Effects of Anisodine Hydrobromide on the Cardiovascular and Respiratory Functions in Conscious Dogs.

PURPOSE: Anisodine hydrobromide (Ani) is isolated from the medicinal plant Anisodus tanguticus (Maxim.) Pascher for clinical use. Although considerable research regarding Ani has been reported, the safety profiles of Ani are currently unknown. This study investigated the cardiorespiratory effects of Ani in conscious dogs to provide clinicians a detailed safety profile of Ani on the cardiorespiratory system. MATERIALS AND METHODS: Using the Latin square design, the study was divided into six phases, where in each phase, six telemetered beagle dogs received one dose of normal saline or sotalol hydrochloride or Ani (0.1, 0.4, 1.6, or 6.4 mg/kg). Electrocardiogram, blood pressure (BP) and respiratory parameters were collected before and after administration for 24 hours. Statistical comparisons were performed at scheduled time-points. RESULTS: The heart rate was significantly increased, PR and QTCV intervals were significantly shortened in Ani 0.4, 1.6, 6.4 mg/kg treatment group after drug administration. Compared with the saline group, a significant increase in heart rate and shortening of PR, QTCV intervals were observed in the Ani 1.6, 6.4 mg/kg treatment groups from 5 min to 4 h time-points. Diastolic and mean BP were significantly increased in Ani 1.6, 6.4 mg/kg from 1 h to 2 h time-points compared to those of the saline control. Accelerated breathing was observed in the first 20 min after Ani 0.4, 1.6, and 6.4 mg/kg treatment, although not statistically significant. Furthermore, no significant differences were observed in any of the corresponding indexes of Ani 0.1 mg/kg treatment group at different time-points compared to those of the saline group. CONCLUSION: Ani may have adverse effects on the cardio-respiratory systems of dogs at doses above 0.4 mg/kg, whereas Ani 0.1 mg/kg was devoid of potentially deleterious effects on cardiorespiratory function.

A Proof-of-Concept Evaluation of JTPc and Tp-Tec as Proarrhythmia Biomarkers in Preclinical Species: A Retrospective Analysis by an HESI-Sponsored Consortium.

INTRODUCTION: Based on the ICH S7B and E14 guidance documents, QT interval (QTc) is used as the primary in vivo biomarker to assess the risk of drug-induced torsades de pointes (TdP). Clinical and nonclinical data suggest that drugs that prolong the corrected QTc with balanced multiple ion channel inhibition (most importantly the l-type calcium, Cav1.2, and persistent or late inward sodium current, Nav1.5, in addition to human Ether-à-go-go-Related Gene [hERG] IKr or Kv11.1) may have limited proarrhythmic liability. The heart rate-corrected J to T-peak (JTpc) measurement in particular may be considered to discriminate selective hERG blockers from multi-ion channel blockers. METHODS: Telemetry data from Beagle dogs given dofetilide (0.3 mg/kg), sotalol (32 mg/kg), and verapamil (30 mg/kg) orally and Cynomolgus monkeys given medetomidine (0.4 mg/kg) orally were retrospectively analyzed for effects on QTca, JTpca, and T-peak to T-end covariate adjusted (Tpeca) interval using individual rate correction and super intervals (calculated from 0-6, 6-12, 12-18, and 18-24 hours postdose). RESULTS: Dofetilide and cisapride (IKr or Kv11.1 blockers) were associated with significant increases in QTca and JTpca, while sotalol was associated with significant increases in QTca, JTpca, and Tpeca. Verapamil (a Kv11.1 and Cav1.2 blocker) resulted in a reduction in QTca and JTpca, however, and increased Tpeca. Medetomidine was associated with a reduction in Tpeca and increase in JTpca. DISCUSSION: Results from this limited retrospective electrocardiogram analysis suggest that JTpca and Tpeca may discriminate selective IKr blockers and multichannel blockers and could be considered in the context of an integrated comprehensive proarrhythmic risk assessment.

