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1.
Toxicol Appl Pharmacol ; 390: 114883, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31981640

RESUMO

Human-based in silico models are emerging as important tools to study the effects of integrating inward and outward ion channel currents to predict clinical proarrhythmic risk. The aims of this study were 2-fold: 1) Evaluate the capacity of an in silico model to predict QTc interval prolongation in the in vivo anesthetized cardiovascular guinea pig (CVGP) assay for new chemical entities (NCEs) and; 2) Determine if a translational pharmacokinetic/pharmacodynamic (tPKPD) model can improve the predictive capacity. In silico simulations for NCEs were performed using a population of human ventricular action potential (AP) models. PatchXpress® (PX) or high throughput screening (HTS) ion channel data from respectively n = 73 and n = 51 NCEs were used as inputs for the in silico population. These NCEs were also tested in the CVGP (n = 73). An M5 pruned decision tree-based regression tPKPD model was used to evaluate the concentration at which an NCE is liable to prolong the QTc interval in the CVGP. In silico results successfully predicted the QTc interval prolongation outcome observed in the CVGP with an accuracy/specificity of 85%/73% and 75%/77%, when using PX and HTS ion channel data, respectively. Considering the tPKPD predicted concentration resulting in QTc prolongation (EC5%) increased accuracy/specificity to 97%/95% using PX and 88%/97% when using HTS. Our results support that human-based in silico simulations in combination with tPKPD modeling can provide correlative results with a commonly used early in vivo safety assay, suggesting a path toward more rapid NCE assessment with reduced resources, cycle time, and animal use.

2.
Circ Res ; 125(9): 855-867, 2019 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-31600125

RESUMO

Given that cardiovascular safety concerns remain the leading cause of drug attrition at the preclinical drug development stage, the National Center for Toxicological Research of the US Food and Drug Administration hosted a workshop to discuss current gaps and challenges in translating preclinical cardiovascular safety data to humans. This white paper summarizes the topics presented by speakers from academia, industry, and government intended to address the theme of improving cardiotoxicity assessment in drug development. The main conclusion is that to reduce cardiovascular safety liabilities of new therapeutic agents, there is an urgent need to integrate human-relevant platforms/approaches into drug development. Potential regulatory applications of human-derived cardiomyocytes and future directions in employing human-relevant platforms to fill the gaps and overcome barriers and challenges in preclinical cardiovascular safety assessment were discussed. This paper is intended to serve as an initial step in a public-private collaborative development program for human-relevant cardiotoxicity tools, particularly for cardiotoxicities characterized by contractile dysfunction or structural injury.

3.
Br J Pharmacol ; 176(19): 3819-3833, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31271649

RESUMO

BACKGROUND AND PURPOSE: Early identification of drug-induced cardiac adverse events is key in drug development. Human-based computer models are emerging as an effective approach, complementary to in vitro and animal models. Drug-induced shortening of the electromechanical window has been associated with increased risk of arrhythmias. This study investigates the potential of a cellular surrogate for the electromechanical window (EMw) for prediction of pro-arrhythmic cardiotoxicity, and its underlying ionic mechanisms, using human-based computer models. EXPERIMENTAL APPROACH: In silico drug trials for 40 reference compounds were performed, testing up to 100-fold the therapeutic concentrations (EFTPCmax ) and using a control population of human ventricular action potential (AP) models, optimised to capture pro-arrhythmic ionic profiles. EMw was calculated for each model in the population as the difference between AP and Ca2+ transient durations at 90%. Drug-induced changes in the EMw and occurrence of repolarisation abnormalities (RA) were quantified. KEY RESULTS: Drugs with clinical risk of Torsade de Pointes arrhythmias induced a concentration-dependent EMw shortening, while safe drugs lead to increase or small change in EMw. Risk predictions based on EMw shortening achieved 90% accuracy at 10× EFTPCmax , whereas RA-based predictions required 100× EFTPCmax to reach the same accuracy. As it is dependent on Ca2+ transient, the EMw was also more sensitive than AP prolongation in distinguishing between pure hERG blockers and multichannel compounds also blocking the calcium current. CONCLUSION AND IMPLICATIONS: The EMw is an effective biomarker for in silico predictions of drug-induced clinical pro-arrhythmic risk, particularly for compounds with multichannel blocking action.

