Journal of pharmacological and toxicological methods最新文献

{"title":"Challenges in adapting a conscious dog haemodynamic systems model to an anaesthetised dog protocol","authors":"Joanne Mahmud ,&nbsp;Chris E. Pollard ,&nbsp;Tariq Abdulla ,&nbsp;Barira Islam ,&nbsp;Yevgeniya E. Koshman ,&nbsp;Michael K. Pugsley ,&nbsp;Will S. Redfern","doi":"10.1016/j.vascn.2025.107831","DOIUrl":"10.1016/j.vascn.2025.107831","url":null,"abstract":"<div><div>Previously¹, the cardiovascular-contractility systems model described by Fu et al.² was evaluated and extended using Simcyp™ Designer, a graphical interface platform and physiologically-based pharmacokinetic tool. Model outputs replicated published dog telemetry data¹. In the present study, the model was adapted for use in pentobarbital-anaesthetised beagle dogs, utilizing cardiovascular (CV) and exposure data for atenolol and atropine³. Model adaptations included removal of circadian rhythm, attenuation of baroreflex negative feedback, and development of a pharmacokinetic model in Simcyp™ Designer to accommodate intravenous dose escalation and hysteresis. Model simulations and experimental data for atenolol (0.3, 1, and 3 mg/kg/30 min) showed a decrease in dP/dt_max from 2300 to 1800 mmHg/s and mean arterial pressure (MAP) from 120 to 110 mmHg. The model predicts a decrease in heart rate (HR) from 110 to 85 bpm; however, bradycardia was not observed experimentally. For atropine (0.01, 0.03, 0.1 mg/kg/30 min), model outputs and experimental data displayed an increase in HR (124 to 164 bpm) and dP/dt_max (2650 to 3000 mmHg/s). While experimental MAP data decreased from 130 to 120 mmHg, model outputs predicted an increase from 105 to 130 mmHg. Several challenges were encountered during the development of the anaesthetised systems model. Firstly, the study design for the anaesthetised dog differs markedly from that of conscious dog telemetry. Secondly, the extent to which the baroreceptor reflex is attenuated by the anesthesia is unknown. Furthermore, the Fu et al. model² employs antagonist K_d values to predict haemodynamic effects, rather than <em>in vivo</em> EC₅₀ values. Finally, the decrease in HR predicted by the model following atenolol administration was not observed in anaesthetised dogs, possibly due to low resting cardiac sympathetic tone under the anesthesia. Despite these limitations, the adapted Fu et al.² model has the potential to maximise the use of anaesthetised CV dog data. Specifically: to interpolate CV effects at intermediate dose levels, derive threshold plasma concentrations for detectable CV effects, and predict the CV effects at doses which cannot be tolerated.</div><div>1. Mahmud et al. (2023) SPS Meeting, Brussels, Belgium</div><div>2. Fu et al. (2022) <span><span>https://doi.org/10.1002/psp4.12774</span><svg><path></path></svg></span></div><div>3. Antic et al. (2024) <span><span>https://doi.org/10.1016/j.vascn.2024.107497</span><svg><path></path></svg></span></div></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":"135 ","pages":"Article 107831"},"PeriodicalIF":1.8,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145095004","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":"Sensitivity of latin square and ascending dose designs for in vivo QT moxifloxacin positive control to support ICH E14/S7B studies","authors":"Rachael Hardman ,&nbsp;Joyce Obeng ,&nbsp;Jill Nichols ,&nbsp;Karim Melliti","doi":"10.1016/j.vascn.2025.107779","DOIUrl":"10.1016/j.vascn.2025.107779","url":null,"abstract":"<div><div>The Latin square crossover is recommended as best practice for in-vivo QT studies. However, an escalating design may instead be required due to compound long half-life, insufficient toxicity data to inform dose level selection, a need to build up tolerance to the test article to enable safe dosing, or logistics (e.g. avoiding use of multiple simultaneous inhalation dose generation systems thereby reducing risk of contamination). We have performed ICH E14/S7B Q&amp;A compliant in vivo QT validation studies with oral moxifloxacin using both a Latin square and an ascending dose design in primate (<em>n</em> = 4), dog (n = 4 for Latin square and <em>n</em> = 6 for ascending dose design) and minipig (<em>n</em> = 8 for Latin square and <em>n</em> = 5 for ascending dose design). The objective of this work was to compare the sensitivity of the dosing designs and to generate positive moxifloxacin QTc data in support of ICH E14/S7B studies when using an ascending dose design. Increases in QTc at the highest doses for the Latin square and ascending dose designs were + 30 and + 26 msec in primate, +40 and + 34 msec in dog, +86 and + 67 msec in minipig, respectively. Root mean square error (RMSE) values ranged from 5.9 to 10.8 msec for Latin square and 4.1 to 7.3 msec for ascending dose in all species. The smallest statistically detectable difference (SSDD) for the Latin square and ascending dose