Journal of pharmacological and toxicological methods最新文献_第3页

Concordance of cardiovascular contractility between FLEXcyte assay and the rat left ventricular catheterized telemetry model 大鼠左心室导管遥测模型与弹性细胞测定的一致性

IF 1.8 4区医学 Q4 PHARMACOLOGY & PHARMACY

Journal of pharmacological and toxicological methods

Pub Date : 2025-09-01 DOI: 10.1016/j.vascn.2025.107827

Jason D. Payseur, Xuejun Wu, Elizabeth Holliday, Shuya Wang, Earl Gordon, Eric I. Rossman, Ajeesh K. Cherian

Changes in cardiac contractility can lead to hemodynamic alterations and abnormal structural changes within the heart, which over time can cause reduced function and failure. Recent advancements in technology, allowing the assessment of cardiac contractility to become easier and more efficient, have resulted in a greater focus on contractility measures within safety pharmacology studies. In particular, the development of in vitro assays such as the FLEXcyte assay, which utilizes human stem cell derived cardiomyocytes (hiPSC-CMs), allows for the ability to screen for potential contractility changes earlier in development. The objective of this study was to explore the concordance of changes in contractility between FLEXcyte measurements and left ventricular pressure measurements (LV + dP/dt_max) in the telemetered rat model. Verapamil and milrinone were selected as tool compounds. For the FLEXcyte, hiPSC-CMs (Cardiosight-S; NEXCEL Co., Ltd.) were maintained in a serum-free culture medium for 7 days with media changes every 48 h. Initially, 30 s of baseline was recorded every 15 min for 1 h. Following baseline, a dose response curve was attained using 5 concentrations. Spontaneous beat rate and contraction amplitude were analyzed to determine the concordance with heart rate and LV + dP/dt_max in the telemetered rat. For the telemetered study, CRL WI(Han) rats were instrumented with HD-S21 (DSI) implants monitoring systemic and LV pressures and placed into a 4 × 4 latin square design for each tool compound. For verapamil, the FLEXcyte showed an increase in beat rate (22.7 % at 33 nM) and a substantial decrease in amplitude (66.5 % at 33 nM), which concords well with the increase in heart rate (25.2 %) and decrease in LV + dP/dt_max (−33.6 %) in the telemetered rat. However, for milrinone, the FLEXcyte did not show any significant changes in beat rate or amplitude while the telemetered rat showed increases in both heart rate (up to 57 %) and LV + dP/dt_max (up to 87.4 %). While more work is needed, this data highlights the importance of conducting both in vitro and in vivo studies to assess the potential effects on cardiac contractility in drug development, allowing for the identification of molecules with diverse mechanisms of action.

心脏收缩力的改变可导致血液动力学的改变和心脏结构的异常变化，随着时间的推移可导致功能下降和衰竭。最近技术的进步，使得心脏收缩力的评估变得更加容易和有效，导致了在安全药理学研究中对收缩力测量的更多关注。特别是，体外 测定法的发展，如利用人类干细胞来源的心肌细胞（hiPSC-CMs）的FLEXcyte测定法，允许在发育早期筛选潜在的收缩性变化。本研究的目的是探讨遥测大鼠模型中屈细胞测量值与左心室压力测量值（LV + dP/dtmax）之间收缩性变化的一致性。选择维拉帕米和米立酮作为工具化合物。对于flexcell， hiPSC-CMs （Cardiosight-S; NEXCEL Co., Ltd）在无血清培养基中维持7 天，每48 h更换一次培养基。最初，每15 min记录30 s基线，持续1 h。在基线之后，用5种浓度获得剂量反应曲线。分析自然心率和收缩幅度，以确定遥测大鼠心率和LV + dP/dtmax的一致性。在遥测研究中，CRL WI（Han）大鼠使用HD-S21 （DSI）植入物监测全身和左室压力，并将每种工具化合物放置在4 × 4拉丁方设计中。维拉帕米的作用下，FLEXcyte的心率增加（在33 nM处增加22.7 %），振幅大幅下降（在33 nM处减少66.5 %），这与遥测大鼠的心率增加（25.2 %）和LV + dP/dtmax下降（- 33.6 %）非常吻合。然而，对于米立酮，FLEXcyte没有显示出任何显著的心率或振幅变化，而遥测大鼠显示心率（高达57 %）和LV + dP/dtmax（高达87.4% %）均增加。虽然还需要做更多的工作，但这一数据强调了进行体外和体内研究的重要性，以评估药物开发中对心脏收缩性的潜在影响，从而确定具有不同作用机制的分子。

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引用次数: 0

Reproducible safety pharmacology echocardiography studies in vehicle-treated canines and NHPs 车辆治疗犬和NHPs的可重复安全性药理学超声心动图研究

IF 1.8 4区医学 Q4 PHARMACOLOGY & PHARMACY

Journal of pharmacological and toxicological methods

Pub Date : 2025-09-01 DOI: 10.1016/j.vascn.2025.107835

Steve R. Roof, Sydney E. St Clair

Echocardiography has emerged as a key component in cardiovascular diagnostics, offering a non-invasive, clinically relevant approach. Echocardiography enables the ability monitor disease progression, therapeutic responses, efficacy and safety of gene therapy interventions, as well as detecting cardiac toxicity. Typical measurements include the assessment of left ventricular (LV) systolic and diastolic function to evaluate overall cardiac performance, LV wall thickness/chamber size, evaluation of mitral and tricuspid regurgitation (MR/TR) to identify valvular defects, quantification of right ventricular performance (TAPSE and pulmonary velocities), left atrial annulus tissue doppler, as well as measurement of left atrial size/function to assess atrial performance. To determine the reproducibility and consistency of echocardiographic measurements, vehicle-treated animals were studied. Canines and non-human primates (NHPs) were sedated with butorphanol (0.2 mg/kg, IV) or ketamine (10 mg/kg, IM), respectively. In the canine study, echocardiographic measurements were collected in 4 animals at baseline and 6 h post vehicle (oral gavage). Of the 43 measured parameters, 40 were within 10 % of baseline. In the NHP study (n = 4), longitudinal vehicle data was collected at baseline, 6- and 12-weeks post intervention and demonstrated similar precision and reproducibility as all parameters were less than 15 % change from baseline at 6 weeks (32 out of 37 under 10 %). At 12 weeks, 35 of 37 were less than 15 % and 29 of 37 were under 10 %. In conclusion, incorporating high-quality and reproducible echocardiographic assessments into safety pharmacology studies offers a significant advantage by providing critical insights into cardiac function. However, the sonographer, ultrasound machine, and technique must be constant to detect a small difference in safety pharmacology studies.

