The ICH E14/S7B Q&As emphasize importance of robust in vivo cardiovascular studies, in appropriate non-rodent (dog/NHP) species, to perform an integrated risk assessment. It is well established that the QT-interval is inversely correlated with heart rate (HR) and a reliable method is required to determine heart rate corrected QT-interval (QTc-interval) for accurate assessment of QT liability in nonclinical studies. The individual animal correction method using QT/HR relationship (slope) from each animal's vehicle or baseline data is the recommended best practice. However, this method may not perform adequately in the presence of large HR changes. We evaluated the performance of two different individual animal corrections 1) using vehicle slope (QTcVeh) and 2) using treatment specific slope (QTcT) for two compounds. Hydralazine, a direct vasodilator, was included as it caused a large HR increase but does not have a QT-liability (hERG negative). Dofetilide was included as it is a direct hERG blocker without confounding HR changes. Each compound was tested separately in standard crossover paradigm in dogs (N = 4) at doses of 0.1, 1 and 3 mg/kg (Hydralazine) or 0.01, 0.03 and 0.15 mg/kg (Dofetilide). QTcVeh was calculated using the 24-h QT/HR slope from the vehicle treatment. QTcT was calculated using the 24-h QT/HR slope from the treatment phase at each dose level. Hydralazine caused marked decreases in blood pressure (−6 to −39 mmHg) and increases in HR (+18 to +81 bpm), with significant decreases in QT (−13 to −29 msec) at ≥1 mg/kg. QTcVeh was also increased (+9 to +11 msec), whereas QTcT did not show any statistically significant increase demonstrating adequate correction. Dofetilide did not cause any change in HR but did exhibit dose dependent increases in QT (+12 to +27 msec) as well as both QTcVeh (+10 to +27 msec) and QTcT (+10 to +28 msec). These results show that individual animal correction using the ‘standard’ vehicle slope does not perform adequately in the presence of large magnitude HR increases. In such instances, an alternative approach (e.g. using treatment specific slope) should be considered to prevent a false positive for QTc prolongation.

Previously published work established the correlation between hiPSC-CM APD90 to clinical concentrations of drugs associated with a + 10 ms change in QTc (Kilfoil et al., PMID 34678241). In this prior work, completed at 28 °C, the relationship (expressed in -Log [M] units) between free plasma exposure causing +10 ms QTc (X) and drug concentration causing threshold rate-corrected cAPD90 increase (Y) was described by the linear equation Y = 0.86× - 0.64 (Pearson r2 = 0.762). Here, we investigated the effect of temperature on hiPSC-CM action potential parameters and impact on in vitro to clinical translation. 14 compounds were retested at 37 °C to investigate the impact of physiological temperature on this relationship.1) Repolarization time (APD90 = 408 ± 20 vs 773 ± 69 ms, p < 0.0001) and spontaneous beat rate (61 ± 5 vs 25 ± 3 beats per min, p < 0.0001) were accelerated at 37 °C compared to 28 °C. When recorded at physiological temperature, these parameters are more physiologically relevant (APD90 200–400 ms, beat rate 50–70 per min). 2) Rate-corrected repolarization was also faster at 37 °C than at 28 °C (cAPD90 = 410 ± 21 vs. 512 ± 25 ms, p < 0.001), reflecting the inability to correct for different heart rates and temperature conditions with one eq. 3) Finally, the cAPD90-QTc relationship at 37 °C is described by the equation Y = 0.98× −0.37 (Pearson r2 = 0.8) which is greater than the relationship described by this same subset of compounds at 28 °C (Pearson r2 = 0.74).These results demonstrate that recording temperature has profound effects on the basal electrophysiological function of these cells, and that the correlation between drug effect on hiPSC-CM repolarization and clinical QTc outcome is enhanced at physiological temperature.

