RESUMO
Several clinical cases of severe bradyarrhythmias have been reported upon co-administration of the Hepatitis-C NS5B Nucleotide Polymerase Inhibitor (HCV-NI) direct-acting antiviral agent, sofosbuvir (SOF), and the Class-III anti-arrhythmic amiodarone (AMIO). We model the cardiac drug-drug interaction (DDI) between AMIO and SOF, and between AMIO and a closely-related SOF analog, MNI-1 (Merck Nucleotide Inhibitor #1), in functional assays of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), to provide mechanistic insights into recently reported clinical cases. AMIO co-applied with SOF or MNI-1 increased beating rate or field potential (FP) rate and decreased impedance (IMP) and Ca(2+) transient amplitudes in hiPSC-CM syncytia. This action resembled that of Ca(2+) channel blockers (CCBs) in the model, but CCBs did not substitute for AMIO in the DDI. AMIO analog dronedarone (DRON) did not substitute for, but competed with AMIO in the DDI. Ryanodine and thapsigargin, decreasing intracellular Ca(2+) stores, and SEA-0400, a Na(+)/Ca(2+) exchanger-1 (NCX1) inhibitor, partially antagonized or suppressed DDI effects. Other agents affecting FP rate only exerted additive or subtractive effects, commensurate with their individual effects. We also describe an interaction between AMIO and MNI-1 on Cav1.2 ion channels in an over-expressing HEK-293 cell line. MNI-1 enhanced Cav1.2 channel inhibition by AMIO, but did not affect inhibition of Cav1.2 by DRON, verapamil, nifedipine, or diltiazem. Our data in hiPSC-CMs indicate that HCV-NI agents such as SOF and MNI-1 interact with key intracellular Ca(2+)-handling mechanisms. Additional study in a Cav1.2 HEK-293 cell-line suggests that HCV-NIs potentiate the inhibitory action of AMIO on L-type Ca(2+) channels.
Assuntos
Amiodarona/farmacologia , Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Canais de Cálcio Tipo L , Células HEK293 , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologiaRESUMO
Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are widely used for drug safety and efficacy testing with various techniques, including high content imaging (HCI). Upon drug treatment, a significant number of hiPSC-CMs grown in regular 96-well plates coated with fibronectin detached from the bottom of the plate, complicating data acquisition. Several cell culture configurations were tested to improve cell adherence, and the effects of these configurations on total cell number, separation of feature values between the negative (DMSO 0.1%) and positive (antimycin, staurosporine) controls, scale of feature value differences, and data variability were statistically calculated. hiPSC-CMs were plated on fibronectin- (in "blanket" configuration) or MaxGel- (in "sandwich" configuration) coated plates and covered with a layer of either HydroMatrix or MaxGel 2, 7, or 11d after plating. After a total of 14d in culture, cells were treated with compounds, labeled with four fluorescent dyes (Hoechst, TMRM, NucView, and RedDot), and imaged with GE INCell2000. Based on the statistical parameters calculated, the MaxGel 25% 7d "sandwich" was superior to all other tested conditions when the cells were treated with 0.3 µM antimycin for 2 h and test compounds 10 µM crizotinib and 30 µM amiodarone for 48 h. For staurosporine treatment, the best culturing condition varied between MaxGel "sandwich" systems, depending on which parameters were under consideration. Thus, cell culturing conditions can significantly affect the ability of high content imaging to detect changes in cellular features during compound treatment and should be thoroughly evaluated before committing to compound testing.
