Molecular determinants of loperamide and N-desmethyl loperamide binding in the hERG cardiac K+ channel.

Abuse of the common anti-diarrheal loperamide is associated with QT interval prolongation as well as development of the potentially fatal arrhythmia torsades de pointes. The mechanism underlying this cardiotoxicity is high affinity inhibition of the human ether-a-go-go-related gene (hERG) cardiac K+ channel. N-Desmethyl loperamide is the major metabolite of loperamide and is a close structural relative of the parent molecule. To date no information is available regarding the affinity of N-desmethyl loperamide for human cardiac ion channels. The effects of N-desmethyl loperamide on various cloned human cardiac ion channels including hERG, KvLQT1/mink and Nav1.5 were studied and compared to that of the parent. N-Desmethyl loperamide was a much weaker (7.5-fold) inhibitor of hERG compared to loperamide. However, given the higher plasma levels of the metabolite relative to the parent, it is likely that N-desmethyl loperamide can contribute, at least secondarily, to the cardiotoxicity observed with loperamide abuse. We used the recently solved cryo-EM structure of the hERG channel together with previously published inhibitors, to understand the basis of the interactions as well as the difference that a single methyl plays in the hERG channel blocking affinities of these two compounds.

Observations on conducting whole-cell patch clamping of the hERG cardiac K+ channel in pure human serum.

Inhibition of the human ether-a-go-go-related gene (hERG) K+ channel by drugs leads to QT prolongation on the electrocardiogram and can result in serious cardiac arrhythmia. For this reason, screening of drugs on hERG is mandatory during the drug development process. Patch clamp electrophysiology in a defined physiological saline solution (PSS) represents the standard method for assaying drug effects on the channel. To make the assay more translatable to clinical studies, we have conducted whole-cell patch clamping of hERG using pure human serum as the extracellular medium. Pure human serum had little effect on the hERG channel waveform or the current-voltage relationship when compared to PSS. hERG current recordings were highly stable in serum at room temperature, but prolonged recordings at the physiological temperature required prior heat inactivation of the serum. Compared to PSS, the IC50 values, conducted at room temperature, of the classic hERG blocking drugs cisapride, moxifloxacin, and terfenadine were shifted to the right by an extent predicted by their known plasma protein binding, but we did not detect any differences in IC50 s between male and female serum. Total plasma levels of these drugs associated with clinical QT prolongation corresponded to small (<15%) inhibition of hERG current in pure serum suggesting that minor inhibition of the channel leads to observable pharmacodynamic effects. Conducting whole-cell patch clamping of hERG in human serum has the potential to make the assay more translatable to clinical studies and improve its predictive value for safety testing. Copyright © 2016 John Wiley & Sons, Ltd.

Proarrhythmic mechanisms of the common anti-diarrheal medication loperamide: revelations from the opioid abuse epidemic.

Loperamide is a µ-opioid receptor agonist commonly used to treat diarrhea and often available as an over-the-counter medication. Recently, numerous reports of QRS widening accompanied by dramatic QT interval prolongation, torsades de pointe arrhythmia, and death have been reported in opioid abusers consuming large amounts of the drug to produce euphoria or prevent opiate withdrawal. The present study was undertaken to determine the mechanisms of this cardiotoxicity. Using whole-cell patch clamp electrophysiology, we tested loperamide on the cloned human cardiac sodium channel (Nav1.5) and the two main repolarizing cardiac K(+) channels cloned from the human heart: KvLQT1/minK and the human ether-a-go-go-related gene (hERG) channel. Loperamide inhibited Nav1.5 with IC50 values of 297 and 239 nM at holding potentials of -90 and -70 mV, respectively. Loperamide was weakly active on KvLQT1/minK producing 17 and 65 % inhibition at concentrations of 1 and 10 µM, respectively. Conversely, loperamide was found to be a very high affinity inhibitor of the hERG channel with an IC50 value of 89 nM at room temperature and 33 nM when measured at physiological temperature. The QRS and QT interval prolongation and the attending arrhythmias, produced by loperamide, derive from high affinity inhibition of Nav1.5 and especially hERG. Since the drug has been widely available and safely used as directed for many years, we believe that the potent inhibition loperamide possesses for cardiac ion channels has only been uncovered because of the excessive misuse of the drug as a consequence of the recent opioid abuse epidemic.