Electrophysiological characteristics and pharmacological sensitivity of two lines of human induced pluripotent stem cell derived cardiomyocytes coming from two different suppliers.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are increasingly used as preclinical tool for predicting drug-induced QT prolongation and arrhythmias. This study was conducted to assess the electrophysiological characteristics and the pharmacological sensitivity of two commercialized hiPSC-CMs. The baseline electrophysiological characteristics measured with a multi-electrode array (MEA) technology differ between Cor.4U and iCell2: higher beat rate (+32bpm) and shorter field potential duration (FPD, -201ms) for Cor.4U. The FPD lengthening after cisapride (100nM: +65% versus +18%), quinidine (10µM: +65% versus +31%), sotalol (30µM: +90% versus +47%) or flecainide (3µM: +76% versus +22%) application appeared earlier in iCell2 as compared to Cor.4U. Arrhythmia occurrence also appeared earlier in iCell2 as compared to Cor.4U for the 3 substances mentioned above. The FPD shortening recorded after verapamil or nifedipine application was similar in both hiPSC-CMs. In conclusion, Cor.4U and iCell2 hiPSC-CMs are both sensitive enough to detect drug-induced delayed or shortened repolarization and arrhythmia and can provide useful predictive cardiac electrophysiology data. Arrhythmias occurred at concentrations higher than clinical free maximum plasma concentrations with an overestimation of the risk with cisapride. However, quantitative differences of baseline electrophysiological characteristics or pharmacological sensitivity of both cell types have to be considered with caution during the interpretation of data. The new chemical entities included within a given drug development program should be evaluated in hiPSC-CMs coming from a single supplier.

Promising approach for the preclinical assessment of cardiac risks using left ventricular pressure-volume loop analyses in anesthetized monkeys.

INTRODUCTION: Load-independent cardiac parameters obtained from the ventricular pressure-volume relationship are recognized as gold standard indexes for evaluating cardiac inotropy. In this study, for better analyses of cardiac risks, load-independent pressure-volume loop parameters were assessed in addition to load-dependent inotropic, hemodynamic and electrocardiographic changes in isoflurane-anesthetized monkeys. METHODS: The animals were given milrinone (a PDE 3 inhibitor), metoprolol (a ß-blocker), or dl-sotalol (a ß+IKr blocker) intravenously over 10min at two dose levels including clinically relevant doses (n=5/drug). RESULTS: Milrinone and metoprolol produced positive and negative inotropy, respectively. These effects were detected as changes in the slope of the preload-recruitable stroke work, which is a load-independent inotropic parameter. However, dl-sotalol did not alter the slope of the preload-recruitable stroke work. That means dl-sotalol produced no inotropy, although it decreased load-dependent inotropic parameters, including maximal upstroke velocity of left ventricular pressure, attributable to decreased heart rate and blood pressure. Other typical pharmacological effects of the compounds tested were also detected. Both ß-blockers produced PR prolongation, decreased left ventricular end-systolic pressure, increased left ventricular end-diastolic pressure, and increased maximal descending velocity of left ventricular pressure and time constant for isovolumic relaxation. dl-Sotalol also prolonged heart-rate-corrected QT interval. Milrinone induced reflex tachycardia, PR shortening, and decreased left ventricular end-diastolic pressure. DISCUSSION: The overall assessment by not only load-dependent inotropic parameters but also load-independent parameters obtained from the ventricular pressure-volume loop analysis using monkeys can provide further appropriate information for the assessment of drug-induced cardiac risks.

Inter-study variability of preclinical in vivo safety studies and translational exposure-QTc relationships--a PKPD meta-analysis.

BACKGROUND AND PURPOSE: Preclinical cardiovascular safety studies (CVS) have been compared between facilities with respect to their sensitivity to detect drug-induced QTc prolongation (ΔQTc). Little is known about the consistency of quantitative ΔQTc predictions that are relevant for translation to humans. EXPERIMENTAL APPROACH: We derived typical ΔQTc predictions at therapeutic exposure (ΔQTcTHER ) with 95% confidence intervals (95%CI) for 3 Kv 11.1 (hERG) channel blockers (moxifloxacin, dofetilide and sotalol) from a total of 14 CVS with variable designs in the conscious dog. Population pharmacokinetic-pharmacodynamic (PKPD) analysis of each study was followed by a meta-analysis (pooling 2-6 studies including 10-32 dogs per compound) to derive meta-predictions of typical ΔQTcTHER . Meta-predictions were used as a reference to evaluate the consistency of study predictions and to relate results to those found in the clinical literature. KEY RESULTS: The 95%CIs of study-predicted ΔQTcTHER comprised in 13 out of 14 cases the meta-prediction. Overall inter-study variability (mean deviation from meta-prediction at upper level of therapeutic exposure) was 30% (range: 1-69%). Meta-ΔQTcTHER predictions for moxifloxacin, dofetilide and sotalol overlapped with reported clinical QTc prolongation when expressed as %-prolongation from baseline. CONCLUSIONS AND IMPLICATIONS: Consistent exposure-ΔQTc predictions were obtained from single preclinical dog studies of highly variable designs by systematic PKPD analysis, which is suitable for translational purposes. The good preclinical-clinical pharmacodynamic correlations obtained suggest that such an analysis should be more routinely applied to increase the informative and predictive value of results obtained from animal experiments.

