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Telemetry‐based methods are recommended as best practice for monitoring changes in cardiovascular (CV) function in conscious nonrodent species during the development of new chemical entities. Such methods allow for unrestrained data collection in animals over extended time periods with high sensitivity to detect small effects. The CV profile of new drugs is also assessed in repeat‐dose toxicology studies, routinely utilizing non‐invasive blood pressure, heart rate, and ECG measurement methods that require physical or chemical restraint. These methods are limited to a short “snapshot” data collection period and incur physiological stress and behavioral excitement from handling/restraint during data collection. The resultant sympathetic activation impacts heart rate, blood pressure, and ECG intervals, causing increased variability and reduced sensitivity. Nonclinical best practices have been defined for standalone CV telemetry studies to support an integrated QTc risk assessment (ICH E14/S7B Q&A 5.1 & 6.1) using non‐restraint telemetry methods; however, the pharmacological and statistical sensitivity of restraint‐based methods used in repeat‐dose toxicology studies is a gap. This paper retrospectively analyzed two case studies (AMG 319 and AMG 337) in which proprietary small molecules were evaluated by both non‐restraint‐based telemetry and restraint‐based methods. AMG 319 and AMG 337 caused QTc interval prolongation and hypotension, respectively, in telemetry studies, which were also observed clinically with these compounds. However, in toxicology studies in which restraint‐based ECG and blood pressure methods were used, CV effects were missed, blunted, or directionally wrong. These case studies highlight the need for the utilization of unrestrained telemetry methods over restraint‐based methods.

Torsades de Pointes (TdP) is a type of ventricular arrhythmia which could be observed as an unwanted drug-induced cardiac side effect, and it is associated with repolarization abnormalities in single cells. The pharmacological evaluations of TdP risk in previous years mainly focused on the hERG channel due to its vital role in the repolarization of cardiomyocytes. However, only considering drug effects on hERG led to false positive predictions since the drug action on other ion channels can also have crucial regulatory effects on repolarization. To address the limitation of only evaluating hERG, the Comprehensive in Vitro Proarrhythmia Assay initiative has proposed to systematically integrate drug effects on multiple ion channels into drug trial to improve TdP risk assessment. It is not clear how many ion channels are sufficient for reliable TdP risk predictions, and whether differences in IC and Hill coefficient values from independent sources can lead to divergent prediction outcomes. The rationale of this work is to investigate the above two questions using a computationally efficient population of human ventricular cells optimized to favor repolarization abnormality. Our blinded results based on two independent data sources confirm that simulations with the optimized population of human ventricular cell models enable efficient in silico drug screening, and also provide direct observation and mechanistic analysis of repolarization abnormality. Our results show that 1) the minimum set of ion channels required for reliable TdP risk predictions are Nav1.5 (peak), Cav1.2, and hERG; 2) for drugs with multiple ion channel blockage effects, moderate IC variations combined with variable Hill coefficients can affect the accuracy of predictions.

The intrinsic characteristics of oligonucleotides pose a challenge for their assessment in conventional primary in vitro cardiac models, which were designed for the acute application of small molecule agents and are not suitable for transfection and extended culture periods. Conversely, human‐induced pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CM) offer a viable platform for the evaluation of agents over prolonged application and recording times. Our previous experiments demonstrated that a chronic protocol of 48 h is necessary to discern the functional effects of a siRNA targeting hERG in a stable cell line heterologously expressing hERG. To investigate whether a targeted hERG siRNA induces delayed repolarization in hiPSC‐CM, we recorded field potentials (FPs) using a multielectrode array. FP duration (FPD) prolongation was noted as early as 10 min after exposure to moxifloxacin, whereas pentamidine required 24 h to induce FPD prolongation. Transfection with hERG‐targeting siRNA reduced mRNA expression at 6 h post‐transfection. However, FPD prolongation was only observed after 24 h post‐transfection, with significantly larger effects at 48 h, which is indicative of the time needed for turnover of the hERG protein on the plasma membrane. Our findings provide compelling evidence that MEA recordings in hiPSC‐CM can accurately detect disruptions in cardiac repolarization due to various mechanisms that impair hERG channel function, including direct channel blockade, inhibition of protein trafficking, and gene silencing via siRNA. The findings also indicate that indirect mechanisms of hERG knockdown, including gene silencing, require assessment at least 48 h following treatment to detect delayed repolarization in the hiPSC‐CM model.

