Göran Duker

Instability and Triangulation of the Action Potential Predict Serious Proarrhythmia, but Action Potential Duration Prolongation Is Antiarrhythmic

BackgroundProlongation of action potential duration (APD) is considered a major antiarrhythmic mechanism (class III), but paradoxically, it frequently is also proarrhythmic (torsade de pointes). Methods and ResultsThe cardiac electrophysiological effects of 702 chemicals (class III or HERG channel block) were studied in 1071 rabbit Langendorff-perfused hearts. Temporal instability of APD, triangulation (duration of phase 3 repolarization), reverse use-dependence, and induction of ectopic beats were measured. Instability, triangulation, and reverse use-dependence were found to be important determinants of proarrhythmia. Agents that lengthened the APD by >50 ms, with induction of instability, triangulation, and reverse use-dependence (n=59), induced proarrhythmia (primarily polymorphic ventricular tachycardia); in their absence (n=19), the same prolongation of APD induced no proarrhythmia but significant antiarrhythmia (P <0.001). Shortening of APD, when accompanied by instability and triangulation, was also markedly proarrhythmic (primarily monomorphic ventricular tachycardia). In experiments in which instability and triangulation were present, proarrhythmia declined with prolongation of APD, but this effect was not large enough to become antiarrhythmic. Only with agents without instability did prolongation of APD become antiarrhythmic. For 20 selected compounds, it was shown that instability of APD and triangulation observed in vitro were strong predictors of in vivo proarrhythmia (torsade de pointes). ConclusionsLengthening of APD without instability or triangulation is not proarrhythmic but rather antiarrhythmic.

QTU-prolongation and torsades de pointes induced by putative class III antiarrhythmic agents in the rabbit: etiology and interventions.

When low doses of clofilium were administered to conscious rabbits, ventricular tachyarrhythmia (VT) with features typical of torsades de pointes developed. This arrhythmia was further studied and characterized in cholralose-anesthetized rabbits. In 20 of 20 rabbits, VT developed after a mean cumulative dose of 0.53 ± 0.04 μmol/kg clofilium, provided an infusion of the α1-agonist methoxamine (15 (μg/kg/min) was given concomitantly. The arrhythmia was preceded by a marked prolongation of the QTU interval and the monophasic action potential duration, as well as signs of early afterdepolarizations (EADs). In seven of 10 rabbits receiving only clofilium (cumulative dose, 20.8 μmol/kg), no VT occurred (p < 0.001 compared to the incidence in animals given both methoxamine and clofilium). In animals given both methoxamine and clofilium, pretreatment with prazosin (1 mg/kg i.v., n = 4) attenuated the arrhythmia, whereas diltiazem (0.5 mg/kg i.v., n = 4) or propranolol (0.5 mg/kg i.v., n = 8) was ineffective. Acute intervention with prazosin, isoproterenol, pinacidil, or magnesium sulfate promptly regularized the rhythm in animals with VT. Prazosin and pinacidil were equally effective in β-blocked rabbits. When other agents known to retard the repolarization currents (sematilide, UK-68,798, LY97119, amperozide, and cesium chloride) were examined, a strong correlation (r = 0.99, p < 0.001) between the potency of the drug to prolong the QTU interval and the proarrhythmic potential was obtained. This experimental model may represent an appropriate alternative for studying the acquired (“pause-dependent”) long QT syndrome.

Application of human stem cell-derived cardiomyocytes in safety pharmacology requires caution beyond hERG

Human embryonic stem cell-derived cardiomyocytes (hESC-CM) have been proposed as a new model for safety pharmacology. So far, a thorough description of their basic electrophysiology and extensive testing, and mechanistic explanations, of their overall pro-arrhythmic ability is lacking. Under standardized conditions, we have evaluated the sensitivity of hESC-CM to proarrhythmic provocations by blockade of hERG and other channels. Using voltage patch clamp, some ion current densities (pA/pF) in hESC-CM were comparable to adult CM: I(Kr) (-12.5 ± 6.9), I(Ks) (0.65 ± 0.12), I(Na,peak) (-72 ± 21), I(Na,late) (-1.10 ± 0.36), and I(Ca,L) (-4.3 ± 0.6). I(f) density was larger (-10 ± 1.1) and I(K1) not existent or very small (-2.67 ± 0.3). The low I(K1) density was corroborated by low KCNJ2 mRNA levels. Effects of pro-arrhythmic compounds on action potential (AP) parameters and provocation of early afterdepolarizations (EADs) revealed that Chromanol293B (100 μmol/l) and Bay K8644 (1 μmol/l) both significantly prolonged APD(90). ATX-II (<1 μmol/l ) and BaCl(2) (10 μmol/l ) had no effect on APD. The only compound that triggered EADs was hERG blocker Cisapride. Computer simulations and AP clamp showed that the immature AP of hESC-CM prevents proper functioning of I(Na)-channels, and result in lower peak/maximal currents of several other channels, compared to the adult situation. Lack of functional I(K1) channels and shifted I(Na) channel activation cause a rather immature electrophysiological phenotype in hESC-CM, and thereby limits the potential of this model to respond accurately to pro-arrhythmic triggers other than hERG block. Maturation of the electrical phenotype is a prerequiste for future implementation of the model in arrhythmogenic safety testing.

