Journal of pharmacological and toxicological methods最新文献_第4页

Intravenous self-administration – Valid experimental designs to evaluate the abuse potential of psychedelics, entactogens and drugs with novel mechanisms 静脉内自我给药——评估致幻剂、致幻剂和具有新机制的药物滥用潜力的有效实验设计

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.2025.107793

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

Intravenous self-administration (IVSA) evaluates whether a CNS drug-candidate produces rewarding effects that could cause psychological dependence in patients and lead to its diversion for abuse. The model was originally developed to investigate powerful reinforcers like opiates and stimulants. For that reason, IVSA is not well adapted to detect the abuse potential of a new generation of drugs with moderate/low abuse potential. Using experience gained from conducting IVSA experiments with many types of drugs of abuse and novel drug-candidates, we offer insights on obtaining translationally predictive results from IVSA experiments, and technical refinements to increase the sensitivity and granularity of the findings. All experiments were conducted in mildly food-restricted, male, Sprague-Dawley rats with implanted with intravenous catheters. Standard IVSA tests were conducted on low fixed ratio (FR) schedules (FR3 or FR5), not FR10 as recommended in CDER/FDA guidance (CDER/FDA, 2017). As a technical refinement, the relative reinforcing effect was assessed by break-point determination on a progressive ratio (PR) schedule of reinforcement. All break-points were determined across a range of reinforcing drug doses to ensure the maximum reinforcing effect was identified. Moderate reinforcers, MDMA (entactogen), butorphanol (κ-agonist/μ-partial agonist), and (−)pentazocine (κ-agonist/μ-antagonist) produced break-points between 25 and 33 lever-presses/infusion. Weak reinforcers, WIN55,212 (CB1/CB2 agonist), diazepam, midazolam and methohexital (all GABA-A receptor positive allosteric modulators [PAMs]) produced mean break-points of 17–22 lever-presses/infusion. In contrast, powerful reinforcers like the opioids, eg heroin, remifentanil, oxycodone, and stimulants, eg cocaine, methylphenidate, supported mean break-points ranging between 41 and 98 lever‑presses/infusion. The break-point for saline (non-reinforcing control) was 10.4 ± 0.8 lever-presses/infusion. The results were used to design and conduct successful IVSA evaluations on cannabidiol (CBD), ulotaront and samidorphan on a FR3 schedule, difelikefalin, dasotraline, centanafadine, and soticlestat on a FR5 schedule, and PR/break-point determinations on samidorphan and naloxone. CNS drug development is in an era where new entactogens, psychedelics and drugs with novel mechanisms are undergoing clinical evaluation. Refinements to IVSA testing that we advocate have proven value based on experiments with known substances of abuse and novel drug-candidates.

静脉内自我给药（IVSA）评估一种中枢神经系统候选药物是否产生可能引起患者心理依赖并导致其转向滥用的奖励效应。该模型最初是为了研究阿片类药物和兴奋剂等强效强化剂而开发的。因此，IVSA不能很好地适应于检测具有中等/低滥用潜力的新一代药物的滥用潜力。 利用从对多种滥用药物和新型候选药物进行IVSA实验中获得的经验，我们提供了从IVSA实验中获得翻译预测结果的见解，以及技术改进以提高结果的敏感性和粒度。 所有实验均在轻度食物限制的雄性Sprague-Dawley大鼠中进行，并植入静脉导管。标准IVSA测试是在低固定比（FR）表（FR3或FR5）上进行的，而不是CDER/FDA指南中推荐的FR10 （CDER/FDA, 2017）。作为一项技术改进，通过 断点确定的渐进比（PR）补强计划来评估相对补强效果。 所有的断点都是在一系列强化药物剂量范围内确定的，以确保确定最大的强化效果。 中等强化剂、MDMA （entacto原）、butorphanol （κ-激动剂/μ-部分激动剂）和（−）pentazocine （κ-激动剂/μ-拮抗剂）在25 - 33次杠杆按压/输注之间产生断点。弱强化剂、win55212 （CB1/CB2激动剂）、地西泮、咪达唑仑和甲氧己ital（均为GABA-A受体阳性变构调节剂[PAMs]）产生17-22次杠杆按压/输注的平均断点。相比之下，像阿片类药物（如海洛因、瑞芬太尼、羟考酮）和兴奋剂（如可卡因、哌醋甲酯）这样的强效强化剂支持41 - 98次杠杆按压/输注之间的平均断点。生理盐水（非强化对照）的断点为10.4 ± 0.8杠杆按压/输注。结果用于设计并进行成功的IVSA评估大麻二酚（CBD）， ulotaront和samidorphan (FR3)，异花素，dasotraline， centanafadine和soticlestat (FR5)，以及samidorphan和naloxone的PR/断点测定。 中枢神经系统药物开发正处于一个新的致幻剂、致幻剂和具有新机制的药物正在进行临床评估的时代。我们提倡的IVSA测试的改进已经通过对已知滥用物质和新型候选药物的实验证明了其价值。

