Effect Of Drugs On HERG Potassium Channels Using Different Test Methodology

Background and objectiveThe human ether-a-go-go related gene?hERG?encodes the inward rectifying voltage gated potassium channel(I_Kr)in the heart,which is involved in cardiac repolarization.Inhibition of the hERG current causes QT interval prolongation resulting in Torsade de Pointes?TdP?.QT interval prolongation is one of the key indicators for new drug safety evaluation and research.In vitro evaluation of the effects of drugs on hERG potassium channel is a key step in the preclinical cardiac safety.The patch clamp technique is the gold standard for studying ion channel characteristics and drug safety testing,which is difficult and complex and requires pay more attention to details.So,only a few GLP institutions in China can carry out this assay under GLP conditions.In addition,the methodology has not been standardized,and different laboratories use different types of cell lines and voltage pulse protocols,so the results vary widely.Our objective was to study the effects of drugs that have high,intermediate and low torsade de pointes risk on the hERG potassium channel by using two voltage stimulation protocols in the GLP laboratory,to determine the detection conditions of different types of drugs.The results in this study can provide reference of hERG assay for domestic GLP laboratories,and then expand this methodology in China if possible.The test methodology should be standardized so as to reduce the differences among laboratories and improve the accuracy and reproducibility of data,which is good for evaluation of arrhythmia risk.At present,the most commonly cell used in hERG assay is mammalian cells that heterologously transfected with hERG potassium channels,which express only single hERG potassium channel and lack complex ion channels in the natural cardiomyocyte.Human induced pluripotent stem cells derived cardiomyocytes?hiPSC-CM?is a in vitro model that is closer to human cardiomyocytes,and hiPSC-CM would express human cardiac ion channels in a native environment,the use of them for cardiac safety assessment is expected to generate results that more accurately predict the relevant in vivo human response.Methods and ResultsPart ?:Using a step-ramp protocol to study the effect of drugs on hERG potassium channel expressed on HEK293 cells.To investigate the blocking effect of eleven clinical drugs that have high?bepridil,quinidine,sotalol,doftilide?,intermediate?ondansetron,cisapride,terfenadine?and low?ranolazine,verapamil,mexiletine and diltiazem?torsade de pointes?TdP?risk on hERG potassium channel.A step-ramp protocol was used to record the change in hERG potassium current(I_Kr)on HEK293 cells that stably expressed hERG potassium channel?hERG-HEK293 steady-state cells?,which was treated with four test concentrations of aforementioned drugs,to study the concentration-dependence and the time-dependence of the effects on I_Kr and the half maximal inhibitory concentration(IC₅₀).The results showed that 11 drugs inhibited the transfected hERG potassium channels expressed in HEK293 cells in a concentration-dependent manner.Some drugs?quinidine,sotalol,ondansetron,ranolazine,diltiazem and mexiletine?have faster kinetics with hERG potassium,and some drugs?bepridil,doftilide,cisapride,terfenadine and verapamil?have slower kinetics with hERG potassium.The IC₅₀0 values of bepridil,quinidine and dofetilide?high TdP risk clinical drugs?are about 98.32 nmol·L^-1,1.95?mol·L^-1 and 52.71 nmol·L^-1,respectively.,and the IC₅₀0 values of sotalol is>300?mol·L^-1;The IC₅₀0 values of ondansetron,cisapride,terfenadine?intermediate TdP risk clinical drugs?are about 0.94?mol·L^-1,39.10 nmol·L^-1,128.58 nmol·L^-1,respectively.The IC₅₀values of ranolazine,verapamil,diltiazem and mexiletine?low TdP risk clinical drugs?are about 9.94?mol·L^-1,235.49 nmol·L^-1,12.40?mol·L^-1 and 65.56?mol·L^-1,respectively.The IC₅₀ values for the 11 clinical drugs corresponded well to those published within the literature.Part ?:Using a step-pulse protocol to study the effect of drugs on hERG potassium channels expressed on HEK293 cells.In this part,nine of eleven clinical drugs in Part I were studied.To investigate the blocking effect of nine drugs that have high?bepridil,quinidine,sotalol?,intermediate?ondansetron,cisapride,terfenadine?and low?ranolazine,diltiazem and mexiletine?torsade de pointes risk on hERG potassium channel.A step-pulse protocol was used to record the change in hERG potassium current(I_Kr)on hERG-HEK293 steady-state cells,which was treated with four test concentrations of aforementioned drugs,to study the concentration-dependence of the effects on I_Kr and the half maximal inhibitory concentration(IC₅₀).The results showed that 9 drugs tested inhibited the transfected hERG potassium channels expressed in HEK293 cells in a concentration-dependent manner.The IC₅₀values of bepridil,quinidine and sotalol?high TdP risk clinical drugs?are about 92.16nmol·L^-1?0.88?mol·L^-1 and 278.65?mol·L^-1,respectively.The IC₅₀0 values of ondansetron,cisapride and terfenadine?intermediate TdP risk clinical drugs?are about0.57?mol·L^-1?37.82 nmol·L^-11 and 45.19 nmol·L^-1,respectively.The IC₅₀0 values of ranolazine,diltiazem and mexiletine?low TdP risk clinical drugs?are about 8.83?mol·L^-1?10.79?mol·L^-11 and 25.23?mol·L^-1,respectively.The IC₅₀ values for the 9 clinical drugs corresponded well to those published within the literature.Part ?:To investigate the effect of drugs on potassium channels on human induced pluripotent stem cells derived cardiomyocytesFirst,we established a method of recording potassium current in hiPSC-CM.We used the established method to record the changes of potassium current in hiPSC-CM by different concentration of bepridil,to study the concentration dependence,half-inhibitory concentration(IC₅₀)and voltage dependence of potassium current and then compare with HEK293.The results showed that bepridil inhibited the K⁺current in hiPSC-CM in a concentration-dependent and voltage-dependent manner,and the IC₅₀ value is about82.23 nmol·L^-1,which is slightly smaller than that of HEK293 cells.Conclusions1.The risk of TdP caused by clinical drugs is related to a variety of ion channels expressed on the heart,which is closely related to the block of hERG channels,but some clinical drugs can reduce risk by blocking sodium channels and calcium channels.2.Different stimulation protocols may affect the effect of drugs on the hERG channel.Therefore,standardized methodology between different laboratories are very important for drug screening.3.Whether hiPSC-CM is more suitable than HEK293 cells that transfected with a single ion channel for evaluation of drug toxicity still needs further research and evaluate.