Rosanne Varkevisser

Comparison of the IKr blockers moxifloxacin, dofetilide and E‐4031 in five screening models of pro‐arrhythmia reveals lack of specificity of isolated cardiomyocytes

Drug development requires the testing of new chemical entities for adverse effects. For cardiac safety screening, improved assays are urgently needed. Isolated adult cardiomyocytes (CM) and human embryonic stem cell‐derived cardiomyocytes (hESC‐CM) could be used to identify pro‐arrhythmic compounds. In the present study, five assays were employed to investigate their sensitivity and specificity for evaluating the pro‐arrhythmic properties of IKr blockers, using moxifloxacin (safe compound) and dofetilide or E‐4031 (unsafe compounds).

Beat-to-beat variability of repolarization as a new biomarker for proarrhythmia in vivo

Pharmacological safety evaluation of (pro) drugs includes cardiac safety assessment of proarrhythmic liability in healthy tissue with emphasis on the rapid component of the delayed rectifier (I(Kr)). The lack of (1) an arrhythmic end point, (2) tests in remodeled, predisposed tissue, and (3) testing chronic drug influence on channel trafficking impairs on the drawn conclusions of these assays regarding drug safety. Moreover, the currently used human ether-à-go-go-related gene assays, action potential duration, prolongation in multicellular preparations, or the QT interval have significant shortcomings in their prediction of an increased risk for drug-induced torsades de pointes arrhythmia. In this review, it will be proposed that beat-to-beat variability of repolarization quantified as short-term variability can (1) discriminate between safe and unsafe drugs even under predisposed and highly arrhythmogenic conditions despite accompanying QT prolongation and (2) identify the individual at risk for subsequent arrhythmic events.

Efficient and specific cardiac IK1 inhibition by a new pentamidine analogue

AIMS In excitable cells, KIR2.x ion-channel-carried inward rectifier current (IK₁) is thought to set the negative and stable resting membrane potential, and contributes to action potential repolarization. Loss- or gain-of-function mutations correlate with cardiac arrhythmias and pathological remodelling affects normal KIR2.x protein levels. No specific IK1 inhibitor is currently available for in vivo use, which severely hampers studies on the precise role of IK1 in normal cardiac physiology and pathophysiology. The diamine antiprotozoal drug pentamidine (P) acutely inhibits IK₁ by plugging the cytoplasmic pore region of the channel. We aim to develop more efficient and specific IK₁ inhibitors based on the P structure. METHODS AND RESULTS We analysed seven pentamidine analogues (PA-1 to PA-7) for IK₁ blocking potency at 200 nM using inside-out patches from KIR2.1 expressing HEK-293 cells. PA-6 showed the highest potency and was tested further. PA-6 blocked KIR2.x currents of human and mouse with low IC₅₀ values (12-15 nM). Modelling indicated that PA-6 had less electrostatic but more lipophilic interactions with the cytoplasmic channel pore than P, resulting in a higher channel affinity for PA-6 (ΔG -44.1 kJ/Mol) than for P (ΔG -31.7 kJ/Mol). The involvement of acidic amino acid residues E224 and E299 in drug-channel interaction was confirmed experimentally. PA-6 did not affect INav1.5, ICa-L, IKv4.3, IKv11.1, and IKv7.1/minK currents at 200 nM. PA-6 inhibited the inward (50 nM 40%; 100 nM 59%; 200 nM 77%) and outward (50 nM 40%; 100 nM 76%; 200 nM 100%) components of IK₁ in isolated canine adult-ventricular cardiomyocytes (CMs). PA-6 prolonged action potential duration of CMs by 8 (n = 9), 26 (n = 5), and 34% (n = 11) at 50, 100, and 200 nM, respectively. Unlike P, PA-6 had no effect on KIR2.1 channel expression at concentrations from 0.1 to 3 μM. However, PA-6 at 10 μM increased KIR2.1 expression levels. Also, PA-6 did not affect the maturation of hERG, except when applied at 10 μM. CONCLUSION PA-6 has higher efficiency and specificity to KIR2.x-mediated current than P, lengthens action potential duration, and does not affect channel trafficking at concentrations relevant for complete IK₁ block.

Structure-activity relationships of pentamidine-affected ion channel trafficking and dofetilide mediated rescue.

