In Silico Human Cardiomyocyte Action Potential Modeling: Exploring Ion Channel Input Combinations.

In silico modeling offers an opportunity to supplement and accelerate cardiac safety testing. With in silico modeling, computational simulation methods are used to predict electrophysiological interactions and pharmacological effects of novel drugs on critical physiological processes. The O'Hara-Rudy's model was developed to predict the response to different ion channel inhibition levels on cardiac action potential duration (APD) which is known to directly correlate with the QT interval. APD data at 30% 60% and 90% inhibition were derived from the model to delineate possible ventricular arrhythmia scenarios and the marginal contribution of each ion channel to the model. Action potential values were calculated for epicardial, myocardial, and endocardial cells, with action potential curve modeling. This study assessed cardiac ion channel inhibition data combinations to consider when undertaking in silico modeling of proarrhythmic effects as stipulated in the Comprehensive in Vitro Proarrhythmia Assay (CiPA). As expected, our data highlight the importance of the delayed rectifier potassium channel (IKr) as the most impactful channel for APD prolongation. The impact of the transient outward potassium channel (Ito) inhibition on APD was minimal while the inward rectifier (IK1) and slow component of the delayed rectifier potassium channel (IKs) also had limited APD effects. In contrast, the contribution of fast sodium channel (INa) and/or L-type calcium channel (ICa) inhibition resulted in substantial APD alterations supporting the pharmacological relevance of in silico modeling using input from a limited number of cardiac ion channels including IKr, INa, and ICa, at least at an early stage of drug development.

The Spontaneous Incidence of Neurological Clinical Signs in Preclinical Species Using Cage-side Observations or High-definition Video Monitoring: A Retrospective Analysis.

When conducting toxicology studies, the interpretation of drug-related neurological clinical signs such as convulsions, myoclonus/myoclonic jerks, tremors, ataxia, and salivation requires an understanding of the spontaneous incidence of those observations in commonly used laboratory animal species. The spontaneous incidence of central nervous system clinical signs in control animals from a single facility using cage-side observations or high definition video monitoring was retrospectively analyzed. Spontaneous convulsions were observed at low incidence in Beagle dogs and Sprague-Dawley rats but were not identified in cynomolgus monkeys and Göttingen minipigs. Spontaneous myoclonic jerks and muscle twitches were observed at low incidence in Beagle dogs, cynomolgus monkeys, and Sprague-Dawley rats but were not seen in Göttingen minipigs. Spontaneous ataxia/incoordination was identified in all species and generally with a higher incidence when using video monitoring. Salivation and tremors were the two most frequent spontaneous clinical signs and both were observed in all species. Data from the current study unveil potential limitations when using control data obtained from a single study for toxicology interpretation related to low incidence neurological clinical signs while providing historical control data from Beagle dogs, cynomolgus monkeys, Sprague-Dawley rats, and Göttingen minipigs.

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.

Polysorbate 80-Induced Anaphylactoid Reaction and the Effects on Cardiovascular Function: Dose Threshold and Species Comparison.

Polysorbate 80 (PS80) is commonly used in pre-clinical formulations. The dose threshold for cardiovascular (CV) changes and hypersensitivity reaction in the dog was assessed and compared to other species. PS80 was administered by intravenous (IV) bolus (.5, 1 mg/kg), IV infusion (.3, .5, 1, 3 mg/kg), subcutaneous (SC) injection (5, 10, 15 mg/kg) and oral gavage (10 mg/kg) to dogs with CV monitoring. Monkeys and minipigs received PS80 by IV infusion at 3 mg/kg. Plasma histamine concentration was measured following PS80 IV infusion and with diphenhydramine pre-treatment in dogs only. In dogs, PS80 was not associated with CV changes at doses up to 15 mg/kg SC and 10 mg/kg oral, but decreased blood pressure and increased heart rate with IV bolus at ≥ .5 mg/kg and IV infusion at ≥ 1.0 mg/kg and decreased body temperature with IV infusion at 3 mg/kg was observed. Transient edema and erythema were noted with all administration routes, in all three species including doses that were devoid of CV effects. In monkeys and minipigs, PS80 did not induce CV, cutaneous or histamine concentration changes. These results suggest that mild, transient skin changes occur following PS80 administration at doses that are not associated with CV effects in the dogs. In dogs, the cardiovascular effect threshold was <.5 mg/kg for IV bolus, .3 mg/kg for IV infusion, 15 mg/kg for SC injection, and 10 mg/kg for oral administration. Monkey and minipig were refractory to PS80-induced histamine release at 3 mg/kg by IV infusion over 15 minutes.

Combined Cardiopulmonary Assessments Using Impedance and Digital Implants in Conscious Freely Moving Cynomolgus Monkeys, Beagle Dogs, and Göttingen Minipigs: Pharmacological Characterization and Social Housing Effects.

