Kirsch Glenn E, Trepakova Elena S, Brimecombe Jessica C, Sidach Serguei S, Erickson Hope D, Kochan Mary C, Shyjka Lisa M, Lacerda Antonio E, Brown Arthur M
ChanTest, Inc., 14656 Neo Parkway Cleveland, OH 44128, USA.
J Pharmacol Toxicol Methods. 2004 Sep-Oct;50(2):93-101. doi: 10.1016/j.vascn.2004.06.003.
In vitro evaluation of drug effects on hERG K(+) channels is a valuable tool for identifying potential proarrhythmic side effects in drug safety testing. Patch-clamp recording of hERG K(+) current in mammalian cells can accurately evaluate drug effects, but the methodology has not been standardized, and results vary widely. Our objective was to evaluate two potential sources of variability: the temperature at which recordings are performed and the voltage pulse protocol used to activate hERG K(+) channels expressed in HEK293 cells.
A panel of 15 drugs that spanned a broad range of potency for hERG inhibition and pharmacological class was evaluated at both room and near-physiological temperatures using several patch-clamp voltage protocols. Concentration-response analysis was performed with three stimulus protocols: 0.5- and 2-s step pulses, or a step-ramp pattern.
Block by 2 of the 15 drugs tested, d,l-sotalol (antiarrhythmic) and erythromycin (antibiotic), was markedly temperature sensitive. hERG inhibition measured using a 2-s step-pulse protocol underestimated erythromycin potency compared with results obtained with a step-ramp protocol. Using conservative acceptance criteria and the step-ramp protocol, the IC(50) values for hERG block differed by less than twofold for 15 drugs.
Data obtained at near-physiological temperatures using a step-ramp pattern are highly repeatable and provide a conservative safety evaluation of hERG inhibition.
在药物安全性测试中，体外评估药物对人乙醚相关基因（hERG）钾离子通道的影响是识别潜在致心律失常副作用的重要工具。通过膜片钳记录哺乳动物细胞中的hERG钾离子电流可以准确评估药物作用，但该方法尚未标准化，结果差异很大。我们的目的是评估两个潜在的变异性来源：记录时的温度以及用于激活HEK293细胞中表达的hERG钾离子通道的电压脉冲方案。
使用几种膜片钳电压方案，在室温和接近生理温度下评估了一组15种药物，这些药物对hERG抑制的效力和药理类别范围广泛。采用三种刺激方案进行浓度-反应分析：0.5秒和2秒的阶跃脉冲，或阶跃-斜坡模式。
所测试的15种药物中有2种，即d，l-索他洛尔（抗心律失常药）和红霉素（抗生素）的阻断作用明显对温度敏感。与使用阶跃-斜坡方案获得的结果相比，使用2秒阶跃脉冲方案测得的hERG抑制作用低估了红霉素的效力。使用保守的接受标准和阶跃-斜坡方案，15种药物的hERG阻断IC50值相差不到两倍。
使用阶跃-斜坡模式在接近生理温度下获得的数据具有高度可重复性，并为hERG抑制提供了保守的安全性评估。
