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INTRODUCTION: The occurrence of early afterdepolarizations (EADs) has been related to the incidence of torsades de pointes in drug-induced long QT (LQT). The generation of EADs may be facilitated by Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase). METHODS AND RESULTS: In the present study, we investigated a possible involvement of Ca(2+)/Calmodulin dependent protein kinase in the generation of sparfloxacin-induced EADs in isolated rabbit Purkinje fibers by means of a calmodulin antagonist W-7. EADs were evident in 8 of the 10 preparations perfused with sparfloxacin at 1 x 10(-4) M and stimulated at 0.2 Hz. The induction of EADs by sparfloxacin was associated with a large prolongation of the duration of the action potential (APD), an increase in the triangulation, and the short-term instability of the repolarization. CaM kinase blockade with the calmodulin antagonist W-7 inhibited sparfloxacin-induced EADs in a concentration-dependent manner (EADs were induced in 3 of 10, 1 of 10, and 0 of 8 preparations in the presence of W-7 at 5 x 10(-7) M, 5 x 10(-6) M, and 5 x 10(-5) M, respectively; P < 0.01 at 5 x 10(-6) M and 5 x 10(-5) M). The inhibition of sparfloxacin-induced EADs by W-7 at 5 x 10(-7) M and 5 x 10(-6) M was associated with a significant decrease in the beat-to-beat instability but not associated with a significant shortening of the APD and reduction of V(max). CONCLUSION: The present findings support the hypothesis that CaM kinase may be a proarrhythmic signaling molecule and demonstrate that CaM kinase may be involved in the generation of EADs in drug-induced LQT and enhanced beat-to-beat instability of repolarization is essential for the genesis of EADs in rabbit in vitro. [ABSTRACT FROM AUTHOR]

A Monte Carlo (MC) programme was written using the dose point kernel method to calculate doses in the roof zone of a building from nearby releases of radioactive gases. A Gaussian Plume Model (GPM) was parameterised to account for near-field building effects on plume spread and reflection from the roof. Rooftop recirculation zones and building-generated plume spread effects were accounted in a novel Dual Gaussian Plume (DGP) formulation used with the MC model, which allowed for the selection of angle of approach flow, plume release height in relation to the building and position of the release point in relation to the leading edge of the building. Three-dimensional wind tunnel concentration field data were used for the parameterisation. The MC code used the parameterised concentration field to calculate the contributions to effective dose from inhalation, cloud immersion from positron/beta particles, and gamma-ray dose for a wide range of receptor dose positions in the roof zone, including receptor positions at different heights above the roof. Broad trends in predicted radiation dose with angle of approach flow, release position in relation to the building and release height are shown. Alternative approaches for the derivation of the concentration field are discussed., (© 2024 Society for Radiological Protection. Published on behalf of SRP by IOP Publishing Limited. All rights reserved.)

Chronic lung disease (CLD) of prematurity, a common cause of morbidity and mortality in preterm-born infants, has a multifactorial aetiology. This review summarizes the current evidence for the effect of the gut and airway microbiota on the development of CLD, highlighting the differences in the early colonisation patterns in preterm-born infants compared to term-born infants. Stool samples from preterm-born infants who develop CLD have less diversity than those who do not develop CLD. Pulmonary inflammation, which is a hallmark in the development of CLD, may potentially be influenced by gut bacteria. The respiratory microbiota is less abundant than the stool microbiota in preterm-born infants. There is a lack of clear evidence for the role of the respiratory microbiota in the development of CLD, with results from individual studies not replicated. A common finding is the presence of a single predominant bacterial genus in the lungs of preterm-born infants who develop CLD. Probiotic preparations have been proposed as a potential therapeutic strategy to modify the gut or lung microbiota with the aim of reducing rates of CLD but additional robust evidence is required before this treatment is introduced into routine clinical practice.

We investigated drug-induced acute neuronal electrophysiological changes using Micro-Electrode arrays (MEA) to rat primary neuronal cell cultures. Data based on 6-key MEA parameters were analyzed for plate-to-plate vehicle variability, effects of positive and negative controls, as well as data from over 100 reference drugs, mostly known to have pharmacological phenotypic and clinical outcomes. A Least Absolute Shrinkage and Selection Operator (LASSO) regression, coupled with expert evaluation helped to identify the 6-key parameters from many other MEA parameters to evaluate the drug-induced acute neuronal changes. Calculating the statistical tolerance intervals for negative-positive control effects on those 4-key parameters helped us to develop a new weighted hazard scoring system on drug-induced potential central nervous system (CNS) adverse effects (AEs). The weighted total score, integrating the effects of a drug candidate on the identified six-pivotal parameters, simply determines if the testing compound/concentration induces potential CNS AEs. Hereto, it uses four different categories of hazard scores: non-neuroactive, neuroactive, hazard, or high hazard categories. This new scoring system was successfully applied to differentiate the new compounds with or without CNS AEs, and the results were correlated with the outcome of in vivo studies in mice for one internal program. Furthermore, the Random Forest classification method was used to obtain the probability that the effect of a compound is either inhibitory or excitatory. In conclusion, this new neuronal scoring system on the cell assay is actively applied in the early de-risking of drug development and reduces the use of animals and associated costs., Competing Interests: All the authors were employed by company Janssen Research and Development, Janssen Pharmaceutical Companies of Johnson & Johnson., (Copyright © 2024 Kreir, Putri, Tekle, Pibiri, d’Ydewalle, Van Ammel, Geys, Teisman, Gallacher and Lu.)

Background: Macrolides, including azithromycin, are increasingly used in preterm-born infants to treat Ureaplasma infections. The baseline carriage of macrolide resistance genes in the preterm stool microbiota is unknown., Objectives: Identify carriage of azithromycin resistant bacteria and the incidence of macrolide resistant genes., Methods: Azithromycin resistant bacteria were isolated from serial stool samples obtained from preterm infants (≤32 weeks' gestation) by culturing aerobically/anaerobically, in the presence/absence of azithromycin. Using quantitative PCR, we targeted 6 common macrolide resistance genes (erm(A), erm(B), erm(C), erm(F), mef(A/E), msr(A)) in DNA extracted from selected bacteria resistant to azithromycin., Results: From 89 stool samples from 37 preterm-born infants, 93.3% showed bacterial growth in aerobic or anaerobic conditions. From the 280 azithromycin resistant isolates that were identified, Staphylococcus (75%) and Enterococcus (15%) species dominated. Macrolide resistance genes were identified in 91% of resistant isolates: commonest were erm(C) (46% of isolates) and msr(A) (40%). Multiple macrolide resistance genes were identified in 18% of isolates., Conclusion: Macrolide resistance is common in the gut microbiota of preterm-born infants early in life, most likely acquired from exposure to the maternal microbiota. It will be important to assess modulation of macrolide resistance, if macrolide treatment becomes routine in the management of preterm infants., Impact Statement: Azithromycin resistance is present in the stool microbiota in the first month of life in preterm infants 91% of azithromycin resistant bacteria carried at least one of 6 common macrolide resistant genes Increasing use of macrolides in the preterm population makes this an important area of study., (© 2023. The Author(s).)

Loperamide has been a safe and effective treatment for diarrhea for many years. However, many cases of cardiotoxicity with intentional abuse of loperamide ingestion have recently been reported. We evaluated loperamide in in vitro and in vivo cardiac safety models to understand the mechanisms for this cardiotoxicity. Loperamide slowed conduction (QRS-duration) starting at 0.3 µM [~1200-fold (×) its human Free Therapeutic Plasma Concentration; FTPC] and reduced the QT-interval and caused cardiac arrhythmias starting at 3 µM (~12,000× FTPC) in an isolated rabbit ventricular-wedge model. Loperamide also slowed conduction and elicited Type II/III A-V block in anesthetized guinea pigs at overdose exposures of 879× and 3802× FTPC. In ion-channel studies, loperamide inhibited hERG (I Kr ) , I Na, and I Ca currents with IC 50 values of 0.390 µM, 0.526 µM, and 4.091 µM, respectively (i.e., >1560× FTPC). Additionally, in silico trials in human ventricular action potential models based on these IC50s confirmed that loperamide has large safety margins at therapeutic exposures (≤600× FTPC) and confirmed repolarization abnormalities in the case of extreme doses of loperamide. The studies confirmed the large safety margin for the therapeutic use of loperamide but revealed that at the extreme exposure levels observed in human overdose, loperamide can cause a combination of conduction slowing and alterations in repolarization time, resulting in cardiac proarrhythmia. Loperamide's inhibition of the I Na channel and hERG-mediated IKr are the most likely basis for this cardiac electrophysiological toxicity at overdose exposures. The cardiac toxic effects of loperamide at the overdoses could be aggravated by co-medication with other drug(s) causing ion channel inhibition.

