New cell models and assays in cardiac safety profiling.

Drug-induced prolongation of the QT interval in the electrocardiogram has been associated with life-threatening ventricular tachycardia of the Torsades de Pointes type. To prevent this risk to patients, all new drug entities must undergo thorough in vitro and preclinical in vivo testing. Because a hERG channel block is the primary reason for ventricular repolarisation, disturbances causing a QT interval prolongation, established in vitro test systems focus on the analysis of drug action on hERG channel function. More sophisticated assays study ventricular repolarisation directly with cardiac tissue preparations. In addition, in the future, novel biological models, such as stem-cell-derived cardiomyocytes and cardiac tissue slices, may allow the design of innovative assay systems to address relevant cardiac safety pharmacology parameters. In this review, established as well as innovative assays and cell models used in these assays are discussed.

The roboocyte: automated electrophysiology based on Xenopus oocytes.

Automated electrophysiological assays are of great importance for modern drug discovery, and various approaches have been developed into practical devices. Here, we describe the automation of two-electrode voltage-clamp (TEVC) recording from Xenopus oocytes using the Roboocyte automated workstation, jointly developed by Multi Channel Systems and Bayer Technology Services. We briefly discuss the technology, including its advantages and limitations relative to patch clamp and other TEVC systems. We provide a step-by-step description of typical operating procedures and show that the Roboocyte represents a practical and highly effective way to perform automated electrophysiology in an industrial setting.

QT-screen: high-throughput cardiac safety pharmacology by extracellular electrophysiology on primary cardiac myocytes.

Cardiac safety pharmacology focuses mostly on the drug-induced prolongation of the QT interval in the electrocardiogram. A prolonged QT interval is an important indicator for an increased risk of severe ventricular arrhythmia. Guidelines demand safety tests addressing QT prolongation in vitro and in vivo before a drug enters clinical trials. If safety risks will be detected not until an advanced stage of preclinical drug development, a considerable sum of money has already been invested into the drug development process. To prevent this, high-throughput systems have been developed to obtain information on the potential toxicity of a substance earlier. We will discuss in this publication that the QT-Screen system, which is based on primary cardiac myocytes, is able to provide a sufficient throughput for secondary screening. With this system, extracellular field potentials can be recorded from spontaneously beating cultures of mammalian or avian ventricular cardiac myocytes simultaneously on 96 channels. The system includes software-controlled and automated eight-channel liquid handling, data acquisition, and analysis. These features allow a user-friendly and unsupervised operation. The throughput is over 100 compounds in six replicates and with full dose-response relationships per day. This equals a maximum of approximately 6,000 data points per day at an average cost for consumables of 0.20 US pennies (U.S.) per data point. The system is intended for a non-good laboratory practice-compliant screening; however, it can be adapted to be used in a good laboratory practice environment.

Automated higher-throughput compound screening on ion channel targets based on the Xenopus laevis oocyte expression system.

As numerous diseases have been shown to be related to dysfunction of ion channels and neurotransmitter receptors and to affect regulatory pathways, ion channels have attracted increasing attention as a target class for drug discovery. The concomitant demand of the pharmaceutical industry for adequate electrophysiological methods to investigate drug effects on specific ion channels in secondary and safety screening has resulted in the development of electrophysiological instrumentation that allows automated monitoring of ion channel function with a higher throughput. Here we tested a fully automated screening system based on the Xenopus laevis oocyte expression system. We addressed the questions of data quality and reproducibility obtained by automated oocyte injection and two-electrode voltage-clamp (TEVC) recording using the Roboocyte (Multi Channel Systems GmbH, Reutlingen, Germany) technology compared to conventional oocyte recording. A gamma-aminobutyric acid (GABA)A-receptor subtype (alpha(1)beta(2)) was chosen as an example for a ligand-gated ion channel, and the slowly activating potassium current I(Ks) as a voltage-activated ion channel. Oocytes were injected with cDNA or cRNA via the Roboocyte injection stage. Ion channel currents were successfully recorded after 2-7 days in about 40% of the oocytes injected with GABA(A) receptor cDNA, and after 2-4 days in about 60% of the oocytes injected with KCNE1 cRNA. EC(50) values for the GABA(A) receptor and IC(50) values for blockers of I(Ks) were comparable to values obtained with conventional TEVC recording techniques. In conclusion, our results show that the Roboocyte is a valuable automated tool for oocyte injection and TEVC recording that can be used in drug screening and target validation to enhance the number of compounds and oocytes tested per day.