Dog Purkinje Fibers Research Articles

Testing the nonclinical Comprehensive In Vitro Proarrhythmia Assay (CiPA) paradigm with an established anti-seizure medication: Levetiracetam case study.

Levetiracetam (LEV), a well-established anti-seizure medication (ASM), was launched before the original ICH S7B nonclinical guidance assessing QT prolongation potential and the introduction of the Comprehensive In Vitro Proarrhythmia Assay (CiPA) paradigm. No information was available on its effects on cardiac channels. The goal of this work was to "pressure test" the CiPA approach with LEV and check the concordance of nonclinical core and follow-up S7B assays with clinical and post-marketing data. The following experiments were conducted with LEV (0.25-7.5 mM): patch clamp assays on hERG (acute or trafficking effects), NaV 1.5, CaV 1.2, Kir 2.1, KV 7.1/mink, KV 1.5, KV 4.3, and HCN4; in silico electrophysiology modeling (Virtual Assay® software) in control, large-variability, and high-risk human ventricular cell populations; electrophysiology measurements in human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes and dog Purkinje fibers; ECG measurements in conscious telemetered dogs after single oral administration (150, 300, and 600 mg/kg). Except a slight inhibition (<10%) of hERG and KV 7.1/mink at 7.5 mM, that is, 30-fold the free therapeutic plasma concentration (FTPC) at 1500 mg, LEV did not affect any other cardiac channels or hERG trafficking. In both virtual and real human cardiomyocytes, and in dog Purkinje fibers, LEV induced no relevant changes in electrophysiological parameters or arrhythmia. No QTc prolongation was noted up to 2.7 mM unbound plasma levels in conscious dogs, corresponding to 10-fold the FTPC. Nonclinical assessment integrating CiPA assays shows the absence of QT prolongation and proarrhythmic risk of LEV up to at least 10-fold the FTPC and the good concordance with clinical and postmarketing data, although this does not exclude very rare occurrence of QT prolongation cases in patients with underlying risk factors.

Cardiac electrophysiological effects of ibuprofen in dog and rabbit ventricular preparations: possible implication to enhanced proarrhythmic risk.

Ibuprofen is a widely used nonsteroidal anti-inflammatory drug, which has recently been associated with increased cardiovascular risk, but its electrophysiological effects have not yet been properly studied in isolated cardiac preparations. We studied the effects of ibuprofen on action potential characteristics and several transmembrane ionic currents using the conventional microelectrode technique and the whole-cell configuration of the patch-clamp technique on cardiac preparations and enzymatically isolated ventricular myocytes. In dog (200 µM; n= 6) and rabbit (100 µM; n= 7) papillary muscles, ibuprofen moderately but significantly prolonged repolarization at 1Hz stimulation frequency. In dog Purkinje fibers, repolarization was abbreviated and maximal rate of depolarization was depressed in a frequency-dependent manner. Levofloxacin (40 µM) alone did not alter repolarization, but augmented the ibuprofen-evoked repolarization lengthening in rabbit preparations (n= 7). In dog myocytes, ibuprofen (250 µM) did not significantly influence IK1, but decreased the amplitude of Ito and IKr potassium currents by 28.2% (60mV) and 15.2% (20mV), respectively. Ibuprofen also depressed INaL and ICa currents by 19.9% and 16.4%, respectively. We conclude that ibuprofen seems to be free from effects on action potential parameters at lower concentrations. However, at higher concentrations it may alter repolarization reserve, contributing to the observed proarrhythmic risk in patients.

Nonclinical Cardiovascular Studies of Prucalopride, a Highly Selective 5-Hydroxytryptamine 4 Receptor Agonist.

