Reliable identification of cardiac liability in drug discovery using automated patch clamp: Experimental and technical considerations for high throughput recordings of NaV1.5 and hERG


M G Rotordam1A Obergrussberger1; N Brinkwirth1; K Takasuna2; N Becker1; A Horváth1; T A Goetze1; M Rapedius1; H Furukawa2; Y Hasegawa2; T Oka2; N Fertig1; S Stoelzle-Feix1
1 Nanion Technologies GmbH, Germany;  2 Axcelead Drug Discovery Partners, Inc., Japan


The aim of the CiPA initiative is to improve specificity compared to in vitro hERG and in vivo QT studies. Automated patch clamp (APC) instruments are increasingly adopted for cardiac safety measurements on hERG, NaV1.5 and CaV1.2, thus requiring standardized experimental protocols and technical specifications, e.g. temperature control, accessibility of recording solutions to allow for sample collection or Liquid Junction Potential (LJP) correction for accurate voltage control. Here, we identified parameters influencing IC50 determination of compounds on hERG and NaV1.5 currents recorded using APC. For example, we found that although voltage protocol did not affect the IC50 of hERG compounds, both voltage protocol and holding potential could affect IC50 on NaV1.5 peak currents. Compounds ranolazine and tamoxifen, two blockers known to block NaV1.5 in the open state, showed more potent IC50s at a holding potential (HP) of -80 mV compared with -95 mV. Temperature could also influence IC50 values of compounds tested on NaV1.5 peak. Temperature affected the Vhalf of inactivation, shifting Vhalf of inactivation by ~7 mV to more negative potentials at 37°C vs room temperature (RT), and also affected the IC50 of mexiletine. Given the influence of temperature on NaV1.5 and hERG biophysics and pharmacology, it is critical to maintain a constant recording temperature of the system either by cooling where room temperature fluctuates, or heating to record at physiological temperature. Experimental parameters such as incubation time also influenced IC50 values for both hERG and NaV1.5 peak currents and whereas compounds such as bepridil reached steady state within approximately 5-6 mins, sticky compounds such as terfenadine required longer incubation times. In keeping with this, the open-well design of the patch clamp chip makes it possible to collect samples directly from the measurement site. This allows the direct measurement of actual compound concentration at the cell.

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