Inhibition of hERG K channels by verapamil at physiological temperature: Implications for the CiPA Initiative

Abstract

The Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative reassesses using the inhibition of hERG channels by drugs as the major determinant for assessment of a drugs potential to cause Torsades de Pointes (TdP) cardiac arrhythmias. Here we report on one phase of CiPA: independent determination of hERG inhibitory properties in order to test the reproducibility of data gathered in different labs and the practicality of using the CiPA-defined recording conditions. We measured inhibition of hERG1a potassium channels stably expressed in HEK293 cells using manual whole-cell patch-clamp electrophysiology recordings at the physiological temperature of 37{degrees}C by verapamil, a known hERG inhibitor. We report that the IC50 for inhibition (180.4nM) and the Hill coefficient (1.4) for verapamil measured in an academic lab were similar to the IC50 (204nM) and Hill coefficient (0.9) measured at the Food and Drug Administration labs, indicating low variability in measurements between labs. We report that the CiPA step voltage protocol, a series of voltage steps characterized by 10 second duration depolarizing pulses at 37{degrees}C, resulted in a very low (5%) experimental success rate per cell in our hands. To circumvent the low success rate, we shortened the duration of the depolarizing pulse to 3 seconds (from 10 seconds) and shortened the duration of interpulse intervals, which is more consistent with the duration of voltage pulses used in biophysical studies of hERG, such that the entire revised protocol was shortened from over 30 minutes to approximately 10 minutes in duration. Using the revised protocol, we found an IC50 of inhibition by verapamil of 210.5 nM and Hill coefficient of 1.2. These values were similar to those generated using the original, longer CiPA step protocol. Furthermore, our success rate using the shortened protocol rose to 25%, an increase of 5-fold over the initial protocol. In summary, we captured key pharmacological data for subsequent analysis in CiPA using a revised, shorter protocol with an enhanced success rate and an overall enhanced feasibility. We propose the shorter protocol is more pragmatic for generation of hERG channel drug inhibition data for CiPA and other regulatory sciences.