超声心动图已成为心血管诊断的关键组成部分，提供了一种非侵入性的临床相关方法。超声心动图能够监测疾病进展、治疗反应、基因治疗干预的有效性和安全性，以及检测心脏毒性。典型的测量包括评估左心室（LV）收缩和舒张功能以评估整体心脏功能，左室壁厚度/室大小，评估二尖瓣和三尖瓣反流（MR/TR）以识别瓣膜缺陷，量化右心室功能（TAPSE和肺流速），左房环组织多普勒以及测量左房大小/功能以评估心房功能。为了确定超声心动图测量结果的重复性和一致性，我们研究了经药物治疗的动物。用丁托啡诺（0.2 mg/kg， IV）或氯胺酮（10 mg/kg， IM）分别对犬和非人灵长类动物（NHPs）进行镇静。在犬类研究中，4只动物在基线时和6只动物在给药（灌胃）后 h收集了超声心动图测量结果。在43个测量参数中，40个在基线的10 %以内。在NHP研究中（n = 4），纵向车辆数据在干预后的基线、6周和12周收集，显示出相似的精度和可重复性，因为所有参数在6 周时比基线变化小于15 %（37人中有32人低于10 %）。在12 周时，37例患者中有35例小于15 %，29例小于10 %。总之，将高质量和可重复的超声心动图评估纳入安全性药理学研究，通过提供对心功能的关键见解，提供了显著的优势。然而，超声仪、超声机器和技术必须是恒定的，才能在安全药理学研究中发现微小的差异。

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引用次数: 0

Assessing the cardiotoxicity of 31 compounds using a multiplexed kinetic image cytometry (KIC)-based assay: Harnessing the predictive power of human iPSC-cardiomyocytes for early drug development 使用基于多路动态图像细胞术（KIC）的检测评估31种化合物的心脏毒性：利用人类ipsc -心肌细胞的预测能力进行早期药物开发

IF 1.8 4区医学 Q4 PHARMACOLOGY & PHARMACY

Journal of pharmacological and toxicological methods

Pub Date : 2025-09-01 DOI: 10.1016/j.vascn.2025.107820

Ranor C.B. Basa , Randall S. Ingermanson , Filiberto Catalan-Perez , Ricardo Serrano , Ariel Wang , Alyson Smith , Jeffrey M. Hilton , Patrick M. McDonough , Cherie Handley , Lilian Harrison , Ameena Pascua , Mark Mercola , Jeffrey H. Price

Human-induced pluripotent stem cell (hiPSC) models are more species-relevant than animal models and are amenable to high-throughput scalability. Previously, our Kinetic Image Cytometry (KIC) proarrhythmia assay examining calcium kinetics in hiPSC-cardiomyocytes (CMs) had a clinical accuracy of ~90 % (Pfeiffer, et al., 2016); when augmented with deep learning, accuracy increased to 95 % (Serrano, et al., 2023). Expanding upon this work, we screened 31 compounds with known clinical effects (effector classes: sarcoplasmic reticulum/calcium release, plasma membrane/ion channel, mitochondrial/myosin, and negative controls) in a 7-point concentration-response format—bracketing clinical exposures—using a panel of patient-derived hiPSC-CMs. We loaded hiPSC-CMs with Cal-520 (a green-channel calcium indicator), BeRST-1 (a far red-channel membrane voltage dye), TMRM (a mitochondrial membrane potential dye), and Hoechst (for nuclei), then exposed them to test compounds acutely. We then acquired interleaved calcium and voltage movies at 60 Hz (30 Hz/channel) as well as single images of TMRM and Hoechst. Vala's image analysis software, CyteSeer, was used to simultaneously measure calcium/action potential (AP) kinetics on—and to derive contraction metrics from—those movies. Furthermore, we mathematically characterized and classified calcium/AP waveforms to quantify compound-induced cardiotoxic effects. Additionally, we assessed mitochondrial health (using TMRM fluorescence) as well as acute cytotoxicity (using nucleus morphology) in the same experiment. By multiplexing these readouts on a single-cell level, we were able to correctly classify 30/31 (96.8 %) compounds on the basis of earliest-observed adverse effects (if any) by primary effector class at clinically-relevant test concentrations. While the data were more complex than initially expected (e.g., due to pleiotropic clinical effects and/or multiple efficacy−/toxicity-related targets), this multiplexed approach has the potential to elucidate mechanistic classes or targets quickly; and some of the more unexpected results from our screen highlight the need for de-risking early in drug development using relatively inexpensive and highly scalable hiPSC models. A larger study is currently underway to examine a library of 300+ compounds in order to comprehensively validate and refine our multiplexed assay.

