RESUMO
BACKGROUND: Determination of a drug's potency in blocking the hERG channel is an established safety pharmacology study. Best practice guidelines have been published for reliable assessment of hERG potency. In addition, a set of plasma concentration and plasma protein binding fraction data were provided as denominators for margin calculations. The aims of the current analysis were five-fold: provide data allowing creation of consistent denominators for the hERG margin distributions of the key reference agents, explore the variation in hERG margins within and across laboratories, provide a hERG margin to 10 ms QTc prolongation based on several newer studies, provide information to use these analyses for reference purposes, and provide recommended hERG margin 'cut-off' values. METHODS: The analyses used 12 hERG IC50 'best practice' data sets (for the 3 reference agents). A group of 5 data sets came from a single laboratory. The other 7 data sets were collected by 6 different laboratories. RESULTS: The denominator exposure distributions were consistent with the ICH E14/S7B Training Materials. The inter-occasion and inter-laboratory variability in hERG IC50 values were comparable. Inter-drug differences were most important in determining the pooled margin variability. The combined data provided a robust hERG margin reference based on best practice guidelines and consistent exposure denominators. The sensitivity of hERG margin thresholds were consistent with the sensitivity described over the course of the last two decades. CONCLUSION: The current data provide further insight into the sensitivity of the 30-fold hERG margin 'cut-off' used for two decades. Using similar hERG assessments and these analyses, a future researcher can use a hERG margin threshold to support a negative QTc integrated risk assessment.
Assuntos
Canais de Potássio Éter-A-Go-Go , Síndrome do QT Longo , Humanos , Medição de Risco/métodos , Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Síndrome do QT Longo/induzido quimicamente , Canal de Potássio ERG1/antagonistas & inibidores , Canal de Potássio ERG1/metabolismo , Laboratórios/normas , Animais , Bloqueadores dos Canais de Potássio/farmacologia , Bloqueadores dos Canais de Potássio/efeitos adversos , Concentração Inibidora 50RESUMO
Chloroquine and hydroxychloroquine have been proposed recently as therapy for SARS-CoV-2-infected patients, but during 3 months of extensive use concerns were raised related to their clinical effectiveness and arrhythmogenic risk. Therefore, we estimated for these compounds several proarrhythmogenic risk predictors according to the Comprehensive in vitro Proarrhythmia Assay (CiPA) paradigm. Experiments were performed with either CytoPatch™2 automated or manual patch-clamp setups on HEK293T cells stably or transiently transfected with hERG1, hNav1.5, hKir2.1, hKv7.1+hMinK, and on Pluricyte® cardiomyocytes (Ncardia), using physiological solutions. Dose-response plots of hERG1 inhibition fitted with Hill functions yielded IC50 values in the low micromolar range for both compounds. We found hyperpolarizing shifts of tens of mV, larger for chloroquine, in the voltage-dependent activation but not inactivation, as well as a voltage-dependent block of hERG current, larger at positive potentials. We also found inhibitory effects on peak and late INa and on IK1, with IC50 of tens of µM and larger for chloroquine. The two compounds, tested on Pluricyte® cardiomyocytes using the ß-escin-perforated method, inhibited IKr, ICaL, INa peak, but had no effect on If. In current-clamp they caused action potential prolongation. Our data and those from literature for Ito were used to compute proarrhythmogenic risk predictors Bnet (Mistry HB, 2018) and Qnet (Dutta S et al., 2017), with hERG1 blocking/unblocking rates estimated from time constants of fractional block. Although the two antimalarials are successfully used in autoimmune diseases, and chloroquine may be effective in atrial fibrillation, assays place these drugs in the intermediate proarrhythmogenic risk group.
