Utility of Normalized TdP Score System in Drug Proarrhythmic Potential Assessment: A Blinded in vitro Study of CiPA Drugs.

Drugs that prolong QT may cause torsade de pointes (TdP). However, translation of nonclinical assessment of QT prolongation or hERG channel, targeted by QT-prolonging drugs, into clinical TdP risk has been insufficient to date. In this blinded study, we confirmed the utility of a Normalized TdP Score System in predicting drug-induced TdP risks among 34 drugs, including 28 with low, intermediate, and high TdP risks under the Comprehensive In Vitro Proarrhythmia Assay (CiPA) initiative plus six compounds with names blinded to the investigators, using the rabbit ventricular wedge assay. Concentration-dependent TdP scores were determined by drug-induced changes in QT, Tp-e , and proarrhythmias. Disclosure of the names and testing concentrations was made after completion of the experiments and report to the sponsors. Drugs' normalized TdP scores were calculated thereafter based on their respective free clinical maximum concentration (Cmax ). Drugs' normalized TdP scores were calculated and ranked for 33 drugs, excluding 1 investigational drug, and the TdP risks of the 28 CiPA drugs were correctly distinguished according to their respective categories of low, intermediate, and high TdP risks under the CiPA initiative. Accordingly, we are able to propose the cutoff values of the normalized TdP scores at 1 × Cmax : ≤ 0, > 0 to < 0.65 and ≥ 0.65, respectively, for low, intermediate, and high risk. This blinded study supports utility of our Normalized TdP Score System in predicting drug-induced TdP risks in 33 drugs, including 28 used for characterization of other assays under the CiPA initiative. However, these results need to be replicated in other laboratories.

Wenxin Keli Regulates Mitochondrial Oxidative Stress and Homeostasis and Improves Atrial Remodeling in Diabetic Rats.

Mitochondrial dysfunction and oxidative stress play an important role in the pathogenesis of both atrial fibrillation (AF) and diabetes mellitus (DM). Wenxin Keli (WXKL), an antiarrhythmic traditional Chinese medicine, has been shown to prevent cardiac arrhythmias through modulation of cardiac ion channels. This study tested the hypothesis that WXKL can improve atrial remodeling in diabetic rats by restoring mitochondrial function. Primary atrial fibroblasts of neonatal SD rats were divided into four groups: control, hydrogen peroxide (H2O2), H2O2+WXKL 1 g/L, and H2O2+WXKL 3 g/L groups. Intracellular mitochondrial membrane potential (MMP), reactive oxygen species (ROS), and mitochondrial oxygen consumption were measured. SD male rats were randomly divided into three groups: control, DM, and DM+WXKL groups. Rats in the DM+WXKL group were treated with daily gavage of WXKL at 3 g/kg. After eight weeks, echocardiography, hemodynamic examination, histology, electrophysiology study, mitochondrial respiratory function, and western blots were assessed. H2O2 treatment led to increased ROS and decreased intracellular MMP and mitochondrial oxygen consumption in primary atrial fibroblasts. WXKL improved the above changes. DM rats showed increased atrial fibrosis, greater left atrial diameter, lower atrial conduction velocity, higher conduction heterogeneity, higher AF inducibility, and lower mitochondrial protein expression, and all these abnormal changes except for left atrial diameter were improved in the DM+WXKL group. WXKL improves atrial remodeling by regulating mitochondrial function and homeostasis and reducing mitochondrial ROS in diabetic rats.

Characterization and Management of Arrhythmic Events in Young Patients With Brugada Syndrome.

BACKGROUND: Information on young patients with Brugada syndrome (BrS) and arrhythmic events (AEs) is limited. OBJECTIVES: The purpose of this study was to describe their characteristics and management as well as risk factors for AE recurrence. METHODS: A total of 57 patients (age ≤20 years), all with BrS and AEs, were divided into pediatric (age ≤12 years; n = 26) and adolescents (age 13 to 20 years; n = 31). RESULTS: Patients' median age at time of first AE was 14 years, with a majority of males (74%), Caucasians (70%), and probands (79%) who presented as aborted cardiac arrest (84%). A significant proportion of patients (28%) exhibited fever-related AE. Family history of sudden cardiac death (SCD), prior syncope, spontaneous type 1 Brugada electrocardiogram (ECG), inducible ventricular fibrillation at electrophysiological study, and SCN5A mutations were present in 26%, 49%, 65%, 28%, and 58% of patients, respectively. The pediatric group differed from the adolescents, with a greater proportion of females, Caucasians, fever-related AEs, and spontaneous type-1 ECG. During follow-up, 68% of pediatric and 64% of adolescents had recurrent AE, with median time of 9.9 and 27.0 months, respectively. Approximately one-third of recurrent AEs occurred on quinidine therapy, and among the pediatric group, 60% of recurrent AEs were fever-related. Risk factors for recurrent AE included sinus node dysfunction, atrial arrhythmias, intraventricular conduction delay, or large S-wave on ECG lead I in the pediatric group and the presence of SCN5A mutation among adolescents. CONCLUSIONS: Young BrS patients with AE represent a very arrhythmogenic group. Current management after first arrhythmia episode is associated with high recurrence rate. Alternative therapies, besides defibrillator implantation, should be considered.