人诱导多能干细胞（hiPSC）模型比动物模型更具有物种相关性，并且具有高通量可扩展性。此前，我们的动态图像细胞术（KIC）检测hipsc -心肌细胞（CMs）中钙动力学的心律失常前检测的临床准确性为~90 % (Pfeiffer等，2016)；当使用深度学习增强时，准确率提高到95% % （Serrano等，2023）。在此基础上，我们使用一组患者源性hiPSC-CMs，以7点浓度-反应格式筛选了31种具有已知临床效应的化合物（效应类：肌浆网/钙释放、质膜/离子通道、线粒体/肌球蛋白和阴性对照）。我们将Cal-520（一种绿色通道钙指示剂）、BeRST-1（一种远红色通道膜电压染料）、TMRM（一种线粒体膜电位染料）和Hoechst（用于细胞核）加载到hiPSC-CMs中，然后将它们暴露在测试化合物中。然后，我们在60 Hz（30 Hz/通道）下获得交错的钙和电压电影以及TMRM和Hoechst的单幅图像。Vala的图像分析软件CyteSeer被用于同时测量钙/动作电位（AP）动力学，并从这些电影中得出收缩指标。此外，我们在数学上表征和分类钙/AP波形，以量化化合物诱导的心脏毒性效应。此外，我们在同一实验中评估了线粒体健康（使用TMRM荧光）以及急性细胞毒性（使用细胞核形态学）。通过在单细胞水平上对这些读数进行多重处理，我们能够根据最早观察到的不良反应（如果有的话），根据主要效应物类别在临床相关测试浓度下对30/31（96.8% %）化合物进行正确分类。虽然数据比最初预期的更复杂（例如，由于多效性临床效应和/或多个疗效/毒性相关靶点），但这种多效性方法有可能快速阐明机制类别或靶点；从我们的筛选中得到的一些更意想不到的结果强调了在药物开发早期使用相对便宜和高度可扩展的hiPSC模型降低风险的必要性。目前正在进行一项更大的研究，以检查300多种化合物的文库，以全面验证和完善我们的多重分析。

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引用次数: 0

SmartHeart: An innovative high throughput assay to generate and assess cardiac micro tissues from hiPSCs answering to the current challenges of drug discovery smarheart：一种创新的高通量检测方法，用于从hipsc中生成和评估心脏微组织，以应对当前药物发现的挑战

IF 1.8 4区医学 Q4 PHARMACOLOGY & PHARMACY

Journal of pharmacological and toxicological methods

Pub Date : 2025-09-01 DOI: 10.1016/j.vascn.2025.107821

Ahmed Khedher , Patricia Davidson , Pauline Thiebaud , Stijn Robben , Cyril Cerveau , Jamie Bhagwan , Mael Le Berre , Rita S.R. RIbeiro

Cardiac adverse events are among the top reasons for discontinuing drugs in early clinical phase studies, where drugs with cardiotoxic liabilities are responsible for one third of regulatory failures. These alarming statistics highlight the widespread occurrence and significant financial burden of ineffective drugs that proceed from preclinical animal studies. Despite advancements in cardiac bioengineering, challenges remain regarding physiological relevance, cost, and throughput. To address these challenges, an innovative 3D cardiac model, the SmartHeart (SH), was developed. This model facilitates the self-assembly and maturation of ring-shaped cardiac tissues and allows for precise in-situ measurements of various parameters (e.g. contraction stress, strain, beating metrics, membrane action potential and calcium signaling). The technology is based on standard 96-well plates coated with a structured hydrogel, which features an array of conical-shaped microwells, each surrounding a central pillar. Within less than 48 h after cell seeding, the tissues (composed of iPSC-derived ventricular cardiomyocytes (Axol) and fibroblasts) demonstrated rhythmic contractions. The contractility stress and strain as well as the beating rate of the tissues were quantified by monitoring the variation of the central pillar's area with known stiffness (12 kPa) with time. After 14 days, the tissues presented morphological signs of maturation and were exposed to several classical drugs. The presence of isoproterenol caused a positive inotropic and chronotropic response. A negative inotropic response was induced by nifedipine and the tissues became quiescent when exposed to high doses of mexiletine, which is consistent with its known pharmacological effects as a sodium channel blocker. The hydrogel's optical transparency allows compatibility with high-resolution image-based techniques. This includes the use of voltage-sensitive fluorescent dyes, such as FluoVolt that showed an intensity spike just before the tissue contraction. Likewise, cellular spatial organization and intracellular morphology, e.g. cardiomyocyte cytoskeletal fiber elongation and striation, showing signs of maturation, could be visualized using immunofluorescence. In conclusion, the SmartHeart 3D-cardiac model provides an advanced solution to the ongoing challenges in drug discovery by enabling precise, real-time monitoring of cardiac tissue function and maturation. This innovative platform offers robust and relevant readouts for high-throughput and high-content screening, significantly enhancing the assessment of drug efficacy and safety.

在早期临床阶段研究中，心脏不良事件是停药的主要原因之一，在早期临床阶段研究中，具有心脏毒性的药物造成了三分之一的监管失败。这些令人震惊的统计数据突出了临床前动物研究中无效药物的广泛发生和巨大的经济负担。尽管心脏生物工程取得了进步，但在生理学相关性、成本和吞吐量方面仍然存在挑战。 为了应对这些挑战，开发了一种创新的3D心脏模型，即smarheart （SH）。该模型有助于环状心脏组织的自组装和成熟，并允许精确的原位测量各种参数（例如收缩应力、应变、跳动指标、膜动作电位和钙信号）。该技术基于涂有结构水凝胶的标准96孔板，其特征是一系列锥形微孔，每个微孔围绕中心柱。在细胞播种后不到48 h内，组织（由ipsc衍生的心室心肌细胞（Axol）和成纤维细胞组成）表现出节律性收缩。通过监测已知刚度（12 kPa）的中心柱面积随时间的变化，量化组织的收缩应力、应变和跳动率。 14 天后，组织出现成熟的形态学迹象，并暴露于几种经典药物。  异丙肾上腺素的存在引起正性肌力和变时反应。硝苯地平引起负性肌力反应，当暴露于高剂量美西汀时，组织变得静止，这与已知的美西汀作为钠通道阻滞剂的药理作用一致。 水凝胶的光学透明性允许与基于高分辨率图像的技术兼容。这包括使用电压敏感的荧光染料，如FluoVolt，它在组织收缩之前显示出强度峰值。同样，细胞的空间组织和细胞内形态，如心肌细胞的细胞骨架纤维伸长和条纹，显示成熟的迹象，可以使用免疫荧光可视化。 总而言之，smarheart 3d心脏模型通过精确、实时监测心脏组织功能和成熟度，为药物发现中的持续挑战提供了先进的解决方案。这个创新的平台为高通量和高含量筛选提供了可靠和相关的读数，显著提高了药物疗效和安全性的评估。