RESUMO
Blockade of the human ether-a-go-go-related gene (hERG) potassium channel, with a consequent possibility of QT prolongation and increased susceptibility to a characteristic polymorphic ventricular arrhythmia, torsade de pointes, is an important cause of withdrawal of drugs from the market. In the aftermath of recent drug withdrawals, regulatory agencies now require in vitro hERG screening of all pharmaceutical compounds that are targeted for human use. To minimize the potential for failure in later-stage drug development, many pharmaceutical and biotechnology companies have begun to use automated patch clamp systems with higher throughput than conventional manual patch-clamp techniques to conduct routine functional hERG screening during drug discovery and early development. We have optimized an automated patch-clamp hERG screening method for the PatchXpress 7000A system (Molecular Devices, Sunnyvale, CA) using potassium fluoride (KF) in the internal recording solution. In this study we show that (1) the biophysical and pharmacological properties of hERG current recorded with KF are similar to those with standard potassium chloride solutions, (2) use of KF significantly improves the success rate of hERG screening using PatchXpress without compromising data quality, and (3) utilization of KF can significantly increase the throughput of hERG screening with PatchXpress.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Canais de Potássio Éter-A-Go-Go/efeitos dos fármacos , Fluoretos , Técnicas de Patch-Clamp/métodos , Bloqueadores dos Canais de Potássio/farmacologia , Compostos de Potássio , Animais , Células CHO , Membrana Celular/efeitos dos fármacos , Cricetinae , Cricetulus , Interpretação Estatística de Dados , EletrofisiologiaRESUMO
We challenged human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) syncytia, mainly, CDI iCells with several classes of well-characterized pharmacological agents (including hERG blocker, Nav1.5 blocker, Cav1.2 blocker and opener, ß-adrenergic agonist, and If blocker) under pacing conditions, utilizing the Cardio-ECR instrument, a non-invasive platform featuring simultaneous and continuous measurement of synchronized beating rate and contractility (both signals were acquired simultaneously and well aligned). We found that: 1) with increasing acute stimulation rates (no pacing; 1, 1.5, and 2Hz), beat interval was gradually shortened mainly in the relaxation phase of each beat cycle; 2) typical responses of iCells hiPSC-CMs to all tested pharmacological agents were either attenuated or even eliminated by pacing, in a concentration- and stimulation rate-dependent manner; and 3) when iCells were influenced by pharmacological agents and cannot follow pacing rates, they still beat regularly at exactly 1/2 or 1/3 of pacing rates. We concluded that when intrinsic syncytial pacing was overcome by faster, external stimulations, beat intervals of hiPSC-CMs were mainly shortened in the relaxation phase, instead of proportionally in each beat cycle, with increasing pacing rates. In addition, in response to pharmacological agents upon pacing, hiPSC-CMs exhibited distinct patterns of refractoriness, manifested by skipped beats in pacing-rate dependent manner, and attenuation (or even abolition) of the typical response evoked under spontaneous beating.
Assuntos
Potenciais de Ação/efeitos dos fármacos , Fármacos Cardiovasculares/farmacologia , Células Gigantes/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Linhagem Celular , Células Gigantes/fisiologia , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Miócitos Cardíacos/fisiologiaRESUMO
INTRODUCTION: The ICH S7B guidelines recommend that all new chemical entities should be subjected to hERG repolarization screening due to its association with life-threatening "Torsades de Pointes" (TdP) arrhythmia. However, it has become evident that not all hERG channel inhibitors result in TdP and not all compounds that induce QT prolongation and TdP necessarily inhibit hERG. In order to address the limitations of the S7B/E14 guidelines, the FDA through a public/private partnership initiated the Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative to examine the possible modification and refinement of the ICH E14/S7B guidelines. One of the main components of the CiPA initiative is to utilize a predictive assay system together with human cardiomyocytes for risk assessment of arrhythmia. METHOD: In this manuscript we utilize the xCELLigence® CardioECR system which simultaneously measures excitation-contraction coupling together with human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) to assess the effect of 8 reference compounds across 3 different independent sites. These 8 compounds were part of Phase I CiPA validation study. RESULTS: Our data demonstrate that hERG channel blockers, such as E4031 and moxifloxacin, prolonged field potential duration (FPD) at low concentration and induced arrhythmic beating activity as measured by field potential (FP) recording and impedance (IMP) recordings at higher concentrations. On the contrary, nifedipine, an inhibitor of calcium channel, didn't disrupt the periodicity of cell beating and weakened cell contractile activity and shortened FPD. Multichannel inhibitors, such as flecainide, quinidine and mexiletine, not only increased FPD and induced arrhythmia but also significantly reduced the amplitude of FP spike. JNJ303, an IKs inhibitor, only affected FPD. Comparison of the compound effect on FPD across the 3 different sites is consistent in terms of trend of the effect with observed 3-10 fold differences in minimal effective concentration at which a minimum of 10% response is detected. In addition, pentamidine, a hERG trafficking inhibitor which induced irregular beating activity over a more prolonged duration of time was readily flagged in this assay system. Taken together, this multi-parameter assay using hiPSC-CMs in conjunction with simultaneous measurement of ion channel activity and contractility can be a reliable approach for risk assessment of proarrhythmic compounds.
