Journal of pharmacological and toxicological methods最新文献

{"title":"Categorizing TdP risk with qNet: Effects of protocol design on HERG models and TMS outcomes","authors":"Leigh A. Korbel ,&nbsp;Mark W. Nowak ,&nbsp;Brian K. Panama ,&nbsp;Shivani Acharya ,&nbsp;Glenna C. Bett ,&nbsp;Randall L. Rasmusson","doi":"10.1016/j.vascn.2025.107815","DOIUrl":"10.1016/j.vascn.2025.107815","url":null,"abstract":"<div><div>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.</div></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":"135 ","pages":"Article 107815"},"PeriodicalIF":1.8,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145094845","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"High throughput automated patch-clamp of cardiac ion channels using physiological fluoride-free internal solution","authors":"Markus Rapedius ,&nbsp;Alison Obergrussberger ,&nbsp;Stephanie Scholz ,&nbsp;Ilka Rinke-Weiss ,&nbsp;Tom A. Goetze ,&nbsp;Nina Brinkwirth ,&nbsp;Maria G. Rotordam ,&nbsp;Tim Strassmaier ,&nbsp;Aaron Randolph ,&nbsp;Søren Friis ,&nbsp;Kefan Yang ,&nbsp;Jan Rathje ,&nbsp;Aiste Liutkute ,&nbsp;Fitzwilliam Seibertz ,&nbsp;Niels Voigt ,&nbsp;Niels Fertig ,&nbsp;Sonja Stoelzle-Feix","doi":"10.1016/j.vascn.2025.107825","DOIUrl":"10.1016/j.vascn.2025.107825","url":null,"abstract":"<div><div>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 &gt;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.</div></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":"135 ","pages":"Article 107825"},"PeriodicalIF":1.8,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145094728","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Feasbility of non-invasive arterial blood pressure estimation using inductive plethysmography in anaesthetised rats","authors":"Leandro Fontana Pires ,&nbsp;Agathe Cambier ,&nbsp;Stéphane Tanguy ,&nbsp;Charles Eynard ,&nbsp;Timothé Flenet ,&nbsp;François Boucher ,&nbsp;Pierre-Yves Gumery","doi":"10.1016/j.vascn.2025.107788","DOIUrl":"10.1016/j.vascn.2025.107788","url":null,"abstract":"<div><div>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.</div></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":"135 ","pages":"Article 107788"},"PeriodicalIF":1.8,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145095181","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Video-EEG for safety pharmacology seizure liability assessment in rabbits","authors":"Magdalena K. Smyk,&nbsp;Henk van der Linde,&nbsp;David J. Gallacher","doi":"10.1016/j.vascn.2025.107846","DOIUrl":"10.1016/j.vascn.2025.107846","url":null,"abstract":"<div><div>Linking brain activity with behavior during video-electroencephalographic (video-EEG) recordings provides a better insight into epileptic phenomena than behavioral observations alone. Detection of non-convulsive seizures (often with subtle behavioral manifestations but clear EEG signatures) or non-brain-derived convulsive-like behavior, makes the technique superior in seizure liability assessment in preclinical safety. The present study was motivated by a need for confirmation of seizurogenic effect of a compound, which was labelled as pro-convulsive in a standard tolerability study with clinical observations in rabbits. Considering contradicting results from extensive experiments performed during development and no proof other than visual observation, the study was recreated with use of the video-EEG methodology. Prior to the exact experiment, the newly-build recording setup for rabbits was validated. Epileptic activity elicited by a single, subcutaneous injection of pentylenetetrazol (PTZ), which dose-dependently evoked both convulsive and non-convulsive seizures and induced a wide range of seizure-related behaviors, was assessed. Female New Zealand White rabbits were implanted with telemetry devices (implant M01, DSI a division of Harvard Bioscience, Inc., USA). After post-surgical recovery, animals were habituated to recording cages equipped with two video cameras allowing front and side views. On a test day, 1-h baseline recordings of EEG, temperature, activity and video signals were performed, after which rabbits received either PTZ (20, 40 or 60 mg/kg) or vehicle (saline) and were recorded for 24 consecutive hours. Synchronized EEG and video signals were analyzed offline by custom-made software, which automatically detected suspicious EEG epochs and linked them with the video fragments for closer inspection. Vice versa, abnormal behavior during the test could be immediately linked to the EEG signal to assess contribution from the central nervous system to the observed behavior. PTZ dose-dependently evoked both types of epileptic phenomena, non-convulsive seizures in the form of spike complexes after the low dose, and convulsions after the middle and high dose. The video-EEG set up allowed reliable, simultaneous recordings of brain activity and behavior of the rabbits and characterization of epileptic activity evoked by a reference compound.