Scientific Review of the Proarrhythmic Risks of Oligonucleotide Therapeutics: Are Dedicated ICH S7B/E14 Studies Needed for Low-Risk Modalities?

Oligonucleotide therapeutics (ONTs) represent a new modality with unique pharmacological and chemical properties that modulate gene expression with a high degree of target specificity mediated by complementary Watson-Crick base pair hybridization. To date, the proarrhythmic assessment of ONTs has been influenced by International Conference on Harmonization (ICH) E14 and S7B guidance. To document current hERG/QTc evaluation practices, we reviewed US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) Approval Packages (source: PharmaPendium.com) and collated preclinical and clinical studies for 17 marketed ONTs. In addition, clinical QTc data from 12 investigational ONTs were obtained from the literature. Of the marketed ONTs, eight were tested in the hERG assay with no inhibitory effect identified at the top concentration (range: 34-3,000 µM) tested. Fourteen of the ONTs were evaluated in nonhuman primate cardiovascular studies with 11 of them in dedicated telemetry studies. No effect on QTc intervals were observed (at high exposure multiples) in all studies. Clinically, four ONTs were evaluated in TQT studies; an additional six ONTs were assessed by concentration-QTc interval analysis, and six by routine safety electrocardiogram monitoring. None of the clinical studies identified a QTc prolongation risk; the same was true for the 12 investigational ONTs. A search of the FDA Adverse Event Database indicated no association between approved ONTs and proarrhythmias. Overall, the collective weight of evidence from 29 ONTs demonstrate no clinical proarrhythmic risk based on data obtained from ICH S7B/E14 studies. Thus, new ONTs may benefit from reduced testing strategies because they have no proarrhythmic risk, a similar cardiac safety profile as monoclonal antibodies, proteins, and peptides.

Assessment of sarcomere shortening and calcium transient in primary human and dog ventricular myocytes.

Understanding translation from preclinical observations to clinical findings is important for evaluating the efficacy and safety of novel compounds. Of relevance to cardiac safety is profiling drug effects on cardiomyocyte (CM) sarcomere shortening and intracellular Ca2+ dynamics. Although CM from different animal species have been used to assess such effects, primary human CM isolated from human organ donor heart represent an ideal non-animal alternative approach. We performed a study to evaluate primary human CM and have them compared to freshly isolated dog cardiomyocytes for their basic function and responses to positive inotropes with well-known mechanisms. Our data showed that simultaneous assessment of sarcomere shortening and Ca2+-transient can be performed with both myocytes using the IonOptix system. Amplitude of sarcomere shortening and Ca2+-transient (CaT) were significantly higher in dog compared to human CM in the basic condition (absence of treatment), while longer duration of sarcomere shortening and CaT were observed in human cells. We observed that human and dog CMs have similar pharmacological responses to five inotropes with different mechanisms, including dobutamine and isoproterenol (ß-adrenergic stimulation), milrinone (PDE3 inhibition), pimobendan and levosimendan (increase of Ca2+sensitization as well as PDE3 inhibition). In conclusion, our study suggests that myocytes obtained from both human donor hearts and dog hearts can be used to simultaneously assess drug-induced effects on sarcomere shortening and CaT using the IonOptix platform.

Best practice considerations for nonclinical in vivo cardiovascular telemetry studies in non-rodent species: Delivering high quality QTc data to support ICH E14/S7B Q&As.

The ICH E14/S7B Questions and Answers (Q&As) guideline introduces the concept of a "double negative" nonclinical scenario (negative hERG assay and negative in vivo QTc study) to demonstrate that a drug does not produce a clinically relevant QT prolongation (i.e., no QT liability). This nonclinical "double negative" data package, along with negative Phase 1 clinical QTc data, may be sufficient to substitute for a clinical Thorough QT (TQT) study in some specific cases. While standalone GLP in vivo cardiovascular studies in non-rodent species are standard practice during nonclinical drug development for small molecule programs, a variety of approaches to the design, conduct, analysis and interpretation are utilized across pharmaceutical companies and contract research organizations (CROs) that may, in some cases, negatively impact the stringent sensitivity needed to fulfill the new Q&As. Subject matter experts from both Pharma and CROs have collaborated to recommend best practices for more robust nonclinical cardiovascular telemetry studies in non-rodent species, with input from clinical and regulatory experts. The aim was to increase consistency and harmonization across the industry and to ensure delivery of high quality nonclinical QTc data to meet the proposed sensitivities defined within the revised ICH E14/S7B Q&As guideline (Q&As 5.1 and 6.1). The detailed best practice recommendations presented here cover the design and execution of the safety pharmacology cardiovascular study, including optimal methods for acquiring, analyzing, reporting, and interpreting the resulting QTc and pharmacokinetic data to allow for direct comparison to clinical exposures and assessment of safety margin for QTc prolongation.