Introduction: Cardiotoxicity is one of the leading causes of compound attrition during drug development. Most in vitro screening platforms aim at detecting acute cardio-electrophysiological changes and drug-induced chronic functional alterations are often not studied in the early stage of drug development. Therefore, we developed an assay using human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) that evaluates both drug-induced acute and delayed electrophysiological and cytotoxic effects of reference compounds with clinically known cardiac outcomes. Methods: hiPSC-CMs were seeded in 48-well multielectrode array (MEA) plates and were treated with four doses of reference compounds (covering and exceeding clinical free plasma peak concentrations -fC max values) and MEA recordings were conducted for 4 days. Functional-electrophysiological (field-potentials) and viability (impedance) parameters were recorded with a MEA machine. Results: To assess this platform, we tested tyrosine-kinase inhibitors with high-cardiac risk profile (sunitinib, vandetanib and nilotinib) and low-cardiac risk (erlotinib), as well as known classic cardiac toxic drugs (doxorubicin and BMS-986094), ion-channel trafficking inhibitors (pentamidine, probucol and arsenic trioxide) and compounds without known clinical cardiotoxicity (amoxicillin, cetirizine, captopril and aspirin). By evaluating the effects of these compounds on MEA parameters, the assay was mostly able to recapitulate different drug-induced cardiotoxicities, represented by a prolongation of the field potential, changes in beating rate and presence of arrhythmic events in acute (<2 h) or delayed phase ≥24 h, and/or reduction of impedance during the delayed phase (≥24 h). Furthermore, a few reference compounds were tested in hiPSC-CMs using fluorescence- and luminescence-based plate reader assays, confirming the presence or absence of cytotoxic effects, linked to changes of the impedance parameters measured in the MEA assay. Of note, some cardiotoxic effects could not be identified at acute time points (<2 h) but were clearly detected after 24 h, reinforcing the importance of chronic drug evaluation. Discussion: In conclusion, the evaluation of chronic drug-induced cardiotoxicity using a hiPSC-CMs in vitro assay can contribute to the early de-risking of compounds and help optimize the drug development process., Competing Interests: Authors CA, KVA, MS, MK, FT, AT, DG, and HRL were employed by Janssen R&D., (Copyright © 2023 Altrocchi, Van Ammel, Steemans, Kreir, Tekle, Teisman, Gallacher and Lu.)

Aerial seed banks facilitate population persistence by extending the temporal range of seed dispersal. Knowing the temporal range of germination will improve our understanding of the relationship between seed germination dynamics and aerial seed bank storage duration. We tested the effects of temperature (12/12 h of 5/10, 10/20, 20/30 and 25/35 °C) and light variation (12 h light/12 h darkness and 24 h darkness per day) on germination of Rumex obtusifolius L. seeds retained in an aerial seed bank for 0, 2, 4, 6, 8 and 10 months. Freshly harvested R. obtusifolius were non-dormant and exhibited germination rates of up to 92%. Overall, seeds of R. obtusifolius germinated reliably at all but the lowest temperature (5/10 °C). Seeds maintained high viability throughout the collection period, indicating that fluctuating weather conditions had little influence on seed germination. Thus, the species can maintain viable seeds in aerial storage for up to 10 months and contribute viable seeds to the soil seed bank year-round. This ability to maintain a renewed soil seed bank contributes to the species' strong resilience in colonizing disturbed areas and makes it a difficult weed to control.

Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) are currently used following the Comprehensive in vitro Proarrhythmic Assay (CiPA) initiative and subsequent recommendations in the International Council for Harmonization (ICH) guidelines S7B and E14 Q&A, to detect drug-induced cardiotoxicity. Monocultures of hiPSC-CMs are immature compared to adult ventricular cardiomyocytes and might lack the native heterogeneous nature. We investigated whether hiPSC-CMs, treated to enhance structural maturity, are superior in detecting drug-induced changes in electrophysiology and contraction. This was achieved by comparing hiPSC-CMs cultured in 2D monolayers on the current standard (fibronectin matrix, FM), to monolayers on a coating known to promote structural maturity (CELLvo™ Matrix Plus, MM). Functional assessment of electrophysiology and contractility was made using a high-throughput screening approach involving the use of both voltage-sensitive fluorescent dyes for electrophysiology and video technology for contractility. Using 11 reference drugs, the response of the monolayer of hiPSC-CMs was comparable in the two experimental settings (FM and MM). The data showed no functionally relevant differences in electrophysiology between hiPSC-CMs in standard FM and MM, while contractility read-outs indicated an altered amplitude of contraction but not changes in time course. RNA profiling for cardiac proteins shows similarity of the RNA expression across the two forms of 2D culture, suggesting that cell-to-matrix adhesion differences may explain account for differences in contraction amplitude. The results support the view that hiPSC-CMs in both 2D monolayer FM and MM that promote structural maturity are equally effective in detecting drug-induced electrophysiological effects in functional safety studies.

Incorporating native shrubs into restoration projects can improve biodiversity conservation and enhance the sustainability of ecosystem functions. Shrubs grow under different forest canopy structures, having varied microclimatic conditions according to forest type and composition. Currently, there is a lack of information on propagation from seed and planting material availability for the utilization of shrubs in forest restoration. In the present study, we evaluated the effects of temperature and light on germination of ten shrub species (Ardisia japonica, Callicarpa cathayana, Callicarpa giraldii var. subcanescens, Deutzia schneideriana, Fraxinus sieboldiana, Hydrangea chinensis, Maesa japonica, Rhododendron simsii, Spiraea japonica var. fortunei and Weigela japonica var. sinica) occurring in subtropical forests in China. No seeds of any species germinated in the coolest thermal regime (5/10 °C), while optimal temperature requirements varied from 10/20 °C to 25/35 °C. Seeds of small-seeded species had higher germination percentages in the light treatments, while larger seeds were not photoblastic. There was no relationship between germination in the light and the seed shape index. Our results may assist in identification of seed traits and suitable shrub species for restoration in specific forest types, thus aiding native forest recovery of structure and composition. Successful recovery leads to enhanced biodiversity, reestablishment of microhabitats and ecological interactions in the forest understorey., (© 2023 German Society for Plant Sciences, Royal Botanical Society of the Netherlands.)

Drug-induced seizure liability is a significant safety issue and the basis for attrition in drug development. Occurrence in late development results in increased costs, human risk, and delayed market availability of novel therapeutics. Therefore, there is an urgent need for biologically relevant, in vitro high-throughput screening assays (HTS) to predict potential risks for drug-induced seizure early in drug discovery. We investigated drug-induced changes in neural Ca 2+ oscillations, using fluorescent dyes as a potential indicator of seizure risk, in hiPSC-derived neurons co-cultured with human primary astrocytes in both 2D and 3D forms. The dynamics of synchronized neuronal calcium oscillations were measured with an FDSS kinetics reader. Drug responses in synchronized Ca 2+ oscillations were recorded in both 2D and 3D hiPSC-derived neuron/primary astrocyte co-cultures using positive controls (4-aminopyridine and kainic acid) and negative control (acetaminophen). Subsequently, blinded tests were carried out for 25 drugs with known clinical seizure incidence. Positive predictive value (accuracy) based on significant changes in the peak number of Ca 2+ oscillations among 25 reference drugs was 91% in 2D vs. 45% in 3D hiPSC-neuron/primary astrocyte co-cultures. These data suggest that drugs that alter neuronal activity and may have potential risk for seizures can be identified with high accuracy using an HTS approach using the measurements of Ca 2+ oscillations in hiPSC-derived neurons co-cultured with primary astrocytes in 2D.

