Journal of pharmacological and toxicological methods最新文献_第10页

A retrospective review of the effects of moxifloxacin in NHP at four different test sites 莫西沙星在四个不同试验点对NHP的影响的回顾性回顾

IF 1.8 4区医学 Q4 PHARMACOLOGY & PHARMACY

Journal of pharmacological and toxicological methods

Pub Date : 2025-09-01 Epub Date: 2025-09-19 DOI: 10.1016/j.vascn.2025.107780

Kevin Norton , Steve Tichenor , Abdel El Amrani , Jared Slain , Nacera Mella , Steve Denham , Matt St Peter , Jill Dalton

With adoption of the ICH E14/S7B Q&A best practice guidance, nonclinical CV data sets can be used in place of clinical data to support of a TQT waiver application. However, as part of this guidance each laboratory is required to demonstrate adequate sensitivity of their specific in vivo model using a known positive control agent (e.g., moxifloxacin) or through retrospective review of historical data sets. These requirements raise questions around how often a positive control study should be conducted and how stable the effects of moxifloxacin are across multiple laboratories. In the current review, we assessed the effects of moxifloxacin in non-human primates (NHP) from four test sites. All four laboratories administered moxifloxacin at 10, 80 and 175 mg/kg by oral administration and evaluated cardiovascular parameters for 24 h post administration. The study designs between labs were highly comparable with each site using a Williams Latin square 4*4 crossover design in male animals, with the exception of site 1 which used female animals. Concentration-QTc modeling was also conducted at all sites with minor site-to-site variances in the blood sample collection methods and timing observed. All sites illustrated dose dependent increases in QTc prolongation. Administration at 175 mg/kg resulted in QTc prolongation of up to 21.9, 25.3, 16.9 and 45.1 msec, relative to control, at test sites 1, 2, 3 and 4 respectively. Whilst a review of study specific least significant difference, ranged from 8.4 to 10.2 msec, and residual error ranged from 4.8 to 5.9 msec; with conc-QT modeling, indicating that concentration ranges of 2444–3938 ng/ml would be expected to induce a 10-msec prolongation. These results indicate that when following best practice methodologies, NHP CV models are highly consistent, independent of laboratory. This suggests that repeat conductance across of positive control studies, across multiple labs is not required and reliance on historical data sets alone should suffice to confirm model suitability.

随着ICH E14/S7B Q&；A最佳实践指南的采用，非临床CV数据集可以代替临床数据来支持TQT豁免申请。然而，作为本指南的一部分，要求每个实验室使用已知的阳性对照剂（例如莫西沙星）或通过对历史数据集的回顾性审查来证明其特定体内模型的足够敏感性。这些要求提出了以下问题：应多久进行一次阳性对照研究，以及莫西沙星在多个实验室中的作用有多稳定。在本综述中，我们从四个试验点评估了莫西沙星对非人灵长类动物（NHP）的影响。所有四个实验室均口服莫西沙星，剂量分别为10、80和175 mg/kg，并评估给药后24 h的心血管参数。实验室之间的研究设计具有高度可比性，每个站点在雄性动物中使用Williams Latin square 4*4交叉设计，但站点1使用雌性动物。还在所有站点进行了浓度- qtc建模，观察到血液样本采集方法和时间的站点间差异较小。所有位点均显示QTc延长的剂量依赖性增加。在试验1、2、3和4位点，175 mg/kg剂量的QTc与对照相比分别延长了21.9、25.3、16.9和45.1 毫秒。而对研究特定的最不显著差异的回顾，范围从8.4到10.2 msec，残差范围从4.8到5.9 msec；通过conc-QT模型，表明2444-3938 ng/ml的浓度范围有望诱导10毫秒的延长。这些结果表明，当遵循最佳实践方法时，NHP CV模型是高度一致的，独立于实验室。这表明，在阳性对照研究中，不需要在多个实验室中重复电导，仅依赖历史数据集就足以确认模型的适用性。

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引用次数: 0

An iPSC-derived neurotoxicity screening platform for HIV Associated Neurocognitive Disorder (HAND) demonstrates compound toxicity on neural progenitor cells and mature neurons, astrocytes, and microglia ipsc衍生的HIV相关神经认知障碍（HAND）的神经毒性筛选平台显示了对神经祖细胞和成熟神经元、星形胶质细胞和小胶质细胞的复合毒性

IF 1.8 4区医学 Q4 PHARMACOLOGY & PHARMACY

Journal of pharmacological and toxicological methods

Pub Date : 2025-09-01 Epub Date: 2025-09-19 DOI: 10.1016/j.vascn.2025.107843

Kara L. Gordon, Christine G. Rines, Nicole A. Suarez, Alyson Smith, Jeff Price, Patrick McDonough

Individuals living with HIV, including pregnant women, receive combination antiretroviral therapy (cART) indefinitely to maintain their health and to prevent perinatal HIV transmission. Clinical studies and preclinical research suggest that cART may affect fetal brain development, and impair adult hippocampal neurogenesis, thus contributing to HIV-Associated Neurocognitive Disorder (HAND) and related neuroafflictions (NeuroHIV). Therefore, it is important to develop predictive assays for assessing the safety of cART. Here, we used cells derived from human induced pluripotent stem cells (hiPSCs) to develop and validate platforms for anti-retroviral (ARV) toxicity testing. The platforms include neural progenitor cells (NPCs) and cultures of mature neurons alone, and neurons in culture with astrocytes and microglia. After treatment with HIV anti-retroviral drugs (ARV), we assessed outcomes such as cell viability, proliferation, neurite length and synapse number, and calcium imaging. We developed assays for NPCs and mature neuron cultures in 384-well plates. In all assays, nuclei were stained with a nuclear dye for single cell identification and viability measurements. Cell proliferation was measured using Click-iT EdU kits and fluorescent calcium dyes were used for imaging calcium transients. Fixed endpoint labeling was performed with antibodies detecting neurites, pre- and post-synaptic markers for neurite measurements and synapse count. High speed imaging and fixed sample imaging was performed on Vala Science's Kinetic Image Cytometer (KIC IC200) and single cell analysis was performed on Vala's CyteSeer Image Analysis software. We observed a decrease in NPC viability and cell proliferation after treatment with the ARVs Elvitegravir (EVG) and Dolutegravir (DTG). These two ARVs also reduced viability, neurite length, and synapse count in mature neuron monocultures. In tri-cultures, we observed toxicity with EVG, with reduced neuronal firing and diminished spike amplitudes. In conclusion, we demonstrated that Elvitegravir and Dolutegravir reduce viability of NPC's and neurons, as well as affect neuron function. The toxic effects of the treatments may have implications for HAND and NeuroHIV. We are also expanding this platform to address HIV infection/replication of microglia within the cultures, so that both safety and efficacy of future ARV compounds can be assessed.