Patients with chronic constipation benefit from treatment with 5-hydroxytryptamine 4 (5-HT4) receptor agonists. However, the first-generation 5-HT4 receptor agonists cisapride and tegaserod were withdrawn from the market owing to rare cardiovascular adverse events that were not 5-HT4-receptor-related but due to the lack of selectivity of these drugs. Here we report the nonclinical cardiovascular profile of the selective 5-HT4 receptor agonist prucalopride. To assess its non-5-HT4 receptor-mediated effects on cardiovascular electrophysiological parameters, in vitro studies were performed in human ether-à-go-go-related gene-transfected cells, guinea pig ventricular myocytes and papillary muscle preparations, rabbit and dog Purkinje fibers, and the Langendorff rabbit heart. In vivo experiments were performed in a rabbit model for drug-induced proarrhythmogenesis, in anesthetized guinea pigs, and anesthetized and conscious dogs. In addition, human platelet aggregation and coronary artery contraction were studied to exclude interactions that have been suggested to mediate the cardiovascular effects of tegaserod. Effects at 5-HT4 receptors were evaluated in piglet and human atrial myocardium, and in anesthetized pigs. Finally, cardiovascular endpoints were investigated in chronic, repeated-dose toxicology studies at very high prucalopride doses in rats and dogs. No relevant effects were observed in any of the cardiovascular studies at concentrations at least 50 times the therapeutic plasma level. Only in pigs were minor and transient increases in heart rate and blood pressure noted upon first exposure to prucalopride, at plasma levels at least 10 times higher than human therapeutic plasma levels. Prucalopride may thus provide therapeutic benefit without the cardiovascular risks reported for other 5-HT4 receptor agonists.

The Effect of a Novel Highly Selective Inhibitor of the Sodium/Calcium Exchanger (NCX) on Cardiac Arrhythmias in In Vitro and In Vivo Experiments.

BackgroundIn this study the effects of a new, highly selective sodium-calcium exchanger (NCX) inhibitor, ORM-10962 were investigated on cardiac NCX current, Ca2+ transients, cell shortening and in experimental arrhythmias. The level of selectivity of the novel inhibitor on several major transmembrane ion currents (L-type Ca2+ current, major repolarizing K+ currents, late Na+ current, Na+/K+ pump current) was also determined.MethodsIon currents in single dog ventricular cells (cardiac myocytes; CM), and action potentials in dog cardiac multicellular preparations were recorded utilizing the whole-cell patch clamp and standard microelectrode techniques, respectively. Ca2+ transients and cell shortening were measured in fluorescent dye loaded isolated dog myocytes. Antiarrhythmic effects of ORM-10962 were studied in anesthetized ouabain (10 μg/kg/min i.v.) pretreated guinea pigs and in ischemia-reperfusion models (I/R) of anesthetized coronary artery occluded rats and Langendorff perfused guinea pigs hearts.ResultsORM-10962 significantly reduced the inward/outward NCX currents with estimated EC50 values of 55/67 nM, respectively. The compound, even at a high concentration of 1 μM, did not modify significantly the magnitude of ICaL in CMs, neither had any apparent influence on the inward rectifier, transient outward, the rapid and slow components of the delayed rectifier potassium currents, the late and peak sodium and Na+/K+ pump currents. NCX inhibition exerted moderate positive inotropic effect under normal condition, negative inotropy when reverse, and further positive inotropic effect when forward mode was facilitated. In dog Purkinje fibres 1 μM ORM-10962 decreased the amplitude of digoxin induced delayed afterdepolarizations (DADs). Pre-treatment with 0.3 mg/kg ORM-10962 (i.v.) 10 min before starting ouabain infusion significantly delayed the development and recurrence of ventricular extrasystoles (by about 50%) or ventricular tachycardia (by about 30%) in anesthetized guinea pigs. On the contrary, ORM-10962 pre-treatment had no apparent influence on the time of onset or the severity of I/R induced arrhythmias in anesthetized rats and in Langendorff perfused guinea-pig hearts.ConclusionsThe present study provides strong evidence for a high efficacy and selectivity of the NCX-inhibitory effect of ORM-10962. Selective NCX inhibition can exert positive as well as negative inotropic effect depending on the actual operation mode of NCX. Selective NCX blockade may contribute to the prevention of DAD based arrhythmogenesis, in vivo, however, its effect on I/R induced arrhythmias is still uncertain.

