Quantitative Profiling of the Effects of Vanoxerine on Human Cardiac Ion Channels and its Application to Cardiac Risk.

Vanoxerine has been in clinical trials for Parkinsonism, depression and cocaine addiction but lacked efficacy. Although a potent blocker of hERG, it produced no serious adverse events. We attributed the unexpected result to offsetting Multiple Ion Channel Effects (MICE). Vanoxerine's effects were strongly frequency-dependent and we repositioned it for treatment of atrial fibrillation and flutter. Vanoxerine terminated AF/AFL in an animal model and a dose-ranging clinical trial. Reversion to normal rhythm was associated with QT prolongation yet absent proarrhythmia markers for Torsade de Pointes (TdP). To understand the QT/TdP discordance, we used quantitative profiling and compared vanoxerine with dofetilide, a selective hERG-blocking torsadogen used for intractable AF, verapamil, a non-torsadogenic MICE comparator and bepridil, a torsadogenic MICE comparator. At clinically relevant concentrations, verapamil blocked hCav1.2 and hERG, as did vanoxerine and bepridil both of which also blocked hNav1.5. In acute experiments and simulations, dofetilide produced early after depolarizations (EADs) and arrhythmias, whereas verapamil, vanoxerine and bepridil produced no proarrhythmia markers. Of the MICE drugs only bepridil inhibited hERG trafficking following overnight exposure. The results are consistent with the emphasis on MICE of the CiPA assay. Additionally we propose that trafficking inhibition of hERG be added to CiPA.

COR-ART: A multicenter, randomized, double-blind, placebo-controlled dose-ranging study to evaluate single oral doses of vanoxerine for conversion of recent-onset atrial fibrillation or flutter to normal sinus rhythm.

BACKGROUND: Restoration of sinus rhythm (SR) in patients with atrial fibrillation/atrial flutter (AF/AFL) is limited principally to direct current cardioversion. The multi-ion channel blocker vanoxerine may prove an effective alternative. OBJECTIVE: The purpose of this study was to assess vanoxerine, a 1,4-dialkylpiperazine derivative, for acute conversion of recent-onset, symptomatic AF and AFL. METHODS: One hundred four subjects with symptomatic AF/AFL for <7 days were randomized sequentially to single oral doses of vanoxerine 200, 300, and 400 mg or placebo. Holter monitors were examined for conversion to SR and proarrhythmia through ≥24 hours. RESULTS: Conversion to SR was dose related: 18.2%, 44.0%, and 52.0% within 4 hours, and 59.1%, 64.0%, and 84.0% within 24 hours, for the 200-, 300-, and 400-mg groups, respectively. This was significantly higher than placebo for the 300- and 400-mg groups within 4 hours (12.5% for placebo; P = .0138 and P = .0028, respectively) and for all doses within 24 hours (31.3% for placebo; P = .0421, P = .0138, P = .0001 for 200-, 300-, and 400-mg vanoxerine groups, respectively). Although vanoxerine caused significant dose-dependent QTcF (QT correction by Fridericia) prolongation, monomorphic or polymorphic ventricular tachycardia did not occur. Adverse events were mild and self-limited, with only the highest dose having a greater frequency than placebo. CONCLUSION: Oral vanoxerine converted AF/AFL to SR at a high rate, was well tolerated, and caused no ventricular proarrhythmia.

Evaluating state dependence and subtype selectivity of calcium channel modulators in automated electrophysiology assays.