艾滋病毒感染者，包括孕妇，无限期地接受抗逆转录病毒联合治疗，以保持健康和预防围产期艾滋病毒传播。临床研究和临床前研究表明，cART可能影响胎儿大脑发育，损害成人海马神经发生，从而导致hiv相关神经认知障碍（HAND）和相关神经疾病（NeuroHIV）。因此，开发预测方法来评估cART的安全性是很重要的。在这里，我们使用来自人类诱导多能干细胞（hiPSCs）的细胞来开发和验证抗逆转录病毒（ARV）毒性测试的平台。该平台包括神经祖细胞（npc）和单独成熟神经元的培养物，以及星形胶质细胞和小胶质细胞培养的神经元。在接受HIV抗逆转录病毒药物（ARV）治疗后，我们评估了诸如细胞活力、增殖、神经突长度和突触数量以及钙成像等结果。我们开发了384孔板中NPCs和成熟神经元培养的检测方法。在所有的实验中，细胞核都用核染料染色，用于单细胞鉴定和活力测定。使用Click-iT EdU试剂盒检测细胞增殖，使用荧光钙染料成像钙瞬态。固定终点标记使用抗体检测神经突，突触前和突触后标记用于神经突测量和突触计数。在Vala Science的Kinetic Image Cytometer （KIC IC200）上进行高速成像和固定样本成像，在Vala的CyteSeer图像分析软件上进行单细胞分析。我们观察到使用抗逆转录病毒药物Elvitegravir （EVG）和Dolutegravir （DTG）治疗后鼻咽癌存活率和细胞增殖下降。这两种抗逆转录病毒药物也降低了成熟神经元单培养的活力、神经突长度和突触计数。在三种培养中，我们观察到EVG的毒性，神经元放电减少，峰值振幅减弱。总之，我们证明Elvitegravir和Dolutegravir降低了NPC和神经元的活力，并影响神经元功能。这些治疗的毒性作用可能对HAND和NeuroHIV有影响。我们还在扩大这一平台，以解决培养物中小胶质细胞的HIV感染/复制问题，以便评估未来ARV化合物的安全性和有效性。

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引用次数: 0

Exploratory continuous interstitial glucose monitoring using the FreeStyle Libre 2 device in cynomolgus monkeys 使用FreeStyle Libre 2设备对食蟹猴间质葡萄糖进行探索性连续监测

IF 1.8 4区医学 Q4 PHARMACOLOGY & PHARMACY

Journal of pharmacological and toxicological methods

Pub Date : 2025-09-01 Epub Date: 2025-09-19 DOI: 10.1016/j.vascn.2025.107776

Julia Popp , Cassandra O'Hara , J.E. Bernal , Jackson Kalanzi , Mariusz Lubomirski , Dina Andrews-Cleavenger , J. Karmel , K.A. Henderson

Glucose is an important endpoint in efficacy and toxicology assessments for cardiometabolic indications. Collection of serial glucose values can inform treatment-related effects and animal health status. Handling stress can cause heart rate (HR) and glucose spikes in nonhuman primates (NHPs), increasing variability and confounding data interpretation. Continuous glucose monitors (CGMs) are sensor-based systems that provide interstitial glucose (IG) readings without venipuncture; thus reducing handling stress and resultant glucose spikes. The purpose of this study was to evaluate the Freestyle®Libre™2 CGM (FL2-CGMs, Abbott) with NHPs for multiple days with simultaneous continuous HR monitoring and collection of serum to determine glucose and insulin levels at key timepoints. FL2-CGMs were pericutaneously applied to the back of jacketed NHPs for IG measurements. FL2-CGMs retain up to 8 h of data in 15 min increments. IG data were collected at 10 h intervals for 72 h after each dose administration. During phase 1, jacketed external telemetry was used for assessment of concurrent HR over 24 h. Animals were administered saline, oral glucose (1 g/kg) or injections of 0.15 U/kg insulin. Serum glucose and insulin were measured to compare with IG data predose, 8, and 24 h postdose. In phase 2, animals were administered 3 g/kg glucose; IG was monitored over 72 h with repeated serum sampling of glucose and insulin within the first 8 h of data collection to compare magnitude and timing of changes between the two glucose collection methods. Higher IG levels (up to 72 mg/dL above predose levels) were observed with FL2-CGMs in response to 3 g/kg glucose, while changes in IG levels in response to 1 g/kg glucose were not reliably detected outside of observed IG variability. FL2-CGM captured lower glucose levels (12 mg/dL below controls) after administration of 0.15 U/kg insulin. HR spikes due to handling were concurrent with increased glucose (IG and serum) and serum insulin. In conclusion, this investigation demonstrates that FL2-CGMs can be successfully used for characterization of continuous glucose levels in NHPs and provides a significant advantage over glucose measured from serum samples collected from restrained animals.