A New Perspective in the Field of Cardiac Safety Testing through the Comprehensive In Vitro Proarrhythmia Assay Paradigm.

For the past decade, cardiac safety screening to evaluate the propensity of drugs to produce QT interval prolongation and Torsades de Pointes (TdP) arrhythmia has been conducted according to ICH S7B and ICH E14 guidelines. Central to the existing approach are hERG channel assays and in vivo QT measurements. Although effective, the present paradigm carries a risk of unnecessary compound attrition and high cost, especially when considering costly thorough QT (TQT) studies conducted later in drug development. The C: omprehensive I: n Vitro P: roarrhythmia A: ssay (CiPA) initiative is a public-private collaboration with the aim of updating the existing cardiac safety testing paradigm to better evaluate arrhythmia risk and remove the need for TQT studies. It is hoped that CiPA will produce a standardized ion channel assay approach, incorporating defined tests against major cardiac ion channels, the results of which then inform evaluation of proarrhythmic actions in silico, using human ventricular action potential reconstructions. Results are then to be confirmed using human (stem cell-derived) cardiomyocytes. This perspective article reviews the rationale, progress of, and challenges for the CiPA initiative, if this new paradigm is to replace existing practice and, in time, lead to improved and widely accepted cardiac safety testing guidelines.

On Becoming a Pharmacologist: Channeling David Triggle.

This Festschrift contribution summarizes the perspectives of two of David Triggle's graduate students. Both share somewhat parallel scientific and career paths-i.e., enrolling in the Biochemical Pharmacology program at the University of Buffalo, pursuing graduate training under the mentorship of David Triggle and subsequently undertaking postdoctoral studies at Baylor College of Medicine, and ultimately transitioning to careers in the pharmaceutical industry, now for the past 20+ years. David Triggle's mentorship and guidance was instrumental in developing scientific careers and thought processes at both professional and personal levels. The article is a chronological view of the lessons learned from David Triggle starting in the early 1980s and continuing to the present.

L-type Ca2+ channel responses to bay k 8644 in stem cell-derived cardiomyocytes are unusually dependent on holding potential and charge carrier.

Human stem cell-derived cardiomyocytes provide a cellular model for the study of electrophysiology in the human heart and are finding a niche in the field of safety pharmacology for predicting proarrhythmia. The cardiac L-type Ca2+ channel is an important target for some of these safety studies. However, the pharmacology of this channel in these cells is altered compared to native cardiac tissue, specifically in its sensitivity to the Ca2+ channel activator S-(-)-Bay K 8644. Using patch clamp electrophysiology, we examined the effects of S-(-)-Bay K 8644 in three separate stem cell-derived cardiomyocyte cell lines under various conditions in an effort to detect more typical responses to the drug. S-(-)-Bay K 8644 failed to produce characteristically large increases in current when cells were held at -40 mV and Ca2+ was used as the charge carrier, although high-affinity binding and the effects of the antagonist isomer, R-(+)-Bay K 8644, were intact. Dephosphorylation of the channel with acetylcholine failed to restore the sensitivity of the channel to the drug. Only when the holding potential was shifted to a more hyperpolarized (-60 mV) level, and external Ca2+ was replaced by Ba2+, could large increases in current amplitude be observed. Even under these conditions, increases in current amplitude varied dramatically between different cell lines and channel kinetics following drug addition were generally atypical. The results indicate that the pharmacology of S-(-)-Bay K 8644 in stem cell-derived cardiomyocytes varies by cell type, is unusually dependent on holding potential and charge carrier, and is different from that observed in primary human heart cells.