Background: Functional network activity is a characteristic for neuronal cells, and the complexity of the network activity represents the necessary substrate to support complex brain functions. Drugs that drastically increase the neuronal network activity may have a potential higher risk for seizures in human. Although there has been some recent considerable progress made using cultures from different types of human-induced pluripotent stem cell (hiPSC) derived neurons, one of the primary limitations is the lack of - or very low - network activity., Method: In the present study, we investigated whether the limited neuronal network activity in commercial hiPSC-neurons (CNS.4U®) is capable of detecting drug-induced potential seizure risks. Therefore, we compared the hiPSC-results to those in rat primary neurons with known high neuronal network activity in vitro., Results: Gene expression and electrical activity from in vitro developing neuronal networks were assessed at multiple time-points. Transcriptomes of 7, 28, and 50 days in vitro were analyzed and compared to those from human brain tissues. Data from measurements of electrical activity using multielectrode arrays (MEAs) indicate that neuronal networks matured gradually over time, albeit in hiPSC this developed slower than rat primary cultures. The response of neuronal networks to neuronal active reference drugs modulating glutamatergic, acetylcholinergic and GABAergic pathways could be detected in both hiPSC-neurons and rat primary neurons. However, in comparison, GABAergic responses were limited in hiPSC-neurons., Conclusion: Overall, despite a slower network development and lower network activity, CNS.4U® hiPSC-neurons can be used to detect drug induced changes in neuronal network activity, as shown by well-known seizurogenic drugs (affecting e.g., the Glycine receptor and Na + channel). However, lower sensitivity to GABA antagonists has been observed., Competing Interests: Declaration of competing interest MK, WF, RP, ADB, AT, DJG, HRL are employees of Janssen Pharmaceutica NV. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Wetland species commonly exhibit a range of strategies to cope with water stress, either through drought tolerance or through avoidance of the period of limited water availability. Natural populations provide a genetic resource for ecological remediation and may also have direct economic value. We investigated the effects of drought stress on the seed germination of wetland species. Nineteen species were germinated in four concentrations of polyethylene glycol 6000 (PEG) and were evaluated daily (12-h light photoperiod) or after 35 days (continuous darkness) to determine seed germination under water stress. Germination percentage decreased with an increase in polyethylene glycol 6000 (PEG) concentration, but species' germination response to PEG concentration varied significantly. Seeds recovered their germinability after the alleviation of water stress, but the extent of recovery was species-dependent., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Bhatt, Daibes, Gallacher, Jarma-Orozco and Pompelli.)

Introduction: Early identification of cardiac risk is essential for reducing late-stage attrition in drug development. We adapted the previously published cardiac hazard risk-scoring system using a calcium transient assay in human stem cell-derived CMs for the identification of cardiac risks recorded from the new hiPSC-CM line and investigated its predictivity and translational value based on the screening of a large number of reference and proprietary compounds. Methods: Evaluation of 55 reference drugs provided the translation of various pharmacological effects into a single hazard label (no, low, high, or very high hazard) using a Ca 2+ -sensitive fluorescent dye assay recorded by -by FDSS/µCell Functional Drug Screening System (Hamamatsu on hiPSC-CM line (FCDI iCell Cardiomyocytes 2 ). Results: Application of the adapted hazard scoring system in the Ca 2+ transient assay, using a second hiPS-CM line, provided comparable scoring results and predictivity of hazard, to the previously published scoring approach, with different pharmacological drug classes, as well as screening new chemical entities (NCE's) using a single hazard label from four different scoring levels (no, low, high, or very high hazard). The scoring system results also showed minimal variability across three different lots of hiPSC-CMs, indicating good reproducibility of the cell line. The predictivity values (sensitivity and specificity) for drug-induced acute cardiac risk for QT-interval prolongation and Torsade de pointes (TdPs) were >95% and statistical modeling confirmed the prediction of proarrhythmic risk. The outcomes of the NCEs also showed consistency with findings in other well-established in vitro and in vivo cardiac risk assays. Conclusion: Evaluation of a large list of reference compounds and internal NCEs has confirmed the applicability of the adaptations made to the previously published novel scoring system for the hiPSC-CMs. The validation also established the predictivity for drug-induced cardiac risks with good translation to other established preclinical in vitro and in vivo assays, confirming the application of this novel scoring system in different stem cell-CM lines for early cardiac hazard identification., Competing Interests: Authors HL, MK, VK, FT, DG, AT and DG are employed by Janssen Pharmaceutica NV., (Copyright © 2022 Lu, Kreir, Karel, Tekle, Geyskens, Teisman and Gallacher.)

Plant species of the Brazilian Caatinga experience seasonal wet and dry extremes, requiring seasonally different leaf characteristics for optimizing water availability. We investigated if Croton blanchetianus Baill exhibits leaf morphoanatomical traits across seasons and positioning in sunlight/natural shade. Leaves of ten 1-3 m tall plants in full sunlight and ten in natural shade were assessed in May, July (wet season), October and December (dry season) 2015 for gas exchange, leaf size, lamina and midrib cross sections (14 parameters), and chloroplast structure (5 parameters). Net photosynthesis was greater during the wet season (21.6 µm -2  s -1 ) compared to the dry season (5.8 µm -2  s -1 ) and was strongly correlated with almost all measured parameters (p < 0.01). Shaded leaves in the wet season had higher specific leaf area (19.9 m 2  kg -1 in full-sun and 23.1 m 2  kg -1 in shade), but in the dry season they did not differ from those in full sun (7.5 m 2  kg -1 and 7.2 m 2  kg -1 ). In the wet season, the expansion of the adaxial epidermis and mesophyll lead to larger and thicker photosynthetic area of leaves. Furthermore, chloroplast thickness, length and area were also significantly larger in full sunlight (2.1 μm, 5.1 μm, 15.2 μm 2 ; respectively) and shaded plants (2.0 μm, 5.2 μm, 14.8 μm 2 ; respectively) during wetter months. Croton blanchetianus exhibits seasonal plasticity in leaf structure, presumably to optimize water use efficiency during seasons of water abundance and deficit. These results suggest that the species is adaptable to the increased drought stress projected by climate change scenarios., (© 2022. The Author(s).)

Human-induced pluripotent stem cell (hiPSC) and stem cell (hSC) derived cardiomyocytes (CM) are gaining popularity as in vitro model for cardiology and pharmacology studies. A remaining flaw of these cells, as shown by single-cell electrophysiological characterization, is a more depolarized resting membrane potential (RMP) compared to native CM. Most reports attribute this to a lower expression of the Kir2.1 potassium channel that generates the I K1 current. However, most RMP recordings are obtained from isolated hSC/hiPSC-CMs whereas in a more native setting these cells are interconnected with neighboring cells by connexin-based gap junctions, forming a syncytium. Hereby, these cells are electrically connected and the total pool of I K1 increases. Therefore, the input resistance (Ri) of interconnected cells is lower than that of isolated cells. During patch clamp experiments pipettes need to be well attached or sealed to the cell, which is reflected in the seal resistance (Rs), because a nonspecific ionic current can leak through this pipette-cell contact or seal and balance out small currents within the cell such as I K1 . By recording the action potential of isolated hSC-CMs and that of hSC-CMs cultured in small monolayers, we show that the RMP of hSC-CMs in monolayer is approximately -20 mV more hyperpolarized compared to isolated cells. Accordingly, adding carbenoxolone, a connexin channel blocker, isolates the cell that is patch clamped from its neighboring cells of the monolayer and depolarizes the RMP. The presented data show that the recorded RMP of hSC-CMs in a syncytium is more negative than that determined from isolated hSC/hiPSC-CMs, most likely because the active pool of Kir2.1 channels increased.

