The JTpeak (JTp) interval has been proposed as a complementary, sensitive, and more specific biomarker than the QT interval for assessing proarrhythmic risk. It aims to improve the differentiation between drugs that selectively inhibit hERG channels (associated with high proarrhythmic risk) and those that affect multiple cardiac ion channels (associated with minimal or no risk).As previously reported by Darpo et al. in human (J. Clin. Pharmacol., 2019;60:125–139), the present study aimed to evaluate the effects of five QT-prolonging reference drugs, namely moxifloxacin, dofetilide, dolasetron, ondansetron and quinine, on JTp interval in telemetered conscious dogs (n = 7). PR and QRS intervals were also measured to reflect the effects on other cardiac ion channels. Levocetirizine, a QT-negative control drug, was included for comparison. Each compound was administered orally using a crossover design at two dose levels, with ECG monitoring conducted over a 24-h period post-dosing. Dedicated pharmacokinetic sessions were performed to determine corresponding plasma exposures, and sensitivity parameters were calculated for the new parameter JTp. JTp interval was individually corrected (JTpc) for heart rate variations.Compared to vehicle, dofetilide (0.03 and 0.15 mg/kg), moxifloxacin (30 and 100 mg/kg), and ondansetron (10 and 30 mg/kg) produced dose-dependent increases in JTpc interval, consistent with predominant hERG channel blockade. In contrast, dolasetron (6 and 20 mg/kg) and quinine (20 mg/kg) did not affect the JTpc interval. Notably, dolasetron increased both QRS and PR intervals, reflecting its inhibitory effects on sodium and calcium currents. Quinine also showed a slight increase in QRS interval. As expected, levocetirizine (2 and 10 mg/kg) showed no effect on ECG intervals, at any dose or timepoint. Total plasma drug concentrations for each compound increased dose-proportionally.In summary, this study demonstrated that drugs with selective hERG-blocking properties specifically prolong the JTp interval. In contrast, drugs with mixed ion channel-blocking activity tend to prolong other ECG intervals, but not the JTp interval. These findings support the relevance of JTp interval assessment as a valuable parameter to complement QT interval measurements in cardiovascular safety pharmacology studies, enhancing the specificity and sensitivity of proarrhythmic risk evaluation.

INTRODUCTION:Although deep learning methods for electroencephalogram (EEG) analysis are rapidly advancing, architectures developed for human multichannel recordings may not be suited to animal EEG, where recordings typically involve substantially fewer channels. This study investigated whether deep learning could detect EEG alterations induced by the NMDAr antagonist MK-801 (dizocilpine), recorded in rats subjected or not to auditory stimulations, to determine the potential of this method for brain circuit-related efficacy and safety assessment. METHODS:EEG recordings were obtained from male Sprague-Dawley rats implanted with telemetry implants studied in a randomized crossover treatment design. Signals from frontal and temporal electrodes were segmented into 0.5-s epochs and processed using a minimal preprocessing pipeline. Several candidate deep-learning architectures were evaluated on classification tasks. RESULTS:EEG Conformer provided the best balance between performance and robustness. On the recording-level test set, the final model reached an F-score close to 0.90 on the recording-level test set. In a complementary leave-one-subject-out (LOSO) cross-validation, it reached a mean weighted F1-score of 0.646 ± 0.034 (n = 5 folds), above the reference chance level and consistent with transfer of a learned MK-801-related EEG signature to unseen animals. In MK-801-treated rats, abnormal-class outputs increased with dose, from 0.17 at 0.025 mg/kg to 0.62 at 0.2 mg/kg. Auditory stimulation increased abnormal classification relative to non-stimulated epochs, indicating improved sensitivity under stimulation conditions. CONCLUSION:These findings support the feasibility of using deep learning to detect compound-related EEG alterations in low-channel rat recordings with limited preprocessing. Two complementary forms of generalization were assessed: transfer to unseen treatment conditions within known subjects in the crossover setting, and generalization to entirely unseen animals in the LOSO setting. The lower LOSO performance is consistent with the more stringent validation conditions. Together, these results suggest that the approach may complement conventional analytical pipelines and support more automated, standardized preclinical EEG workflows for both safety and efficacy pharmacology studies.