Improving the in Vivo QTc assay: The value of implementing best practices to support an integrated nonclinical-clinical QTc risk assessment and TQT substitute.

Recent updates and modifications to the clinical ICH E14 and nonclinical ICH S7B guidelines, which both relate to the evaluation of drug-induced delayed repolarization risk, provide an opportunity for nonclinical in vivo electrocardiographic (ECG) data to directly influence clinical strategies, interpretation, regulatory decision-making and product labeling. This opportunity can be leveraged with more robust nonclinical in vivo QTc datasets based upon consensus standardized protocols and experimental best practices that reduce variability and optimize QTc signal detection, i.e., demonstrate assay sensitivity. The immediate opportunity for such nonclinical studies is when adequate clinical exposures (e.g., supratherapeutic) cannot be safely achieved, or other factors limit the robustness of the clinical QTc evaluation, e.g., the ICH E14 Q5.1 and Q6.1 scenarios. This position paper discusses the regulatory historical evolution and processes leading to this opportunity and details the expectations of future nonclinical in vivo QTc studies of new drug candidates. The conduct of in vivo QTc assays that are consistently designed, executed and analyzed will lead to confident interpretation, and increase their value for clinical QTc risk assessment. Lastly, this paper provides the rationale and basis for our companion article which describes technical details on in vivo QTc best practices and recommendations to achieve the goals of the new ICH E14/S7B Q&As, see Rossman et al., 2023 (this journal).

Evaluation of levocetirizine in beagle dog and cynomolgus monkey telemetry assays: Defining the no QTc effect profile by timepoint and concentration-QTc analysis.

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.

Time Is a Critical Factor When Evaluating Oligonucleotide Therapeutics in hERG Assays.

In accord with International Conference on Harmonization S7B guidelines, an in vitro human ether-a-go-go-related gene (hERG) assay is one component of an integrated risk assessment for delayed ventricular repolarization. Function of hERG could be affected by direct (acute) mechanisms, or by indirect (chronic) mechanisms. Some approved oligonucleotide therapeutics had submitted hERG data to regulatory agents, which were all collected with the same protocol used for small-molecule testing (incubation time <20 min; acute), however, oligonucleotides have unique mechanisms and time courses of action (indirect). To reframe the hERG testing strategy for silencing RNA (siRNA), an investigation was performed to assess the time course for siRNA-mediated inhibition of hERG function and gene expression. Commercially available siRNAs of hERG were evaluated in a stable hERG-expressed cell line by whole-cell voltage clamp using automated electrophysiology and polymerase chain reaction. In the acute hERG study, no effects were observed after treatment with 100 nM siRNA for 20 min. The chronic effects of 100 nM siRNAs on hERG function were evaluated and recorded over 8-48 h following transfection. At 8 h there was no significant effect, whereas 77% reduction was observed at 48 h. Measurement of hERG mRNA levels demonstrated a 79% and 93% decrease of hERG mRNA at 8 and 48 h, respectively, consistent with inhibition of hERG transcription. The results indicate that an anti-hERG siRNA requires a long exposure time (48 h) in the hERG assay to produce a maximal reduction in hERG current; short exposures (20 min-8 h) had no effect. These findings imply that off-target profiling of novel oligonucleotides could benefit from using hERG protocol with long incubation times to de-risk potential off-target (indirect) effects on the hERG channel. This hERG assay modification may be important to consider if the findings are used to support an integrated nonclinical-clinical risk assessment for QTc (the duration of the QT interval adjusted for heart rate) prolongation.

The Challenges of Predicting Drug-Induced QTc Prolongation in Humans.