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引用次数: 0

Comparisons of hERG data for 30 drugs generated by automated and manual patch clamp systems by the same laboratory 同一实验室自动和手动膜片钳系统生成的30种药物的hERG数据比较

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.2025.107813

Jun Zhao, Wendy W. Wu

hERG block is the most common mechanism of drug-induced QT_C prolongation and the rare but potentially fatal arrhythmia Torsade de Pointes. Accordingly, hERG results are used to support first-in-human studies (ICH S7B), and new pathways have been developed to use hERG data generated following best practices (ICH S7B Q&A 2.1) to complement clinical QT_C studies and to inform labeling (ICH E14 Q&As 5.1 and 6.1). High-throughput, automated patch clamp systems (APC) can efficiently evaluate hERG block for new drug candidates. However, elements in APC experimental design and conduct, such as the use of non-physiological fluoride and recording temperature, have raised concerns regarding their impact on hERG pharmacology. This study compared hERG block potencies for 30 drugs collected using an APC to the same laboratory's manual patch clamp (MPC) data generated following best practices. MPC data are presented in a companion abstract by Alvarez-Baron et al. Recordings were conducted using SyncroPatch384 at 36 °C and 21 °C using fluoride-based internal solution and the same voltage protocol as the MPC. There was no systematic difference in IC₅₀s between the 36 °C APC and 37 ± 2 °C MPC data. Twenty-seven drugs (90 %) had APC IC₅₀ values within a range closely matching the MPC data. Reducing nonspecific binding by adding BSA or saturating binding sites with repeated drug applications lowered these drugs' IC₅₀s, suggesting that the largest differences are attributed to more drug loss in the APC experiments. Comparisons of the 36 °C and 21 °C APC data showed no systematic effect of temperature. Data from APC and MPC at near physiological temperature align well. Ongoing experiments are testing whether hERG data variability, determined by repeatedly obtaining IC₅₀ for the same drug, is drug-specific. The IC₅₀s variability needs to be accounted for when using the hERG safety margin to identify the likelihood of clinical QT_C prolongation. Empirically determining this using MPC is not practical. The ability to determine hERG data variability for different drugs rapidly is an advantage of APC that could lead to clearer comparisons of hERG safety margins of the investigational product and reference products.

hERG阻滞是药物诱导QTC延长和罕见但潜在致命性心律失常Torsade de Pointes的最常见机制。 因此，hERG结果用于支持首次人体研究（ICH S7B），并且已经开发出新的途径，使用最佳实践（ICH S7B Q&A 2.1）生成的hERG数据来补充临床QTC研究并告知标签（ICH E14 Q&As 5.1和6.1）。高通量、自动化膜片钳系统（APC）可以有效地评估hERG阻滞用于新药候选。然而，APC实验设计和实施中的一些因素，如使用非生理性氟化物和记录温度，引起了人们对其对hERG药理学影响的担忧。 本研究比较了使用APC收集的30种药物的hERG阻滞效力与同一实验室根据最佳实践生成的手动膜片钳（MPC）数据。MPC数据由Alvarez-Baron等人提供。使用SyncroPatch384在36 °C和21 °C的温度下进行记录，使用基于氟化物的内部溶液和与MPC相同的电压协议。 36 °C APC和37 ± 2 °C MPC数据的ic50无系统差异。27种药物（90% %）的APC IC50值在与MPC数据密切匹配的范围内。通过添加BSA或通过反复给药使结合位点饱和来减少非特异性结合降低了这些药物的ic50，这表明APC实验中最大的差异是由于更多的药物损失。36 °C和21 °C APC数据的比较显示温度没有系统影响。 在接近生理温度下APC和MPC的数据吻合良好。正在进行的实验正在测试hERG数据变异性是否具有药物特异性（通过反复获得同一药物的IC50来确定）。当使用hERG安全边际来确定临床QTC延长的可能性时，需要考虑ic50的可变性。经验性地使用MPC来确定这一点是不实际的。快速确定不同药物的hERG数据可变性的能力是APC的一个优势，可以更清楚地比较研究产品和参比产品的hERG安全边际。

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引用次数: 0

Home cage analysis in double deck rat cages for safety pharmacology: A validation study 双层大鼠笼安全药理学的家庭笼分析：一项验证研究