Quantified proarrhythmic potential of selected human embryonic stem cell-derived cardiomyocytes

To improve proarrhythmic predictability of preclinical models, we assessed whether human ventricular-like embryonic stem cell-derived cardiomyocytes (hESC-CMs) can be selected following a standardized protocol. Also, we quantified their arrhythmogenic response and compared this to a contemporary used rabbit Purkinje fiber (PF) model. Multiple transmembrane action potentials (AP) were recorded from 164 hESC-CM clusters (9 different batches), and 12 isolated PFs from New Zealand White rabbits. AP duration (APD), early afterdepolarizations (EADs), triangulation (T), and short-term variability of repolarization (STV) were determined on application of the I(Kr) blocker E-4031 (0.03/0.1/0.3/1 muM). Isoproterenol (0.1 muM) was used to assess adrenergic response. To validate the phenotype, RNA isolated from atrial- and ventricular-like clusters (n=8) was analyzed using low-density Taqman arrays. Based on initial experiments, slow beating rate (<50 bpm) and long APD (>200 ms) were used to select 31 ventricular-like clusters. E-4031 (1 muM) prolonged APD (31/31) and induced EADs only in clusters with APD90>300 ms (11/16). EADs were associated with increased T (1.6+/-0.2 vs 2.0+/-0.3) and STV (2.7+/-1.5 vs 6.9+/-1.9). Rabbit PF reacted in a similar way with regards to EADs (5/12), increased T (1.3+/-0.1 vs 1.9+/-0.4), and STV (1.2+/-0.9 vs 7.1+/-5.6). According to ROC values, hESC-CMs (STV 0.91) could predict EADs at least equivalent to PF (STV 0.69). Isoproterenol shortened APD and completely suppressed EADs. Gene expression analysis revealed that HCN1/2, KCNA5, and GJA5 were higher in atrial/nodal-like cells, whereas KCNJ2 and SCN1B were higher in ventricular-like cells (P<0.05). Selection of hESC-CM clusters with a ventricular-like phenotype can be standardized. The proarrhythmic results are qualitatively and quantitatively comparable between hESC-CMs and rabbit PF. Our results indicate that additional validation of this new safety pharmacology model is warranted.

Blocking Characteristics of hERG, hNav1.5, and hKvLQT1/hminK after Administration of the Novel Anti-Arrhythmic Compound AZD7009

Introduction: AZD7009 is a novel anti‐arrhythmic compound under development for short‐ and long‐term management of atrial fibrillation and flutter. Electrophysiological studies in animals have shown high anti‐arrhythmic efficacy, predominant action on atrial electrophysiology, and low proarrhythmic activity. The main aim of this study was to characterize the blocking effects of AZD7009 on the human ether‐a‐go‐go‐related gene (hERG), the hNav1.5, and the hKvLQT1/hminK currents.

Gender disparity in cardiac electrophysiology: Implications for cardiac safety pharmacology

BACKGROUND Gender differences in cardiac electrophysiology were reported for the first time almost a century ago. The importance for safety pharmacology became significant when modern medicine came into use and women appeared to be more susceptible to drug-induced Torsade de Pointes (TdP). To unravel the underlying mechanisms, the effect of sex hormones on cardiac electrophysiology has been studied in humans, animals and cell models. In this review, these data have been summarized and discussed in regard to possible consequences for safety pharmacology testing. RESULTS In man, electrophysiological differences become apparent during adolescence when the QTc interval shortens in males. This protective effect for long-QT related arrhythmias can be correlated to testosterone levels. Testosterone likely suppresses I(Ca,L) and enhances I(K) which increases the repolarization reserve. Though progesterone may have similar effects in women, these effects are probably balanced out by the small but opposite effects of estrogen. Progesterone levels, however, vary importantly throughout the different phases of the human menstrual cycle, implying that the sensitivity for drug-induced TdP changes too. The consequences for drug safety testing and TdP have not been assessed. CONCLUSION The testosterone-mediated increase in repolarization reserve in men is a likely cause for their lower susceptibility to drug-induced TdP. For the female population, the shifting balance in estrogen and progesterone creates temporal variation in the lability of repolarization to drug-induced TdP. This is a possible confounding factor in the evaluation and comparison of drugs that has to be further tested.