Assuntos
Antivirais/efeitos adversos , Arritmias Cardíacas/induzido quimicamente , Cloroquina/farmacologia , Hidroxicloroquina/efeitos adversos , Potenciais de Ação/efeitos dos fármacos , Bioensaio , Simulação por Computador , Correlação de Dados , Relação Dose-Resposta a Droga , Canal de Potássio ERG1/agonistas , Canal de Potássio ERG1/antagonistas & inibidores , Canal de Potássio ERG1/metabolismo , Células HEK293 , Humanos , Concentração Inibidora 50 , Canal de Potássio KCNQ1/antagonistas & inibidores , Canal de Potássio KCNQ1/metabolismo , Cinética , Miócitos Cardíacos/efeitos dos fármacos , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Técnicas de Patch-Clamp , Canais de Potássio Corretores do Fluxo de Internalização/antagonistas & inibidores , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Medição de Risco , SARS-CoV-2/efeitos dos fármacos , Tratamento Farmacológico da COVID-19RESUMO
The human ether-a-go-go-related voltage-gated cardiac ion channel (commonly known as hERG) conducts the rapid outward repolarizing potassium current in cardiomyocytes (IKr). Inadvertent blockade of this channel by drug-like molecules represents a key challenge in pharmaceutical R&D due to frequent overlap between the structure-activity relationships of hERG and many primary targets. Building on our previous work, together with recent cryo-EM structures of hERG, we set about to better understand the energetic and structural basis of promiscuous blocker-hERG binding in the context of Biodynamics theory. We propose a two-step blocker binding process consisting of: The initial capture step: diffusion of a single fully solvated blocker copy into a large cavity lined by the intra-cellular cyclic nucleotide binding homology domain (CNBHD). Occupation of this cavity is a necessary but insufficient condition for ion current disruption.The IKr disruption step: translocation of the captured blocker along the channel axis, such that: The head group, consisting of a quasi-rod-shaped moiety, projects into the open pore, accompanied by partial de-solvation of the binding interface.One tail moiety packs along a kink between the S6 helix and proximal C-linker helix adjacent to the intra-cellular entrance of the pore, likewise accompanied by mutual de-solvation of the binding interface (noting that the association barrier is comprised largely of the total head + tail group de-solvation cost).Blockers containing a highly planar moiety that projects into a putative constriction zone within the closed channel become trapped upon closing, as do blockers terminating prior to this region.A single captured blocker copy may conceivably associate and dissociate to/from the pore many times before exiting the CNBHD cavity. Lastly, we highlight possible flaws in the current hERG safety index (SI), and propose an alternate in vivo-relevant strategy factoring in: Benefit/risk.The predicted arrhythmogenic fractional hERG occupancy (based on action potential (AP) simulations of the undiseased human ventricular cardiomyocyte).Alteration of the safety threshold due to underlying disease.Risk of exposure escalation toward the predicted arrhythmic limit due to patient-to-patient pharmacokinetic (PK) variability, drug-drug interactions, overdose, and use for off-label indications in which the hERG safety parameters may differ from their on-label counterparts.
Assuntos
Canal de Potássio ERG1/química , Canal de Potássio ERG1/metabolismo , Bloqueadores dos Canais de Potássio/farmacologia , Arritmias Cardíacas , Sítios de Ligação , Microscopia Crioeletrônica , Humanos , Modelos Biológicos , Modelos Moleculares , Bloqueadores dos Canais de Potássio/química , Conformação Proteica , Domínios ProteicosRESUMO
INTRODUCTION: Screening compounds for activity on the hERG channel using patch clamp is a crucial part of safety testing. Automated patch clamp (APC) is becoming widely accepted as an alternative to manual patch clamp in order to increase throughput whilst maintaining data quality. In order to standardize APC experiments, we have investigated the effects on IC50 values under different conditions using several devices across multiple sites. METHODS: APC instruments SyncroPatch 384i, SyncroPatch 384PE and Patchliner, were used to record hERG expressed in HEK or CHO cells. Up to 27 CiPA compounds were used to investigate effects of voltage protocol, incubation time, labware and time between compound preparation and experiment on IC50 values. RESULTS: All IC50 values of 21 compounds recorded on the SyncroPatch 384PE correlated well with IC50 values from the literature (Kramer et al., 2013) regardless of voltage protocol or labware, when compounds were used immediately after preparation, but potency of astemizole decreased if prepared in Teflon or polypropylene (PP) compound plates 2-3 h prior to experiments. Slow acting compounds such as dofetilide, astemizole, and terfenadine required extended incubation times of at least 6 min to reach steady state and therefore, stable IC50 values. DISCUSSION: Assessing the influence of different experimental conditions on hERG assay reliability, we conclude that either the step-ramp protocol recommended by CiPA or a standard 2-s step-pulse protocol can be used to record hERG; a minimum incubation time of 5 min should be used and although glass, Teflon, PP or polystyrene (PS) compound plates can be used for experiments, caution should be taken if using Teflon, PS or PP vessels as some adsorption can occur if experiments are not performed immediately after preparation. Our recommendations are not limited to the APC devices described in this report, but could also be extended to other APC devices.
