Assessment of Contractility in Human iPS Cell-Derived Cardiomyocytes Using Motion Vector Analysis.

Human-induced pluripotent stem cell (iPSC) technology paves the way for next-generation drug-safety assessment. In particular, human iPSC-derived cardiomyocytes, which exhibit electrical activity, are useful as a human cell model for assessing QT-interval prolongation and the risk of the lethal arrhythmia Torsade de Pointes (TdP). In addition to proarrhythmia assay, contractile behavior has received increased attention in drug development. In this study, we developed a novel high-throughput in vitro assay system using motion vectors to evaluate the contractile activity of iPSC-derived cardiomyocytes as a physiologically relevant human platform. The methods presented here highlight the use of commercially available iPSC-derived cardiomyocytes, iCell cardiomyocytes, for contractility evaluation recorded by the motion vector system.

Assessment of Safety Margin of an Antipsychotic Drug Haloperidol for Torsade de Pointes Using the Chronic Atrioventricular Block Dogs.

Since an antipsychotic drug haloperidol has been clinically reported to induce QT interval prolongation and torsade de pointes, in this study its risk stratification for the onset of torsade de pointes was performed by using the chronic atrioventricular block canine model with a Holter electrocardiogram. Haloperidol in a dose of 3 mg kg-1 p.o. prolonged the QT interval, but it did not induce torsade de pointes during the observation period of 21 h (n = 4), indicating that the dose would be safe. Meanwhile, haloperidol in a dose of 30 mg kg-1 p.o. significantly increased the short-term variability in beat-to-beat analysis of QT interval (n = 4), and it induced torsade de pointes in 4 animals out of 4, showing that the dose could be torsadogenic. Since 3 mg kg-1 p.o. of haloperidol in this study can be estimated to provide about 8 times higher plasma concentrations than its therapeutic level, haloperidol may be used safely for most of the patients, as long as its plasma drug concentration is kept within the therapeutic range.

A new paradigm for drug-induced torsadogenic risk assessment using human iPS cell-derived cardiomyocytes.

INTRODUCTION: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are anticipated to be a useful tool for conducting proarrhythmia risk assessments of drug candidates. However, a torsadogenic risk prediction paradigm using hiPSC-CMs has not yet been fully established. METHODS: Extracellular field potentials (FPs) were recorded from hiPSC-CMs using the multi-electrode array (MEA) system. The effects on FPs were evaluated with 60 drugs, including 57 with various clinical torsadogenic risks. Actual drug concentrations in medium were measured using the equilibrium dialysis method with a Rapid Equilibrium Dialysis device. Relative torsade de pointes (TdP) scores were determined for each drug according to the degree of FP duration prolongation and early afterdepolarization occurrence. The margins were calculated from the free concentration in medium and free effective therapeutic plasma concentration. Each drug's results were plotted on a two-dimensional map of relative TdP risk scores versus margins. RESULTS: Each drug was categorised as high, intermediate, or low risk based on its location within predefined areas of the two-dimensional map. We categorised 19 drugs as high risk; 18 as intermediate risk; and 17 as low risk. We examined the concordance between our categorisation of high and low risk drugs against the torsadogenic risk categorisation in CredibleMeds®. Our system demonstrated high concordance, as reflected in a sensitivity of 81%, specificity of 87%, and accuracy of 83%. DISCUSSION: These results indicate that our torsadogenic risk assessment is reliable and has a potential to replace the hERG assay for torsadogenic risk prediction, however, this system needs to be improved for the accurate of prediction of clinical TdP risk. Here, we propose a novel drug induced torsadogenic risk categorising system using hiPSC-CMs and the MEA system.

Electrophysiological Characteristics of Human iPSC-Derived Cardiomyocytes for the Assessment of Drug-Induced Proarrhythmic Potential.