designs were 10 and 6.6 msec in primate, 10.2 and 5.4 msec in dog, and 11.4 and 10 msec in minipig, respectively. While the SSDD values cannot directly be compared due to the differing N values for dog and minipig, SSDD values were generally low for all species and study designs (6.6–11.4 msec). In conclusion, these data demonstrate that the sensitivity to detect a moxifloxacin-induced increase in QTc was similar with use of either an ascending dose or Latin square design. However, it is important to note that day effect is confounded with treatment effect in an ascending dose design, risking bias in the estimation of a treatment-related effect. Therefore, while sensitivity values were similar between designs, when feasible, Latin square is preferred over ascending dose.</div></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":"135 ","pages":"Article 107779"},"PeriodicalIF":1.8,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145095211","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 the effects of moxifloxacin in NHP at four different test sites","authors":"Kevin Norton ,&nbsp;Steve Tichenor ,&nbsp;Abdel El Amrani ,&nbsp;Jared Slain ,&nbsp;Nacera Mella ,&nbsp;Steve Denham ,&nbsp;Matt St Peter ,&nbsp;Jill Dalton","doi":"10.1016/j.vascn.2025.107780","DOIUrl":"10.1016/j.vascn.2025.107780","url":null,"abstract":"<div><div>With adoption of the ICH E14/S7B Q&amp;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 <em>in vivo</em> 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.</div></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":"135 ","pages":"Article 107780"},"PeriodicalIF":1.8,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145095214","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 the effects of moxifloxacin in dogs at four different test sites","authors":"Kevin Norton ,&nbsp;Michael Scott ,&nbsp;Abdel El Amrani ,&nbsp;Jared Slain ,&nbsp;Nacera Mella ,&nbsp;Steve Denham ,&nbsp;Matt St Peter ,&nbsp;Jill Dalton","doi":"10.1016/j.vascn.2025.107781","DOIUrl":"10.1016/j.vascn.2025.107781","url":null,"abstract":"<div><div>With adoption of the ICH E14/S7B Q&amp;A best practice guidance, non-clinical cardiovascular 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 <em>in vivo</em> 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 dogs, from four test sites. All four laboratories administered moxifloxacin at 10, 30 and 90 mg/kg by oral administration and evaluated cardiovascular parameters for 24 h post administration. The study designs between labs differed slightly, with test sites 1 and 2 utilizing a traditional 4*4 cross-over design in a single housing paradigm, test site 3 utilizing a 4*4 cross-over design with paired housing, and test site 4 utilizing a modified cross-over design with only two dose levels assessed per day. Additionally test site 1, used female animals, with other sites using males. Concentration-QTc modeling was also conducted at two sites with minor site-to-site variances in the blood sample collection methods and timing observed. All sites illustrated comparable dose dependent increases in QTc prolongation. Administration at 90 mg/kg resulted in QTc prolongation of up to 30.5, 35.6 33.2 and 31.2 msec, relative to control, at test sites 1, 2, 3 and 4 respectively. Whilst a review of study specific least significant difference, ranged from 6.4 to 11.1 msec, and residual error ranged from 4.1 to 6.4 msec; with conc-QT modeling, at test site 1 and 4,indicating that concentration ranges of 2867 to 2937 ng/ml would be expected to induce a 10-msec prolongation. These results indicate that when following best practice methodologies, dog CV models are highly consistent, independent of laboratory. This suggests that repeated administration of moxifloxacin across all laboratories is not essential and reliance on historical data sets alone should suffice to confirm model suitability.</div></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":"135 ","pages":"Article 107781"},"PeriodicalIF":1.8,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145095217","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":"Analysis of baroreflex and heart rate variability – Tools in cardiovascular pre-clinical safety studies","authors":"Olivera Antic ,&nbsp;Yevgeniya E. Koshman ,&nbsp;Amanda S. Wilsey ,&nbsp;Brandan M. Bird ,&nbsp;Geena Jasiek ,&nbsp;Rebecca Kohnken ,&nbsp;Scott W. Mittelstadt ,&nbsp;Harald M. Stauss ,&nbsp;Charles M. Foley","doi":"10.1016/j.vascn.2025.107783","DOIUrl":"10.1016/j.vascn.2025.107783","url":null,"abstract":"<div><div>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.</div></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":"135 ","pages":"Article 107783"},"PeriodicalIF":1.8,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145095276","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":"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","authors":"Kara L. Gordon,&nbsp;Christine G. Rines,&nbsp;Nicole A. Suarez,&nbsp;Alyson Smith,&nbsp;Jeff Price,&nbsp;Patrick McDonough","doi":"10.1016/j.vascn.2025.107843","DOIUrl":"10.1016/j.vascn.2025.107843","url":null,"abstract":"<div><div>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.