Voltage-gated Ca2+ channels play essential roles in control of neurosecretion and muscle contraction. The pharmacological significance of Cav channels stem from their identification as the molecular targets of calcium blockers used in the treatment of cardiovascular diseases, such as hypertension, angina, and arrhythmia, and neurologic diseases, such as pain and seizure. It has been proposed that state-dependent Cav inhibitors, that is, those that preferentially bind to channels in open or inactivated states, may improve the therapeutic window over relatively state-independent Cav inhibitors. High-throughput fluorescent-based functional assays have been useful in screening chemical libraries to identify Cav inhibitors. However, hit confirmation, mechanism of action, and subtype selectivity are better suited to automated patch clamp assays that have sufficient capacity to handle the volume of compounds identified during screening, even of modest sized libraries (≤500,000 compounds), and the flexible voltage control that allows evaluation of state-dependent drug blocks. IonWorks Barracuda (IWB), the newest generation of IonWorks instruments, provides the opportunity to accelerate the Cav drug discovery studies in an automated patch clamp platform in 384-well format capable of medium throughput screening and profiling studies. We have validated hCav1.2, hCav2.1, hCav2.2, and hCav3.2 channels assays on the IWB platform (population patch clamp mode) and demonstrated that the biophysical characteristics of the channels (activation, inactivation, and steady-state inactivation) obtained with the IWB system are consistent with known subtype-specific characteristics. Using standard reference compounds (nifedipine, BAY K8644, verapamil, mibefradil, and pimozide), we demonstrated subtype-selective and state- and use-dependent characteristics of drug-channel interactions. Here we describe the design and validation of novel robust high-throughput Cav channel assays on the IWB platform. The assays can be used to screen focused compound libraries for state-dependent Cav channel antagonists, to prioritize compounds for potency or to counterscreen for Cav subtype selectivity.

MICE models: superior to the HERG model in predicting Torsade de Pointes.

Drug-induced block of the cardiac hERG (human Ether-à-go-go-Related Gene) potassium channel delays cardiac repolarization and increases the risk of Torsade de Pointes (TdP), a potentially lethal arrhythmia. A positive hERG assay has been embraced by regulators as a non-clinical predictor of TdP despite a discordance of about 30%. To test whether assaying concomitant block of multiple ion channels (Multiple Ion Channel Effects or MICE) improves predictivity we measured the concentration-responses of hERG, Nav1.5 and Cav1.2 currents for 32 torsadogenic and 23 non-torsadogenic drugs from multiple classes. We used automated gigaseal patch clamp instruments to provide higher throughput along with accuracy and reproducibility. Logistic regression models using the MICE assay showed a significant reduction in false positives (Type 1 errors) and false negatives (Type 2 errors) when compared to the hERG assay. The best MICE model only required a comparison of the blocking potencies between hERG and Cav1.2.

Multi-parameter in vitro toxicity testing of crizotinib, sunitinib, erlotinib, and nilotinib in human cardiomyocytes.

Tyrosine kinase inhibitors (TKi) have greatly improved the treatment and prognosis of multiple cancer types. However, unexpected cardiotoxicity has arisen in a subset of patients treated with these agents that was not wholly predicted by pre-clinical testing, which centers around animal toxicity studies and inhibition of the human Ether-à-go-go-Related Gene (hERG) channel. Therefore, we sought to determine whether a multi-parameter test panel assessing the effect of drug treatment on cellular, molecular, and electrophysiological endpoints could accurately predict cardiotoxicity. We examined how 4 FDA-approved TKi agents impacted cell viability, apoptosis, reactive oxygen species (ROS) generation, metabolic status, impedance, and ion channel function in human cardiomyocytes. The 3 drugs clinically associated with severe cardiac adverse events (crizotinib, sunitinib, nilotinib) all proved to be cardiotoxic in our in vitro tests while the relatively cardiac-safe drug erlotinib showed only minor changes in cardiac cell health. Crizotinib, an ALK/MET inhibitor, led to increased ROS production, caspase activation, cholesterol accumulation, disruption in cardiac cell beat rate, and blockage of ion channels. The multi-targeted TKi sunitinib showed decreased cardiomyocyte viability, AMPK inhibition, increased lipid accumulation, disrupted beat pattern, and hERG block. Nilotinib, a second generation Bcr-Abl inhibitor, led to increased ROS generation, caspase activation, hERG block, and an arrhythmic beat pattern. Thus, each drug showed a unique toxicity profile that may reflect the multiple mechanisms leading to cardiotoxicity. This study demonstrates that a multi-parameter approach can provide a robust characterization of drug-induced cardiomyocyte damage that can be leveraged to improve drug safety during early phase development.

A history of the role of the hERG channel in cardiac risk assessment.