Treating patients affected by schizophrenia (SZ) remains highly challenging for drug developers. While available antipsychotics mostly reduce positive symptoms, their effects on cognitive and social deficits as well as their underlying pathophysiology remain insufficient. Disinhibition in SZ patients' cortex is considered a key player in the genesis of these symptoms and would result from N-methyl-D-aspartate receptor (NMDAr) hypofunction on interneurons. Besides, electroencephalography-based biomarkers of these mechanisms including spontaneous and evoked gamma oscillations have been highlighted. These strongly depend on NMDAr function, associate with symptoms and therefore represent translational tools for drug development. However, reversing these circuit abnormalities is challenging for the field and finding drugs engaging them would constitute a major milestone. Here, we tested three different drugs whose features attract scientists for restoration of excitation/inhibition balance: cariprazine (D2/D3 receptor partial agonist and 5-HT2A receptor antagonist), LY379268 (mGlur2/3 receptor agonist) and donepezil (acetylcholinesterase inhibitor). We investigated their effects on spontaneous oscillations and auditory steady-state responses (ASSR, measuring evoked gamma oscillations) in a rat model of SZ induced by MK-801, a selective NMDAr blocker. MK-801 increased locomotor activity, impaired spontaneous and evoked gamma oscillations and altered spontaneous oscillations in delta, alpha and beta frequencies of male Sprague Dawley rats. Remarkably, both cariprazine and LY379268 normalized spontaneous gamma oscillations. Moreover, cariprazine normalized alpha and beta waves and LY379268 selectively corrected delta activity. Interestingly, only cariprazine mitigated the ASSR deficit. These differential normalization profiles, including cariprazine's unique broad-spectrum signature, reveal distinct patterns of circuit engagement and provide a translational pharmaco-EEG framework for circuit-level target engagement in SZ drug development.

Safety pharmacology is a relatively young discipline dedicated to identifying adverse pharmacodynamic effects of new drugs that may impact human safety. Since the publication of the ICH S7A guideline in 2000, the field has made significant efforts in implementing the 3Rs principles—Replacement, Reduction, and Refinement of animal use. This review provides a comprehensive overview of key milestones and innovations that have shaped the application of the 3Rs in safety pharmacology over the past 25 years. Typical examples are the use of non-invasive jacket telemetry for real-time cardiovascular monitoring in conscious freely moving animals and the introduction of the CiPA (Comprehensive in Vitro Proarrhythmia Assessment) paradigm in 2014. The review explores 3Rs-driven advances across core organ systems—cardiovascular, central nervous, and respiratory—as well as supplemental systems including gastrointestinal and renal. Furthermore, it examines the evolving global regulatory landscape and the emergence of new approach methodologies (NAMs), such as artificial intelligence tools and complex microphysiological systems (MPS), and their integration into drug development pipelines. Finally, the review discusses future directions and potential challenges in achieving broader adoption of 3Rs-compliant strategies and NAMs in safety pharmacology.

Guinea-pig (GP) has been used for many years as a good surrogate species in the early stages of drug development, in particular to predict compound-related QTc prolongation. Implementation of the ICH E14/S7B Q&As emphasized the use of a concentration-QTc analysis (c-QT) in non-rodent GLP telemetry studies, as it is done in clinical trials. We aimed at performing such c-QT analysis in an anaesthetized GP model to evaluate its translational relevance. GP were anaesthetized with sodium pentobarbital and prepared for blood pressure, left ventricular pressure and ECG measurements (lead II). Six GP received 2 successive infusions of increasing doses of compound UCB-X whilst 6 others received 2 successive infusions of vehicle. Treatments were administered as iv infusions over 20 min followed by a 30-min washout period. Blood samples were collected 5, 10, 15 and 20 min after the start of each infusion, for total blood concentration measurement of UCB-X. UCB-X induced a dose-dependent and marked increased in QTc (corrected for heart rate by Bazett formula, QTcB). Maximum increases in QTcB were 9 ± 2 % and 14 ± 3 % at the end of the first and second infusion dose, respectively, from a mean baseline QTcB value of 312 ± 9 ms. At the end of the washout period, QTcB remained markedly increased (18 ± 3 %). Concentration versus vehicle adjusted delta QTcB were plotted, and the best fit correlation was calculated. Logarithmic transformation led to a linear correlation that allowed to estimate the concentration of compound inducing a 10-ms increase (4703 ng/mL; intercept upper CI90). Furthermore, the comparison of the c-QT sensitivity analysis between GP and monkey will be presented. This additional way of analyzing QTc effects in anaesthetized GP by means of plasma concentration modeling may offer further insight in early derisking studies before starting regulatory GLP studies in a fully integrated proarrhythmia risk assessment.