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.2025.107794

David Waiz , Rowland R. Sillito , Douglas Armstrong , Andrea Greiter-Wilke

Home cage analysis (HCA) technologies offer significant 3Rs benefits by measuring individual rodent behavior and physiological parameters within group housed animals. In recent years HCA approaches have been adopted in Safety Pharmacology in single deck cages. Increasingly animal facilities (and rack/cage suppliers) are providing ‘enriched’ double deck cage options for rats. We explored the feasibility of adapting HCA technology to the more complex environment in double deck cage systems and performed a single dose validation study. We adapted a previously published RFID and IR camera-based HCA system to fit a rat double deck cage. Additional RFID detectors covered the mezzanine area. Amphetamine at 0, 1, 3 and 10 mg/kg (n = 6–8 / dose) was tested as an initial reference compound in male Wistar Han rats, dosed AM (3 h into the inactive/light phase) or PM (30 min prior to entering active/dark phase) and effects measured for 24 h after both dosing events. We were able to track each individual animal on the cage base as well as along the mezzanine level including the vertical transitions between the two spatial zones. We could also measure a range of other parameters including activity, body temperature, drinking, rearing, and social proximity. Amphetamine induced dose-dependent increases in general activity, rearing, and body temperature in line with previously reported effects. Specific to the double deck cage, we observed significant increases in transitions between floor levels: a > 6-fold increase (10 mg/kg) after AM dose (next 6 h inactive/light phase), and > 4-fold (10 mg/kg) and > 2-fold (3 mg/kg) increases after PM dose (first 6 h active/dark phase). Results aligned well with data from a parallel IRWIN examination. We show that HCA methods can be adapted to handle more complex environments such as double deck cages for rats. The core parameters obtained reproduce previous published findings for amphetamine. The more complex environment allows us to capture richer behaviors including increased numbers of transitions between the upper and lower decks which all showed dose dependent responses most striking during the PM dosed animals.

家庭笼分析（HCA）技术通过测量群体饲养动物的个体行为和生理参数，提供了显著的3Rs效益。近年来，HCA方法已被应用于单层笼的安全药理学研究。越来越多的动物设施（和机架/笼子供应商）正在为老鼠提供“丰富”的双层笼子选择。我们探索了在双层笼系统中使HCA技术适应更复杂环境的可行性，并进行了单剂量验证研究。 我们改编了先前发表的基于RFID和IR摄像机的HCA系统，以适应双层鼠笼。额外的射频识别探测器覆盖了夹层区域。在雄性Wistar Han大鼠中测试了0、1、3和10 mg/kg （n = 6-8 /剂量）的安非他明作为初始参考化合物，分别给药AM（3 h进入非活性/光明期）或PM（30 min进入活性/黑暗期），并在两种给药事件发生后24 h测量效果。我们能够跟踪笼子基座上的每只动物，以及沿着夹层层，包括两个空间区域之间的垂直过渡。我们还可以测量一系列其他参数，包括活动、体温、饮酒、养育和社会亲近度。 安非他明引起的一般活动、饲养和体温的剂量依赖性增加与先前报道的效果一致。特定于双层笼子,我们观察到显著的地板之间的过渡 级别:一个 祝辞 倍增长之后(10 毫克/公斤)是剂量(下一个6 h不活跃/光阶段), 和 祝辞 4倍( 10毫克/公斤)和 祝辞 2倍(3 毫克/公斤)增加剂量后点(第一个6 h主动/黑暗阶段)。 结果与平行IRWIN检查的数据吻合良好。 我们表明，HCA方法可以适应处理更复杂的环境，如双层鼠笼。获得的核心参数重现了以前发表的安非他明的发现。更复杂的环境使我们能够捕捉到更丰富的行为，包括在上层和下层甲板之间增加的过渡数量，这些过渡在PM给药的动物中都显示出最显著的剂量依赖性反应。

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引用次数: 0

Physiologic corrections for ECG intervals beyond QT 心电图间期超过QT的生理校正

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.2025.107785

Sarah N. Freeman, Michael J. Murray-Busher, Alexa M. Spencer, Kenneth R. Kearney

Quantitative assessment of ECG intervals informs investigators of drug activation within the heart. A limitation to understanding a test article's influence on specific ion channels can be due to extreme alterations in physiologic parameters, namely heart rate (HR) and body temperature (BT). While it's well understood that ECG intervals exhibit an expected inverse relationship to HR, it may be difficult to determine if changes in ECG intervals are disproportionately shortened or prolonged at extreme HRs. As QT prolongation is a known biomarker for increased Torsadogenic potential, in alignment with ICHS7B, HR correction formulae are employed to correct for QT interval duration. However, no direct guidance directs the correction of alternate intervals. Due to this, investigators are left to correlative comparisons between magnitude of change in HR and magnitude of change in the PR interval. While greater risks are known to be associated with ventricular arrhythmias, disproportionate shortening or prolongation of the PR interval may represent AV node dissociation, which could lead to AV Nodal reentry and subsequent potential of more significant ventricular arrhythmias. Recent studies have also suggested increased risk of atrial arrhythmias, heart failure, and mortality in patients with PR prolongation. In addition to direct HR influence, associations between BT and QT duration have been characterized in the dog (Van der Linde, 2008), but have not been completely profiled in the non-human primate (NHP). To address these gaps in physiologic corrections, an investigation of historical data was performed to determine if corrections were possible. Internal review (~60 animals/species – Beagle Dogs, NHPs) was utilized to establish the relationships between PR vs RR and QT vs BT intervals. Through this investigation, a species-specific correction for BT was established for NHP and exhibited equitable concordance to an individual animal correction. Therefore, this correction was believed to be an adequate precursory correction formula to adopt during acquisition to monitor for QT/BT. Contrarily, population-based corrections for PR vs RR exhibited variance, and individual study-based corrections were found to be most applicable. It is believed that employment of these additional physiological corrections may better profile the associated risks of new chemical entities.

