Journal of pharmacological and toxicological methods最新文献

{"title":"Determination of moxifloxacin QTc sensitivity in NHP to support ICH E14/S7B Q&A compliant studies using a parallel ascending or an ascending dose design","authors":"Ninette Andersen,&nbsp;Rachael Hardman,&nbsp;Tomas Mow,&nbsp;Louise Bang-Lauritsen,&nbsp;Sophie Strindberg,&nbsp;Anette Grossi,&nbsp;Karim Melliti","doi":"10.1016/j.vascn.2026.108466","DOIUrl":"10.1016/j.vascn.2026.108466","url":null,"abstract":"<div><div>Alternative dose design may be required for long-acting compounds when the best practice cross-over design is not feasible. The objective of this study was to determine QTc sensitivity with moxifloxacin in telemetry implanted NHPs to support ICH E14/S7B Q&amp;A compliant cardiovascular studies using a parallel ascending (<em>N</em> = 12) or an ascending (<em>N</em> = 6) dose design.</div><div>A control group of 6 animals was treated with vehicle on Days 1, 8, 15 and 22 while a treatment group of another 6 animals received vehicle on Day 1, and moxifloxacin at 25, 50 and 100 mg/kg on Days 8, 15 and 22, respectively. Analysis for the parallel ascending design was done by comparing time-matched data between the 2 groups on Days 8, 15 and 22. Analysis for the ascending design involved comparing the treatment group data on Days 8, 15, and 22 with the control data on Day 1 in the same animals. Full moxifloxacin pharmacokinetic assessment (without telemetric recording) was performed for the moxifloxacin treatment group on Days 29, 36 and 43. These data were used, in combination with the QTc data from the ascending design, for concentration-QTc modeling.</div><div>Respective maximum increases in QTc following 25, 50 and 100 mg/kg moxifloxacin were 10, 17 and 26 msec with the parallel ascending design and 9, 18 and 26 msec with the ascending design. The sensitivity (LSD) of QTc was 9.7 msec (RMSE 6.6 msec) for the parallel ascending design and 8.4 msec (RMSE 6.8 msec) for the ascending design. Using concentration-QTc analysis with a Linear Mixed Effect (LME) model, at the human critical concentration (1120 ng/mL unbound), moxifloxacin produced a 3 msec increase in ΔΔQTcI with the ascending design, with a 10 msec effect in NHP occurring at a concentration of 2983 ng/mL (unbound), that is within 2.6-fold of that of human. The LSD range for the LME model was 2.4–2.6 msec, confirming that drug exposure-based analysis is more sensitive than by-time analysis.</div><div>In conclusion, these data demonstrate adequate moxifloxacin QTc sensitivity in NHP using a parallel ascending or ascending dose design to support ICH E14/S7B Q&amp;A compliant studies.</div></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":"140 ","pages":"Article 108466"},"PeriodicalIF":2.1,"publicationDate":"2026-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148731197","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 multiparametric cardiotoxicity screening using optical electrophysiology in hiPSC-CM spheroids","authors":"Dunia Garcia Cruz,&nbsp;Ping Yan,&nbsp;Joseph Adewuyi,&nbsp;Les Loew,&nbsp;Corey Acker","doi":"10.1016/j.vascn.2026.108496","DOIUrl":"10.1016/j.vascn.2026.108496","url":null,"abstract":"<div><div>Cardiotoxicity continues to be a leading cause of drug attrition, underscoring the need for predictive, human-relevant preclinical assays. Human iPSC-derived cardiomyocytes (hiPSC-CMs) offer an improved platform over traditional hERG assays by capturing the integrated electrophysiological response of the heart. In this study, we established a 3D hiPSC-CM spheroid model compatible with ultra-high-throughput screening to assess cardiac drug liability using concurrent optical voltage and calcium recordings. Using nifedipine and E-4031 as reference compounds, we evaluated acute, chronic, and recovery responses. Multiparametric metrics such as action potential duration, beat interval, calcium transient duration, amplitude, and triangulation were quantified. The assay revealed distinct, physiologically relevant drug effects and recovery patterns. Together, these findings demonstrate that our 3D optical electrophysiology platform enables scalable, multiparametric cardiac safety testing, aligning with FDA Modernization Act 2.0 initiatives to enhance predictivity and reduce reliance on animal models.