Drug interference with normal hERG protein trafficking substantially reduces the channel density in the plasma membrane and thereby poses an arrhythmic threat. The chemical substructures important for hERG trafficking inhibition were investigated using pentamidine as a model drug. Furthermore, the relationship between acute ion channel block and correction of trafficking by dofetilide was studied.

Vernakalant is devoid of proarrhythmic effects in the complete AV block dog model.

The anesthetized chronic AV-blocked dog (cAVB) and methoxamine-sensitized rabbit model are widely used to determine pro-arrhythmic properties of drugs. In general, both models show similar results. However, conflicting data have also been reported; K201 and AZD1305 induced Torsade de Pointes (TdP) exclusively in cAVB dogs. Vernakalant, an antiarrhythmic drug that blocks several ion channels has been approved only in Europe. Its propensity to induce repolarization-dependent TdP arrhythmias has been evaluated solely in the methoxamine-sensitized rabbits. We therefore assessed the proarrhythmic potential of vernakalant in the cAVB dog model. Vernakalant was evaluated in 10 mongrel dogs (sinus rhythm (SR) 2mg/kg; chronic AV block (cAVB) 2+3mg/kg). The same dogs were challenged with dofetilide (25 μg/kg) to evaluate TdP inducibility. During the serial experiments the animals were paced from the right ventricular apex (60 beats/min). Short-term variability of repolarization (STV) was quantified for proarrhythmic risk. In SR (n=8) vernakalant prolonged QT (265 ± 11 to 311 ± 18 ms P<0.01(**)) but not PQ or QRS. In cAVB (n=8), 2mg/kg vernakalant prolonged QT (391 ± 43 to 519 ± 73 ms(**)) and QRS (103 ± 24 to 108 ± 23 ms(**)). After a 30 min lag-time, 3mg/kg vernakalant (n=4) increased QT to a lesser extent (413 ± 34 to 454 ± 27 ms(**)) while maintaining QRS prolongation (114 ± 18 to 122 ± 20 ms(**)). Neither dose increased STV or caused arrhythmias. Dofetilide prolonged QT (398 ± 51 to 615 ± 71 ms(**)), increased STV (1.0 ± 0.4 to 2.2 ± 1.0 ms P<0.05(⁎)) and induced TdP arrhythmias in 6/8(⁎) cAVB dogs. Vernakalant did not induce arrhythmias in the cAVB dog model. Higher dosages (3mg/kg) did not prolong repolarization further whereas negative inotropic effects were starting to become apparent precluding further increases in dose.

AV-block and conduction slowing prevail over TdP arrhythmias in the methoxamine-sensitized pro-arrhythmic rabbit model

The methoxamine‐sensitized rabbit model is widely used to screen drugs for proarrhythmic properties, especially repolarization‐dependent TdP arrhythmias. With the change of anesthesia and/or sensitizing agent, conduction disturbances have been reported as well. Therefore, we compared currently available in‐house anesthetics in order to preserve arrhythmia sensitivity and preclude conduction disturbances.

Short-term variability of repolarization is superior to other repolarization parameters in the evaluation of diverse antiarrhythmic interventions in the chronic atrioventricular block dog

Short-term variability (STV), to quantify beat-to-beat variability of repolarization (BVR), is a surrogate parameter that reliably identifies proarrhythmic risk in preclinical models. Examples include not only the use in the chronic atrioventricular block (CAVB) dog model whereby it was developed, but also in vulnerable patients with heart failure or drug-induced long QT syndrome. In the CAVB dog model, STV can specifically distinguish between safe and unsafe drugs in proarrhythmic screening. Conversely, this dog model also offers the possibility to evaluate antiarrhythmic strategies in a setting of Torsades de Pointes (TdP) induction with a standard IKr inhibitor. The different antiarrhythmic interventions studied in suppression and prevention of drug-induced TdP in vivo in the CAVB dog model and in vitro in canine ventricular cardiomyocytes are described in this overview. We provide evidence that STV predicts the magnitude of antiarrhythmic effect against TdP better than other repolarization parameters in both suppression and prevention conditions. Moreover, suppression and prevention experiments revealed the same level of antiarrhythmic efficacy, while cellular experiments appear more sensitive in comparison to drug testing in vivo. Together, these observations suggest that STV could be used as a consistent indicator to rank efficacy of antiarrhythmic interventions in a number of conditions.This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND) , where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal.Abstract: Short-term variability (STV), to quantify beat-to-beat variability of repolarization, is a surrogate parameter that reliably identifies proarrhythmic risk in preclinical models. Examples include not only the use in the chronic atrioventricular block (CAVB) dog model whereby it was developed but also in vulnerable patients with heart failure or drug-induced long QT syndrome. In the CAVB dog model, STV can specifically distinguish between safe and unsafe drugs in proarrhythmic screening. Conversely, this dog model also offers the possibility to evaluate antiarrhythmic strategies in a setting of Torsades de Pointes (TdP) induction with a standard IKr inhibitor. The different antiarrhythmic interventions studied in suppression and prevention of drug-induced TdP in vivo in the CAVB dog model and in vitro in canine ventricular cardiomyocytes are described in this overview. We provide evidence that STV predicts the magnitude of antiarrhythmic effect against TdP better than other repolarization parameters in both suppression and prevention conditions. Moreover, suppression and prevention experiments revealed the same level of antiarrhythmic efficacy, whereas cellular experiments seem more sensitive in comparison with drug testing in vivo. Together, these observations suggest that STV could be used as a consistent indicator to rank efficacy of antiarrhythmic interventions in a number of conditions.

