Accurate in silico simulation of the rabbit Purkinje fiber electrophysiological assay to facilitate early pharmaceutical cardiosafety assessment: Dream or reality?

As a branch of quantitative systems toxicology, in silico simulations are of growing attractiveness to guide preclinical cardiosafety risk assessments. Traditionally, a cascade of in vitro/in vivo assays has been applied in pharmaceutical research to screen out molecules at risk for cardiac side effects and prevent subsequent risk for patients. Drug cardiosafety assessments typically employ early mechanistic, hazard-oriented in silico/in vitro assays for compound inhibition of cardiac ion channels, followed by induced pluripotent stem cells (iPSCs) or tissue-based models such as the rabbit Purkinje fiber assay, which includes the major mechanisms contributing to action potential (AP) genesis. Additionally, multiscale simulation techniques based on mathematical models have become available, which are performed in silico 'at the heart' of compound triage to substitute Purkinje tests and increase translatability through mechanistic interpretability. To adhere to the 3R principle and reduce animal experiments, we performed a comparative benchmark and investigated a variety of mathematical cardiac AP models, including a newly developed minimalistic model specifically tailored to the AP of rabbit Purkinje cells, for their ability to substitute experiments. The simulated changes in AP duration (dAPD90) at increasing drug concentrations were compared to experimental results from 588 internal Purkinje fiber studies covering 555 different drugs with diverse modes of action. Using our minimalistic model, 80% of the Purkinje experiments could be quantitatively reproduced. This result allows for significant saving of experimental effort in early research and justifies the embedding of electrophysiological simulations into the DMTA (Design, Make, Test, Analyze) cycle in pharmaceutical compound optimization.

In vitro ion channel profile and ex vivo cardiac electrophysiology properties of the R(-) and S(+) enantiomers of hydroxychloroquine.

Hydroxychloroquine (HCQ) is a derivative of the antimalaria drug chloroquine primarily prescribed for autoimmune diseases. Recent attempts to repurpose HCQ in the treatment of corona virus disease 2019 has raised concerns because of its propensity to prolong the QT-segment on the electrocardiogram, an effect associated with increased pro-arrhythmic risk. Since chirality can affect drug pharmacological properties, we have evaluated the functional effects of the R(-) and S(+) enantiomers of HCQ on six ion channels contributing to the cardiac action potential and on electrophysiological parameters of isolated Purkinje fibers. We found that R(-)HCQ and S(+)HCQ block human Kir2.1 and hERG potassium channels in the 1 µM-100 µM range with a 2-4 fold enantiomeric separation. NaV1.5 sodium currents and CaV1.2 calcium currents, as well as KV4.3 and KV7.1 potassium currents remained unaffected at up to 90 µM. In rabbit Purkinje fibers, R(-)HCQ prominently depolarized the membrane resting potential, inducing autogenic activity at 10 µM and 30 µM, while S(+)HCQ primarily increased the action potential duration, inducing occasional early afterdepolarization at these concentrations. These data suggest that both enantiomers of HCQ can alter cardiac tissue electrophysiology at concentrations above their plasmatic levels at therapeutic doses, and that chirality does not substantially influence their arrhythmogenic potential in vitro.

SAR340835, a Novel Selective Na+/Ca2+ Exchanger Inhibitor, Improves Cardiac Function and Restores Sympathovagal Balance in Heart Failure.

