The JTpeak (JTp) interval has been proposed as a complementary, sensitive, and more specific biomarker than the QT interval for assessing proarrhythmic risk. It aims to improve the differentiation between drugs that selectively inhibit hERG channels (associated with high proarrhythmic risk) and those that affect multiple cardiac ion channels (associated with minimal or no risk).As previously reported by Darpo et al. in human (J. Clin. Pharmacol., 2019;60:125–139), the present study aimed to evaluate the effects of five QT-prolonging reference drugs, namely moxifloxacin, dofetilide, dolasetron, ondansetron and quinine, on JTp interval in telemetered conscious dogs (n = 7). PR and QRS intervals were also measured to reflect the effects on other cardiac ion channels. Levocetirizine, a QT-negative control drug, was included for comparison. Each compound was administered orally using a crossover design at two dose levels, with ECG monitoring conducted over a 24-h period post-dosing. Dedicated pharmacokinetic sessions were performed to determine corresponding plasma exposures, and sensitivity parameters were calculated for the new parameter JTp. JTp interval was individually corrected (JTpc) for heart rate variations.Compared to vehicle, dofetilide (0.03 and 0.15 mg/kg), moxifloxacin (30 and 100 mg/kg), and ondansetron (10 and 30 mg/kg) produced dose-dependent increases in JTpc interval, consistent with predominant hERG channel blockade. In contrast, dolasetron (6 and 20 mg/kg) and quinine (20 mg/kg) did not affect the JTpc interval. Notably, dolasetron increased both QRS and PR intervals, reflecting its inhibitory effects on sodium and calcium currents. Quinine also showed a slight increase in QRS interval. As expected, levocetirizine (2 and 10 mg/kg) showed no effect on ECG intervals, at any dose or timepoint. Total plasma drug concentrations for each compound increased dose-proportionally.In summary, this study demonstrated that drugs with selective hERG-blocking properties specifically prolong the JTp interval. In contrast, drugs with mixed ion channel-blocking activity tend to prolong other ECG intervals, but not the JTp interval. These findings support the relevance of JTp interval assessment as a valuable parameter to complement QT interval measurements in cardiovascular safety pharmacology studies, enhancing the specificity and sensitivity of proarrhythmic risk evaluation.

The recently approved ICH E14/S7B Q&As allow sponsors to forgo a dedicated clinical QT study together with submission of high-quality in vitro hERG (human Ether-à-go-go-Related Gene) and in vivo QTc (corrected QT interval) data that support a nonclinical double-negative, when paired with satisfactory clinical QTc data. This has heightened the interest of drug developers and regulators in utilizing nonclinical data from in vivo QT studies to complement clinical ECG data for an integrated assessment of proarrhythmia risk. In this scenario, it's crucial to demonstrate no QTc prolongation in a nonclinical study with sufficient power to detect an effect of a similar magnitude as a dedicated clinical QT study to mitigate against a potential false negative result, particularly for E14 Q6.1. While some nonclinical in vivo QTc study designs may have limited ability to achieve this level of sensitivity/power based on statistical methods, the utilization of concentration-QTc (C-QT) analysis of nonclinical data may enhance sensitivity to enable exclusion of a QTc effect of regulatory concern. Moreover, in instances where in vivo QTc prolongation is observed, C-QT will facilitate translation to clinical assessment and estimation of a clinical safety margin. To improve consistency and harmonization across the industry for the conduct of nonclinical C-QT analysis, subject matter experts from Pharma and CROs (Contract Research Organizations) are collaborating to review current methodologies and pros/cons/limitations to establish best practices for this analysis. These will be presented along with responses from a survey to spotlight the current state-of-the-science to position use of nonclinical double-negative data together with Phase 1 ECG data for substitution of the dedicated clinical QT study per ICH E14/S7B Q&As. Most safety pharmacology groups have some experience with C-QT modeling within their companies for internal decision making. The Linear Mixed Effect Model (LME) is most commonly used with ΔQTc (change from predose) as the dependent variable extracted to match 5–7 PK sample timepoints collected from the same animals in a separate dosing/PK phase. These or similar methods have been utilized to perform C-QT analysis across species with positive (e.g. moxifloxacin) and negative (e.g. levocetirizine) QTc-prolonging drugs.

