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).

The incidence of spontaneous arrhythmias in telemetered beagle dogs, Göttingen Minipigs and Cynomolgus non-human primates: A HESI consortium retrospective analysis.

INTRODUCTION: Characterization of the incidence of spontaneous arrhythmias to identify possible drug-related effects 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), Göttingen 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 transmural 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.

A comparison of activity data generated from cardiovascular telemetry studies - With quantitative open field locomotor studies in Wistar Han rats.

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.

Use of high throughput ion channel profiling and statistical modeling to predict off-target arrhythmia risk - One pharma's experience and perspective.

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, Cav1.2, and Nav1.5 (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 ∼10% compounds and trended with passive cellular permeability. hERG and Cav1.2 potency did not correlate for proprietary compounds, with more potent hERG compounds showing selectivity versus Cav1.2. For clinical compounds where hERG and Cav1.2 activity was more balanced, an analysis of TdP risk versus hERG/Cav1.2 ratio demonstrated low TdP probability when the hERG/Cav1.2 potency ratios were < 1. Modeling of clinical compound data revealed a lack of impact of the Nav1.5 (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.

Comprehensive Nonclinical Safety Assessment of Nirmatrelvir Supporting Timely Development of the SARS-COV-2 Antiviral Therapeutic, Paxlovid™.

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.

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.

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.

An Industry Survey With Focus on Cardiovascular Safety Pharmacology Study Design and Data Interpretation.

INTRODUCTION: The Safety Pharmacology Society (SPS) conducted a membership survey to examine industry practices related mainly to cardiovascular (CV) safety pharmacology (SP). METHODS: Questions addressed nonclinical study design, data analysis methods, drug-induced effects, and conventional and novel CV assays. RESULTS: The most frequent therapeutic area targeted by drugs developed by the companies/institutions that employ survey responders was oncology. The most frequently observed drug-mediated effects included an increased heart rate, increased arterial blood pressure, hERG (IKr) block, decreased arterial blood pressure, decreased heart rate, QTc prolongation, and changes in body temperature. Broadly implemented study practices included Latin square crossover study design with n = 4 for nonrodent CV studies, statistical analysis of data (eg, analysis of variance), use of arrhythmia detection software, and the inclusion of data from all study animals when integrating SP studies into toxicology studies. Most responders frequently used individual animal housing conditions. Responders commonly evaluated drug effects on multiple ion channels, but in silico modeling methods were used much less frequently. Most responders rarely measured the J-Tpeak interval in CV studies. Uncertainties relative to Standard for Exchange of Nonclinical Data applications for data derived from CV SP studies were common. Although available, the use of human induced pluripotent stem cell cardiomyocytes remains rare. The respiratory SP study was rarely involved with identifying drug-induced functional issues. Responders indicated that the study-derived no observed effect level was more frequently determined than the no observed adverse effect level in CV SP studies; however, a large proportion of survey responders used neither.

General Principles for the Validation of Proarrhythmia Risk Prediction Models: An Extension of the CiPA In Silico Strategy.

This white paper presents principles for validating proarrhythmia risk prediction models for regulatory use as discussed at the In Silico Breakout Session of a Cardiac Safety Research Consortium/Health and Environmental Sciences Institute/US Food and Drug Administration-sponsored Think Tank Meeting on May 22, 2018. The meeting was convened to evaluate the progress in the development of a new cardiac safety paradigm, the Comprehensive in Vitro Proarrhythmia Assay (CiPA). The opinions regarding these principles reflect the collective views of those who participated in the discussion of this topic both at and after the breakout session. Although primarily discussed in the context of in silico models, these principles describe the interface between experimental input and model-based interpretation and are intended to be general enough to be applied to other types of nonclinical models for proarrhythmia assessment. This document was developed with the intention of providing a foundation for more consistency and harmonization in developing and validating different models for proarrhythmia risk prediction using the example of the CiPA paradigm.

Preclinical to Clinical Translation of Hemodynamic Effects in Cardiovascular Safety Pharmacology Studies.

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.