</div></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":"140 ","pages":"Article 108496"},"PeriodicalIF":2.1,"publicationDate":"2026-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148730087","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":"Emerging fluid-based safety biomarkers for advanced modality therapeutic-related neurotoxicity in nonhuman primates","authors":"Catherine F. Ruff,&nbsp;Tony Tony Kam-Thong,&nbsp;Svetlana Lyalina,&nbsp;Tien Nguyen,&nbsp;Diana Lac,&nbsp;Tanja Zabka","doi":"10.1016/j.vascn.2026.108447","DOIUrl":"10.1016/j.vascn.2026.108447","url":null,"abstract":"<div><div>There have been exciting advances in neurologic therapeutics, but their progression to clinical trials has been disappointing. Despite impressive efficacy demonstrated in preclinical studies, delayed onset of drug-related neurotoxicity has become a major obstacle for many of these novel therapeutic modalities. Neurotoxicity can be challenging to identify and neurobehavioral tests are only minimally sensitive and insufficient at detecting early neurodegeneration. Therefore, long (≥ 8-week) in vivo preclinical studies with histopathology evaluation are required, which are resource and time intensive. More efficient, sensitive and quantitative methods are needed to better identify the neurotoxic potential, both in dose level and time of onset, of drug candidates early in preclinical safety assessment. Fluid-based biomarkers have emerged as a potential tool for improving neurotoxicity detection across species. Here, we evaluated a subset of safety biomarkers for nervous system injury in preclinical toxicity studies in nonhuman primates administered a single intrathecal dose of an advanced modality therapeutic molecule with a 60 or 113 day observation period. We show dose-dependent and time-dependent increases in neurofilament light (NfL), total microtubule associated protein (tTau), and to a lesser extent ubiquitin carboxy-terminal hydrolase L1 (UCHL1) in the cerebrospinal fluid, but less compelling responses in plasma. Elevations in these biomarkers in the CSF above the receiver operator curve (ROC)-defined threshold of concern correlated with microscopic evidence of neuronal necrosis/loss, and preceded any clinical observations or abnormalities reported on neurofunctional tests. These results demonstrate that NfL and tTau are sensitive and specific safety biomarkers for advanced modality therapeutic-associated neurotoxicity in preclinical studies. Furthermore, these biomarkers have the potential to accelerate screening for drugs with neurologic indications and help create a therapeutic index for forward translation into the clinic.</div></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":"140 ","pages":"Article 108447"},"PeriodicalIF":2.1,"publicationDate":"2026-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148730174","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":"A comparative review of cardiovascular historical control data sets in dogs across three different test facilities applying similar best practice data collection and analysis procedures","authors":"Kevin Norton,&nbsp;Nacera Mella,&nbsp;Jennifer Shaffer,&nbsp;Courtney Jenkins,&nbsp;Anson Philips,&nbsp;Jared Slain,&nbsp;Jill Dalton","doi":"10.1016/j.vascn.2026.108472","DOIUrl":"10.1016/j.vascn.2026.108472","url":null,"abstract":"<div><div>We have previously reported that when applying best practice procedures for cardiovascular data collection and analysis in dogs that the effects of Moxifloxacin on QTc interval duration are comparable across different test sites even when slight differences in study designs exist and that there is minimal inter-site variability. In the current study we compared historical data sets from three different facilities, compromising of a total of 43 dog studies which represent 12, 11 and 20 studies from facilities 1, 2 and 3, respectively. Although slight differences in study design were observed across the 43 studies, each study followed a 4*4 Latin square cross over design with a minimum of 24 h of continuous blood pressure, heart rate, ECG interval duration and body temperature assessment, following each dose, using DSI™ L-series telemetry implants. The data analysis included minimal detectable difference (MDD) and root mean square error (RMSE), an indicator of variability, within each site's historical control data set. Additionally, for all 43 individual studies the least significant difference (LSD) and RMSE were compared across facilities. The MDD