Electrophysiological characterization of drug response in hSC-derived cardiomyocytes using voltage-sensitive optical platforms

Introduction: Voltage-sensitive optical (VSO) sensors offer a minimally invasive method to study the time course of repolarization of the cardiac action potential (AP). This Comprehensive in vitro Proarrhythmia Assay (CiPA) cross-platform study investigates protocol design and measurement variability of VSO sensors for preclinical cardiac electrophysiology assays. Methods: Three commercial and one academic laboratory completed a limited study of the effects of 8 blinded compounds on the electrophysiology of 2 commercial lines of human induced pluripotent stem-cell derived cardiomyocytes (hSC-CMs). Acquisition technologies included CMOS camera and photometry; fluorescent voltage sensors included di-4-ANEPPS, FluoVolt and genetically encoded QuasAr2. The experimental protocol was standardized with respect to cell lines, plating and maintenance media, blinded compounds, and action potential parameters measured. Serum-free media was used to study the action of drugs, but the exact composition and the protocols for cell preparation and drug additions varied among sites. Results: Baseline AP waveforms differed across platforms and between cell types. Despite these differences, the relative responses to four selective ion channel blockers (E-4031, nifedipine, mexiletine, and JNJ 303 blocking IKr, ICaL, INa, and IKs, respectively) were similar across all platforms and cell lines although the absolute changes differed. Similarly, four mixed ion channel blockers (flecainide, moxifloxacin, quinidine, and ranolazine) had comparable effects in all platforms. Differences in repolarisation time course and response to drugs could be attributed to cell type and experimental method differences such as composition of the assay media, stimulated versus spontaneous activity, and single versus cumulative compound addition. Discussion: In conclusion, VSOs represent a powerful and appropriate method to assess the electrophysiological effects of drugs on iPSC-CMs for the evaluation of proarrhythmic risk. Protocol considerations and recommendations are provided toward standardizing conditions to reduce variability of baseline AP waveform characteristics and drug responses

The roles of human induced pluripotent stem cell-derived cardiomyocytes in drug discovery: managing in vitro safety study expectations

Introduction: Human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) preparations are increasingly employed in in vitro cardiac safety studies to support candidate drug selection and regulatory submissions. The value of hiPSC-CM-based approaches depends on their ability to recapitulate the cellular mechanisms responsible for cardiotoxicity as well as overall assay characteristics (thus defining model performance). Different expectations at different drug development stages define the utility of these human-derived models. Areas covered: Herein, the authors review the importance of understanding the functional characteristics of the evolving spectrum of simpler (2D) and more complex (co-cultures, 3D constructs, and engineered tissues) human-derived cardiac preparations, and how their performance may be evaluated based on analytical sensitivity, variability, and reproducibility in order to correctly match preparations with expectations of different safety assays. The need for consensus clinical examples of electrophysiologic, contractile, and structural cardiotoxicities essential for benchmarking human-derived models is also discussed. Expert opinion: It is helpful (but not essential) that hiPSC-CMs preparations fully recapitulate pharmacological responses of native adult human ventricular myocytes when evaluating cardiotoxicity in vitro. Further calibration and model standardization (aligning concordance with clinical findings) are necessary to understand the role of hiPSC-CMs in guiding cardiotoxicity assessments in early drug discovery efforts

T vector velocity: A new ECG biomarker for identifying drug effects on cardiac ventricular repolarization.

BackgroundWe present a new family of ECG biomarkers for assessing drug effects on ventricular repolarization. We show that drugs blocking inward (depolarizing) ion currents cause a relative increase of the T vector velocity (TVV) and accelerate repolarization, while drugs blocking outward ion currents cause a relative decrease of the TVV and delay repolarization. The results suggest a link between the TVV and the instantaneous change of the cellular action potentials that may contribute to bridge the gap between the surface ECG and myocardial cellular processes.MethodsWe measure TVV as the time required to reach X% of the total Trajectory length of the T vector loop, denoted as TrX. Applied to data from two FDA funded studies (22+22 subjects, 5232+4208 ECGs) which target ECG effects of various ion-channel blocking drugs, the TrX effect profiles indicate increasingly delayed electrical activity over the entire repolarization process for drugs solely reducing outward potassium current (dofetilide, moxifloxacin). For drugs eliciting block of the inward sodium or calcium currents (mexiletine, lidocaine), the TrX effect profiles were consistent with accelerated electrical activity in the initial repolarization phase. For multichannel blocking drugs (ranolazine) or drug combinations blocking multiple ion currents (dofetilide + mexiletine, dofetilide + lidocaine), the overall TrX effect profiles indicate a superposition of the individual TrX effect profiles.ResultsThe parameter Tr40c differentiates pure potassium channel blocking drugs from multichannel blocking drugs with an area under the ROC curve (AUC) of 0.90, CI = [0.88 to 0.92]. This is significantly better than the performance of J-Tpeakc (0.81, CI = [0.78 to 0.84]) identified as the best parameter in the second FDA study. Combining the ten parameters Tr10c to Tr100c in a logistic regression model further improved the AUC to 0.94, CI = [0.92 to 0.96].ConclusionsTVV analysis substantially improves assessment of drug effects on cardiac repolarization, providing a plausible and improved mechanistic link between drug effects on ionic currents and overall ventricular repolarization reflected in the body surface ECG. TVV contributes to an enhanced appraisal of the proarrhythmic risk of drugs beyond QTc prolongation and J-Tpeakc