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引用次数: 0

Ventricular electromechanics in a cardiac glycoside arrhythmia model in isoflurane-anaesthetised New Zealand White (NZW) rabbits 异氟醚麻醉新西兰白兔（NZW）心脏糖苷性心律失常模型的心室电力学

IF 1.8 4区医学 Q4 PHARMACOLOGY & PHARMACY

Journal of pharmacological and toxicological methods

Pub Date : 2025-09-01 DOI: 10.1016/j.vascn.2025.107830

Gail E. Geist, Deborah Calantropio-Covington, Lindsay Bates, Angelica Cabrera

Cardiac glycoside (CG) intoxication models are well-established tools to evaluate non-torsadogenic arrhythmic liabilities in vivo. However, preparations using practical inhalant anesthetics, particularly in rabbits, a highly translational species for preclinical electrophysiology and functional assessments, are seldom described. In this study, we characterize CG-induced cardiovascular responses under isoflurane anesthesia in an optimized in vivo rabbit preparation. Ventilated male NZW rabbits (n = 4–6/group, 6-month old) were administered either digoxin (DIG, 40 μg/kg/min IV) or ouabain (OUA, 15 μg/kg/min IV) after anesthetic stabilization under isoflurane inhalant (1.5–2 %) and butorphanol (0.2 mg/kg/h IV). Animals were vagotomized and instrumented for lead-II ECG, catheter-based arterial (MAP) and left-ventricular pressures (LVP), and pericardiectomized to permit electrophysiological mapping. Electromechanical window (EMW) was derived from ECG and LVP signals. The effects of concurrent beta-adrenergic stimulation with dobutamine (+DOB: 2.5 μg/kg/min) were evaluated in a subset of OAU rabbits. At baseline (BL), heart rate (HR: 266 ± 44 bpm), hemodynamics (MAP: 60 ± 10 mmHg), ECG intervals (QT: 146 ± 21 msec), and electromechanical indices (dPdtmax: 2551 ± 1144 mmHg/s, EMW: 46 ± 11 ms) were reflective of normal physiology in rabbits. Under CG administration, sustained ventricular ectopy (SVE, >30 s) and ventricular fibrillation (VF) were reliably and successively induced (DIG SVE: 22.2 ± 3 min, VF: 34.7 ± 5 min; OUA SVE: 8.5 ± 2 min, VF: 16 ± 2 min). Prior to SVE and consistent with CG toxicity, animals demonstrated robust increases in indices of inotropy (dPdtmax: DIG: +92 ± 47 %, OUA: +116 ± 43 %), afterload (MAP: DIG: +58 ± 34 %; OUA:+50 ± 14 %), and serum potassium ([K+]: DIG: +40 ± 11 %; OUA: +47 ± 15 %). Heart rate was equivalent to baseline in both preparations, aligning to pre-existing vagal withdrawal (DIG: +2 ± 11 %, OUA: -0.4 ± 3 %). EMW was variable but remained positive in all animals before SVE (EMW DIG: 44 ± 21 msec; OUA: 40 ± 11 msec) with nominal QT change (DIG: -2 ± 11 %, OUA: +5 ± 6 % vs. BL). Beta-adrenergic stimulation accelerated the time to OUA-induced SVE and VF (+DOB: −19 % and − 10 %, respectively vs. OUA). Taken together, these data strongly suggest that rabbits instrumented under a contemporary isoflurane anesthetic regimen constitute a viable model of cardiac glycoside induced ventricular arrhythmias, characterized by predictable and progressive ectopy that is sensitive to pharmacological modulation.

心糖苷（CG）中毒模型是一种完善的工具，用于评估体内非反体源性心律失常。然而，使用实际吸入麻醉剂的制备，特别是在兔，一个高度转化的物种，用于临床前电生理和功能评估，很少被描述。在这项研究中，我们在优化的兔体内制剂中表征了异氟醚麻醉下cg诱导的心血管反应。在异氟烷吸入剂（1.5-2 %）和丁托啡诺（0.2 mg/kg/h IV）麻醉稳定后，给予通气雄性NZW兔（n = 4-6 /组，6月龄）或地高辛（DIG, 40 μg/kg/min IV）或瓦阿因（OUA, 15 μg/kg/min IV）。动物迷走神经切除并测量导联- ii心电图、导管动脉（MAP）和左心室压（LVP），心包切除以进行电生理测绘。机电窗口（EMW）是由ECG和LVP信号推导而来的。多巴酚丁胺并发β -肾上腺素能刺激（+DOB: 2.5 μg/kg/min）对非统组织家兔的影响进行了评估。在基线(提单)、心率(HR: 266 ± 44 bpm),血液动力学(地图:60 ± 10 毫米汞柱),心电图间隔(QT: 146 ± 21 msec),和机电指数(dPdtmax: 2551 ±1144  mmHg / s, EMW: 46 ±11  ms)在兔子正常生理的反射。在CG管理,持续室性异位(SVE祝辞30 年代)和心室颤动(VF)可靠地先后诱导(SVE挖掘:22.2 ± 3 min, VF: 34.7 ± 5 分钟;OUA SVE: 8.5 ± 2 min, VF: 16 ±2  min)。SVE和符合CG毒性之前,动物展示了强劲增长指数inotropy (dPdtmax:挖掘:+ 92 ± 47 %,OUA: + 116 ± 43 %),后负荷(地图:挖掘:+ 58 ± 34 %;OUA: 50 + ± 14 %),和血清钾([K +]:挖:+ 40 ±11  %;OUA: + 47 ± 15 %)。两种制剂的心率与基线相当，与先前存在的迷走神经退缩一致（DIG: +2 ± 11 %,OUA: -0.4 ± 3 %）。EMW变量,但仍是积极的在所有动物SVE (EMW挖:44 ± 21 msec; OUA: 40 ± 11 msec)与名义QT变化(挖:2 ± 11 %,OUA: + 5 ± 6 %和提单)。β -肾上腺素能刺激加速了到OUA诱导的SVE和VF的时间（与OUA相比，+DOB分别为- 19 %和 - 10 %）。综上所述，这些数据强烈表明，在当代异氟醚麻醉方案下，家兔构成了一种可行的心脏糖苷性室性心律失常模型，其特征是可预测和进行性异位，对药物调节敏感。