The human ether-a-go-go-related gene (hERG, Kv11.1) K(+) channel plays an important role in cardiac repolarization. Following its cloning and expression it was established that inhibition of this channel was the molecular mechanism for many non-antiarrhythmic drugs that produce torsades de pointes associated with QT prolongation. Therefore the study of in vitro drug-hERG interactions has become an important part of modern safety pharmacology. Manual and automated patch clamp electrophysiology, in silico modeling, and hERG trafficking assays have been developed to aid in this study. The correlation between in vitro hERG IC50, drug exposure, QT prolongation in the thorough QT clinical trial and risk of TdP has greatly reduced drug withdrawals due to TdP. However a significant association with Type 1 errors in particular remains and may have a negative impact on drug development. Combining hERG data with other non-clinical and clinical markers of proarrhythmia will increase the specificity and sensitivity of cardiac risk assessment. hERG will continue to play an important role in drug development and safety pharmacology in the future.

Oral vanoxerine prevents reinduction of atrial tachyarrhythmias: preliminary results.

BACKGROUND: Vanoxerine is a promising, new, investigational antiarrhythmic drug. The purpose of this study was to test the hypothesis that oral dosing of vanoxerine would first terminate induced atrial flutter (AFL) and atrial fibrillation (AF), and then prevent their reinduction. METHODS: In 5 dogs with sterile pericarditis, on the fourth day after creating the pericarditis, we performed electrophysiologic (EP) studies at baseline, measuring atrial excitability, refractoriness (AERP), and conduction time (CT) when pacing from the right atrial appendage, Bachmann's bundle (BB), and the posteroinferior left atrium at cycle lengths (CLs) of 400, 300, and 200 ms. Then, after induction of AFL or AF, all dogs received hourly oral doses of vanoxerine: 90 mg, followed by 180 mg and 270 mg. Blood was obtained to determine plasma vanoxerine concentrations at baseline, every 30 minutes, when neither AFL nor AF were inducible, and, finally, 1 hour after the 270 mg dose. Then we repeated the baseline EP studies. RESULTS: Four dogs had inducible, sustained AFL, and 1 dog only had induced, nonsustained AF. In 4 AFL episodes, oral vanoxerine terminated the AFL and then rendered it noninducible after an average of 111 minutes (range 75-180 minutes) after the first dose was administered. The mean vanoxerine plasma level at the point of noninducibility was 84 ng/mL, with a narrow range of 76-99 ng/mL. In the dog with induced, nonsustained AF, it was no longer inducible at a drug level of 75 ng/mL. Vanoxerine did not significantly (1) prolong the AERP except at BB, and then only at the faster pacing CLs; (2) change atrial excitability thresholds; (3) prolong atrial conduction time, the PR interval, the QRS complex or the QT interval. CONCLUSIONS: Orally administered vanoxerine effectively terminated AFL and rendered it noninducible. It also suppressed inducibility of nonsustained AF. These effects occurred at consistent plasma drug levels. Vanoxerine's insignificant or minimal effects on measured electrophysiologic parameters are consistent with little proarrhythmic risk.

Increased cardiac risk in concomitant methadone and diazepam treatment: pharmacodynamic interactions in cardiac ion channels.

Methadone, a synthetic opioid for treatment of chronic pain and withdrawal from opioid dependence, has been linked to QT prolongation, potentially fatal torsades de pointes, and sudden cardiac death. Concomitant use of diazepam or other benzodiazepines in methadone maintenance treatment can increase the risk of sudden death. Therefore, we determined the effects of methadone and diazepam singly and in combination on cardiac action potentials (APs) and on the major ion channels responsible for cardiac repolarization. Using patch clamp recording in human stem cell-derived cardiomyocytes and stably transfected mammalian cells, we found that methadone produced concentration-dependent AP prolongation and ion channel block at low micromolar concentrations: hERG (IC50 = 1.7 µM), hNav1.5 (11.2 µM tonic block; 5.5 µM phasic block), and hCav1.2 (26.7 µM tonic block; 7.7 µM phasic block). Methadone was less potent in hKv4.3/hKChIP2.2 (IC50 = 39.0 µM) and hKvLQT1/hminK (53.3 µM). In contrast, diazepam blocked channels only at much higher concentrations and had no effect on AP duration at 1 µM. However, coadministration of 1-µM diazepam with methadone caused a statistically significant increase in AP duration and a 4-fold attenuation of hNav1.5 block (IC50 values were 44.2 µM and 26.6 µM, respectively, for tonic and phasic block), with no significant effect on methadone-induced block of hERG, hCav1.2, hKv4.3/hKChIP2.2, and hKvLQT1/hminK channels. Thus, although diazepam alone does not prolong the QT interval, the relief of methadone-induced Na channel block may leave hERG K channel block uncompensated, thereby increasing cardiac risk.