Rechanneling the cardiac proarrhythmia safety paradigm: A meeting report from the Cardiac Safety Research Consortium

, with the intention of moving toward consensus on defining a new paradigm in the field of cardiac safety in which proarrhythmic risk would be primarily assessed using nonclinical in vitro human models based on solid mechanistic considerations of torsades de pointes proarrhythmia. This new paradigm would shift the emphasis from the present approach that strongly relies on QTc prolongation (a surrogate marker of proarrhythmia) and could obviate the clinical Thorough QT study during later drug development. These discussions represent current thinking and suggestions for furthering our knowledge and understanding of the public health case for adopting a new, integrated nonclinical in vitro/in silico paradigm, the Comprehensive In Vitro Proarrhythmia Assay, for the assessment of a candidate drug's proarrhythmic liability, and for developing a public-private collaborative program to characterize the data content, quality, and approaches required to assess proarrhythmic risk in the absence of a Thorough QT study. This paper seeks to encourage multistakeholder input regarding this initiative and does not represent regulatory guidance. (Am Heart J 2014;0:1-9.) A Think Tank sponsored by Cardiac Safety Research Consortium (CSRC), Health and Environmental Sciences Institute (HESI), and Food and Drug Administration (FDA) was convened at FDA Headquarters on July 23, 2013, to discuss a potential new approach to assessing druginduced proarrhythmic risk. The current safety testing paradigm is based primarily on the predictive link between drug-induced in vitro hERG channel blockade and in vivo/clinical QT interval prolongation and torsades de pointes (TdP). Prolongation of the QT interval has been considered as an initiating factor in clinical TdP. Although the current paradigm has largely eliminated new drugs entering the market with unanticipated potential for torsade, it has important limitations and may have led to stopping the development of potentially valuable therapeutics. Therefore, a Comprehensive In vitro Proarrhythmia Assay (CiPA) was proposed as a new paradigm. It should be emphasized that the Think Tank was not designed to seek consensus endorsement of a fully defined and vetted new approach ready for immediate implementation. Rather, its goals were to suggest components of such a paradigm, identify weaknesses and areas for refinement, facilitate transparent stakeholder input and discussions, propose potential member organizations of a collaborative group to develop the specifics that would be needed, and consider the first pragmatic steps. These goals were accomplished, and such next steps are underway. Based on the principles of the US FDA Critical Path Initiative, the CSRC 1 was created to facilitate collaborations among academicians, industry professionals, and regulators to develop consensus approaches addressing cardiac and vascular safety issues that can arise in the development of new medical products. Executive summary The present cardiac safety paradigm (ICH S7B nonclinical guidance Although the present paradigm has largely eliminated the unanticipated discovery of new torsadogenic drugs entering the market, important limitations of the present approach include that block of I Kr alone is often insufficient in predicting delayed repolarization (itself a surrogate marker of proarrhythmia); increases in the QTc interval are highly sensitive but not very specific for predicting ventricular proarrhythmia risk; and there are clinically important drugs that block I Kr at therapeutic plasma concentrations that are not proarrhythmic. The bulk of the presentations and discussions, therefore, revolved around the following proposition: A new cardiac safety paradigm utilizing a novel array of nonclinical proarrhythmia assessments, combined with in silico predictive modelling of cellular electrophysiological effects, could make drug discovery and development efforts more efficient, move the major clinical/regulatory analysis concerning arrhythmogenic potential earlier in the drug discovery and development continuum, enhance the accuracy with which existing and/or new drugs are labelled relative to actual proarrhythmic risks, and increase the output of new chemical entities that benefit patients. The proposed paradigm, labeled the "Comprehensive In vitro Proarrhythmia Assay" (CiPA), is based on an established mechanistic understanding of TdP. To assess overall proarrhythmic risk, CiPA relies upon (a) characterization of electrophysiological effects of evolving or existing drugs on multiple human cardiac currents measured in heterologous expression systems, whose electrophysiological effects will then be integrated in silico by computer models reconstructing human cellular ventricular electrophysiology, and (b) confirmation of the electrophysiological effects in a myocyte assay such as human induced pluripotent stem cell-derived cardiomyocytes. Evaluations of hemodynamic and electrocardiographic (ECG) effects from standard nonclinical cardiovascular in vivo studies (as described in ICH S7A and S7B) will remain part of the new paradigm, along with careful ECG assessment in phase 1 studies to evaluate a drug's effects on ECG intervals (QTc, PR, and QRS durations), atrioventricular conduction, and heart rate. These later studies would confirm that there were no unanticipated clinical ECG changes as compared with the nonclinical testing; if unanticipated changes are found, the reasons for the discrepancy would need to be understood. With this new paradigm in place, the ICH S7B guideline 4 defining hERG as the primary ion channel of focus for proarrhythmia would need to be revised, and the Thorough QT (TQT) study described in ICH E14 guideline