Respiratory monitoring, using impedance with implanted telemetry in socially housed animals, was not possible until the recent development of digital signal transmission. The objective of this study was to evaluate digital telemetry monitoring of cardiopulmonary parameters (respiratory rate, tidal volume, minute volume, electrocardiography (DII), systemic arterial blood pressure, physical activity, and body temperature) in conscious, single-housed, non-rodent species commonly used in toxicology studies following administration of positive/negative controls (saline, dexmedetomidine, morphine, amphetamine, and doxapram), and also, the effects of various social housing arrangements in untreated female and/or male cynomolgus monkeys, Beagle dogs, and Göttingen minipigs (n = 4 per species). Aggressions were observed in socially housed male minipigs, however, which prevented pair-housed assessments in this species. All tested pharmacological agents significantly altered more than one organ system, highlighting important inter-organ dependencies when analyzing functional endpoints. Stress-related physiological changes were observed with single-housing or pair-housing with a new cage mate in cynomolgus monkeys and Beagle dogs, suggesting that stable social structures are preferable to limit variability, especially around dosing. Concomitant monitoring of cardiovascular and respiratory parameters from the same animals may help reduce the number of animals (3 Rs) needed to fulfill the S7A guidelines and allows for identification of organ system functional correlations. Globally, the data support the use of social housing in non-rodents for safety pharmacology multi-organ system (heart and lungs) monitoring investigations.

Confounders and Pharmacological Characterization When Using the QT, JTp, and Tpe Intervals in Beagle Dogs.

INTRODUCTION: Corrected QT (QTc) interval is an essential proarrhythmic risk biomarker, but recent data have identified limitations to its use. The J to T-peak (JTp) interval is an alternative biomarker for evaluating drug-induced proarrhythmic risk. The aim of this study was to evaluate pharmacological effects using spatial magnitude leads and DII electrocardiogram (ECG) leads and common ECG confounders (ie, stress and body temperature changes) on covariate adjusted QT (QTca), covariate adjusted JTp (JTpca), and covariate adjusted T-peak to T-end (Tpeca) intervals. METHODS: Beagle dogs were exposed to body hyper- (42 °C) or hypothermic (33 °C) conditions or were administered epinephrine to assess confounding effects on heart rate corrected QTca, JTpca, and Tpeca intervals. Dofetilide (0.1, 0.3, 1.0 mg/kg), ranolazine (100, 140, 200 mg/kg), and verapamil (7, 15, 30, 43, 62.5 mg/kg) were administered to evaluate pharmacological effects. RESULTS: Covariate adjusted QT (slope -12.57 ms/°C) and JTpca (-14.79 ms/°C) were negatively correlated with body temperature but Tpeca was minimally affected. Epinephrine was associated with QTca and JTpca shortening, which could be related to undercorrection in the presence of tachycardia, while minimal effects were observed for Tpeca. There were no significant ECG change following ranolazine administration. Verapamil decreased QTca and JTpca intervals and increased Tpeca, whereas dofetilide increased QTca and JTpca intervals but had inconsistent effects on Tpeca. CONCLUSION: Results highlight potential confounders on QTc interval, but also on JTpca and Tpeca intervals in nonclinical studies. These potential confounding effects may be relevant to the interpretation of ECG data obtained from nonclinical drug safety studies with Beagle dogs.

Reprint of "EEG: Characteristics of drug-induced seizures in rats, dogs and non-human primates".

Seizures are amongst the most frequent neurological issues encountered in pre-clinical safety testing. The objective was to characterize EEG morphologies and premonitory signs in drug-induced seizures in preclinical species. A comparative (inter-species) retrospective analysis for drug-induced seizures recorded by video-telemetry was conducted in rats (n = 53), dogs (n = 195), and non-human primates (n = 234). The most frequent premonitory signs were, in rats, myoclonus (100%), tremors (93%), salivation (75%), partial ptosis (58%) and chewing/bruxism (58%); in dogs, tremors (77%), ataxia/uncoordination (60%), myoclonus (45%), salivation (43%), excessive licking (38%), high vocalization (38%) and decreased activity (34%); in non-human primates, tremors (79%), decreased activity (70%), myoclonus (57%), retching/emesis (37%), hunched posture (30%) and ataxia/uncoordination (27%). Seizure duration ranged from 3 s to 14 min with an average of 46 ±â€¯21 s, comparable across species. At seizure onset, spike frequency averaged 9.4 Hz for the three species compared to 4.3 Hz at seizure end. Peak average amplitudes were attained at mid-seizure and amplitudes at seizure end decreased from peak but remained higher than onset amplitudes. Spike duration was inversely correlated with frequency and presented a crescendo pattern. Morphological characteristics can serve to refine automated EEG analysis. From a regulatory perspective, the most common paradigm is to use the most sensitive species in seizure liability studies but translational potential and clinical relevance may be under represented in the decision making process in some cases. EEG morphologies during drug-induced seizures presented remarkable similarities between species and tremors were identified as a predominant premonitory clinical sign in all species.

Incidence of spontaneous arrhythmias in freely moving healthy untreated Sprague-Dawley rats.