The gold standard methods used to assess cardiorespiratory system in rats, whether telemetry implants or whole-body plethysmography (WBP), have their shortcomings. One method requires surgery, while the other involves the isolation of rats. In a 3Rs approach, alternatives such as the use of non-invasive telemetry jackets, allowing simultaneous cardiorespiratory assessment, without hemodynamic parameters evaluation have been considered. No peer-reviewed publication to date has investigated the concordance between cardiorespiratory parameters recorded simultaneously via the jackets and the reference methods, following the administration of a pharmacological compound. Goal: to test and potentially validate the telemetry jackets by comparing data generated using this device with those obtained using gold standard methods at the same time, in the same animal, in an integrative interpretation approach. We compared cardiac (heart rate (HR)) and respiratory (respiratory rate (RR), minute ventilation (MV), tidal volume (TV)) parameters recorded via the invasive (DSI implant), non-invasive (Jacket DECRO) telemetry systems, and via the WBP (Buxco + Notocord HEM), simultaneously in the same male rats (n = 8rats/ pharmacological compound in a cross-over design). The recording lasted 7 h after the per-os administration of vehicle or pharmacological reference compounds tested (Ivabradine or Theophylline) at 3 doses. A generalized linear model was used to assess the effects of the pharmacological compounds. The Bland-Altman method was used to study the agreement between the jackets and the two reference methods. The jackets and reference methods both captured the expected pharmacological effects. For Ivabradine, a significant dose-dependent decrease in HR was observed, while no changes were noted in respiratory variables. Theophylline induced a dose-dependent increase in heart rate and RR. No significant change on TV and MV was noticed with the WBP, whereas a significant increase was shown with the jackets. Bland-Altman analysis revealed an increasing discrepancy in high TV, RR and MV values between the two devices under the experimental conditions of the studies. Jackets can be used as an alternative to implanted telemetry for recording HR without hemodynamic parameters. Although the two respiratory methods detect pharmacological effects, further investigation is needed to determine what is the current state of the respiratory parameters.

Since their implementation, the ICH S7B and E14 guidelines have been successful in that no new approved drugs have been withdrawn from the market due to unanticipated risk of Torsade de Pointe (TdP). While hERG block and QTc prolongation biomarkers are indeed sensitive, they are not specific as multiple drugs block hERG and/or prolong QTc but do not cause TdP. The J-Tpeak interval (JTp) of the ECG has recently been proposed as a novel clinical biomarker to differentiate selective hERG blockers from multi cardiac ion channels inhibitors. Therefore, the present study aimed at evaluating the effects of dofetilide (pure hERG blocker) and dolasetron (balanced ion-channel blocking drug) on QT, JTp and Tpeak-to-Tend (TpTe) Intervals in telemetered conscious dogs, in comparison with levocetirizine, used as a QT negative control drug. The experiments were carried out using 7 dogs previously instrumented with a telemetry implant. Using a cross-over design, each drug was administered at two doses (0.03/0.15, 6/20, 2/10 mg/kg p.o. for dofetilide, dolasetron and levocetirizine, respectively) and ECG monitored over 24 h post-dosing. Dedicated PK sessions were performed to measure corresponding plasma exposure. QT, JTp and TpTe intervals were individually corrected (c) for heart rate variations. Total plasma drug concentrations increased dose-proportionally. Compared to vehicle, dofetilide induced dose-dependent, marked, and long-lasting increases in both QTc and JTpc intervals, confirming predominant hERG blockage by dofetilide. However, no effect was observed on TpTec interval. In contrast, QTc prolongation associated with dolasetron was moderate and transient and no effect on JTpc was observed; interestingly, dolasetron slightly increased TpTec and QRS intervals, as well as PR interval, reflecting its inhibitory effect on calcium and sodium currents. As expected, levocetirizine had no effect, at any dose or timepoint post-dosing. To summarize, pure hERG-blocking drug (such as dofetilide) prolonged both QTc and JTpc, whereas drug with mixed ion channel blockage (such as dolasetron) prolonged QTc but not JTpc. In conclusion, evaluating the effects of a drug on the J-Tpeak interval may be relevant in cardiovascular safety pharmacology studies to complement the QT interval as a sensitive and more specific biomarker for proarrhythmic risk.