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引用次数: 0

Analyzing atrial human iPSC-cardiomyocyte responses to GPCR and ion channel modulators: Introducing a chamber-specific cell model for preclinical testing 分析心房人ipsc -心肌细胞对GPCR和离子通道调节剂的反应：引入用于临床前测试的室特异性细胞模型

IF 1.8 4区医学 Q4 PHARMACOLOGY & PHARMACY

Journal of pharmacological and toxicological methods

Pub Date : 2025-09-01 DOI: 10.1016/j.vascn.2025.107832

Bettina Lickiss , Peter Linder , Jan Hunker , Steven D. Broadbent , Jamie R. Bhagwan , Jan Turner , Elena Dragicevic , Sonja Stoelzle-Feix , Matthias Gossmann

Over the past decade, commercial human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have emerged as vital tools for preclinical cardiac risk assessment, thanks to their human origin and limitless reproducibility. Commonly, mixed cell populations comprising ventricular, atrial, and nodal cardiomyocytes are used. However, diseases such as atrial fibrillation, impacting over 33 million individuals globally, underscore the urgent need for cardiac subtype-specific commercial cell lines. Here, we characterize commercially available hiPSC-derived atrial cardiomyocytes (atrial hiPSC-CMs) and compare them to hiPSC-derived ventricular cardiomyocytes (ventricular hiPSC-CMS) (both Axol Biosciences) regarding their contractile properties using FLEXcyte 96 technology. The cells were seeded on flexible 96-well plates mimicking physiological human heart conditions in vitro. Pre-compound beat characteristics were analyzed regarding beat rate and amplitude. Compound-induced effects of GPCR agonists acetylcholine and carbachol were tested regarding contractile properties including beat rate, amplitude and beat duration using a concentration range of 1 μM – 100 μM. Furthermore, ion channel modulators including S-Bay K8644, ivabradine, vernakalant and 4-Aminopyridine were assessed at 4 different concentrations ranging from 100 nM – 1 μM. Pre-compound analysis reveals cell type-specific beat shapes analogous to the respective cardiac action potential, in which atrial cells show a higher beat rate and less pronounced contraction force compared to ventricular cells. Pharmacological analysis demonstrates a higher susceptibility of atrial hiPSC-CMs towards GPCR agonists acetylcholine and carbachol than ventricular cells. Ion channel modulator S-Bay K8644 induces reversed inotropic and chronotropic effects in atrial and ventricular cells, while ivabradine causes a pronounced negative chronotropic effect in atrial cells alone. 4-Aminopyridine reveals prolonged contraction duration and reduced chronotropy in atrial hiPSC-CMs, while vernakalant induces opposing reactions in chronotropy of chamber-specific cardiomyocytes. These results underscore the significant pharmacological responses of atrial hiPSC-CMs and their utility on a multiwell contractility platform for enhancing in vitro cardiac liability studies and disease modeling.

在过去的十年中，商业人类诱导多能干细胞衍生的心肌细胞（hiPSC-CMs）由于其人类起源和无限的可重复性，已成为临床前心脏风险评估的重要工具。通常，混合细胞群包括心室、心房和结型心肌细胞。然而，影响全球3300多万人的房颤等疾病强调了对心脏亚型特异性商业细胞系的迫切需求。在这里，我们描述了市售的hipsc来源的心房心肌细胞（心房hiPSC-CMs），并使用FLEXcyte 96技术将它们与hipsc来源的心室心肌细胞（心室hiPSC-CMs）（都是Axol Biosciences）的收缩特性进行了比较。将细胞接种在体外模拟人类生理心脏条件的96孔板上。从拍频和幅值两个方面分析了预复合拍频特性。在1 μM - 100 μM的浓度范围内，测试了GPCR激动剂乙酰胆碱和氨基苯酚的复合诱导效应，包括跳动速率、振幅和跳动持续时间。此外，离子通道调节剂包括S-Bay K8644、伊伐布雷定、vernakalant和4-氨基吡啶在100 nM - 1 μM范围内的4种不同浓度下进行了评估。预复合分析揭示了细胞类型特异性的搏动形状类似于各自的心脏动作电位，其中心房细胞比心室细胞表现出更高的搏动率和更不明显的收缩力。药理学分析表明，心房hiPSC-CMs对GPCR激动剂乙酰胆碱和氨基酚的敏感性高于心室细胞。离子通道调节剂S-Bay K8644在心房和心室细胞中诱导逆转的肌力和变时作用，而伊伐布雷定仅在心房细胞中引起明显的负性变时作用。4-氨基吡啶显示心房hiPSC-CMs的收缩持续时间延长和慢变性降低，而vernakalant在心室特异性心肌细胞的慢变性中诱导相反的反应。这些结果强调了心房hiPSC-CMs的显著药理学反应及其在多孔收缩性平台上的应用，以加强体外心脏负荷研究和疾病建模。