Wenxin Keli suppresses ventricular triggered arrhythmias via selective inhibition of late sodium current.

BACKGROUND: Wenxin Keli is a popular Chinese herb extract that approximately five million Asians are currently taking for the treatment of a variety of ventricular arrhythmias. However, its electrophysiological mechanisms remain poorly understood. METHODS AND RESULTS: The concentration-dependent electrophysiological effects of Wenxin Keli were evaluated in the isolated rabbit left ventricular myocytes and wedge preparation. Wenxin Keli selectively inhibited late sodium current (INa) with an IC50 of 3.8 ± 0.4 mg/mL, which was significantly lower than the IC50 of 10.6 ± 0.9 mg/mL (n = 6, P < 0.05) for the fast INa. Wenxin Keli produced a small but statistically significant QT prolongation at 0.3 mg/mL, but shortened the QT and Tp-e interval at concentrations ≥ 1 mg/mL. Wenxin Keli increased QRS duration by 10.1% from 34.8 ± 1.0 ms to 38.3 ± 1.1 ms (n = 6, P < 0.01) at 3 mg/mL at a basic cycle length of 2,000 ms. However, its effect on the QRS duration exhibited weak use-dependency, that is, QRS remained less changed at increased pacing rates than other classic sodium channel blockers, such as flecainide, quinidine, and lidocaine. On the other hand, Wenxin Keli at 1-3 mg/mL markedly reduced dofetilide-induced QT and Tp-e prolongation by attenuation of its reverse use-dependence and abolished dofetilide-induced early afterdepolarization (EAD) in four of four left ventricular wedge preparations. It also suppressed digoxin-induced delayed after depolarization (DAD) and ventricular tachycardias without changing the positive staircase pattern in contractility at 1-3 mg/mL in a separate experimental series (four of four). CONCLUSIONS: Wenxin Keli suppressed EADs, DADs, and triggered ventricular arrhythmias via selective inhibition of late INa.

Differentiating electrophysiological effects and cardiac safety of drugs based on the electrocardiogram: a blinded validation.

BACKGROUND: The ventricular components (QRS and QT) on the electrocardiogram (ECG) depend on the properties of ventricular action potentials that can be modulated by drugs via specific ion channels. However, the correlation of ECG ventricular waveforms with underlying ion actions is not well established and has been extensively debated. OBJECTIVE: To conduct a blinded in vitro assessment of the ionic mechanisms for drug-induced ECG changes. METHODS AND RESULTS: Fourteen cardiac and noncardiac drugs with known effects on cardiac ion channels were selected by the study sponsor, and were tested in the rabbit left ventricular wedge preparation with recording of the ECG and contractility. The investigators who performed the experiments and analyzed the data were blinded to names, concentrations, and molecular weights of the drugs. The compounds were prepared by the sponsor and sent to the investigators as 56 stock solutions. The effects of I(Kr), I(Ks), I(Ca,L), I(Na) blocker, and I(KATP) opener on QRS, QT, and T(p-e), were evaluated. Disclosure of the names and concentrations after completion of the study revealed that there were highly correlated ECG changes with underlying ionic mechanisms and proarrhythmic potential of drugs that, respectively, target I(Kr), I(Ks), I(Ca,L), I(Na), and I(KATP). Among ECG parameters, T(p-e) was more useful in differentiating drugs' actions. CONCLUSIONS: Specific electrophysiological action and the consequent proarrhythmic potential of a drug can be accurately determined by analysis of drug-induced changes in ECG in the rabbit left ventricular wedge preparation. Change in T(p-e) provides the most relevant information.

Electrophysiological properties of HBI-3000: a new antiarrhythmic agent with multiple-channel blocking properties in human ventricular myocytes.