Assuntos
Arritmias Cardíacas/tratamento farmacológico , Benchmarking/métodos , Fármacos Cardiovasculares/farmacologia , Descoberta de Drogas/métodos , Coração/efeitos dos fármacos , Técnicas de Patch-Clamp/métodos , Animais , Arritmias Cardíacas/metabolismo , Astemizol/farmacologia , Células CHO , Calibragem , Fármacos Cardiovasculares/química , Linhagem Celular , Cricetulus , Avaliação Pré-Clínica de Medicamentos/métodos , Canal de Potássio ERG1/metabolismo , Células HEK293 , Humanos , Fenetilaminas/farmacologia , Polipropilenos/química , Politetrafluoretileno/química , Padrões de Referência , Reprodutibilidade dos Testes , Sulfonamidas/farmacologia , Terfenadina/farmacologiaRESUMO
INTRODUCTION: Long QT syndrome (LQTS) mutation carriers have elevated the risk of cardiac events even in the absence of QTc prolongation; however, mutation penetrance in patients with normal QTc may be reflected in abnormal T-wave shape, particularly in KCNH2 mutation carriers. We aimed to assess whether the magnitude of a three-dimensional T-wave vector (TwVM) will identify KCNH2-mutation carriers with normal QTc at risk for cardiac events. METHODS: Adult LQT2 patients with QTc < 460 ms in men and <470 ms in women (n = 113, age 42 ± 16 years, 43% male) were compared with genotype-negative family members (n = 1007). The TwVM was calculated using T-wave amplitudes in leads V6, II, and V2 as the square root of (TV62 + TII2 + (0.5*TV2)2 ). Cox regression analysis adjusted for gender and time-dependent beta-blocker use was performed to assess cardiac event (CE) risk, defined as syncope, aborted cardiac arrest, implantable cardioverter-defibrillator therapy, or sudden death. RESULTS: Dichotomized by median of 0.30 mV, lower TwVM was associated with elevated CE risk compared to those with high TwVM (HR = 2.95, 95% CI, 1.25-6.98, P = .014) and also remained significant after including sex and time-dependent beta-blocker usage in the Cox regression analysis (HR = 2.64, 95% CI, 1.64-4.24, P < .001). However, these associations were found only in women but not in men who had low event rates. CONCLUSION: T-wave morphology quantified as repolarization vector magnitude using T-wave amplitudes retrieved from standard 12-lead electrocardiogram predicts cardiac events risk in LQT2 women and appears useful for risk stratification of KCNH2-mutation carriers without QTc prolongation.