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引用次数: 0

Discriminating evidence – Use and misuse of the drug discrimination test in abuse potential assessments of novel CNS drugs 鉴别证据-新型中枢神经系统药物滥用潜力评估中药物鉴别试验的使用和误用

IF 1.8 4区医学 Q4 PHARMACOLOGY & PHARMACY

Journal of pharmacological and toxicological methods

Pub Date : 2025-09-01 DOI: 10.1016/j.vascn.2025.107792

David J. Heal , Jane Gosden , Sharon L. Smith

Drug-discrimination is a mandatory test for evaluating abuse potential of CNS drug‑candidates. The major challenge is designing translationally valid protocols for drug-candidates with novel pharmacological mechanisms and no affinity for targets mediating the psychoactive effects of known substances of abuse. We assessed predictive validity of 3 experimental protocols using our and other published data. Experimental protocols: (1) Drug‑candidate tested in animals trained to discriminate a selected abused substance from vehicle, (2) Drug-candidate investigated in multiple studies with animals trained to recognize representatives from major abused drug classes; (3) Drug-candidate used as training cue with back-testing of various abused substances. Female rats were trained to discriminate d-amphetamine (0.25–0.5 mg/kg,ip) from saline. Full, Partial, and No Generalization: ≥75 %, ≥60 % and < 25 % drug‑associated lever-presses. Microdialysis measurements of extracellular dopamine in accumbens (ACB) or striatum (STR) were made in freely‑moving rats. Generalization to d-amphetamine (mg/kg[route]): lisdexamfetamine (3.4[po]); phentermine (3.0[po]); methylphenidate (5.0[po]); bupropion (30[po]); methamphetamine (0.5[ip]); fencamfamine (3.0[ip]); cocaine (10[ip]). Partial generalization <60 %: sibutramine (3.0[ip]); modafinil (100[ip]). No generalization: atomoxetine (10[po]); desipramine (5.0[ip]); venlafaxine (10[ip]). Only drugs markedly increasing dopamine neurotransmission in ACC/STR generalized to d‑amphetamine. Drugs producing small/moderate dopamine increases in ACB/STR and/or increased dopamine in prefrontal cortex did not produce full or ≥ 60 % generalization to d-amphetamine. Dopamine D₂ antagonists blocked d‑amphetamine's discriminative cue and its reinforcing effect in humans [1,2] showing the pharmacological specificity of both effects. Drug-discrimination findings with other substances of abuse, e.g. cannabinoids, psychedelics, GABA-A positive allosteric modulators (PAMs), glutamatergic ligands, confirm the hypothesis. Results obtained using the drug‑candidate as training cue showed this experimental approach is not viable [3,4]. Evaluation of approaches 1–3: (1) Has weaknesses, but highest predictive validity. (2) Invalid, except when drug-candidate has pharmacology shared with selected abused substances. Behavioral similarity does not produce generalization or have predictive validity. (3) Invalid. No evidence that generalization to drug-candidate would generate any meaningful information or predict abuse potential.

[1] Lile et al., 2005; [2] Rush et al., 2003; [3] Swedberg et al., 2014; [4] Swedberg & Raboisson, 2014.

药物歧视是评估中枢神经系统候选药物滥用可能性的强制性测试。主要的挑战是为 候选药物 设计翻译有效的方案，这些方案具有新的药理学机制，并且对介导已知滥用物质的精神活性作用的靶点没有亲和力。 我们使用我们和其他已发表的数据评估了3个实验方案的预测有效性。实验方案：(1)候选药物在经过训练的动物中进行测试，以区分选定的滥用物质和载体；(2)候选药物在经过训练的动物中进行多项研究，以识别主要滥用药物类别的代表；(3)以候选药物为训练线索，对各种滥用药物进行回测。 雌性大鼠被训练区分d-安非他明（0.25-0.5 mg/kg,ip）和生理盐水。完全、部分和不可概括：≥75 %、≥60 %和 <； 25 %与药物相关的杠杆按压。在自由运动大鼠中进行伏隔体（ACB）或纹状体（STR）的细胞外多巴胺微透析测量。 推广到d-安非他明（mg/kg[路线]）：利地胺非他明（3.4[po]）；芬特明(3.0 (po));(po)哌醋甲酯(5.0);安非他酮(30 (po));(ip)甲基苯丙胺(0.5);(ip) fencamfamine (3.0);可卡因(10 (ip))。部分概括<；60 %：西布曲明（3.0[ip]）；莫达非尼(100 (ip))。无推广：托莫西汀（10[po]）；(ip)去郁敏(5.0);文拉法辛(10 (ip))。只有药物能显著增加ACC/STR患者的多巴胺神经传递。产生ACB/STR小/中度多巴胺增加 和/或前额皮质多巴胺增加的药物对d-安非他明的泛化不完全或 ≥ 60 %。多巴胺D2拮抗剂阻断了d -安非他明的鉴别提示及其在人体内的强化作用[1,2]，显示了这两种作用的药理学特异性。与其他滥用物质（如大麻素、致幻剂、GABA-A阳性变构调节剂（pam）、谷氨酸能配体）的药物鉴别结果证实了这一假设。使用候选药物作为训练线索获得的结果表明，这种实验方法是不可行的[3,4]。 方法1 - 3的评价： (1)有缺点，但预测效度最高。 (2)除候选药物与选定的滥用药物具有共同药理作用外，无效。行为相似性不会产生概括或具有预测有效性。 (3)无效。没有证据表明将候选药物普遍化会产生任何有意义的信息或预测滥用的可能性Lile et al., 2005；[2] Rush et al., 2003；[3] Swedberg et al., 2014；[4] Swedberg & Raboisson, 2014。

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引用次数: 0

Are current rat studies sensitive to detect blood pressure changes per the new FDA draft clinical blood pressure guidance? 根据新的FDA临床血压指南草案，目前的大鼠研究对检测血压变化是否敏感？

