hERG Channel Blockers Research Articles

HERG Channel Blocking Ipecac Alkaloids Identified by Combined In Silico - In Vitro Screening.

Human ether-a-go-go-related gene channel blocking is associated with QT interval prolongation and increased risk of potentially fatal arrhythmias. As natural products keep increasing in popularity, there is an urgent need for studies assessing human ether-a-go-go-related gene channel-related cardiotoxic risks. We selected 49 plant species based on the results of a pharmacophore-based virtual screening campaign, in parallel with a literature data survey concerning highly consumed herbal medicines with reported cardiac liabilities. Lead-like enhanced extracts were prepared, an initial in vitro screening was performed at 100 µg/mL by voltage clamp on Xenopus oocytes, and five human ether-a-go-go-related gene channel blocking extracts were identified. In accordance to the six virtually predicted alkaloids, the root extract of Carapichea ipecacuanha inhibited human ether-a-go-go-related gene channel currents by 32.5 %. A phytochemical workflow resulted in the isolation and identification of five out of the six virtually predicted alkaloids. All isolates blocked human ether-a-go-go-related gene channel currents to different extents. The major ipecac constituents emetine (1) and cephaeline (2) showed IC50 values of 21.4 and 5.3 µM, respectively, measured by whole-cell patch clamp in HEK293 cells. This is the first report on human ether-a-go-go-related gene channel blockers from C. ipecacuanha. Its roots and rhizomes are used to produce different pharmacopeial ipecac preparations that are mainly used as emetics for poisoning treatment. Our findings raise further questions regarding the safety and over-the-counter appropriateness of these herbal products.

Development of potent small-molecule inhibitors to drug the undruggable steroid receptor coactivator-3

Protein-protein interactions (PPIs) play a central role in most biological processes, and therefore represent an important class of targets for therapeutic development. However, disrupting PPIs using small-molecule inhibitors (SMIs) is challenging and often deemed as "undruggable." We developed a cell-based functional assay for high-throughput screening to identify SMIs for steroid receptor coactivator-3 (SRC-3 or AIB1), a large and mostly unstructured nuclear protein. Without any SRC-3 structural information, we identified SI-2 as a highly promising SMI for SRC-3. SI-2 meets all of the criteria of Lipinski's rule [Lipinski et al. (2001) Adv Drug Deliv Rev 46(1-3):3-26] for a drug-like molecule and has a half-life of 1 h in a pharmacokinetics study and a reasonable oral availability in mice. As a SRC-3 SMI, SI-2 can selectively reduce the transcriptional activities and the protein concentrations of SRC-3 in cells through direct physical interactions with SRC-3, and selectively induce breast cancer cell death with IC50 values in the low nanomolar range (3-20 nM), but not affect normal cell viability. Furthermore, SI-2 can significantly inhibit primary tumor growth and reduce SRC-3 protein levels in a breast cancer mouse model. In a toxicology study, SI-2 caused minimal acute cardiotoxicity based on a hERG channel blocking assay and an unappreciable chronic toxicity to major organs based on histological analyses. We believe that this work could significantly improve breast cancer treatment through the development of "first-in-class" drugs that target oncogenic coactivators.

Assessing the in Vitro Cardiotoxicity of Superparamagnetic Ion Oxide Nanoparticles (SPIONs)

Understanding the mechanism of toxicity of nanoparticles (NPs) represents a major challenge for biomedical application. Since the cardiotoxicity of drugs is mainly due to blockade of the human ether-a-go-go related gene (hERG) protein, the inhibition of hERG channel is a first step in a non-clinical testing strategy. Superparamagnetic iron oxide nanoparticles (SPIONs) are composed of either a magnetite (Fe3O4) or maghemite (γ-Fe2O3) core coated with a biocompatible polymer. Both maghemite and magnetite are ferrimagnetic in nature, and when the size of the particles enters the nanometric scale (below ca. 80-120 nm) they assume a single domain magnetic structure and superparamagnetic feature. SPIONs are suitable for biological application for the following reasons: i) Fe is a naturally occurring metal in the human body; ii) they can be utilized by the body in subsequent metabolic processes; iii) their magnetic behavior allows them to be used in various biomedical applications, e.g. magnetic fluid hyperthermia.In this work we describe the interaction of SPIONs, coated with different ligands, with hERG channel. The NPs were synthesized by thermal decomposition and investigated by XRD, TEM and DLS analysis; the superparamagnetic behavior was confirmed by SQUID magnetometric analysis. We functionalized the NPs by a coating exchange reaction with several ligands and analyzed them by DLS and Z-Potential analysis. The interaction between SPIONs and hERG channel, expressed in HEK cells, was investigated by patch-clamp. We found that NPs coated with polyacrylic acid (PAA) or 2,3-dimercaptosuccinic acid (DMSA) blocked hERG channels. On the contrary, 3-aminopropyl phosphonic acid (APPA)-stabilized NPs or non-coated NPs had no effect on hERG channel, suggesting an important role of charge density of NPs in hERG binding interactions. A mode of action fitting the experimental evidence has been proposed for the observed hERG blocking activity.