Evaluation of cardiovascular changes in dogs administered three positive controls using jacketed external telemetry-blood pressure (JET-BP).

INTRODUCTION: Nonclinical safety studies are increasingly incorporating cardiac safety endpoints to discover potential cardiovascular liabilities. This trend for more thorough cardiovascular nonclinical safety evaluation is prudent given the high attrition rate of potential therapeutics due to unexpected cardiovascular liabilities discovered in late-stage clinical trials or post-market approval. In particular, the causal relationship of blood pressure changes that lead to risk of major adverse cardiac events suggests hemodynamic changes should be critically evaluated in preclinical studies of novel therapeutics. METHODS: Jacketed external telemetry with an implanted miniature blood pressure transmitter (JET-BP) was used to characterize the tolerability, functionality, and sensitivity of this study design in dogs. Thirty-six male or female beagles (n=6 dogs/sex/group) were administered vehicle control (reverse osmosis water) or etilefrine (1, 10mg/kg), sotalol (3, 30mg/kg), and hydralazine (1, 10mg/kg) on separate days. Telemetry data were evaluated for positive control article-related changes and retrospective power analysis was also completed. Animals were evaluated for instrumentation-related changes in clinical and anatomic pathology endpoints. RESULTS: All three positive controls elicited the expected pharmacologic responses that were statistically different at high and low doses. Retrospective power analysis confirmed this study design was able to statistically differentiate minor (approximately 5 to 15%) changes in electrocardiography and blood pressure values. This study also demonstrated the potential advantages of combining cardiovascular data across sex when the test article exposure and pharmacodynamics were consistent. Data collection using miniature telemetry blood pressure transmitters did not result in anatomic or clinical pathology findings that would prevent their use in general toxicology studies. DISCUSSION: This characterization study indicates that JET-BP in dogs offers a scientifically-robust method to evaluate novel therapeutics for potential cardiovascular liabilities.

Analysis of unipolar electrograms in rabbit heart demonstrated the key role of ventricular apicobasal dispersion in arrhythmogenicity.

Reduced repolarization reserve and increased transmural dispersion of repolarization (TDR) are known risk factors for Torsade de Pointes development, but less is known about the role of apex-to-base (apicobasal) repolarization in arrhythmogenesis. Three needles were inserted in rabbit left ventricle to record unipolar electrograms from endocardium to epicardium and base to apex. Total repolarization interval (TRI) and peak-to-end repolarization interval (Tp) were assessed after quinidine (n = 6) and D,L-sotalol (n = 6) perfusion in combination with the IKs inhibitor chromanol 293B. About 30 µM D,L-sotalol increased TRI and Tp more at the base (TRI + 40 ± 4 %; Tp +89 ± 11 %) relative to the apex (TRI + 28 ± 3 %, Tp + 30 ± 8 %). Similar results were obtained with quinidine: TRI and Tp increased more at the base compared to the apex. No significant differences were recorded from the endocardium to the epicardium. Our results show that combined IKr + IKs block prolonged TRI and Tp significantly more at the ventricular base than at the apex, in the absence of transmural dispersion of refractoriness. Regional changes in TRI and Tp indicate the contribution of apicobasal dispersion to arrhythmogenicity compared to TDR in a rabbit heart model.

In vitro model for assessing arrhythmogenic properties of drugs based on high-resolution impedance measurements.

