Reliable identification of cardiac liability in drug discovery using automated patch clamp: Benchmarking best practices and calibration standards for improved proarrhythmic assessment
Reliable identification of cardiac liability in drug discovery using automated patch clamp: Benchmarking best practices and calibration standards for improved proarrhythmic assessment
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DOI：
10.1016/j.vascn.2020.106884
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发表时间：
2020-09-01
影响因子：
1.9
通讯作者：
Stoelzle-Feix, Sonja
中科院分区：
文献类型：
--
作者：
Brinkwirth, Nina;Takasuna, Kiyoshi;Stoelzle-Feix, Sonja
引言：使用膜片钳筛选化合物对hERG通道的活性是安全性试验的关键部分。自动膜片钳（APC）作为手动膜片钳的替代方法正在被广泛接受，以提高通量，同时保持数据质量。为了使APC实验标准化，我们研究了在不同条件下使用跨多个站点的几种设备对IC 50值的影响。方法：使用APC仪器SyncroPatch 384i、SyncroPatch 384PE和Patchliner记录HEK或CHO细胞中表达的hERG。结果：在SyncroPatch 384 PE上记录的21种化合物的所有IC 50值与来自文献的IC 50值良好相关（克雷默等人，2013），无论电压方案或实验室器皿，当化合物在制备后立即使用时，但是如果在实验前2 - 3小时在Teflon或聚丙烯（PP）化合物板中制备，则阿司咪唑的效力降低。缓慢作用的化合物，如多非利特，阿司咪唑，和特非那定需要至少6分钟的延长孵育时间，以达到稳态，因此，稳定的IC50 values.Discussion：评估不同的实验条件对hERG测定可靠性的影响，我们得出结论，无论是由CiPA推荐的阶梯斜坡协议或标准的2秒阶梯脉冲协议可以用来记录hERG;应使用5 min的最小孵育时间，尽管玻璃、Teflon、PP或聚苯乙烯（PS）化合物板可用于实验，但如果使用Teflon、PS或PP容器，则应小心，因为如果制备后不立即进行实验，可能会发生一些吸附。我们的建议不仅限于本报告中描述的APC设备，还可以扩展到其他APC设备。
Introduction: Screening compounds for activity on the hERG channel using patch clamp is a crucial part of safety testing. Automated patch clamp (APC) is becoming widely accepted as an alternative to manual patch clamp in order to increase throughput whilst maintaining data quality. In order to standardize APC experiments, we have investigated the effects on IC50 values under different conditions using several devices across multiple sites.Methods: APC instruments SyncroPatch 384i, SyncroPatch 384PE and Patchliner, were used to record hERG expressed in HEK or CHO cells. Up to 27 CiPA compounds were used to investigate effects of voltage protocol, incubation time, labware and time between compound preparation and experiment on IC50 values.Results: All IC50 values of 21 compounds recorded on the SyncroPatch 384PE correlated well with IC50 values from the literature (Kramer et al., 2013) regardless of voltage protocol or labware, when compounds were used immediately after preparation, but potency of astemizole decreased if prepared in Teflon or polypropylene (PP) compound plates 2-3 h prior to experiments. Slow acting compounds such as dofetilide, astemizole, and terfenadine required extended incubation times of at least 6 min to reach steady state and therefore, stable IC50 values.Discussion: Assessing the influence of different experimental conditions on hERG assay reliability, we conclude that either the step-ramp protocol recommended by CiPA or a standard 2-s step-pulse protocol can be used to record hERG; a minimum incubation time of 5 min should be used and although glass, Teflon, PP or polystyrene (PS) compound plates can be used for experiments, caution should be taken if using Teflon, PS or PP vessels as some adsorption can occur if experiments are not performed immediately after preparation. Our recommendations are not limited to the APC devices described in this report, but could also be extended to other APC devices.