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.

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.

From the Cover: Evaluation of the Effects of Tanezumab, a Monoclonal Antibody Against Nerve Growth Factor, on the Sympathetic Nervous System in Adult Cynomolgus Monkeys (Macaca fascicularis): A Stereologic, Histomorphologic, and Cardiofunctional Assessment.

Tanezumab, a humanized monoclonal antibody against nerve growth factor is in development for treatment of chronic pain. Three nonclinical studies assessed effects of clinically relevant and supratherapeutic doses of tanezumab on the sympathetic nervous system (SNS) of adult nonhuman primates. Study 1 evaluated potential effects of subcutaneous (SC) tanezumab (1.2 mg/kg every 8 weeks [Q8W]) on SNS in cynomolgus monkeys for 3 or 6 months and reversibility or persistence of any effects through a nondosing/recovery period. Study 2 evaluated whether neuronal cell death occurs shortly after a single SC tanezumab injection (1.2 mg/kg). Assessments for these two studies included evaluations of superior cervical and cervicothoracic ganglia for neuronal cell death and morphology. Study 3 evaluated effects of SC tanezumab (1.2 mg/kg Q8W and 30 mg/kg/week) over 6 months on sympathetic control of cardiovascular function. Tanezumab exposure was associated with stereologic changes in sympathetic ganglia, including smaller ganglion volume, and smaller average neuron size/area beginning at 2 weeks and reaching maximal levels by 1 month with no further progression through 6 months. These changes were not associated with clinical signs, completely reversed upon tanezumab withdrawal, and were not considered adverse. Tanezumab had no adverse effects on sympathetic control of cardiovascular function. These data support the conclusion that tanezumab administration for up to 6 months has no adverse effects on SNS morphology or function and does not cause neuronal cell death in adult nonhuman primates.

Assessment of Cardiac Troponin I Responses in Nonhuman Primates during Restraint, Blood Collection, and Dosing in Preclinical Safety Studies.

Limited information has been published on the use of cardiac troponin I (cTnI) as a biomarker of cardiac injury in monkeys. The purpose of these studies was to characterize the cTnI response seen in cynomolgus macaques during routine dosing and blood collection procedures typically used in preclinical safety studies and to better understand the pathogenesis of this response. We measured cTnI using two different methods, the Siemens Immulite cTnI assay and the more sensitive Siemens Troponin I-Ultra assay. We were able to demonstrate that after oral, subcutaneous, or intravenous dosing of common vehicles, as well as serial chair restraint for venipuncture blood collection, that minimal to mild transient increases in cTnI could be detected in monkeys with both assays. cTnI values typically peaked at 2, 3, 4, or 6 hr after sham dosing and returned to baseline at 22 or 24 hr. In addition, marked increases in heart rate (HR) and blood pressure (BP) occurred in monkeys during the restraint procedures, which likely initiated the cTnI release in these animals. Monkeys that were very well acclimated to the chairing procedures and had vascular access ports for blood sampling did not have marked increases in HRs and BP or increases in cTnI.

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.

Validation and utility of the PhysioTel™ Digital M11 telemetry implant for cardiovascular data evaluation in cynomolgus monkeys and Beagle dogs.

INTRODUCTION: The cardiovascular liability of candidate compounds can be evaluated by a number of methods including implanted telemetry, jacketed telemetry and surface lead electrocardiogram (ECG). The utility of the new PhysioTel™ Digital M11 cardiovascular telemetry implant was evaluated in monkeys and dogs. METHODS: Eight monkeys and dogs (4 males and 4 females per species) were implanted with the M11 device utilizing a femoral blood pressure catheter and periosteal ECG leads. The signal quality of the ECGs was determined as a percentage of software-matched waveforms and as a percentage of signal loss during the recording periods. To investigate sensitivity for detecting changes in QT/QTc and HR/BP, moxifloxacin and doxazosin were administered to monkeys and dogs implanted with the M11 device. Additionally, histopathological evaluation of the implant site was completed. RESULTS: For both monkey and dog, the percentage of recognizable waveforms was high (65% and 85%, respectively), while the average amount of signal loss was low (1% and 3%, respectively), indicating that the M11 implants delivered data of sufficient quality. In monkeys, moxifloxacin (90mg/kg) induced QT and QTc prolongation up to 22 and 12ms, respectively, while at 30mg/kg in dogs, the maximal increases in QT and QTc were 13 and 16ms, respectively. Doxazosin (1.5 and 1.0mg/kg) produced HR increases up to 35 and 29bpm with decreases in blood pressure up to -14 and -26mmHg in monkeys and dogs, respectively. The histopathological impact of the implant, catheter and biopotential leads was limited to expected minor local inflammatory changes as assessed at necropsy and with microscopic examination. DISCUSSION: Based upon the results of this study, the PhysioTel™ Digital M11 is a suitable technology for assessing cardiovascular parameters in monkeys and dogs, and because of the size and limited invasiveness of the implant, is well positioned for use on toxicology studies.