BACKGROUND/AIMS: Cardiac dysfunction is one of the main cause of drug candidate failures in the preclinical and/or clinical studies and responsible for the retraction of large number of drugs from the market. The prediction of arrhythmic risk based on preclinical trials during drug development remains limited despite intensive and costly investigation. Moreover, methods for analyzing beating behavior of cardiomyocytes (CMs) in culture to diagnose arrhythmias are not well developed. METHODS: In this study, we combined two emerging technologies, induced pluripotent stem (iPS) cell-derived CMs and impedance-based real-time (xCELLigence RTCA Cardio Instrument) monitoring of CM electrical activity, to assess the effect of drugs known affect cardiac activity such as isoproterenol, carbachol, terfenadine, sotalol and doxorubicin. Cells were exposed to a drug in a single dose or repeated dose scenarios and data were analyzed using RTCA Cardio software, Poincaré plot and detrended fluctuation analysis. RESULTS: The results revealed significant changes in beating parameters of iPS-CMs induced by reference compounds. Heptanol, gap junction blocker, completely disrupted the synchronous beating pattern of iPS-CMs. Decrease of beating rate, amplitude and beat-to-beat signal variations of iPS-CMs monolayer observed in the presence of doxorubicin revealed severe abnormality detected by the system. Additionally, the irregular beating rhythms recorded in the presence of Terfenadine and Sotalol at high concentration, reflect abnormalities in cell contraction and/or relaxation which may lead to arrhythmia. CONCLUSIONS: All these results indicated that xCELLigence RTCA Cardio system combined with iPS cells, has the potential to be an attractive high-throughput tool for studying CMs during prolonged culture times and to screen potential drugs for cardiotoxic side effects.

Electrophysiologic profile of dronedarone on the ventricular level: beneficial effect on postrepolarization refractoriness in the presence of rapid phase 3 repolarization.

BACKGROUND: Dronedarone (D) is developed to maintain sinus rhythm in patients suffering from atrial fibrillation. The aim of the present study was to investigate, whether dronedarone also has an antiarrhythmic potential in the ventricle and to elucidate the mechanisms for its low proarrhythmic potential in an experimental whole heart model. METHODS AND RESULTS: Thirty-five rabbits underwent chronic treatment with D (n = 15; 50 mg · kg(-1) · d(-1)) and amiodarone (A; n = 20; 50 mg · kg(-1) · d(-1)). Hearts were perfused on a Langendorff apparatus. Results were compared with hearts acutely treated with sotalol (S; 50-100 µM; n = 14). A 12-lead electrocardiogram and up to 8 ventricular epi- and endocardial monophasic action potentials showed a significant prolongation of QT interval (D: +24 milliseconds, A: +28 milliseconds, S: +35 milliseconds (50 µM), +56 milliseconds (100 µM); P < 0.02) compared with baseline. In contrast to D and A, S led to a significant increase in dispersion of repolarization and exhibited reverse use dependence. D, A, and S increased refractory period, resulting in a significant increase in postrepolarization refractoriness (effective refractory period minus action potential duration; D = +12 milliseconds; A = +14 milliseconds; S = +25 milliseconds; P < 0.05). S led to a triangular action potential configuration, whereas D and A caused a fast phase 3 prolongation. After lowering of potassium concentration, 50% of S-treated hearts showed torsade de pointes, in contrast to an absence of torsade de pointes in D and A. CONCLUSIONS: Prolongation of myocardial repolarization and postrepolarization refractoriness by D may act antiarrhythmic. A fast phase 3 repolarization in the absence of both increased dispersion of repolarization and reverse use dependence prevents proarrhythmia.

Cardiovascular and respiratory safety pharmacology in Göttingen minipigs: Pharmacological characterization.

INTRODUCTION: Similarities between pigs and humans support the relevance of Göttingen minipigs for regulatory safety pharmacology. The minipig is the species of choice for cardiovascular safety pharmacology when pivotal repeat toxicology studies are conducted in this species. METHODS: 4 male Göttingen minipigs with cardiovascular telemetry transmitters received intravenous saline, esmolol (0.5, 1, 2, 4 and 8mg/kg), medetomidine (0.04mg/kg), remifentanil (0.5, 1, 2, 4, 8 and 16µg/kg) and dopamine (2, 8, 10, 20, 30 and 50µg/kg/min) and oral sotalol (3 and 10mg/kg). Respiratory monitoring was conducted in 3 male and 3 female Göttingen minipigs receiving intravenous saline and methacholine (0, 3.4, 13.5 and 68µg/kg). RESULTS: Heart rate (HR) corrected QT was optimal with a method based on analysis of covariance (QTca) followed by Fridericia's standard formula. Esmolol induced a decrease in HR. Medetomidine was associated with an initial hypertension with bradycardia followed by sustained hypotension, bradycadia and prolonged QTc. Remifentanil induced a dose-dependent QTc shortening with an increase in arterial pressures. Sotalol caused a decrease in HR and systolic arterial pressure with an increase in PR and QTc intervals. Dopamine induced an increase in arterial and pulse pressures. Methacholine increased tidal volume, respiratory rate and minute volume. DISCUSSION: The results suggest that the minipig is a valid alternative to other non-rodent species for cardiovascular and respiratory safety pharmacology studies when this species is justified.