OBJECTIVE:Nutrient-stimulated gut hormone peptide YY3-36 (PYY3-36) selectively activates the neuropeptide Y2 receptor (NPY2R) and reduces energy intake in humans. We describe the discovery and pharmacology of the long-acting NPY2R agonist BI 1820237 and its potential bodyweight-lowering efficacy alone and in combination with the glucagon receptor (GCGR)/glucagon-like peptide-1 receptor (GLP-1R) dual agonist survodutide. METHODS & RESULTS:BI 1820237 dose-dependently reduced food intake and gastric emptying in lean mice. Significant bodyweight reductions were not observed with BI 1820237 alone in diet-induced obese mice, however combination with survodutide led to bodyweight reduction of 22% which was significantly (p < 0.01) greater than the 17% bodyweight reduction with survodutide alone. Regression-based interaction analysis demonstrated that BI 1820237 increased the efficacy of survodutide by 265% at an ED50 of 11.7 nmol/kg over a range of dose combinations. CONCLUSION:Synergistic NPY2R and GCGR/GLP-1R agonism provides an attractive mode of action for clinically relevant weight loss in patients with obesity.

The assessment of drug-induced QT interval prolongation and associated proarrhythmic risks, such as Torsades de Pointes (TdP), has evolved significantly over the past decades. This review traces the development of nonclinical QT evaluation, highlighting key milestones and innovations that have shaped current practices in cardiac safety assessment. The emergence of regulatory guidelines, including International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) S7B, established a nonclinical framework for evaluating drug effects on cardiac repolarization, addressing concerns raised by drug withdrawals in the 1990s. Advances in in vitro, in vivo, and in silico models have enhanced the predictive accuracy of nonclinical studies, with the hERG assay and telemetry-based animal models becoming gold standards. Recent initiatives, such as the Comprehensive in vitro Proarrhythmia Assay (CiPA) and the Japan iPS Cardiac Safety Assessment (JiCSA), emphasize integrating mechanistic insights from human-derived cardiomyocyte models and computational approaches to refine risk predictions. The 2020s mark a shift toward integrated nonclinical-clinical risk assessments, as exemplified by the ICH E14/S7B Questions and Answers. These highlight the need of best practices for study design, data analysis, and interpretation to support regulatory decision-making. Furthermore, the adoption of New Approach Methodologies (NAMs) and reinforced adherence to 3Rs principles (Reduce, Refine, Replace) reflect a commitment to ethical and innovative safety science. This review underscores the importance of harmonized and translational approaches in cardiac safety evaluation, providing a foundation for advancing drug development while safeguarding patient safety. Future directions include further integration of advanced methodologies and regulatory harmonization to streamline nonclinical and clinical risk assessments.

The ICH E14/S7B Q&As highlighted the need for best practices concerning the design, execution, analysis, interpretation, and reporting of the in vivo non‐rodent QT assay as a component of the integrated risk assessment to potentially support a TQT waiver or substitute. We conducted a dog telemetry study to assess the effects on QTc of six reference compounds (five positive and one negative) previously evaluated by Darpo et al. (2015) in humans. The sensitivity of the assay to detect QTc increases was determined, and exposure–response analysis was performed, as done in clinical practice. By‐timepoint analysis showed QTc prolongation induced by moxifloxacin, dofetilide, dolasetron, ondansetron, and quinine within human relevant plasma exposures ranges. Moreover, a hysteresis was observed for quinine. As expected, levocetirizine showed no statistically significant effect on QTc across a range of exposure, well exceeding the therapeutic Cmax. Power analyses confirmed the study ability to detect statistically significant QTc changes of less than 10 milliseconds with 80% probability, even with a sample size as low as n = 4 animals. Finally, concentration‐QTc modeling enabled to predict the minimal plasma concentration needed to detect a 10 milliseconds QTc prolongation, including for quinine. The comparison with clinical available data supported the relevance of dogs under these experimental conditions as a robust translational predictor of drug‐induced QTc prolongation in humans as a key pillar of the integrated risk assessment.