The content of this article derives from a Health and Environmental Sciences Institute (HESI) consortium with a focus to improve cardiac safety during drug development. A detailed literature review was conducted to evaluate the concordance between nonclinical repolarization assays and the clinical thorough QT (TQT) study. Food and Drug Administration and HESI developed a joint database of nonclinical and clinical data, and a retrospective analysis of 150 anonymized drug candidates was reviewed to compare the performance of 3 standard nonclinical assays with clinical TQT study findings as well as investigate mechanism(s) potentially responsible for apparent discrepancies identified. The nonclinical assays were functional (IKr) current block (Human ether-a-go-go related gene), action potential duration, and corrected QT interval in animals (in vivo corrected QT). Although these nonclinical assays demonstrated good specificity for predicting negative clinical QT prolongation, they had relatively poor sensitivity for predicting positive clinical QT prolongation. After review, 28 discordant TQT-positive drugs were identified. This article provides an overview of direct and indirect mechanisms responsible for QT prolongation and theoretical reasons for lack of concordance between clinical TQT studies and nonclinical assays. We examine 6 specific and discordant TQT-positive drugs as case examples. These were derived from the unique HESI/Food and Drug Administration database. We would like to emphasize some reasons for discordant data including, insufficient or inadequate nonclinical data, effects of the drug on other cardiac ion channels, and indirect and/or nonelectrophysiological effects of drugs, including altered heart rate. We also outline best practices that were developed based upon our evaluation.

Key Characteristics of Cardiovascular Toxicants.

BACKGROUND: 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. OBJECTIVES: 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. METHODS: An expert working group was convened to discuss mechanisms associated with CV toxicity. RESULTS: 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. DISCUSSION: 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 ≤2.5µm in aerodynamic diameter (PM2.5)] 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.

Validation of using cardioplegic solutions for preserving cardiac function in isolated rabbit heart assays.

INTRODUCTION: Cardioplegic solutions were first developed to preserve heart function during cardiac surgeries and heart transplants but have application in the nonclinical setting. Due to lack of lab space in the vivarium, cardioplegic solution was used to conserve cardiac function for ex-vivo studies performed in a separate building. All studies in this report were conducted with isolated female rabbit hearts (IRHs) via retrograde perfusion using the Langendorff apparatus to investigate if cardioplegia usage affects cardiac function. METHODS: Cardioplegia was achieved with a hyperkalemia (27 mM KCL) solution kept at 4 °C. Cardiac function was assessed by measuring ECG parameters, left ventricular contractility, and coronary flow under constant perfusion pressure. IRHs were cannulated with Krebs Henseleit buffer (KH) either fresh or after cardioplegic solution storage (C-IRH). Three comparisons were performed with and without cardioplegia; (i) direct side-by side studies of cardiac function; (ii) pharmacological responses to typical ion channels blockers, dofetilide, flecainide, and diltiazem; (iii) retrospective evaluation of cardiac functions in a large sample of hearts. RESULTS: In the side-by-side comparisons, cardioplegia-stored IRHs (C-IRH; storage time 90 min) had similar electrocardiographic (ECG) and hemodynamic parameters to fresh-cannulated hearts with KH buffer (KH-IRH). In addition, responses to dofetilide, flecainide, and diltiazem, were similar for C-IRH and KH-IRH hearts. Over the years (2006-2011), baseline data was collected from 79 hearts without cardioplegia and 100 hearts with cardioplegia (C-IRH; storage time 15 min), which showed no meaningful differences in a retrospective analysis. DISCUSSION: Cardiac function was preserved after cardioplegic treatment, however, coronary flow rates were decreased (-19.3%) in C-IRH hearts which indicated an altered coronary vascular tone. In conclusion, storage in cardioplegic solution preserves rabbit cardiac function, a practice that enables heart tissues to be collected at one site (e.g., vivarium) and transported to a laboratory in a separate location.

Evaluation of moxifloxacin in canine and non-human primate telemetry assays: Comparison of QTc interval prolongation by timepoint and concentration-QTc analysis.

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.

Comprehensive in vitro pro-arrhythmic assays demonstrate that omecamtiv mecarbil has low pro-arrhythmic risk.

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.

The in vivo QTc core assay: An evaluation of QTc variability, detection sensitivity and implications for the improvement of conscious dog and non-human primate telemetry studies.