HBI-3000 (sulcardine sulfate) has been shown to suppress various ventricular arrhythmias in animal models. The electrophysiological properties of HBI-3000 were investigated using standard microelectrode and patch-clamp techniques in single human ventricular myocytes. HBI-3000 led to concentration-dependent suppression of dofetilide-induced early afterdepolarizations in single nonfailing human ventricular myocytes and early afterdepolarizations seen in failing ventricular myocytes. The concentration-dependent prolongation of action potential duration (APD) by HBI-3000 was bell shaped with maximum response occurring around 10 µM. Interestingly, HBI-3000 at the concentration of 10 µM modestly prolonged the APD at all 3 basic cycle lengths. The slope of APD-cycle length curve of HBI-3000 was only slightly steeper than that of control (88.8 ± 7.7 ms/s vs. 78.9 ± 5.2 ms/s in control, n = 8, P > 0.05). HBI-3000 only showed a minimal use-dependent prolongation of the APD in human ventricular myocytes. HBI-3000 inhibited fast sodium current (INa-F), late sodium channel (INa-L), L-type calcium current (ICa-L), and rapidly activating delayed rectifier K current (IKr) in single human ventricular myocytes. The estimated half-maximal inhibitory concentration values of INa-F, INa-L, ICa-L, and IKr were 48.3 ± 3.8, 16.5 ± 1.4, 32.2 ± 2.9, and 22.7 ± 2.5 µM, respectively. The ion channel profile and electrophysiological properties of HBI-3000 are similar to those of ranolazine and chronic amiodarone (reduced INa-F, INa-L, ICa-L, and IKr). HBI-3000 may be a promising antiarrhythmic agent with low proarrhythmic risk.

Predicting drug-induced slowing of conduction and pro-arrhythmia: identifying the 'bad' sodium current blockers.

BACKGROUND AND PURPOSE: The regulatory guidelines (ICHS7B) for the identification of only drug-induced long QT and pro-arrhythmias have certain limitations. EXPERIMENTAL APPROACH: Conduction time (CT) was measured in isolated Purkinje fibres, left ventricular perfused wedges and perfused hearts from rabbits, and sodium current was measured in Chinese hamster ovary cells, transfected with Na(v)1.5 channels. KEY RESULTS: A total of 355 compounds were screened for their effects on CT: 32% of these compounds slowed conduction, 65% had no effect and 3% accelerated conduction. Lidocaine and flecainide, which slow conduction, were tested in more detail as reference compounds. In isolated Purkinje fibres, flecainide largely slowed conduction and markedly increased triangulation, while lidocaine slightly slowed conduction and did not produce significant triangulation. Also in isolated left ventricular wedge preparations, flecainide largely slowed conduction in a rate-dependent manner, and elicited ventricular tachycardia (VT). Lidocaine slightly slowed conduction, reduced Tp-Te and did not induce VT. Similarly in isolated hearts, flecainide markedly slowed conduction, increased Tp-Te and elicited VT or ventricular fibrillation (VF). The slowing of conduction and induction of VT/VF with flecainide was much more evident in a condition of ischaemia/reperfusion. Lidocaine abolished ischaemia/reperfusion-induced VT/VF. Flecainide blocked sodium current (I(Na)) preferentially in the activated state (i.e. open channel) with slow binding and dissociation rates in a use-dependent manner, and lidocaine weakly blocked I(Na). CONCLUSION AND IMPLICATIONS: Slowing conduction by blocking I(Na) could be potentially pro-arrhythmic. It is possible to differentiate between compounds with 'good' (lidocaine-like) and 'bad' (flecainide-like) I(Na) blocking activities in these models.

Vanoxerine: cellular mechanism of a new antiarrhythmic.

INTRODUCTION: There remains an unmet need for safe and effective antiarrhythmic drugs, especially for the treatment of atrial fibrillation. Vanoxerine is a drug that is free of adverse cardiac events in normal volunteers, yet is a potent blocker of the hERG (hK(v)11.1) cardiac potassium channel. Consequently,we hypothesized that vanoxerine might also be a potent blocker of cardiac calcium (Ca) and sodium (Na) currents, and would not affect transmural dispersion of repolarization. METHODS: The whole cell patch clamp technique was used to measure currents from cloned ion channels overexpressed in stable cell lines and single ventricular myocytes. We measured intracellular action potentials from canine ventricular wedges and Purkinje fibers using sharp microelectrode technique. RESULTS: We found that vanoxerine was a potent hK(v)11.1 blocker, and at submicromolar concentrations, it blocked Ca and Na currents in a strongly frequency-dependent manner. In the canine ventricular wedge preparation vanoxerine did not significantly affect transmural action potential waveforms, QT interval or transmural dispersion of repolarization. CONCLUSIONS: Vanoxerine (1) is a potent blocker of cardiac hERG, Na and Ca channels; (2) block is strongly frequency-dependent especially for Na and Ca channels; and (3) transmural dispersion of ventricular repolarization is unaffected. The multichannel block and repolarization uniformity resemble the effects of amiodarone, the exemplar atrial fibrillation drug. Vanoxerine is a completely different chemical and has none of amiodarone's toxic effects. Vanoxerine has characteristics of a potentially effective and safe antiarrhythmic.