</div></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":"135 ","pages":"Article 107843"},"PeriodicalIF":1.8,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145095284","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":"Exploratory continuous interstitial glucose monitoring using the FreeStyle Libre 2 device in cynomolgus monkeys","authors":"Julia Popp ,&nbsp;Cassandra O'Hara ,&nbsp;J.E. Bernal ,&nbsp;Jackson Kalanzi ,&nbsp;Mariusz Lubomirski ,&nbsp;Dina Andrews-Cleavenger ,&nbsp;J. Karmel ,&nbsp;K.A. Henderson","doi":"10.1016/j.vascn.2025.107776","DOIUrl":"10.1016/j.vascn.2025.107776","url":null,"abstract":"<div><div>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.</div></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":"135 ","pages":"Article 107776"},"PeriodicalIF":1.8,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145095340","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":"HERG block in HEK cells incorporated in real time into computer models of in silico cardiac repolarization using dynamic clamp","authors":"Mark W. Nowak ,&nbsp;Brian K. Panama ,&nbsp;Leigh Korbel ,&nbsp;Michael Hines ,&nbsp;Nicholas T. Carnavale ,&nbsp;Randall L. Rasmusson ,&nbsp;Glenna C.L. Bett","doi":"10.1016/j.vascn.2025.107819","DOIUrl":"10.1016/j.vascn.2025.107819","url":null,"abstract":"<div><div>Using dynamic clamp in Synthetic Cell Mode, we input experimentally-obtained HERG current, in real time, into <em>in-silico</em> cardiomyocytes. <em>In-silico</em> models were either generated by the dynamic clamp system or by interfacing with NEURON software (<span><span>www.neuron.yale.edu/neuron/</span><svg><path></path></svg></span>). The <em>in-silico</em> 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 <em>in-silico</em> cardiomyocytes. The objective was to examine the experimental effect of HERG block by dofetilide on AP morphology in the <em>in-silico</em> 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 <em>in-silico</em> ventricular cardiomyocyte consisting of electronically-expressed I_Na, I_K1, I_KS, I_{Ca_L,} and I_to or an <em>in-silico</em> 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 <em>in-silico</em> 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 (<em>n</em> = 6). For the <em>in-silico</em> ventricular cardiomyocytes, dofetilide prolonged the AP (APD90 baseline: 442 ± 21 ms, 100 nM: 974 ± 104 ms, <em>p</em> &lt; 0.01, n = 6). For the <em>in-silico</em> 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, <em>p</em> &lt; 0.001, n = 6). Using dynamic clamp in Synthetic Cell Mode, experimentally-expressed ion channel currents can be incorporated in real time into <em>in-silico</em> 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.</div></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":"135 ","pages":"Article 107819"},"PeriodicalIF":1.8,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145094612","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":"Effects of temperature, compound concentration verification, and internal fluoride on hERG","authors":"Aimee L. Bielinski,&nbsp;Mark T. Zafiratos,&nbsp;Andrew S. Zielonka,&nbsp;Ruth L. Martin","doi":"10.1016/j.vascn.2025.107823","DOIUrl":"10.1016/j.vascn.2025.107823","url":null,"abstract":"<div><div>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 &gt;5 min). At 25 °C, in the presence of internal fluoride, we saw a range of targeted IC₅₀s from &lt;1 to &gt;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 &lt;1 μM to &gt;30 μM and with correction for verification of concentrations was 0.2 μM to &gt;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.</div></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":"135 ","pages":"Article 107823"},"PeriodicalIF":1.8,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145094723","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":"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","authors":"Jean-Marie Chambard ,&nbsp;Tanya Goncalves ,&nbsp;Michel de Waard ,&nbsp;Michael Kurz ,&nbsp;Stefan De Waard ,&nbsp;Jerome Montnach ,&nbsp;Francoise Chesney ,&nbsp;Remy Beroud ,&nbsp;Denis Servent ,&nbsp;Michel Partiseti ,&nbsp;Evelyne Benoit","doi":"10.1016/j.vascn.2025.107811","DOIUrl":"10.1016/j.vascn.2025.107811","url":null,"abstract":"<div><div>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 <em>Poecilotheria subfusca</em> 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.</div></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":"135 ","pages":"Article 107811"},"PeriodicalIF":1.8,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145094837","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}