Introduction: People with epilepsy are at heightened risk of sudden death compared to the general population. The leading cause of epilepsy-related premature mortality is a sudden unexpected death in epilepsy (SUDEP). The mechanism of SUDEP remains largely unresolved and the lack of preclinical models to study the potential mechanism underlying SUDEP is a problem., Method: By combining electroencephalographic (EEG) and electrocardiogram (ECG) measurements within a well described LQT1 dog model, we investigated the effect of the proconvulsive compound pentylenetetrazol (PTZ), and its link to the induction of Torsades de Pointes (TdP)., Results: Pre-treatment with the potent and selective I Ks blocker JNJ 282 induced a pronounced QT (QTc) prolongation in anaesthetized dogs (Long QT syndrome type 1 or LQT1 group) compared to dogs that were not treated (control group). Subsequent PTZ administration induced spiking on the EEG signal and seizures in both groups, but only R-on-T, salvo and TdP were observed in dogs of the LQT1 group., Conclusion: Our results show that a proconvulsive drug can trigger TdP-like cardiac arrhythmias, in conditions of compromised repolarization in the heart (I ks blockade). In man, TdP arrythmia's can often lead to ventricular fibrillation (VF) and sudden death. This observation suggests that long QT-intervals (genetic or drug induced) could potentially be one of the risk factors for SUDEP in epileptic patients., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Drugs that prolong QT may cause torsade de pointes (TdP). However, translation of nonclinical assessment of QT prolongation or hERG channel, targeted by QT-prolonging drugs, into clinical TdP risk has been insufficient to date. In this blinded study, we confirmed the utility of a Normalized TdP Score System in predicting drug-induced TdP risks among 34 drugs, including 28 with low, intermediate, and high TdP risks under the Comprehensive In Vitro Proarrhythmia Assay (CiPA) initiative plus six compounds with names blinded to the investigators, using the rabbit ventricular wedge assay. Concentration-dependent TdP scores were determined by drug-induced changes in QT, T p-e , and proarrhythmias. Disclosure of the names and testing concentrations was made after completion of the experiments and report to the sponsors. Drugs' normalized TdP scores were calculated thereafter based on their respective free clinical maximum concentration (C max ). Drugs' normalized TdP scores were calculated and ranked for 33 drugs, excluding 1 investigational drug, and the TdP risks of the 28 CiPA drugs were correctly distinguished according to their respective categories of low, intermediate, and high TdP risks under the CiPA initiative. Accordingly, we are able to propose the cutoff values of the normalized TdP scores at 1 × C max : ≤ 0, > 0 to < 0.65 and ≥ 0.65, respectively, for low, intermediate, and high risk. This blinded study supports utility of our Normalized TdP Score System in predicting drug-induced TdP risks in 33 drugs, including 28 used for characterization of other assays under the CiPA initiative. However, these results need to be replicated in other laboratories., (© 2020 The Authors. Clinical Pharmacology & Therapeutics © 2020 American Society for Clinical Pharmacology and Therapeutics.)

Objectives: Improvements in human stem cell-derived cardiomyocyte (hSC-CM) technology have promoted their use for drug testing and disease investigations. Several in silico hSC-CM models have been proposed to augment interpretation of experimental findings through simulations. This work aims to assess the response of three hSC-CM in silico models (Koivumäki2018, Kernik2019, and Paci2020) to simulated drug action, and compare simulation results against in vitro data for 15 drugs. Methods: First, simulations were conducted considering 15 drugs, using a simple pore-block model and experimental data for seven ion channels. Similarities and differences were analyzed in the in silico responses of the three models to drugs, in terms of Ca 2+ transient duration (CTD 90 ) and occurrence of arrhythmic events. Then, the sensitivity of each model to different degrees of blockage of Na + (I Na ), L-type Ca 2+ (I CaL ), and rapid delayed rectifying K + (I Kr ) currents was quantified. Finally, we compared the drug-induced effects on CTD 90 against the corresponding in vitro experiments. Results: The observed CTD 90 changes were overall consistent among the in silico models, all three showing changes of smaller magnitudes compared to the ones measured in vitro . For example, sparfloxacin 10 µM induced +42% CTD 90 prolongation in vitro , and +17% (Koivumäki2018), +6% (Kernik2019), and +9% (Paci2020) in silico . Different arrhythmic events were observed following drug application, mainly for drugs affecting I Kr . Paci2020 and Kernik2019 showed only repolarization failure, while Koivumäki2018 also displayed early and delayed afterdepolarizations. The spontaneous activity was suppressed by Na + blockers and by drugs with similar effects on I CaL and I Kr in Koivumäki2018 and Paci2020, while only by strong I CaL blockers, e.g. nisoldipine, in Kernik2019. These results were confirmed by the sensitivity analysis. Conclusion: To conclude, The CTD 90 changes observed in silico are qualitatively consistent with our in vitro data, although our simulations show differences in drug responses across the hSC-CM models, which could stem from variability in the experimental data used in their construction., Competing Interests: MP and Tampere University collaborated with InSilicoTrials Technologies SRL for developing a software for predicting drug cardiac safety risk based on an in silico model of human induced pluripotent stem cell-derived cardiomyocyte. HRL, and DJG are employees of Janssen Pharmaceutica NV. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Paci, Koivumäki, Lu, Gallacher, Passini and Rodriguez.)

The cardiac Nav1.5 mediated sodium current (I Na ) generates the upstroke of the action potential in atrial and ventricular myocytes. Drugs that modulate this current can therefore be antiarrhythmic or proarrhythmic, which requires preclinical evaluation of their potential drug-induced inhibition or modulation of Nav1.5. Since Nav1.5 assembles with, and is modulated by, the auxiliary β1-subunit, this subunit can also affect the channel's pharmacological response. To investigate this, the effect of known Nav1.5 inhibitors was compared between COS-7 cells expressing Nav1.5 or Nav1.5+β1 using whole-cell voltage clamp experiments. For the open state class Ia blockers ajmaline and quinidine, and class Ic drug flecainide, the affinity did not differ between both models. For class Ib drugs phenytoin and lidocaine, which are inactivated state blockers, the affinity decreased more than a twofold when β1 was present. Thus, β1 did not influence the affinity for the class Ia and Ic compounds but it did so for the class Ib drugs. Human stem cell-derived cardiomyocytes (hSC-CMs) are a promising translational cell source for in vitro models that express a representative repertoire of channels and auxiliary proteins, including β1. Therefore, we subsequently evaluated the same drugs for their response on the I Na in hSC-CMs. Consequently, it was expected and confirmed that the drug response of I Na in hSC-CMs compares best to I Na expressed by Nav1.5+β1., (Copyright © 2019 Van de Sande, Kopljar, Teisman, Gallacher, Snyders, Lu and Labro.)

