Taro Hihara

A novel method of selecting human embryonic stem cell-derived cardiomyocyte clusters for assessment of potential to influence QT interval.

Physiologically relevant assessment of delayed repolarization is necessary in drug development. In our preliminary experiments on the evaluation using a multielectrode recording system, we had found that the responsiveness of field potential duration (FPD), as QT-like intervals, to hERG channel blockers differed greatly from non-responders to excessive responders in human embryonic stem cell-derived cardiomyocyte clusters. Thus, we report a novel method of selecting clusters suitable for evaluating compounds for the assessment. Clusters were treated with cisapride, a hERG channel blocker, at 100nM, and selected with criteria of 5-20% of corrected FPD (FPDc) prolongation. Then, selected clusters were treated with reference compounds. FPDc was prolonged by blockade of the hERG channel (E-4031 and dl-sotalol) and KvLQT1 channel (chromanol 293B and HMR1556), and by activation of the sodium channel (veratridine) and calcium channel (Bay K8644). FPDc was shortened by calcium channel blockage (verapamil, nifedipine and diltiazem) and by K(ATP) channel activation (pinacidil). Class Ia antiarrhythmic drugs, quinidine and disopyramide, prolonged FPDc. Selected clusters are appropriate for assessing the effects of compounds on ion channels affecting QT intervals. This is the first report of the establishment of an assessment system of potential to influence QT interval, using pharmacologically selected clusters.

E2012-Induced Cataract and Its Predictive Biomarkers

E2012, a gamma secretase modulator without affecting Notch processing, aimed at Alzheimers disease by reduction of amyloid β-42, induced cataract following repeated doses in the rat. Cataract appeared first at week 10-11 of treatment as a posterior subcapsular area with granular/punctate opaque or shiny dots along the suture line, characterized histologically as lenticular fiber degeneration, which eventually coalesced to form a triangular or circular opacity. It was associated with prolonged and sustained elevation of lenticular desmosterol (24-dehydrocholesterol), the final precursor of cholesterol, and decrease in lenticular cholesterol. In vitro studies to investigate the effect of E2012 on cholesterol metabolism demonstrated that E2012 inhibits 3β-hydroxysterol Δ24-reductase (DHCR24) at the final step in the cholesterol biosynthesis. In vivo lenticular concentration of E2012 after 13-week repeated dose with cataract was well above those where inhibition was observed in vitro. There was no cataract formation at doses where desmosterol did not accumulate in the lens. The elevation of desmosterol and decreased cholesterol levels were also seen in the liver and plasma and preceded those in the lens. These results demonstrate that E2012 induces cataract in the rat by inhibiting DHCR24 at the final step of cholesterol synthesis with associated elevation in desmosterol within the lens, preceded by desmosterol changes that would serve as a predictive safety biomarker for lenticular opacity.

Probucol and the cholesterol synthesis inhibitors simvastatin and triparanol regulate I ks channel function differently.

Channels responsible for slowly activating delayed-rectifier potassium current (I Ks) are composed of KCNQ1 and KCNE1 subunits, and these channels play a role in the repolarization of cardiac action potentials. Recently, we showed that the antihyperlipidemic drug probucol, which induces QT prolongation, decreases the I Ks after 24-h treatment. In the present study, we investigated the effects of three cholesterol-lowering agents (probucol, an enhancer of cholesterol efflux; simvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor; and triparanol, a 3β-hydroxysterol-▵24-reductase inhibitor) on cholesterol synthesis, the KCNQ1 current (I KCNQ1), and the I Ks to clarify the differences in the modes of action of these agents on the I Ks. Probucol did not inhibit cholesterol synthesis and had no effect on I KCNQ1, while I Ks decreased after 24-h treatment. Simvastatin inhibited cholesterol synthesis and decreased I KCNQ1 and I Ks. Additionally, the activation kinetics of I Ks became faster, compared with that of control I Ks. Triparanol inhibited cholesterol synthesis but did not reduce I KCNQ1 and I Ks. However, the activation kinetics of I Ks became faster. Our data indicated that the mechanism by which probucol inhibits I Ks was not mediated by the inhibition of cholesterol synthesis but depended on an interaction with the KCNQ1/KCNE1 complex. Meanwhile, the reduction in cholesterol induced by simvastatin and triparanol is one of the mechanisms that affects the kinetics of I ks.