Comparison of the IKr blockers moxifloxacin, dofetilide and E‐4031 in five screening models of pro‐arrhythmia reveals lack of specificity of isolated cardiomyocytes

Drug development requires the testing of new chemical entities for adverse effects. For cardiac safety screening, improved assays are urgently needed. Isolated adult cardiomyocytes (CM) and human embryonic stem cell‐derived cardiomyocytes (hESC‐CM) could be used to identify pro‐arrhythmic compounds. In the present study, five assays were employed to investigate their sensitivity and specificity for evaluating the pro‐arrhythmic properties of IKr blockers, using moxifloxacin (safe compound) and dofetilide or E‐4031 (unsafe compounds).

Beat-by-beat QT interval variability, but not QT prolongation per se, predicts drug-induced torsades de pointes in the anaesthetised methoxamine-sensitized rabbit

INTRODUCTION Accumulating evidence suggest that drug-induced QT prolongation per se poorly predicts repolarisation-related proarrhythmia liability. We examined whether beat-by-beat variability of the QT interval may be a complementary proarrhythmia marker to QT prolongation. METHODS Anaesthetised rabbits sensitized towards developing torsades de pointes (TdP) were infused for 30 min maximum with explorative antiarrhythmic compounds characterised as mixed ion channel blockers. Based on the outcome in this model the compounds were classified as having a low (TdPlow; n=5), intermediate (TdPintermediate; n=7) or high (TdPhigh; n=10) proarrhythmic potential. Dofetilide (n=4) was included as a representative of a selective IKr-blocking antiarrhythmic with known high proarrhythmic potential. QT interval prolongation and beat-by-beat QT variability (quantified as the short-term variability, STV) were continuously assessed during the infusion or up to the point where ventricular proarrhythmias were induced. RESULTS All compounds significantly prolonged the QT interval. For TdPlow and TdPhigh compounds the QT interval maximally increased from 169 ± 14 to 225 ± 28 ms (p<0.05) and from 186 ± 21 to 268 ± 42 ms (p<0.01), respectively. Likewise, in the dofetilide-infused rabbits the QT interval maximally increased from 177 ± 11 to 243 ± 25 ms (p<0.01). In contrast, whereas the STV in rabbits administered the TdPhigh compounds or dofetilide significantly increased prior to proarrhythmia induction (from 1.6 ± 0.4 to 10.5 ± 5.6 ms and from 1.6 ± 0.5 to 5.9 ± 1.8 ms, p<0.01) it remained unaltered in the TdPlow group (1.3 ± 0.6 to 2.2 ± 0.9 ms). In the TdPintermediate group, rabbits experiencing TdP had a similar maximal QT prolongation as the non-susceptible rabbits whereas the change in the STV was significantly different (from 0.9 ± 0.5 to 8.7 ± 7.3 ms vs 0.8 ± 0.3 to 2.5 ± 1.1 ms). DISCUSSION It is concluded from the present series of experiments in a sensitive rabbit model of TdP that increased beat-by-beat QT interval variability precedes drug-induced TdP. In addition, assessment of this potential proarrhythmia marker may be useful in discriminating highly proarrhythmic compounds from compounds with a low proarrhythmic potential.

Report and recommendations of the workshop of the European Centre for the Validation of Alternative Methods for Drug-Induced Cardiotoxicity.

Cardiotoxicity is among the leading reasons for drug attrition and is therefore a core subject in non-clinical and clinical safety testing of new drugs. European Centre for the Validation of Alternative Methods held in March 2008 a workshop on “Alternative Methods for Drug-induced Cardiotoxicity” in order to promote acceptance of alternative methods reducing, refining or replacing the use of laboratory animals in this field. This review reports the outcome of the workshop. The participants identified the major clinical manifestations, which are sensitive to conventional drugs, to be arrhythmias, contractility toxicity, ischaemia toxicity, secondary cardiotoxicity and valve toxicity. They gave an overview of the current use of alternative tests in cardiac safety assessments. Moreover, they elaborated on new cardiotoxicological endpoints for which alternative tests can have an impact and provided recommendations on how to cover them.

Prolonged Action Potential Duration and Positive Inotropy Induced by the Novel Class III Antiarrhythmic Agent H 234/09 (Almokalant) in Isolated Human Ventricular Muscle

The electromechanical properties of H 234/09 (Almokalant), a novel class III antiarrhythmic agent, was examined in isolated human ventricular muscle strips excised from patients undergoing mitral valve replacement. Using transmembrane microelectrode recording techniques, we demonstrated that H 234/09 markedly prolonged the action potential duration (APD) without affecting the maximal rate of depolarization or action potential amplitude. At 75 and 90% repolarization APD was prolonged to a similar extent, whereas the lengthening at 50% repolarization was somewhat less marked. In isometrically contracting muscle strips, H 234/09 increased peak developed force and its maximal rate of rise (dF/dt) and fall (–dF/dt) in a concentration-dependent manner, whereas time to peak developed force was unaltered. We conclude from these studies that H 234/09 is a class III agent in human ventricular muscle and that the class III effect is linked with a positive inotropic response.