In failing hearts, Na+/Ca2+ exchanger (NCX) overactivity contributes to Ca2+ depletion, leading to contractile dysfunction. Inhibition of NCX is expected to normalize Ca2+ mishandling, to limit afterdepolarization-related arrhythmias, and to improve cardiac function in heart failure (HF). SAR340835/SAR296968 is a selective NCX inhibitor for all NCX isoforms across species, including human, with no effect on the native voltage-dependent calcium and sodium currents in vitro. Additionally, it showed in vitro and in vivo antiarrhythmic properties in several models of early and delayed afterdepolarization-related arrhythmias. Its effect on cardiac function was studied under intravenous infusion at 250,750 or 1500 µg/kg per hour in dogs, which were either normal or submitted to chronic ventricular pacing at 240 bpm (HF dogs). HF dogs were infused with the reference inotrope dobutamine (10 µg/kg per minute, i.v.). In normal dogs, NCX inhibitor increased cardiac contractility (dP/dtmax) and stroke volume (SV) and tended to reduce heart rate (HR). In HF dogs, NCX inhibitor significantly and dose-dependently increased SV from the first dose (+28.5%, +48.8%, and +62% at 250, 750, and 1500 µg/kg per hour, respectively) while significantly increasing dP/dtmax only at 1500 (+33%). Furthermore, NCX inhibitor significantly restored sympathovagal balance and spontaneous baroreflex sensitivity (BRS) from the first dose and reduced HR at the highest dose. In HF dogs, dobutamine significantly increased dP/dtmax and SV (+68.8%) but did not change HR, sympathovagal balance, or BRS. Overall, SAR340835, a selective potent NCX inhibitor, displayed a unique therapeutic profile, combining antiarrhythmic properties, capacity to restore systolic function, sympathovagal balance, and BRS in HF dogs. NCX inhibitors may offer new therapeutic options for acute HF treatment. SIGNIFICANCE STATEMENT: HF is facing growing health and economic burden. Moreover, patients hospitalized for acute heart failure are at high risk of decompensation recurrence, and no current acute decompensated HF therapy definitively improved outcomes. A new potent, Na+/Ca2+ exchanger inhibitor SAR340835 with antiarrhythmic properties improved systolic function of failing hearts without creating hypotension, while reducing heart rate and restoring sympathovagal balance. SAR340835 may offer a unique and attractive pharmacological profile for patients with acute heart failure as compared with current inotrope, such as dobutamine.

Cannabidiol inhibits multiple cardiac ion channels and shortens ventricular action potential duration in vitro.

Cannabidiol (CBD) is a non-psychoactive component of Cannabis which has recently received regulatory consideration for the treatment of intractable forms of epilepsy such as the Dravet and the Lennox-Gastaut syndromes. The mechanisms of the antiepileptic effects of CBD are unclear, but several pre-clinical studies suggest the involvement of ion channels. Therefore, we have evaluated the effects of CBD on seven major cardiac currents shaping the human ventricular action potential and on Purkinje fibers isolated from rabbit hearts to assess the in vitro cardiac safety profile of CBD. We found that CBD inhibits with comparable micromolar potencies the peak and late components of the NaV1.5 sodium current, the CaV1.2 mediated L-type calcium current, as well as all the repolarizing potassium currents examined except Kir2.1. The most sensitive channels were KV7.1 and the least sensitive were KV11.1 (hERG), which underly the slow (IKs) and rapid (IKr) components, respectively, of the cardiac delayed-rectifier current. In the Purkinje fibers, CBD decreased the action potential (AP) duration more potently at half-maximal than at near complete repolarization, and slightly decreased the AP amplitude and its maximal upstroke velocity. CBD had no significant effects on the membrane resting potential except at the highest concentration tested under fast pacing rate. These data show that CBD impacts cardiac electrophysiology and suggest that caution should be exercised when prescribing CBD to carriers of cardiac channelopathies or in conjunction with other drugs known to affect heart rhythm or contractility.

The oral Ca/calmodulin-dependent kinase II inhibitor RA608 improves contractile function and prevents arrhythmias in heart failure.