Cardiovascular safety pharmacology profile of etamicastat, a novel peripheral selective dopamine-ß-hydroxylase inhibitor

Etamicastat, a peripheral reversible dopamine-ß-hydroxylase inhibitor, blocked the hERG current amplitude with an IC50 value of 44.0µg/ml in HEK 293 cells. At 0.3 and 3µg/ml, etamicastat had no effects on the action potential (AP) in male dog Purkinje fibers. At 30µg/ml, etamicastat significantly affected resting membrane potential (+4%), AP amplitude (−4%), AP duration at 60% (−14%) and AP duration at 90% (+5%) repolarization, and AP triangulation (+79%). In the telemetered conscious male dog, etamicastat (up to 20mg/kg) had no effects on arterial blood pressure, heart rate and the PR interval. At 10 and 20mg/kg, the QTc interval was slightly prolonged (8–9% max, P<0.05). No arrhythmia or other changes in the morphology of the ECG were observed. The maximum observed plasma concentrations (Cmax) of etamicastat (i.e. 3h post-administration) were 1.4 and 3.7µg/ml at 10 and 20mg/kg, respectively. No deleterious effects, including ECG disturbance were observed in male and female dogs dosed by gavage with etamicastat (up to 20mg/kg/day) for 28 days. Mean plasma Cmax etamicastat levels ranged between 2.4 and 6.3µg/ml on Day 1 and Day 28 of treatment, respectively. It is concluded that the blockade of the delayed rectifier potassium channels by etamicastat together with the QTc interval prolongation observed in conscious dogs can be considered as modest with respect to the measured plasmatic concentrations. These findings suggest that etamicastat is not likely to prolong the QT interval at therapeutic doses (~0.2µg/ml).

Abstract 504: Cardiac Safety Profile of Etamicastat, a Novel Peripheral Selective Dopamine-ß-Hydroxylase Inhibitor, in the Dog

Etamicastat, a peripheral reversible dopamine-ß-hydroxylase inhibitor, blocked the hERG current amplitude with an IC50 value of 35.4 μg/ml in HEK 293 cells. At 0.3 and 3 μg/ml, etamicastat had no effects on the action potential (AP) in dog Purkinje fibers. At 30 μg/ml, etamicastat significantly affected resting membrane potential (+4%), AP amplitude (-4%), AP duration at 60% (-14%) and AP duration at 90% (+5%) repolarization, and AP triangulation (+79%). In the telemetered conscious dog, etamicastat (up to 20 mg/kg) had no effects on arterial blood pressure, heart rate and the PR interval. At 10 and 20 mg/kg, the QTc interval was slightly prolonged (8 to 9% max, P &lt; 0.05). No arrhythmia or other changes in the morphology of the ECG were observed. The maximum observed plasma concentrations (Cmax) of etamicastat (i.e. 3h post administration) were 1.4 and 3.7 μg/ml at 10 and 20 mg/kg, respectively. No deleterious effects, including ECG disturbance were observed in male and female dogs dosed by gavage with etamicastat (up to 20 mg/kg/day) for 28 days. Mean plasma Cmax etamicastat levels ranged between 2.4 and 6.3 μg/ml on Day 1 and Day 28 of treatment, respectively. It is concluded that the blockade of the delayed rectifier potassium channels by etamicastat together with the QTc interval prolongation observed in conscious dogs can be considered as modest with respect to the measured plasmatic concentrations. These findings suggest that etamicastat is not likely to prolong the QT interval at therapeutic doses (~ 0.2 μg/ml).

Novel blockers of hyperpolarization‐activated current with isoform selectivity in recombinant cells and native tissue

BACKGROUND AND PURPOSE Selective hyperpolarization activated, cyclic nucleotide-gated channel (HCN) blockers represent an important therapeutic goal due to the wide distribution and multiple functions of these proteins, representing the molecular correlate of f- and h-current (I(f) or I(h) ). Recently, new compounds able to block differentially the homomeric HCN isoforms expressed in HEK293 have been synthesized. In the present work, the electrophysiological and pharmacological properties of these new HCN blockers were characterized and their activities evaluated on native channels. EXPERIMENTAL APPROACH HEK293 cells expressing mHCN1, mHCN2 and hHCN4 isoforms were used to verify channel blockade. Selected compounds were tested on native guinea pig sinoatrial node cells and neurons from mouse dorsal root ganglion (DRG) by patch-clamp recordings and on dog Purkinje fibres by intracellular recordings. KEY RESULTS In HEK293 cells, EC18 was found to be significantly selective for HCN4 and MEL57A for HCN1 at physiological membrane potential. When tested on guinea pig sinoatrial node cells, EC18 (10 µM) maintained its activity, reducing I(f) by 67% at -120 mV, while MEL57A (3 µM) reduced I(f) by 18%. In contrast, in mouse DRG neurons, only MEL57A (30 and 100 µM) significantly reduced I(h) by 60% at -80 mV. In dog cardiac Purkinje fibres, EC18, but not MEL57A, reduced the amplitude and slowed the slope of the spontaneous diastolic depolarization. CONCLUSIONS Our results have identified novel and highly selective HCN isoform blockers, EC18 and MEL57A; the selectivity found in recombinant system was maintained in various tissues expressing different HCN isoforms.