The in vivo correct QT (QTc) assay is used by the pharmaceutical industry to characterize the potential for delayed ventricular repolarization and is a core safety assay mentioned in International Conference on Harmonization (ICH) S7B guideline. The typical telemetry study involves a dose‐response analysis of QTc intervals over time using a crossover (CO) design. This method has proven utility but does not include direct integration of pharmacokinetic (PK) data. An alternative approach has been validated and is used routinely in the clinical setting that pairs pharmacodynamic (PD) responses with PK exposure (e.g., concentration‐QTc (C‐QTc) analysis. The goal of our paper was to compare the QTc sensitivity of two experimental approaches in the conscious dog and non‐human primate (NHP) QTc assays. For timepoint analysis, a conventional design using eight animals (8 × 4 CO) to detect moxifloxacin‐induced QTc prolongation was compared to a PK/PD design in a subset (N = 4) of the same animals. The findings demonstrate that both approaches are equally sensitive in detecting threshold QTc prolongation on the order of 10 ms. Both QTc models demonstrated linearity in the QTc prolongation response to moxifloxacin dose escalation (6 to 46 ms). Further, comparison with human QTc findings with moxifloxacin showed agreement and consistent translation across the three species: C‐QTc slope values were 0.7‐ (dog) and 1.2‐ (NHP) fold of the composite human value. In conclusion, our data show that dog and NHP QTc telemetry with an integrated PK arm (C‐QTc) has the potential to supplement clinical evaluation and improve integrated QTc risk assessment.

Omecamtiv mecarbil (OM) is a myosin activator (myotrope), developed as a potential therapeutic agent for heart failure with reduced ejection fraction. To characterize the potential pro‐arrhythmic risk of this novel sarcomere activator, we evaluated OM in a series of International Conference on Harmonization S7B core and follow‐up assays, including an in silico action potential (AP) model. OM was tested in: (i) hERG, Nav1.5 peak, and Cav1.2 channel assays; (ii) in silico computation in a human ventricular AP (hVAP) population model; (iii) AP recordings in canine cardiac Purkinje fibers (PF); and (iv) electrocardiography analysis in isolated rabbit hearts (IRHs). OM had low potency in the hERG (half‐maximal inhibitory concentration [IC50] = 125.5 µM) and Nav1.5 and Cav1.2 assays (IC50 > 300 µM). These potency values were used as inputs to investigate the occurrence of repolarization abnormalities (biomarkers of pro‐arrhythmia) in an hVAP model over a wide range of OM concentrations. The outcome of hVAP analysis indicated low pro‐arrhythmia risk at OM concentration up to 30 µM (100‐fold the effective free therapeutic plasma concentration). In the isolated canine PF assay, OM shortened AP duration (APD)60 and APD90 significantly from 3 to 30 µM. In perfused IRH, ventricular repolarization (corrected QT and corrected JT intervals) was decreased significantly at greater than or equal to 1 µM OM. In summary, the comprehensive proarrhythmic assessment in human and non‐rodent cardiac models provided data indicative that OM did not delay ventricular repolarization at therapeutic relevant concentrations, consistent with clinical findings.