The Kv7 family of voltage-dependent non-inactivating potassium channels is composed of five members, of which four are expressed in the CNS. Kv7.2, 7.3 and 7.5 are responsible for the M-current, which plays a critical role in the regulation of neuronal excitability. Stimulation of M 1 muscarinic acetylcholine receptor, M 1 receptor, increases neuronal excitability by suppressing the M-current generated by the Kv7 channel family. The M-current modulation via M 1 receptor is well-described in in vitro assays using cell lines and in native rodent tissue. However, this mechanism was not yet reported in human induced pluripotent stem cells (hiPSC) derived neurons. In the present study, we investigated the effects of both agonists and antagonists of Kv7.2/7.3 channel and M 1 receptor in hiPSC derived neurons and in primary rat cortical neuronal cells. The role of M 1 receptors in the modulation of neuronal excitability could be demonstrated in both rat primary and hiPSC neurons. The M 1 receptors agonist, xanomeline, increased neuronal excitability in both rat cortical and the hiPSC neuronal cells. Furthermore, M 1 receptor agonist-induced neuronal excitability in vitro was reduced by an agonist of Kv7.2/7.3 in both neuronal cells. These results show that hiPSC derived neurons recreate the modulation of the M-current by the muscarinic receptor in hiPSC neurons similarly to rat native neurons. Thus, hiPSC neurons could be a useful human-based cell assay for characterization of drugs that affect neuronal excitability and/or induce seizure activity by modulation of M 1 receptors or inhibition of Kv7 channels., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The goal of this research consortium including Janssen, MSD, Ncardia, FNCR/LBR, and Health and Environmental Sciences Institute (HESI) was to evaluate the utility of an additional in vitro assay technology to detect potential drug-induced long QT and torsade de pointes (TdP) risk by monitoring cytosolic free Ca2+ transients in human stem-cell-derived cardiomyocytes (hSC-CMs). The potential proarrhythmic risks of the 28 comprehensive in vitro proarrhythmia assay (CiPA) drugs linked to low, intermediate, and high clinical TdP risk were evaluated in a blinded manner using Ca2+-sensitive fluorescent dye assay recorded from a kinetic plate reader system (Hamamatsu FDSS/µCell and FDSS7000) in 2D cultures of 2 commercially available hSC-CM lines (Cor.4U and CDI iCell Cardiomyocytes) at 3 different test sites. The Ca2+ transient assay, performed at the 3 sites using the 2 different hSC-CMs lines, correctly detected potential drug-induced QT prolongation among the 28 CiPA drugs and detected cellular arrhythmias-like/early afterdepolarization in 7 of 8 high TdP-risk drugs (87.5%), 6 of 11 intermediate TdP-risk drugs (54.5%), and 0 of 9 low/no TdP-risk drugs (0%). The results were comparable among the 3 sites and from 2 hSC-CM cell lines. The Ca2+ transient assay can serve as a user-friendly and higher throughput alternative to complement the microelectrode array and voltage-sensing optical action potential recording assays used in the HESI-CiPA study for in vitro assessment of drug-induced long QT and TdP risk., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have emerged as a promising cardiac safety platform, demonstrated by numerous validation studies using drugs with known cardiac adverse effects in humans. However, the challenge remains to implement hiPSC-CMs into cardiac de-risking of new chemical entities (NCEs) during preclinical drug development. Here, we used the calcium transient screening assay in hiPSC-CMs to develop a hazard score system for cardiac electrical liabilities. Tolerance interval calculations and evaluation of different classes of cardio-active drugs enabled us to develop a weighted scoring matrix. This approach allowed the translation of various pharmacological effects in hiPSC-CMs into a single hazard label (no, low, high, or very high hazard). Evaluation of 587 internal NCEs and good translation to ex vivo and in vivo models for a subset of these NCEs highlight the value of the cardiac hazard scoring in facilitating the selection of compounds during early drug safety screening., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: Preventing sudden cardiac death (SCD) is one of the main goals in hypertrophic cardiomyopathy (HCM). Many variables have been proposed, however the European and American guidelines do not incorporate any ECG or Holter monitoring derived variables other than the presence of ventricular arrhythmia in their risk stratification models. In the present study we evaluated electrocardiographic parameters in risk stratification of HCM., Methods and Results: Novel electrocardiographic parameters including the index of cardio-electrophysiological balance (iCEB), individualized QT correction (QTi) and QT rate dependence were evaluated along with established risk factors. A composite endpoint of SCD was defined as out of hospital cardiac arrest, appropriate ICD shock and sustained ventricular tachycardia. Cox regression analysis was used to evaluate predictors of SCD. Out of the 466 HCM patients, 31 reached the composite endpoint during a follow up of 75 ± 86 months. In a multivariate model, nor iCEB, QTi or QT rate dependence were predictors of SCD. Only male gender (p < 0.01; OR 13.1; CI 1.74-98.83), negative T waves in the inferior leads (p = 0.04; OR 2.51; CI 1.03-6.13) and familial sudden death (p < 0.01; OR 3.03; CI 1.39-6.59) were significant predictors. On top of either the ESC risk score or the 3 traditional 'American risk factors', only male gender was a significant predictor of SCD., Conclusion: No ECG or Holter monitoring parameters added in risk stratification for SCD in HCM. However, male gender and negative T waves in the inferior leads are promising novel markers to evaluate in larger cohorts., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Drug-induced seizures contribute to the high attrition rate of pharmaceutical compounds in development. The assessment of drug-induced seizure liability generally occurs in later phases of development using low throughput and intensive in vivo assays. In the present study, we evaluated the potential of an in vitro assay for detecting drug-induced seizure risk compared to evaluation in rats in vivo. We investigated the effects of 8 reference drugs with a known seizurogenic risk using micro-electrode array (MEA) recordings from freshly-dissociated rat primary neurons cultured on 48-well dishes for 28 days, compared to their effects on the EEG in anesthetized rats. In addition, we evaluated functional responses and mRNA expression levels of different receptors in vitro to understand the potential mechanisms of drug-induced seizure risk. Combining the functional MEA in vitro data with concomitant gene expression allowed us to identify several potential molecular targets that might explain the drug-induced seizures occurring in both rats and humans. Our data 1) demonstrate the utility of a group of MEA parameters for detecting potential drug-induced seizure risk in vitro; 2) suggest that an in vitro MEA assay with rat primary neurons may have advantages over an in vivo rat model; and 3) identify potential mechanisms for the discordance between rat assays and human seizure risk for certain seizurogenic drugs., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Introduction: Calcium-based screening of hiPS-CMs is a useful preclinical safety evaluation platform with the ability to generate robust signals that facilitates high-throughput screening and data analysis. However, due to the potential inherent toxicities, it is important to understand potential effects of different calcium-sensitive dyes on the hiPS-CMs model., Methods: We compared three calcium-sensitive fluorescence dyes (Cal520, ACTOne and Calcium 5) for their impact on the variability, the beating properties and the pharmacological responses of hiPS-CMs using the Hamamatsu FDSS/μCell imaging platform. Direct effects of three dyes on the electrophysiological properties of hiPS-CMs were evaluated with the multi-electrode array (MEA) Axion Maestro platform., Results: We propose a specific experimental protocol for each dye which gives the most optimal assay conditions to minimize variability and possible adverse effects. We showed that Cal520 had the smallest effect on hiPS-CMs together with the longest-lasting stable amplitude signal (up to 4 h). Although all dyes had a (minor) acute effect on hiPS-CMs, in the form of reduced beat rate and prolonged field potential duration, the selection of the dye did not influence the pharmacological response of four cardioactive drugs (dofetilide, moxifloxacin, nimodipine and isoprenaline)., Discussion: In conclusion, we have documented that different calcium sensitive dyes have only minor direct (acute) effects on hiPS-CMs with Cal520 showing the least effects and the longest lasting signal amplitude. Importantly, drug-induced pharmacological responses in hiPS-CMs were comparable between the three dyes. These findings should help further improve the robustness of the hiPS-CMs-based calcium transient assay as a predictive, preclinical cardiac safety evaluation tool., (Copyright © 2018 Elsevier Inc. All rights reserved.)

A single pot dipolar cycloaddition reaction/Cope elimination sequence was developed to access novel 1,4,6,7-tetrahydro-5H-[1,2,3]triazolo[4,5-c]pyridine P2X7 antagonists that contain a synthetically challenging chiral center. The structure-activity relationships of the new compounds are described. Two of these compounds, (S)-(2-fluoro-3-(trifluoromethyl)phenyl)(1-(5-fluoropyrimidin-2-yl)-6-methyl-1,4,6,7-tetrahydro-5H-[1,2,3]triazolo[4,5-c]pyridin-5-yl)methanone (compound 29) and (S)-(3-fluoro-2-(trifluoromethyl)pyridin-4-yl)(1-(5-fluoropyrimidin-2-yl)-6-methyl-1,4,6,7-tetrahydro-5H-[1,2,3]triazolo[4,5-c]pyridin-5-yl)methanone (compound 35), were found to have robust P2X7 receptor occupancy at low doses in rat with ED 50 values of 0.06 and 0.07 mg/kg, respectively. Compound 35 had notable solubility compared to 29 and showed good tolerability in preclinical species. Compound 35 was chosen as a clinical candidate for advancement into phase I clinical trials to assess safety and tolerability in healthy human subjects prior to the initiation of proof of concept studies for the treatment of mood disorders.

Background and Purpose: In the pharmaceutical industry risk assessments of chronic cardiac safety liabilities are mostly performed during late stages of preclinical drug development using in vivo animal models. Here, we explored the potential of human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) to detect chronic cardiac risks such as drug-induced cardiomyocyte toxicity., Experimental Approach: Video microscopy-based motion field imaging was applied to evaluate the chronic effect (over 72 h) of cardiotoxic drugs on the contractile motion of hiPS-CMs. In parallel, the release of cardiac troponin I (cTnI), heart fatty acid binding protein (FABP3) and N-terminal pro-brain natriuretic peptide (NT-proBNP) was analysed from cell medium, and transcriptional profiling of hiPS-CMs was done at the end of the experiment., Key Results: Different cardiotoxic drugs altered the contractile motion properties of hiPS-CMs together with increasing the release of cardiac biomarkers. FABP3 and cTnI were shown to be potential surrogates to predict cardiotoxicity in hiPS-CMs, whereas NT-proBNP seemed to be a less valuable biomarker. Furthermore, drug-induced cardiotoxicity produced by chronic exposure of hiPS-CMs to arsenic trioxide, doxorubicin or panobinostat was associated with different profiles of changes in contractile parameters, biomarker release and transcriptional expression., Conclusion and Implications: We have shown that a parallel assessment of motion field imaging-derived contractile properties, release of biomarkers and transcriptional changes can detect diverse mechanisms of chronic drug-induced cardiac liabilities in hiPS-CMs. Hence, hiPS-CMs could potentially improve and accelerate cardiovascular de-risking of compounds at earlier stages of drug discovery., Linked Articles: This article is part of a themed section on New Insights into Cardiotoxicity Caused by Chemotherapeutic Agents. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.21/issuetoc., (© 2017 The British Pharmacological Society.)