葡萄糖是心脏代谢适应症疗效和毒理学评估的重要终点。一系列葡萄糖值的收集可以告知治疗相关效果和动物健康状况。处理压力会导致非人类灵长类动物（NHPs）的心率（HR）和葡萄糖峰值，增加可变性并混淆数据解释。连续血糖监测仪（cgm）是一种基于传感器的系统，可以在没有静脉穿刺的情况下提供间质血糖（IG）读数；从而减少处理压力和由此产生的葡萄糖峰值。本研究的目的是对使用NHPs的Freestyle®Libre™2 CGM （fl2 -CGM，雅培）进行为期多天的评估，同时进行持续的HR监测和血清收集，以确定关键时间点的葡萄糖和胰岛素水平。将fl2 - cgm经皮应用于夹套NHPs的背部进行IG测量。FL2-CGMs以15 min的增量保留高达8 h的数据。IG数据在每次给药后每隔10 h收集72 h。在第一阶段，使用夹套外部遥测技术评估24 h以上的并发HR。分别给予生理盐水、口服葡萄糖（1 g/kg）或注射胰岛素（0.15 U/kg）。在给药前、给药后8和24 h，测定血清葡萄糖和胰岛素与IG数据进行比较。在第二阶段，动物被给予3 g/kg葡萄糖；在72 h内监测IG，在数据收集的前8 h内反复抽取血清葡萄糖和胰岛素，以比较两种葡萄糖收集方法的变化幅度和时间。当葡萄糖浓度为3 g/kg时，fl2 - cgm的IG水平升高（高于给药前水平72 mg/dL），而在葡萄糖浓度为1 g/kg时，IG水平的变化在观察到的IG变变性之外无法可靠地检测到。在给药0.15 U/kg胰岛素后，FL2-CGM捕获到较低的葡萄糖水平（比对照组低12 mg/dL）。处理引起的HR峰值与葡萄糖（IG和血清）和血清胰岛素升高同时发生。总之，本研究表明fl2 - cgm可以成功地用于表征NHPs中的连续葡萄糖水平，并且比从受限动物的血清样本中测量葡萄糖具有显着优势。

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引用次数: 0

Analysis of baroreflex and heart rate variability – Tools in cardiovascular pre-clinical safety studies 压力反射和心率变异性分析——心血管临床前安全性研究的工具

IF 1.8 4区医学 Q4 PHARMACOLOGY & PHARMACY

Journal of pharmacological and toxicological methods

Pub Date : 2025-09-01 Epub Date: 2025-09-19 DOI: 10.1016/j.vascn.2025.107783

Olivera Antic , Yevgeniya E. Koshman , Amanda S. Wilsey , Brandan M. Bird , Geena Jasiek , Rebecca Kohnken , Scott W. Mittelstadt , Harald M. Stauss , Charles M. Foley

Cardiovascular reflexes and drug effects on the autonomic nervous system may contribute to hypotension/hypertension and bradycardia/tachycardia and pose a safety risk for patients. Thus, there is a need to assess autonomic and cardiovascular reflex responses to pharmacologic compounds in pre-clinical safety studies. To validate techniques assessing baroreflex function and cardiac autonomic modulation based on hemodynamic parameters commonly recorded in pre-clinical safety studies. Following three escalating oral doses of the vasodilator hydralazine or the α₁‑agonist midodrine hemodynamic responses were monitored for 24 h via telemetry (PhysioTel Digital L21, DSI, St. Paul, MN) in conscious male beagle dogs, and compared to vehicle control data. Baroreceptor-HR reflex function was assessed by the gain of the transfer function between systolic blood pressure (BP) and heart rate (HR). Cardiac autonomic modulation was assessed by low frequency (LF, sympathetic and parasympathetic modulation) and high frequency (HF, parasympathetic modulation) spectral power of inter-beat interval variability and the time-domain HR variability parameters SDNN (sympathetic and parasympathetic modulation) and RMSSD (parasympathetic modulation). As expected, hydralazine and midodrine caused opposite dose-dependent effects on BP and HR. Hydralazine at mid and high doses caused prolonged tachycardia beyond the hypotensive response. Tachycardia was explained by reduced cardiac parasympathetic modulation as demonstrated by marked reductions in SDNN, RMSSD, LF and HF spectral powers, and LF/HF ratio. Despite pronounced hypertensive responses to midodrine at mid and high doses, only low and mid doses caused bradycardia as expected from baroreflex responses. Interestingly, the low midodrine dose increased the gain of the BP-HR transfer function, while the mid and high doses decreased transfer function gain. Thus, bradycardia at the low midodrine dose is explained by augmentation of baroreflex function, while the lack of bradycardia at the high dose may be related to inhibition of baroreflex function. The results of this study demonstrate that assessing baroreceptor reflex function and autonomic effects on the cardiovascular system can provide important insights on hemodynamic effects of pharmacological compounds. Adding such assessments to pre-clinical safety studies may expand evaluation of complex autonomic inputs to help understand drug-induced cardiovascular responses.