Preclinical assessment of drug-induced proarrhythmias: role of the arterially perfused rabbit left ventricular wedge preparation.

Drug-induced torsade de pointes (TdP) is a rare but lethal side effect of many cardiovascular and non-cardiovascular drugs. It has led to black box warnings or even withdrawal of many useful compounds from the market and is one of the major stumbling blocks for new drug development. The critical need for a better test that can predict the TdP liability of a candidate drug has led to the development of multiple preclinical models. Each of these models has it own merits and limitations in preclinical testing for TdP liability; however, most of these models have not been adequately validated, so their precise sensitivity and specificity remain largely unknown. Recent blinded validation studies have demonstrated that the rabbit left ventricular wedge preparation can predict drug-induced TdP with an extremely high sensitivity and specificity. As a matter of fact, the wedge technique was initially developed primarily for studying the electrical heterogeneity of myocardium and the cellular basis of QT prolongation and TdP. Naturally then, the electrophysiological data obtained from the wedge takes into account every critical factor associated with the development of TdP. The TdP scores generated using the wedge technique have been shown to assess the torsadogenic potential of the drugs in a predictable fashion. This review elaborates on the current and prospective role of the rabbit left ventricular wedge preparation in preclinical assessment of drug-induced proarrhythmias including but not limited to TdP.

Blinded validation of the isolated arterially perfused rabbit ventricular wedge in preclinical assessment of drug-induced proarrhythmias.

BACKGROUND: The development of preclinical models with high predictive value for the identification of drugs with a proclivity to induce Torsade de Pointes (TdP) in the clinic has long been a pressing goal of academia, industry and regulatory agencies alike. The present study provides a blinded appraisal of drugs, in an isolated arterially-perfused rabbit ventricular wedge preparation, with and without the potential to produce TdP. METHODS AND RESULTS: Thirteen compounds were tested for their potential for TdP using the rabbit left ventricular wedges. All investigators were blinded to the names, concentrations and molecular weights of the drugs. The compounds were prepared by the study sponsor and sent to the investigator as 4 sets of 13 stock solutions with the order within each set being assigned by a random number generator. Each compound was scored semi-quantitatively for its relative potential for TdP based on its effect on ventricular repolarization measured as QT interval, dispersion of repolarization measured as T(p-e)/QT ratio and early afterdepolarizations. Disclosure of the names and concentrations after completion of the study revealed that all compounds known to be free of TdP risk received a score of less or equal to 0.25, whereas those with known TdP risk received a score ranging from 1.00 to 7.25 at concentrations less than 100X their free therapeutic plasma C(max). CONCLUSIONS: Our study provides a blinded evaluation of the isolated arterially-perfused rabbit wedge preparation demonstrating both a high sensitivity and specificity in the assessment of 13 agents with varying propensity for causing TdP.

Preclinical strategies to assess QT liability and torsadogenic potential of new drugs: the role of experimental models.

The recognition of QT prolongation and torsade de pointes (TdP) in humans has resulted in the re-labeling of some drugs and the removal of others from the market in the past decade. Recent regulatory guidelines have recommended a battery of preclinical tests to assess a new drug for the QT liability in humans. The assessment includes the effect of a drug on: 1) the ionic current in stable cell lines expressing hERG channel; 2) action potential duration (APD) measured in isolated ventricular tissues; 3) the QTc interval and TdP in animals in vivo; and 4) APD, the QT interval, transmural dispersion of repolarization (TDR) and TdP potential in the isolated arterially-perfused ventricular wedge preparation. Because a noncardiac drug with an incidence of TdP even less than 0.1% can be potentially removed from the market, the experimental models used for preclinical testing have to be high sensitive and specific to the signals related to TdP. Among available experimental models, the rabbit left ventricle wedge preparation exhibits a high sensitivity and a high specificity in the identification of compounds positive and negative for QT prolongation and TdP. This is attributed to the fact that the preparation demonstrates strong signals related to QT prolongation in response to even a weaker QT prolonging agent. Signals specifically pertinent to the development of TdP, ie, early afterdepolarization (EAD) and an increase in TDR can be detected as well. The preclinical data obtained from the wedge preparation correlate well with clinical outcomes.