Direct effects of arsenic trioxide on action potentials in isolated cardiac tissues: Importance of the choice of species, type of cardiac tissue and perfusion time

IntroductionThe aim of the present study was to evaluate direct/acute effects of arsenic trioxide on action potentials (APs) in isolated cardiac tissues, and to investigate if the choice of species and tissue and the duration of the perfusion play a role in arsenic-induced acute/direct prolongation of AP/QT. Methods and resultsDirect electrophysiological effects of arsenic trioxide were measured in cardiac tissues isolated from four different species using micro-electrode recording. Arsenic (after 30 to 95min perfusion at 10μM) significantly prolonged APD90, increased triangulation of the AP and elicited early afterdepolarizations (EADs) only in isolated guinea-pig and dog Purkinje fibers but not in rabbit and porcine (minipig) Purkinje fibers. Arsenic induced a prolongation of the APD90 and increases in triangulation and the occurrence of EADs was not observed in papillary muscles of guinea-pigs and rabbits. Arsenic at 4 increasing concentrations from 0.1μM to 10μM at the standard perfusion-time of 15min per concentration, and after a continuous 90-min perfusion at 1μM and 1Hz did not induce these direct effects on APD90, triangulation and EADs in isolated guinea-pig Purkinje fibers, but it at 1 µM elicited EADs in 2 out of 7 preparations after 90min at 0.2Hz. DiscussionThe present study demonstrates that the choice of species and cardiac tissue as well as perfusion-time play important roles in arsenic-induced direct/acute effects on APD90 and induction of EADs in vitro.

Mitigating hERG Inhibition: Design of Orally Bioavailable CCR5 Antagonists as Potent Inhibitors of R5 HIV-1 Replication.

A series of CCR5 antagonists representing the thiophene-3-yl-methyl ureas were designed that met the pharmacological criteria for HIV-1 inhibition and mitigated a human ether-a-go-go related gene (hERG) inhibition liability. Reducing lipophilicity was the main design criteria used to identify compounds that did not inhibit the hERG channel, but subtle structural modifications were also important. Interestingly, within this series, compounds with low hERG inhibition prolonged the action potential duration (APD) in dog Purkinje fibers, suggesting a mixed effect on cardiac ion channels.

Electrophysiological effects of ivabradine in dog and human cardiac preparations: Potential antiarrhythmic actions

Ivabradine is a novel antianginal agent which inhibits the pacemaker current. The effects of ivabradine on maximum rate of depolarization (V max), repolarization and spontaneous depolarization have not yet been reported in human isolated cardiac preparations. The same applies to large animals close to human in heart size and spontaneous frequency. Using microelectrode technique action potential characteristics and by applying patch-clamp technique ionic currents were studied. Ivabradine exerted concentration-dependent (0.1–10 μM) decrease in the amplitude of spontaneous diastolic depolarization and reduction in spontaneous rate of firing of action potentials and produced a concentration- and frequency-dependent V max block in dog Purkinje fibers while action potential duration measured at 50% of repolarization was shortened. In the presence of ivabradine, at 400 ms cycle length, V max block developed with an onset kinetic rate constant of 13.9 ± 3.2 beat −1 in dog ventricular muscle. In addition to a fast recovery of V max from inactivation (τ = 41–46 ms) observed in control, a second slow component for recovery of V max was expressed (offset kinetics of V max block) having a time constant of 8.76 ± 1.34 s. In dog after attenuation of the repolarization reserve ivabradine moderately but significantly lengthened the repolarization. In human, significant prolongation of repolarization was only observed at 10 μM ivabradine. Ivabradine in addition to the Class V antiarrhythmic effect also has Class I/C and Class III antiarrhythmic properties, which can be advantageous in the treatment of patients with ischemic heart disease liable to disturbances of cardiac rhythm.