心血管反射和药物对自主神经系统的影响可能导致低血压/高血压和心动过缓/心动过速，并对患者构成安全风险。因此，有必要在临床前安全性研究中评估药物化合物的自主神经和心血管反射反应。验证基于临床前安全性研究中通常记录的血流动力学参数评估压力反射功能和心脏自主调节的技术。通过遥测（PhysioTel Digital L21， DSI, St. Paul， MN）监测清醒雄性比格犬24 h的血流动力学反应，并与对照数据进行比较。通过收缩压（BP）和心率（HR）之间传递函数的增益来评估压力感受器-心率反射功能。通过低频（LF，交感神经和副交感神经调节）和高频（HF，副交感神经调节）心跳间隔变异性谱功率和时域HR变异性参数SDNN（交感神经和副交感神经调节）和RMSSD（副交感神经调节）评估心脏自主调节。正如预期的那样，肼嗪和米多定对血压和心率产生相反的剂量依赖性作用。中剂量和高剂量的海氮嗪可引起长时间的心动过速，而不是低血压反应。通过SDNN、RMSSD、LF和HF频谱功率以及LF/HF比值的显著降低，可以解释心动过速的原因是心脏副交感神经调节的减少。尽管中剂量和高剂量的midodrine对高血压有明显的反应，但只有低剂量和中剂量的midodrine引起了从压力反射反应中预期的心动过缓。有趣的是，低剂量的米多定增加了BP-HR传递函数的增益，而中、高剂量则降低了传递函数的增益。因此，低剂量midodrine时的心动过缓可以通过增强血压反射功能来解释，而高剂量时心动过缓的缺失可能与抑制血压反射功能有关。本研究结果表明，评估压力感受器反射功能和自主神经对心血管系统的影响可以为药理化合物的血流动力学作用提供重要的见解。将此类评估添加到临床前安全性研究中，可能会扩大对复杂自主神经输入的评估，以帮助了解药物诱导的心血管反应。

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引用次数: 0

HERG block in HEK cells incorporated in real time into computer models of in silico cardiac repolarization using dynamic clamp 利用动态钳将HEK细胞中的HERG阻滞实时纳入计算机模型

IF 1.8 4区医学 Q4 PHARMACOLOGY & PHARMACY

Journal of pharmacological and toxicological methods

Pub Date : 2025-09-01 Epub Date: 2025-09-19 DOI: 10.1016/j.vascn.2025.107819

Mark W. Nowak , Brian K. Panama , Leigh Korbel , Michael Hines , Nicholas T. Carnavale , Randall L. Rasmusson , Glenna C.L. Bett

Using dynamic clamp in Synthetic Cell Mode, we input experimentally-obtained HERG current, in real time, into in-silico cardiomyocytes. In-silico models were either generated by the dynamic clamp system or by interfacing with NEURON software (www.neuron.yale.edu/neuron/). The in-silico action potential (AP) voltage generated in real time was applied to HEK cells expressing the cloned HERG current. The experimental HERG current was then input into the in-silico cardiomyocytes. The objective was to examine the experimental effect of HERG block by dofetilide on AP morphology in the in-silico cardiomyocytes. HERG currents were recorded from a stable HERG_A: HEK cell line in the whole cell ruptured patch configuration (200B amplifier, Molecular Devices). Using dynamic clamp (Cybercyte DC-1, Cytocybernetics), experimental HERG currents were incorporated into either an in-silico ventricular cardiomyocyte consisting of electronically-expressed I_Na, I_K1, I_KS, I_{Ca_L,} and I_to or an in-silico NEURON atrial cardiomyocyte (Courtemanche et al., 1998; Jacobson, 1998). HERG current magnitude was adjusted so AP duration (APD) ranged from 300 to 500 ms (ventricular) and 200–300 ms (atrial) for the in-silico cardiomyocytes. HERG current block with dofetilide (10–100 nM) on AP morphology was examined. Dofetilide blocked HERG current with an IC₅₀ of 22.5 ± 0.9 nM and a Hill coefficient of 1.03 ± 0.05 (n = 6). For the in-silico ventricular cardiomyocytes, dofetilide prolonged the AP (APD90 baseline: 442 ± 21 ms, 100 nM: 974 ± 104 ms, p < 0.01, n = 6). For the in-silico atrial cardiomyocytes, the NEURON model I_Kr was replaced with HERG current. Dofetilide block of HERG current (30 nM dofetilide: 62 ± 8 %, n = 6) increased the APD90 (baseline: 263 ± 13 ms, 30 nM dofetilide: 347 ± 15 ms, p < 0.001, n = 6). Using dynamic clamp in Synthetic Cell Mode, experimentally-expressed ion channel currents can be incorporated in real time into in-silico cardiomyocytes, and the effects of drugs targeting the expressed ion channel on AP morphology/behavior assessed. Dynamic Clamp Synthetic Cell Mode can readily be implemented on automated patch clamp systems, allowing for HT screening of drugs targeting cardiac ion channels with AP morphology as the readout. These studies were supported, in part, by NIH NS011613, 1R43NS125749 and 5R44MH119842.

利用合成细胞模式下的动态箝位，我们将实验获得的HERG电流实时输入到硅心肌细胞中。通过动态夹紧系统或通过与NEURON软件（www.neuron.yale.edu/neuron/）接口生成的硅模型。实时产生的硅动作电位（AP）电压作用于表达克隆HERG电流的HEK细胞。然后将实验HERG电流输入到硅质心肌细胞中。目的是观察多非利特阻断HERG对硅片心肌细胞AP形态的实验影响。 记录了稳定的HERG_A: HEK细胞系在全细胞破裂斑块构型下的HERG电流（200B放大器，Molecular Devices）。使用动态钳（Cybercyte DC-1, Cytocybernetics），实验HERG电流被纳入由电子表达的INa、IK1、IKS、ICa_L和Ito组成的硅基心室心肌细胞或硅基NEURON心房心肌细胞（Courtemanche等，1998;Jacobson, 1998）。调整HERG电流大小，使硅化心肌细胞的AP持续时间（APD）在300 ~ 500 ms（心室）和200 ~ 300 ms（心房）之间变化。用多非利特（10-100 nM）阻断HERG电流，观察其对AP形态的影响。 多非利特阻断HERG电流的IC50为22.5 ± 0.9 nM， Hill系数为1.03 ± 0.05 （n = 6）。in-silico心室心肌细胞,dofetilide长期美联社(APD90基线:442 ± 21 女士,100 海里:974 ±104  女士,p & lt; 0.01 n = 6)。对于人工心房心肌细胞，用HERG电流代替神经元模型IKr。Dofetilide HERG当前块(30 海里Dofetilide: 62 ± 8 % n = 6)增加了APD90(基线:263 ± 13 女士,30 nM Dofetilide: 347 ± 15 女士,p & lt; 0.001 n = 6)。 在合成细胞模式下使用动态箝位，可以将实验表达的离子通道电流实时整合到硅心肌细胞中，并评估靶向表达离子通道的药物对AP形态/行为的影响。动态钳合成细胞模式可以很容易地在自动膜片钳系统上实现，允许以AP形态为读数的靶向心脏离子通道的药物HT筛选。 这些研究得到了NIH NS011613、1R43NS125749和5R44MH119842的部分支持。