Assuntos
Potenciais de Ação , Canal de Potássio ERG1/genética , Sistema de Condução Cardíaco/fisiopatologia , Frequência Cardíaca , Síndrome do QT Longo/diagnóstico , Síndrome do QT Longo/genética , Mutação , Vetorcardiografia , Adulto , Estudos de Casos e Controles , Canal de Potássio ERG1/metabolismo , Feminino , Predisposição Genética para Doença , Humanos , Síndrome do QT Longo/fisiopatologia , Síndrome do QT Longo/terapia , Masculino , Pessoa de Meia-Idade , Fenótipo , Valor Preditivo dos Testes , Prognóstico , Estudos Prospectivos , Sistema de Registros , Medição de Risco , Fatores de Risco , Fatores Sexuais , Fatores de TempoRESUMO
Human ether-a-go-go-related gene (hERG) trafficking inhibition is known to be one of the mechanisms of indirect hERG inhibition, resulting in QT prolongation and lethal arrhythmia. Pentamidine, an antiprotozoal drug, causes QT prolongation/Torsades de Pointes (TdP) via hERG trafficking inhibition, but 17-AAG, a geldanamycin derivative heat shock protein 90 (Hsp90) inhibitor, has not shown torsadogenic potential clinically, despite Hsp90 inhibitors generally being hypothesized to cause TdP by hERG trafficking inhibition. In the present study, we investigated the underlying mechanisms of both drugs' actions on hERG channels using hERG-overexpressing CHO cells (hERG-CHOs) and human embryonic stem cell-derived cardiomyocytes (hES-CMs). The effects on hERG tail current and protein levels were evaluated using population patch clamp and Western blotting in hERG-CHOs. The effects on field potential duration (FPD) were recorded by a multi-electrode array (MEA) in hES-CMs. Neither drug affected hERG tail current acutely. Chronic treatment with each drug inhibited hERG tail current and decreased the mature form of hERG protein in hERG-CHOs, whereas the immature form of hERG protein was increased by pentamidine but decreased by 17-AAG. In MEA assays using hES-CMs, pentamidine time-dependently prolonged FPD, but 17-AAG shortened it. The FPD prolongation in hES-CMs upon chronic pentamidine exposure is relevant to its clinically reported arrhythmic risk. Cav1.2 or Nav1.5 current were not reduced by chronic application of either drug at a relevant concentration to hERG trafficking inhibition in human embryonic kidney (HEK293) cells. Therefore, the reason why chronic 17-AAG shortened the FPD despite the hERG trafficking inhibition occur is still unknown.
Assuntos
Benzoquinonas/farmacologia , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Lactamas Macrocíclicas/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Pentamidina/farmacologia , Segurança , Células-Tronco/citologia , Animais , Benzoquinonas/efeitos adversos , Células CHO , Canais de Cálcio Tipo L/metabolismo , Cricetulus , Canal de Potássio ERG1/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Lactamas Macrocíclicas/efeitos adversos , Miócitos Cardíacos/citologia , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Pentamidina/efeitos adversosRESUMO
To predict drug-induced serious adverse events (SAE) in clinical trials, a model using a panel of cells derived from human induced pluripotent stem cells (hiPSCs) of individuals with different susceptibilities could facilitate major advancements in translational research in terms of safety and pharmaco-economics. However, it is unclear whether hiPSC-derived cells can recapitulate interindividual differences in drug-induced SAE susceptibility in populations not having genetic disorders such as healthy subjects. Here, we evaluated individual differences in SAE susceptibility based on an in vitro model using hiPSC-derived cardiomyocytes (hiPSC-CMs) as a pilot study. hiPSCs were generated from blood samples of ten healthy volunteers with different susceptibilities to moxifloxacin (Mox)-induced QT prolongation. Different Mox-induced field potential duration (FPD) prolongation values were observed in the hiPSC-CMs from each individual. Interestingly, the QT interval was significantly positively correlated with FPD at clinically relevant concentrations (r > 0.66) in multiple analyses including concentration-QT analysis. Genomic analysis showed no interindividual significant differences in known target-binding sites for Mox and other drugs such as the hERG channel subunit, and baseline QT ranges were normal. The results suggest that hiPSC-CMs from healthy subjects recapitulate susceptibility to Mox-induced QT prolongation and provide proof of concept for in vitro preclinical trials.
Assuntos
Fenômenos Eletrofisiológicos/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/fisiologia , Potenciais de Ação/efeitos dos fármacos , Alelos , Diferenciação Celular , Canal de Potássio ERG1/genética , Canal de Potássio ERG1/metabolismo , Eletrocardiografia , Perfilação da Expressão Gênica , Frequência do Gene , Voluntários Saudáveis , Sistema de Condução Cardíaco/efeitos dos fármacos , Humanos , Masculino , Mutação , Miócitos Cardíacos/citologia , Polimorfismo de Nucleotídeo ÚnicoRESUMO
The estimation of the cardiotoxicity of compounds is an important task for the drug discovery as well as for the risk assessment in ecological aspect. The experimental estimation of the above endpoint is complex and expensive. Hence, the theoretical computational methods are very attractive alternative of the direct experiment. A model for cardiac toxicity of 400 hERG blocker compounds (pIC50) is built up using the Monte Carlo method. Three different splits into the visible training set (in fact, the training set plus the calibration set) and invisible validation sets examined. The predictive potential is very good for all examined splits. The statistical characteristics for the external validation set are (i) the coefficient of determination r(2)=(0.90-0.93); and (ii) root-mean squared error s=(0.30-0.40).