Treating people with schizophrenia still represents a major challenge for neuropsychiatric drug development companies. While available atypical antipsychotics are mainly effective on positive symptoms of schizophrenia, their effects on cognitive and social-cognitive deficits remain insufficient and poorly characterized. For instance, a modest improvement of cognitive functions has been described following clozapine treatment. Nevertheless, it remains unclear whether this outcome is due to a direct effect on the neural circuits underlying cognition or to an indirect effect mediated by an overall reduction in positive symptoms. To address this question, we sought to measure mismatch negativity (MMN) responses in telemetered rats. MMN constitutes an electroencephalography-based biomarker of sensory, pre-attentional and predictive coding processes, functions whose disruptions highly influence certain aspects of patients' cognitive symptoms. MMN was measured under N-methyl-d-aspartate receptor (NMDAr) pharmacological inhibition by MK-801 (dizocilpine), a model based on the glutamatergic hypothesis of schizophrenia, and we tested whether clozapine could improve MMN under this condition or not. We found that MK-801 dose-dependently reduced the MMN peak amplitude in rats, aligning with the MMN response deficit seen in schizophrenia patients. Strikingly, clozapine was able to mitigate this electrophysiological deficit, an unprecedented observation that has the potential to inspire new treatment strategies aimed towards unaddressed schizophrenia symptoms.

Since their implementation, the ICH S7B and E14 guidelines have been successful in that no new approved drugs have been withdrawn from the market due to unanticipated risk of Torsade de Pointe (TdP). While hERG block and QTc prolongation biomarkers are indeed sensitive, they are not specific as multiple drugs block hERG and/or prolong QTc but do not cause TdP. The J-Tpeak interval (JTp) of the ECG has recently been proposed as a novel clinical biomarker to differentiate selective hERG blockers from multi cardiac ion channels inhibitors. Therefore, the present study aimed at evaluating the effects of dofetilide (pure hERG blocker) and dolasetron (balanced ion-channel blocking drug) on QT, JTp and Tpeak-to-Tend (TpTe) Intervals in telemetered conscious dogs, in comparison with levocetirizine, used as a QT negative control drug. The experiments were carried out using 7 dogs previously instrumented with a telemetry implant. Using a cross-over design, each drug was administered at two doses (0.03/0.15, 6/20, 2/10 mg/kg p.o. for dofetilide, dolasetron and levocetirizine, respectively) and ECG monitored over 24 h post-dosing. Dedicated PK sessions were performed to measure corresponding plasma exposure. QT, JTp and TpTe intervals were individually corrected (c) for heart rate variations. Total plasma drug concentrations increased dose-proportionally. Compared to vehicle, dofetilide induced dose-dependent, marked, and long-lasting increases in both QTc and JTpc intervals, confirming predominant hERG blockage by dofetilide. However, no effect was observed on TpTec interval. In contrast, QTc prolongation associated with dolasetron was moderate and transient and no effect on JTpc was observed; interestingly, dolasetron slightly increased TpTec and QRS intervals, as well as PR interval, reflecting its inhibitory effect on calcium and sodium currents. As expected, levocetirizine had no effect, at any dose or timepoint post-dosing. To summarize, pure hERG-blocking drug (such as dofetilide) prolonged both QTc and JTpc, whereas drug with mixed ion channel blockage (such as dolasetron) prolonged QTc but not JTpc. In conclusion, evaluating the effects of a drug on the J-Tpeak interval may be relevant in cardiovascular safety pharmacology studies to complement the QT interval as a sensitive and more specific biomarker for proarrhythmic risk.