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引用次数: 0

Effects of temperature, compound concentration verification, and internal fluoride on hERG 温度、化合物浓度验证和内氟对hERG的影响

IF 1.8 4区医学 Q4 PHARMACOLOGY & PHARMACY

Journal of pharmacological and toxicological methods

Pub Date : 2025-09-01 Epub Date: 2025-09-19 DOI: 10.1016/j.vascn.2025.107823

Aimee L. Bielinski, Mark T. Zafiratos, Andrew S. Zielonka, Ruth L. Martin

Are automated electrophysiology platform (EP) data appropriately rigorous to be included in regulatory filings? Automated EP platforms have been in use for more than 20 years yet are still primarily used for initial screening of compounds against hERG and other ion channels. In this study we investigated the effects of 20 known compounds with a wide range of potencies against hERG. We conducted all experiments using SyncroPatch 384i and cryo-preserved assay-ready hERG cells. We compare results for temperature effects (25 °C vs. 35 °C) and the presence of fluoride in the internal solution. Verification of well compound concentrations via LC-MS/MS analysis were determined for all variations of the experiment. We used a standard 2-step voltage protocol (Vh = −80 mV, Vs = +20 mV for 5 s, Vt = −50 mV for 5 s, repeated once every 15 s for >5 min). At 25 °C, in the presence of internal fluoride, we saw a range of targeted IC₅₀s from <1 to >30 μM. When we corrected the data after verification of compound well concentrations, the range of IC₅₀s was now 0.1 μM to 38 μM. At 35 °C, in the presence of internal fluoride, the targeted range of IC₅₀s was <1 μM to >30 μM and with correction for verification of concentrations was 0.2 μM to >38 μM. We conclude that for this set of compounds, temperature (25 °C vs 35 °C) has no meaningful effect on IC₅₀s nor on the verification of concentrations. We repeated this set of experiments, now in the absence of internal fluoride. Targeted IC₅₀s were comparable to those determined in the presence of internal fluoride and verification of concentrations did not change that result. We conclude that for this set of compounds, the presence or absence of internal fluoride has no meaningful effect on IC₅₀s nor on the verification of concentrations, although it has a very large effect on experimental success rate. We hope this study will further the conversation on steps needed to implement automated electrophysiology platforms as an accepted tool used in hERG investigations for regulatory filings.

自动电生理平台（EP）数据是否严格到可以纳入监管文件？自动化EP平台已经使用了20多年 ，但仍然主要用于抗hERG和其他离子通道的化合物的初步筛选。在这项研究中，我们研究了20种已知化合物对hERG的作用，这些化合物具有广泛的效力。我们使用SyncroPatch 384i和冷冻保存的hERG细胞进行所有实验。我们比较了温度效应的结果（25 °C vs. 35 °C）和内溶液中氟的存在。通过LC-MS/MS分析确定了所有实验变量的井化合物浓度验证。 我们使用一个标准的两步电压协议(Vh = −80 mV, Vs = + 20 mV 5 年代,Vt = −50 mV 5 年代,重复每隔15 年代祝辞 5分钟)。在25 °C下，在内部氟化物存在的情况下，我们看到目标ic50范围从<；1到>；30 μM。当我们在验证复合井浓度后对数据进行校正时，ic50的范围现在为0.1 μM至38 μM。在35 °C时，在内氟存在的情况下，ic50的目标范围为<；1 μM ~ >30 μM，校正浓度验证范围为0.2 μM ~ >38 μM。我们得出结论，对于这组化合物，温度（25 °C vs 35 °C）对ic50和浓度验证没有显著影响。 我们重复了这组实验，在没有氟化物的情况下。目标ic50与体内氟化物存在时确定的ic50相当，对浓度的验证并未改变这一结果。我们得出的结论是，对于这组化合物，内部氟化物的存在或不存在对ic50和浓度验证没有意义的影响，尽管它对实验成功率有很大的影响。 我们希望这项研究将进一步讨论实现自动化电生理平台作为hERG调查中用于监管备案的公认工具所需的步骤。

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引用次数: 0

High throughput automated patch-clamp of cardiac ion channels using physiological fluoride-free internal solution 使用生理无氟内溶液的高通量自动膜片钳心脏离子通道

IF 1.8 4区医学 Q4 PHARMACOLOGY & PHARMACY

Journal of pharmacological and toxicological methods

Pub Date : 2025-09-01 Epub Date: 2025-09-19 DOI: 10.1016/j.vascn.2025.107825

Markus Rapedius , Alison Obergrussberger , Stephanie Scholz , Ilka Rinke-Weiss , Tom A. Goetze , Nina Brinkwirth , Maria G. Rotordam , Tim Strassmaier , Aaron Randolph , Søren Friis , Kefan Yang , Jan Rathje , Aiste Liutkute , Fitzwilliam Seibertz , Niels Voigt , Niels Fertig , Sonja Stoelzle-Feix