AIMS: Excessive activation of Ca/calmodulin-dependent kinase II (CaMKII) is of critical importance in heart failure (HF) and atrial fibrillation. Unfortunately, lack of selectivity, specificity, and bioavailability have slowed down development of inhibitors for clinical use. We investigated a novel CaMKIIδ/CaMKIIÉ£-selective, ATP-competitive, orally available CaMKII inhibitor (RA608) on right atrial biopsies of 119 patients undergoing heart surgery. Furthermore, we evaluated its oral efficacy to prevent deterioration of HF in mice after transverse aortic constriction (TAC). METHODS AND RESULTS: In human atrial cardiomyocytes and trabeculae, respectively, RA608 significantly reduced sarcoplasmic reticulum Ca leak, reduced diastolic tension, and increased sarcoplasmic reticulum Ca content. Patch-clamp recordings confirmed the safety of RA608 in human cardiomyocytes. C57BL6/J mice were subjected to TAC, and left ventricular function was monitored by echocardiography. Two weeks after TAC, RA608 was administered by oral gavage for 7 days. Oral RA608 treatment prevented deterioration of ejection fraction. At 3 weeks after TAC, ejection fraction was 46.1 ± 3.7% (RA608) vs. 34.9 ± 2.6% (vehicle), n = 9 vs. n = 12, P < 0.05, ANOVA, which correlated with significantly less CaMKII autophosphorylation at threonine 287. Moreover, a single oral dose significantly reduced inducibility of atrial and ventricular arrhythmias in CaMKIIδ transgenic mice 4 h after administration. Atrial fibrillation was induced in 6/6 mice for vehicle vs. 1/7 for RA608, P < 0.05, 'n - 1' χ2 test. Ventricular tachycardia was induced in 6/7 for vehicle vs. 2/7 for RA608, P < 0.05, 'n - 1' χ2 test. CONCLUSIONS: RA608 is the first orally administrable CaMKII inhibitor with potent efficacy in human myocytes. Moreover, oral administration potently inhibits arrhythmogenesis and attenuates HF development in mice in vivo.

Comprehensive in vitro Proarrhythmia Assay (CiPA): Pending issues for successful validation and implementation.

INTRODUCTION: The Comprehensive in vitro Proarrhythmia Assay (CiPA) is a nonclinical Safety Pharmacology paradigm for discovering electrophysiological mechanisms that are likely to confer proarrhythmic liability to drug candidates intended for human use. TOPICS COVERED: Key talks delivered at the 'CiPA on my mind' session, held during the 2015 Annual Meeting of the Safety Pharmacology Society (SPS), are summarized. Issues and potential solutions relating to crucial constituents [e.g., biological materials (ion channels and pluripotent stem cell-derived cardiomyocytes), study platforms, drug solutions, and data analysis] of CiPA core assays are critically examined. DISCUSSION: In order to advance the CiPA paradigm from the current testing and validation stages to a research and regulatory drug development strategy, systematic guidance by CiPA stakeholders is necessary to expedite solutions to pending and newly arising issues. Once a study protocol is proved to yield robust and reproducible results within and across laboratories, it can be implemented as qualified regulatory procedure.

The isolated rabbit heart and Purkinje fibers as models for identifying proarrhythmic liability.

INTRODUCTION: Delayed ventricular repolarization is associated with rare, but often fatal, polymorphic tachyarrhythmias named Torsades de Pointes. ICH S7B guideline recommends an integrated approach for cardiovascular preclinical evaluation of new drug candidates, including action potential assays (as a Purkinje fiber test) but also proarrhythmia models. The aim of this preliminary study was to compare the respective value of two preclinical in vitro rabbit cardiac preparations-the Purkinje fiber and the isolated perfused heart (Langendorff method)-based on effects of dofetilide, a selective IKr inhibitor. METHODS: Transmembrane action potentials from rabbit Purkinje fibers were recorded using a conventional intracellular glass microelectrode. Electrocardiograms from rabbit isolated hearts were evaluated for QRS, QT and T wave durations (Tpeak-Tend). The pacing protocol was the same for both preparations (basal rate of 80 bpm and pacing of 40, 60 and 140 bpm). Dofetilide was tested in both systems at concentrations of 1, 3 and 10 nmol/L. RESULTS: In Purkinje fibers dofetilide induced a concentration- and reverse use-dependent increase in action potential durations measured at 50 and 90% of repolarization. At 10 nmol/L, only 3/10 fibers showed early after depolarizations. In the isolated heart model, dofetilide also induced a similar concentration- and reverse use-dependent increase in QT-interval. From 3 nmol/L, major changes in T wave morphology, R-on-T extrasystoles and TdP were observed, mainly at low rate. Prior to arrhythmias, T wave shape and duration were markedly altered suggesting an increase in the heterogeneity of cardiac ventricular repolarization. CONCLUSIONS: The effects of dofetilide were comparable in the two models for delayed repolarization but the isolated heart appears to be a better predictor for arrhythmias and a unique in vitro model to assess arrhythmogenic potential of QT prolonging compounds at least when associated with IKr/hERG inhibition.