across all three facilities were highly consistent with an MDD of 6.4, 7.2 and 5.5 mmHg observed for mean arterial pressure, 11.4, 11.8 and 11.5 bpm for heart rate, 5.7, 6.3 and 4.8 msec for QTc; and 0.29, 0.29 and 0.23 °C for body temperature for facilities 1, 2 and 3, respectively. Additionally, there was a large degree of overlap in individual study LSD values across all facilities with median LSD values at each site of 4.7, 5.2 and 4.0 mmHg observed for mean arterial pressure, 7.6, 8.6 and 8.4 bpm for heart rate, 4.2 4.6 and 3.5 msec for QTc; and 0.20, 0.21 and 0.22 °C for body temperature for sites 1, 2 and 3, respectively. This data set further illustrates that when best practice methods are followed for data collection and analysis, model sensitivity is highly consistent and reproducible across different facilities.</div></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":"140 ","pages":"Article 108472"},"PeriodicalIF":2.1,"publicationDate":"2026-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148730672","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":"Assessment of sensitivity of invasive telemetry to detect a prolongation of the QTc interval in the conscious Göttingen minipig using moxifloxacin as a positive control","authors":"Coralie Perret,&nbsp;David Coquard,&nbsp;Marie Latzko,&nbsp;Léa Di Gennaro,&nbsp;Stéphane Baudet","doi":"10.1016/j.vascn.2026.108473","DOIUrl":"10.1016/j.vascn.2026.108473","url":null,"abstract":"<div><div>ICH E14/S7B guidelines evolved to re-consider the need to conduct Thorough QT trials (TQT) to evaluate the clinical “QT risk”, based on the outcome of non-clinical ECG investigations in telemetered non-rodents (“CV assay”), using an integrated risk assessment. In addition to generate standard QT/QTc interval duration, CV assays should now provide values for Least Significant Difference (LSD) and Root Mean Square Error (RMSE) as indices of study sensitivity, using ANOVA on super-interval (SI), performed on time-matched, vehicle-subtracted QTc values (DQTc), and a cross-over, Latin square (XO-LS) design. If neither QTc prolongation nor hERG inhibition are detected, with demonstration of suitable study sensitivity, drug developers may apply for a waiver, for not conducting TQT, thereby reducing drug exposure of healthy volunteers and allowing faster progression of promising therapeutics. Minipigs were selected as an increasingly used non-rodent species, better ethically accepted than NHPs and dogs. However, by comparison with ample information gathered from the latter two species, data on the ability of minipig CV assays to detect QTc prolongation using a XO-LS design are scarce. Therefore, in agreement with our Ethical Committee, we applied XO-LS design to test QT‑prolonging effects of moxifloxacin at 10, 30, and 100 mg/kg, given orally and once weekly to four M11-implanted male Göttingen minipigs. Arterial blood pressure, body temperature, ECG, and activity were recorded for 1.5 h predose and 24 h postdose. Moxifloxacin dose‑dependently prolonged Holzgrefe-corrected QT interval (QTcH) for 24 h, with maximum increases in DQTcH of 15 msec at 10 mg/kg (SI: [0 h–4 h]), 20 msec at 30 mg/kg (SI: [8 h–18 h]) and 71 msec at 100 mg/kg (SI: [8 h–18 h]); in-study sensitivity values for DQTcH LSD and RMSE were 24 msec and 13 msec, respectively. Moxifloxacin exerted no relevant effects on the other telemetry parameters. This study demonstrates the capacity of our facility to conduct a cross-over, Latin square study design with a sensitivity high enough to detect drug-induced QTc prolongation in minipigs, and further supports their use for CV assays, as an alternative species to dogs and NHPs, in the context of regulatory evolutions about human QT risk evaluation.</div></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":"140 ","pages":"Article 108473"},"PeriodicalIF":2.1,"publicationDate":"2026-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148730673","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":"Delayed IKs/Ikr trafficking effect inducing Torsade de Pointes in dogs","authors":"Henk J. van der Linde,&nbsp;David J. Gallacher","doi":"10.1016/j.vascn.2026.108475","DOIUrl":"10.1016/j.vascn.2026.108475","url":null,"abstract":"<div><div>A test compound induced extreme long QT intervals with negative T-waves in a 1-month repeated-dose oral toxicity study in the beagle dog. The purpose of this follow-up study