On the Relation between HERG Channel Block in Cell Line and Action Potential Prolongation in Human iPSC Cardiomyocytes

Blockade of rapid delayed rectifier current (IKr) can lead to cardiac arrhythmia, which is a major concern in drug discovery and development. Drug safety tests involve the voltage clamp measurements on HEK cells expressing hERG potassium channels (Kv11.1) and high affinity hERG channel inhibition is widely accepted predictor of Torsade de Point arrhythmias (TdP). There is, however, evidence that multiple ion channel block prevent EADs and TdP arrhythmias. To address this question we estimated the IC50 values of the established hERG channel inhibitor dofetilide and 13 derivatives using a planar patch clamp system and HEK cells expressing hERG (Kv11.1) channels. Additionally we examined action potential prolongation in human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) in response to dofetilide and its derivatives. The arrhythmogenic potency was assessed by estimating the concentration required to prolong the action potential (AP) to 150% of control duration. In 11 out of 14 cases hERG inhibition was accompanied by a prolongation of AP the concentration of drugs inducing 150% increase of the AP duration correlated well with IC50 of hERG channels block (r = 0.94, p 200 µM). We conclude that the simultaneous block of depolarizing L-type calcium currents or cardiac sodium current offsets the effect of hERG block. Experimental data are supported by simulations of drug-induced changes in hiPSC-CMs action potentials. Our data support the new cardiac safety paradigm (CiPA) that discrimination between safe and unsafe drugs should address hERG inhibition and additionally their action on inward currents.

Environment-Sensitive Fluorescent Probe for the Human Ether-a-go-go-Related Gene Potassium Channel.

A novel environment-sensitive probe S2 with turn-on switch for Human Ether-a-go-go-Related Gene (hERG) potassium channel was developed herein. After careful evaluation, this fluorescent probe showed high binding affinity with hERG potassium channel with an IC50 value of 41.65 nM and can be well applied to hERG channel imaging or cellular distribution study for hERG channel blockers. Compared with other imaging techniques, such as immunofluorescence and fluorescent protein-based approaches, this method is convenient and affordable, especially since a washing procedure is not needed. Meanwhile, this environment-sensitive turn-on design strategy may provide a good example for the probe development for these targets that have no reactive or catalytic activity.

Evaluation of drug-mediated arrhythmic changes in spontaneous beating cardiomyocytes by AFM.

Arrhythmia caused by drug-induced cardiotoxicity is among the leading reasons for late-stage drug attrition and is therefore a core subject in safety testing of new compounds. Alternative methods such as surface and interface characterization approaches for assessing the drug-mediated cardiotoxicity should be promoted, in order to reduce, refine and replace the use of laboratory animals. Here, we investigate the possibility of using known human Ether-à-go-go-Related Gene (hERG) channel blockers to induce irregular beating patterns in the mouse and human induced pluripotent stem cell-derived (miPSC and hiPSC) cardiomyocyte (CM) model systems. An AFM-based approach was developed to precisely monitor the beating interval and rate of cardiomyocytes. Verification was implemented using three individual cardiovascular compounds to validate the potential application of this AFM approach as a complementary drug screening tool. Consistent with previous reports, isoproterenol increased the beating rate, with a more pronounced effect in the mouse CMs, nifedipine increased the beating rate in a dose-dependent manner, and sotalol induced arrhythmias with a significant variation in beating interval responses at increasing concentrations. The results of this initial study show that accurate analysis of individual drug-mediated effects can be achieved using our method, comparable to previous reports, and that a well-controlled AFM test of ion channel manipulation on human and murine-derived cardiomyocytes can be performed for investigation on multi-compound effects. Preliminary results indicate that the hERG blocker E-4031 can stimulate irregular, arrhythmic beating patterns in the cardiomyocytes from both species, and that immediate subsequent treatment with the hERG enhancer nicorandil can rescue these back to regular beating patterns, similar to those observed when the cells were untreated. This approach has not been extensively reported, and the use of our AFM system provides a platform to further investigate compound-induced ion channel effects in cardiomyocytes for potential application in pre-screening drug development stages.