Fluoride has been used in the internal recording solution for manual and automated patch clamp experiments for decades because it helps to improve the seal resistance and promotes longer lasting recordings. In manual patch clamp, fluoride has been used to record voltage-gated Na (Na_V) channels where seal resistance and access resistance are critical for good voltage control. In automated patch clamp, suction is applied from underneath the patch clamp chip to attract a cell to the hole and obtain a good seal. Since the patch clamp aperture cannot be moved to improve the seal like the patch clamp pipette in manual patch clamp, automated patch clamp manufacturers use internal fluoride to improve the success rate for obtaining GΩ seals. However, internal fluoride can affect voltage-dependence of activation and inactivation, as well as affecting internal second messenger systems and therefore, it is desirable to have the option to perform experiments using physiological, fluoride-free internal solution. We have developed an approach for high throughput fluoride-free recordings on a 384-well based automated patch clamp system with success rates >40 % for GΩ seals and have also extended the approach to include the medium throughput device, the Patchliner. We demonstrate the method using hERG expressed in HEK cells, as well as Na_V1.5, and K_Ca3.1 expressed in CHO cells. Although the voltage dependence of activation of hERG was not affected by internal fluoride, the voltage-dependence of activation and inactivation of Na_V1.5 was shifted to more negative potentials when fluoride was present (V_half,Act = −25.2 ± 0.5 mV (241) in fluoride-free versus − 37.5 ± 0.4 mV (484) in standard internal; V_half,Inact = −53.6 ± 0.6 mV (165) in fluoride-free versus − 68.4 ± 0.4 mV (728) in standard internal), whereas activation of K_Ca3.1 by internal calcium was more robust when physiological internal solution was used. In preliminary recordings we could successfully use the fluoride-free approach for experiments involving stem cell-derived cardiomyocytes. Therefore, APC data are more comparable to recordings done using manual patch clamp and physiological solutions. Ultimately, this will enhance ion channel characterization and cardiac safety testing for cell lines, induced pluripotent stem cells and primary cells.

几十年来，氟化物一直用于手动和自动膜片钳实验的内部记录溶液中，因为它有助于提高密封阻力并促进更持久的记录。在手动膜片钳中，氟化物已用于记录电压门控钠（NaV）通道，其中密封电阻和接入电阻对于良好的电压控制至关重要。在自动膜片钳中，从膜片钳芯片下方施加吸力以吸引细胞到孔中并获得良好的密封。由于膜片钳的孔径不能像手动膜片钳中的膜片钳移液管那样移动以改善密封性，因此自动化膜片钳制造商使用内部氟化物来提高获得GΩ密封性的成功率。然而，内部氟化物会影响激活和失活的电压依赖性，以及影响内部第二信使系统，因此，希望有选择使用生理的，无氟的内部溶液进行实验。我们开发了一种基于384口井的自动化膜片钳系统的高通量无氟记录方法，对GΩ密封的成功率为>；40 %，并且还将该方法扩展到中通量设备Patchliner。我们使用HEK细胞中表达的hERG，以及CHO细胞中表达的NaV1.5和KCa3.1来演示该方法。虽然激活的电压依赖的hERG并不影响内部氟化物,激活和失活的voltage-dependence NaV1.5转移到更多的氟化物在场时负电位(Vhalf 行动=  −25.2±0.5  mV (241) fluoride-free与 −  37.5±0.4  mV在标准内部(484);Vhalf,非活性的 =  −53.6±0.6  mV (165) fluoride-free与 −  68.4±0.4  mV(728)在标准内),而激活的KCa3.1内部钙时更健壮的生理内部的解决方案是使用。在初步记录中，我们可以成功地使用无氟方法进行涉及干细胞衍生心肌细胞的实验。 因此，APC数据与使用手动膜片钳和生理溶液的记录更具可比性。最终，这将增强细胞系、诱导多能干细胞和原代细胞的离子通道表征和心脏安全性测试。

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引用次数: 0

Categorizing TdP risk with qNet: Effects of protocol design on HERG models and TMS outcomes 用qNet分类TdP风险：方案设计对HERG模型和TMS结果的影响

IF 1.8 4区医学 Q4 PHARMACOLOGY & PHARMACY

Journal of pharmacological and toxicological methods

Pub Date : 2025-09-01 Epub Date: 2025-09-19 DOI: 10.1016/j.vascn.2025.107815

Leigh A. Korbel , Mark W. Nowak , Brian K. Panama , Shivani Acharya , Glenna C. Bett , Randall L. Rasmusson

The Comprehensive in vitro Proarrhythmia Assay (CiPA) requires assessing drug effects on multiple ionic currents to measure pro-arrhythmic risk and a detailed model of drug binding to HERG channels. Manual patch clamp recordings of hERG current are performed using the Milnes protocol. The CiPA procedure has strong predictive ability, but extracting HERG model parameters is challenging at the experimental and analytical levels. We examined a two-voltage pulse (2P) protocol to categorize TdP risk using the qNet metric and Torsade Metric Score (TMS) in simulated and experimental data using improved perfusion techniques. We also explored whether these shorter and simpler experimental protocols could produce qNet/TMS results similar to those of the Milnes protocol. We tested the simulated predictions for dofetilide block of hERG current expressed in HEK cells (room and physiological temperatures) using the FDA-CiPAORdv1.0 drug-binding model. Our CYBERQ-qNET analysis software calculated the qNet and TMS values and compared the TdP risk category to those from the Milnes protocol. There were no significant differences in the qNET, TMS values, and TdP risk categories when using simulated/experimental data from either the two voltage pulses or the Milnes protocol. Using simulated data, the TMS/TdP risk values for the 2P protocol (using fractional block across the inactivation pulse) vs. Milnes protocol: dofetilide (0.0473/2 vs. 0.0524/2), bepridil (0.0453/2 vs. 0.0452/2), terfenadine (0.0635/1 vs. 0.0605/1) and diltiazem (0.0888/0 vs. 0.092/0), respectively. The experimental dofetilide data resulted in correct TdP categorization across the activation and inactivation pulses at room temperature (0.0481/2 and 0.0305/2) and physiological temperature (0.0309/2 and − 0.0189/2). However, the physiological temperature data displayed significantly more block than the room temperature and published Milnes data, which were comparable. Combining both pulses produced a miscategorization for the room temperature data (0.0625/1) but not the physiological temperature data (0.0440/2). Simulated data had consistent TMS scores. In contrast, experimental data showed more variable TMS scores depending on the pulse analyzed. These results suggest that model development can be coupled to simpler and less demanding experimental protocols for assessing arrhythmogenic potential using CiPA.