IF 1.8 4区医学 Q4 PHARMACOLOGY & PHARMACY

Journal of pharmacological and toxicological methods

Pub Date : 2025-09-01 DOI: 10.1016/j.vascn.2025.107777

Kamila J. Sadko , Derek J. Leishman , Hannah Garver , Gregory Fink , Marc B. Bailie , Adam Lauver

Released in 2022, the FDA's draft guidance “Assessment of Pressor Effects of Drugs”, proposes that a dedicated clinical study for chronic use drugs should be powered to rule out a potential systolic arterial blood pressure (BP) increase of 3 mmHg over 24 h. Given the resource commitment of clinical studies, sensitive nonclinical prediction of potentially meaningful BP increases would be valuable. This study aimed to determine the utility of long-term rat studies for detecting acute and chronic blood pressure changes. We hypothesized that studies using rats have adequate stability and variability to detect BP changes ≥3 mmHg over several weeks. Available data from a historic rodent assessment of 80 days duration (N = 9) in vehicle treated Sprague Dawley rats was used. The data were assessed for minimal detectable differences (MDDs), least significant differences (LSDs), and changes from baseline for all BP measures in light and dark 12 h, days, and weeks. Day 15 was used as the reference day and Day 43 as the comparison for day-to-day and light and dark changes from baseline to simulate a 4-week study with a week-long baseline assessment. In week comparison, week 2 was compared to week 5. A one-way t-test was conducted against an assumed mean difference of 0 to evaluate for significance. Study average difference from baseline for the dark cycle were 1.80 ± 1.42 (mean ± standard deviation), 1.99 ± 1.34, and 1.86 ± 1.25 mmHg for diastolic (Dia), systolic (Sys), and mean arterial pressure (MAP), respectively. Light cycle differences were 0.54 ± 1.23, −0.04 ± 1.101, and 0.143 ± 0.91 mmHg for Dia, Sys, and MAP . Day-to-day differences of 1.21 ± 1.29, 0.89 ± 1.12, and 0.931 ± 1.01 mmHg for Dia, Sys, and MAP were seen. Week-to-week differences were − 2.55 ± 1.47 mmHg for Sys, 2.30 ± 1.29 mmHg for MAP, and 1.87 ± 1.22 mmHg for Dia. Results were not significantly different from 0. Overall, rats demonstrated small insignificant changes from baseline in their blood pressure measures across a longitudinal study. The low variability observed was sufficient to encourage further evaluation of the minimal detectable differences which is currently underway.

FDA于2022年发布的指南草案“药物降压效应评估”建议，应针对慢性用药进行专门的临床研究，以排除24 h内潜在的收缩压（BP）升高3 mmHg的可能性。鉴于临床研究的资源承诺，敏感的非临床预测潜在有意义的血压升高将是有价值的。本研究旨在确定长期大鼠研究对检测急性和慢性血压变化的效用。我们假设使用大鼠的研究具有足够的稳定性和可变性，可以在数周内检测血压变化≥3 mmHg。本研究使用的是一项历史啮齿类动物评估的数据，该评估为80 天持续时间（N = 9）。评估数据的最小可检测差异（mdd），最小显著差异（lsd），以及在光照和黑暗12 h，天数和周内所有BP测量值与基线的变化。第15天作为参考日，第43天作为对照日，对比从基线开始的昼夜变化，模拟为期4周的研究，并进行为期一周的基线评估。在周比较中，将第2周与第5周进行比较。假设平均差为0，进行单向t检验以评估显著性。研究黑暗周期是1.80的平均差异从基线 ±1.42 (平均 ± 标准差),1.99 ± 1.34,和1.86 ±1.25  毫米汞柱,舒张压(Dia)、收缩压(Sys)和平均动脉压(MAP),分别。Dia、Sys和MAP的光周期差异分别为0.54 ± 1.23、- 0.04 ± 1.101和0.143 ± 0.91 mmHg。Dia、Sys和MAP的日差异分别为1.21 ± 1.29、0.89 ± 1.12和0.931 ± 1.01 mmHg。周而复始的差异 −  2.55±1.47  Sys mmHg, 2.30±1.29  毫米汞柱的地图,和1.87 ±1.22 Dia 毫米汞柱。结果与0无显著差异。总体而言，在一项纵向研究中，大鼠的血压测量值与基线相比发生了微不足道的变化。观察到的低变异性足以鼓励对目前正在进行的可检测到的最小差异进行进一步评价。

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引用次数: 0

Validation of in vivo QT ICH E14/S7B Q&A guidance in minipig 小型猪体内QT ICH E14/S7B问答指南的验证

IF 1.8 4区医学 Q4 PHARMACOLOGY & PHARMACY

Journal of pharmacological and toxicological methods

Pub Date : 2025-09-01 DOI: 10.1016/j.vascn.2025.107778

Rachael Hardman , Joyce Obeng , Jill Nichols , Karim Melliti

The E14/S7B Q&A guidelines introduce the concept of “double negative” (negative hERG and negative in vivo QTc) non-clinical data which can be used along with negative Phase 1 clinical QTc data to substitute for a clinical Thorough QT study in specific cases (Q&As 5.1 and 6.1). The non-clinical data are to be generated using “best practice” designs in order to support data quality and consistency across the industry. For the in vivo QT assay, best practice recommendations require characterization of individual study sensitivity, verification of independence QTc from heart rate, demonstration of test facility sensitivity and pharmacological translation to human using a positive control. We have previously validated E14/S7B Q&A compliant in vivo QT assays in dog and non-human primate. The objective of this work was to perform the validation in the minipig as the third non-rodent species commonly used for cardiovascular safety assessment. A positive control telemetry study was performed with moxifloxacin (30, 120, 300 mg/kg) using a double Latin square crossover study design (n = 8), followed by an ascending dose design (n = 5). A single PK sample was drawn during the telemetry phase, and a separate full PK phase was included at all dose levels for PK/PD assessment to determine translation to human. Moxifloxacin-induced increases in QTc were observed, with a maximal increase at the highest dose of 86 and 67 msec for the double cross-over and ascending dose designs, respectively. Smallest statistically detectable differences (SSDD) for QTc were 11 msec for the double cross-over and 10 msec for the ascending dose design. Based on the free plasma exposures and magnitude of the QTc effects from the crossover data, a 10 msec effect was observed at 0.77× of the free moxifloxacin concentration known to produce a 10 msec QTc effect in human. In conclusion, we have validated an E14/S7B Q&A compliant in vivo QT assay in the minipig for both cross-over and ascending dose designs and shown good translation to human, completing the Labcorp™ offering for in-vivo E14/S7B Q&A compliant studies.

