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1.
Cardiovasc Res ; 76(2): 247-56, 2007 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-17669388

RESUMO

OBJECTIVE: Congenital loss of function and drug-induced inhibition of the slowly-activating delayed-rectifier K(+) current (I(Ks)) cause impaired cardiac repolarization. beta-Adrenergic-receptor stimulation contributes to sympathetically-induced torsades de pointes (TdP). An in vivo model of long-QT1 (LQT1) syndrome and TdP in a species with I(Ks) characteristics relevant to man is lacking. We investigated the in vivo mechanisms of TdP in a novel canine model of drug-induced LQT1 syndrome. METHODS: Adult beagle dogs (n=30; F/M) were anesthetized with lofentanil (0.075 mg/kg i.v.) and etomidate (1.5 mg/kg/hour). ECGs, left- (LV) and right-ventricular (RV) monophasic action potentials (MAPs), and intracavitary pressures were recorded simultaneously. Infusion of the I(Ks) blocker HMR1556 (0.025-0.050 mg/kg/min) mimicked LQT1, and bolus injections of isoproterenol (1.25-5 microg/kg) reproducibly triggered TdP in 94% of dogs (defibrillated if necessary). RESULTS: Isoproterenol evoked paradoxical repolarization prolongation during heart rate accelerations. Beat-to-beat variability [QT, LV MAP duration (MAPD(90))] and spatial dispersion of repolarization (T(peak)-T(end) interval, endo-minus epicardial MAPD(90), LV-RVMAPD(90)) were significantly increased. Early afterdepolarizations occurred predominantly in the endocardium and not the epicardium. During isoproterenol, secondary systolic contractions (aftercontractions; peak 25+/-6 mm Hg) arose in the LV (not RV) when TdP ensued. Prevention of TdP by esmolol (1.25 mg/kg), verapamil (0.4 mg/kg) or mexiletine (5 mg/kg) was only successful when repolarization prolongation was contained and aftercontractions remained absent. CONCLUSIONS: beta-Adrenergic challenges trigger TdP in a reproducible manner in this model of drug-induced LQT1. Paradoxical prolongation and increased temporal and spatial dispersion of repolarization precipitate TdP. Incremental LV systolic aftercontractions precede TdP, suggesting abnormal cellular Ca(2+) handling contributes to the arrhythmogenic mechanism.


Assuntos
Modelos Animais de Doenças , Síndrome do QT Longo/induzido quimicamente , Torsades de Pointes/induzido quimicamente , Potenciais de Ação/efeitos dos fármacos , Animais , Antiarrítmicos/farmacologia , Cálcio/metabolismo , Cromanos/farmacologia , Cães , Feminino , Isoproterenol/farmacologia , Masculino , Reprodutibilidade dos Testes , Sulfonamidas/farmacologia
2.
Artigo em Inglês | MEDLINE | ID: mdl-29421525

RESUMO

INTRODUCTION: Calcium-based screening of hiPS-CMs is a useful preclinical safety evaluation platform with the ability to generate robust signals that facilitates high-throughput screening and data analysis. However, due to the potential inherent toxicities, it is important to understand potential effects of different calcium-sensitive dyes on the hiPS-CMs model. METHODS: We compared three calcium-sensitive fluorescence dyes (Cal520, ACTOne and Calcium 5) for their impact on the variability, the beating properties and the pharmacological responses of hiPS-CMs using the Hamamatsu FDSS/µCell imaging platform. Direct effects of three dyes on the electrophysiological properties of hiPS-CMs were evaluated with the multi-electrode array (MEA) Axion Maestro platform. RESULTS: We propose a specific experimental protocol for each dye which gives the most optimal assay conditions to minimize variability and possible adverse effects. We showed that Cal520 had the smallest effect on hiPS-CMs together with the longest-lasting stable amplitude signal (up to 4 h). Although all dyes had a (minor) acute effect on hiPS-CMs, in the form of reduced beat rate and prolonged field potential duration, the selection of the dye did not influence the pharmacological response of four cardioactive drugs (dofetilide, moxifloxacin, nimodipine and isoprenaline). DISCUSSION: In conclusion, we have documented that different calcium sensitive dyes have only minor direct (acute) effects on hiPS-CMs with Cal520 showing the least effects and the longest lasting signal amplitude. Importantly, drug-induced pharmacological responses in hiPS-CMs were comparable between the three dyes. These findings should help further improve the robustness of the hiPS-CMs-based calcium transient assay as a predictive, preclinical cardiac safety evaluation tool.