综合体外心律失常检测（CiPA）需要评估药物对多种离子电流的影响，以测量致心律失常的风险，并建立药物与HERG通道结合的详细模型。使用Milnes协议进行手动膜片钳记录hERG电流。CiPA方法具有较强的预测能力，但提取HERG模型参数在实验和分析层面都具有挑战性。我们研究了一种双电压脉冲（2P）方案，使用qNet度量和Torsade度量评分（TMS）在模拟和实验数据中使用改进的灌注技术对TdP风险进行分类。我们还探讨了这些更短、更简单的实验方案是否可以产生与Milnes方案相似的qNet/TMS结果。我们使用FDA-CiPAORdv1.0药物结合模型测试了多非利特阻断HEK细胞（室温和生理温度）中hERG电流表达的模拟预测。我们的CYBERQ-qNET分析软件计算了qNet和TMS值，并将TdP风险类别与Milnes协议的风险类别进行了比较。当使用来自两个电压脉冲或Milnes协议的模拟/实验数据时，qNET， TMS值和TdP风险类别没有显着差异。使用模拟数据，2P方案（在失活脉冲上使用分数块）与Milnes方案的TMS/TdP风险值分别为：多非利特（0.0473/2 vs. 0.0524/2）、贝普地尔（0.0453/2 vs. 0.0452/2）、特非那定（0.0635/1 vs. 0.0605/1）和地尔硫平（0.0888/0 vs. 0.092/0）。实验结果表明，在室温（0.0481/2和0.0305/2）和生理温度（0.0309/2和 − 0.0189/2）下，多非利特的激活和失活脉冲的TdP分类是正确的。然而，生理温度数据比室温和已发表的Milnes数据显示出明显更大的阻滞，两者具有可比性。结合这两种脉冲产生了对室温数据（0.0625/1）的错误分类，但没有对生理温度数据（0.0440/2）的错误分类。模拟数据具有一致的TMS评分。相比之下，实验数据显示，根据所分析的脉冲，TMS评分变化更多。这些结果表明，模型开发可以与使用CiPA评估心律失常电位的更简单、要求更低的实验方案相结合。

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引用次数: 0

Spinning natural venoms into drug discovery: The journey of a new potent peptide blocker of the human Cav1.2 channel subtype from Poecilotheria subfusca spider 将天然毒液转化为药物发现：来自Poecilotheria subfusca蜘蛛的人类Cav1.2通道亚型的一种新的有效肽阻断剂的旅程

IF 1.8 4区医学 Q4 PHARMACOLOGY & PHARMACY

Journal of pharmacological and toxicological methods

Pub Date : 2025-09-01 Epub Date: 2025-09-19 DOI: 10.1016/j.vascn.2025.107811

Jean-Marie Chambard , Tanya Goncalves , Michel de Waard , Michael Kurz , Stefan De Waard , Jerome Montnach , Francoise Chesney , Remy Beroud , Denis Servent , Michel Partiseti , Evelyne Benoit

Animal venoms have been explored as rich and valuable sources of new peptide modulators that target a large variety of ion channel subtypes. Over the last two decades, natural peptides have been identified with the ability to modulate several targets at a time. Post-discovery studies are used to then refine the identity of the best target. We employed a similar strategy to identify a novel and highly potent modulator of the human Cav1.2 channel; a high throughput screening of a large library of 200 animal venoms was initiated through automated whole-cell patch‐clamp experiments using engineered cells overexpressing human voltage-gated sodium NaV1.7 or NaV1.5 channel subtypes. Out of several positive venom fractions, one bioactive peptide, poecitoxin‐1a (PecTx‐1a) from the Poecilotheria subfusca spider venom, was purified for its higher potency to block NaV1.7 over NaV1.5 and identified based on mass spectrometry sequencing. The peptide is 35 amino acids long and belongs to the inhibitor cystine knot (ICK) structural family. Next, the synthetic peptide was generated and found identical to the native one, which allowed further characterization on a large set of voltage-gated calcium, voltage-gated potassium and inward-rectifier potassium (hKir) channel subtypes overexpressed in recombinant cells. This ion channel selectivity profiling uncovered hCaV1.2 as the best target of PecTx‐1a (IC50 of 24 nM) making this peptide the best-known inhibitor of this cardiac channel isoform to date. Further characterization of the peptide on the action potential and calcium currents of human induced pluripotent stem cell-derived cardiomyocytes indicates the pharmacological value of this peptide in modulating cardiac excitability. In conclusion, PecTx‐1a is the first high affinity ICK spider toxin that targets the L-type hCaV1.2 channel with high affinity and should therefore represent a valuable tool to study the CaV1.2 subtype physiological and pharmacological functions in healthy and pathological models.