The aims of this study were to (1) characterize basic electrophysiological elements of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) that correspond to clinical properties such as QT-RR relationship, (2) determine the applicability of QT correction and analysis methods, and (3) determine if and how these in-vitro parameters could be used in risk assessment for adverse drug-induced effects such as Torsades de pointes (TdP). Field potential recordings were obtained from commercially available hiPSC-CMs using multi-electrode array (MEA) platform with and without ion channel antagonists in the recording solution. Under control conditions, MEA-measured interspike interval and field potential duration (FPD) ranged widely from 1049 to 1635 ms and from 334 to 527 ms, respectively and provided positive linear regression coefficients similar to native QT-RR plots obtained from human electrocardiogram (ECG) analyses in the ongoing cardiovascular-based Framingham Heart Study. Similar to minimizing the effect of heart rate on the QT interval, Fridericia's and Bazett's corrections reduced the influence of beat rate on hiPSC-CM FPD. In the presence of E-4031 and cisapride, inhibitors of the rapid delayed rectifier potassium current, hiPSC-CMs showed reverse use-dependent FPD prolongation. Categorical analysis, which is usually applied to clinical QT studies, was applicable to hiPSC-CMs for evaluating torsadogenic risks with FPD and/or corrected FPD. Together, this results of this study links hiPSC-CM electrophysiological endpoints to native ECG endpoints, demonstrates the appropriateness of clinical analytical practices as applied to hiPSC-CMs, and suggests that hiPSC-CMs are a reliable models for assessing the arrhythmogenic potential of drug candidates in human.

Assessment of the Anti-anginal Effect of Tetramethylpyrazine Using Vasopressin-Induced Angina Model Rats.

Intravenous tetramethylpyrazine has been widely used in China as a complementary and/or alternative medicine to treat patients with ischemic heart disease. We assessed the anti-anginal effect of tetramethylpyrazine (10 mg/kg, intravenously (i.v.), n=6) in comparison with that of its vehicle, saline (1 mL/kg, i.v., n=6), using vasopressin-induced angina model rats. First, Donryu rats were anesthetized with pentobarbital sodium (60 mg/kg, intraperitoneally (i.p.)), and the surface lead I electrocardiogram was continuously monitored. Administration of vasopressin (0.5 IU/kg, i.v.) to the rats depressed the S-wave level of the electrocardiogram, indicating the onset of subendocardial ischemia. However, pretreatment with tetramethylpyrazine suppressed the vasopressin-induced depression of the S-wave level, which was not observed following pretreatment with its vehicle alone (saline), suggesting that tetramethylpyrazine ameliorated the vasopressin-induced subendocardial ischemia in vivo. These results may provide experimental evidence for the empirically known clinical efficacy of tetramethylpyrazine against ischemic heart disease, and could provide clues to better understanding its in vivo mechanism of action.

Assessment of testing methods for drug-induced repolarization delay and arrhythmias in an iPS cell-derived cardiomyocyte sheet: multi-site validation study.

A prospective comparison study across 3 independent research laboratories of a pure IKr blocker E-4031 was conducted by using the same batch of human iPS cell-derived cardiomyocytes in order to verify the utility and reliability of our original standard protocol. Field potential waveforms were recorded with a multi-electrode array system to measure the inter-spike interval and field potential duration. The effects of E-4031 at concentrations of 1 to 100 nM were sequentially examined every 10 min. In each facility, E-4031 significantly prolonged the field potential duration corrected by Fridericia's formula and caused early afterdepolarizations occasionally resulting in triggered activities, whereas it tended to decrease the rate of spontaneous contraction. These results were qualitatively and quantitatively consistent with previous non-clinical in vitro and in vivo studies as well as clinical reports. There were inter-facility differences in some absolute values of the results, which were not observed when the values were normalized as percentage change. Information described in this paper may serve as a guide when predicting the drug-induced repolarization delay and arrhythmias with this new technology of stem cells.

Microminipig, a non-rodent experimental animal optimized for life science research: preface.

Fuji Micra Inc. has recently achieved success in a challenging and prospective project that produces the smallest pig in the world, the "Microminipig", at a breeding farm at the foothills of Mt. Fuji in Japan. Microminipigs weigh approximately 7.0 kg at 6 months of age when they are mature. Microminipigs have been provided to several research organizations in Japan as a non-rodent experimental animal optimized for life science research.