A Proof-of-Concept Evaluation of JTPc and Tp-Tec as Proarrhythmia Biomarkers in Preclinical Species: A Retrospective Analysis by an HESI-Sponsored Consortium.

INTRODUCTION: Based on the ICH S7B and E14 guidance documents, QT interval (QTc) is used as the primary in vivo biomarker to assess the risk of drug-induced torsades de pointes (TdP). Clinical and nonclinical data suggest that drugs that prolong the corrected QTc with balanced multiple ion channel inhibition (most importantly the l-type calcium, Cav1.2, and persistent or late inward sodium current, Nav1.5, in addition to human Ether-à-go-go-Related Gene [hERG] IKr or Kv11.1) may have limited proarrhythmic liability. The heart rate-corrected J to T-peak (JTpc) measurement in particular may be considered to discriminate selective hERG blockers from multi-ion channel blockers. METHODS: Telemetry data from Beagle dogs given dofetilide (0.3 mg/kg), sotalol (32 mg/kg), and verapamil (30 mg/kg) orally and Cynomolgus monkeys given medetomidine (0.4 mg/kg) orally were retrospectively analyzed for effects on QTca, JTpca, and T-peak to T-end covariate adjusted (Tpeca) interval using individual rate correction and super intervals (calculated from 0-6, 6-12, 12-18, and 18-24 hours postdose). RESULTS: Dofetilide and cisapride (IKr or Kv11.1 blockers) were associated with significant increases in QTca and JTpca, while sotalol was associated with significant increases in QTca, JTpca, and Tpeca. Verapamil (a Kv11.1 and Cav1.2 blocker) resulted in a reduction in QTca and JTpca, however, and increased Tpeca. Medetomidine was associated with a reduction in Tpeca and increase in JTpca. DISCUSSION: Results from this limited retrospective electrocardiogram analysis suggest that JTpca and Tpeca may discriminate selective IKr blockers and multichannel blockers and could be considered in the context of an integrated comprehensive proarrhythmic risk assessment.

Can non-clinical repolarization assays predict the results of clinical thorough QT studies? Results from a research consortium.

BACKGROUND AND PURPOSE: Translation of non-clinical markers of delayed ventricular repolarization to clinical prolongation of the QT interval corrected for heart rate (QTc) (a biomarker for torsades de pointes proarrhythmia) remains an issue in drug discovery and regulatory evaluations. We retrospectively analysed 150 drug applications in a US Food and Drug Administration database to determine the utility of established non-clinical in vitro IKr current human ether-à-go-go-related gene (hERG), action potential duration (APD) and in vivo (QTc) repolarization assays to detect and predict clinical QTc prolongation. EXPERIMENTAL APPROACH: The predictive performance of three non-clinical assays was compared with clinical thorough QT study outcomes based on free clinical plasma drug concentrations using sensitivity and specificity, receiver operating characteristic (ROC) curves, positive (PPVs) and negative predictive values (NPVs) and likelihood ratios (LRs). KEY RESULTS: Non-clinical assays demonstrated robust specificity (high true negative rate) but poor sensitivity (low true positive rate) for clinical QTc prolongation at low-intermediate (1×-30×) clinical exposure multiples. The QTc assay provided the most robust PPVs and NPVs (ability to predict clinical QTc prolongation). ROC curves (overall test accuracy) and LRs (ability to influence post-test probabilities) demonstrated overall marginal performance for hERG and QTc assays (best at 30× exposures), while the APD assay demonstrated minimal value. CONCLUSIONS AND IMPLICATIONS: The predictive value of hERG, APD and QTc assays varies, with drug concentrations strongly affecting translational performance. While useful in guiding preclinical candidates without clinical QT prolongation, hERG and QTc repolarization assays provide greater value compared with the APD assay.

Cardiac sodium channel antagonism - Translation of preclinical in vitro assays to clinical QRS prolongation.

