Novel predictive approaches for drug-induced convulsions in non-human primates using machine learning and heart rate variability analysis.

Drug-induced convulsions are a major challenge to drug development because of the lack of reliable biomarkers. Using machine learning, our previous research indicated the potential use of an index derived from heart rate variability (HRV) analysis in non-human primates as a biomarker for convulsions induced by GABAA receptor antagonists. The present study aimed to explore the application of this methodology to other convulsants and evaluate its specificity by testing non-convulsants that affect the autonomic nervous system. Telemetry-implanted males were administered various convulsants (4-aminopyridine, bupropion, kainic acid, and ranolazine) at different doses. Electrocardiogram data gathered during the pre-dose period were employed as training data, and the convulsive potential was evaluated using HRV and multivariate statistical process control. Our findings show that the Q-statistic-derived convulsive index for 4-aminopyridine increased at doses lower than that of the convulsive dose. Increases were also observed for kainic acid and ranolazine at convulsive doses, whereas bupropion did not change the index up to the highest dose (1/3 of the convulsive dose). When the same analysis was applied to non-convulsants (atropine, atenolol, and clonidine), an increase in the index was noted. Thus, the index elevation appeared to correlate with or even predict alterations in autonomic nerve activity indices, implying that this method might be regarded as a sensitive index to fluctuations within the autonomic nervous system. Despite potential false positives, this methodology offers valuable insights into predicting drug-induced convulsions when the pharmacological profile is used to carefully choose a compound.

Identifying the dataset to define the optimal timing of histopathological examination for central nervous system toxicity in MPTP-induced Parkinson's disease monkey model.

Determining the optimal timing for histopathological examination following exposure to a test article is crucial for assessing neurotoxicity. However, no study has focused on identifying an ideal dataset to define the optimal timing for histopathological examination of central nervous system (CNS) toxicity in monkeys. Therefore, this study aimed to define a predictive endpoint that would guide us in selecting the optimal timing for histopathological examination of CNS toxicity in monkeys. Four cynomolgus monkeys were administered 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intravenously at a dosage of 0.6 mg/kg twice at 1-week intervals. Necropsies were performed 1 week after the final dose. The Parkinsonian rating (PR) score and temporal changes in neurofilament light chain and glial fibrillary acidic protein concentrations in the cerebrospinal fluid (CSF) and serum were evaluated and compared with the histopathological findings in the brain. The PR score of all animals administered MPTP increased from days 10 to 11, with some degree of individual variability. Microscopically, all animals showed axonal swelling and vacuolation, with or without microgliosis in the nigrostriatal bundle. However, substantial neurodegenerative findings were observed only in animals with high PR scores at necropsy. A slight increase in CSF biomarker levels at necropsy was also observed in animals with high PR scores. However, their correlation with microscopic findings in these animals was unclear. These data suggest that comprehensive clinical observations, such as PR score alone or combined with other CSF biomarkers, could be further evaluated as potential indicators for triggering anatomic CNS evaluations in monkeys following toxic insults.

Motor-evoked potentials after focal electrical stimulation predict drug-induced convulsion potentials in rats.

Drug-induced convulsions-often caused by the inhibition of GABA receptors and stimulation of glutamate receptors-are difficult to predict in animals. In this study, we attempted to detect the proconvulsant potential using motor-evoked potentials (MEPs) after focal electrical stimulation or upon using a functional observational battery (FOB). Pentylenetetrazole, kainic acid, and pilocarpine were used as convulsion-inducing drugs, and baclofen was used as a negative control. First, each compound was administered to male rats, and the FOB tests were performed. All drugs induced behavioral changes, but no commonality was found. Single electrical stimulation train MEPs were recorded under anesthesia for 60 min (at 5 min intervals) after administration of each drug. A dose-dependent increase in MEPs was observed for each convulsion-inducing drug. Moreover, paired electrical stimulation (conditioned and test) of the cerebral motor cortex was conducted with a 1-15 ms interstimulus interval (ISI), 10 min after administration of the drug. All convulsion-inducing drugs inhibited the short-interval intracortical inhibition (ISI: 3 ms), which may be associated with GABA. Intracortical facilitation (ISI: 11 ms), related to glutamate, was not enhanced by any drug but was inhibited by pilocarpine. Dose correlation was not found in short-interval intracortical inhibition or intracortical facilitation in any drugs. No changes in MEPs were observed after baclofen administration. These results suggest that it is possible to evaluate the convulsion potential and associated mechanisms using MEP, independent of the behavioral changes. The early identification of convulsion potential using this model will lead to more efficient drug development.

[Technology and evaluation best practices for in vivo cardiovascular safety pharmacology studies].

In vivo cardiovascular experiments as part of safety pharmacology studies have been developed for small molecule drug candidates to maximize detection power for potential undesirable pharmacodynamic effects of a drug candidate on physiological functions, and have been established with appropriate expertise. Conscious freely-moving telemeterized non-rodents are generally used for the in vivo cardiovascular experiments. The technology and evaluation best practices for the experiments have been optimized by multiple researchers and as a result, the experiments considerably contribute to the estimation of cardiovascular risks for humans. In addition, as described in ICH E14&S7B Q&A draft, non-clinical studies are gaining importance in the integrated risk assessment for QT prolongation in humans, and high quality data obtained in non-clinical studies are being required. This manuscript introduces actual technology and evaluation for in vivo cardiovascular safety pharmacology studies based on Japan activity for Improvement of Cardiovascular Evaluation by Telemetry system (J-ICET), which is one of the working groups hosted by Japanese Safety Pharmacology Society.