The assessment of drug-induced QT interval prolongation and associated proarrhythmic risks, such as Torsades de Pointes (TdP), has evolved significantly over the past decades. This review traces the development of nonclinical QT evaluation, highlighting key milestones and innovations that have shaped current practices in cardiac safety assessment. The emergence of regulatory guidelines, including International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) S7B, established a nonclinical framework for evaluating drug effects on cardiac repolarization, addressing concerns raised by drug withdrawals in the 1990s. Advances in in vitro, in vivo, and in silico models have enhanced the predictive accuracy of nonclinical studies, with the hERG assay and telemetry-based animal models becoming gold standards. Recent initiatives, such as the Comprehensive in vitro Proarrhythmia Assay (CiPA) and the Japan iPS Cardiac Safety Assessment (JiCSA), emphasize integrating mechanistic insights from human-derived cardiomyocyte models and computational approaches to refine risk predictions. The 2020s mark a shift toward integrated nonclinical-clinical risk assessments, as exemplified by the ICH E14/S7B Questions and Answers. These highlight the need of best practices for study design, data analysis, and interpretation to support regulatory decision-making. Furthermore, the adoption of New Approach Methodologies (NAMs) and reinforced adherence to 3Rs principles (Reduce, Refine, Replace) reflect a commitment to ethical and innovative safety science. This review underscores the importance of harmonized and translational approaches in cardiac safety evaluation, providing a foundation for advancing drug development while safeguarding patient safety. Future directions include further integration of advanced methodologies and regulatory harmonization to streamline nonclinical and clinical risk assessments.

The ICH E14 guideline issued in 2005 made the assessment of drug-induced QT-prolongation a mandatory step in the development of all new therapeutics. The guidance provided a clear requirement, however the means to achieve this requirement within the specific space of oncology clinical development remained challenging. Several updates to the guidelines, mainly in the form of Q&As published in 2012, 2015 and 2022 helped to clarify regulator expectations and thus determine the strategies available in a field where a straightforward investigation performed in Healthy Subjects (i.e. the dedicated TQT trial) is not feasible for reasons of drug tolerability ; a dedicated trial in the patient population also poses significant ethical questions in addition to logistical hurdles. Today, following the different updates to ICH E14 we feel that a clear strategy may be implemented by sponsors developing new treatments in the field of oncology. These strategies, which include in vitro and in vivo testing and a specific approach for ECG recordings in phase I, are followed by several companies, but are not yet universally included in development plans from their start. We thus believe that a simplified roadmap should hold value for professionals looking to develop novel oncology therapeutics. The focused and precise assessment of drug-induced QT prolongation also meets the needs of clinicians looking to assess this risk in complex clinical setting with multiple risk factors1, 2. The purpose of our abstract will be to provide a clear history of the evolution of the ICH E14 guideline and illustrate what could be the current practice implemented for new therapeutics in the field of oncology. Our main interest will be for NCEs who cannot be safely administered to Healthy Participants however approaches for NBEs will also be described, with illustrations of results. This will include a description of the in vivo preclinical and clinical strategies which can be followed. Louis-Guillaume Marquier, Frederique Lemaux-Duvillier, Stephanie Le Goaller, Philippe L'Hostis, Eric Delpy, Mathieu Grace, Jean-Louis Pinquier. History and summary of current practices for assessment of drug-induced QT-prolongation in oncology drug development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2512.

The ICH E14/S7B Q&As highlighted the need for best practices concerning the design, execution, analysis, interpretation, and reporting of the in vivo non‐rodent QT assay as a component of the integrated risk assessment to potentially support a TQT waiver or substitute. We conducted a dog telemetry study to assess the effects on QTc of six reference compounds (five positive and one negative) previously evaluated by Darpo et al. (2015) in humans. The sensitivity of the assay to detect QTc increases was determined, and exposure–response analysis was performed, as done in clinical practice. By‐timepoint analysis showed QTc prolongation induced by moxifloxacin, dofetilide, dolasetron, ondansetron, and quinine within human relevant plasma exposures ranges. Moreover, a hysteresis was observed for quinine. As expected, levocetirizine showed no statistically significant effect on QTc across a range of exposure, well exceeding the therapeutic Cmax. Power analyses confirmed the study ability to detect statistically significant QTc changes of less than 10 milliseconds with 80% probability, even with a sample size as low as n = 4 animals. Finally, concentration‐QTc modeling enabled to predict the minimal plasma concentration needed to detect a 10 milliseconds QTc prolongation, including for quinine. The comparison with clinical available data supported the relevance of dogs under these experimental conditions as a robust translational predictor of drug‐induced QTc prolongation in humans as a key pillar of the integrated risk assessment.