INTRODUCTION: Cardiac sodium channel antagonists have historically been used to treat cardiac arrhythmias by preventing the reentry of the electrical impulse that could occur following myocardial damage. However, clinical studies have highlighted a significant increase in mortality associated with such treatment. Cardiac sodium channel antagonist activity is now seen as an off-target pharmacology that should be mitigated during the drug development process. The aim of this study was to examine the correlation between in vitro/ex vivo assays that are routinely used to measure Nav1.5 activity and determine the translatability of the individual assays to QRS prolongation in the clinic. METHODS: A set of clinical compounds with known Nav1.5 activity was profiled in several in vitro/ex vivo assays (binding, membrane potential, patch clamp and the Langendorff isolated heart). Clinical data comprising compound exposure levels and changes in QRS interval were obtained from the literature. Sensitivity/specificity analysis was performed with respect to the clinical outcome. RESULTS: The in vitro assays showed utility in predicting QRS prolongation in the clinic. Optimal thresholds were defined for each assay (binding: IC20; membrane potential: IC10; patch clamp: IC20) and sensitivity (69-88%) and specificity (53-84%) values were shown to be similar between assay formats. DISCUSSION: The data provide clear statistical insight into the translatability of Nav1.5 antagonism data generated in vitro to potential clinical outcomes. These results improve our ability to understand the liability posed by such activity in novel development compounds at an early stage.

Cellular and functional actions of tofacitinib related to the pathophysiology of hibernoma development.

Tofacitinib is an oral JAK inhibitor for the treatment of rheumatoid arthritis. In the 2-year carcinogenicity study with tofacitinib, increased incidence of hibernoma (a neoplasm of brown adipose tissue [BAT]) was noted in female rats at ≥30 mg/kg/day (≥41x human exposure multiples). Thus, signaling pathways within BAT were investigated by measuring BAT: weight, cell proliferation biomarkers, content of basal and prolactin-induced phosphorylated Signal Transducer and Activator of Transcription (STAT), and uncoupling protein 1 (UCP-1). The relationship between cardiovascular hemodynamics and plasma norepinephrine (NE) levels was also investigated. Tofacitinib administered to female rats at doses of 10, 30, or 75 mg/kg/day for 14 days increased BAT weight at 75 mg/kg/day and cell proliferation at ≥30 mg/kg/day. JAK inhibition, observed as lower pSTAT3 and pSTAT5 in BAT, was noted at ≥10 mg/kg/day, while lower activity of BAT was observed as lower UCP-1 protein at ≥30 mg/kg/day. In cultured brown adipocytes, prolactin-induced increase in pSTAT5 and pSTAT3 were inhibited by tofacitinib in a concentration-dependent manner. Tofacitinib lowered blood pressure, increased heart rate, and resulted in dose-dependent increases in circulating NE. Thus, JAK/STAT inhibition in BAT and sympathetic stimulation are two factors which might contribute to the genesis of hibernomas by tofacitinib in rats.

Use of Rat Primary Mesenteric Cells for the Prediction of PDE4 Inhibitor Drug-Induced Vascular Injury.

Drug-induced vascular injury (DIVI) in preclinical studies can delay, if not terminate, a drug development program. Clinical detection of DIVI can be very difficult as there are no definitive biomarkers known to reliably detect this disorder in all instances. The preclinical identification of DIVI requires detailed microscopic examination of a wide range of tissues although one of the most commonly affected areas in rats is the mesenteric vasculature. The reason for this predisposition of mesenteric arteries in rats as well as the exact mechanism and cell types involved in the initial development of these lesions have not been fully elucidated. We hypothesized that by using a mixed culture of cells from rat mesenteric tissue, we would be able to identify an RNA expression signature that could predict the invivo development of DIVI. Five compounds designed to inhibit Phosphodiesterase 4 activity (PDE4i) were chosen as positive controls. PDE4i's are well known to induce DIVI in the mesenteric vasculature of rats and there is microscopic evidence that this is associated, at least in part, with a proinflammatory mechanism. We surveyed, by qRT-PCR, the expression of 96 genes known to be involved in inflammation and using a Random-Forest model, identified 12 genes predictive of invivo DIVI outcomes in rats. Using these genes, we were able to cross-validate the ability of the Random-Forest modeling to predict the concentration at which PDE4i caused DIVI invivo.