Electrophysiologic effects of SB-237376: a new antiarrhythmic compound with dual potassium and calcium channel blocking action.

Combined potassium and calcium channel blocking activities are suggested to be the basis for antiarrhythmic efficacy with low proarrhythmic risk. The electrophysiologic effects of SB-237376 were investigated in single myocytes and arterially perfused wedge preparations of canine or rabbit left ventricles. The concentration-dependent prolongation of action potential duration (APD) and QT interval by SB-237376 was bell-shaped and the maximum response occurred at 1-3 microM SB-237376 inhibited rapidly activating delayed rectifier K current (I(Kr) ) with an IC50 of 0.42 microM and use-dependently blocked L-type Ca current (I (Ca,L) ) at high concentrations. The SB-237376 (3 microM) induced phase-2 early afterdepolarizations (EADs) in five of six rabbit wedge preparations but none of six canine wedge preparations. This is probably due to larger increases of APD, QT interval, and transmural dispersion of repolarization (TDR) in rabbits than dogs. Based on the drug effects on QT interval, TDR, and EAD in rabbit ventricular wedge preparations, a scoring system predicted lower proarrhythmic risk for SB-237376 than for dl-sotalol, a specific I blocker. In conclusion, SB-237376 increases APD, QT interval, and TDR mainly by I (Kr) inhibition. These effects are self-limited due to SB-237376-induced I(Ca,L) blockade at high concentrations, which may explain its lower proarrhythmic risk than dl-sotalol.

Effect of epicardial or biventricular pacing to prolong QT interval and increase transmural dispersion of repolarization: does resynchronization therapy pose a risk for patients predisposed to long QT or torsade de pointes?

BACKGROUND: The present study examined pacing site-dependent changes in QT interval and transmural dispersion of repolarization (TDR) and their potential role in the development of torsade de pointes (TdP). METHODS AND RESULTS: In humans, the QT interval, JT interval, and TDR were measured in 29 patients with heart failure during right ventricular endocardial pacing (RVEndoP), biventricular pacing (BiVP), and left ventricular epicardial pacing (LVEpiP). In animal experiments, pacing site--dependent changes in ventricular repolarization were examined with a rabbit left ventricular wedge preparation in which action potentials from endocardium and epicardium could be simultaneously recorded with a transmural ECG. In humans, LVEpiP and BiVP led to significant QT and JT prolongation. LVEpiP also enhanced TDR. Frequent R-on-T extrasystoles generated by BiVP and LVEpiP but completely inhibited by RVEndoP occurred in 4 patients, of whom 1 developed multiple episodes of nonsustained polymorphic ventricular tachycardia and another suffered incessant TdP. In rabbit experiments, switching from endocardial to epicardial pacing produced a net increase in QT interval and TDR by 17+/-5 and 22+/-5 ms, respectively (n=6, P<0.01), without parallel increases in ventricular transmembrane action potential durations. Epicardial pacing facilitated transmural propagation of early afterdepolarization, leading to the development of R-on-T extrasystoles and TdP in the presence of action potential duration-prolonging agents. CONCLUSIONS: LVEpiP and BiVP increase QT, JT, and TDR by altering the transmural sequence of activation of the intrinsically heterogeneous ventricular myocardium. Our data suggest that the resultant exaggeration of arrhythmic substrates can lead to the development of TdP in a subset of patients.

Unique topographical distribution of M cells underlies reentrant mechanism of torsade de pointes in the long-QT syndrome.

BACKGROUND: Specific ion channel mutations underlie the congenital long-QT syndrome (LQTS). However, the mechanisms by which dysfunction at the molecular level translates into functional electrical instability leading to torsade de pointes (TdP) in LQTS are poorly understood. METHODS AND RESULTS: The cellular basis of TdP was investigated using a novel approach of transmural optical imaging in the canine wedge preparation (n=14). The spatial organization of repolarization and arrhythmogenesis were determined in a surrogate model of LQT2. Action potentials were recorded simultaneously from 128 sites spanning the transmural wall of the left ventricle. In LQT2, QT interval prolongation was paralleled by an abrupt rise in transmural dispersion of repolarization (DOR) from 2.7 plus/minus 0.9 ms/mm (controls) to 12.2 plus/minus 2.1 ms/mm (LQT2). Islands of midmyocardial (M) cells formed zones of increased refractoriness in LQT2, producing steep spatial gradients of repolarization that were directly responsible for conduction block and self-sustained intramural reentrant circuits underlying TdP. CONCLUSIONS: These data provide direct evidence supporting the functional expression of M cells in intact myocardium and a central role for M cells in the development of reentrant TdP arrhythmias in LQTS.