4.
J Pharmacol Toxicol Methods ; 98: 106593, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31158459

RESUMO

This commentary highlights and expands upon the thoughts conveyed in the lecture by Dr. Alan S. Bass, recipient of the 2017 Distinguished Service Award from the Safety Pharmacology Society, given on 27 September 2017 in Berlin, Germany. The lecture discussed the societal, scientific, technological, regulatory and economic events that dramatically impacted the pharmaceutical industry and ultimately led to significant changes in the strategic operations and practices of safety pharmacology. It focused on the emerging challenges and opportunities, and considered the lessons learned from drug failures and the influences of world events, including the financial crisis that ultimately led to a collapse of the world economies from which we are now recovering. Events such as these, which continue to today, challenge the assumptions that form the foundation of our discipline and dramatically affect the way that safety pharmacology is practiced. These include the latest scientific and technological developments contributing to the design and advancement of safe medicines. More broadly, they reflect the philosophical mission of safety pharmacology and the roles and responsibilities served by safety pharmacologists. As the discipline of Safety Pharmacology continues to evolve, develop and mature, the reader is invited to reflect on past experiences as a framework towards a vision of the future of the field.


Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Indústria Farmacêutica/métodos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/prevenção & controle , Animais , Humanos , Sociedades
5.
JRSM Cardiovasc Dis ; 8: 2048004019854919, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31217965

RESUMO

Objective: We investigated if there is IKs, and if there is repolarization reserve by IKs in human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Design: We used a specific KCNQ1/KCNE1 channel blocker, L-000768673, with an IC50 of 9 nM, and four hERG-specific blockers, astemizole, cisapride, dofetilide, and E-4031 to investigate the issue. Results: L-000768673 concentration-dependently prolonged feature point duration (FPD)-a surrogate signal of action potential duration-from 1 to 30 nM without pacing or paced at 1.2 Hz, resulting from IKs blockade in hiPSC-CMs. At higher concentrations, the effect of L-000768673 on IKs was mitigated by its effect on ICa-L, resulting in shortened FPD, reduced impedance amplitude, and increased beating rate at 1 µM and above, recapitulating the self-limiting properties of L-000768673 on action potentials. All four hERG-specific blockers prolonged FPD as expected. Co-application of L-000768673 at sub-threshold (0.1 and 0.3 nM) and threshold (1 nM) concentrations failed to synergistically enhance the effects of hERG blockers on FPD prolongation, rather it showed additive effects, inconsistent with the repolarization reserve role of IKs in mature human myocytes that enhanced IKr response, implying a difference between hiPSC-CMs used in this study and mature human cardiomyocytes. Conclusion: There was IKs current in hiPSC-CMs, and blockade of IKs current caused prolongation of action potential of hiPSC-CMs. However, we could not demonstrate any synergistic effects on action potential duration prolongation of hiPSC-CMs by blocking hERG current and IKs current simultaneously, implying little or no repolarization reserve by IKs current in hiPSC-CMs used in this study.

6.
J Pharmacol Toxicol Methods ; 99: 106598, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31201864

RESUMO

We investigated whether human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) created from diverse origins could have qualitatively (not just quantitatively) different responses to pharmacological reagents. Specifically, we challenged six hiPSC-CM syncytia made from a female Caucasian, a female black non-Hispanic, a female white non-Hispanic, a male Caucasian non-Hispanic, a male Asian Indian, and a male-Asian, respectively, with eight different classes of pharmacological reagents (hERG channel blocker cisapride and dofetilide, calcium channel opener FPL64176, ß-adrenergic agonist Isoproterenol, HCN channel blocker Ivabradine, IKs current blocker L-000768673, sodium channel blocker tetrodotoxin, and calcium channel blocker verapamil). We focused our analysis and comparison on qualitative differences (e.g., yes or no), and, found the following: hiPSC-CMs from female donors were uniformly more sensitive to dofetilide or cisapride, whereas those from male donors of all races were less sensitive to the two typical hERG blockers; isoproterenol had no chronotropic effect at all in one line; and two lines reacted to tetrodotoxin at very low concentrations and were more sensitive to external stimulation. We conclude that not all hiPSC-CMs are suitable for drug testing in terms of cardiac safety assessment, and pre-set acceptance criteria need to be established before any hiPSC-CMs can be used in CiPA-style study to evaluate cardiac liabilities of drug candidates.

7.
Toxicol Rep ; 6: 305-320, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31011540

RESUMO

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.