Novel 3-fluoro-6-methoxyquinoline derivatives as inhibitors of bacterial DNA gyrase and topoisomerase IV.

Novel (non-fluoroquinolone) inhibitors of bacterial type II topoisomerases (NBTIs) are an emerging class of antibacterial agents. We report an optimized series of cyclobutylaryl-substituted NBTIs. Compound 14 demonstrated excellent activity both in vitro (S. aureus MIC90=0.125µg/mL) and in vivo (systemic and tissue infections). Enhanced inhibition of Topoisomerase IV correlated with improved activity in S. aureus strains with mutations conferring resistance to NBTIs. Compound 14 also displayed an improved hERG IC50 of 85.9µM and a favorable profile in the anesthetized guinea pig model.

L-type calcium channel antagonism - Translation from in vitro to in vivo.

INTRODUCTION: Although therapeutically beneficial in the treatment of certain diseases, L-type calcium channel antagonism can result in unwanted off-target pharmacology leading to adverse drug reactions and to the termination of the development of otherwise promising compounds. In the present study three marketed calcium channel inhibitors, nifedipine, verapamil and diltiazem were profiled in a series of in vitro and ex-vivo assays in an effort to determine the ability of these assays to discriminate, between dihydropyridine versus non-dihydropyridine-like compounds, and how well they can predict the cardiovascular effects observed in a conscious telemetered rat model. METHODS: Standard calcium channel antagonists were profiled in radioligand binding, patch clamp and calcium flux assays. In addition, cardiovascular endpoints related to calcium channel activity were also examined in ex vivo tissue bath preparations, including relaxation of pre-constricted rat aorta and the guinea pig Langendorff isolated heart model. The data generated were correlated with in vivo blood pressure and heart rate data from conscious telemetered rats. RESULTS: Our results show that the binding, FLIPR and aorta assays allow differentiation of the compounds in two distinct classes of L-type calcium channel antagonists, and are good predictors of in vivo outcomes. DISCUSSION: These results suggest that in vitro and ex vivo profiling remains a valuable tool in predicting potential in vivo cardiovascular safety issues, and can aid in the selection of novel development compounds that show inherent inhibitory activity against L-type calcium channels.

Statistical power analysis of cardiovascular safety pharmacology studies in conscious rats.

UNLABELLED: Cardiovascular (CV) toxicity and related attrition are a major challenge for novel therapeutic entities and identifying CV liability early is critical for effective derisking. CV safety pharmacology studies in rats are a valuable tool for early investigation of CV risk. Thorough understanding of data analysis techniques and statistical power of these studies is currently lacking and is imperative for enabling sound decision-making. METHODS: Data from 24 crossover and 12 parallel design CV telemetry rat studies were used for statistical power calculations. Average values of telemetry parameters (heart rate, blood pressure, body temperature, and activity) were logged every 60s (from 1h predose to 24h post-dose) and reduced to 15min mean values. These data were subsequently binned into super intervals for statistical analysis. A repeated measure analysis of variance was used for statistical analysis of crossover studies and a repeated measure analysis of covariance was used for parallel studies. Statistical power analysis was performed to generate power curves and establish relationships between detectable CV (blood pressure and heart rate) changes and statistical power. Additionally, data from a crossover CV study with phentolamine at 4, 20 and 100mg/kg are reported as a representative example of data analysis methods. RESULTS: Phentolamine produced a CV profile characteristic of alpha adrenergic receptor antagonism, evidenced by a dose-dependent decrease in blood pressure and reflex tachycardia. Detectable blood pressure changes at 80% statistical power for crossover studies (n=8) were 4-5mmHg. For parallel studies (n=8), detectable changes at 80% power were 6-7mmHg. Detectable heart rate changes for both study designs were 20-22bpm. DISCUSSION: Based on our results, the conscious rat CV model is a sensitive tool to detect and mitigate CV risk in early safety studies. Furthermore, these results will enable informed selection of appropriate models and study design for early stage CV studies.