定量评估心电图间隔告知研究者心脏内的药物激活。由于生理参数，即心率（HR）和体温（BT）的极端变化，对测试品对特定离子通道的影响的理解受到限制。虽然心电间隔与心率呈反比关系是众所周知的，但很难确定在极端心率下，心电间隔的变化是否不成比例地缩短或延长。由于QT间期延长是一种已知的生物标记物，表明扭转性电位增加，因此与ICHS7B一致，采用HR校正公式来校正QT间期持续时间。然而，没有直接的指导指导交替间隔的校正。因此，研究者只能对HR变化幅度和PR区间变化幅度进行相关比较。虽然已知更大的风险与室性心律失常有关，但PR间隔的不成比例缩短或延长可能代表房室结分离，这可能导致房室结再入和随后更严重的室性心律失常的潜在危险。最近的研究也表明，PR延长患者发生房性心律失常、心力衰竭和死亡率的风险增加。除了HR的直接影响外，BT和QT持续时间之间的关系已经在狗身上得到了表征（Van der Linde, 2008），但在非人灵长类动物（NHP）中还没有得到完全的描述。为了解决生理校正中的这些空白，对历史数据进行了调查，以确定是否有可能进行校正。利用内部回顾（约60只动物/物种-比格犬，NHPs）来建立PR / RR和QT / BT间期之间的关系。通过这项调查，建立了针对NHP的BT的物种特异性校正，并显示出与单个动物校正的公平一致性。因此，这种校正被认为是一种适当的先兆校正公式，可在采集期间用于监测QT/BT。相反，基于人群的PR和RR校正显示出差异，而基于个体研究的校正被发现是最适用的。据信，使用这些额外的生理校正可以更好地描述新化学实体的相关风险。

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引用次数: 0

Off-target of a novel TYK2 inhibitor is associated with myocardial necrosis mediated by hemodynamic changes in rats 一种新型TYK2抑制剂的脱靶与大鼠血流动力学改变介导的心肌坏死有关

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.2025.107834

Rebecca Kohnken, Stacey Fossey, Wayne R. Buck, Jason Segreti, Jessica Treadway, Jonathon Green, Yevgeniya E. Koshman, Mark Zafiratos, Scott Mittelstadt, Eric Blomme, Charles Michael Foley

After an observation of myocardial necrosis and inflammation in rats administered an experimental TYK2 inhibitor (ABBV-712), investigative studies were performed to identify the potential mechanism. Telemetry instrumented rats were administered ABBV-712 with or without atenolol to assess hemodynamic changes and cardiac pathology. In vitro studies included cytotoxicity evaluation in human induced pluripotent stem cell-derived cardiomyocytes and assessment of relaxation of an isolated rat aorta model. Off-target pharmacology was evaluated by binding and inhibition screening assays. Finally, TYK2 knockout (KO) mice were instrumented with telemetry to determine hemodynamic changes as compared to wildtype animals following administration of ABBV-712. In these studies in rats, ABBV-712 resulted in decreased mean arterial pressure and increased heart rate that was prevented by pre-dosing atenolol. ABBV-712-induced myocardial pathology was also prevented by atenolol, consistent with a mechanistic link between hemodynamic changes and cardiac injury. The myocardial necrosis observed was determined to be unrelated to direct cytotoxicity on cardiomyocytes as demonstrated in vitro. Further, a compound-related effect on vascular relaxation mediated by endothelial cells was found in the aortic ring model. Overall the toxicity was considered an off-target effect, as suggested by similar hemodynamic responses between TYK2 KO and wildtype mice administered ABBV-712, as well as by the lack of hemodynamic changes in the KO mouse at baseline. Inhibition of the off-targets that were identified in the screening effort were considered unlikely to be the cause of the hemodynamic changes based on canonical pharmacology. In this study, a novel TYK2 small molecule inhibitor resulted in decreased mean arterial pressure, increased heart rate, and secondary myocardial necrosis in rats. These cardiovascular effects were unrelated to TYK2 inhibition. This report is an example of a cross-functional mechanistic investigation into the pharmacologic cause of an observation of cardiovascular toxicity.

在观察实验性TYK2抑制剂（ABBV-712）对大鼠心肌坏死和炎症的影响后，进行了调查研究，以确定其潜在的机制。 遥测仪器大鼠给予ABBV-712加或不加阿替洛尔，以评估血液动力学变化和心脏病理。体外研究包括对人诱导多能干细胞衍生的心肌细胞的细胞毒性评估和对离体大鼠主动脉模型松弛的评估。通过结合和抑制筛选试验评价脱靶药理学。最后，用遥测法测定TYK2敲除（KO）小鼠在给药ABBV-712后与野生型小鼠相比的血流动力学变化。在这些大鼠研究中， ABBV-712导致平均动脉压降低和心率增加，这是通过预先给药阿替洛尔预防的。阿替洛尔也能预防abbv -712诱导的心肌病理，这与血流动力学改变和心脏损伤之间的机制联系一致。经体外实验证实，心肌坏死与心肌细胞的直接细胞毒性无关。此外，在主动脉环模型中发现了内皮细胞介导的血管舒张的化合物相关作用。总的来说，毒性被认为是脱靶效应，这表明TYK2 KO和给予ABBV-712的野生型小鼠之间的血流动力学反应相似，并且在基线时KO小鼠缺乏血流动力学变化。在筛选过程中发现的脱靶抑制被认为不太可能是基于标准药理学的血流动力学改变的原因。在这项研究中， 新型TYK2小分子抑制剂导致大鼠平均动脉压降低，心率增加和继发性心肌坏死。这些心血管效应与TYK2抑制无关。 本报告是对心血管毒性观察的药理学原因进行跨功能机制调查的一个例子。