Increases in arterial blood pressure (BP) contribute to adverse cardiovascular (CV) outcomes in patients; preclinical effects of a drug on BP are routinely evaluated during the safety pharmacology assessments as outlined in the ICH S7A guidance. A Health and Environmental Sciences Institute (HESI) Consortium initiated a multi-site study with the objective to assess the ability of the standard conscious telemetry instrumented CV dog model to detect drug-induced changes in BP and evaluate translation to human data. The goal of these studies is also to determine the reproducibility and consistency of BP assessment when measured across different laboratories using the same study protocol and recording methodology to detect drug-induced changes in hemodynamics using drugs known to clinically elevate and reduce BP. Animals were chronically instrumented with a BP catheter and ECG electrodes for telemetric collection of hemodynamic and ECG endpoints, respectively. Study endpoints include systolic, diastolic, and mean BP, heart rate, electrocardiogram (ECG), body temperature, and locomotor activity. Drugs evaluated include midodrine (alpha-1 agonist), nifedipine (calcium channel blocker), hydralazine (direct-acting smooth muscle relaxant), prazosin (alpha-1 blocker) and milrinone (phosphodiesterase-3 inhibitor). Drugs were selected based on known pharmacological mechanisms of action, primary cardiovascular effects as well as availability of clinical effect and exposure data. Drugs were evaluated in beagle dogs using a double (8 × 4) Latin square design and administered orally at 3 doses selected to match clinical exposure data with a vehicle control. A full pharmacokinetic profile for each drug was conducted in dogs at doses selected using automated blood sampling (ABS). Initial analysis shows that all 5 positive control drugs show consistent hemodynamic profiles (e.g., BP elevation or reduction) in the dog as seen in humans. These data sets with additional testing at multiple sites will be amenable to further statistical analysis, super-interval analysis and follow-up study endpoint evaluation such as pressure waveform analysis. The results from this chronically instrumented conscious dog model will provide essential information about accuracy and consistency in blood pressure measurement across multiple sites and translation of preclinical BP data to clinical outcomes.

Safety pharmacology (SP) evaluations are required for small molecule drug candidates (ICH-S7A) to identify effects on the cardiovascular, central nervous (CNS), and respiratory systems. Gastrointestinal (GI) effects are common in drug development, manifesting preclinically as emesis (large animals), nonspecific clinical observations, and/or histopathological abnormalities in GI tissues (toxicology studies). Decreases in locomotor activity (LA) are a common finding in rodent CNS SP studies; however, it is often not possible to differentiate primary CNS effects from secondary GI effects, particularly given that rodents are non-emetic. The relationships between GI-related clinical signs and GI pathology in rodent toxicity studies and decreased quantitative LA in rodent SP studies was quantified via contingency tables using Chi-Squared tests. A binary logistic regression was computed to describe the log odds of a decrease in LA when certain GI predictors are present. Data evaluated across similar dose levels for each compound (n = 65) showed a correlation between the presence of GI clinical signs and GI pathology (X^2 = 5.454, p-value = 0.01952. Sensitivity = 50 %, Specificity = 76.23 %), as well as the presence of GI clinical signs and decreased LA (X^2 = 23.265, p-value = 1.411e-06. Sensitivity = 45.45 %, Specificity = 83.06 %). Although the GI pathology and decreased LA correlation was not significant, the specificity for this association was high at 94.54 %, indicating that the there is a strong correlation between negative LA and negative pathology findings (X^2 = 0.50847, p-value = 0.4758. Sensitivity = 7.79 %, Specificity = 94.54 %). A final regression model (reduced to the lowest Akaike Information Criterion [AIC]) identified food consumption and distended abdomen as predictors. Food consumption was a significant predictor of decreased LA (p = 2.16e-05), suggesting that compounds that impact food consumption could also impact animal behavior, including activity levels. The final regression model has an AUC of 0.621 (95 % CI: 0.5533–0.6797); while not exceptionally strong, it is slightly better than random at distinguishing occurrences of decreased LA. When present, the potential contribution of GI effects to decreased LA in SP assessments should be considered in addition to direct effects on the CNS.