The available antipsychotics for schizophrenia (SZ) only reduce positive symptoms and do not significantly modify SZ neurobiology. This has raised the question of the robustness and translational value of methods employed during drug development. Electroencephalography (EEG)-based measures like evoked and spontaneous gamma oscillations are considered robust translational biomarkers as they can be recorded in both patients and animal models to probe a key mechanism underlying all SZ symptoms: the excitation/inhibition imbalance mediated by N-methyl-D-aspartate receptor (NMDAr) hypofunction. Understanding the effects of commercialized atypical antipsychotics on such measures could therefore contribute to developing better therapies for SZ. Yet, the effects of such drugs on these EEG readouts are unknown. Here, we studied the effect of the atypical antipsychotic aripiprazole on the gamma-band auditory steady-state response (ASSR), spontaneous gamma oscillations and behavioral features in a SZ rat model induced by the NMDAr antagonist MK-801. Interestingly, we found that aripiprazole could not normalize MK-801-induced abnormalities in ASSR, spontaneous gamma oscillations or social interaction while it still improved MK-801-induced hyperactivity. Suggesting that aripiprazole is unable to normalize electrophysiological features underlying SZ symptoms, our results might explain aripiprazole's inefficacy towards the social interaction deficit in our model but also its limited efficacy against social symptoms in patients.

Background Porphyromonas gingivalis exacerbates tissue hypoxia and worsens periodontal inflammation. This study investigated the effect of a therapeutic oxygen carrier (M101), derived from Arenicola marina, on hypoxia and associated inflammation in the context of periodontitis. Methods The effect of M101 on GLUT-1, GLUT-3, HIF-1 alpha, and MMP-9 expression, hypoxia, and antioxidant status in oral epithelial cells (EC) exposed to CoCl2 (1000 mu M), P. gingivalis (MOI 100), and CoCl2 + P. gingivalis was evaluated through hypoxia detection fluorescence assay, antioxidant concentration colorimetric assay, and RTqPCR. Evaluation of M101 on EC proliferation was evaluated in an in vitro wound assay. In experimental periodontitis, periodontal wound healing and osteoclastic activity were compared among natural wound healing, placebo, and gels containing M101 (1 and 2 g/L) groups through histomorphometry and TRAP (tartrate-resistant acid phosphatase activity assay) assay respectively. The expression of HIF-1 alpha, MMP-9, and NF kappa B in periodontal tissues was also evaluated through immunofluorescence studies. Results M101 downregulated GLUT-1, GLUT-3, HIF-1 alpha, and MMP-9 levels in EC exposed to CoCl2, P. gingivalis, and CoCl2 + P. gingivalis (p < 0.05). Fluorescence and colorimetric analyses confirmed hypoxia reduction and antioxidant capacity improvement in such EC upon M101 treatment. Moreover, M101 improved significantly the in vitro wound closure. In vivo, the attachment level was significantly improved, and osteoclastic activity was reduced in mice treated with M101 gels compared to placebo and natural wound healing groups (p < 0.05). HIF-1 alpha, MMP-9, and NF kappa B expression in periodontal tissues was reduced in M101 gels treated mice compared to the controls. Conclusion M101 showed promise in resolving hypoxia and associated inflammation-mediated tissue degradation. Its potential in the clinical management of periodontitis must be further investigated.