Effects of Tannic Acid, Green Tea and Red Wine on hERG Channels Expressed in HEK293 Cells.

Tannic acid presents in varying concentrations in plant foods, and in relatively high concentrations in green teas and red wines. Human ether-à-go-go-related gene (hERG) channels expressed in multiple tissues (e.g. heart, neurons, smooth muscle and cancer cells), and play important roles in modulating cardiac action potential repolarization and tumor cell biology. The present study investigated the effects of tannic acid, green teas and red wines on hERG currents. The effects of tannic acid, teas and red wines on hERG currents stably transfected in HEK293 cells were studied with a perforated patch clamp technique. In this study, we demonstrated that tannic acid inhibited hERG currents with an IC50 of 3.4 μM and ~100% inhibition at higher concentrations, and significantly shifted the voltage dependent activation to more positive potentials (Δ23.2 mV). Remarkably, a 100-fold dilution of multiple types of tea (green tea, oolong tea and black tea) or red wine inhibited hERG currents by ~90%, and significantly shifted the voltage dependent activation to more positive potentials (Δ30.8 mV and Δ26.0 mV, respectively). Green tea Lung Ching and red wine inhibited hERG currents, with IC50 of 0.04% and 0.19%, respectively. The effects of tannic acid, teas and red wine on hERG currents were irreversible. These results suggest tannic acid is a novel hERG channel blocker and consequently provide a new mechanistic evidence for understanding the effects of tannic acid. They also revealed the potential pharmacological basis of tea- and red wine-induced biology activities.

Electrophysiological analysis of mammalian cells expressing hERG using automated 384-well-patch-clamp.

BackgroundAn in vitro electrophysiological assay system, which can assess compound effects and thus show cardiotoxicity including arrhythmia risks of test drugs, is an essential method in the field of drug development and toxicology.MethodsIn this study, high-throughput electrophysiological recordings of human embryonic kidney (HEK 293) cells and Chinese hamster ovary (CHO) cells stably expressing human ether-a-go-go related gene (hERG) were performed utilizing an automated 384-well-patch-clamp system, which records up to 384 cells simultaneously. hERG channel inhibition, which is closely related to a drug-induced QT prolongation and is increasing the risk of sudden cardiac death, was investigated in the high-throughput screening patch-clamp system.ResultsIn the automated patch-clamp measurements performed here, Kv currents were investigated with high efficiency. Various hERG channel blockers showed concentration-dependent inhibition, the 50 % inhibitory concentrations (IC50) of those blockers were in good agreement with previous reports.ConclusionsThe high-throughput patch-clamp system has a high potential in the field of pharmacology, toxicology, and cardiac physiology, and will contribute to the acceleration of pharmaceutical drug development and drug safety testing.

An investigation into consumed South African plant species as potential hERG channel blockers

hERG (human Ether-à-go-go Related Gene) is a gene that encodes the pore-forming α-subunit of a voltage-gated potassium K+ channel expressed in the heart and in nervous tissue [1]. It is responsible for channels mediating the 'rapid' delayed rectifier K+ current (IKI) which plays a critical role in ventricular repolarisation phase of the cardiac action potential [2].

Pre-clinical pharmacokinetics of the hERG blocking iboga alkaloid voacangine from Voacanga africana

The bark of Voacanga africana Stapf ex Scott Elliot and related species is not only used in African folk medicine, but also as legal high with increasing popularity in Europe. Voacangine (1), its major iboga-type alkaloid, was recently identified as potent hERG channel blocker in vitro which might point towards possible cardiotoxicity [1]. Since there is no in vivo characterisation of 1, the aim of this study was to assess its pharmacokinetics after oral administration of both, the pure compound and an ethanol extract of V. africana bark (VABE) containing 9.71% of 1, to allow for a critical discussion of its potential cardiotoxic risk in vivo. A precise and sensitive LC-MS/MS method was developed and validated according to FDA guidelines to detect 1 in plasma of male Wistar rats. Within the investigated drug concentration, 1 showed a high plasma protein binding of 98.7 ± 0.29%. Pharmacokinetics was evaluated by comparing four groups (G1-G4; n = 5 – 7/group). G1 received a single 5 mg/kg i.v. bolus of 1; G2 and G3 received single 25 mg/kg or 50 mg/kg doses of 1 p.o., resp.; G4 received a single oral dose of 500 mg/kg of VABE. Non compartmental analysis showed a volume of distribution (Vz) of 6.1 ± 3.4 L/kg after i.v. dosing, a clearance (CL) of 1.4 ± 1 L/h/kg, and an average half-life (t1/2) of 6.0 ± 2.0h. No statistical differences were observed in CL, Vz and t1/2 after oral application of 1, and indicated linear pharmacokinetics. Oral bioavailability (F) of 1 was 12% when given as pure compound. In VABE, a decrease of F was observed for 1 (8%). These findings provide first data to estimate the in vivo hERG channel related cardiotoxic risk of 1.