Early prediction of cardiotoxicity is critical for drug development. Current animal models raise ethical and translational questions, and have limited accuracy in clinical risk prediction. Human-based computer models constitute a fast, cheap and potentially effective alternative to experimental assays, also facilitating translation to human. Key challenges include consideration of inter-cellular variability in drug responses and integration of computational and experimental methods in safety pharmacology. Our aim is to evaluate the ability of in silico drug trials in populations of human action potential (AP) models to predict clinical risk of drug-induced arrhythmias based on ion channel information, and to compare simulation results against experimental assays commonly used for drug testing. A control population of 1,213 human ventricular AP models in agreement with experimental recordings was constructed. In silico drug trials were performed for 62 reference compounds at multiple concentrations, using pore-block drug models (IC 50 /Hill coefficient). Drug-induced changes in AP biomarkers were quantified, together with occurrence of repolarization/depolarization abnormalities. Simulation results were used to predict clinical risk based on reports of Torsade de Pointes arrhythmias, and further evaluated in a subset of compounds through comparison with electrocardiograms from rabbit wedge preparations and Ca 2+ -transient recordings in human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs). Drug-induced changes in silico vary in magnitude depending on the specific ionic profile of each model in the population, thus allowing to identify cell sub-populations at higher risk of developing abnormal AP phenotypes. Models with low repolarization reserve (increased Ca 2+ /late Na + currents and Na + /Ca 2+ -exchanger, reduced Na + /K + -pump) are highly vulnerable to drug-induced repolarization abnormalities, while those with reduced inward current density (fast/late Na + and Ca 2+ currents) exhibit high susceptibility to depolarization abnormalities. Repolarization abnormalities in silico predict clinical risk for all compounds with 89% accuracy. Drug-induced changes in biomarkers are in overall agreement across different assays: in silico AP duration changes reflect the ones observed in rabbit QT interval and hiPS-CMs Ca 2+ -transient, and simulated upstroke velocity captures variations in rabbit QRS complex. Our results demonstrate that human in silico drug trials constitute a powerful methodology for prediction of clinical pro-arrhythmic cardiotoxicity, ready for integration in the existing drug safety assessment pipelines.

Introduction: Human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) are emerging as new and human-relevant source in vitro model for cardiac safety assessment that allow us to investigate a set of 20 reference drugs for predicting cardiac arrhythmogenic liability using optical action potential (oAP) assay., Methods: Here, we describe our examination of the oAP measurement using a voltage sensitive dye (Di-4-ANEPPS) to predict adverse compound effects using hiPS-CMs and 20 cardioactive reference compounds. Fluorescence signals were digitized at 10kHz and the records subsequently analyzed off-line. Cells were exposed to 30min incubation to vehicle or compound (n=5/dose, 4 doses/compound) that were blinded to the investigating laboratory. Action potential parameters were measured, including rise time (T rise ) of the optical action potential duration (oAPD)., Results: Significant effects on oAPD were sensitively detected with 11 QT-prolonging drugs, while oAPD shortening was observed with I Ca -antagonists, I Kr -activator or ATP-sensitive K + channel (K ATP )-opener. Additionally, the assay detected varied effects induced by 6 different sodium channel blockers. The detection threshold for these drug effects was at or below the published values of free effective therapeutic plasma levels or effective concentrations by other studies., Discussion: The results of this blinded study indicate that OAP is a sensitive method to accurately detect drug-induced effects (i.e., duration/QT-prolongation, shortening, beat rate, and incidence of early after depolarizations) in hiPS-CMs; therefore, this technique will potentially be useful in predicting drug-induced arrhythmogenic liabilities in early de-risking within the drug discovery phase., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Methods used to convert wind tunnel and ADMS concentration field data for a complex building array into effective radiation dose were developed based on simulations of a site in central London. Pollutant source terms were from positron emitting gases released from a cyclotron and clinical PET radiotracer facility. Five years of meteorological data were analysed to determine the probability distribution of wind direction and speed. A hemispherical plume cloud model (both static and moving) was developed which enabled an expression of gamma-ray dose, taking into account build-up factors in air, in terms of analytic functions in this geometry. The standard building wake model is presented, but this is extended and developed in a new model to cover the concentration field in the vicinity of a roof top structure recirculation zone, which is then related to the concentration in the main building wake zone. For all models presented the effective dose was determined from inhalation, positron cloud immersion and gamma ray plume contributions. Results of applying these models for determination of radiation dose for a particular site are presented elsewhere.

A radiological assessment was carried out on the release of positron-emitting radioactive gases from a roof-level stack at a central London site. Different modelling approaches were performed to investigate the range of radiation doses to representative persons. Contributions from plume inhalation, gamma shine and immersion to effective dose were taken into account. Dry and wet surface deposition on the roof, and exposure from contamination on the skin of roof-workers, added only a mean 4.7% to effective dose and were neglected. A 1:200 scale model, consisting of the stack and surrounding buildings, was tested in a wind tunnel to simulate pollutant dispersion in the near-field region i.e. rooftop. Concentration field measurements in the wind tunnel were converted into effective dose, including for roof-workers installing glass cladding to the stack building. Changes in the building shape, from addition of the cladding layer, were investigated in terms of the near-field flow pattern and significant differences found between the two cases. Pollutant concentrations were also modelled using Air Dispersion Modelling System (ADMS) and the results used to calculate the effective dose using the same meteorological data set and source release terms. Sector averaged wind tunnel dose estimates were greater than the ADMS figure by approximately a factor of two to three. Different stack release heights were investigated in the wind tunnel and ADMS simulations in order to determine the best height for the replacement flue stack for the building. Other techniques were investigated: building wake models, modified Gaussian plume methods and uniform dilution into a hemispherical volume to show the wide variation in predicted dose possible with different approaches. Large differences found between simpler analytic approaches indicated that more robust radiological assessments, based on more complex modelling approaches, were required to achieve satisfactory estimates of radiation dose to representative groups in adjacent buildings and on the building rooftop.

Drug-induced QTc interval prolongation ( Δ QTc) is a main surrogate for proarrhythmic risk assessment. A higher in vivo than in vitro potency for hERG-mediated QTc prolongation has been suggested. Also, in vivo between-species and patient populations' sensitivity to drug-induced QTc prolongation seems to differ. Here, a systems pharmacology model integrating preclinical in vitro (hERG binding) and in vivo (conscious dog Δ QTc) data of three hERG blockers (dofetilide, sotalol, moxifloxacin) was applied (1) to compare the operational efficacy of the three drugs in vivo and (2) to quantify dog-human differences in sensitivity to drug-induced QTc prolongation (for dofetilide only). Scaling parameters for translational in vivo extrapolation of drug effects were derived based on the assumption of system-specific myocardial ion channel densities and transduction of ion channel block: the operational efficacy (transduction of hERG block) in dogs was drug specific (1-19% hERG block corresponded to ≥10 msec Δ QTc). System-specific maximal achievable Δ QTc was estimated to 28% from baseline in both dog and human, while %hERG block leading to half-maximal effects was 58% lower in human, suggesting a higher contribution of hERG-mediated potassium current to cardiac repolarization. These results suggest that differences in sensitivity to drug-induced QTc prolongation may be well explained by drug- and system-specific differences in operational efficacy (transduction of hERG block), consistent with experimental reports. The proposed scaling approach may thus assist the translational risk assessment of QTc prolongation in different species and patient populations, if mediated by the hERG channel.

Most small-molecule inhibitors of voltage-gated ion channels display poor subtype specificity because they bind to highly conserved residues located in the channel's central cavity. Using a combined approach of scanning mutagenesis, electrophysiology, chemical ligand modification, chemical cross-linking, MS/MS-analyses and molecular modelling, we provide evidence for the binding site for adamantane derivatives and their putative access pathway in Kv7.1/KCNE1 channels. The adamantane compounds, exemplified by JNJ303, are highly potent gating modifiers that bind to fenestrations that become available when KCNE1 accessory subunits are bound to Kv7.1 channels. This mode of regulation by auxiliary subunits may facilitate the future development of potent and highly subtype-specific Kv channel inhibitors.