动物毒液已被探索为针对多种离子通道亚型的新肽调节剂的丰富和有价值的来源。在过去的二十年中，天然肽已被确定具有一次调节几个目标的能力。发现后的研究用于确定最佳目标。我们采用了类似的策略来鉴定一种新的高效的人类Cav1.2通道调制器；通过自动化全细胞膜片钳实验，使用过表达人电压门控钠NaV1.7或NaV1.5通道亚型的工程细胞，对200种动物毒液的大型文库进行了高通量筛选。在几种阳性毒液组分中，从Poecilotheria subfusca蜘蛛毒液中纯化出一种生物活性肽，poecitoxin‐1a (PecTx‐1a)，其阻断NaV1.7的效力高于NaV1.5，并基于质谱测序进行了鉴定。该肽长35个氨基酸，属于抑制剂胱氨酸结（ICK）结构家族。接下来，合成肽被生成，并发现与天然肽相同，从而可以进一步表征重组细胞中过表达的大量电压门控钙、电压门控钾和内向整流钾（hKir）通道亚型。这种离子通道选择性分析揭示了hCaV1.2是PecTx‐1a的最佳靶点（IC50为24 nM），使该肽成为迄今为止最知名的心脏通道亚型抑制剂。该肽对人诱导多能干细胞衍生心肌细胞的动作电位和钙电流的进一步表征表明，该肽在调节心脏兴奋性方面具有药理价值。总之，PecTx‐1a是第一个高亲和力的ICK蜘蛛毒素，以高亲和力靶向l型hCaV1.2通道，因此应该代表一个有价值的工具来研究CaV1.2亚型在健康和病理模型中的生理和药理功能。

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引用次数: 0

Feasbility of non-invasive arterial blood pressure estimation using inductive plethysmography in anaesthetised rats 麻醉大鼠无创动脉血压测量方法的可行性

IF 1.8 4区医学 Q4 PHARMACOLOGY & PHARMACY

Journal of pharmacological and toxicological methods

Pub Date : 2025-09-01 Epub Date: 2025-09-19 DOI: 10.1016/j.vascn.2025.107788

Leandro Fontana Pires , Agathe Cambier , Stéphane Tanguy , Charles Eynard , Timothé Flenet , François Boucher , Pierre-Yves Gumery

Inductive plethysmography (IP) has proven effective in detecting changes in cardiac output and stroke volume, enabling non-invasive hemodynamic monitoring using jacketed telemetry systems. However, the deployment of this modality in safety pharmacology is limited by its current inability to measure arterial blood pressure (AP). Recent studies have demonstrated the feasibility of estimating AP using mathematical formulas describing the relationship between AP and the ‘time delay’ required for a pulse to travel a certain distance in the arterial tree (‘pulse transit time’ or PTT). From a physiological perspective, AP estimated from an average PTT can be correlated with the mean arterial pressure (MAP). The aim of this study was to assess the feasibility of estimating arterial blood pressure (AP’) in rats by measuring PTT between the abdominal and thoracic plethysmographic signals recorded by the DECRO jacketed telemetry device and to compare it with a reference measurement during a pharmacological challenge. The pharmacological protocol consisted of a continuous intravenous (i.v.) infusion of 12.5 μg/ml dobutamine (beta-agonist) administered at increasing infusion rates (4, 6 and 8 ml/h) in six anaesthetised (2 %/2.5 % isoflurane) male Wistar rats (10 weeks, 355 g) under spontaneous ventilation. Arterial pressure was measured with an Edwards probe through a catheter inserted into the left carotid artery, and MAP was calculated. Animals were equipped with the DECRO device and AP’ was estimated from the PTT using a logarithmic model. The two variables were compared using Pearson's correlation coefficient and a Bland-Altman analysis to evaluate agreement and bias. Both methods detected a statistically significant decrease respectively in the AP’ and MAP. The estimation of AP’ by the PTT calculation algorithm adapted to plethysmographic data correlated with the measurement of MAP throughout the pharmacological protocol (correlation coefficient of 0.94). A mean difference of 1.4 % and 95 % limits of agreement ranging from −3.29 % to +6.09 % were found between the two methods. These results demonstrate the potential of this non-invasive modality for estimating blood pressure changes in preclinical situations. Capabilities to conduct a differentiated estimation of Systolic and diastolic pressure changes and implementation in other conditions remain to be explored.

电感式脉搏波描记术（IP）已被证明在检测心输出量和搏量变化方面是有效的，可以使用夹套遥测系统进行无创血流动力学监测。然而，由于目前无法测量动脉血压（AP），这种方法在安全药理学中的应用受到限制。最近的研究表明，使用数学公式来描述AP与脉冲在动脉树中传播一定距离所需的“时间延迟”（“脉冲传递时间”或PTT）之间的关系，可以估计AP的可行性。从生理学角度来看，平均PTT估计的AP可以与平均动脉压（MAP）相关。本研究的目的是评估通过测量DECRO夹套遥测装置记录的腹部和胸部容积脉搏波信号之间的PTT来估计大鼠动脉血压（AP）的可行性，并将其与药理学挑战期间的参考测量进行比较。药理学方案包括在自发呼吸条件下，对6只麻醉（2 %/2.5 %异氟醚）雄性Wistar大鼠（10 周，355 g）连续静脉（i.v）输注12.5 μg/ml多巴酚丁胺（β激动剂），以递增的输注速率（4、6和8 ml/h）给药。通过导管插入左颈动脉，用爱德华兹探头测量动脉压，计算MAP。动物配备DECRO装置，并使用对数模型从PTT估计AP '。使用Pearson相关系数和Bland-Altman分析对两个变量进行比较，以评估一致性和偏倚。两种方法均检测到AP′和MAP均有统计学意义的降低。在整个药理学治疗过程中，采用适用于容积描记数据的PTT计算算法估计的AP′与MAP测量相关（相关系数为0.94）。两种方法的平均差异为1.4 %和95 %，一致性范围为−3.29 %至+6.09 %。这些结果证明了这种非侵入性方法在估计临床前血压变化方面的潜力。对收缩压和舒张压变化进行差异化评估的能力以及在其他情况下的应用仍有待探索。

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