Simultaneous assessment of pharmacokinetics of pilsicainide transdermal patch and its electropharmacological effects on atria of chronic atrioventricular block dogs.

Pharmacokinetics of pilsicainide transdermal patch and its electropharmacological effects were simultaneously assessed using chronic atrioventricular block dogs. After application of the patch (9.8 mg/kg), pilsicainide was continuously absorbed through the skin with a C(max) of 0.49 +/- 0.13 microg/ml, while its plasma concentration was kept above the clinically reported minimum effective plasma concentration for 2 - 8 h. Inter-atrial conduction time was significantly prolonged, whereas statistically significant prolongation was not detected in the atrial effective refractory period. Prolongation of the cycle length of atrial fibrillation and anti-fibrillatory action were confirmed. Thus, pilsicainide can be absorbed transdermally to exert long-lasting electropharmacological effects leading to anti-atrial fibrillatory action.

Halothane-anaesthetized, closed-chest, guinea-pig model for assessment of drug-induced QT-interval prolongation.

For the halothane-anaesthetized, closed-chest, guinea-pig model, corrected QT interval (QTc) has been empirically used to estimate the extent of drug-induced QT-interval prolongation. In the present study, we employed an atrial pacing method to clarify a net effect of a drug on the QT interval in this model. The atrial pacing catheter was inserted via the jugular vein with a minimal surgical invasion, and the effects of d-sotalol (0.3 and 3 mg/kg, intravenously) and verapamil (0.01 and 0.1 mg/kg, intravenously) on electrocardiogram parameters were assessed under the sinus rhythm and during the atrial pacing of 200 and 240 beats/min. d-Sotalol significantly prolonged the QT interval in a reverse use-dependent manner and decreased the heart rate, while verapamil prolonged the PR interval without affecting the heart rate or QT interval, indicating the sensitivity and specificity of this model in assessing the pharmacodynamics of the drug-induced QT-interval prolongation. Using the QT/RR relationship under the sinus rhythm, we obtained the following two types of QT-interval correcting formulae; namely, QTc = QT - 0.207(RR - 300) by a linear regression method; and QTc = QT/(RR/300)0.332 by a non-linear regression method, the latter of which is equal to 0.67 times of Fridericia's formula, providing rationale for the use of mathematical correction in this model. Thus, the halothane-anaesthetized, closed-chest, guinea-pig model may be highly useful for assessing the drug-induced QT-interval prolongation, which may become an alternative to current models for the in vivo QT assay.

In vivo experimental approach for the risk assessment of fluoroquinolone antibacterial agents-induced long QT syndrome.

The proarrhythmic effects of fluoroquinolone antibacterial agents, sitafloxacin, gatifloxacin and moxifloxacin, were compared using three in vivo models. In the halothane-anesthetized dogs (n=5), intravenous 10-min infusion of gatifloxacin and moxifloxacin (1-3 mg/kg) prolonged the ventricular effective refractory period and the repolarization period to a similar extent, whereas sitafloxacin (1-3 mg/kg) prolonged the former only. No significant change was detected in other cardiovascular parameters. In the chronic complete atrioventricular block dogs (n=4), oral administration of 100 mg/kg of gatifloxacin (2 of 4) and moxifloxacin (3 of 4) induced torsades de pointes, which was not observed by sitafloxacin. In the alpha-chloralose-anesthetized rabbits (n=5), intravenous 20-min infusion of 60 mg/kg of gatifloxacin induced torsades de pointes (1 of 5) in the presence of methoxamine infusion, which was not observed by sitafloxacin or moxifloxacin. Thus, the halothane-anesthetized model is suitable for assessing QT prolongation, whereas the chronic complete atrioventricular block model is sensitive for detecting torsadogenic action of drugs. The alpha-chloralose-anesthetized model is the simplest and least expensive method, but its sensitivity to detect proarrhythmic action may be less great.