A new ECG biomarker for drug toxicity: a combined signal processing and computational modeling study.

QT prolongation is the only clinically proven, yet insufficient, electrocardiogram (ECG) biomarker for drug-induced cardiac toxicity. The goal of this study is to evaluate whether JT area, i.e., total area of the T-wave, can serve as an ECG biomarker for drug-induced cardiac toxicity using both signal processing and computational modeling approaches. An ECG dataset that contained recordings from patients under control and sotalol condition was analyzed. In order to relate sotalol-induced ECG changes to its effect on ion channel level, i.e., blockade of the rapid component of the delayed rectifier potassium channel (I(Kr)), varied degrees of I(Kr) blockade were simulated in a slab of ventricular tissue. The mean JT area increased by 36.5% following the administration of sotalol in patients. Simulations in the slab tissue showed that sotalol increased action potential duration preferentially in the midmyocardium, which led to increased transmural dispersion of repolarization and JT area. In conclusion, JT area reflects the transmural dispersion of repolarization and may be a potentially useful surrogate/supplemental ECG biomarker to assess drug safety.

Human and murine embryonic stem cell-derived cardiomyocytes serve together as a valuable model for drug safety screening.

AIMS: Screening of drug safety is typically performed in diverse non-human healthy species with an intact repolarization reserve. Nevertheless, these drugs are later applied in diseased humans with a reduced repolarization reserve. It would be optimal to set up a preclinical screening tool to estimate the proarrhythmic potential of drugs in human cardiac tissue with a reduced repolarization reserve in vitro. METHODS AND RESULTS: In our study spontaneously beating human embryonic stem cell-derived cardiomyocytes clusters (hESCM) and murine ES cell-derived cardiomyocytes (mESCMs) were plated onto micro-electrode arrays (MEAs) to record the extracelluar field potentials (FPs) as well as effects of several antiarrhythmic drugs. In line with clinical observations the class III antiarrhythmic drugs (+/-)-sotalol, E4031 and class I antiarrhythmic drug quinidine led to a prolongation of the cardiac repolarization phase (FP duration, FPdur) and a decrease of the FP frequency. Verapamil (a class IV antiarrhythmic drug) decreased the FP frequency and shortened FPdur. Both, quinidine and verapamil, but not (+/-)-sotalol or E4031 decreased conduction velocities in hESCM clusters. Moreover, (+/-)-sotalol exerted stronger effects on FPdur in early developmental stages of hESCMs, as proof for a reduced repolarization reserve. The EC(50) of the (+/-)-sotalol-induced prolongation of the FPdur was higher in mESCMs than in hESCMs implying species-dependent differences in cardiac repolarization. Likewise, the incidence of drug-induced early recurrent depolarization (ERDs) was higher in mESCMs than hESCMs. CONCLUSION: The combined measurement of drug effects on FP parameters in hESCMs and mESCMs serves as a reliable in vitro model for preclinical studies of drug safety.

d,l-Sotalol reverses abbreviated atrial refractoriness and prevents promotion of atrial fibrillation in a canine model with left ventricular dysfunction induced by atrial tachypacing.