Pivotal cardiovascular (CV) safety pharmacology studies using telemetered non-rodent (dog and nonhuman primate (NHP)) models provide key data that enable development of novel therapeutics. Statistical power calculations demonstrate the sensitivity of an experimental model as well as provide rationale for study design including sample size selection. The power of a statistical test is the probability of detecting a signal (e.g. a CV effect) when there truly is a signal. Robust understanding of statistical sensitivity also underpins the confidence in study results, yet systematic power analysis of standard CV studies is currently lacking. We analyzed pooled data from CV telemetry studies in standard cynomolgus monkeys (n = 21) and beagle dog (n = 27), separately, to determine the statistical power of these experimental models. Studies typically utilized a 4 × 4 (dog) or 8 × 4 (NHP) vehicle +3 dose level crossover paradigm. Data were collected for approximately 24 h, and derived results were binned into time intervals for statistical analysis using a linear ANOVA model. The minimum detectable differences (MDD) with 80 % statistical power were calculated for standard parameters (e.g. blood pressure (BP), heart rate (HR), ECG intervals etc). MDDs for dogs, using a N = 4 crossover design, were: BP (5–7 mmHg), HR (10 bpm), QT-interval (9 msec), and QTc-interval (6 msec). MDDs for NHP, using a N = 8 crossover design, were: BP (4–5 mmHg), HR (11 bpm), QT-interval (13 msec), and QTc-interval (9 msec). Additionally, we also report MDDs for alternate groups sizes (e.g. N = 4, 8 and 12) as well as reference intervals of root mean square error (RMSE) as a measure of variability in the studies. Using the 2.5th and 97.5th percentiles of the RMSE, we also report the lower and upper bounds of the MDDs for each parameter. Overall, our results indicate that the nonrodent CV model is a sensitive tool to detect CV risk in early safety studies. Furthermore, the results also demonstrate assay sensitivity of functional endpoints (e.g. QTc MDD <10 msec) and support use of data in the context of ICH E14/S7B Q&As. Lastly, these results will enable informed selection of appropriate models and study designs for CV studies.

Introduction: Corrected QT interval (QTc)is an established biomarker for drug-induced Torsade de Pointe (TdP), but with concerns for a false positive signal. Clinically, JTpc and TpTec have emerged as ECG sub-intervals to differentiate predominant hERG vs. mixed ion channel blocking drugs that prolong QTc. Methods: In a multicentric, prospective, controlled study, different proarrhythmic drug effects on QTc, JTpc and TpTec were characterized with cynomolgus monkeys using telemetry in a Lead II configuration for internal and external telemetry.Drugs and vehicle were administered orally (PO) to group size of 4 to 8 animals, in 4 laboratories. Results: In monkeys, dofetilide (0.03-0.3 mg/kg) was associated with exposure dependent QTc and JTpc increase, but no significant TpTec effect. Similarly, quinidine (2-50 mg/kg) increased QTc and JTpc but did not change TpTec. Mexiletine (1-15 mg/kg) and verapamil (50 mg/kg) did not induce any significant effect on QTc, JTpc or TpTec. Discussion: Clinically, predominant hERG blockers (dofetilide and quinidine) prolong QTc, JTpc and TpTec and are associated with increased risk for TdP. Results from this study demonstrate that ECG changes after dofetilide and quinidine administration to telemetered monkeys differ from the clinical response, lacking the expected effects on TpTec. Potential explanations for the lack of translation include physio-pharmacology species differences or ECG recording and analysis methodology variations. Mixed ion channel blockers verapamil and mexiletine administered to monkeys showed no significant QTc, JTpc or TpTec prolongation as expected based on the similar clinical response for these agents.

Cardiovascular (CV) safety-related attrition is an important contributor to the loss of promising drug candidates during development. CV safety pharmacology studies are conducted to identify these safety effects. Understanding translation of CV endpoints (specifically, heart rate [HR], and blood pressure [BP]) across preclinical animal models and to the clinic is critical in developing a robust CV derisking strategy. To this end, we investigated translation of HR and BP endpoints using data from 83 compounds that were tested in telemetry studies in rat and large animal (LA; dog or monkey) and 79 compounds that were tested in LA telemetry studies and human phase I clinical trials. Sensitivity, specificity as well as predictive values were calculated for rat to LA model comparison and for LA to human studies comparison. The rat CV model showed good concordance (sensitivity = 84% and specificity = 71%) for LA BP and HR changes. Similarly, LA CV measures of HR and BP showed good concordance (sensitivity = 78% and specificity = 79%) to clinical changes. The CV effects generally occurred within 0.3-3× free plasma concentration across species. Directionality of BP and HR change was conserved between LA to humans. However, for rat to LA comparisons the directionality of change was opposite for 23%-26% compounds. In conclusion, these data establish the translation of HR and BP from preclinical to clinical studies and emphasize the importance of preclinical animal models in the examination of CV safety of drugs.

