Use of arterially perfused rabbit ventricular wedge in predicting arrhythmogenic potentials of drugs.

INTRODUCTION: A growing number of drugs have reportedly been associated with delayed ventricular repolarization and a potentially fatal but rare arrhythmia, torsade de pointes (TdP). There is obviously a call for a validated proarrhythmia model that distinguishes proarrhythmic drugs from nonarrhythmogenic drugs. METHODS: In this article, we validated the arterially perfused rabbit left ventricular wedge preparation model and examined its use in predicting proarrhythmic potentials of drugs. A fairly detailed methodological description about this technically challenging model was given, aiming to help others establish the assay successfully. Parameters commonly used in the action potential studies were verified and critical experimental conditions (e.g. stability and reproducibility of recordings) were examined. Six commercially available compounds with various proarrhythmic potentials were administered in the model to evaluate their correlations with individual clinical outcomes. RESULTS: Our study indicated that, in a successful experiment, the action potential duration (APD) can be stably maintained for several hours without intervention. Dofetilide, DL-sotalol, cisapride, risperidone and moxifloxacin increased endo- and epicardial APD(90), QT interval and T(P-E) (peak-to-end time of the T wave) in a reverse use-dependent manner within clinically relevant concentration ranges. Phase 2 early afterdepolarizations (EADs) were observed at 1.6, 2.3, 16.7, 37.5 and 7.9 fold, respectively, their corresponding unbound therapeutic concentrations. In contrast, fluoxetine at up to 3 microM (approximately 35 fold unbound therapeutic mean plasma concentration after 60 mg/day, p.o. for 5 weeks) had only a mild prolonging effect on APD(90) and QT with essentially no effect on T(P-E). DISCUSSION: Our results strongly support the usefulness of this model in predicting a compound's arrhythmogenic potential in humans within clinically relevant concentration ranges, and the experimental results with this model need to be interpreted in light of each drug's pharmacokinetic and pharmacodynamic behavior in clinic.

QT prolongation and proarrhythmia by moxifloxacin: concordance of preclinical models in relation to clinical outcome.

Moxifloxacin, a fluoroquinolone antibiotic associated with QT prolongation, has been recommended as a positive control by regulatory authorities to evaluate the sensitivity of both clinical and preclinical studies to detect small but significant increases in QT interval measurements. In this study, we investigated effects of moxifloxacin on the hERG current in HEK-293 cells, electrocardiograms in conscious telemetered dogs, and repolarization parameters and arrhythmogenic potentials in the arterially perfused rabbit ventricular wedge model. Moxifloxacin inhibited the hERG current with an IC50 of 35.7 microM. In conscious telemetered dogs, moxifloxacin significantly prolonged QTc at 30 and 90 mg kg(-1), with mean serum Cmax of 8.52 and 22.3 microg ml(-1), respectively. In the wedge preparation, moxifloxacin produced a concentration-dependent prolongation of the action potential duration, QT interval, and the time between peak and end of the T wave, an indicator for transmural dispersion of repolarization. Phase 2 early after-depolarizations were observed in one of five experiments at 30 microM and five of five experiments at 100 microM. The arrhythmogenic potential was also concentration-dependent, and 100 microM ( approximately 18-fold above the typical unbound Cmax exposure in clinical usage) appeared to have a high risk of inducing torsade de pointes (TdP). Our data indicated a good correlation among the concentration-response relationships in the three preclinical models and with the available clinical data. The lack of TdP report by moxifloxacin in patients without other risk factors might be attributable to its well-behaved pharmacokinetic profile and other dose-limiting effects.

Novel potent human ether-a-go-go-related gene (hERG) potassium channel enhancers and their in vitro antiarrhythmic activity.