BACKGROUND: This study evaluated antiarrhythmic effects of d,l-sotalol in a canine atrial fibrillation (AF) model with left ventricular dysfunction. METHODS AND RESULTS: Thirteen beagles (Sotalol group n=7 and Control group n=6) were subjected to atrial tachypacing (ATP) (400 beats/min) with intact atrioventricular conduction for 4 weeks. Oral d,l-sotalol (2 mg/kg) was administered 1 week after starting ATP and continued throughout the experiment. One week after starting ATP, atrial effective refractory periods (AERPs) were shortened in both groups. However, d,l-sotalol treatment gradually prolonged AERP, resulting in a significant prolongation of AERP compared with the Control group at 4 weeks (Control 76 +/-4 and Sotalol 126 +/-5 ms, p<0.01). d,l-Sotalol treatment showed lower AF inducibility and shorter AF duration at 4 weeks. In the control group, expressions of L-type Ca(2+) channel alpha1c and Kv4.3 mRNA were downregulated by 46.2% and 43.0%, respectively, after 4 weeks of ATP; d,l-sotalol treatment did not affect these changes. CONCLUSIONS: d,l-Sotalol treatment prolonged AERP, even after atrial electrical remodeling had developed, and prevented AF perpetuation without affecting downregulated expression of L-type Ca(2+) channel alpha1c and Kv4.3 mRNA in an ATP-induced canine AF model.

Pharmacological enhancement of cardiac gap junction coupling prevents arrhythmias in canine LQT2 model.

Gap junctions contribute to the transmural heterogeneity of repolarization in the normal heart and under conditions of prolonged QT interval in the diseased heart. This study examined whether enhancing of gap junction coupling can reduce transmural dispersion of repolarization (TDR) and prevent torsade de pointes (TdP) in a canine LQT2 model. Canine left ventricular wedge preparations were perfused with delayed rectifier potassium current (IKr) blocker d-sotalol to mimic LQT2 and the antiarrhythmic peptide 10 (AAP10) was used as a gap junction coupling enhancer. As compared with the control group, the LQT2 group had significantly augmented TDR and higher incidence of TdP associated with increased nonphosphorylated connexin 43 (Cx43). AAP10 prevented augmentation of TDR and induction of TdP while rescuing Cx43 phosphorylation. There was no significant change in the quantity and spatial distribution of Cx43. These data indicate that gap junction enhancer AAP10 can prevent augmentation of TDR and suppress TdP by preventing dephosphorylation of Cx43 in a LQT2 model.

Evaluation of drug-induced QT prolongation in a halothane-anesthetized monkey model: effects of sotalol.

INTRODUCTION: Cynomolgus monkeys are used in in vivo models of safety pharmacological studies to evaluate the effects of drug candidates on the cardiovascular system. Models using halothane-anesthetized animals have been used for the detection of drug-induced QT interval prolongation, but few studies with anesthetized monkeys have been reported. METHODS: The electrophysiological changes induced by dl-sotalol, a representative class III antiarrhythmic drug, were assessed in halothane-anesthetized monkeys (n = 4) or conscious and unrestrained monkeys (n = 4). RESULTS: In terms of basal characteristics, the QT interval was longer and the heart rate (HR) was lower under anesthesia than those under conscious conditions. Intravenous administration of 0.1 to 3 mg/kg dl-sotalol to anesthetized monkeys decreased the HR and prolonged the QT interval, monophasic action potential (MAP) duration and ventricular effective refractory period in a dose-dependent manner. In addition, reverse use-dependent prolongation of MAP duration was detected by electrical pacing, whereas the terminal repolarization period was hardly affected at any dose. Oral administration of 3 to 30 mg/kg dl-sotalol to conscious monkeys also decreased the HR and prolonged the QT interval in a dose-dependent manner. When compared at similar plasma concentrations of sotalol, the extent of QT interval prolongation under halothane anesthesia was equal to or greater than that under conscious conditions. DISCUSSION: The sensitivity for detection of drug-induced QT prolongation under halothane anesthesia may be satisfactory compared with that under conscious conditions. The present examinations indicated the usefulness of a model using halothane-anesthetized monkeys for evaluation of drug-induced QT interval prolongation.

Dispersion of repolarization and arrhythmogenesis.