Inhibition of hERG potassium channel by the antiarrhythmic agent mexiletine and its metabolite m-hydroxymexiletine.

Mexiletine is a sodium channel blocker, primarily used in the treatment of ventricular arrhythmias. Moreover, recent studies have demonstrated its therapeutic value to treat myotonic syndromes and to relieve neuropathic pain. The present study aims at investigating the direct blockade of hERG potassium channel by mexiletine and its metabolite m-hydroxymexiletine (MHM). Our data show that mexiletine inhibits hERG in a time- and voltage-dependent manner, with an IC50 of 3.7 ± 0.7 μmol/L. Analysis of the initial onset of current inhibition during a depolarizing test pulse indicates mexiletine binds preferentially to the open state of the hERG channel. Looking for a possible mexiletine alternative, we show that m-hydroxymexiletine (MHM), a minor mexiletine metabolite recently reported to be as active as the parent compound in an arrhythmia animal model, is a weaker hERG channel blocker, compared to mexiletine (IC50 = 22.4 ± 1.2 μmol/L). The hERG aromatic residues located in the S6 helix (Tyr652 and Phe656) are crucial in the binding of mexiletine and the different affinities of mexiletine and MHM with hERG channel are interpreted by modeling their corresponding binding interactions through ab initio calculations. The simulations demonstrate that the introduction of a hydroxyl group on the meta-position of the aromatic portion of mexiletine weakens the interaction of the drug xylyloxy moiety with Tyr652. These results provide further insights into the molecular basis of drug/hERG interactions and, in agreement with previously reported results on clofilium and ibutilide analogs, support the possibility of reducing hERG potency and related toxicity by modifying the aromatic pattern of substitution of clinically relevant compounds.

Inter-study variability of preclinical in vivo safety studies and translational exposure-QTc relationships--a PKPD meta-analysis.

Preclinical cardiovascular safety studies (CVS) have been compared between facilities with respect to their sensitivity to detect drug-induced QTc prolongation (ΔQTc). Little is known about the consistency of quantitative ΔQTc predictions that are relevant for translation to humans. We derived typical ΔQTc predictions at therapeutic exposure (ΔQTcTHER ) with 95% confidence intervals (95%CI) for 3 Kv 11.1 (hERG) channel blockers (moxifloxacin, dofetilide and sotalol) from a total of 14 CVS with variable designs in the conscious dog. Population pharmacokinetic-pharmacodynamic (PKPD) analysis of each study was followed by a meta-analysis (pooling 2-6 studies including 10-32 dogs per compound) to derive meta-predictions of typical ΔQTcTHER . Meta-predictions were used as a reference to evaluate the consistency of study predictions and to relate results to those found in the clinical literature. The 95%CIs of study-predicted ΔQTcTHER comprised in 13 out of 14 cases the meta-prediction. Overall inter-study variability (mean deviation from meta-prediction at upper level of therapeutic exposure) was 30% (range: 1-69%). Meta-ΔQTcTHER predictions for moxifloxacin, dofetilide and sotalol overlapped with reported clinical QTc prolongation when expressed as %-prolongation from baseline. Consistent exposure-ΔQTc predictions were obtained from single preclinical dog studies of highly variable designs by systematic PKPD analysis, which is suitable for translational purposes. The good preclinical-clinical pharmacodynamic correlations obtained suggest that such an analysis should be more routinely applied to increase the informative and predictive value of results obtained from animal experiments.

Human Ether-à-go-go Related Gene (hERG) Channel Blocking Aporphine Alkaloids from Lotus Leaves and Their Quantitative Analysis in Dietary Weight Loss Supplements.