AIM:To describe the biomarker strategy that was applied to select survodutide (BI 456906), BI 456908 and BI 456897 from 19 dual glucagon receptor (GCGR)/ glucagon-like peptide-1 receptor (GLP-1R) agonists for in-depth pharmacological profiling, which led to the qualification of survodutide as the clinical development candidate. MATERIALS AND METHODS:Potencies to increase cyclic adenosine monophosphate (cAMP) were determined in Chinese hamster ovary (CHO)-K1 cells stably expressing human GCGR and GLP-1R. Agonism for endogenously expressed receptors was investigated in insulinoma cells (MIN6) for mouse GLP-1R, and in rat primary hepatocytes for the GCGR. In vivo potencies to engage the GLP-1R or GCGR were determined, measuring improvement in oral glucose tolerance (30 nmol/kg) and increase in plasma fibroblast growth factor-21 (FGF21) and liver nicotinamide N-methyltransferase (NNMT) mRNA expression (100 nmol/kg), respectively. Body weight- and glucose-lowering efficacies were investigated in diet-induced obese (DIO) mice and diabetic db/db mice, respectively. RESULTS:Upon acute dosing in lean mice, target engagement biomarkers for the GCGR and GLP-1R demonstrated a significant correlation (Spearman correlation coefficient with p < 0.05) to the in vitro GCGR and GLP-1R potencies for the 19 dual agonists investigated. Survodutide, BI 456908 and BI 456897 were selected for in-depth pharmacological profiling based on the significant improvement in acute oral glucose tolerance achieved (area under the curve [AUC] of 54%, 57% and 60% vs. vehicle) that was comparable to semaglutide (AUC of 45% vs. vehicle), while showing different degrees of in vivo GCGR engagement, as determined by hepatic NNMT mRNA expression (increased by 15- to 17-fold vs. vehicle) and plasma FGF21 concentrations (increased by up to sevenfold vs. vehicle). In DIO mice, survodutide (30 nmol/kg/once daily), BI 456908 (30 nmol/kg/once daily) and BI 456897 (10 nmol/kg/once daily) achieved a body weight-lowering efficacy from baseline of 25%, 27% and 26%, respectively. In db/db mice, survodutide and BI 456908 (10 and 20 nmol/kg/once daily) significantly lowered glycated haemoglobin (0.4%-0.6%); no significant effect was observed for BI 456897 (3 and 7 nmol/kg/once daily). CONCLUSIONS:Survodutide was selected as the clinical candidate based on its balanced dual GCGR/GLP-1R pharmacology, engaging the GCGR for robust body weight-lowering efficacy exceeding that of selective GLP-1R agonists, while achieving antidiabetic efficacy that was comparable to selective GLP-1R agonism. Survodutide is currently being investigated in Phase 3 clinical trials in people living with obesity.

Conducting safety evaluations of new drugs using conscious animals has been a specialty of our working group for thirty years. In this article, we review the various technical challenges and solutions dealt with over the years to improve both the data quality and the well being of our animal subjects. Of particular interest for us has been the use of telemetry-based data acquisition for conducting studies on cardiovascular (CV) function. This includes the evolving technical aspects of the studies, as well as the development of new applications that take advantage of this technical approach.

Cardiovascular (CV) effects represent a major safety issue during drug development. Typically, this risk is mitigated by preclinical in vivo CV studies, based on which measured CV readouts are analyzed independently. Here, we apply a regression approach to simultaneously integrate CV readouts, i.e., heart rate (HR), mean arterial pressure (MAP) and QT from five dog telemetry studies. These CV studies comprise data on verapamil, captopril, dofetilide, pimobendan, and formoterol, and are combined with the respective dog pharmacokinetic (PK) profiles. A published PK/CV model structure for rats is extended by a semi-mechanistic parameterization of the interaction between HR and QT specific to dogs. This semi-mechanistic modelling approach allows differentiation between compound-independent system-specific parameters (e.g., HR baseline) and compound-specific parameters (e.g., EC50). Compared to previous results in rodents, estimated parameters for dogs indicate stronger dependency of stroke volume on HR, slower HR response, faster QT response and steeper concentration-response relationships. In addition, we illustrate how to practically apply the PK/CV model to derive concentration-response relationships for CV readouts. This approach allows a more detailed quantitative evaluation based on the maximum effect on CV effects (Emax), the EC50, and the steepness of this relation (Hill coefficient) especially for HR-independent effects on QT interval duration (QTc) while taking the systemic feedback into account. This approach also allows to derive plasma concentrations associated with relevant CV effects ("threshold concentration"; CTHRESH). The presented modelling analysis highlights the potential of an integrative evaluation of CV data and provides a framework for obtaining quantitative insights from safety pharmacology evaluations.