Class I/B antiarrhythmic property of ranolazine, a novel antianginal agent, in dog and human cardiac preparations

The aim of this study was to investigate the cellular electrophysiological effects of ranolazine on action potential characteristics. The experiments were carried out in dog and human cardiac preparations using the conventional microelectrode technique. In dog Purkinje fibres ranolazine produced a concentration- and frequency-dependent depression of the maximum rate of depolarization (Vmax) while action potential duration (APD) was shortened. In dog and human right ventricular papillary muscle ranolazine exerted no significant effect on APD, while it produced, like mexiletine, use-dependent depression of Vmax with relatively fast onset and offset kinetics. In dog midmyocardial preparations the drug did not exert statistically significant effect on repolarization at 10μM, although a tendency toward prolongation was observed at 20μM. A moderate lengthening of APD90 by ranolazine was noticed in canine atrial preparations obtained from dogs in sinus rhythm and in tachypacing induced remodelled preparations. Use-dependent depression of Vmax was more pronounced in atria from dogs in sinus rhythm than those in remodelled atria or in the ventricle. These findings indicate that ranolazine, in addition to its known late sodium current blocking effect, also depresses peak INa with class I/B antiarrhythmic characteristics. Although peak INa inhibition by ranolazine is stronger in the atria, it is also substantial (at fast stimulation frequencies) in ventricular preparations. Ranolazine also decreased the dispersion of ventricular repolarization (the difference in APD90 values between Purkinje fibres and papillary muscles), which can contribute to the antiarrhythmic property of the drug.

Properties and Molecular Composition of Mouse and Canine Cardiac Purkinje Fiber ATP-Sensitive K + Channels

ATP-sensitive potassium (KATP) channels consist of pore-forming (Kir6.1 and Kir6.2) and sulfonylurea receptor (SUR1, SUR2A or SUR2B) subunits. We compared the biophysical and pharmacological properties, as well as the molecular composition of KATP channels in cardiac Purkinje cells and ventricular myocytes. Mouse Purkinje cells and myocytes were enzymatically isolated from Cntn2-EGFP Purkinje cell reporter mice. KATP channels recorded from Purkinje cells (inside-out patch clamp configuration) were inhibited by ATP with an IC50 of 125±18µM (Hill coefficient of 2.6), which was significantly higher than that of the ventricular KATP channel (68±10µM). The slope conductance of the Purkinje cell KATP channel in symmetric 140 mM K+ was also lower (57±3pS versus 76±1pS in the ventricle). Kinetic analysis showed similar mean open (respectively 2.2±0.16 and 2.9±0.95ms) and closed times (respectively 0.4±0.02 and 0.4±0.02ms) in Purkinje cells and ventricular myocytes. The Purkinje fiber KATP channel was activated preferentially by tolbultamide (200µM) and less by levcromakalim (30µM), whereas the reverse pharmacological profile was observed for the ventricular KATP channel. Using real-time semi-quantitative RT-PCR, we detected expression of Kir6.1, Kir6.2, and SUR2 mRNA in both mouse cell types. Purkinje fibers expressed more Kir6.1 and less total SUR2 than ventricular myocytes. Conventional RT-PCR demonstrated increased SUR2B expression relative to SUR2A in Purkinje cells. Western blotting confirmed expression of Kir6.1, Kir6.2, SUR2A and SUR2B protein in both dog Purkinje fibers and ventricular tissues with Kir6.1 and SUR2B levels elevated in the Purkinje fiber. Consistently, our confocal microscopy imaging data demonstrated the presence of Kir6.1, Kir6.2, and SUR2B subunits on the Purkinje cell. In summary, KATP channels in cardiac Purkinje cells manifest unique channel properties and molecular composition. They may have a differential electrophysiological response (eg. conduction velocity and arrhythmia) during periods of ischemia.