</div></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":"135 ","pages":"Article 107846"},"PeriodicalIF":1.8,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145095264","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Differential QTc prolonging effects of dofetilide and HMR1556 in rabbits and guinea pigs","authors":"William Salvail,&nbsp;Annie Bouchard,&nbsp;Helene Mouilleron,&nbsp;Dany Salvail","doi":"10.1016/j.vascn.2025.107771","DOIUrl":"10.1016/j.vascn.2025.107771","url":null,"abstract":"<div><div>The sensitivity to QT-prolonging drugs varies between species due to differences cardiac electrophysiology and ion channel expression. Rabbits are often used in cardiac safety pharmacology because their depolarization and repolarization characteristics are similar to humans. Rabbits tend to be sensitive to QT-prolonging drugs due to their reliance on IKr for repolarization. Guinea-pigs have a different cardiac electrophysiological profile; They exhibit a greater reliance on IKs rather than IKr. ECG telemetry probes were implanted in 8 adult male guinea pigs and 8 rabbits followed by 5 days of recovery. The IKr blocker dofetilide (10 μg/kg i.v.) and the IKs blocker HMR1556 (400 ng/kg i.v.) were administered by 10-min infusion alone or in combination. Dofetilide alone caused a 77 ms (rabbit) and 27 ms (guinea pig) peak QTc (Bazett's) prolongation. HMR1556 alone caused a 15 ms (rabbit) and 11 ms (guinea pig) peak QTc prolongation. Torsades de pointes (TdP) were recorded in 3/8 rabbits (dofetilide) and 4/8 guinea pig (HMR1556), and lasted 21 s on average. Administered together, dofetilide and HMR1556 caused 81 ms (rabbit) and 41 ms (guinea pig) peak QTc prolongations. TdP were observed in 5/8 rabbits and 6/8 guinea pigs after 10-min infusions of the combined blockers. In all cases, the TdP self-arrested within 170 s. None of the animals died. These results illustrate the additivity of ion channel inhibition across two species, which suggests that the more QT-sensitive rabbit is particularly useful to assess the impact of known IKr blockers while guinea pigs offer a broader screening potential, especially for drugs that might affect multiple ion channels. Results from rabbit studies should be more predictive of human responses to IKr blockers.</div></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":"135 ","pages":"Article 107771"},"PeriodicalIF":1.8,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145095363","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Improving the safety pharmacology standard: CNS behavioral investigations using anticholinergics as a comparator against the standard Irwin test","authors":"Hayley Woods,&nbsp;Kayleigh Roebuck,&nbsp;Victoria Ascough,&nbsp;Shamila Griffiths,&nbsp;Sharon Rowton","doi":"10.1016/j.vascn.2025.107840","DOIUrl":"10.1016/j.vascn.2025.107840","url":null,"abstract":"<div><div>The ICH S7A Safety Pharmacology guidelines require a novel test article to be assessed for central nervous system (CNS) effects as a part of the Core Battery. Although follow-up behavioral studies are advised, these are rarely seen as part of the GLP safety pharmacology package of work, with an Irwin screen alone conducted to address this requirement. The objective of this investigation was to provide examples using the anticholinergic drugs scopolamine and atropine, where the standard behavioral assessment (Irwin test) is limited in terms of identifying certain undesirable behavioral effects of a novel drug and in some instances, which may mistakenly lead to a conclusion of test subject animals as ‘normal’. A series of behavioral tests were conducted following intraperitoneal administration of 10 mg/kg atropine or 0.25, 1 or 4 mg/kg scopolamine to male C57BL/6J mice or male Han® Wistar rats. Memory assessments were