Assuntos
Potenciais de Ação/efeitos dos fármacos , Arritmias Cardíacas/induzido quimicamente , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Arritmias Cardíacas/fisiopatologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos/métodos , Guias como Assunto , Humanos , Células-Tronco Pluripotentes Induzidas , Bloqueadores dos Canais de Potássio/farmacologia , Segurança , Torsades de Pointes/induzido quimicamenteRESUMO
The human Ether-à-go-go related gene (hERG) potassium channel has been widely used to counter screen potential pharmaceuticals as a biomarker to predict clinical QT prolongation. Thus, higher throughput assays of hERG are valuable for early in vitro screening of drug candidates to minimize failure in later-stage drug development due to this potentially adverse cardiac risk. We have developed a novel method utilizing potassium fluoride to improve throughput of hERG counter screening with an automated patch clamp system, PatchXpress 7000A. In that method, â¼50% substitution of internal Cl(-) with F(-) greatly increases success rate without substantially altering the biophysical properties of the hERG channel or compromising data quality. However, effect of F(-) or other halide ions on hERG channel properties has not been studied in detail. In this study, we examined effects of complete replacement of Cl(-) in internal solution with halide ions, F(-), or Br(-). We found that (1) F(-) slightly shifts the voltage dependence of hERG channel activation to more positive voltages, while Br(-) shifts it to more negative voltages; (2) Br(-) shifts to more positive voltages both the inactivation-voltage relationship and the peak position of channel full activation of hERG; (3) F(-) slows hERG activation, while both F(-) and Br(-) make the channel close faster; (4) neither F(-) nor Br(-) have any effect on hERG inactivation kinetics. In conclusion, compared to Cl(-), F(-) has subtle effect on hERG activation, while Br(-) has distinct effects on certain, but not all biophysical properties of hERG channel.
Assuntos
Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Canais de Potássio Éter-A-Go-Go/fisiologia , Halogênios/farmacologia , Ativação do Canal Iônico/fisiologia , Potenciais da Membrana/fisiologia , Técnicas de Patch-Clamp/métodos , Bloqueadores dos Canais de Potássio/farmacologia , Animais , Células CHO , Cricetinae , Cricetulus , Avaliação Pré-Clínica de Medicamentos/métodos , Canal de Potássio ERG1 , Citometria de Fluxo/métodos , Análise de Injeção de Fluxo/métodos , Ativação do Canal Iônico/efeitos dos fármacos , Íons , Potenciais da Membrana/efeitos dos fármacosRESUMO
INTRODUCTION: Ca(v)1.2 channels play an important role in shaping the cardiac action potential. Screening pharmaceutical compounds for Ca(v)1.2 block is very important in developing drugs without cardiac liability. Ca(v)1.2 screening has been traditionally done using fluorescence assays, but these assays have some limitations. Patch clamping is considered the gold standard for ion channel studies, but is very labor intensive. The purpose of this study was to develop a robust medium throughput Ca(v)1.2 screening assay in PatchXpress 7000A by optimizing cell isolation conditions, recording solutions and experimental parameters. Under the conditions established, structurally different standard Ca(v)1.2 antagonists and an agonist were tested. METHODS: HEK-293 cells stably transfected with hCa(v)1.2 L-type Ca channel were used. For experiments, cells were isolated using 0.05% Trypsin. Currents were recorded in the presence of 30 mM extracellular Ba2+ and low magnesium intracellular recording solution to minimize rundown. Ca(v)1.2 currents were elicited from a holding potential of -60 mV at 0.05 Hz to increase pharmacological sensitivity and minimize rundown. Test compounds were applied at increasing concentrations for 5 min followed by a brief washout. RESULTS: Averaged peak Ca(v)1.2 current amplitudes were increased from 10 pA/pF to 15 pA/pF by shortening cell incubation and trypsin exposure time from 2.5 min at 37 degrees C to 1 min at room temperature and adding 0.2 mM cAMP to the intracellular solution. Rundown was minimized from 2%/min to 0.5%/min by reducing the intracellular free Mg2+ from 2.7 mM to 0.2 mM and adding 100 nM Ca2+. Under the established conditions, we tested 8 structurally different antagonists and an agonist. The IC(50) values obtained ranked well against published values and results obtained using traditional clamp experiments performed in parallel using the expressed cell line and native myocytes. DISCUSSION: This assay can be used as a reliable pharmacological screening tool for Ca(v)1.2 block to assess compounds for cardiac liability during lead optimization.