Effect of 4‐Aminopyridine on Action Potential Parameters in Isolated Dog Purkinje Fibers

Introduction4-Aminopyridine (fampridine), a potassium channel blocker, has demonstrated efficacy in improving lower extremity strength and walking speed in patients with multiple sclerosis. Since in vitro electrophysiologic studies are recommended for evaluating a drug's potential to prolong the QT interval and induce such cardiac arrhythmias as Torsades de Pointes, we examined the electrophysiologic effects of 4-aminopyridine (0.5, 5.0, 50, and 500 µM) on isolated canine Purkinje fibers.MethodsMicroelectrodes monitored the resting membrane potential, overshoot, amplitude of action potential (AP), and maximal rate of depolarization of the AP upstroke in Purkinje fibers stimulated at 0.5 and 1.0 Hz.ResultsNone of the above variables were altered in the presence of 4-aminopyridine. The AP duration at 30%, 50%, and 90% repolarization was also monitored, with only the 500-µM concentration at the 1.0-Hz frequency significantly increasing these values with respect to baseline (P < 0.05). However, the small sample size (N = 4) was small. The proportional increases, and their 95% confidence intervals, were 90.8% (−36.4%, 218.0%), 25.8% (11.9%, 39.7%), and 22.0% (14.9%, 29.1%) for APD 30%, 50%, and 90% repolarization, respectively. Reverse rate dependence was not observed, suggesting inhibition of ion channels other than those contributing to QT interval prolongation.

Comparison of dog myocytes and purkinje fibres for assessment of drug-induced changes in action potential duration

Comparison of dog myocytes and purkinje fibres for assessment of drug-induced changes in action potential duration

Electrophysiological profile of propiverine – relationship to cardiac risk

Drugs that prolong the QT interval by blocking human ether-a-go-go (HERG) channels may enhance the risk of ventricular arrhythmia. The spasmolytic drug propiverine is widely used for the therapy of overactive bladder (OAB). Here, we have investigated the effects of propiverine on cardiac ion channels and action potentials as well as on contractile properties of cardiac tissue, in order to estimate its cardiac safety profile, because other drugs used in this indication had to be withdrawn due to safety reasons. Whole-cell patch clamp technique was used to record the following cardiac ion currents: rapidly and slowly activating delayed rectifier K+ current (I(Kr), I(Ks)), ultra rapidly activating delayed rectifier K+ current (I(Kur)), inwardly rectifying K+ current I(K1), transient outward K+ current (I(to)), and L-type Ca2+ current (I(Ca,L)). Action potentials in cardiac tissue biopsies were recorded with conventional microelectrodes. The torsade de pointes screening assay (TDPScreen) was used for drug scoring. Propiverine blocked in a concentration-dependent manner HERG channels expressed in HEK293 cells, as well as native I(Kr) current in ventricular myocytes of guinea pig (IC50 values: 10 microM and 1.8 microM respectively). At high concentrations (100 microM), propiverine suppressed I(Ks). I(K1) and the transient outward current I(to) and I(Kur) were not affected. In guinea-pig ventricular and human atrial myocytes, propiverine also blocked I(Ca,L) (IC50 values: 34.7 microM and 41.7 microM, respectively) and reduced force of contraction. Despite block of I(Kr), action potential duration was not prolonged in guinea-pig and human ventricular tissue, but decreased progressively until excitation failed altogether. Similar effects were observed in dog Purkinje fibers. Propiverine obtained a low score in the TDPScreen. In conclusion, in vitro and in vivo studies of propiverine do not provide evidence for an enhanced cardiovascular safety risk. We propose that lack of torsadogenic risk of propiverine is related to enhancement of repolarization reserve by block of I(Ca,L).

Comparison of guinea-pig ventricular myocytes and dog Purkinje fibres for in vitro assessment of drug-induced delayed repolarization

Introduction QT interval prolongation and Torsade de Pointes (TdP) arrhythmias are recognised as a potential risk with many drugs, most of which delay cardiac repolarization by inhibiting the rapidly activating K + current ( I Kr). The objective of this study was to compare the effects of compounds on cardiac action potentials recorded from guinea-pig ventricular myocytes and dog Purkinje fibres. Methods and results Effects of dofetilide, sotalol, cisapride, terfenadine, haloperidol and sparfloxacin, compounds known to cause QT prolongation (positive controls), and nifedipine and verapamil, not associated with QT prolongation (negative controls) were studied on intracellular action potentials recorded from guinea-pig isolated ventricular myocytes (VM) and dog isolated Purkinje fibres (PF). Prolongation of action potential duration (APD) by sotalol, dofetilide and sparfloxacin was concentration-dependent and of greater magnitude in dog PF compared to guinea-pig VM. The maximum prolongation of APD in guinea-pig VM at 0.5 and 1 Hz was ∼ 25% and this was associated with complete inhibition of I Kr by dofetilide. Effects on APD of cisapride and haloperidol in both preparations, and terfenadine in guinea-pig VM, were biphasic, consistent with inhibition of multiple ion channels. There was no effect of terfenadine on APD in dog PF. Haloperidol increased APD by more than 25% in guinea-pig VM, consistent with effects on additional repolarizing currents. The negative controls shortened APD to a greater extent in guinea-pig VM compared to dog PF. In general, the positive control drugs increased action potential triangulation (APD 40–90) to a greater extent than APD 90. Conclusion Guinea-pig isolated VM may be more sensitive for detecting APD prolongation with compounds inhibiting multiple ion channels and action potential triangulation (APD 40–90). Effects on repolarizing currents other than I Kr were also distinguished in guinea-pig VM.