Blockage of the human ether-à-go-go related gene (hERG) channel can result in life-threatening ventricular tachyarrhythmia. In an in vitro screening of herbal materials for hERG blockers using an automated two-microelectrode voltage clamp assay on Xenopus oocytes, an alkaloid fraction of Nelumbo nucifera Gaertn. (lotus) leaves induced ∼50% of hERG current inhibition at 100 μg/mL. Chromatographic separation resulted in the isolation and identification of (-)-asimilobine, 1, nuciferine, 2, O-nornuciferine, 3, N-nornuciferine, 4, and liensinine, 5. In agreement with in silico predicted ligand-target interactions, 2, 3, and 4 revealed distinct in vitro hERG blockages measured in HEK293 cells with IC50 values of 2.89, 7.91, and 9.75 μM, respectively. Because lotus leaf dietary weight loss supplements are becoming increasingly popular, the identified hERG-blocking alkaloids were quantitated in five commercially available products. Results showed pronounced differences in the content of hERG-blocking alkaloids ranging up to 992 μg (2) in the daily recommended dose.

Novel Voltage Protocols for Determining hERG Channel Kinetics

Drug interactions with cardiac ion channels can cause disturbances to normal electrical activity in the heart. In particular, hERG channel block has been linked with QT prolongation and increased pro-arrhythmic risk. Consequently, hERG channel screening, quantifying drug block at different drug concentrations, forms a routine part of cardiovascular safety assessment. Recent initiatives led by the US Food and Drug Administration (FDA) aim to reform the current safety assessment guidelines and in silico approaches are envisaged to form an integral part of the new regime (Sager et al., Am Heart J., 2014).Mathematical ion channel representations, embedded within action potential models, are typically constructed through fitting to patch clamp data that are acquired by applying a series of voltage-step protocols. But different models exhibit disparate behaviour under non-standard protocols; including physiologically relevant action potential clamps, or clamps mimicking arrhythmogenic behaviour. We demonstrate the influence the choice of hERG channel representation can have on action potentials simulated from a mathematical ventricular myocyte model when predicting drug-induced effects; indicating the necessity of including accurate descriptions of hERG channel kinetics.We present novel rapidly fluctuating voltage-clamp protocols, designed mathematically to enable a robust model of hERG channel kinetics to be determined, on a cell-specific basis. The protocols have been tested in manual patch clamp experiments, using hERG-1a transfected HEK 293 cells. The approach will be extended to determine compound-specific models describing hERG channel kinetics in the presence of different drugs, which may enhance the predictive ability of in silico approaches used for cardiovascular safety assessment.

Effect of mutating amino acids considered critical for hERG channel blocker activity on the profile of a hERG channel activator

Effect of mutating amino acids considered critical for hERG channel blocker activity on the profile of a hERG channel activator

The 'Lotus Leave Diet' – A life-threatening experience? hERG channel blocking aporphine alkaloids from Nelumbo nucifera Gaertn

The 'Lotus Leave Diet' – A life-threatening experience? hERG channel blocking aporphine alkaloids from Nelumbo nucifera Gaertn

In vitro chronic effects on hERG channel caused by the marine biotoxin azaspiracid-2

Azaspiracids (AZAs) are marine biotoxins produced by the dinoflagellate Azadinium spinosum that accumulate in many shellfish species. Azaspiracid poisoning caused by AZA-contaminated seafood consumption is primarily manifested by diarrhea in humans. To protect human health, AZA-1, AZA-2 and AZA-3 content in seafood has been regulated by food safety authorities in many countries. Recently AZAs have been reported as a low/moderate hERG channel blockers. Furthermore AZA-2 has been related to arrhythmia appearance in rats, suggesting potential heart toxicity. In this study AZA-2 in vitro effects on hERG channel after chronic exposure are analyzed to further explore potential cardiotoxicity. The amount of hERG channel in the plasma membrane, hERG channel trafficking and hERG currents were evaluated up to 12 h of toxin exposure. In these conditions AZA-2 caused an increase of hERG levels in the plasma membrane, probably related to hERG retrograde trafficking impairment. Although this alteration did not translate into an increase of hERG channel-related current, more studies will be necessary to understand its mechanism and to know what consequences could have in vivo. These findings suggest that azaspiracids might have chronic cardiotoxicity related to hERG channel trafficking and they should not be overlooked when evaluating the threat to human health.

Experimentally validated HERG pharmacophore models as cardiotoxicity prediction tools.