Assuntos
Potenciais de Ação/efeitos dos fármacos , Cálcio/metabolismo , Corantes Fluorescentes/farmacologia , Ensaios de Triagem em Larga Escala/métodos , Miócitos Cardíacos/efeitos dos fármacos , Cálcio/química , Fármacos Cardiovasculares/farmacologia , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos/métodos , Eletrodos , Corantes Fluorescentes/química , Ensaios de Triagem em Larga Escala/instrumentação , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Miócitos Cardíacos/fisiologia , Fatores de Tempo
3.
Front Physiol ; 8: 668, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28955244

RESUMO

Early prediction of cardiotoxicity is critical for drug development. Current animal models raise ethical and translational questions, and have limited accuracy in clinical risk prediction. Human-based computer models constitute a fast, cheap and potentially effective alternative to experimental assays, also facilitating translation to human. Key challenges include consideration of inter-cellular variability in drug responses and integration of computational and experimental methods in safety pharmacology. Our aim is to evaluate the ability of in silico drug trials in populations of human action potential (AP) models to predict clinical risk of drug-induced arrhythmias based on ion channel information, and to compare simulation results against experimental assays commonly used for drug testing. A control population of 1,213 human ventricular AP models in agreement with experimental recordings was constructed. In silico drug trials were performed for 62 reference compounds at multiple concentrations, using pore-block drug models (IC50/Hill coefficient). Drug-induced changes in AP biomarkers were quantified, together with occurrence of repolarization/depolarization abnormalities. Simulation results were used to predict clinical risk based on reports of Torsade de Pointes arrhythmias, and further evaluated in a subset of compounds through comparison with electrocardiograms from rabbit wedge preparations and Ca2+-transient recordings in human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs). Drug-induced changes in silico vary in magnitude depending on the specific ionic profile of each model in the population, thus allowing to identify cell sub-populations at higher risk of developing abnormal AP phenotypes. Models with low repolarization reserve (increased Ca2+/late Na+ currents and Na+/Ca2+-exchanger, reduced Na+/K+-pump) are highly vulnerable to drug-induced repolarization abnormalities, while those with reduced inward current density (fast/late Na+ and Ca2+ currents) exhibit high susceptibility to depolarization abnormalities. Repolarization abnormalities in silico predict clinical risk for all compounds with 89% accuracy. Drug-induced changes in biomarkers are in overall agreement across different assays: in silico AP duration changes reflect the ones observed in rabbit QT interval and hiPS-CMs Ca2+-transient, and simulated upstroke velocity captures variations in rabbit QRS complex. Our results demonstrate that human in silico drug trials constitute a powerful methodology for prediction of clinical pro-arrhythmic cardiotoxicity, ready for integration in the existing drug safety assessment pipelines.

4.
J Biomol Screen ; 10(2): 168-81, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15799960

RESUMO

The authors used the PatchXpress 7000A system to measure compound activity at the hERG channel using procedures that mimicked the "gold-standard" conventional whole-cell patch clamp. A set of 70 compounds, including hERG antagonists with potencies spanning 3 orders of magnitude, were tested on hERG302-HEK cells using protocols aimed at either identifying compound activity at a single concentration or obtaining compound potency from a cumulative concentration dependence paradigm. After exposure to compounds and subsequent washout of the wells to determine reversibility of the block, blockade by a reference compound served as a quality control. Electrical parameters and voltage dependence were similar to those obtained using a conventional whole-cell patch clamp. Rank order of compound potency was also comparable to that determined by conventional methods. One exception was flunarizine, a particularly lipophilic compound. The PatchXpress accurately identified the activity of 29 moderately potent antagonists, which only weakly displace radiolabeled astemizole and are false negatives in the binding assay. Finally, no false hits were observed from a collection of relatively inactive compounds. High-quality data acquisition by PatchXpress should help accelerate secondary screening for ion channel modulators and the drug discovery process.


Assuntos
Técnicas de Patch-Clamp/instrumentação , Técnicas de Patch-Clamp/métodos , Canais de Potássio/metabolismo , Linhagem Celular , Eletrofisiologia , Canais de Potássio Éter-A-Go-Go , Humanos , Concentração Inibidora 50 , Reprodutibilidade dos Testes
5.
Br J Pharmacol ; 166(4): 1490-502, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22300168

RESUMO

BACKGROUND AND PURPOSE: Terfenadine has been reported to cause cardiac death. Hence, we investigated its pro-arrhythmic potential in various in vitro models. EXPERIMENTAL APPROACH: Pro-arrhythmic effects of terfenadine were investigated in rabbit isolated hearts and left ventricular wedge preparations. Also, using whole-cell patch-clamp recording, we examined its effect on the human ether-à-go-go-related gene (hERG) current in HEK293 cells transfected with hERG and on the I(Na) current in rabbit ventricular cells and human atrial myocytes. KEY RESULTS: Terfenadine concentration- and use-dependently inhibited I(Na) in rabbit myocytes and in human atrial myocytes and also inhibited the hERG. In both the rabbit left ventricular wedge and heart preparations, terfenadine at 1 µM only slightly prolonged the QT- and JT-intervals but at 10 µM, it caused a marked widening of the QRS complex, cardiac wavelength shortening, incidences of in-excitability and non-TdP-like ventricular tachycardia/fibrillation (VT/VF) without prolongation of the QT/JT-interval. At 10 µM terfenadine elicited a lower incidence of early afterdepolarizations versus non- Torsades de Pointes (TdP)-like VT/VF (100% incidence), and did not induce TdPs. Although the concentration of terfenadine in the tissue-bath was low, it accumulated within the heart tissue. CONCLUSION AND IMPLICATIONS: Our data suggest that: (i) the induction of non-TdP-like VT/VF, which is caused by slowing of conduction via blockade of I(Na) (like Class Ic flecainide), may constitute a more important risk for terfenadine-induced cardiac death; (ii) although terfenadine is a potent hERG blocker, the risk for non-TdP-like VT/VF exceeds the risk for TdPs; and (iii) cardiac wavelength (λ) could serve as a biomarker to predict terfenadine-induced VT/VF.