was to determine whether the QT effects observed at 28-days post-dose were I_Kr and/or I_Ks ion channel related in a repeat dose telemetry safety study. Six conscious female beagle dogs were administered at daily dosages of 400 mg/kg body weight/day and a similar group of four dogs received the vehicle (PEG400), according to the same regimen. Relative to the vehicle, the compound did not induce dose-dependent or physiologically relevant effects on heart rate, RR interval, PQ interval, QRS interval, S1 and S2 amplitude (from a cardiophonogram), S1/S2 index and QS2 interval on any of the dosing days. Moreover, the compound did not induce any relevant effects on QT (QTc) up to Day 3 of dosing. Starting at Day 4, and maximal at Day 9, QT (QTc) was massively increased (Δ + 54%). In addition, starting at Day 4 a decrease in the QS2 interval (Δ-110 ms) was noted and T-waves were inverted from positive to negative in all dogs. Unfortunately, one dog was found dead in the morning at Day 8 and a second dog died suddenly after a staff member entered the animal house at Day 9. Dosing was immediately stopped, and remaining dogs were transferred into the anesthetized dog lab for a b-adrenoceptor stimulation, to investigate the potential risk for Torsades de Pointes (TdP) induction. After stabilization under anesthesia, all dogs received isoproterenol at a dosage of 5 μg/kg i.v. In the vehicle treated dogs (<em>n</em> = 4) isoproterenol induced an increase in heart rate, contractility and some ectopic beats and/or ventricular tachycardia, but in all compound-treated dogs (<em>n</em> = 3) isoproterenol, induced TdP leading to ventricular fibrillation and sudden cardiac death. The test compound induced a massive delayed QT (QTc) prolongation (at levels beyond which classical hERG blockers would produce) upon which sympathetic stimulation induced TdP, a strong indication that the prolongation is induced primarily by interfering with I_Ks ion channel trafficking.</div></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":"140 ","pages":"Article 108475"},"PeriodicalIF":2.1,"publicationDate":"2026-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148730677","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":"A retrospective review of multi-lead jacketed telemetry collections in 2023 for quality endpoints","authors":"Katarina S. Cummins,&nbsp;David Holdsworth,&nbsp;Steve Tichenor,&nbsp;Jill Dalton","doi":"10.1016/j.vascn.2026.108470","DOIUrl":"10.1016/j.vascn.2026.108470","url":null,"abstract":"<div><div>With the growing number of biologics being developed in recent years, the use of jacketed telemetry within general toxicology studies has become an increasingly popular alternative to standalone cardiovascular evaluations. However, this approach has some drawbacks, including the potential for reduced data quality and/or data loss compared to implanted studies. This study evaluated the quality of all ECG datasets collected in 2023 at our facility utilizing a multi‑lead Jacketed External Telemetry (DSI-JET™) device. Data quality was determined based on 1) occurrences of signal loss, 2) use of only measured (non-calculated) leads for analysis, and 3) use of additional lead analysis due to an inability to adequately analyze data across all intervals of a study using a single lead. Additionally, the data were analyzed statistically to determine if bodyweight, species, or analyst contributed to the lead choice. Signal loss occurrence was 10.96%, a single measured lead was used for 97.64% of cases (Lead I: 57.25%; Lead II: 40.39%), and additional lead analysis was limited (1.57%). It was also observed that if a multi‑lead JET device lost connection to a particular lead during collection, another lead could potentially be analyzed, which could limit the rate of total signal loss (i.e., no usable data). Additionally with a multi‑lead view, arrhythmias are represented in all leads which allows for lead comparison to rule out noise or verify arrhythmic data during qualitative assessment. No statistically significant differences in bodyweight or species were observed for lead selection; however, the analyst designation was statistically significant for lead choice decision (<em>p</em> <em>&lt;</em> <em>0.01</em>). The minimal signal loss and high usage of Lead I and Lead II for data analysis in 2023 support consistent high-quality JET data output to meet cardiovascular safety pharmacology requirements when incorporated into toxicology and alternative study designs. This study shows that usage of multi‑lead JET provides advantages that single lead devices do not have, and lead choice is generally based on discretion of trained individuals instead of bodyweight or species.