conducted using the Y-Maze and novel object recognition (NOR) tests; the elevated plus maze (EPM), and light/dark box tests for anxiety; and nest building was assessed as an activity of daily living which is influenced by the general well-being of the animals. A modified Irwin was also conducted. Scopolamine and atropine produced statistically significant deficits on memory retrieval within the NOR or Y-Maze; anxiogenic effects within the EPM and light/dark box; and inferior nest building quality, possibly associated with the marked anxiogenic effect of these drugs resulting in lack of desire/motivation to build a nest. None of these effects were quantifiable within the Irwin test. The endpoints investigated are by no mean exhaustive but serve to provide examples of relatively simple tests which lend themselves to be incorporated as additional endpoints within repeat-dose toxicology studies or within stand-alone safety pharmacology studies, and in doing so, add value to the overall characterization of the novel drug. The actual endpoints for consideration should be determined by the on- and/or off target effects of the drug, e.g., for a serotonin activator, the inclusion of a head twitch response test would be appropriate to rule out or support 5-HT_2A activation.</div></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":"135 ","pages":"Article 107840"},"PeriodicalIF":1.8,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145095378","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Video-based detection of pentylenetetrazol induced severe convulsions in cynomolgus monkeys","authors":"Hiroya Konno ,&nbsp;Andrew Z. Summers ,&nbsp;Noriaki Iwata ,&nbsp;Hiroaki Miida ,&nbsp;Yoshimi Tsuchiya","doi":"10.1016/j.vascn.2025.107841","DOIUrl":"10.1016/j.vascn.2025.107841","url":null,"abstract":"<div><div>Central nervous system (CNS) toxicity is one of the important toxicities in nonclinical studies for drug development. Abnormal behaviors often serve as indicators of CNS abnormalities. However, the exact timing of such symptoms' occurrence is usually uncertain, and accurate detection of abnormal behaviors is difficult since real-time observation or long-time observation of video data is required. To address this, there is a need for automated continuous monitoring methods that can assist observers and enhance detection sensitivity. In this study, we attempted to develop an advanced model for detecting drug-induced convulsions using video data of cynomolgus monkeys (<em>n</em> = 7) after administration of pentylenetetrazol, a GABA receptor antagonist, at a dose of 70 mg/kg. To detect convulsions, we tested various algorithms including classical image processing techniques, supervised learning models that use frequency information derived from wavelet transformation of each body part based on pose estimation, and unsupervised learning models that identify abnormalities through training only on videos of animals exhibiting ordinary behavior. Our results showed that the most feasible approach was to track regularly sampled points on the animal's body using optical flow. This method calculates the apparent motion of pixels between consecutive frames by analyzing the intensity patterns. This enabled the detection of convulsions based on characteristic frequencies in the tracking point trajectories, observed via Fourier transformation. Despite the limitation of available data, our model captured several convulsion patterns and demonstrated encouraging scores (accuracy: 77.8 %, false positive rate: 1.7 per hour). In conclusion, we have established a prototype of the convulsion detection model although fine-tuning is still necessary. The use of optical flow, which is also employed in human seizure detection models, as a benchmark for automatically detecting convulsions in monkeys holds potential in combination with other methods and analysis, with the possibility of further improvement with larger datasets.