Periodontitis is an inflammatory disease associated with anaerobic bacteria leading to the destruction of tooth supporting tissues. Porphyromonas gingivalis is a keystone anaerobic pathogen involved in the development of severe lesions. Periodontal treatment aims to suppress subgingival biofilms and to restore tissue homeostasis. However, hypoxia impairs wound healing and promotes bacterial growth within periodontal pocket. This study aimed to evaluate the potential of local oxygen delivery through the local application of a hydrogel containing Arenicola marina's hemoglobin (M101). To this end, a hydrogel (xanthan (2%), hyaluronic acid (1%)) containing M101 (1-2 g/L) (Xn(2%)-HA(1%)-M101) was prepared and characterized. Rheological tests revealed the occurrence of high deformation without the loss of elastic properties. Dialysis experiment revealed that incorporation of M101 within the gel did not modify its oxygen transportation properties. Samples of release media of the gels (1 g/L (10%) and 2 g/L (10%) M101) decreased significantly the growth of P. gingivalis after 24 h validating its antibacterial effect. Metabolic activity measurement confirmed the cytocompatibility of Xn(2%)HA(1%)-M101. This study suggests the therapeutic interest of Xn(2%)-HA(1%)-M101 gel to optimize treatment of periodontitis with a non-invasive approach.

The amanitins (namely α- and β-amanitin) contained in certain mushrooms are bicyclic octapeptides that, when ingested, are responsible for potentially lethal hepatotoxicity. M101 is an extracellular hemoglobin extracted from the marine worm Arenicola marina. It has intrinsic Cu/Zn-SOD-like activity and is currently used as an oxygen carrier in organ preservation solutions. Our present results suggest that M101 might be effective in reducing amanitin-induced hepatotoxicity and may have potential for therapeutic development.

BACKGROUND:M101 is an extracellular hemoglobin isolated from a marine lugworm and is present in the medical device HEMO2 life®. The clinical investigation OXYOP was a paired kidney analysis (n = 60) designed to evaluate the safety and performance of HEMO2 life® used as an additive to preservation solution in renal transplantation. The secondary efficacy endpoints showed less delayed graft function (DGF) and better renal function in the HEMO2 life® group but due to the study design cold ischemia time (CIT) was longer in the contralateral kidneys.METHODS:An additional analysis was conducted including OXYOP patients and patients from the ASTRE database (n = 6584) to verify that the decrease in DGF rates observed in the HEMO2 life® group may not be due solely to the shorter CIT but also to HEMO2 life® performance. Kaplan-Meier estimate curves of cumulative probability of achieving a creatinine level below 250 µmol/L were generated and compared in both groups. A Cox model was used to test the effect of the explanatory variables (use of HEMO2 life® and CIT). Finally, a bootstrap strategy was used to randomly select smaller samples of patients and test them for statistical comparison in the ASTRE database.RESULTS:Kaplan-Meier estimate curves confirmed the existence of a relation between DGF and CIT and Cox analysis showed a benefit in the HEMO2 life® group regardless of the associated CIT. Boostrap analysis confirmed these results.CONCLUSIONS:The present study suggested that the better recovery of renal function observed among kidneys preserved with HEMO2 life® in the OXYOP study is a therapeutic benefit of this breakthrough innovative medical device.

The combined impact of an increasing demand for liver transplantation and a growing incidence of nonalcoholic liver disease has provided the impetus for the development of innovative strategies to preserve steatotic livers. A natural oxygen carrier, HEMO2life®, which contains M101 that is extracted from a marine invertebrate, has been used for static cold storage (SCS) and has shown superior results in organ preservation. A total of 36 livers were procured from obese Zucker rats and randomly divided into three groups, i.e., control, SCS-24H and SCS-24H + M101 (M101 at 1 g/L), mimicking the gold standard of organ preservation. Ex situ machine perfusion for 2 h was used to evaluate the quality of the livers. Perfusates were sampled for functional assessment, biochemical analysis and subsequent biopsies were performed for assessment of ischemia-reperfusion markers. Transaminases, GDH and lactate levels at the end of reperfusion were significantly lower in the group preserved with M101 (p < 0.05). Protection from reactive oxygen species (low MDA and higher production of NO2-NO3) and less inflammation (HMGB1) were also observed in this group (p < 0.05). Bcl-1 and caspase-3 were higher in the SCS-24H group (p < 0.05) and presented more histological damage than those preserved with HEMO2life®. These data demonstrate, for the first time, that the addition of HEMO2life® to the preservation solution significantly protects steatotic livers during SCS by decreasing reperfusion injury and improving graft function.