Frederick Sannajust

Cardiac drug-drug interaction between HCV-NS5B pronucleotide inhibitors and amiodarone is determined by their specific diastereochemistry

Severe bradycardia/bradyarrhythmia following coadministration of the HCV-NS5B prodrug sofosbuvir with amiodarone was recently reported. Our previous preclinical in vivo experiments demonstrated that only certain HCV-NS5B prodrugs elicit bradycardia when combined with amiodarone. In this study, we evaluate the impact of HCV-NS5B prodrug phosphoramidate diastereochemistry (D-/L-alanine, R-/S-phosphoryl) in vitro and in vivo. Co-applied with amiodarone, L-ala,SP prodrugs increased beating rate and decreased beat amplitude in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), but D-ala,RP produgs, including MK-3682, did not. Stereochemical selectivity on emerging bradycardia was confirmed in vivo. Diastereomer pairs entered cells equally well, and there was no difference in intracellular accumulation of L-ala,SP metabolitesu2009±u2009amiodarone, but no D-ala,RP metabolites were detected. Cathepsin A (CatA) inhibitors attenuated L-ala,SP prodrug metabolite formation, yet exacerbated L-ala,SPu2009+u2009amiodarone effects, implicating the prodrugs in these effects. Experiments indicate that pharmacological effects and metabolic conversion to UTP analog are L-ala,SP prodrug-dependent in cardiomyocytes.

Determination of proarrhythmic effects of compounds in human iPSC-derived cardiomyocytes using FDSS/μCell imaging platform

• Overall, by monitoring Ca2+ transient parameters and the morphology of the Ca2+ and MP signal that elucidate EADs when there is potential for arrhythmias, we were able to predict the proarrhythmic cardiac effect of a variety of drugs. FDSS/μCell is a high-speed acquisition imaging platform (Hamamatsu Ltd., Japan) that allows for simultaneous high-throughput reading under controlled conditions. We evaluated the Ca2+ transients or optical membrane potential changes of hiPSC-CMs (iCells®) in the presence or absence of pharmacological agents known to interfere with cardiac ion channels (e.g. hERG, IKs, Nav1.5, Cav1.2). Ca2+sensitive fluorescence dyes (Codex ACTOne® and EarlyTox®) and a membrane potential dye (FLIPR MP®) were tested. We were able to detect acute and delayed drug effects, quantify and report druginduced early-after depolarizations (EAD)-like waveforms, ectopic cardiomyocyte beats and changes in beating rate, from a variety of agents. Cardiovascular drugs, such as dofetilide and D,L-sotalol, exhibited EAD-like signals at 3nM and 10μM, respectively. CNS drugs, such as haloperidol and sertindole, exhibited EAD-like signals and ectopic beats at 30nM and 1μM, respectively. Other drugs, such as astemizole, solifenacin, and moxifloxacin, exhibited similar arrhythmias at 30nM, 3μM, and 300μM, respectively. Our data suggest that the membrane potential and intracellular Ca2+ signal are tightly coupled, supporting the idea that the EAD-like signals reported are the accurate representation of an EAD signal of the cardiac action potential. Finally, the EAD Ca2+ signal was well correlated to reported clinical TdP arrhythmias at relevant concentrations.