Establishment And Application Of HERG Evaluation Model In Vitro For Drug-induced QT Interval Prolongation

Many drugs in clinical use can cause cardiac QT interval prolongation and lead to the development of severe ventricular arrhythmia, torsade de pointes. In the last decade, it has become the number one safety issue in the development of pharmaceuticals, superseding liver injury in being the primary cause of drug withdrawals. The electrocardiographic QT interval represents the duration of the ventricular action potential (APD) plus the time associated with transmission across the myocardium at the cellular level. Cardiac APD is controlled importantly by the repolarization of the membrane potential. The rapidly activating delayed rectifier potassium current (IKr) is the major outward K+ current in the repolarization phase of the action potential. The HERG (human ether-a-go-go-related gene) is now widely accepted as encoding theα-subunit of IKr and the HERG product was found to be a functional K+ channel with properties resembling those of IKr. Almost all drugs that induce TdP and cause QT interval prolongation in humans block IKr/HERG channel more or less. In 2005, the ICH S7B (International Conference on Harmonization) clearly state the need for an in vitro IKr/HERG assay to identify the potential of a test substance to delay ventricular repolarization. Therefore, in vitro HERG assays with patch-clamp electrophysiology form a key element of an integrated assessment of TdP liability.Currently, HERG assays form an important component of early pre-clinical screens and cardiac safety-pharmacology during drug development. However, the relevant testing is not widely carried out in our country, especially there is little background data concerning about Chinese herbal medicines. In recent years, cardiovascular adverse effects of various herbs have been widely reported. Based on the above situation, the establishment of HERG evaluation model in vitro for drug-induced QT interval prolongation, which can be used to assess the cardiac toxicity and study the mechanism of traditional Chinese medicine (TCM), is of important theoretical and practical significance. Therefore, the present study was designed to establish HERG ion channel evaluation model in vitro with patch clamp technique, choose Cyclovirobuxine D and Dauricine as the representative TCM with significant cardiovascular pharmacological activity, and assess their effect and mechanism of inhibition.First, the HERG evaluation model was established base on whole-cell patch clamp recording technique. Using primary cultured cardiomyocytes, after the formation of whole-cell mode, we recorded the voltage-gated sodium, potassium and calcium current. By recombinant HERG channels stably expressed in human embryonic kidney cells (HEK-293), HERG current was recorded. The HERG current blockade by drug can be directly observed with this model. The current-voltage relationship for HERG current exhibited the characteristic bell-shape that increased from -40 mV to +10 mV and decreased from +10 mV to +40 mV because of fast C-type inactivation. The tail currents increased with voltage and then plateaued at test potential positive to +10 mV. These features are similar to HERG channel current in the literature. Moreover, the sensitivity of our transfected HERG channels to the positive drug (E-4031 and terfenadine) was studied. E-4031 and terfenadine significantly inhibited HERG current in a concentration-dependent manner, IC50 values (concentration at 50% of maximal inhibition) were 0.042μM and 0.038μM respectively, and the results are consistent with the previous studies. The electrophysiological and pharmacological properties confirm that the currents are IKr currents, indicating that the HERG evaluation model has been successfully established.Secondly, this study examined the HERG current blockade by Cyclovirobuxine D and Dauricine using the established HERG evaluation model. Amongst early cardiac safety screens, peak tail amplitudes were used to quantify the degree of HERG current inhibition. If the IC50 less than 30μM, it suggest that the drug may have the potential to induce QT interval prolongation and need further in vivo QT assays. We found that Cyclovirobuxine D inhibited HERG current in a concentration-dependent manner with an IC50 of 19.7μM, the blockade reached a nearly steady-state level within 5~6 min, the depressant effect was partially reversible by washing with drug free solution, indicating that Cyclovirobuxine D has the potential to induce APD prolongation, which is probably one of the pro-arrhythmic mechanisms of Cyclovirobuxine D in overdose. Dauricine directly inhibited HERG current in a concentration-dependent manner with an IC50 of 3.5μM, the blockade occurred rapidly to reach a steady-state level within 1~3 min, the depressant effect was partially reversible upon washout. Because Dauricine inhibits a variety of other ion channels, the effect would result in a lengthening of the refractory period and a decrease of cardiac excitability, thus contributing to its anti-arrhythmic effects.Finally, the possible mechanisms of HERG current inhibition by Cyclovirobuxine D and Dauricine were investigated. We used some sophisticated electrophysiological protocols to study the voltage, time, channel kinetics, channel state and gating properties. Cyclovirobuxine D reduced the step current at some potentials and tail current between -10 mV and +60 mV, reduced instantaneous currents between -50 mV and +20 mV, the fully activated current showed the reversal potential was not changed. Using the 10 s voltage pulse and envelope voltage protocol, the blockade by Cyclovirobuxine D did show time-dependence, with blockade developing over the first 400~500 ms of the pulse. The inhibition was relieved by depolarization to +80 mV that favored HERG channel inactivation. These findings suggested that Cyclovirobuxine D inhibit HERG channels in the open states. Cyclovirobuxine D had no effect on HERG current kinetics (activation, onset of inactivation, recovery from inactivation and steady-state inactivation). Dauricine reduced the step current between -10 mV and +40 mV and tail current between -10 mV and +60 mV, the inhibition was stronger at positive potentials between +20 mV and +60 mV than at potentials between -20 mV to +10 mV, indicating voltage-dependence. Dauricine reduced instantaneous currents between -50 mV and +20 mV, reduced the fully activated current from -100 mV to +20 mV. The reversal potential changed from -75 mV to -65 mV, suggestive of an alteration to ion selectivity. Using the 10 s voltage pulse and envelope voltage protocol, the inhibition by Dauricine developed progressively over the first ~4 s of the pulse and showed a time-dependence. Dauricine produced a leftward shift in activation curves, the values of half-maximal activation voltage (V1/2) were -14.85±0.2 mV before and -23.13±1.1 mV after. The onset of inactivation was accelerated in the presence of Dauricine, at -40 mV, from 14.1±0.7 ms of control to 9.1±0.5 ms. Dau decreased the time constant of recovery from inactivation, at -50 mV, from 10.1±0.5 ms of control to 8.1±0.5 ms. We found depolarization to +80 mV further increased fractional block. These findings suggested that Dauricine inhibited HERG current in the open and inactivated states.Based on the results of this study, the following conclusions could be made under the present experimental condition:①HERG evaluation model in vitro for drug-induced QT interval prolongation has been successfully established. The electrophysiological and pharmacological properties confirm that the currents are IKr.②Cyclovirobuxine D and Dauricine inhibited HERG current in a concentration-dependent manner, indicating that they have the potential to induce APD prolongation.③HERG current blockade by Cyclovirobuxine D required channel activation, suggesting an open state block.④ Development of block by Dauricine was fast. It inhibited HERG current in the open and inactivated states.⑤Taking IC50 and Cmax into account, Cyclovirobuxine D might have a less powerful effect on QT interval prolongation within its therapeutic dose range, but in patients receiving Dauricine overdose or co-treatment with other QT interval-prolonging drugs, the inhibition by Dauricine is of greater concern.⑥HERG evaluation model can be used directly to evaluate the inhibitory effect of drugs. HERG assays form an important component of early cardiac toxicity screens and drug-safety evaluation.