The inward rectifier current inhibitor PA‐6 terminates atrial fibrillation and does not cause ventricular arrhythmias in goat and dog models

The density of the inward rectifier current (IK1) increases in atrial fibrillation (AF), shortening effective refractory period and thus promoting atrial re‐entry. The synthetic compound pentamidine analogue 6 (PA‐6) is a selective and potent IK1 inhibitor. We tested PA‐6 for anti‐AF efficacy and potential proarrhythmia, using established models in large animals.

Dehydroevodiamine and hortiamine, alkaloids from the traditional Chinese herbal drug Evodia rutaecarpa, are IKr blockers with proarrhythmic effects in vitro and in vivo

Graphical abstract Figure. No caption available. ABSTRACT Evodiae fructus is a widely used herbal drug in traditional Chinese medicine. Evodia extract was found to inhibit hERG channels. The aim of the current study was to identify hERG inhibitors in Evodia extract and to investigate their potential proarrhythmic effects. Dehydroevodiamine (DHE) and hortiamine were identified as IKr (rapid delayed rectifier current) inhibitors in Evodia extract by HPLC‐microfractionation and subsequent patch clamp studies on human embryonic kidney cells. DHE and hortiamine inhibited IKr with IC50s of 253.2 ± 26.3 nM and 144.8 ± 35.1 nM, respectively. In dog ventricular cardiomyocytes, DHE dose‐dependently prolonged the action potential duration (APD). Early afterdepolarizations (EADs) were seen in 14, 67, 100, and 67% of cells after 0.01, 0.1, 1 and 10 &mgr;M DHE, respectively. The proarrhythmic potential of DHE was evaluated in 8 anesthetized rabbits and in 8 chronic atrioventricular block (cAVB) dogs. In rabbits, DHE increased the QT interval significantly by 12 ± 10% (0.05 mg/kg/5 min) and 60 ± 26% (0.5 mg/kg/5 min), and induced Torsade de Pointes arrhythmias (TdP, 0.5 mg/kg/5 min) in 2 rabbits. In cAVB dogs, 0.33 mg/kg/5 min DHE increased QT duration by 48 ± 10% (P < 0.05*) and induced TdP in 2/4 dogs. A higher dose did not induce TdP. In human induced pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CMs), methanolic extracts of Evodia, DHE and hortiamine dose‐dependently prolonged APD. At 3 &mgr;M DHE and hortiamine induced EADs. hERG inhibition at submicromolar concentrations, APD prolongation and EADs in hiPSC‐CMs and dose‐dependent proarrhythmic effects of DHE at micromolar plasma concentrations in cAVB dogs should increase awareness regarding proarrhythmic effects of widely used Evodia extracts.

Selective late sodium current inhibitor GS‐458967 suppresses Torsades de Pointes by mostly affecting perpetuation but not initiation of the arrhythmia

Enhanced late sodium current (late INa) in heart failure and long QT syndrome type 3 is proarrhythmic. This study investigated the antiarrhythmic effect and mode of action of the selective and potent late INa inhibitor GS‐458967 (GS967) against Torsades de Pointes arrhythmias (TdP) in the chronic atrioventricular block (CAVB) dog.