The action potential and comparative pharmacology of stem cell-derived human cardiomyocytes.

INTRODUCTION: The cardiac action potential (CAP) of stem cell-derived human cardiomyocytes (SC-hCMs) is potentially the most powerful preclinical biomarker for cardiac safety and efficacy in humans. Our experiments tested this hypothesis by examining the CAP and relevant pharmacology of these cells. METHODS: The electrophysiological and pharmacological profiles of SC-hCMs were compared to rabbit and canine Purkinje fibers (PFs). Ventricular SC-hCMs provided the dominant electrophysiological phenotype (approximately 82%) in a population of ventricular, atrial and nodal cardiomyocytes (CMs). The effects of reference compounds were measured in SC-hCMs using perforated patch, current clamp recording. Selective inhibitors of I(Kr), I(Ks), I(Ca,L), and I(Na), and norepinephrine (NE), were tested on SC-hCM action potentials (APs). RESULTS: AP prolongation was observed upon exposure to hERG channel blockers (terfenadine, quinidine, cisapride, sotalol, E-4031 and verapamil), with significantly shorter latencies than in PF assays. For the torsadogenic compounds, terfenadine and quinidine, SC-hCM AP prolongation occurred at significantly lower concentrations than in canine or rabbit PF APs. Moreover, the I(Ks) blocker chromanol 293B prolonged APs from SC-hCMs, whereas both rabbit and canine PF assays are insensitive to I(Ks) blockers in the absence of adrenergic preconditioning. Early afterdepolarizations (EADs) were induced by 100 nM E-4031 and 100 nM cisapride in the SC-hCM assay, but not in the canine or rabbit PF assay. Selective inhibition of I(Na) and I(Ca,L) slowed V(max) and shortened AP duration, respectively. NE prolonged the AP duration of SC-hCMs. DISCUSSION: The CAP of SC-hCMs has been validated as a powerful preclinical biomarker for cardiac safety and efficacy. In addition to its human nature, the SC-hCM AP assay removes diffusion delays, reduces test compound consumption, demonstrates an overall pharmacological sensitivity that is greater than conventional rabbit or canine PF assays, and accurately predicts cardiac risk of known torsadogenic compounds.

Vanoxerine: cellular mechanism of a new antiarrhythmic.

INTRODUCTION: There remains an unmet need for safe and effective antiarrhythmic drugs, especially for the treatment of atrial fibrillation. Vanoxerine is a drug that is free of adverse cardiac events in normal volunteers, yet is a potent blocker of the hERG (hK(v)11.1) cardiac potassium channel. Consequently,we hypothesized that vanoxerine might also be a potent blocker of cardiac calcium (Ca) and sodium (Na) currents, and would not affect transmural dispersion of repolarization. METHODS: The whole cell patch clamp technique was used to measure currents from cloned ion channels overexpressed in stable cell lines and single ventricular myocytes. We measured intracellular action potentials from canine ventricular wedges and Purkinje fibers using sharp microelectrode technique. RESULTS: We found that vanoxerine was a potent hK(v)11.1 blocker, and at submicromolar concentrations, it blocked Ca and Na currents in a strongly frequency-dependent manner. In the canine ventricular wedge preparation vanoxerine did not significantly affect transmural action potential waveforms, QT interval or transmural dispersion of repolarization. CONCLUSIONS: Vanoxerine (1) is a potent blocker of cardiac hERG, Na and Ca channels; (2) block is strongly frequency-dependent especially for Na and Ca channels; and (3) transmural dispersion of ventricular repolarization is unaffected. The multichannel block and repolarization uniformity resemble the effects of amiodarone, the exemplar atrial fibrillation drug. Vanoxerine is a completely different chemical and has none of amiodarone's toxic effects. Vanoxerine has characteristics of a potentially effective and safe antiarrhythmic.