The Drugs Safety Evaluation On The Cardiac Repolarization: An In Vitro Study Based On The HERG Gene Expression Model

Drug-induced QT interval prolongation has become an important public health problem.And Drug Safety Authouities and Drug companies pay more and more attention to the drug cardiac toxicity. Many drugs induced QT interval prolongation by directly blocked the the rapidly activating delayed rectifier potassium current (ⅠKr).Besides, other drugs can inhibit potassium channel protein expression, which indirect reduceⅠKr.Indeed, many drugs have been withdrawn from the market by FDA because they lead to long QT syndrome and ventricular arrhythmias. Regulatory guidelines [CPMP/986/96 (1997) and ICH S7B (2005)] recommend conducting preclinical in vivo and in vitro studies to detect the QT-prolonging effects and arrhythmogenic potential of new drugs.The investigation ofⅠKr has become an indicator for whether the drug can cause QT interval prolongation and proarrhythmia. The present study is to build up an in vitro method to detect the QT-prolonging effects base on ion channel currents and protein expression, and then evaluate drugs effects. The study is divided into three parts.Part 1. To build up a HERG-HEK293 cell model by transfected of HERG gene into HEK293, and build up a stably transfected HERG-HEK 293 cells line.Part 2. To evaluate the electrophysiogical effects of cisapride on the rapidly activating delayed rectifier potassium current (ⅠKr) encoded by the HERG gene with a whole-cell patch clamp technique, and study the effects of cisapride on the HERG protein trafficking by Western blot analysis.Part 3. To investigate the effects of Guanfu base A (GFA) on the amplitude, concentration and voltage dependence, and the kinetics of HERG current. An HERG mutant at this position F656C, was also evaluated in this study. The HERG current was measured by the whole-cell patch clamp method. The effects of GFA on HERG protein trafficking were investigated by Western blot analysis. Part 1. The transfection of HERG gene into HEK293 and the certification of HERG channel currents and proteinObjective:To set up the HERG-HEK293 cell line by transfected HERG gene into HEK293 cells, which can express the the rapidly activating delayed-rectifier K+ current (ⅠKr) and HERG protein.Methods:The HEK293 cells were transient transfected by the pcDNA3.0-HERG gene and green fluorescin protein (GFP) using Lipofectamine 2000, and theⅠKr was measured by whole cell patch clamp techniques. Then screen by G418 to achieve stable transfection, and the HERG channel protein was detected by the Western blot analysis.Results:Lipofectamine 2000 can transfected the pcDNA3.0-HERG gene inth HEK293 cells and the transient transfection efficiency was between 30-70%. Patch clamp study confirmed that HERG gene expressedⅠKr. G418 can be successfully screened HERG-HEK293 positive clone cells, making the cells stable expressed HERG protein and can be used for Western blot study Conclusions:Both the patch clamp study and Western blot analysis prove that this is an effective method for transfecting the HERG gene into HEK293 and expressing the HERG channel currents and protein.Part 2. The effects of cisapride on the HERG K+ channel current and protein traffickingObjective To evaluate the electrophysiogical effects of cisapride on the rapidly activating delayed rectifier potassium current (ⅠKr) encoded by the HERG gene with a whole-cell patch clamp technique, and study the effects of cisapride on the HERG protein trafficking by Western blot analysis. Methods Wild-type HERG cDNA plasmids were transfected into human embryonic kidney (HEK293) cells by lipofectamine method. The whole-cell patch clamp method was used to record the IKr and kinesics of channel gating. Used G418 to build up a stably transfected HERG-HEK 293 cells line, and studied the effects of cisapride on the protein expression.Results Cisapride produced a concentration-and voltage-dependent blockage of theⅠHERG andⅠtail with IC50 (50% inhibitory concentration) of 14.5 and 3.9 nmol/L, respectively. Cisapride degraded peak current potential ofⅠHERG andⅠtail without altered activation threshold potential. The activation curve was shifted in a negative direction and accelerated channel inactivation by cisapride, but the time constants of recovery from inactivation were not significant change. Cisapride did not present significant influence on the IKr channel protein trafficking.Conclusion Cisapride blocked HERG K+ channel via inactivated and open state, not influenced on the protein trafficking, which was the mechanism of drug inducing proarrhythmia.Part 3. The effect of the novel anti-arrhythmia Guanfu base A on HERG K+ channel current and proteinObjective To evaluate the electrophysiogical effects of Guanfu base A (GFA) on the rapid delayed rectifier potassium current (ⅠKr) encoded by the HERG gene using patch clamp whole cell recording techniques, and study the effects of acehytisine hydrochloride on the HERG protein trafficking.Methods Wild-type HERG cDNA plasmids were transfected into human embryonic kidney(HEK293) cells by lipofectamine method. The whole cell patch clamp method was used to record theⅠKr. Using Site-directed mutagenesis techniques to make a F656C mutation of HERG gene, and then study the effects of Guanfu base A on theⅠKr by patch clamp. The Western blot analysis was used to study the effects of Guanfu base A on the protein expression.Results1. GFA produced a concentration-dependent and voltage-dependent blockade of theⅠHERG with an IC50 (50% inhibitory concentration) of 465.95μmol/L. GFA (400 and 1000μmol/L) degraded peak current potential ofⅠHERG without altered activation threshold potential. GFA (25,100,400μmol/L) presented no significant inhibition effects ong theⅠtail, wheres, GFA at concentration of 1,2.5 and 5mmol/L inhibitedⅠtaii by 34.9%,58% and 70.5%, respectively. The IC50 forⅠtail was 1.64mmol/L. GFA (lmmol/L) produced voltage-dependent blockage of theⅠtail, degraded degraded peak current potential ofⅠtail without altered activation threshold potential. GFA showed no time-dependent blockade of HERG currents.2. The activation curve was shifted in a negative direction and accelerated channel inactivation by GFA. Moreover, GFA slowed channel recovery from inactivation at above-60mV without no significant effects on the deactivation time constants.3. GFA produced a concentration-dependent blockage of the F656C mutation HERG currents, including theⅠHERG andⅠtail.The activation curve was shifted in a negative direction by GFA. However, the F656C mutation in the S6 domain attenuated acehytisine hydrochloride inhibition of HERG current.4. High concentration of GFA inhibited HERG K+channel protein trafficking, and F656C mutation enhanced the inhibition of protein trafficking.Conclusion 25-400μmol/L GFA had little inhibitory effects onⅠKr current. High concentration of GFA blocked the HERG currents. GFA blocked HERG channel via inactivated and activated states. GFA inhibited HERG K+ channel current via S6 domain. In addtition, acehytisine hydrochloride didn't inhibit protein synthesis, but could inhibit protein trafficking at high concentration.