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引用次数: 0

Automated patch-clamp for high throughput characterization of subtype-specific induced pluripotent stem cell derived cardiomyocytes 用于高通量表征亚型特异性诱导多能干细胞衍生心肌细胞的自动膜片钳

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.2025.107824

Sonja Stoelzle-Feix , Fitzwilliam Seibertz , Marcus Lluis Gerloff , Markus Rapedius , Aiste Liutkute , Lukas Cyganek , Claudia Haarmann , Michael George , Niels Voigt , Niels Fertig

Atrial fibrillation (AF) represents the most prevalent cardiac arrhythmia observed in clinical practice. The efficacy of therapeutic interventions for AF remains suboptimal, and the exploration of ion channel remodeling mechanisms in AF is restricted by both the lack of atrial specificity in standard cell lines and the low throughput of ‘gold standard’ methodologies. Significant progress in the development of high throughput automated patch-clamp (APC) systems could be extremely beneficial for accelerating electrical investigations into the mechanisms of atrial-specific disorders such as AF. Human induced pluripotent stem cells (iPSC) were derived from dermal fibroblasts of a healthy male donor. Factor-free cardiac differentiation into iPSC derived cardiomyocytes (iPSC-CM) was achieved via small molecule temporal modulation of wnt signaling. Atrial specificity was induced through Retinoic acid (1 μM) application between day 3 and 6 of cardiac cell differentiation as has been previously shown (Seibertz et al., 2023). We describe the application of a high throughput APC device (SyncroPatch 384) to characterize key ionic currents and action potentials (AP) in human atrial- and ventricular-specific iPSC-CM. Thin borosilicate glass 384-well planar chips (1xS-type NPC-384 T) were used for all experiments. Recordings were excluded if they showed a seal resistance of <250 MΩ or a peak current of <50 pA. All measurements were carried out at room temperature. Robust current-clamp measurements of cardiac action potential revealed subtype-specific characteristics with shorter action potentials recorded in atrial iPSC-CM. High throughput voltage-clamp measurements of sodium current (INa), L-type calcium current (ICa,L), the rapid component of the delayed rectifier (IKr), and basal inward rectifier potassium current (IK1) were able to be measured in both cell types. In addition, activation of the atrial-specific acetylcholine-activated inward rectifier potassium current (IK,ACh) was exclusively observed in atrial but not in ventricular iPSC-CM following application of the M-receptor agonist carbachol. The successful application of a high throughput APC-system for the recording and characterization of atrial and ventricular APs and ionic currents in highly scalable iPSC-CM models implies that APC represents a powerful tool for future studies of atrial-specific pathologies such as AF.

心房颤动（AF）是临床观察到的最常见的心律失常。房颤治疗干预的疗效仍然不理想，房颤离子通道重构机制的探索受到标准细胞系缺乏心房特异性和“金标准”方法低通量的限制。高流量自动膜片钳（APC）系统的发展取得了重大进展，对于加速心房特异性疾病（如房颤）机制的电调查非常有益。 人类诱导多能干细胞（iPSC） 来源于健康男性供体的真皮成纤维细胞。 无因子心脏分化为iPSC来源的心肌细胞（iPSC- cm）是通过wnt信号的小分子时间调节实现的。 心房特异性是通过维甲酸（1 μM）在心脏细胞分化的第3天至第6天之间诱导的，如先前所示 （Seibertz et al., 2023）。 我们描述了高通量APC设备（SyncroPatch 384）的应用，以表征人类心房和心室特异性iPSC-CM中的关键离子电流和动作电位（AP）。 所有实验均采用薄硼硅玻璃384孔平面芯片（1xs型NPC-384 T）。如果记录显示密封电阻为<；250 MΩ 或 ，峰值电流为<；50 pA，则排除记录。所有测量均在室温下进行。 稳健的电流钳测量心脏动作电位揭示了亚型特异性特征，心房iPSC-CM记录的动作电位较短。高通量电压箝位测量钠电流（INa）， L型钙电流（ICa，L），延迟整流器的快速分量（IKr）和基底内向整流器钾电流（IK1）都能够在两种电池类型中测量。此外，应用m受体激动剂carbachol后，心房特异性乙酰胆碱激活的内向整流钾电流（IK,ACh）仅在心房观察到激活，而在心室iPSC-CM中没有观察到。 高通量APC系统在高度可扩展iPSC-CM模型中用于记录和表征心房和心室ap和离子电流的成功应用意味着APC代表了未来心房特异性病理（如房颤）研究的强大工具。