Assuntos
Simulação por Computador , Canal de Potássio ERG1/antagonistas & inibidores , Cardiopatias/induzido quimicamente , Modelos Biológicos , Método de Monte Carlo , Bloqueadores dos Canais de Potássio/toxicidade , Cardiotoxicidade , Relação Dose-Resposta a Droga , Canal de Potássio ERG1/metabolismo , Cardiopatias/metabolismo , Humanos , Modelos Estatísticos , Estrutura Molecular , Bloqueadores dos Canais de Potássio/química , Relação Quantitativa Estrutura-Atividade , Reprodutibilidade dos Testes , Medição de Risco , SoftwareRESUMO
In vitro screening of hERG channels are recommended under ICH S7B guidelines to predict drug-induced QT prolongation and Torsade de Pointes (TdP), whereas proarrhythmia is known to be evoked by blockage of other ion channels involved in cardiac contraction and compensation mechanisms. A consortium for drug safety assessment using human iPS cells-derived cardiomyocytes (hiPS-CMs), CSAHi, has been organized to establish a novel in vitro test system that would enable better prediction of drug-induced proarrhythmia and QT prolongation. Here we report the inter-facility and cells lot-to-lot variability evaluated with FPDc (corrected field potential duration), FPDc10 (10% FPDc change concentration), beat rate and incidence of arrhythmia-like waveform or arrest on hiPS-CMs in a multi-electrode array system. Arrhythmia-like waveforms were evident for all test compounds, other than chromanol 293B, that evoked FPDc prolongation in this system and are reported to induce TdP in clinical practice. There was no apparent cells lot-to-lot variability, while inter-facility variabilities were limited within ranges from 3.9- to 20-folds for FPDc10 and about 10-folds for the minimum concentration inducing arrhythmia-like waveform or arrests. In conclusion, the new assay model reported here would enable accurate prediction of a drug potential for proarrhythmia.
Assuntos
Arritmias Cardíacas/induzido quimicamente , Diferenciação Celular , Canal de Potássio ERG1/antagonistas & inibidores , Frequência Cardíaca/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Microeletrodos , Miócitos Cardíacos/efeitos dos fármacos , Bloqueadores dos Canais de Potássio/toxicidade , Testes de Toxicidade/instrumentação , Potenciais de Ação , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/fisiopatologia , Bioensaio , Cardiotoxicidade , Técnicas de Cultura de Células , Células Cultivadas , Relação Dose-Resposta a Droga , Canal de Potássio ERG1/metabolismo , Desenho de Equipamento , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Japão , Miócitos Cardíacos/metabolismo , Observação , Reprodutibilidade dos Testes , Medição de Risco , Testes de Toxicidade/métodos , Testes de Toxicidade/normasRESUMO
The Kv11.1 or hERG potassium channel is responsible for one of the major repolarising currents (IKr) in cardiac myocytes. Drug binding to hERG can result in reduction in IKr, action potential prolongation, acquired long QT syndrome and fatal cardiac arrhythmias. The current guidelines for pre-clinical assessment of drugs in development is based on the measurement of the drug concentration that causes 50% current block, i.e., IC50. However, drugs with the same apparent IC50 may have very different kinetics of binding and unbinding, as well as different affinities for the open and inactivated states of Kv11.1. Therefore, IC50 measurements may not reflect the true risk of drug induced arrhythmias. Here we have used an in silico approach to test the hypothesis that drug binding kinetics and differences in state-dependent affinity will influence the extent of cardiac action potential prolongation independent of apparent IC50 values. We found, in general that drugs with faster overall kinetics and drugs with higher affinity for the open state relative to the inactivated state cause more action potential prolongation. These characteristics of drug-hERG interaction are likely to be more arrhythmogenic but cannot be predicted by IC50 measurement alone. Our results suggest that the pre-clinical assessment of Kv11.1-drug interactions should include descriptions of the kinetics and state dependence of drug binding. Further, incorporation of this information into sophisticated in silico models should be able to better predict arrhythmia risk and therefore more accurately assess safety of new drugs in development.