Spontaneous arrhythmia characterization in healthy rats can support interpretation when studying novel therapies. Male (n = 55) and female (n = 40) Sprague-Dawley rats with telemetry transmitters for a derivation II ECG. Arrhythmias were assessed from continuous ECG monitoring over a period of 24-48 h, and data analyzed using an automated detection algorithm with 100% manual over-read. While a total of 1825 spontaneous ventricular premature beats (VPB) were identified, only 7 rats (or 7.4%) did not present with any over the recording period. Spontaneous episode(s) of ventricular tachycardia (VT) were noted in males (27%) and females (3%). The incidence of VPB was significantly higher (p < 0.01) during the night time (7 pm-7 am) compared to daytime, while males presented with significantly (p < 0.001) more VPB than females. Most VPB were observed as single ectopic beats, followed by salvos (2 or 3 consecutive VPBs), and VT (i.e. 4 consecutive VPBs). Most VPBs were single premature ventricular contractions (PVCs) (57%), while the remaining were escape complexes (43%). Spontaneous premature junctional complexes (PJC) were also observed and were significantly more frequent during the night, and in males. Lastly, 596 episodes of spontaneous 2nd-degree atrioventricular (AV) block were identified and were significantly more frequent during the day time in males. Most 2nd-degree AV block episodes were Mobitz type I (57%), with a significantly (p < 0.05) higher incidence in males. This work emphasizes the importance of obtaining sufficient baseline data when undertaking arrhythmia analysis in safety study and provides a better understanding of both sex- and time- dependent effects of spontaneous arrhythmias in rats.

EEG: Characteristics of drug-induced seizures in rats, dogs and non-human primates.

Seizures are amongst the most frequent neurological issues encountered in pre-clinical safety testing. The objective was to characterize EEG morphologies and premonitory signs in drug-induced seizures in preclinical species. A comparative (inter-species) retrospective analysis for drug-induced seizures recorded by video-telemetry was conducted in rats (n = 53), dogs (n = 195), and non-human primates (n = 234). The most frequent premonitory signs were, in rats, myoclonus (100%), tremors (93%), salivation (75%), partial ptosis (58%) and chewing/bruxism (58%); in dogs, tremors (77%), ataxia/uncoordination (60%), myoclonus (45%), salivation (43%), excessive licking (38%), high vocalization (38%) and decreased activity (34%); in non-human primates, tremors (79%), decreased activity (70%), myoclonus (57%), retching/emesis (37%), hunched posture (30%) and ataxia/uncoordination (27%). Seizure duration ranged from 3 s to 14 min with an average of 46 ±â€¯21 s, comparable across species. At seizure onset, spike frequency averaged 9.4 Hz for the three species compared to 4.3 Hz at seizure end. Peak average amplitudes were attained at mid-seizure and amplitudes at seizure end decreased from peak but remained higher than onset amplitudes. Spike duration was inversely correlated with frequency and presented a crescendo pattern. Morphological characteristics can serve to refine automated EEG analysis. From a regulatory perspective, the most common paradigm is to use the most sensitive species in seizure liability studies but translational potential and clinical relevance may be under represented in the decision making process in some cases. EEG morphologies during drug-induced seizures presented remarkable similarities between species and tremors were identified as a predominant premonitory clinical sign in all species.

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 contractility: Correction strategies applied to telemetry data from a HESI-sponsored consortium.

INTRODUCTION: QT has a long history of heart rate (HR) correction but limited investigations have been undertaken to assess the impact of cardiovascular parameters on left ventricular (LV) contractility in drug safety testing. Cardiac contractility is affected by preload (Cyon-Frank-Starling law), afterload (Anrep effect) and HR (Bowditch effect). We evaluated multi-parameter correction methods to help with dP/dtmax interpretation. METHODOLOGY: Modeling was undertaken using data from dogs in single or double 4×4 Latin square studies. Correction models (16 fitting formulas×2 modeling approaches (universal and individualized)×2 correction approaches (linear or proportional)) were evaluated. 3D/2D cloud analysis of the beat-to-beat data for the control, pimobendan, and either itraconazole or atenolol groups were used to evaluate correlations between parameters and derive an optimal correction method. RESULTS: Cardiac contractility (i.e., dP/dtmax) was best correlated to HR and systolic LV pressure with a correlation coefficient of 0.8. In decreasing order, dP/dtmin, mean arterial blood pressure (BP), systolic BP, diastolic BP, arterial pulse pressure and LV end diastolic pressure (LVEDP) showed a reduced correlation to dP/dtmax. Subject-specific models improved the correction by up to 14% when compared to universal correction models. The non-linear correction model was superior to the linear model. DISCUSSION: Results suggest that the optimal correction formula for dP/dtmax would be subject-specific, non-linear and would include HR and LV systolic pressure. Correcting contractility for HR and systolic LV pressure may enhance data interpretation in non-clinical drug safety assessments. Similar correction methods could be evaluated for other species used in safety pharmacology.