In prior clinical studies, levocetirizine (LEVO) has demonstrated no effect on ventricular repolarization (QTc intervals), therefore it is a relevant negative control to assess in nonclinical assays to define low proarrhythmic risk. LEVO was tested in beagle dog and cynomolgus monkey (nonhuman primate [NHP]) telemetry models to understand the nonclinical‐clinical translation of this negative control. One oral dose of vehicle, LEVO (10 mg/kg/species) or moxifloxacin (MOXI; 30 mg/kg/dog; 80 mg/kg/NHP) was administered to instrumented animals (N = 8/species) using a cross‐over dosing design; MOXI was the in‐study positive control. Corrected QT interval values (QTcI) were calculated using an individual animal correction factor. Blood samples were taken for drug exposure during telemetry and for pharmacokinetic (PK) analysis (same animals; different day) for exposure‐response (C‐QTc) modeling. Statistical analysis of QTc‐by‐timepoint data showed that LEVO treatment was consistent with vehicle, thus no effect on ventricular repolarization was observed over 24 h in both species. PK analysis indicated that LEVO‐maximum concentration levels in dogs (range: 12,300–20,100 ng/ml) and NHPs (range: 4090–12,700 ng/ml) were ≥4‐fold higher than supratherapeutic drug levels in clinical QTc studies. Slope analysis values in dogs (0.00019 ms/ng/ml) and NHPs (0.00016 ms/ng/ml) were similar to the human C‐QTc relationship and indicated no relationship between QTc intervals and plasma levels of LEVO. MOXI treatment caused QTc interval prolongation (dog: 18 ms; NHP: 29 ms). The characterization of LEVO in these non‐rodent telemetry studies further demonstrates the value and impact of the in vivo QTc assay to define a “no QTc effect” profile and support clinical safety assessment.

In October 2008, in a public forum organized by the Cardiac Safety Research Consortium and the Health and Environmental Sciences Institute, leaders from government, the pharmaceutical industry, and academia convened in Bethesda, MD, to discuss current challenges in evaluation of short- and long-term cardiovascular safety during drug development. The current paradigm for premarket evaluation of cardiac safety begins with preclinical animal modeling and progresses to clinical biomarker or biosignature assays. Preclinical evaluations have clear limitations but provide an important opportunity to identify safety hazards before administration of potential new drugs to human subjects. Discussants highlighted the need to identify, develop, and validate serum and electrocardiogram biomarkers indicative of early drug-induced myocardial toxicity and proarrhythmia. Specifically, experts identified a need to build consensus regarding the use and interpretation of troponin assays in preclinical evaluation of myocardial toxicity. With respect to proarrhythmia, the panel emphasized a need for better qualitative and quantitative biomarkers for arrhythmogenicity, including more streamlined human thorough QT study designs and a universal definition of the end of the T wave. Toward many of these ends, large shared data repositories and a more seamless integration of preclinical and clinical testing could facilitate the development of novel approaches to both cardiac safety biosignatures. In addition, more thorough and efficient early clinical studies could enable better estimates of cardiovascular risk and better inform phase II and phase III trial design. Participants also emphasized the importance of establishing formal guidelines for data standards and transparency in postmarketing surveillance. Priority pursuit of these consensus-based directions should facilitate both safer drugs and accelerated access to new drugs, as concomitant public health benefits.

Purpose: Doxorubicin-related heart failure has been recognized as a serious complication of cancer chemotherapy. This paper describes a cardiovascular safety pharmacology study with chronic dosing of doxorubicin in a non-human primate model designed to characterize the onset and magnitude of left ventricular dysfunction (LVD) using invasive and non-invasive methods. Methods: Cynomolgus monkeys ( N = 12) were given repeated intravenous injections of doxorubicin over 135 days (19 weeks) with dosing holidays when there was evidence of significantly decreased hematopoiesis; a separate group ( N = 12) received vehicle. Arterial and left ventricular pressure telemetry and cardiac imaging by echocardiography allowed regular hemodynamic assessments and determination of LVD. Blood samples were collected for hematology, clinical chemistry, and assessment of cardiac troponin (cTnI) and N-terminal pro b-type natriuretic peptide (NT-proBNP). Myocardial histopathology was a terminal endpoint. Results: There was variable sensitivity to the onset of treatment effects, for example 25% of doxorubicin-treated animals exhibited LVD (e.g., decreases in ejection fraction) following 50–63 days (cumulative dose: 8–9 mg/kg) on study. All animals deteriorated into heart failure with additional dosing 135 days (total cumulative dose: 11–17 mg/kg). Reductions in arterial pressure and cardiac contractility, as well as QTc interval prolongation, was evident following doxorubicin-treatment. Both cTnI and NT-proBNP were inconsistently higher at the end of the study in animals with LVD. Measurements collected from control animals were consistent and stable over the same time frame. Minimal to mild, multifocal, vacuolar degeneration of cardiomyocytes was observed in 7 of 12 animals receiving doxorubicin and 0 of 12 animals receiving vehicle. Conclusions: This repeat-dose study of doxorubicin treatment in the cynomolgus monkey demonstrated a clinically relevant pattern of progressive heart failure. Importantly, the study revealed how both telemetry and non-invasive echocardiography measurements could track the gradual onset of LVD.