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.

Ca²âº channel activators reveal differential L-type Ca²âº channel pharmacology between native and stem cell-derived cardiomyocytes.

Human stem cell-derived cardiomyocytes provide new models for studying the ion channel pharmacology of human cardiac cells for both drug discovery and safety pharmacology purposes. However, detailed pharmacological characterization of ion channels in stem cell-derived cardiomyocytes is lacking. Therefore, we used patch-clamp electrophysiology to perform a pharmacological survey of the L-type Ca²âº channel in induced pluripotent and embryonic stem cell-derived cardiomyocytes and compared the results with native guinea pig ventricular cells. Six structurally distinct antagonists [nifedipine, verapamil, diltiazem, lidoflazine, bepridil, and 2-[(cis-2-phenylcyclopentyl)imino]-azacyclotridecane hydrochloride (MDL 12330)] and two structurally distinct activators [methyl 2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)phenyl]-1,4-dihydropyridine-3-carboxylate (Bay K8644) and 2,5-dimethyl-4-[2-(phenylmethyl)benzoyl]-1H-pyrrole-3-carboxylic acid methyl ester (FPL 64176)] were used. The IC50 values for the six antagonists showed little variability between the three cell types. However, whereas Bay K8644 produced robust increases in Ca²âº channel current in guinea pig myocytes, it failed to enhance current in the two stem cell lines. Furthermore, Ca²âº channel current kinetics after addition of Bay K8644 differed in the stem cell-derived cardiomyocytes compared with native cells. FPL 64176 produced consistently large increases in Ca²âº channel current in guinea pig myocytes but had a variable effect on current amplitude in the stem cell-derived myocytes. The effects of FPL 64176 on current kinetics were similar in all three cell types. We conclude that, in the stem cell-derived myocytes tested, L-type Ca²âº channel antagonist pharmacology is preserved, but the pharmacology of activators is altered. The results highlight the need for extensive pharmacological characterization of ion channels in stem cell-derived cardiomyocytes because these complex proteins contain multiple sites of drug action.

Mephedrone, a new designer drug of abuse, produces acute hemodynamic effects in the rat.

Mephedrone (4-methylmethcathinone) is a new and popular drug of abuse widely available on the Internet and still legal in some parts of the world. Clinical reports are now emerging suggesting that the drug displays sympathomimetic toxicity on the cardiovascular system but no studies have yet explored its cardiovascular effects. Therefore we examined the effects of mephedrone on the cardiovascular system using a combination of in vitro electrophysiology and in vivo hemodynamic and echocardiographic measurements. Patch clamp studies revealed that mephedrone, up to 30 µM, had little effect on the major voltage-dependent ion channels of the heart or on action potentials recorded in guinea pig myocytes. Subcutaneous administration of mephedrone (3 and 15 mg/kg) to conscious telemetry-implanted rats produced dose-dependent increases in heart rate and blood pressure which persisted after pre-treatment with reserpine. Echocardiographic analysis demonstrated that intravenous injection of mephedrone (0.3 and 1mg/kg) increased cardiac function, including cardiac output, ejection fraction, and stroke volume, similar to methamphetamine (0.3mg/kg). We conclude that mephedrone is not directly pro-arrhythmic, but induces substantial increases in heart rate, blood pressure and cardiac contractility and this activity contributes to the cardiovascular toxicity in people who abuse the drug.

Oversulfated chondroitin sulfate and OSCS-contaminated heparin cause dose- and route-dependent hemodynamic effects in the rat.

