.