The concept of chemical agents having properties that confer potential hazard called key characteristics (KCs) was first developed to identify carcinogenic hazards. Identification of KCs of cardiovascular (CV) toxicants could facilitate the systematic assessment of CV hazards and understanding of assay and data gaps associated with current approaches. We sought to develop a consensus-based synthesis of scientific evidence on the KCs of chemical and nonchemical agents known to cause CV toxicity along with methods to measure them. An expert working group was convened to discuss mechanisms associated with CV toxicity. The group identified 12 KCs of CV toxicants, defined as exogenous agents that adversely interfere with function of the CV system. The KCs were organized into those primarily affecting cardiac tissue (numbers 1-4 below), the vascular system (5-7), or both (8-12), as follows: 1) impairs regulation of cardiac excitability, 2) impairs cardiac contractility and relaxation, 3) induces cardiomyocyte injury and death, 4) induces proliferation of valve stroma, 5) impacts endothelial and vascular function, 6) alters hemostasis, 7) causes dyslipidemia, 8) impairs mitochondrial function, 9) modifies autonomic nervous system activity, 10) induces oxidative stress, 11) causes inflammation, and 12) alters hormone signaling. These 12 KCs can be used to help identify pharmaceuticals and environmental pollutants as CV toxicants, as well as to better understand the mechanistic underpinnings of their toxicity. For example, evidence exists that fine particulate matter [PM in aerodynamic diameter ( )] air pollution, arsenic, anthracycline drugs, and other exogenous chemicals possess one or more of the described KCs. In conclusion, the KCs could be used to identify potential CV toxicants and to define a set of test methods to evaluate CV toxicity in a more comprehensive and standardized manner than current approaches. https://doi.org/10.1289/EHP9321.

Omecamtiv mecarbil (OM) is a selective cardiac myosin activator (myotrope), currently in Phase 3 clinical investigation as a novel treatment for heart failure with reduced ejection fraction. OM increases cardiac contractility by enhancing interaction between myosin and actin in a calcium‐independent fashion. This study aims to characterize the mechanism of action by evaluating its simultaneous effect on myocyte contractility and calcium‐transients (CTs) in healthy canine ventricular myocytes. Left ventricular myocytes were isolated from canines and loaded with Fura‐2 AM. With an IonOptix system, contractility parameters including amplitude and duration of sarcomere shortening, contraction and relaxation velocity, and resting sarcomere length were measured. CT parameters including amplitude at systole and diastole, velocity at systole and diastole, and duration at 50% from peak were simultaneously measured. OM was tested at 0.03, 0.1, 0.3, 1, and 3 µmol concentrations to simulate therapeutic human plasma exposure levels. OM and isoproterenol (ISO) demonstrated differential effects on CTs and myocyte contractility. OM increased contractility mainly by prolonging duration of contraction while ISO increased contractility mainly by augmenting the amplitude of contraction. ISO increased the amplitude and velocity of CT, shortened duration of CT concurrent with increasing myocyte contraction, while OM did not change the amplitude, velocity, and duration of CT up to 1 µmol Decreases in relaxation velocity and increases in duration were present only at 3 µmol In this translational myocyte model study, therapeutically relevant concentrations of OM increased contractility but did not alter intracellular CTs, a mechanism of action distinct from traditional calcitropes. OM increases cardiac contractility at clinically relevant concentrations without altering intracellular Ca‐transients, a mechanism distinct from traditional calcitropes.