AbstractThe human voltage‐gated sodium channel Nav1.5 (hNav1.5/SCN5A) plays a critical role in the initiation and propagation of action potentials in cardiac myocytes, and its modulation by various drugs has significant implications for cardiac safety. Drug‐dependent block of Nav1.5 current (INa) can lead to significant alterations in cardiac electrophysiology, potentially resulting in conduction slowing and an increased risk of proarrhythmic events. This review aims to provide a comprehensive overview of the mechanisms by which various pharmacological agents interact with Nav1.5, focusing on the molecular determinants of drug binding and the resultant electrophysiological effects. We discuss the structural features of Nav1.5 that influence drug affinity and specificity. Special attention is given to the concept of state‐dependent block, where drug binding is influenced by the conformational state of the channel, and its relevance to therapeutic efficacy and safety. The review also examines the clinical implications of INa block, highlighting case studies of drugs that have been associated with adverse cardiac events, and how the Vaughan‐Williams Classification system has been employed to qualify “unsafe” sodium channel block. Furthermore, we explore the methodologies currently used to assess INa block in nonclinical and clinical settings, with the hope of providing a weight of evidence approach including in silico modeling, in vitro electrophysiological assays and in vivo cardiac safety studies for mitigating proarrhythmic risk early in drug discovery. This review underscores the importance of understanding Nav1.5 pharmacology in the context of drug development and cardiac risk assessment.

Compound-mediated locomotion changes, conducted via open field infrared photobeam breaks, are an important common component of neurological assessments conducted in safety pharmacology studies. In addition to open field locomotor activity assessments, activity data (derived from changes in signal strength) from cardiovascular (CV) telemetry studies can also be an alternative method potentially used to assess locomotor effects. However, comparisons of these two methods have not been extensively characterized. The goal of this work was to compare these two methodologies to assess activity in rats using reference compounds known to have central nervous system (CNS)-stimulant (preladenant) or CNS-depressant (chlorpromazine) effects. Open field activity was conducted using the Kinder Scientific Motor Monitor system and data were collected for 30 min at each drug's expected time of maximum plasma exposure (Tmax). Telemetry-based CV assessment data were continuously acquired using DSI radiotelemetry instrumented animals for 24 h postdose (HPD). Drugs were administered during the lights-on period for both study types. Administration of preladenant caused increases in activity within 0.5-2 HPD for both methods. While administration of chlorpromazine caused decreases in activity in the infrared beam-based open field assessment (1.0-1.5 HPD), there was no effect on telemetry-derived activity during a similar time period. However, telemetry-derived decreases in activity were observed during the lights-off period (16-20 HPD), suggesting CNS-depressant compounds may be mischaracterized if the optimal dose administration time is not selected based on the light/dark cycle and pharmacokinetics. Overall, these results suggest that telemetry-based activity assessment is capable of detecting CNS-stimulant effects of compounds.

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.

