Kohji Tanaka

CSAHi study: Evaluation of multi-electrode array in combination with human iPS cell-derived cardiomyocytes to predict drug-induced QT prolongation and arrhythmia — Effects of 7 reference compounds at 10 facilities

INTRODUCTION Drug-induced QT prolongation is a major safety issue during drug development because it may lead to lethal ventricular arrhythmias. In this study, we evaluated the utility of multi-electrode arrays (MEA) with human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) to predict drug-induced QT prolongation and arrhythmia. METHODS Ten facilities evaluated the effects of 7 reference drugs (E-4031, moxifloxacin, flecainide, terfenadine, chromanol 293B, verapamil, and aspirin) using a MED64 MEA system with commercially available hiPS-CMs. Field potential duration (FPD), beat rate, FPD corrected by Fridericias formula (FPDc), concentration inducing FPDc prolongation by 10% (FPDc10), and incidence of arrhythmia-like waveform were evaluated. RESULTS The inter-facility variability of absolute values before drug application was similar to the intra-facility variability for FPD, beat rate, and FPDc. The inter-facility variability of FPDc10 for 5 reference drugs ranged from 1.8- to 5.8-fold. At all 10 facilities, E-4031, moxifloxacin, and flecainide prolonged FPDc and induced arrhythmia-like waveforms at concentrations 1.8- to 6.1-fold higher than their FPDc10. Terfenadine prolonged FPDc and induced beating arrest at 8.0 times the FPDc10. The average FPDc10 values for E-4031, moxifloxacin, and terfenadine were comparable to reported plasma concentrations that caused QT prolongation or Torsade de Pointes in humans. Chromanol 293B, a IKs blocker, also prolonged FPDc but did not induce arrhythmia-like waveforms, even at 7.4 times the FPDc10. In contrast, verapamil shortened FPDc and aspirin did not affect FPDc or FP waveforms. DISCUSSION MEA with hiPS-CMs can be a generalizable method for accurately predicting both QT prolongation and arrhythmogenic liability in humans.

Syk-dependent signaling pathways in neutrophils and macrophages are indispensable in the pathogenesis of anti-collagen antibody-induced arthritis

Spleen tyrosine kinase (Syk) is associated with Fcγ receptors (FcγRs) and transmits activation signals through FcγRs in myeloid cells. Thus, application of drugs to inhibit Syk activity can affect the development of immune diseases mediated by autoantibodies, while unexpected systemic effects by the inhibition may be concerned because Syk has multiple physiological functions. We used tamoxifen-inducible systemic conditional Syk knockout (KO) mice to evaluate the role of Syk in the pathogenesis of autoimmune arthritis and to investigate the systemic effects of Syk deletion. In a collagen antibody-induced arthritis model, Syk KO mice were almost completely protected from disease induction and showed significantly attenuated accumulation of neutrophils and macrophages in the joints. Syk-deleted macrophages showed less IL-6 and MCP-1 production upon FcγR ligation and exhibited reduced FcγR-mediated phagocytosis in vitro. Syk-deleted macrophages produce more RANTES upon FcγR ligation, indicating a Syk-independent signaling through the FcγR. We further found that both wild-type and Syk-deleted macrophages induced neutrophil chemotaxis upon FcγR ligation in vitro, and air-pouch model demonstrated that Syk-deleted neutrophils have a potential to infiltrate into local tissues in response to collagen and anti-collagen antibodies. However, Syk-deleted neutrophils exhibited greatly decreased neutrophil extracellular traps formation and FcγR-mediated phagocytosis. Our results indicated that Syk deficiency rendered mice completely unresponsive to immune activation by anti-collagen antibodies with disabling one pathway of FcγR-mediated signaling that was crucial for arthritis induction.

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

ABSTRACT 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. HIGHLIGHTShiPS‐CMs/MEA accurately predicted reference compounds potential for arrhythmia.Gene expressions showed high correlation among the facility and inter‐facility.This system is suitable as a new non‐clinical testing of drugs cardiac liabilities.

Posterior Lens Rupture in Rats

Rupture of the posterior lens capsule with extrusion of lens fibers or entire lens cortex and nucleus was found in Sprague-Dawley rats. The lesion was detected during an ophthalmoscopic examination as round shadows that interfered with the view of the fundus. By slit-lamp biomicroscopic and dissecting microscopic examinations, the shadows were related to opaque plaques located on the posterior capsule. By light microscopic examination, the posterior lens capsule was ruptured and lens fibers extruded into the vitreous. Mononuclear and multinucleated giant cells were present at the site of rupture, however, few other inflammatory responses were present. Although the incidence and pathogenesis of the lesion is unknown, the lesion should be considered along with other spontaneous ocular changes when evaluating safety assessment studies.

Down regulation of epidermal growth factor receptors in rat hepatocytes treated with clofibric acid

The effect of clofibric acid (CA), a peroxisome proliferator and a non-genotoxic hepatocarcinogen was investigated on epidermal growth factor (EGF) receptors in hepatocytes of female Sprague-Dawley rats treated at a dose of 9000 ppm in a diet for up to 13 weeks. Hepatocyte plasma membranes were isolated in Weeks 1 and 13, and assayed with [125I]EGF. The binding of EGF to the hepatocyte plasma membranes was reduced in Week 1 as a result of decreased number of low-affinity receptors. The fall of binding capacity was further evident in Week 13, which was associated with decreased numbers of both high- and low-affinity receptors. The equilibrium dissociation constant remained unchanged either in Week 1 or 13. These results were in agreement with previous observations of a decreased hepatocyte response to mitogens after prolonged treatment with CA. This suggested that the CA-associated liver tumor promoting effect is related to its ability to decrease the number of EGF receptors and the resultant aberrant growth environment.