</div></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":"135 ","pages":"Article 107841"},"PeriodicalIF":1.8,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145095379","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Use of hemodynamic profiling to explain toxicity observed following chronic dosing in rats","authors":"Peter B. Senese,&nbsp;Charles B. Dean,&nbsp;Kimberly R. Doherty,&nbsp;Melissa Zammit,&nbsp;Michelle Johnson,&nbsp;Michael R. Gralinski","doi":"10.1016/j.vascn.2025.107833","DOIUrl":"10.1016/j.vascn.2025.107833","url":null,"abstract":"<div><div>This GLP study investigated a hemodynamic relationship to toxicity previously observed in a 28-day rat safety study following administration of Compound A. Naïve Sprague Dawley rats (5 M, 5 F) were instrumented with Data Sciences International HDS-11 implantable radiotelemetry devices to measure arterial blood pressure (BP), heart rate (HR) and body temperature (BT). Rats were dosed qd for 5 consecutive days with each treatment: vehicle (DI-water), 60 mg/kg/day (mpk) or 100 mpk of Compound A; up to 10 days of washout occurred between dose escalations. Hemodynamics were monitored (Ponemah v5.41) from 2 h prior to the first dose through 24 h after the final dose at each escalation. Data were analyzed as 1-h epochs; RANCOVA was done to determine statistical significance (<em>p</em> &lt; 0.05). BP (mean; MAP, systolic; SAP; diastolic; DAP), pulse pressure (PP), HR and BT were relatively stable following all doses of vehicle. Administration of 60 and 100 mpk Compound A led to significant increases in MAP, SAP, DAP and PP in male rats, with a higher frequency of significant differences noted on earlier dosing days, suggesting tachyphylaxis associated with Compound A. In female rats, statistically significant MAP, SAP and DAP changes were bidirectional and intermittent following 60 mpk dosing and were predominantly increases after 100 mpk administration. Infrequent HR changes occurred in male rats following 60 mpk Compound A while more frequent significant HR decreases were seen on Days 3–5 following 100 mpk Compound A. Significant HR decreases occurred in females during the first 2 h after Compound A, with more frequent HR decreases noted on later dosing days following 100 mpk administration. Significant BT decreases occurred following dosing with both 60 and 100 mpk Compound A in male and female rats; magnitude and duration of significant decreases appeared dose-dependent. In conclusion, Compound A caused statistically significant changes in BP, HR and BT. The changes in hemodynamics observed during the first 5 days of dosing with Compound A are a possible contributing factor in the toxicity observed during prior 28-day rat safety assessments, demonstrating the sensitivity of this methodology to capture a dose-dependent response.</div></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":"135 ","pages":"Article 107833"},"PeriodicalIF":1.8,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145094587","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Implementation of an electric cell-substrate impedance sensing (ECIS) protocol to parametrize 2D induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) cultures","authors":"Ronald J. Knox ,&nbsp;Ronald Knox ,&nbsp;Carlos Obejero-Paz ,&nbsp;Joseph Wu ,&nbsp;Martin Ziller ,&nbsp;Sonja Stölzle-Feix","doi":"10.1016/j.vascn.2025.107822","DOIUrl":"10.1016/j.vascn.2025.107822","url":null,"abstract":"<div><div>The ECIS technique was developed to investigate the barrier function of confluent epithelial and endothelial cells by measuring the impedance of cell cultures in a wide range of frequencies. The physical interpretation of the data is based on equivalent circuit models. One model developed by Giaever and Keese (1991, GK model) assumes that confluent cells interact with each other and the substrate creating two defined resistances: Rb, the resistance of the space between cells and a, the resistance space occupied by the substrate between the cell and the electrode. The model also includes the cell membrane capacitance (Cm). The goal of this research was 1) to establish whether cardiomyocyte phenotypes can be defined by the strength of interaction between cells (Rb), the strength of interaction with the substrate (a), and the amount of cell membrane (Cm), and 2) whether these parameters define the electrophysiological properties of the 2D culture. Four iPSC-CM lines derived from healthy donors were differentiated using current protocols and transferred (100,000 cells/well) to 0.6 mm electrode CardioExcyte96 plates. The CardioExcyte96 was used to record extracellular field potentials and the AtlaZ was used to measure impedance values in the 0.1 to 100 kHz range. The GK model was fitted using an R script. The impedance measured at 10 kHz growth