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引用次数: 0

Evaluation of the J-Tpeak and Tpeak-to-tend intervals of the ECG as proarrhythmia biomarkers in telemetered dogs 遥测犬心电图J-Tpeak和Tpeak-to-tend间隔作为心律失常前生物标志物的评价

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.2025.107772

Bétat Anne-Marie , Delaunois Annie , Loiseau Mathilde , Poizat Gwendoline , Maurin Anne , Drieu la Rochelle Christophe , Martel Eric , Valentin Jean-Pierre , Delpy Eric

Since their implementation, the ICH S7B and E14 guidelines have been successful in that no new approved drugs have been withdrawn from the market due to unanticipated risk of Torsade de Pointe (TdP). While hERG block and QTc prolongation biomarkers are indeed sensitive, they are not specific as multiple drugs block hERG and/or prolong QTc but do not cause TdP. The J-Tpeak interval (JTp) of the ECG has recently been proposed as a novel clinical biomarker to differentiate selective hERG blockers from multi cardiac ion channels inhibitors. Therefore, the present study aimed at evaluating the effects of dofetilide (pure hERG blocker) and dolasetron (balanced ion-channel blocking drug) on QT, JTp and Tpeak-to-Tend (TpTe) Intervals in telemetered conscious dogs, in comparison with levocetirizine, used as a QT negative control drug. The experiments were carried out using 7 dogs previously instrumented with a telemetry implant. Using a cross-over design, each drug was administered at two doses (0.03/0.15, 6/20, 2/10 mg/kg p.o. for dofetilide, dolasetron and levocetirizine, respectively) and ECG monitored over 24 h post-dosing. Dedicated PK sessions were performed to measure corresponding plasma exposure. QT, JTp and TpTe intervals were individually corrected (c) for heart rate variations. Total plasma drug concentrations increased dose-proportionally. Compared to vehicle, dofetilide induced dose-dependent, marked, and long-lasting increases in both QTc and JTpc intervals, confirming predominant hERG blockage by dofetilide. However, no effect was observed on TpTec interval. In contrast, QTc prolongation associated with dolasetron was moderate and transient and no effect on JTpc was observed; interestingly, dolasetron slightly increased TpTec and QRS intervals, as well as PR interval, reflecting its inhibitory effect on calcium and sodium currents. As expected, levocetirizine had no effect, at any dose or timepoint post-dosing. To summarize, pure hERG-blocking drug (such as dofetilide) prolonged both QTc and JTpc, whereas drug with mixed ion channel blockage (such as dolasetron) prolonged QTc but not JTpc. In conclusion, evaluating the effects of a drug on the J-Tpeak interval may be relevant in cardiovascular safety pharmacology studies to complement the QT interval as a sensitive and more specific biomarker for proarrhythmic risk.

自实施以来，ICH S7B和E14指南取得了成功，因为没有新批准的药物由于未预料到的TdP风险而退出市场。虽然hERG阻断和QTc延长的生物标志物确实是敏感的，但它们不是特异性的，因为多种药物阻断hERG和/或延长QTc，但不会引起TdP。最近，心电图的J-Tpeak interval （JTp）被提出作为一种新的临床生物标志物，用于区分选择性hERG阻滞剂和多种心脏离子通道抑制剂。因此，本研究旨在评估多非利特（纯hERG阻滞剂）和多拉司琼（平衡离子通道阻断药物）对遥测清醒犬QT、JTp和ttp -to- tend （TpTe）间隔的影响，并与作为QT阴性对照药物的左西替利嗪进行比较。实验是在7只狗身上进行的，这些狗之前被植入了遥测植入物。采用交叉设计，每种药物分别以两种剂量给药（分别为0.03/0.15、6/20、2/10 mg/kg p.o.，多非利特、多拉司琼和左西替利嗪），并在给药后24 小时监测心电图。进行了专门的PK会话来测量相应的血浆暴露。QT、JTp和TpTe间期分别校正(c)心率变化。血浆总药物浓度按剂量比例增加。与对照药相比，多非利特诱导QTc和JTpc时间间隔的剂量依赖性、显著性和持续性增加，证实了多非利特对hERG的主要阻断作用。然而，对TpTec间隔没有影响。相比之下，dolasetron相关的QTc延长是中度和短暂的，对JTpc没有影响；有趣的是，多拉司琼略微增加了TpTec和QRS间期，以及PR间期，这反映了它对钙和钠电流的抑制作用。正如预期的那样，左西替利嗪在给药后的任何剂量或时间点都没有效果。综上所述，纯heg阻断药物（如多非利特）可延长QTc和JTpc，而混合离子通道阻断药物（如多拉司琼）可延长QTc，但不能延长JTpc。总之，评估药物对J-Tpeak间期的影响可能与心血管安全药理学研究相关，以补充QT间期作为心律失常风险的敏感和更特异性的生物标志物。

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引用次数: 0

The translation of hERG block and APD90 in vitro to clinical QTc prolongation in man 体外hERG阻滞和APD90对人类临床QTc延长的翻译