</div></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":"140 ","pages":"Article 108470"},"PeriodicalIF":2.1,"publicationDate":"2026-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148730758","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":"Recording ten-fold larger IKr conductances with automated patch clamping using equimolar Cs+ solutions","authors":"Meye Bloothooft,&nbsp;Bente Verbruggen,&nbsp;Fitzwilliam Seibertz,&nbsp;Marcel A.G. Van der Heyden,&nbsp;Niels Voigt,&nbsp;Teun P. De Boer","doi":"10.1016/j.vascn.2026.108512","DOIUrl":"10.1016/j.vascn.2026.108512","url":null,"abstract":"<div><div>The rapid delayed rectifier potassium current (I_Kr) is important for cardiac repolarization and is most often involved in drug-induced arrhythmias. However, accurately measuring this current can be challenging in human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes because of its small current density. Interestingly, the ion channel conducting I_Kr, hERG channel, is not only permeable to K⁺ ions, but also to Cs⁺ ions when present in equimolar concentrations inside and outside of the cell.</div><div>In this study I_hERG was measured from Chinese Hamster Ovary (CHO)-hERG cells using either Cs⁺ or K⁺ as charge carrier. Equimolar Cs⁺ has been used in literature in manual patch-clamp experiments, here we apply this approach using automated patch-clamp systems. Four different (pre)clinical drugs were tested to compare their effects on Cs⁺- and K⁺ based currents.</div><div>Using equimolar Cs⁺ solutions gave rise to approximately ten-fold larger hERG currents. Comparison of Cs⁺-and K⁺-mediated currents upon application of dofetilide, desipramine, moxifloxacin, or LUF7244 revealed many similarities in inhibition or activation properties of the drugs studied. Using equimolar Cs⁺ solutions gave rise to approximately ten-fold larger hERG conductances. In hiPSC-CM the Cs⁺-based conductance is larger compared to known K⁺-based conductance and the Cs⁺ hERG conductance can be inhibited similarly to the K⁺-based conductance.</div><div>Using equimolar Cs⁺ instead of K⁺ for I_hERG measurements in an automated patch-clamp system gives rise to a new method by which, for example, quick scans can be performed on effects of drugs on hERG currents. This application is specifically relevant when such experiments are performed using cells which express small I_Kr current densities in combination with small membrane capacitances.</div></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":"140 ","pages":"Article 108512"},"PeriodicalIF":2.1,"publicationDate":"2026-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148730713","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":"Live tracking of cellular metabolic activation using a fluorescent AMPK biosensor for drug screening and modulation of muscle function","authors":"Branimir Berecic,&nbsp;Wolfram-Hubertus Zimmermann,&nbsp;Malte Tiburcy","doi":"10.1016/j.vascn.2026.108514","DOIUrl":"10.1016/j.vascn.2026.108514","url":null,"abstract":"<div><div>As a central regulator of energy homeostasis, the AMP-activated protein kinase (AMPK) controls the cellular response to metabolic stresses such as nutrient starvation, inhibition of the mitochondrial respiratory chain and protein recycling through autophagy. AMPK activity can be increased through indirect and direct stimulation, but specifically in muscle the physiological effects may vary with dose and treatment duration. We hypothesized that time and concentration dependent AMPK activation could be assessed using a fluorescent biosensor.