Introduction: Characterization of the incidence of spontaneous arrhythmias to identify possible drug-related ef-fects is often an important part of the analysis in safety pharmacology studies using telemetry.Methods: A retrospective analysis in non-clinical species with and without telemetry transmitters was conducted. Electrocardiograms (24 h) from male and female beagle dogs (n = 131), Go center dot ttingen minipigs (n = 108) and cynomolgus non-human primates (NHP; n = 78) were analyzed.Results: Ventricular tachycardia (VT) was observed in 3% of the dogs but was absent in minipigs and NHPs. Ventricular fibrillation (VF) was not observed in the 3 species. Ventricular premature beats (VPBs) were more frequent during daytime and atrioventricular blocks (AVBs) were more frequent at night in all species. A limited number of animals exhibited a high arrhythmia frequency and there was no correlation between animals with higher frequency of an arrhythmia type and the frequency of other arrythmias in the same animals. Clinical chemistry or hematology parameters were not different with or without telemetry devices. NHP with a trans-mural left ventricular pressure (LVP) catheter exhibited a greater incidence of VPBs and PJCs compared to telemetry animals without LVP. Discussion: All species were similar with regards to the frequency of ventricular ectopic beats (26-46%) while the dog seemed to have more frequent junctional complexes and AVB compared to NHP and minipigs. Arrhythmia screening may be considered during pre-study evaluations, to exclude animals with abnormally high arrhythmia incidence.

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).

Introduction: The use of high throughput patch clamp profiling to determine mixed ion channel-mediated arrhythmia risk was assessed using profiling data generated using proprietary internal and clinical reference compounds. We define the reproducibility of the platform and highlight inherent platform issues. The data generated was used to develop predictive models for cardiac arrhythmia risk, specifically Torsades de Pointes (TdP). Methods: A retrospective analysis was performed using profiling data generated over a 3-year period, including patch clamp data from hERG, Ca(v)1.2, and Na(v)1.5 (peak/late), together with hERG binding. Results: Assay reproducibility was robust over the 3-year period examined. High throughput hERG patch IC50 values correlated well with GLP-hERG data (Pearson = 0.87). A disconnect between hERG binding and patch was observed for similar to 10% compounds and trended with passive cellular permeability. hERG and Ca(v)1.2 potency did not correlate for proprietary compounds, with more potent hERG compounds showing selectivity versus Ca(v)1.2. For clinical compounds where hERG and Ca(v)1.2 activity was more balanced, an analysis of TdP risk versus hERG/Ca(v)1.2 ratio demonstrated low TdP probability when the hERG/Ca(v)1.2 potency ratios were < 1. Modeling of clinical compound data revealed a lack of impact of the Na(v)1.5 (late) current in predicting TdP. Moreover, models using hERG binding data (ROC AUC = 0.876) showed an improved ability to predict TdP risk versus hERG patch clamp (ROC AUC = 0.787). Discussion: The data highlight the value of high throughput patch clamp data in the prediction of TdP risk, as well as some potential limitations with this approach.

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.

COVID-19 is a potentially fatal infection caused by the SARS-CoV-2 virus. The SARS-CoV-2 3CL protease (Mpro) is a viral enzyme essential for replication and is the target for nirmatrelvir. Paxlovid (nirmatrelvir co-administered with the pharmacokinetic enhancer ritonavir) showed efficacy in COVID-19 patients at high risk of progressing to hospitalization and/or death. Nonclinical safety studies with nirmatrelvir are essential in informing benefit-risk of Paxlovid and were conducted to support clinical development. In vivo safety pharmacology assessments included a nervous system/pulmonary study in rats and a cardiovascular study in telemetered monkeys. Potential toxicities were assessed in repeat dose studies of up to 1 month in rats and monkeys. Nirmatrelvir administration (1,000 mg/kg, p.o.) to male rats produced transient increases in locomotor activity and respiratory rate but did not affect behavioral endpoints in the functional observational battery. Cardiovascular effects in monkeys were limited to transient increases in blood pressure and decreases in heart rate, observed only at the highest dose tested (75 mg/kg per dose b.i.d; p.o.). Nirmatrelvir did not prolong QTc-interval or induce arrhythmias. There were no adverse findings in repeat dose toxicity studies up to 1 month in rats (up to 1,000 mg/kg daily, p.o.) or monkeys (up to 600 mg/kg daily, p.o.). Nonadverse, reversible clinical pathology findings without clinical or microscopic correlates included prolonged coagulation times at ≥60 mg/kg in rats and increases in transaminases at 600 mg/kg in monkeys. The safety pharmacology and nonclinical toxicity profiles of nirmatrelvir support clinical development and use of Paxlovid for treatment of COVID-19.