The ICH S7B guideline describes the requirement to conduct an in vitro IKr (hERG) and in vivo QTc assay for human risk assessment of new drug products, but the guidance is devoid of recommendations on study execution or quality. In the absence of standard practice, multiple study designs and experimental approaches have been utilized, especially with the nonclinical QTc assay. Since 2009, our approach to the in vivo QTc assay has been consistent for small molecules and yields reproducible and sensitive levels for QTc signal detection. Our database and experience indicate that nonrodent telemetry studies can achieve high sensitivity and a calculated metric of study power can be used to indicate study quality. Using a retrospective statistical power analysis of multiple studies (n = 14 dog; n = 6 NHP), the detection sensitivity for a specific study design (N = 8; double Latin square cross-over) was determined. The output of the power analysis is the minimal detectable effect at 80% power and a 95% probability level. The design provided an average sensitivity to detect a 4.7 (2.0%) and 6.5 (1.9%) msec QTcI change in dog and NHP, respectively. These findings suggest that this experimental approach has a consistent and reproducible sensitivity to enable a robust QTcI risk evaluation and can be used confidently to support an integrated nonclinical-clinical pro-arrhythmia risk assessment. The inclusion of power analysis (i.e., QTc sensitivity) data in a regulatory submission provides key information to critical stakeholders about the quality of the in vivo QTc assessment and its value for human safety testing.

The Effects of Repeat-Dose Doxorubicin on Cardiovascular Functional Endpoints and Biomarkers in the Telemetry-Equipped Cynomolgus Monkey.

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.

Time for a Fully Integrated Nonclinical-Clinical Risk Assessment to Streamline QT Prolongation Liability Determinations: A Pharma Industry Perspective.

Defining an appropriate and efficient assessment of drug-induced corrected QT interval (QTc) prolongation (a surrogate marker of torsades de pointes arrhythmia) remains a concern of drug developers and regulators worldwide. In use for over 15 years, the nonclinical International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) S7B and clinical ICH E14 guidances describe three core assays (S7B: in vitro hERG current & in vivo QTc studies; E14: thorough QT study) that are used to assess the potential of drugs to cause delayed ventricular repolarization. Incorporating these assays during nonclinical or human testing of novel compounds has led to a low prevalence of QTc-prolonging drugs in clinical trials and no new drugs having been removed from the marketplace due to unexpected QTc prolongation. Despite this success, nonclinical evaluations of delayed repolarization still minimally influence ICH E14-based strategies for assessing clinical QTc prolongation and defining proarrhythmic risk. In particular, the value of ICH S7B-based "double-negative" nonclinical findings (low risk for hERG block and in vivo QTc prolongation at relevant clinical exposures) is underappreciated. These nonclinical data have additional value in assessing the risk of clinical QTc prolongation when clinical evaluations are limited by heart rate changes, low drug exposures, or high-dose safety considerations. The time has come to meaningfully merge nonclinical and clinical data to enable a more comprehensive, but flexible, clinical risk assessment strategy for QTc monitoring discussed in updated ICH E14 Questions and Answers. Implementing a fully integrated nonclinical/clinical risk assessment for compounds with double-negative nonclinical findings in the context of a low prevalence of clinical QTc prolongation would relieve the burden of unnecessary clinical QTc studies and streamline drug development.

Effects of omecamtiv mecarbil on calcium-transients and contractility in a translational canine myocyte model.

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\L 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\L. Decreases in relaxation velocity and increases in duration were present only at 3 µmol\L. 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.

Inotropic assessment in engineered 3D cardiac tissues using human induced pluripotent stem cell-derived cardiomyocytes in the BiowireTM II platform.

To develop therapeutics for cardiovascular disease, especially heart failure, translational models for assessing cardiac contractility are necessary for preclinical target validation and lead optimization. The availability of stem cell-derived cardiomyocytes (SC-CM) has generated a great opportunity in developing new in-vitro models for assessing cardiac contractility. However, the immature phenotype of SC-CM is a well-recognized limitation in inotropic evaluation, especially regarding the lack of or diminished positive inotropic response to ß-adrenergic agonists. Recent development of 3D engineered cardiac tissues (ECTs) using human induced pluripotent stem cell derived-cardiomyocytes (hiPSC-CM) in the BiowireTM II platform has shown improved maturation. To evaluate their suitability to detect drug-induced changes in cardiac contractility, positive inotropes with diverse mechanisms, including ß-adrenergic agonists, PDE3 inhibitors, Ca2+-sensitizers, myosin and troponin activators, and an apelin receptor agonist, were tested blindly. A total of 8 compounds were evaluated, including dobutamine, milrinone, pimobendan, levosimendan, omecamtiv mecarbil, AMG1, AMG2, and pyr-apelin-13. Contractility was evaluated by analyzing the amplitude, velocity and duration of contraction and relaxation. All tested agents, except pyr-apelin-13, increased contractility by increasing the amplitude of contraction and velocity. In addition, myosin and troponin activators increase contraction duration. These results indicate that ECTs generated in the BiowireTM II platform can identify inotropes with different mechanisms and provides a human-based in-vitro model for evaluating potential therapeutics.