A variety of drugs has been reported to cause acquired long QT syndrome through inhibition of the IKr channel. Screening compounds in early discovery and development stages against their ability to inhibit IKr or the hERG channel has therefore become an indispensable procedure in the pharmaceutical industry. In contrast to numerous hERG channel blockers discovered during screening, only (3R,4R)-4-[3-(6-methoxyquinolin-4-yl)-3-oxo-propyl]-1-[3-(2,3,5-trifluoro-phenyl)-prop-2-ynyl]-piperidine-3-carboxylic acid (RPR260243) has been reported so far to enhance the hERG current. In this article, we describe several potent mechanistically distinct hERG channel enhancers. One example is PD-118057 (2-{4-[2-(3,4-dichloro-phenyl)-ethyl]-phenylamino}-benzoic acid) which produced average increases of 5.5 +/- 1.1, 44.8 +/- 3.1, and 111.1 +/- 21.7% in the peak tail hERG current at 1, 3, and 10 muM, respectively, in human embryonic kidney 293 cells. PD-118057 did not affect the voltage dependence and kinetics of gating parameters, nor did it require open conformation of the channel. In isolated guinea pig cardiomyocytes, PD-118057 showed no major effect on I(Na), I(Ca,L), I(K1), and I(Ks). PD-118057 shortened the action potential duration and QT interval in arterially perfused rabbit ventricular wedge preparation in a concentration-dependent manner. The presence of 3 muM PD-118057 prevented action potential duration and QT prolongation caused by dofetilide. "Early after-depolarizations" induced by dofetilide were also completely eliminated by 3 microM PD-118057. Although further investigation is warranted to evaluate the therapeutic value and safety profile of these compounds, our data support the notion that hERG activation by pharmaceuticals may offer a new approach in the treatment of delayed repolarization conditions, which may occur in patients with inherited or acquired long QT syndrome, congestive heart failure, and diabetes.

Pentamidine reduces hERG expression to prolong the QT interval.

Pentamidine, an antiprotozoal agent, has been traditionally known to cause QT prolongation and arrhythmias; however, its ionic mechanism has not been illustrated. In a stable HEK-293 cell line, we observed a concentration-dependent inhibition of the hERG current with an IC50 of 252 microM. In freshly isolated guinea-pig ventricular myocytes, pentamidine showed no effect on the L-type calcium current at concentrations up to 300 microM, with a slight prolongation of the action potential duration at this concentration. Since the effective concentrations of pentamidine on the hERG channel and APD were much higher than clinically relevant exposures (approximately 1 microM free or lower), we speculated that this drug might not prolong the QT interval through direct inhibition of I(Kr) channel. We therefore incubated hERG-HEK cells in 1 and 10 microM pentamidine-containing media (supplemented with 10% serum) for 48 h, and examined the hERG current densities in the vehicle control and pentamidine-treated cells. In all, 36 and 85% reductions of the current densities were caused by 1- and 10-microM pentamidine treatment (P<0.001 vs control), respectively. A similar level of reduction of the hERG polypeptides and a reduced intensity of the hERG protein on the surface membrane in treated cells were observed by Western blot analysis and laser-scanning confocal microscopy, respectively. Taken together, our data imply that chronic administration of pentamidine at clinically relevant exposure reduces the membrane expression of the hERG channel, which may most likely be the major mechanism of QT prolongation and torsade de pointes reported in man.

Role of a KCNH2 polymorphism (R1047 L) in dofetilide-induced Torsades de Pointes.

Various drugs are reported to prolong the QT-interval on the surface ECG, thereby increasing the risk of developing a potentially fatal arrhythmia known as Torsades de Pointes (TdP). TdP case reports for these drugs have often been associated with risk factors such as overdosing, concomitant drugs and/or existing pathophysiological conditions. A few cases appear to be devoid of these factors. To determine what role genetic variation in the hERG gene plays in drug-induced arrhythmias, we screened DNA samples collected from 105 atrial-fibrillation patients treated with dofetilide for polymorphisms, seven of whom developed TdP. An uncommon missense change, R1047L, was identified in two of seven patients who experienced TdP as compared with five of 98 individuals who were free of TdP. Included in the affected individuals was the only subject homozygous for this SNP. Cellular electrophysiological studies revealed a 10-mV positive shift in the steady-state activation curve of the 1047L hERG channel stably expressed in HEK-293 cells as compared with the wild-type (WT) channel. The activation and inactivation kinetics of the 1047L current were significantly slower than the WT (P < 0.05) at given membrane potentials. A computer simulation using a rabbit ventricular myocyte model indicated that same extent of changes in the I(Kr) channel may result in an approximately 15% prolongation in the action potential duration. Our study suggests that 1047L leads to a functional impairment of the hERG channel, which may contribute to the higher incidence of TdP in 1047L carriers when challenged with a channel blocker.