Assuntos
Antagonistas não Sedativos dos Receptores H1 da Histamina/farmacologia , Síndrome do QT Longo/induzido quimicamente , Miócitos Cardíacos/efeitos dos fármacos , Taquicardia Ventricular/etiologia , Terfenadina/farmacologia , Torsades de Pointes/induzido quimicamente , Fibrilação Ventricular/etiologia , Potenciais de Ação/efeitos dos fármacos , Animais , Antiarrítmicos/efeitos adversos , Antiarrítmicos/metabolismo , Antiarrítmicos/farmacologia , Apêndice Atrial/citologia , Apêndice Atrial/efeitos dos fármacos , Apêndice Atrial/metabolismo , Transporte Biológico , Células Cultivadas , Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Canais de Potássio Éter-A-Go-Go/genética , Canais de Potássio Éter-A-Go-Go/metabolismo , Feminino , Células HEK293 , Ventrículos do Coração/citologia , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/metabolismo , Antagonistas não Sedativos dos Receptores H1 da Histamina/efeitos adversos , Antagonistas não Sedativos dos Receptores H1 da Histamina/metabolismo , Humanos , Técnicas In Vitro , Síndrome do QT Longo/metabolismo , Síndrome do QT Longo/fisiopatologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Concentração Osmolar , Coelhos , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/metabolismo , Bloqueadores dos Canais de Sódio/efeitos adversos , Bloqueadores dos Canais de Sódio/metabolismo , Bloqueadores dos Canais de Sódio/farmacologia , Terfenadina/efeitos adversos , Terfenadina/metabolismo , Torsades de Pointes/metabolismo , Torsades de Pointes/fisiopatologia
6.
J Pharmacol Toxicol Methods ; 60(1): 1-10, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19439185

RESUMO

The problem of drug-induced hERG channel blockade, which can lead to acquired long QT syndrome and potentially fatal arrhythmias, has exercised drug developers and regulatory authorities for over 10 years, and exacting guidelines have been put into place to test for this liability both preclinically (ICH S7B) and clinically (ICH E14). However, the I(Ks) channel, which along with the transient outward current (I(to)) is the other main potassium channel affecting cardiac repolarisation and thus the length of the QT interval, has received little attention, and potent I(Ks) blocking drugs with serious side effects could potentially enter into human testing without being detected by the existing regulatory core battery and standard screening strategies. Here we review the pharmacology of cardiac I(Ks) channel blockade and describe the discovery of a potent I(Ks) blocker whose activity was not detected by standard hERG or invitro action potential screens, but subsequently evoked unprovoked torsades de pointes (TdP) invivo in our anaesthetised dog model. We have exploited this molecule to develop a ligand binding assay to detect I(Ks) blockade at an earlier stage in drug discovery, and note that several other laboratories developing new drugs have also developed higher throughput screens to detect I(Ks) blockade (e.g., [Trepakova, E. S., Malik, M. G., Imredy, J. P., Penniman, J. R., Dech, S. J., & Salata, J. J. (2007) Application of PatchXpress planar patch clamp technology to the screening of new drug candidates for cardiac KCNQ1/KCNE1 (I(Ks)) activity. Assay Drug Development Technology 5, 617-627]). Because of the presence of I(Ks) channels in other tissues, including blood vessels and in the epithelia of intestine, kidney, lung and the cochlea, I(Ks) blockade has the potential to cause extensive side effects in addition to QT prolongation and arrhythmias. We therefore suggest that compounds selected for development should also be examined for I(Ks) liability before testing in humans. The possibility of undetected I(Ks) blockade is therefore an additional gap to that identified earlier [Lu, H. R., Vlaminckx, E., Hermans, A. N., Rohrbacher, J., Van Ammel, K., Towart, R., et al. (2008) Predicting drug-induced changes in QT interval and arrhythmias: QT-shortening drugs point to gaps in the ICH S7B Guidelines. British Journal of Pharmacology, 154, 1427-1438] in the ICH S7B regulatory guidelines.


Assuntos
Arritmias Cardíacas/induzido quimicamente , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Canal de Potássio KCNQ1/antagonistas & inibidores , Potenciais de Ação/efeitos dos fármacos , Animais , Arritmias Cardíacas/fisiopatologia , Sistema Cardiovascular/efeitos dos fármacos , Sistema Cardiovascular/fisiopatologia , Relação Dose-Resposta a Droga , Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos , Eletrocardiografia , Canais de Potássio Éter-A-Go-Go/metabolismo , Humanos , Técnicas de Patch-Clamp , Torsades de Pointes/induzido quimicamente , Torsades de Pointes/fisiopatologia
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