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.2025.107804

Derek J. Leishman , Najah Abi-Gerges

The core ICH S7B in vitro assay is a hERG assessment which has associated best practice recommendations. Three reference agents are being used to compare margins based on concentrations associated with 10 ms QTc prolongation in man. The objective of the current analyses was to compare the margins for best practice hERG patch-clamp data with those for other in vitro assessments. The unbound critical concentrations shared as part of the Training Materials were used as the denominator throughout. The numerators were: the hERG IC₅₀ generated in a study conforming to current best practice guidance, the pKi from published dofetilide-binding data, the concentrations associated with either published rate corrected APD₉₀ prolongation in isolated IPD-derived human cardiomyocytes, or with a 25 ms change in APD₉₀ in human ventricular trabeculae tissue. The respective margins for dofetilide, moxifloxacin and ondansetron in the patch-clamp study were 45, 32 and 4.2. The dofetilide-binding displacement pKi, gave margins of 11, 15 and 10. In isolated cardiomyocytes the margins were 0.5, 5.2 and 1.3. In human trabeculae tissue the margins were 14, 4.5 and 9. There is an underlying assumption that for selective hERG blocking drugs the amount of hERG block (and associated hERG margin) would be similar for the same 10 ms change in QTc. The consistent margin determined using the dofetilide-binding displacement pKi and the concentration associated with a 25 ms APD90 prolongation in human trabeculae would tend to support that assessment. There was more variability in isolated cardiomyocytes where there was an 11-fold difference between dofetilide and moxifloxacin margins while the ondansetron margin lay in between these margins. The hERG patch-clamp study had skewed variability with a margin for ondansetron 11-fold and 8-fold different from the margins for dofetilide and moxifloxacin, respectively. The small margin for ondansetron caused wider confidence intervals for the pooled margin of 18-fold. Overall, these data suggest that any of these assays might realistically be used to predict a QTc prolongation in man. The hERG patch-clamp study is the required study. Where there are ‘ondansetron-like’ blocking characteristics an additional assay may be warranted to clarify the prediction.

核心ICH S7B体外检测是hERG评估，具有相关的最佳实践建议。目前正在使用三种参考药物来比较与人类10 ms QTc延长相关的浓度。当前分析的目的是比较最佳实践hERG膜片钳数据与其他体外评估数据的差额。作为培训材料的一部分，未结合的临界浓度被用作分母。分子是：在符合当前最佳实践指南的研究中产生的hERG IC50，来自已发表的dofetilide结合数据的pKi，与分离的ipd衍生的人心肌细胞中已发表的率校正APD90延长相关的浓度，或与人心室小梁组织中APD90 25 ms变化相关的浓度。膜片钳研究中，多非利特、莫西沙星和昂丹司琼的边际值分别为45、32和4.2。多肽结合位移pKi，给出了11、15和10的边界。离体心肌细胞的边缘分别为0.5、5.2和1.3。人小梁组织的边缘分别为14、4.5和9。有一个潜在的假设，对于选择性hERG阻断药物，对于相同的10 ms QTc变化，hERG阻断量（和相关的hERG边缘）将是相似的。使用多肽结合位移pKi和与人类小梁中25 ms APD90延长相关的浓度确定的一致边际将倾向于支持该评估。在分离的心肌细胞中有更多的可变性，在多非利特和莫西沙星边缘之间有11倍的差异，而昂丹司琼边缘位于这些边缘之间。hERG膜片钳研究存在偏斜变异性，昂丹司琼与多非利特和莫西沙星的差异分别为11倍和8倍。昂丹司琼的边际较小，使得合并边际的置信区间更宽，达到18倍。总的来说，这些数据表明，这些检测中的任何一种都可能实际地用于预测人类QTc的延长。hERG膜片钳研究是必要的研究。当存在“昂丹司琼样”阻断特性时，可能需要额外的检测来澄清预测。

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引用次数: 0

The translation of QTc across species - Impact of subject number and exposure multiple tested on discriminatory sensitivity QTc的跨物种翻译——受试者数量和暴露倍数对区分敏感性的影响

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.2025.107774

Derek J. Leishman

Small, relatively insensitive studies can be useful in safety assessment when multiples of the therapeutic clinical concentration are tested. This is a fundamental principle in safety testing in animals which is equally valid for early clinical evaluations in healthy volunteers. It is often less practical to increase the number of test subjects than to increase the exposure tested. Both, when combined with the analysis method, can have an impact on the sensitivity to detect an effect. The objective is that the relationship between statistical power, analysis method, number of animals and exposure multiple explored can be illustrated using the example of QTc assessment in animals. The statistical power to detect an effect on the electrocardiogram QTc interval in nonhuman primates (NHP) for different analyses methods was known. The concentration-QTc relationship was also known for reference agents in NHP. Lastly, the critical concentration associated with a 10 ms QTc interval change in man was known for these same reference agents. This information was combined to illustrate how doubling the number of NHP used or increasing the exposure tested would support a conclusion concerning the presence or absence of an effect on the QTc interval for a test agent. In NHP, the most sensitive analysis methods have >80 % power (at p < 0.05) to detect an effect of the reference agent at the critical concentration using only 4 animals. Less sensitive techniques can detect an effect with the same power when either more animals are used or where higher multiples of the critical concentration are tested. This illustrates the principle that even with only 4 animals and an insensitive technique an effect can be detected provided higher exposures are tested. Conversely, a study using more animals, or a more sensitive analysis needn't require a higher exposure in animals to exclude an effect in man. Rather than focusing on a fixed QTc threshold sensitivity regardless of experimental design these analyses demonstrate that investigators have the flexibility to use the simplest available combination of exposures, animal numbers and analysis to achieve an effective QTc assessment.