A non-human primate model for investigating drug-induced risk of orthostatic hypotension and sympathetic dysfunction: Preclinical correlate to a clinical test.

INTRODUCTION: Drug induced orthostatic hypotension (OH) is an important clinical concern and can be an unexpected hurdle during drug development. OH is defined as an abnormal decrease in blood pressure (BP) triggered by a rapid postural change. The sympathetic nervous system is critical for controlling normal cardiovascular function and compensatory responses to changes in posture. Thus, OH can also serve as a surrogate indicator of sympathetic dysfunction. However, preclinical conscious models for investigating risk of OH and/or sympathetic dysfunction are lacking. Herein, we describe a conscious nonhuman primate (NHP) model which mimics the widely used clinical tilt table test for OH. METHODS: Male, Cynomolgus NHPs (n = 7-8) implanted with radio-telemetry transmitters were placed in modified tilt chairs in a supine position. Subsequently, a 90° head up tilt was performed for 3 min followed by return to the supine position. BP and heart rate were continuously monitored. Test compounds were administered either intravenously or via oral gavage in a crossover design, with blood samples collected at the end of the each tilt to assess total drug concentrations. RESULTS: Tilt responses were assessed following treatment with positive control compounds that cause sympathetic dysfunction; hexamethonium (ganglionic blocker) and prazosin (alpha-1 adrenergic receptor antagonist). Both compounds induced marked OH as evidenced by robust and sustained BP reduction in response to a head up tilt (decrease of 25-35 mmHg for hexamethonium, decrease of 21-44 mmHg for prazosin). OH incidence rates increased in a dose-dependent manner. OH incidences following treatment with minoxidil (vasodilator) were markedly lower to those observed with hexamethonium and prazosin indicating the role of sympathetic dysfunction in causing OH. DISCUSSION: These data demonstrate that the NHP tilt test is a valuable model for investigating OH risk. This model fills an important preclinical gap for assessing such a safety concern and can be applied to programs where a sympathetic deficit and/or OH are anticipated or clinically observed.

Impact of study design on proarrhythmia prediction in the SCREENIT rabbit isolated heart model.

INTRODUCTION: Prediction of the propensity of a compound to induce Torsades de Pointes continues to be a formidable challenge to the pharmaceutical industry. Development of an in vitro model for assessment of proarrhythmic potential offers the advantage of higher throughput and reduced compound quantity requirements when compared to in vivo studies. A rabbit isolated heart model (SCREENIT) has been reported to identify compounds with proarrhythmic potential based on the observance of compound-induced triangulation and instability of the monophasic action potential (MAP), ectopic beats, and reverse-use dependence of prolongation of the MAP duration. Previous reports have indicated that this model qualitatively identifies proarrhythmic compounds and suggest the use of this model to assign safety margins for human clinical use. The intent of this series of studies was to evaluate the impact of study design on the proarrhythmic concentration predicted by this model. METHODS: Nine compounds of varying proarrhythmic potential and a negative control were tested in a blinded fashion using a series of different experimental protocols: Compounds were tested at multiple concentration ranges and extended perfusion times were also evaluated. RESULTS: In general when the dataset is viewed as a whole, the model did identify proarrhythmic compounds, however the concentration at which action potential prolongation, triangulation, instability, reverse-use dependence and ectopic beats occurred often varied based on the concentration range selected. Further analysis using extended compound perfusion times demonstrated that variability may be due in part to lack of adequate equilibration of compound with the cardiac tissue. DISCUSSION: We report that the model correctly identified proarrhythmic agents in a qualitative manner, but that study design impacts the proarrhythmic concentration derived from the model.

Fluorescently labeled analogues of dofetilide as high-affinity fluorescence polarization ligands for the human ether-a-go-go-related gene (hERG) channel.