E14/S7B Q&；A指南引入了“双阴性”（hERG阴性和体内QTc阴性）非临床数据的概念，可与临床1期QTc阴性数据一起使用，以替代特定病例的临床彻底QT研究（Q&As 5.1和6.1）。非临床数据将使用“最佳实践”设计生成，以支持整个行业的数据质量和一致性。对于体内QT测定，最佳实践建议需要对个体研究敏感性进行表征，验证QTc与心率的独立性，证明测试设备的敏感性，并使用阳性对照对人类进行药理学翻译。 我们之前已经在狗和非人灵长类动物中验证了E14/S7B Q&；A符合体内QT测定。这项工作的目的是在小型猪中进行验证，作为第三种非啮齿动物，通常用于心血管安全性评估。采用双拉丁方交叉研究设计（n = 8）对莫西沙星（30、120、300 mg/kg）进行阳性对照遥测研究，然后采用递增剂量设计（n = 5）。在遥测阶段抽取一个单一的PK样本，并在所有剂量水平下包括一个单独的完整的PK阶段，以进行PK/PD评估，以确定对人的转化。 观察到莫西沙星诱导的QTc增加，在双交叉和上升剂量设计中，最高剂量分别为86和67 msec时最大增加。双交叉组QTc的最小统计可检测差异（SSDD）为11 msec，上升剂量组为10 msec。根据自由血浆暴露和交叉数据中QTc效应的大小，在已知产生10 msec QTc效应的游离莫西沙星浓度的0.77倍时，观察到10 msec效应。总之，我们已经在小型猪身上验证了E14/S7B Q&；A兼容的体内QT试验，用于交叉和上升剂量设计，并显示出良好的人类翻译，完成了Labcorp™提供的E14/S7B Q&；A兼容的体内研究。

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引用次数: 0

Evaluation of laser optoporation-induced action-potential-like measurements on iPSC-cardiomyocytes and the assessment of drug-induced effects on APD and contractility 评价激光手术诱导的ipsc心肌细胞动作电位样测量及药物诱导对APD和收缩性的影响

IF 1.8 4区医学 Q4 PHARMACOLOGY & PHARMACY

Journal of pharmacological and toxicological methods

Pub Date : 2025-09-01 DOI: 10.1016/j.vascn.2025.107812

Jin Chang

Physiological relevance and accuracy in cardiac safety pharmacology studies is of the utmost importance, as supported by the recent changes in ICH guidelines and the CiPA committee's efforts. Although most of the studies focused on hERG-affecting molecules and the arrhythmic risk related to Action Potential elongation, iPSC-derived cardiomyocytes have the potential to be a more thorough assay with more physiological relevance. In this study, we demonstrate how laser optoporation technology opens transient nanopores within the cell's membrane and allows for Action Potential (AP) recordings from cardiac cells and compare the results to traditional MEA-based Field Potential Measurements. In particular, we show how the response to a potassium channel blocker (dofetilide) is comparable when looking at the APD90(>30 % increase) but that the APD30 is shortened (>10 % decrease) which we would not be able to measure with traditional MEA. The response to an Ica blocker (Nifedipine) reveals how the drug affects the depolarization currents, which can not be measured with traditional MEA. In addition, Beta-adrenergic (isoproterenol) receptor response demonstrated how the AP-amplitude remains unchanged while the contractility is increased (>30 %). We also show how the technology is non-invasive and label-free, allowing for reliable and stable measurements over 3 weeks, and how the results are comparable to previous validated data. With these results, we suggest further exploration of this technique in its use in safety pharmacology to predict cardiotoxic effects, potentially serving as an upgrade on previous technology which was limited to Field Potential Duration elongation effects.

心脏安全药理学研究的生理学相关性和准确性至关重要，最近ICH指南的变化和CiPA委员会的努力支持了这一点。虽然大多数研究都集中在影响heg的分子和与动作电位延伸相关的心律失常风险上，但ipsc衍生的心肌细胞有可能成为一种更彻底的检测方法，具有更多的生理相关性。在这项研究中，我们展示了激光光穿孔技术如何在细胞膜内打开瞬态纳米孔，并允许从心脏细胞中记录动作电位（AP），并将结果与传统的基于mea的场电位测量结果进行比较。特别是，我们展示了在观察APD90（>；30 %增加）时，对钾通道阻滞剂（多非利特）的响应是如何可比的，但APD30缩短了（>；10 %减少），这是我们无法用传统的MEA测量的。对Ica阻滞剂（硝苯地平）的反应揭示了药物如何影响传统MEA无法测量的去极化电流。此外，β -肾上腺素能（异丙肾上腺素）受体反应表明，当收缩力增加时，ap振幅保持不变（>30 %）。我们还展示了该技术如何是非侵入性和无标签的，允许在3 周内进行可靠和稳定的测量，以及结果如何与先前验证的数据相比较。根据这些结果，我们建议进一步探索该技术在安全药理学中的应用，以预测心脏毒性效应，可能作为先前仅限于场电位持续时间延长效应的技术的升级。

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