CSAHi study-2: Validation of multi-electrode array systems (MEA60/2100) for prediction of drug-induced proarrhythmia using human iPS cell-derived cardiomyocytes: Assessment of reference compounds and comparison with non-clinical studies and clinical information.

With the aim of reconsidering ICH S7B and E14 guidelines, a new in vitro assay system has been subjected to worldwide validation to establish a better prediction platform for potential drug-induced QT prolongation and the consequent TdP in clinical practice. In Japan, CSAHi HEART team has been working on hiPS-CMs in the MEA (hiPS-CMs/MEA) under a standardized protocol and found no inter-facility or lot-to-lot variability for proarrhythmic risk assessment of 7 reference compounds. In this study, we evaluated the responses of hiPS-CMs/MEA to another 31 reference compounds associated with cardiac toxicities, and gene expression to further clarify the electrophysiological characteristics over the course of culture period. The hiPS-CMs/MEA assay accurately predicted reference compounds potential for arrhythmogenesis, and yielded results that showed better correlation with target concentrations of QTc prolongation or TdP in clinical setting than other current in vitro and in vivo assays. Gene expression analyses revealed consistent profiles in all samples within and among the testing facilities. This report would provide CiPA with informative guidance on the use of the hiPS-CMs/MEA assay, and promote the establishment of a new paradigm, beyond conventional in vitro and in vivo assays for cardiac safety assessment of new drugs.

CSAHi study: Validation of multi-electrode array systems (MEA60/2100) for prediction of drug-induced proarrhythmia using human iPS cell-derived cardiomyocytes -assessment of inter-facility and cells lot-to-lot-variability.

In vitro screening of hERG channels are recommended under ICH S7B guidelines to predict drug-induced QT prolongation and Torsade de Pointes (TdP), whereas proarrhythmia is known to be evoked by blockage of other ion channels involved in cardiac contraction and compensation mechanisms. A consortium for drug safety assessment using human iPS cells-derived cardiomyocytes (hiPS-CMs), CSAHi, has been organized to establish a novel in vitro test system that would enable better prediction of drug-induced proarrhythmia and QT prolongation. Here we report the inter-facility and cells lot-to-lot variability evaluated with FPDc (corrected field potential duration), FPDc10 (10% FPDc change concentration), beat rate and incidence of arrhythmia-like waveform or arrest on hiPS-CMs in a multi-electrode array system. Arrhythmia-like waveforms were evident for all test compounds, other than chromanol 293B, that evoked FPDc prolongation in this system and are reported to induce TdP in clinical practice. There was no apparent cells lot-to-lot variability, while inter-facility variabilities were limited within ranges from 3.9- to 20-folds for FPDc10 and about 10-folds for the minimum concentration inducing arrhythmia-like waveform or arrests. In conclusion, the new assay model reported here would enable accurate prediction of a drug potential for proarrhythmia.

Repairability of skeletal alterations induced by sodium valproate in rats.

The present study aimed at examining postnatal repairability of sodium valproate-induced skeletal alterations in rats. Sodium valproate (400 mg/kg) or the vehicle (distilled water) was orally administrated to pregnant Sprague-Dawley rats from gestation days 9 to 11. Fetuses and pups were obtained on gestation day 21 and postnatal day 11, respectively, and their skeletons were stained with Alizarin red S and Alcian blue and examined. Sodium valproate-induced costal and vertebral alterations in the fetuses included discontinued rib cartilage, fused rib, full or short supernumerary rib, bipart ossification of thoracic centrum, supernumerary lumbar vertebrae, and lumbarization. In pups, however, discontinued rib cartilage was not observed, and the incidence of a short supernumerary rib was significantly lower than that in the fetuses, suggesting that these alterations are postnatally repairable.

Improvement of acquisition and analysis methods in multi-electrode array experiments with iPS cell-derived cardiomyocytes.

INTRODUCTION: Multi-electrode array (MEA) systems and human induced pluripotent stem (iPS) cell-derived cardiomyocytes are frequently used to characterize the electrophysiological effects of drug candidates for the prediction of QT prolongation and proarrhythmic potential. However, the optimal experimental conditions for obtaining reliable experimental data, such as high-pass filter (HPF) frequency and cell plating density, remain to be determined. METHODS: Extracellular field potentials (FPs) were recorded from iPS cell-derived cardiomyocyte sheets by using the MED64 and MEA2100 multi-electrode array systems. Effects of HPF frequency (0.1 or 1Hz) on FP duration (FPD) were assessed in the presence and absence of moxifloxacin, terfenadine, and aspirin. The influence of cell density on FP characteristics recorded through a 0.1-Hz HPF was examined. The relationship between FP and action potential (AP) was elucidated by simultaneous recording of FP and AP using a membrane potential dye. RESULTS: Many of the FP waveforms recorded through a 1-Hz HPF were markedly deformed and appeared differentiated compared with those recorded through a 0.1-Hz HPF. The concentration-response curves for FPD in the presence of terfenadine reached a steady state at concentrations of 0.1 and 0.3µM when a 0.1-Hz HPF was used. In contrast, FPD decreased at a concentration of 0.3µM with a characteristic bell-shaped concentration-response curve when a 1-Hz HPF was used. The amplitude of the first and second peaks in the FP waveform increased with increasing cell plating density. The second peak of the FP waveform roughly coincided with AP signal at 50% repolarization, and the negative deflection at the second peak of the FP waveform in the presence of E-4031 corresponded to early afterdepolarization and triggered activity. DISCUSSION: FP can be used to assess the QT prolongation and proarrhythmic potential of drug candidates; however, experimental conditions such as HPF frequency are important for obtaining reliable data.

Postnatal changes in vagal control of esophageal muscle contractions in rats.

AIMS: Replacement of smooth muscles by striated muscles occurs in the esophagus during the early postnatal period. The aim of this study was to clarify postnatal changes in vagal control of esophageal muscle contractions in rats. MAIN METHODS: An isolated segment of the neonatal rat esophagus was placed in an organ bath and the contractile responses were recorded using a force transducer. KEY FINDINGS: Electrical stimulation of the vagus trunk evoked a biphasic contractile response in the neonatal esophageal segment. The first and second components of the contractions were inhibited by α-bungarotoxin and atropine, respectively. Ganglion blockers, hexamethonium and mecamylamine, did not affect vagally mediated contractions. The first component gradually enlarged with age in days, whereas the second component declined during the first week after birth. Application of d-tubocurarine or acetylcholine caused an apparent contraction in the esophageal striated muscle at postnatal day 0, but responses to these drugs were not observed at 1 week after birth. The neonatal esophagus expressed the γ-subunit of nicotinic acetylcholine receptors. In contrast, the ε-subunit was dominantly expressed in the adult esophagus. SIGNIFICANCE: The vagus nerves directly innervate both the esophageal striated muscles and smooth muscles in the early neonatal period. During the process of muscle rearrangement, the property of the striated muscles is altered substantially. The specific features of striated muscles in the neonatal rat esophagus might compensate for immature formation of neuromuscular junctions. Unsuccessful conversion of the striated muscle property during postnatal muscle rearrangement would be related to disorders of esophageal motility.