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.

Cardiovascular adverse effects in drug development are a major source of compound attrition. Characterization of blood pressure (BP), heart rate (HR), stroke volume (SV), and QT-interval prolongation are therefore necessary in early discovery. It is, however, common practice to analyze these effects independently of each other. High-resolution time courses are collected via telemetric techniques, but only low-resolution data are analyzed and reported. This ignores codependencies among responses (HR, BP, SV, and QT-interval) and separation of system (turnover properties) and drug-specific properties (potencies, efficacies). An analysis of drug exposure-time and high-resolution response- time data of HR and mean arterial blood pressure was performed after acute oral dosing of ivabradine, sildenafil, dofetilide, and pimobendan in HanWistar rats. All data were modeled jointly, including different compounds and exposure and response time courses, using a nonlinear mixed- effects approach. Estimated fractional turnover rates [h(-1), relative standard error (%RSE) within parentheses] were 9.45 (15), 30.7 (7.8), 3.8 ( 13), and 0.115 (1.7) for QT, HR, total peripheral resistance, and SV, respectively. Potencies (nM, %RSE within parentheses) were IC50 = 475 (11), IC50 = 4.01 (5.4), EC50 = 50.6 (93), and IC50 = 47.8 (16), and efficacies (%RSE within parentheses) were I-max = 0.944 (1.7), I-max = 1.00 (1.3), E-max = 0.195 (9.9), and I-max = 0.745 (4.6) for ivabradine, sildenafil, dofetilide, and pimobendan. Hill parameters were estimated with good precision and below unity, indicating a shallow concentration-response relationship. An equilibrium concentration-biomarker response relationship was predicted and displayed graphically. This analysis demonstrates the utility of a model-based approach integrating data from different studies and compounds for refined preclinical safety margin assessment. SIGNIFICANCE STATEMENT A model-based approach was proposed utilizing biomarker data on heart rate, blood pressure, and QT-interval. A pharmacodynamic model was developed to improve assessment of highresolution telemetric cardiovascular safety data driven by different drugs (ivabradine, sildenafil, dofetilide, and pimobondan), wherein system- (turnover rates) and drug-specific parameters (e.g., potencies and efficacies) were sought. The model-predicted equilibrium concentration-biomarker response relationships and was used for safety assessment (predictions of 20% effective concentration, for example) of heart rate, blood pressure, and QT-interval.

We previously demonstrated that intracardiac delivery of autologous peripheral blood‐derived CD34+ stem cells (SCs), mobilized by granulocyte‐colony stimulating factor (G‐CSF) and collected by leukapheresis after myocardial infarction, structurally and functionally repaired the damaged myocardial area. When used for cardiac indication, CD34+ cells are now considered as Advanced Therapy Medicinal Products (ATMPs). We have industrialized their production by developing an automated device for ex vivo CD34+‐SC expansion, starting from a whole blood (WB) sample. Blood samples were collected from healthy donors after G‐CSF mobilization. Manufacturing procedures included: (a) isolation of total nuclear cells, (b) CD34+ immunoselection, (c) expansion and cell culture recovery in the device, and (d) expanded CD34+ cell immunoselection and formulation. The assessment of CD34+ cell counts, viability, and immunophenotype and sterility tests were performed as quality tests. We established graft acceptance criteria and performed validation processes in three cell therapy centers. 59.4 × 106 ± 36.8 × 106 viable CD34+ cells were reproducibly generated as the final product from 220 ml WB containing 17.1 × 106 ± 8.1 × 106 viable CD34+ cells. CD34+ identity, genetic stability, and telomere length were consistent with those of basal CD34+ cells. Gram staining and mycoplasma and endotoxin analyses were negative in all cases. We confirmed the therapeutic efficacy of both CD34+‐cell categories in experimental acute myocardial infarct (AMI) in immunodeficient rats during preclinical studies. This reproducible, automated, and standardized expansion process produces high numbers of CD34+ cells corresponding to the approved ATMP and paves the way for a phase I/IIb study in AMI, which is currently recruiting patients. Stem Cells Translational Medicine 2019;8:822&832