Cardiovascular Safety of Gadoterate Meglumine (Gd-DOTA)

Gadolinium complexes are not considered to be a drug class at high risk for prolonging cardiac repolarization, which can lead to potentially life-threatening arrhythmias such as torsade de pointes. However, only limited robust data are available on these compounds despite their extensive use as contrast enhancers in magnetic resonance imaging. We present an overview of recent cardiovascular safety data obtained on gadoterate meglumine (Gd-DOTA). Cardiovascular safety was evaluated by "state-of-the-art" nonclinical ex vitro (dog Purkinje fibers) and in vivo studies in both normal (dogs) and sensitized animal models (rabbits) and in patients with various diseases in a specific clinical trial. In all of these studies, Gd-DOTA did not show any direct deleterious effect on cardiac electrophysiology and especially on ventricular repolarization. These results confirmed the good safety profile of Gd-DOTA derived from postmarketing evaluations. Nonspecific gadolinium complexes used for magnetic resonance contrast enhancement do not constitute a class-at-risk for drug-related arrhythmias.

Comparison of K + currents in cardiac Purkinje cells isolated from rabbit and dog

The repolarization reserve determines the ability of drugs to prolong the cardiac action potential duration. Differences in K + currents between rabbit and dog cardiac Purkinje cells were studied by recording the transient outward K + current ( I to) as well as the delayed rectifier K + currents ( I Ks and I Kr) during repolarization. Purkinje fibers were dissected from dog and rabbit hearts and exposed to enzymatic digestion until isolated cells were obtained. Whole cell voltage clamp methods were used to measure K + currents in both cell types. Action potential (AP) recordings from Purkinje cells displayed a rapid phase 1 repolarization due to a prominent I to with densities of 13.3 ± 2.3 and 9.6 ± 0.6 pA/pF at + 40 mV in dog and rabbit respectively. I Ks tail currents were significantly larger in dog Purkinje cells. I Kr tail current densities were comparable in Purkinje cell from both species. Rabbit ventricular and Purkinje cell AP waveforms were used for action potential clamp experiments in TSA201 cells expressing human ether a go–go related gene (HERG). HERG currents elicited by the ventricular waveform reached its maximum amplitude during phase 3 repolarization. In contrast, Purkinje cell AP waveform elicited markedly smaller HERG currents even though the action potential duration was longer. The observations suggest that the fast phase 1 and negative plateau of the Purkinje cell AP limits the contribution of I Kr to repolarization. These results provide evidence that rabbit Purkinje cells have a smaller repolarization reserve and provide a biophysical explanation for a previously observed higher sensitivity to QT prolonging drugs in rabbit than dog Purkinje fibers.

Female Gender is a Risk Factor for Drug-Induced Long QT and Early Afterdepolarizations (EADS) in Anesthetized Dogs

