Investigation of in vitro IKr/hERG assays under physiological temperature conditions using the semi-automated patch-clamp system QPatch compact with temperature control system

Cardiac ion channel activity is crucial for generating cardiac action potentials with proper timing and duration. Drug-induced impairment of these ion channels can cause abnormal cardiac activity, including QT interval prolongation, ventricular arrhythmia, and, in the most severe cases, sudden death. These adverse effects are among the leading reasons for drug withdrawal from the market or the denial of regulatory approval for new therapeutic candidates. The ICH E14/S7B Q&A released in August 2022 provided recommended conditions for best practices for in vitro assay of IKr/hERG to maintain reproducibility and consistency in evaluations. These recommendations include testing under physiological temperature conditions, as well as considering factors such as voltage protocols. In this study, we have investigated whole-cell patch-clamp measurements of hERG currents under physiological temperature conditions (36–37 °C) using the semi-automated patch-clamp system QPatch compact using the recommended best practices. Whole-cell patch-clamp recordings in hERG channel-expressing cells were performed using the QPatch Compact automated patch-clamp system with a temperature control system and the voltage protocol recommended by the CiPA project. Compared to room temperature conditions, the rise time of the hERG current was shorter and its amplitude larger under physiological temperature conditions. The tail current decay rate was also slower. The overall duration of the current was prolonged. These findings imply that temperature influences the dynamics of hERG channels, providing a more accurate reproduction of their physiological function. Furthermore, when testing the temperature-dependent effects of erythromycin, the current inhibition rate at the highest applied concentration of 1000 μM was around 50 % under room temperature conditions (25 °C). In contrast, under physiological temperature conditions, the IC50 was approximately 60 μM, and a nearly complete blockade of hERG currents was achieved at 1000 μM. This result confirms that the inhibitory effect of erythromycin is more pronounced under physiological temperature conditions. Additionally, we have tested other reference compounds, such as dofetilide, ondansetron, and moxifloxacin, to assess their temperature sensitivity. These insights are expected to improve our understanding of the influence of temperature on drug effects and enhance the reliability of testing protocols in accordance with ICH guidelines.