BACKGROUND: The relation between induction of arrhythmias and dispersion of repolarization is not completely understood. OBJECTIVE: The purpose of this study was to study the relation between heterogeneity in repolarization and arrhythmogenesis under conditions of selective regional action potential prolongation and shortening. METHODS: Pig hearts were perfused in a Langendorff setup. The left anterior descending artery (LAD) was cannulated and perfused. Sotalol (220 microM) was infused in the aortic cannula, and pinacidil (20 microM) was infused through the LAD, causing a gradient in repolarization time between the two myocardial regions. Premature stimulation was performed from the LAD region. RESULTS: No transmural repolarization gradients developed after infusion of the drugs. High-density epicardial activation/repolarization mapping (176 unipolar electrodes, 2-mm interelectrode spacing) revealed a maximum repolarization gradient of approximately 120 ms over 14 mm. The critical parameter for differentiating between the occurrence of reentry and the mere occurrence of a line of activation block between the two myocardial regions (and no reentry) was not the magnitude of the repolarization gradient but the timing of arrival of the premature activation wave at the distal side of the line of activation block relative to the repolarization time of the premature beat proximal to the line of block. No spontaneous arrhythmias were observed despite the presence of the repolarization gradient. CONCLUSION: It is not the repolarization gradient but the restitution characteristics of the tissue with the shorter action potential, in combination with the time of arrival of the premature wavefront at the distal side of the line of block, that determines the occurrence of reentry.

Calculation of QT shift in non clinical safety pharmacology studies.

INTRODUCTION: Drug-induced QT interval prolongation is a major concern in new drug candidate development. This study presents a method of assessment of drug-induced QT interval prolongation without need for QT correction in conscious Beagle dogs and Cynomolgus monkeys monitored by telemetry. Accuracy and reliability are analysed by comparison with a reference QT correction method (Holzgrefe) from experiments performed with reference substances terfenadine, thioridazine and sotalol. METHODS: The QT shift method principle is assessment of any drug-induced QT interval shift directly from the individual QT/RR relationship. The individual QT/RR relationship is built from a treatment-free 24-hour recording period. QT and RR intervals are determined from a beat-to-beat analysis. A probabilistic method is used to define the individual QT/RR relationships. Checks were performed to compare results obtained with the QT shift method and the QT correction methods. The robustness of the QT shift method was tested under various conditions of drug-induced heart rate change (i.e. normal, bradycardia and tachycardia). RESULTS: The extent of agreement with the used reference QT correction method, Holzgrefe formula, was excellent (3-4 ms) in both animal species under the various drug induced effects on heart rate. The statistical sensitivity threshold for detection of QT prolongation according to a standard safety pharmacology study design was 7-8 ms. DISCUSSION: When combined with the probabilistic determination of individual QT/RR relationships, this simple method provides a direct assessment of a drug-induced effect on QT interval, without any curve fitting or application of correction formula. Despite noticeably different shapes in QT/RR relationships, the QT shift method is applicable to both Beagle dogs and Cynomolgus monkeys. It is likely that the QT shift method will be particularly helpful in problematic cases, enabling detection of drug-induced prolongation of less than 10 ms.

d,l-Sotalol at therapeutic concentrations facilitates the occurrence of long-lasting non-stationary reentry during ventricular fibrillation in isolated rabbit hearts.

BACKGROUND: The effects of d,l-sotalol at therapeutic concentrations (

In vitro electrophysiological drug testing using human embryonic stem cell derived cardiomyocytes.

Pro-arrhythmia (development of cardiac arrhythmias as a pharmacological side effect) has become the single most common cause of the withdrawal or restrictions of previously marketed drugs. The development of new medications, free from these side effects, is hampered by the lack of an in vitro assay for human cardiac tissue. We hypothesized that human embryonic stem cell-derived cardiomyocytes (hESC-CMs) assessed with a combination of single cell electrophysiology and microelectrode array (MEA) mapping can serve as a novel model for electrophysiological drug screening. Current-clamp studies revealed that E-4031 and Sotalol (IKr blockers) significantly increased hESC-CM's action potential duration and also induced after-depolarizations (the in vitro correlates of increased arrhythmogenic potential). Multicellular aggregates of hESC-CMs were then analyzed with the MEA technique. Application of class I (Quinidine, Procaineamide) and class III (Sotalol) antiarrhythmic agents, E-4031, and Cisapride (a noncardiogenic agent known to lengthen QT) resulted in dose-dependent prolongation of the corrected field potential duration (cFPD). We next utilized the MEA technique to also assess pharmacological effects on conduction. Activation maps demonstrated significant conduction slowing following administration of Na channel blockers (Quinidine and Propafenone) and of the gap junction blocker (1-heptanol). While most attention has been focused on the prospects of using hESC-derived cardiomyocytes for regenerative medicine, this study highlights the possible utilization of these unique cells also for cardiac electrophysiological studies, drug screening, and target validation.