Cardiovascular response to small-molecule APJ activation.

Heart failure (HF) remains a grievous illness with poor prognosis even with optimal care. The apelin receptor (APJ) counteracts the pressor effect of angiotensin II, attenuates ischemic injury, and has the potential to be a novel target to treat HF. Intravenous administration of apelin improves cardiac function acutely in patients with HF. However, its short half-life restricts its use to infusion therapy. To identify a longer acting APJ agonist, we conducted a medicinal chemistry campaign, leading to the discovery of potent small-molecule APJ agonists with comparable activity to apelin by mimicking the C-terminal portion of apelin-13. Acute infusion increased systolic function and reduced systemic vascular resistance in 2 rat models of impaired cardiac function. Similar results were obtained in an anesthetized but not a conscious canine HF model. Chronic oral dosing in a rat myocardial infarction model reduced myocardial collagen content and improved diastolic function to a similar extent as losartan, a RAS antagonist standard-of-care therapy, but lacked additivity with coadministration. Collectively, this work demonstrates the feasibility of developing clinical, viable, potent small-molecule agonists that mimic the endogenous APJ ligand with more favorable drug-like properties and highlights potential limitations for APJ agonism for this indication.

Clinical Implications and Translation of an Off-Target Pharmacology Profiling Hit: Adenosine Uptake Inhibition In Vitro.

Off-target activities of drug candidates observed during in vitro pharmacological profiling frequently do not translate to adverse events (AEs) in human. This could be because off-target activities do not have functional consequences, are not observed at exposures achieved during clinical testing, or may not translate into clinical outcomes. We report clinical consequences of an off-target activity observed during profiling of AMG 337, a selective inhibitor of the mesenchymal-epithelial transition factor being evaluated for treatment of solid tumors. In our screen of 151 potential off-targets, AMG 337 inhibited only adenosine transporter (AT). During clinical trials, headache emerged as the dose-limiting AE in the first-in-human trial. It was thought that headache was caused by extracellular accumulation of adenosine from inhibition of AT by AMG 337 and subsequent adenosine-mediated vasodilation through adenosine receptors (ARs). Further nonclinical studies were performed to evaluate this hypothesis. AMG 337 inhibited AT function in dog and human cells in vitro and dog and human arteries ex vivo. In a dog telemetry study, AMG 337 caused hypotension, which was reduced by pretreatment with theophylline, an AR antagonist. Overall, nonclinical and clinical data suggested that headache was due to cerebral vasorelaxation caused by AMG 337-mediated inhibition of AT. When subjects were advised to drink coffee, an AR antagonist, prior to AMG 337, the severity of headaches was reduced, allowing them to continue treatment. These findings demonstrate the importance of carefully evaluating clinical observations during early drug development and the value of translational nonclinical studies to investigate the mechanism of action driving clinical observations.

Nonclinical safety evaluation of erenumab, a CGRP receptor inhibitor for the prevention of migraine.

Calcitonin gene-related peptide (CGRP) and its receptor have been implicated as a key mediator in the pathophysiology of migraine. Thus, erenumab, a monoclonal antibody antagonist of the CGRP receptor, administered as a once monthly dose of 70 or 140 mg has been approved for the preventive treatment of migraine in adults. Due to the species specificity of erenumab, the cynomolgus monkey was used in the pharmacology, pharmacokinetics, and toxicology studies to support the clinical program. There were no effects of erenumab on platelets in vitro (by binding, activation or phagocytosis assays). Specific staining of human tissues with erenumab did not indicated any off-target binding. There were no erenumab-related findings in a cardiovascular safety pharmacology study in cynomolgus monkeys or in vitro in human isolated coronary arteries. Repeat-dose toxicology studies conducted in cynomolgus monkeys at dose levels up to 225 mg/kg (1 month) or up to 150 mg/kg (up to 6 months) with twice weekly subcutaneous (SC) doses showed no evidence of erenumab-mediated adverse toxicity. There were no effects on pregnancy, embryo-fetal or postnatal growth and development in an enhanced pre-postnatal development study in the cynomolgus monkey. There was evidence of placental transfer of erenumab based on measurable serum concentrations in the infants up to 3 months post birth. The maternal and developmental no-observed-effect level (NOEL) was the highest dose tested (50 mg/kg SC Q2W). These nonclinical data in total indicate no safety signal of concern to date and provide adequate margins of exposure between the observed safe doses in animals and clinical dose levels.