Johnson Ashley A, Trudeau Matthew C
Department of Physiology, University of Maryland School of Medicine, Baltimore, MD 21201, United States of America.
Department of Physiology, University of Maryland School of Medicine, Baltimore, MD 21201, United States of America.
J Pharmacol Toxicol Methods. 2024 Nov-Dec;130:107562. doi: 10.1016/j.vascn.2024.107562. Epub 2024 Sep 26.
The Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative reassesses using the inhibition of hERG potassium channels by drugs as the major determinant for the potential to cause drug-induced Torsades de Pointes (TdP) cardiac arrhythmias. Here we report our findings on the next phase of CiPA: Determination of hERG inhibitory properties using the standard CiPA-defined data acquisition protocol, here called the standard protocol, at physiological temperature (37 degrees Celsius). To do this, we measured inhibition of hERG1a potassium channels stably expressed in HEK293 cells by the small molecule verapamil, using manual whole-cell patch-clamp electrophysiology recordings with the standard protocol, which is characterized, in part, by a series of 10 s duration voltage steps to 0 mV, ultimately leading to a cumulative recording time of approximately 30 min. Using the standard protocol, we measured an IC for verapamil of 225 nM, a Hill coefficient of 1, and time constant of inhibition at 0 mV of 0.64 s. But, using the standard protocol resulted in a very low (5 %) experimental success rate per cell, which had low practicality for future experiments. To address the 5 % success rate, we generated a revised protocol characterized, in part, by a series of 3 s duration voltage steps to 0 mV, leading to a cumulative recording time of approximately 10 min. Using the revised protocol, we found an IC for verapamil of 252 nM, a Hill coefficient of 0.8, and time constant of inhibition at 0 mV of 0.67 s. The values measured with the revised protocol were similar to those measured using the standard protocol and, furthermore, our success rate using the revised protocol rose to 25 %, an increase of 5-fold over the standard protocol, and more in line with the success rate for biophysical studies. In summary, we captured key pharmacological data for subsequent analysis in CiPA using a revised protocol with an increased success rate and an overall enhanced feasibility and practicality. We propose that the revised protocol may be more pragmatic for generation of some hERG channel drug inhibition data for CiPA and other regulatory sciences.
综合体外致心律失常试验（CiPA）计划重新评估将药物对人醚 - 去极化相关基因（hERG）钾通道的抑制作用作为药物诱发尖端扭转型室性心动过速（TdP）心律失常可能性的主要决定因素。在此，我们报告CiPA下一阶段的研究结果：在生理温度（37摄氏度）下，使用标准CiPA定义的数据采集方案（此处称为标准方案）测定hERG抑制特性。为此，我们使用标准方案通过手动全细胞膜片钳电生理记录，测量小分子维拉帕米对稳定表达于HEK293细胞中的hERG1a钾通道的抑制作用，该标准方案的部分特征是一系列持续10秒的电压阶跃至0 mV，最终累积记录时间约为30分钟。使用标准方案，我们测得维拉帕米的半数抑制浓度（IC）为225 nM，希尔系数为1，在0 mV时的抑制时间常数为0.64秒。但是，使用标准方案导致每个细胞的实验成功率非常低（5%），这对未来实验的实用性很低。为了解决5%的成功率问题，我们制定了一个修订方案，其部分特征是一系列持续3秒的电压阶跃至0 mV，导致累积记录时间约为10分钟。使用修订方案，我们发现维拉帕米的IC为252 nM，希尔系数为0.8，在0 mV时的抑制时间常数为0.67秒。用修订方案测得的值与使用标准方案测得的值相似，此外，我们使用修订方案的成功率提高到了25%，比标准方案增加了5倍，更符合生物物理研究的成功率。总之，我们使用修订方案捕获了关键药理学数据，用于CiPA后续分析，该方案成功率提高，整体可行性和实用性增强。我们建议，修订方案对于为CiPA和其他监管科学生成一些hERG通道药物抑制数据可能更实用。