Clinical observations and experimental data in isolated rabbit and dog Purkinje fibers and in vivo in rabbits indicate that female gender is more sensitive to drug-induced long QT and cardiac arrhythmia. However, to-date there has been no demonstration of a gender difference in vivo in dogs and therefore male dogs tend to be the chosen sex for cardiovascular safety pharmacology tests. As such, this may potentially lead to wrong conclusions on the QT-related arrhythmogenic risk of a new chemical entity. Methods and Results We evaluated potential gender differences in the following variables, in neuromuscular blocked, mechanically ventilated anesthetized dogs: ventricular repolarization (ECG lead II, QT and QTcV; right ventricular endocardial monophasic action potential (MAP), duration at 90% repolarization, APD90 and APD90cV), spatial dispersion of the T wave (Tp-Te), instability of QTend (QT TI: total instability) and occurrence of EADs on the MAP signal. These parameters were derived from continuous recording of ECG and MAP signals. 20 female and 18 male dogs were treated with dofetilide (0.05 mg/kg i.v. infusion over 10 min). At baseline, there was no statistically significant gender difference observed in the values of the various parameters, while dofetilide infusion produced marked prolongation. Median maximum percentage changes of baseline in females versus males were respectively: heart rate (+3% / +6%, ns ), QT (+23% / +14%, p = 0.003 ), QTcV (+20% / +15%, p = 0.03 ), APD90 (+26% / +17%, p = 0.006 ), and APD90cV (+24% / +18%, p = 0.009 ). In addition, dofetilide tended to differently increase Tp-Te (+40% / +23%, ns ) and QT TI (+150% / +104%, ns ), more in female than in male animals. Furthermore, dofetilide infusion elicited more incidences of EADs on the MAP signal in female than in male dogs (73% versus 48%). Conclusion The present study confirms that female gender may be a risk factor for drug-induced long QT in the dog. Indeed, since significant alterations in additional markers beyond QT interval itself are more pronounced in female dogs and EADs occur more frequently in this sex, female dogs could be more sensitive to induction of polymorphic ventricular tachycardia (TdP). As such, consideration should be given to incorporation of female dogs into standard cardiovascular safety evaluations, or at least be the chosen single sex for drug safety evaluation for the identification of QT/TdP related adverse effects.

Female Gender is a Risk Factor for Drug-Induced Long QT and Torsade de Point (TdP) in Anesthetized, Beagle Dogs

The female gender is known to be more sensitive to drug-induced long QT and cardiac arrhythmia, based on clinical observations, experimental in vitro data, in isolated rabbit and dog Purkinje fibers, and also in vivo data in rabbits. However, to-date there has been no demonstration of a gender difference in vivo in dogs. As such, the aim of this study was to try and elucidate whether a gender difference is present in this species in vivo. This knowledge is important to drug safety evaluation since conventionally the male dog is the chosen sex for safety pharmacology studies. We evaluated potential gender differences in the following variables in neuromuscular blocked, mechanically ventilated anesthetized dogs: ventricular repolarization (ECG lead II, QT and qtcv; right ventricular endocardial monophasic action potential (MAP), duration at 90% repolarization, (APD90), spatial dispersion of the T wave (Tp-Te), instability of qtend (QT TI: total instability), occurrence of eads and/or tdps. These parameters were derived from continuous beat-to-beat recordings of ECG and MAP signals. 20 female and 18 male dogs were treated with dofetilide (0.05 mg/kg i.v. Infusion over 10 min). At baseline, no statistically significant gender difference was observed on the various parameters, whilst dofetilide infusion produced greater electrophysiological alterations in females than in males. The respective median maximum percentage changes from baseline in females versus males were: heart rate (+3% / +6%, ns), QT (+23% / +14%, p = 0.003), qtcv (+20% / +15%, p = 0.03) and APD90 (+26% / +17%, p = 0.006). Dofetilide tended to differently increase Tp-Te (+40% vs +23%, ns) and QT TI (+150% vs +104%), more in female than in male animals. Additionally, dofetilide infusion elicited more incidences of eads on the MAP signal in female than in male dogs (73% versus 48%). Furthermore, the ikr blocker dofetilide (0.05 mg/kg iv) combined with Iks blocker HMR1556 (0.5 mg/kg iv) elicited tdps in 3 out of 4 female dogs in our preliminary investigations. Similar to man, the present study suggests that the female gender may be a risk factor for drug-induced long QT. Indeed, since significant alterations in additional markers beyond QT interval itself appear to be more pronounced in female dogs and eads occur more frequently in this sex, female dogs could be more sensitive to induction of polymorphic ventricular tachycardia (tdp). As such, consideration should be given to incorporation of female dogs into standard cardiovascular safety evaluations, or at least be the chosen single sex for drug safety evaluation, since the use of males may potentially lead to an underestimate of the QT-related pro-arrhythmogenic risk of a new chemical entity.