The goal of this study was to design, experimentally validate, and apply a virtual screening workflow to identify novel hERG channel blockers. The hERG channel is an important antitarget in drug development since cardiotoxic risks remain as a major cause of attrition. A ligand-based pharmacophore model collection was developed and theoretically validated. The seven most complementary and suitable models were used for virtual screening of in-house and commercially available compound libraries. From the hit lists, 50 compounds were selected for experimental validation through bioactivity assessment using patch clamp techniques. Twenty compounds inhibited hERG channels expressed in HEK 293 cells with IC50 values ranging from 0.13 to 2.77 μM, attesting to the suitability of the models as cardiotoxicity prediction tools in a preclinical stage.

Long-term channel block is required to inhibit cellular transformation by human ether-à-go-go-related gene (hERG1) potassium channels.

Both human ether-à-go-go-related gene (hERG1) and the closely related human ether-à-go-go (hEAG1) channel are aberrantly expressed in a large proportion of human cancers. In the present study, we demonstrate that transfection of hERG1 into mouse fibroblasts is sufficient to induce many features characteristic of malignant transformation. An important finding of this work is that this transformation could be reversed by chronic incubation (for 2-3 weeks) with the hERG channel blocker dofetilide (100 nM), whereas more acute applications (for 1-2 days) were ineffective. The hERG1 expression resulted in a profound loss of cell contact inhibition, multiple layers of overgrowing cells, and high saturation densities. Cells also changed from fibroblast-like to a more spindle-shaped morphology, which was associated with a smaller cell size, a dramatic increase in cell polarization, a reduction in the number of actin stress fibers, and less punctate labeling of focal adhesions. Analysis of single-cell migration and scratch-wound closure clearly demonstrated that hERG1-expressing cells migrated more rapidly than vector-transfected control cells. In contrast to previous studies on hEAG1, there were no increases in rates of proliferation, or loss of growth factor dependency; however, hERG1-expressing cells were capable of substrate-independent growth. Allogeneic transplantation of hERG1-expressing cells into nude mice resulted in an increased incidence of tumors. In contrast to hEAG1, the mechanism of cellular transformation is dependent on ion conduction. Trafficking-deficient and conduction-deficient hERG1 mutants also prevented cellular transformation. These results provide evidence that hERG1 expression is sufficient to induce cellular transformation by a mechanism distinct from hEAG1. The most important conclusion of this study is that selective hERG1 channel blockers have therapeutic potential in the treatment of hERG1-expressing cancers.

Sa2023 Vibrating Capsule for the Treatment of Chronic Idiopathic Constipation (CIC) and Constipation Predominant Irritable Bowel Syndrome (C-IBS) -Safety and Efficacy

Background: Available therapies for chronic constipation (CC) have limited efficacy. Gastrointestinal prokinetics represent an attractive option, but both cisapride and tegaserod, the two agents with proved efficacy in CC, have been withdrawn because of cardiac adverse events. Prucalopride is the first selective, high-affinity 5-HT4 agonist, without any significant activity towards other (5-HT and non 5-HT) receptors or channels (e.g. hERG) at the therapeutic doses. In vitro binding studies have shown that its affinity towards the hERG is more than 250 times lower than that displayed against 5-HT4-receptors. Aim: To evaluate the Number Needed to Treat (NNT) and the Number Needed to Harm (NNH) for prucalopride, at the recommended dose (2 mg daily), in randomized clinical trials (RCTs), carried out in patients with CC. Effective and safe treatments are characterized as having low NNBs (2-6) and relatively high NNHs. Methods: MEDLINE, EMBASE, the Cochrane Central Register of Controlled Trials as well as abstracts from the major American, European and Asian meetings were searched up to November 2012. Large (≥ 250 patients) randomized controlled trials (RCTs) in adult patients with CC, treated with prucalopride, were included. NNT and NNH with 95% CI were calculated from the pooled risk difference, obtained using a random effect model in order to provide a more conservative estimate. The outcomes assessed (at 12 weeks) were the number of patients with an average increase of ≥3 SCBM, the number of patients with an average increase of ≥1 SCBM or SBMs as well as increase of QT corrected according to Bazett's Formula (QTcB) > 500 ms in patients that had a baseline QTcB < 450 ms Results are summarized in Table 1. Conclusions: The NNH for QTcB prolongation largely exceeds the NNT for each of the efficacy end-points analyzed. These results confirm experimental data suggesting that, at clinically relevant concentrations, prucalopride is unlikely to block hERG channels. Therefore, at the recommended therapeutic doses, the pro-arrhythmic potential of this highly selective 5-HT4 agonist is minimal, if any. Table 1