During 2007-2008, serious adverse events were reported following iv administration of certain batches of commercially available heparin in humans. Anaphylactoid reactions with acute hypotension were the hallmark of these cases. Subsequently, it was shown that a contaminant, oversulfated chondroitin sulfate (OSCS), was responsible for these adverse events. The present study was undertaken to further elucidate the risks related to OSCS-contaminated heparin preparations. Using an anesthetized rat hemodynamic model, marked diastolic blood pressure drops were induced with a single iv injection of a contaminated heparin (1000 IU/kg; 34% wt/wt OSCS). OSCS alone (0.8 and 20 mg/kg) or in combination (0.8-1.7 mg/kg) with uncontaminated heparin produced a similar hypotensive effect, whereas heparin spiked with 0.2 or 0.4 mg/kg OSCS produced no hemodynamic changes. In conscious rats, acute hypotensive effects were seen following single iv administration of OSCS-spiked heparin (1.7 or 3.0 mg/kg). Conversely, no hemodynamic effects were observed with same doses when administered sc. Pretreatment with a bradykinin-2 receptor antagonist (HOE140) fully abolished the hypotensive response after iv OSCS (1.7 mg/kg) administration, whereas pretreatment with the histamine (H1) receptor antagonist cetirizine did not. In vitro, OSCS (25 and 250 µg/ml) induced a robust, dose-related increase in kallikrein activity in rat and human plasma with a lower amplitude of response in dog and pig. The data suggest that the adverse events associated with OSCS-contaminated heparin are dependent upon the concentration of contaminant and its route of administration. Furthermore, the kallikrein-kinin system plays a pivotal role in the initiation of OSCS-related vascular effects.

Manual whole-cell patch-clamping of the HERG cardiac K+ channel.

Delayed ventricular repolarization, as measured by a prolongation of the QT interval on the electrocardiogram, is a major safety issue in the drug development process. It is now recognized that most cases of drug-induced QT prolongation arise from direct pharmacological inhibition of the human ether-a-go-go-related gene (HERG) cardiac K+ channel. It is standard practice to test a drug's ability to interact with the HERG channel prior to entry into clinical trials. This testing is used, as part of a larger battery of tests, to help predict the cardiac safety profile of a drug. Manual whole-cell patch-clamping provides the most sensitive and accurate way to examine the biophysical and pharmacological properties of the HERG cardiac K+ channel.

In vitro electrocardiographic and cardiac ion channel effects of (-)-epigallocatechin-3-gallate, the main catechin of green tea.

Epigallocatechin-3-gallate (EGCG) is the major catechin found in green tea. EGCG is also available for consumption in the form of concentrated over-the-counter nutritional supplements. This compound is currently undergoing clinical trials for the treatment of a number of diseases including multiple sclerosis, and a variety of cancers. To date, few data exist regarding the effects of EGCG on the electrophysiology of the heart. Therefore, we examined the effects of EGCG on the electrocardiogram recorded from Langendorff-perfused guinea pig hearts and on cardiac ion channels using patch-clamp electrophysiology. EGCG had no significant effects on the electrocardiogram at concentrations of 3 and 10 microM. At 30 microM, EGCG prolonged PR and QRS intervals, slightly shortened the QT interval, and altered the shape of the ST-T-wave segment. The ST segment merged with the upstroke of the T wave, and we noted a prolongation in the time from the peak of the T wave until the end. Patch-clamp studies identified the KvLQT1/minK K(+) channel as a target for EGCG (IC(50) = 30.1 microM). In addition, EGCG inhibited the cloned human cardiac Na(+) channel Na(v)1.5 in a voltage-dependent fashion. The L-type Ca(2+) channel was inhibited by 20.8% at 30 microM, whereas the human ether-a-go-go-related gene and Kv4.3 cardiac K(+) channels were less sensitive to inhibition by EGCG. ECGC has a number of electrophysiological effects in the heart, and these effects may have clinical significance when multigram doses of this compound are used in human clinical trials or through self-ingestion of large amounts of over-the-counter products enriched in EGCG.

In Vitro electrophysiological activity of nerispirdine, a novel 4-aminopyridine derivative.