after cardiomyocyte plating, following a single exponential function stabilizing after 7 days. The normalized spectrum of the four cell lines showed a peak between 15 and 25 kHz. Rb ranged from 1.8 to 9.3 Wcm², a ranged from 5.1 to 7.4 W^0.5 cm and Cm ranged from 0.74 to 1.82 mFcm⁻². Rb was the most discriminative parameter between phenotypes and correlated with the Sodium Spike Amplitude but not with Field Potential duration or Beating Rate. This study suggests that ECIS parameters, particularly Rb, can differentiate cardiomyocyte phenotypes based on the strength of cell-cell interactions. This finding underscores the potential utility of ECIS in characterizing cellular behavior and electrophysiological properties in cardiomyocyte cultures for disease modeling and drug discovery.</div></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":"135 ","pages":"Article 107822"},"PeriodicalIF":1.8,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145094720","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Assessing variability of hERG data generated using a mock action potential waveform and automated patch clamp platforms – A HESI-coordinated, multi-laboratory comparison of 28 drugs across 3 platforms","authors":"Manni Mashaee ,&nbsp;Sonja Stoelzle-feix ,&nbsp;John Ridley ,&nbsp;Stefano Stabilini ,&nbsp;Sarah Williams ,&nbsp;Juha Kammonen ,&nbsp;Adam Hyman ,&nbsp;Muthukrishnan Renganathan ,&nbsp;Diane Werth ,&nbsp;Jennifer Wesley ,&nbsp;Jun Zhao ,&nbsp;Lars Johannesen ,&nbsp;Claudia Alvarez Baron ,&nbsp;Jennifer Pierson ,&nbsp;Wendy Wu ,&nbsp;Jose Vicente","doi":"10.1016/j.vascn.2025.107817","DOIUrl":"10.1016/j.vascn.2025.107817","url":null,"abstract":"<div><div>New ICH E14/S7B Q&amp;As describe how nonclinical data, including cardiac ion channel pharmacology studies generated following best practices, can support clinical interpretation of QT studies as part of an integrated proarrhythmic risk assessment. Automated patch clamp (APC) systems offer high-throughput capabilities and remove technical barriers to generate cardiac ion channel pharmacology data compared to the traditional, manual patch clamp technique. These advantages are achieved by adjusting experimental aspects such as including the use of fluoride to reduce leak current, fixed duration recordings across all cells, and the use of multi-hole recording chips to optimize catching a cell. Literature search revealed limited information regarding the consequences of these changes on cardiac ion channels pharmacology. Additionally, variability of APC data generated using the same voltage protocol across different laboratories operating either the same or different platforms is unclear. This study is a part of a HESI-coordinated international effort to assess block potencies of 28 drugs on cardiac hERG current using APC systems. Four laboratories participated in the study: one operated QPatch 48; one, SyncroPatch 384; and two, Qube 384. The voltage protocol resembled a ventricular action potential (ICH S7B Q&amp;A 2.1), and was presented at 0.2 Hz. All laboratories generated data at ambient or room temperature (RT). Two laboratories additionally generated data at near physiological temperature (PT). Two laboratories tested single-hole recording chips; one laboratory multi-hole; and one laboratory tested both. Solution samples were collected by two laboratories for concentration verification. Overall variability of the hERG data was estimated by first removing drug-specific effects and laboratory-specific effects, and then pooling the adjusted values. Laboratory-specific systematic tendencies were identified. Comparison of RT to near PT data revealed systematically lower potencies at room temperature across all drugs. Concentration verification of 13 drugs by one laboratory revealed a relationship between the amount of drug lost during the assay and the hydrophobicity of the drug. Variability measures will be shared on the poster. Results of this study will inform the expected variability of hERG current data under best practice recommendations feasible for APC platforms and using these data to support integrated nonclinical risk assessment.</div></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":"135 ","pages":"Article 107817"},"PeriodicalIF":1.8,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145094847","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}