Novel fluorescent derivatives of dofetilide (1) have been synthesized. Analogues that feature a fluorescent probe attached through an aliphatic spacer to the central tertiary nitrogen of 1 have high affinity for the hERG channel, and affinity is dependent on both linker length and pendent dye. These variables have been optimized to generate Cy3B derivative 10e, which has hERG channel affinity equivalent to that of dofetilide. When bound to cell membranes expressing the hERG channel, 10e shows a robust increase in fluorescence polarization (FP) signal. In a FP binding assay using 10e as tracer ligand, Ki values for several known hERG channel blockers were measured and excellent agreement with the literature Ki values was observed over an affinity range of 2 nM to 3 muM. 10e blocks hERG channel current in electrophysiological patch clamp experiments, and computational docking experiments predict that the dofetilide core of 10e binds hERG channel in a conformation similar to that previously predicted for 1. These analogues enable high-throughput hERG channel binding assays that are rapid, economical, and predictive of test compounds' potential for prolonged QT liabilities.

Involvement of beta-site APP cleaving enzyme 1 (BACE1) in amyloid precursor protein-mediated enhancement of memory and activity-dependent synaptic plasticity.

The amyloid precursor protein (APP) undergoes sequential cleavages to generate various polypeptides, including the amyloid-beta protein (Abeta), which forms amyloid plaques in Alzheimer's disease (AD), secreted APPalpha (sAPPalpha) which enhances memory, and the APP intracellular domain (AICD), which has been implicated in the regulation of gene transcription and calcium signaling. The beta-site APP cleaving enzyme 1 (BACE1) cleaves APP in an activity-dependent manner to form Abeta, AICD, and secreted APPbeta. Because this neural activity was shown to diminish synaptic transmission in vitro [Kamenetz F, Tomita T, Hsieh H, Seabrook G, Borchelt D, Iwatsubo T, Sisodia S, Malinow R (2003) Neuron 37:925-937], the prevailing notion has been that this pathway diminishes synaptic function. Here we investigated the role of this pathway in vivo. We studied transgenic mice overproducing APP that do not develop AD pathology or memory deficits but instead exhibit enhanced spatial memory. We showed enhanced synaptic plasticity in the hippocampus that depends on prior synaptic activity. We found that the enhanced memory and synaptic plasticity are abolished by the ablation of one or both copies of the BACE1 gene, leading to a significant decrease in AICD but not of any other APP cleavage products. In contrast to the previously described negative effect of BACE1-mediated cleavage of APP on synaptic function in vitro, our in vivo work indicates that BACE1-mediated cleavage of APP can facilitate learning, memory, and synaptic plasticity.

Early evaluation of compound QT prolongation effects: a predictive 384-well fluorescence polarization binding assay for measuring hERG blockade.

INTRODUCTION: A large number of drugs from a variety of pharmacological classes have been demonstrated to cause adverse effects on cardiac rhythm, including the life-threatening arrhythmia Torsades de Pointes. These side effects are often associated with prolongation of the QT interval and are mediated via blockade of the human ether-a-go-go related gene (hERG) encoded potassium channel. In order to manage this risk in the pharmaceutical industry it is desirable to evaluate QT prolongation as early as possible in the drug discovery process. METHODS: Here we describe the development of a 384-well fluorescence polarization (FP) binding assay compatible with high-throughput assessment of compound blockade of the hERG channel during the lead optimisation process. To characterise the fluorescent ligand that was developed, competition binding studies, kinetic studies and electrophysiology studies were performed. Furthermore, to validate the assay as a key screening method a series of competition binding studies were performed and correlated with functional data obtained via patch-clamp. RESULTS: Evaluation of the assay indicates that high quality data is obtained (Z'>0.6), that the K(i) values determined are equivalent to more traditional radiometric methods and that it is predictive for functional hERG blockade as assessed by patch clamp. DISCUSSION: Whilst FP assays, utilizing a variety of fluors, have become well established for the evaluation of G-protein-coupled receptor (GPCRs) and kinase ligand interactions, this technique has not been applied widely to the study of ion channels. Therefore, this represents a novel assay format that is amenable to the evaluation of thousands of compounds per day. Whilst other assay formats have proven predictive or high throughput, this assay represents one of few that combines both attributes, moreover it represents the most cost effective assay, making it truly amenable to early assessment of hERG blockade.