8.
Toxicol Sci ; 170(2): 345-356, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31020317

RESUMO

The goal of this research consortium including Janssen, MSD, Ncardia, FNCR/LBR, and Health and Environmental Sciences Institute (HESI) was to evaluate the utility of an additional in vitro assay technology to detect potential drug-induced long QT and torsade de pointes (TdP) risk by monitoring cytosolic free Ca2+ transients in human stem-cell-derived cardiomyocytes (hSC-CMs). The potential proarrhythmic risks of the 28 comprehensive in vitro proarrhythmia assay (CiPA) drugs linked to low, intermediate, and high clinical TdP risk were evaluated in a blinded manner using Ca2+-sensitive fluorescent dye assay recorded from a kinetic plate reader system (Hamamatsu FDSS/µCell and FDSS7000) in 2D cultures of 2 commercially available hSC-CM lines (Cor.4U and CDI iCell Cardiomyocytes) at 3 different test sites. The Ca2+ transient assay, performed at the 3 sites using the 2 different hSC-CMs lines, correctly detected potential drug-induced QT prolongation among the 28 CiPA drugs and detected cellular arrhythmias-like/early afterdepolarization in 7 of 8 high TdP-risk drugs (87.5%), 6 of 11 intermediate TdP-risk drugs (54.5%), and 0 of 9 low/no TdP-risk drugs (0%). The results were comparable among the 3 sites and from 2 hSC-CM cell lines. The Ca2+ transient assay can serve as a user-friendly and higher throughput alternative to complement the microelectrode array and voltage-sensing optical action potential recording assays used in the HESI-CiPA study for in vitro assessment of drug-induced long QT and TdP risk.

9.
Toxicol Sci ; 167(2): 573-580, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30365015

RESUMO

Calcium channel blockers (CCBs), such as diltiazem, nifedipine, and verapamil, cause tachycardia effects on several commercially available human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), when tested in culture media provided by suppliers, rather than bradycardia effects, as seen in vivo. We found that in test conditions where Na+ current of hiPSC-CMs was reduced to certain threshold by either specific Na+ channel blocker tetrodotoxin (TTX), or by voltage-dependent inactivation using elevated extracellular potassium concentrations, CCBs produced bradycardia effects on hiPSC-CMs. However, elevated extracellular potassium concentrations or the presence of TTX did not change other pharmacological responses of hiPSC-CMs, including CCBs' effects on contraction intensity and duration; beating rate change by calcium channel opener FPL64176, HCN blocker ivabradine, and ß-adrenergic agonist isoproterenol; and action potential duration prolongation by hERG channel blocker dofetilide. We concluded that action potentials of hiPSC-CMs, with regards to the CCB phenotype, were Na+ current driven. When Na+ channel availability was reduced to a critical level, their action potentials became Ca2+ current driven, and their responses to CCBs correlated well to those seen in vivo. Importantly, the corrected bradycardia effect of calcium channel block with our defined conditions will provide more reliable results in cardiac safety readouts of test compounds that integrate multiple effects including calcium channel inhibition.


Assuntos
Potenciais de Ação/efeitos dos fármacos , Bloqueadores dos Canais de Cálcio/farmacologia , Avaliação Pré-Clínica de Medicamentos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Canais de Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Células Cultivadas , Meios de Cultura , Frequência Cardíaca/efeitos dos fármacos , Humanos , Potássio/farmacologia , Bloqueadores dos Canais de Sódio/farmacologia , Tetrodotoxina/farmacologia
18.
Artigo em Inglês | MEDLINE | ID: mdl-29330131

RESUMO

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/fisiologia
19.
Sci Rep ; 7: 44820, 2017 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-28327633

RESUMO

Severe bradycardia/bradyarrhythmia following coadministration of the HCV-NS5B prodrug sofosbuvir with amiodarone was recently reported. Our previous preclinical in vivo experiments demonstrated that only certain HCV-NS5B prodrugs elicit bradycardia when combined with amiodarone. In this study, we evaluate the impact of HCV-NS5B prodrug phosphoramidate diastereochemistry (D-/L-alanine, R-/S-phosphoryl) in vitro and in vivo. Co-applied with amiodarone, L-ala,SP prodrugs increased beating rate and decreased beat amplitude in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), but D-ala,RP produgs, including MK-3682, did not. Stereochemical selectivity on emerging bradycardia was confirmed in vivo. Diastereomer pairs entered cells equally well, and there was no difference in intracellular accumulation of L-ala,SP metabolites ± amiodarone, but no D-ala,RP metabolites were detected. Cathepsin A (CatA) inhibitors attenuated L-ala,SP prodrug metabolite formation, yet exacerbated L-ala,SP + amiodarone effects, implicating the prodrugs in these effects. Experiments indicate that pharmacological effects and metabolic conversion to UTP analog are L-ala,SP prodrug-dependent in cardiomyocytes.


Assuntos
Amiodarona/química , Amiodarona/farmacologia , Antiarrítmicos/química , Antiarrítmicos/farmacologia , Antivirais/química , Antivirais/farmacologia , Interações de Medicamentos , Amiodarona/farmacocinética , Animais , Antiarrítmicos/farmacocinética , Antivirais/farmacocinética , Canais de Cálcio Tipo L/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Linhagem Celular , Relação Dose-Resposta a Droga , Cobaias , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Estrutura Molecular , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Pró-Fármacos/química , Pró-Fármacos/farmacologia , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/antagonistas & inibidores
20.
Toxicol Appl Pharmacol ; 308: 66-76, 2016 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-27520758

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/citologia
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