In the present study, we investigated an impact of the stimulation rate on the detection of the proarrhythmic potential of 10 reference compounds with effects on different cardiac ion channels in the isolated arterially-perfused rabbit left ventricular wedge preparation. The compounds were tested in the wedge model using two distinct protocols; including baseline stimulation at 1-Hz followed by a brief period at 0.5-Hz, either without an additional brief period of 2-Hz stimulation (i.e. Protocol 1) or with 2-Hz stimulation (i.e. Protocol 2). As expected, QT-prolonging drugs (ibutilide and quinidine) prolonged the QT interval, similarly increased the Torsades de Pointes (TdP) score, and elicited early afterdepolarizations (EADs) in both protocols. HMR1556 and JNJ-303 (IKs blockers) also prolonged the QT interval up to 1μM similarly in both protocols. Nifedipine (Ca(2+) antagonist) shortened the QT interval, and reduced force of contraction similarly in both protocols. However, Na(+) channel blockers (Ia, Ib, Ic) widened the QRS duration more in Protocol 2 than in Protocol 1. Furthermore, it was only possible to detect non-TdP-like ventricular tachycardia/fibrillation (VT/VF) induced by Na(+) blockers and by QT-shortening drugs (levcromakalim and mallotoxin) using the 2-Hz stimulation (Protocol 2). Our data suggest that the inclusion of a brief period of fast stimulation at 2Hz is critical for detecting drug-induced slowing of conduction (QRS widening), QT shortening and associated (non-TdP-like) VT/VF, which are distinct from the QT prolongation/TdP proarrhythmia in isolated, arterially-perfused rabbit left ventricular wedges., (Copyright © 2016 Elsevier Inc. All rights reserved.)

In England and Wales, 19% of live births in 2012 were at 37-38 weeks' gestation, equating to nearly 140 000 early-term births each year. Since caesarean sections (CS) are often performed at early-term gestations, this accounts for some of the increased proportion of the early-term births. Infants born early-term are at an increased risk of neonatal respiratory morbidity particularly if they are delivered by caesarean section. The long term lung function data are limited but available data suggest that early-term delivery is associated with respiratory morbidity in childhood. CS also appears to be associated with increased neonatal morbidity and future development of respiratory symptoms. However, future studies need to confirm the independent effects of caesarean sections and early-term deliveries particularly for long term outcomes as both are likely to affect the respiratory system differently., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: Recently a new risk marker for drug-induced arrhythmias called index of cardio-electrophysiological balance (iCEB), measured as QT interval divided by QRS duration, was evaluated in an animal model. It was hypothesized that iCEB is equivalent to the cardiac wavelength λ (λ = effective refractory period (ERP) x conduction velocity) and that an increased or decreased value of iCEB would potentially predict an increased susceptibility to TdP or non-TdP mediated VT/VF, respectively., Methods: First, the correlation between QT interval and ERP was evaluated by invasively measuring ERP during a ventricular stimulation protocol in humans (N = 40). Then the effect of administration of sotalol and flecainide on iCEB was measured in 40 patients with supraventricular tachycardias. Finally iCEB was assessed in carriers of a long QT syndrome (LQTS, N = 70) or Brugada syndrome (BrS, N = 57) mutation and compared them with genotype negative family members (N = 65)., Results: The correlation between QT interval and ERP was established (Pearson R(2) = 0.25) which suggests that iCEB≈ERPxCV≈QT/QRS. Sotalol administration increased iCEB (+ 0.23; P = 0.01), while it decreased with the administration of flecainide (-0.21, P = 0.03). In the LQTS group iCEB was increased (5.22 ± 0.93, P < 0.0001) compared to genotype negative family members (4.24 ± 0.5), while it was decreased in the BrS group (3.52 ± 0.43, P < 0.0001)., Conclusions: Our data suggest that iCEB (QT/QRS) is a simple but effective ECG surrogate of cardiac wavelength. iCEB is increased in situations that predispose to TdP and is decreased in situations that predispose to non-TdP mediated VT/VF. Therefore, iCEB might serve as a noninvasive and readily measurable marker to detect increased arrhythmic risk., (© 2015 Wiley Periodicals, Inc.)

Unlabelled: Histone deacetylase (HDAC) inhibitors possess therapeutic potential to reverse aberrant epigenetic changes associated with cancers, neurological diseases, and immune disorders. Unfortunately, clinical studies with some HDAC inhibitors displayed delayed cardiac adverse effects, such as atrial fibrillation and ventricular tachycardia. However, the underlying molecular mechanism(s) of HDAC inhibitor-mediated cardiotoxicity remains poorly understood and is difficult to detect in the early stages of preclinical drug development because of a delayed onset of effects. In the present study, we show for the first time in human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) that HDAC inhibitors (dacinostat, panobinostat, vorinostat, entinostat, and tubastatin-a) induce delayed dose-related cardiac dysfunction at therapeutic concentrations associated with cardiac adverse effects in humans. HDAC inhibitor-mediated delayed effects on the beating properties of hiPS-CMs developed after 12 hours by decreasing the beat rate, shortening the field potential duration, and inducing arrhythmic behavior under form of sustained contractions and fibrillation-like patterns. Transcriptional changes that are common between the cardiotoxic HDAC inhibitors but different from noncardiotoxic treatments identified cardiac-specific genes and pathways related to structural and functional changes in cardiomyocytes. Combining the functional data with epigenetic changes in hiPS-CMs allowed us to identify molecular targets that might explain HDAC inhibitor-mediated cardiac adverse effects in humans. Therefore, hiPS-CMs represent a valuable translational model to assess HDAC inhibitor-mediated cardiotoxicity and support identification of better HDAC inhibitors with an improved benefit-risk profile., Significance: Histone deacetylase (HDAC) inhibitors are a promising class of drugs to treat certain cancers, autoimmune, and neurodegenerative diseases. However, treated patients can experience various cardiac adverse events such as hearth rhythm disorders. This study found that human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) can predict cardiac adverse events in patients caused by HDAC inhibitors. Furthermore, transcriptional changes at the level of gene expression supported the effects on the beating properties of hiPS-CMs and highlight targets that might cause these cardiac adverse effects. hiPS-CMs represent a valuable translational model to assess HDAC inhibitor-mediated cardiotoxicity and to support development of safer HDAC inhibitors., (©AlphaMed Press.)

Key Points: Respiratory distress is a common presenting feature among newborn infants.Prompt investigation to ascertain the underlying diagnosis and appropriate subsequent management is important to improve outcomes.Many of the underlying causes of respiratory distress in a newborn are unique to this age group.A chest radiograph is crucial to assist in diagnosis of an underlying cause., Educational Aims: To inform readers of the common respiratory problems encountered in neonatology and the evidence-based management of these conditions.To enable readers to develop a framework for diagnosis of an infant with respiratory distress. The first hours and days of life are of crucial importance for the newborn infant as the infant adapts to the extra-uterine environment. The newborn infant is vulnerable to a range of respiratory diseases, many unique to this period of early life as the developing fluid-filled fetal lungs adapt to the extrauterine environment. The clinical signs of respiratory distress are important to recognise and further investigate, to identify the underlying cause. The epidemiology, diagnostic features and management of common neonatal respiratory conditions are covered in this review article aimed at all healthcare professionals who come into contact with newborn infants.