小规模的、相对不敏感的研究可以用于安全性评估，当测试治疗性临床浓度的倍数时。这是动物安全试验的基本原则，同样适用于健康志愿者的早期临床评估。增加测试对象的数量往往比增加被测试的暴露程度更不实际。当两者与分析方法相结合时，会对检测效果的灵敏度产生影响。目的是通过动物QTc评估的例子来说明统计功效、分析方法、动物数量和所探讨的暴露倍数之间的关系。已知不同分析方法对非人灵长类动物（NHP）心电图QTc间期影响的统计能力。浓度- qtc关系在NHP的参考药物中也是已知的。最后，已知这些相同的参考药物与人类10 ms QTc间隔变化相关的临界浓度。将这些信息结合起来说明，将使用的NHP的数量加倍或增加所测试的暴露量将如何支持有关对测试剂的QTc间隔是否存在影响的结论。在NHP中，仅使用4只动物，最敏感的分析方法在检测临界浓度下对照剂的影响时具有>；80 %的功率（p <； 0.05）。当使用更多的动物或在测试临界浓度的更高倍数时，灵敏度较低的技术可以以相同的功率检测到效果。这说明了即使只有4只动物和一种不敏感的技术，只要测试更高的暴露量，也可以检测到效果的原理。相反，一项使用更多动物的研究，或者一项更敏感的分析，不需要在动物中使用更高的剂量来排除对人类的影响。这些分析表明，研究人员可以灵活地使用暴露、动物数量和分析的最简单的可用组合来实现有效的QTc评估，而不是将重点放在固定的QTc阈值灵敏度上。

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引用次数: 0

Comprehensive protocol for culturing and functionally characterizing primary mixed neural cells from the neonatal rat cortex 从新生大鼠皮层中培养和功能表征初级混合神经细胞的综合方案。

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.2025.108390

Thaynan Silva Ramos , Matheus Heidemann Tempone , Hercules Rezende Freitas , Monique Fonseca-Teixeira , Patricia Fernanda Schuck , Ricardo Augusto de Melo Reis , Gustavo Costa Ferreira

In vitro models using purified neurons or glial cells are crucial for studying neurological functions but often overlook intercellular interactions. Mixed neural cell cultures offer a more physiologically relevant system by preserving cell-to-cell communication and providing deeper insights into neural behavior. Here, we present a protocol for culturing mixed primary cells from the neonatal rat cerebral cortex and functionally characterizing them via calcium imaging. This method enables cell phenotyping, spatial distribution analysis, and activity monitoring in response to stimuli. Our model maintained a cellular composition resembling the native rat cortex, with 35.4 % neurons, 44.3 % astrocytes, and 20.3 % other cell types. Calcium imaging showed that ATP (100 μM) and BzATP (100 μM) evoked stronger calcium transients than KCl (50 mM). BzATP induced a sustained response mediated by P2X7 receptor activation, while ATP activated a broader range of P2 receptors. Unlike purified or enriched cultures, this mixed-cell system better replicates the cellular environment of the brain, ensuring reproducibility and biological relevance. This protocol provides a straightforward platform for investigating neuron-glia interactions and neural signaling, bridging the gap between simplified in vitro models and the complexity of neural networks. Its applications may advance research into neurobiological disease mechanisms and therapeutic development.

使用纯化的神经元或神经胶质细胞的体外模型对于研究神经功能至关重要，但往往忽略了细胞间的相互作用。混合神经细胞培养通过保持细胞间的通信和对神经行为的更深入的了解，提供了一个更生理相关的系统。在这里，我们提出了一种从新生大鼠大脑皮层培养混合原代细胞的方案，并通过钙成像对它们进行功能表征。这种方法使细胞表型，空间分布分析和活动监测响应刺激。我们的模型保持了与天然大鼠皮层相似的细胞组成，神经元占35.4% %，星形胶质细胞占44.3% %，其他细胞占20.3% %。钙成像显示ATP（100 μM）和BzATP（100 μM）比KCl（50 mM）诱发更强的钙瞬变。BzATP诱导了P2X7受体激活介导的持续反应，而ATP激活了更广泛的P2受体。与纯化或富集培养不同，这种混合细胞系统更好地复制了大脑的细胞环境，确保了可重复性和生物学相关性。该协议为研究神经元-胶质细胞相互作用和神经信号提供了一个简单的平台，弥合了简化的体外模型和神经网络复杂性之间的差距。它的应用可能会推动神经生物学疾病机制和治疗发展的研究。

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