Summary 1. The non-selective K(+) channel blocker 4-aminopyridine (4-AP) has shown clinical efficacy in the treatment of neurological disorders such as multiple sclerosis. The clinical usefulness of 4-AP is hampered by its ability to produce seizures. Nerispirdine, an analogue of 4-AP, is currently under clinical investigation for the treatment of multiple sclerosis. In contrast with 4-AP, nerispirdine is not proconvulsant, suggesting mechanistic differences between the two drugs. 2. Using whole-cell patch-clamp electrophysiology, we compared the effects of 4-AP and nerispirdine on the cloned human K(+) channels K(v)1.1 and K(v)1.2, expressed in Chinese hamster ovary cells, and on voltage-dependent Na(+) channels recorded from human SH-SY5Y cells. 3. Nerispirdine inhibited K(v)1.1 and K(v)1.2 with IC(50) values of 3.6 and 3.7 micromol/L, respectively. 4-Aminopyridine was approximately 50-fold less potent at blocking these channels. Nerispirdine also inhibited voltage-dependent Na(+) channel currents recorded from human SH-SY5Y cells with an IC(50) of 11.9 micromol/L when measured from a -70 mV holding potential. In contrast, 4-AP had no effect on Na(+) channel currents. 4. The results demonstrate that nerispirdine, like 4-AP, can inhibit axonal K(+) channels and that this mechanism may underlie the ability of the drug to enhance neuronal conduction. Unlike 4-AP, nerispirdine can also inhibit neuronal Na(+) channels, a mechanism that may explain why nerispirdine lacks proconvulsant activity.

Functional interaction between DPI 201-106, a drug that mimics congenital long QT syndrome, and sevoflurane on the guinea-pig cardiac action potential.

1. Sevoflurane produces QT prolongation on the electrocardiogram, predominantly via inhibition of the slow delayed rectifier K(+) current. DPI 201-106 is an experimental drug that produces QT prolongation by reducing Na(+) channel inactivation, thereby mimicking congenital long QT syndrome type 3 (LQT3). The present study explores the electrophysiological consequences of administration of sevoflurane in the presence of impaired Na(+) channel activity. 2. We examined the effects of sevoflurane and DPI 201-106, alone and in combination, on the cardiac action potential of guinea-pig ventricular myocytes using standard microelectrode techniques. 3. Both sevoflurane and DPI-201-106 prolonged action potential duration, with the combination of the two drugs producing greater than additive effects. Similarly, instability and triangulation of the action potential waveform, measures of pro-arrhythmia, were more pronounced when both drugs were combined. 4. Sevoflurane treatment significantly alters cardiac action potential waveforms when administered in the presence of impaired Na(+) channel inactivation. These results indicate the potential for ventricular arrhythmia when sevoflurane is administered to LQT3 patients and suggests caution when using sevoflurane in this population.

ILSI-HESI cardiovascular safety subcommittee initiative: evaluation of three non-clinical models of QT prolongation.

INTRODUCTION: Drugs that delay cardiac repolarization pose potential safety risks to patients and cause serious regulatory concern because of the link between QT interval prolongation and the potentially fatal arrhythmia torsades de pointes (TdP). Predicting which drugs will cause TdP is an inexact and difficult science. The utility of non-clinical assays was not well understood due in part to variability in methods, species, and consistency in the assays reported in the literature. The Health and Environmental Sciences Institute of the International Life Sciences Institute (ILSI/HESI) outlined a set of studies to determine how well selected commonly used non-clinical assays identified compounds known to cause TdP and prolong QT interval in humans. METHODS: Compounds known to prolong ventricular repolarization and compounds considered safe by years of clinical use were tested in three assays: HERG ionic current, Purkinje fiber repolarization, and in vivo QT studies in conscious telemeterized dogs. RESULTS: The data from each of these assays demonstrate that compounds that may pose a proarrhythmia risk for patients can be distinguished from those that are considered safe. DISCUSSION: Taken collectively, the in-vitro and in-vivo preclinical results can be integrated to develop an accurate preclinical risk assessment to support clinical safety.