</div><div>We utilized a ratiometric biosensor, whose fluorescence intensity ratio changes depending on AMPK phosphorylation status. Biosensor activity and the corresponding increase in phosphorylated AMPK abundance were validated in HEK cells by testing a range of concentrations of 2-deoxy-<span>d</span>-glucose (0.1–100 μmol/L), a potent indirect activator of AMPK. Further, the efficacy of indirect and novel direct AMPK activating compounds was tested to determine the physiological concentration ranges and limits of the temporal patterns of their activity. The direct AMPK activators, O-304 and MK8722, elicited a fast change in biosensor fluorescence ratio (O-304 time to 50% activity = 3.9 ± 1.23 min [<em>n</em> = 9], MK8722 time to 50% activity = 3.07 ± 1.08 min [n = 9]) with an increase in fluorescence intensity that changed with increasing drug concentrations (O-304 up to 67% ± 17 [n = 9], MK8722 up to 49% ± 12 [n = 9] increase in fluorescence intensity). The data also showed a correlation between fluorescence intensity and p-AMPK abundance across the tested concentrations. The indirect activator, metformin, had a delayed effect on biosensor fluorescence and only at high concentrations (300 μmol/L). From the tested compounds, the direct activators O-304 and MK8722 were chosen for screening in iPS derived cardiomyocytes and engineered heart muscle tissues (EHM). These screens indicate two distinctly different effects on muscle function, with MK8722 having a reversible concentration-dependent effect on muscle contractility (% change vs vehicle, O-304, 10 μmol/L = −2.7% ± 9.8 [<em>n</em> = 10], MK8722, 10 μmol/L = −100% [n = 10], MK8722, 1 μmol/L = −39.8% ± 8.95 [n = 10]).</div><div>We established a new approach to screening metabolic states using a novel AMPK biosensor in human cells and tissues to enable screening of AMPK activators that may be beneficial in muscle disease.</div></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":"140 ","pages":"Article 108514"},"PeriodicalIF":2.1,"publicationDate":"2026-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148730714","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 cardiac liability of compounds using new approach methodologies (NAMs)","authors":"Amit Chouhan,&nbsp;Eddie Burns,&nbsp;Stefano Stabilini,&nbsp;Russell Burley,&nbsp;Dario Magnani,&nbsp;Kevin Norton,&nbsp;Mariangela Iovino","doi":"10.1016/j.vascn.2026.108505","DOIUrl":"10.1016/j.vascn.2026.108505","url":null,"abstract":"<div><div>Animal studies have been used extensively to gain information about the toxicity of new compounds. Although these studies have proved beneficial, they are limited by translational challenges at times. To overcome limitations associated with animal studies, there has been a growing interest to develop faster, less expensive, and more informative new approach methodologies (NAMs). Comprehensive <em>in vitro</em> Proarrhythmia assay (CiPA) initiative provides a potential NAMs for assessment of the proarrhythmic potential of new drugs that has improved specificity compared with the hERG assay plus Thorough QT study.</div><div>To assess cardiac liability of CiPA validated compounds in acute (30 min) and prolonged (24 h) exposure conditions, using human induced pluripotent stem cells (hiPSCs)-derived cardiomyocytes (CMs) and multi-electrode array (MEA) platform (NAMs).</div><div>Commercially available hiPSC-derived ventricular CMs were used for this study. hiPSC-derived CMs were cultured for up to 10 days using standard culture medium and sterile conditions. Cardiac activity was recorded every other day, starting at Days in vitro (DIV)- 3. Data were processed using the cardiac analysis tool (Axion BioSystems) and GeneData software.</div><div>We evaluated cardiac liability, upon acute and prolonged exposure of multiple CiPA validated compounds (Bepridil, Quinidine, Ondansetron &amp; Verapamil) linked to low, intermediate, and high torsades de pointes (TdP) risk categories. Data suggests, acute and prolonged exposure of both Quinidine (≥3 μM) &amp; Ondansetron (≥3 μM) results in arrhythmias. Furthermore, acute exposure of certain CiPA validated compounds (Bepridil, 1 μM) can inhibit spontaneous hiPSCs-derived CMs contractions, that recovers upon prolonged exposure, enabling cardiac liability assessment.</div><div>We successfully demonstrate that New Approach Methodologies (NAMs) incorporating hiPSCs-derived CMs, and MEA assay provides a valuable platform to determine cardiac liability of small molecules both in acute and prolonged exposure conditions. This in-vitro platform serves as a translational and accessible resource for determining safety pharmacology of novel compounds during drug discovery efforts.</div></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":"140 ","pages":"Article 108505"},"PeriodicalIF":2.1,"publicationDate":"2026-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148730744","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}