The respiratory tract, once believed to be sterile, harbors diverse bacterial communities. The role of microorganisms within health and disease is slowly being unraveled. Evidence points to the neonatal period as a critical time for establishing stable bacterial communities and influencing immune responses important for long-term respiratory health. This review summarizes the evidence of early airway and lung bacterial colonization and the role the microbiome has on respiratory health in the short and long term. The challenges of neonatal respiratory microbiome studies and future research directions are also discussed.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) are emerging as a powerful in vitro model for cardiac safety assessment which may allow for better identification of compounds with poor arrhythmogenic liability profiles early in the drug discovery process. Here, we describe our examination of the Kinetic Image Cytometer (KIC) system's ability to predict adverse compound effects using hiPS-CMs and a library of 53 compounds, the majority of which are known to be cardioactive compounds, and several negative controls. The KIC provides a high throughput method for analyzing intracellular calcium transients. In the cardiomyocyte, intracellular calcium transients integrate the electrochemical signals of the action potential (AP) with the molecular signaling pathways regulating contraction. Drug-induced alterations in the shape and duration of AP result in changes to the shape and duration of the intracellular calcium transient. By examining calcium transient dynamics in hiPS-CMs, KIC can be used as a phenotypic screen to assess compound effects across multiple ion channel types (MITs), detecting MITs, calcium handling and signaling effects. The results of this blinded study indicate that using hiPS-CMs, KIC is able to accurately detect drug-induced changes in Ca(2+) transient dynamics (ie, duration and beat rate) and therefore, may be useful in predicting drug-induced arrhythmogenic liabilities in early de-risking within the drug discovery phase., (© The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Introduction: With the recent development of more sensitive biomarkers to assess kidney injury preclinically, a survey was designed i) to investigate what strategies are used to investigate renal toxicity in both ICH S7A compliant Safety Pharmacology (SP) studies after a single dose of a compound and within repeat-dose toxicity studies by large pharmaceutical companies today; ii) to understand whether renal SP studies have impact or utility in drug development and/or if it may be more appropriate to assess renal effects after multiple doses of compounds; iii) to ascertain how much mechanistic work is performed by the top 15 largest pharmaceutical companies (as determined by R&D revenue size); iv) to gain an insight into the impact of the validation of DIKI biomarkers and their introduction in the safety evaluation paradigm; and v) to understand the impact of renal/urinary safety study data on progression of projects., Methods: Two short anonymous surveys were submitted to SP leaders of the top 15 pharmaceutical companies, as defined by 2012 R&D portfolio size. Fourteen multiple choice questions were designed to explore the strategies used to investigate renal effects in both ICH S7A compliant SP studies and within toxicology studies., Results: A 67% and 60% response rate was obtained in the first and second surveys, respectively. Nine out of ten respondent companies conduct renal excretory measurements (eg. urine analysis) in toxicology studies whereas only five out of ten conduct specific renal SP studies; and all of those 5 also conduct the renal excretory measurements in toxicology studies. These companies measure and/or calculate a variety of parameters as part of these studies, and also on a case by case basis include regulatory qualified and non-qualified DIKI biomarkers. Finally, only one company has used renal/urinary functional data alone to stop a project, whereas the majority of respondents combine renal data with other target organ assessments to form an integrated decision-making set., Conclusion: These short surveys highlighted areas of similarity: a) urinary measurements are most commonly taken on repeat-dose toxicity studies, and b) renal SP studies are less often utilised. The two major differences are a) lack of consistent use of DIKI biomarkers in urinary safety studies and b) the way large pharmaceutical companies assess renal function. Finally, suggestions were made to improve the safety assessment methods for determining the safety of compounds with potential renal liability., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background and Purpose: Preclinical cardiovascular safety studies (CVS) have been compared between facilities with respect to their sensitivity to detect drug-induced QTc prolongation (ΔQTc). Little is known about the consistency of quantitative ΔQTc predictions that are relevant for translation to humans., Experimental Approach: We derived typical ΔQTc predictions at therapeutic exposure (ΔQTcTHER ) with 95% confidence intervals (95%CI) for 3 Kv 11.1 (hERG) channel blockers (moxifloxacin, dofetilide and sotalol) from a total of 14 CVS with variable designs in the conscious dog. Population pharmacokinetic-pharmacodynamic (PKPD) analysis of each study was followed by a meta-analysis (pooling 2-6 studies including 10-32 dogs per compound) to derive meta-predictions of typical ΔQTcTHER . Meta-predictions were used as a reference to evaluate the consistency of study predictions and to relate results to those found in the clinical literature., Key Results: The 95%CIs of study-predicted ΔQTcTHER comprised in 13 out of 14 cases the meta-prediction. Overall inter-study variability (mean deviation from meta-prediction at upper level of therapeutic exposure) was 30% (range: 1-69%). Meta-ΔQTcTHER predictions for moxifloxacin, dofetilide and sotalol overlapped with reported clinical QTc prolongation when expressed as %-prolongation from baseline., Conclusions and Implications: Consistent exposure-ΔQTc predictions were obtained from single preclinical dog studies of highly variable designs by systematic PKPD analysis, which is suitable for translational purposes. The good preclinical-clinical pharmacodynamic correlations obtained suggest that such an analysis should be more routinely applied to increase the informative and predictive value of results obtained from animal experiments., (© 2015 The British Pharmacological Society.)

Introduction: Preclinical proarrhythmic risk assessment of drug candidates is focused predominantly on arrhythmias arising from repolarization abnormalities. However, drug-induced cardiac conduction slowing is associated with significant risk of life-threatening ventricular arrhythmias, particularly in a setting of cardiac ischemia. Therefore, we optimized and characterized an anesthetized dog model to evaluate the potential proarrhythmic risk of drug candidates in ischemic heart disease patients., Methods: Anesthetized dogs were instrumented with atrial and ventricular epicardial electrodes for pacing and measurement of conduction times, and a balloon occluder and flow probe placed around the left anterior descending coronary artery (LAD) distal to the first branch. Conduction times, ECG intervals and incidence of arrhythmias were assessed serially at the end of each dose infusion (flecainide: 0.32, 0.63, 1.25, 2.5 and 5mg/kg, i.v.; dofetilide:1.25, 2.5, 5, 10 and 20 μg/kg, i.v.; or vehicle; n=6/group) both during normal flow (with and without rapid pacing) and during 5-min LAD occlusion (with and without rapid pacing). Compound X, a development candidate with mild conduction slowing activity, was also evaluated., Results: Flecainide produced pronounced, dose-dependent slowing of conduction that was exacerbated during ischemia and rapid pacing. In addition, ventricular tachycardia (VT) and fibrillation (VF) occurred in 4 of 6 dogs (3 VF @ 0.63 mg/kg; 1VT @ 2.5mg/kg). In contrast, no animals in the vehicle group developed arrhythmias. Dofetilide, a potent IKr blocker that does not slow conduction, prolonged QT interval but did not cause further conduction slowing during ischemia with or without pacing and there were no arrhythmias. Compound X, like flecainide, produced marked conduction slowing and arrhythmias (VT, VF) during ischemia and pacing., Discussion: This model may be useful to more accurately define shifts in safety margins in a setting of ischemia and increased cardiac demand for drugs that slow conduction., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Introduction: Preclinical concentration-effect (pharmacokinetic-pharmacodynamic, PKPD) modeling has successfully quantified QT effects of several drugs known for significant QT prolongation. This study investigated its sensitivity for detecting small magnitudes of QT-prolongation in a typical preclinical cardiovascular (CV) safety study in the conscious telemetered dog (crossover study in 4-8 animals receiving a vehicle and three dose levels). Results were compared with conventional statistical analysis (analysis of covariance, ANCOVA)., Methods: A PKPD model predicting individual QTc was first developed from vehicle arms of 28 typical CV studies and one positive control study (sotalol). The model quantified between-animal, inter-occasion and within-animal variability and described QTc over 24h as a function of circadian variation and drug concentration. This "true" model was used to repeatedly (n = 500) simulate studies with typical drug-induced QTc prolongation (∆QTc) of 1 to 12 ms at high-dose peak concentrations. Simulated studies were re-analyzed by both PKPD analysis (with varying complexity) and ANCOVA. Sensitivity (power) was calculated as the percentage of studies in which a significant (α = 0.05) drug effect was found. One simulation scenario did not include a concentration-effect relationship and served to investigate false-positive rates. Exposure-effect relationships were derived from both PKPD analysis (linear concentration-effect) and ANCOVA (linear trend test for dose) and compared., Results: PKPD analysis/ANCOVA had a sensitivity of 80% to detect the effects of 7/13 ms (n = 4), 5/10 ms (n = 6) and 4.5/8 ms (n = 8), respectively. The false-positive rate was much higher using ANCOVA (40%) compared to PKPD analysis (1%). Typical drug effects were more precisely predicted using estimated concentration-effect slopes (± 1.5-2.8 ms) than dose-effect slopes (± 3.3-3.7 ms)., Discussion: Preclinical PKPD analysis can increase the confidence in the quantification of small QTc effects and potentially allow reducing the number of animals while maintaining the required study sensitivity. This underscores the value of PKPD modeling in preclinical safety testing., (Copyright © 2014 Elsevier Inc. All rights reserved.)