Mechanisms underlying the QT interval-prolonging effects of sevoflurane and its interactions with other QT-prolonging drugs.

BACKGROUND: Sevoflurane prolongs ventricular repolarization in patients, but the mechanisms are not fully characterized. The effects of sevoflurane on many cloned human cardiac ion channels have not been studied, and the interactions between sevoflurane and other drugs that prolong cardiac repolarization have not been detailed. METHODS: The effects of sevoflurane on action potentials and L-type Ca channels in guinea pig myocytes were examined. Sevoflurane's effects on cloned human cardiac K channels and the cloned human cardiac Na channel were studied. The consequences of combining sevoflurane and the class III antiarrhythmic drugs sotalol or dofetilide on action potential duration were also examined. RESULTS: Sevoflurane produced an increase in action potential duration at concentrations of 0.3-1 mm. Contrary to most drugs that delay ventricular repolarization, sevoflurane was without effect on the human ether-a-go-go-related gene cardiac potassium channel but instead produced a reduction in KvLQT1/minK K channel currents and inhibited the Kv4.3 K channel by speeding its apparent rate of inactivation. Sevoflurane had little effect on Na and Ca channel currents at concentrations of 1 mm or less. When the authors coadministered sevoflurane with sotalol or dofetilide, synergistic effects on repolarization were observed, resulting in large increases in action potential duration (up to 66%). CONCLUSION: Prolonged ventricular repolarization observed with administration of sevoflurane results from inhibition of KvLQT1/minK and Kv4.3 cardiac K channels. Combining sevoflurane with class III antiarrhythmic drugs results in supra-additive effects on action potential duration. The results indicate that sevoflurane, when administered with this class of drug, could result in excessive delays in ventricular repolarization. The results suggest the need for further clinical studies.

Predictive models for hERG potassium channel blockers.

We report here a general method for the prediction of hERG potassium channel blockers using computational models generated from correlation analyses of a large dataset and pharmacophore-based GRIND descriptors. These 3D-QSAR models are compared favorably with other traditional and chemometric based HQSAR methods.

Discovery of a small molecule activator of the human ether-a-go-go-related gene (HERG) cardiac K+ channel.

Many drugs inhibit the human ether-a-go-go-related gene (HERG) cardiac K+ channel. This leads to action potential prolongation on the cellular level, a prolongation of the QT interval on the electrocardiogram, and sometimes cardiac arrhythmia. To date, no activators of this channel have been reported. Here, we describe the in vitro electrophysiological effects of (3R,4R)-4-[3-(6-methoxyquinolin-4-yl)-3-oxo-propyl]-1-[3-(2,3,5-trifluoro-phenyl)-prop-2-ynyl]-piperidine-3-carboxylic acid (RPR260243), a novel activator of HERG. Using patch-clamp electrophysiology, we found that RPR260243 dramatically slowed current deactivation when applied to cells stably expressing HERG. The effects of RPR260243 on HERG channel deactivation were temperature- and voltage-dependent and occurred over the concentration range of 1 to 30 microM. RPR260243-modified HERG currents were inhibited by dofetilide (IC50 = 58 nM). RPR260243 had little effect on HERG current amplitude and no significant effects on steady-state activation parameters or on channel inactivation processes. RPR260243 displayed no activator-like effects on other voltage-dependent ion channels, including the closely related erg3 K+ channel. RPR260243 enhanced the delayed rectifier current in guinea pig myocytes but, when administered alone, had little effect on action potential parameters in these cells. However, RPR260243 completely reversed the action potential-prolonging effects of dofetilide in this preparation. Using the Langendorff heart method, we found that 5 microM RPR260243 increased T-wave amplitude, prolonged the PR interval, and shortened the QT interval. We believe RPR260243 represents the first known HERG channel activator and that the drug works primarily by inhibiting channel closure, leading to a persistent HERG channel current upon repolarization. Compounds like RPR260243 will be useful for studying the physiological role of HERG and may one day find use in treating cardiac disease.