Prediction of GABA receptor antagonist-induced convulsion in cynomolgus monkeys by combining machine learning and heart rate variability analysis.

Drug-induced convulsion is a severe adverse event; however, no useful biomarkers for it have been discovered. We propose a new method for predicting drug-induced convulsions in monkeys based on heart rate variability (HRV) and a machine learning technique. Because autonomic nervous activities are altered around the time of a convulsion and such alterations affect HRV, they may be predicted by monitoring HRV. In the proposed method, anomalous changes in multiple HRV parameters are monitored by means of a convulsion prediction model, and convulsion alarms are issued when abnormal changes in HRV are detected. The convulsion prediction model is constructed based on multivariate statistical process control (MSPC), a well-known anomaly detection algorithm in machine learning. In this study, HRV data were collected from four cynomolgus monkeys administered with multiple doses of pentylenetetrazol (PTZ) and picrotoxin (PTX), which are GABA receptor antagonists, as convulsant agents. In addition, low doses of pilocarpine (PILO) were administered as a negative control. Twelve HRV parameters in three hours after drug administration were monitored by means of the prediction model. The number and duration of convulsion alarms from HRV increased at medium and high doses of PTZ and PTX (1/3 or 1/4 of convulsion dose). On the other hand, the frequency of convulsion alarms did not increase with PILO. Although vomiting was observed in all drugs, convulsion alarms were not associated with the vomiting. Thus, convulsion alarms can be used as a biomarker for convulsions induced by GABA receptor antagonists.

Blockade of Motor Cortical Long-Term Potentiation Induction by Glutamatergic Dysfunction Causes Abnormal Neurobehavior in an Experimental Subarachnoid Hemorrhage Model.

Subarachnoid hemorrhage (SAH) is a life-threatening condition that can also lead to permanent paralysis. However, the mechanisms that underlying neurobehavioral deficits after SAH have not been fully elucidated. As theta burst stimulation (TBS) can induce long-term potentiation (LTP) in the motor cortex, we tested its potential as a functional evaluation tool after experimentally induced SAH. Motor cortical inter-neuronal excitability was evaluated in anesthetized rats after 200 Hz-quadripulse TBS (QTS5), 200 Hz-quadripulse stimulation (QPS5), and 400 Hz-octapulse stimulation (OPS2.5). Furthermore, correlation between motor cortical LTP and N-methyl-D-aspartate-receptor activation was evaluated using MK-801, a NMDA-receptor antagonist. We evaluated inhibition-facilitation configurations [interstimulus interval: 3 ms; short-latency intracortical inhibition (SICI) and 11 ms; intracortical facilitation (ICF)] with paired electrical stimulation protocols and the effect of TBS paradigm on continuous recording of motor-evoked potentials (MEPs) for quantitative parameters. SAH and MK-801 completely blocked ICF, while SICI was preserved. QTS5, QPS5, and OPS2.5 facilitated continuous MEPs, persisting for 180 min. Both SAH and MK-801 completely blocked MEP facilitations after QPS5 and OPS2.5, while MEP facilitations after QTS5 were preserved. Significant correlations were found among neurological scores and 3 ms-SICI rates, 11 ms-ICF rates, and MEP facilitation rates after 200 Hz-QTS5, 7 days after SAH (R2 = 0.6236; r = -0.79, R2 = 0.6053; r = -0.77 and R2 = 0.9071; r = 0.95, p < 0.05, respectively). Although these findings need to be verified in humans, our study demonstrates that the neurophysiological parameters 3 ms-SICI, 11 ms-ICF, and 200 Hz-QTS5-MEPs may be useful surrogate quantitative biomarkers for assessing inter-neuronal function after SAH.

Video-based assessment of drug-induced effects on contractile motion properties using human induced pluripotent stem cell-derived cardiomyocytes.

INTRODUCTION: Drug-induced inotropic change is a risk factor in drug development; thus, de-risking is desired in the early stages of drug development. Unlike proarrhythmic risk assessment using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), few in vitro models were validated to predict cardiac contractility. Motion field imaging (MFI), a high-resolution block matching-based optical flow technique, was expected to possess high quantitative predictivity in the detection of contraction speed. We aimed to establish an in vitro model to assess drug-induced contractile changes using hiPSC-CMs and MFI. METHODS: MFI was designed to noninvasively characterize cardiomyocyte contractile behavior by analyzing light microscope video images, and maximum contraction speed (MCS) was used as the index of contractility. Using MFI, 9 inactive compounds, 10 negative inotropes, and 10 positive inotropes were tested. Two negative chronotropes, ZD7288 and ivabradine, were also tested. To determine the sensitivity and specificity of the assay, the minimum effective concentration of the MCS was compared with the human effective total therapeutic concentration for 28 compounds in clinical use. RESULTS: For 8 negative and 8 positive inotropes, the effects observed in in vivo and clinical studies were detected in MFI assay. MFI assay showed negative chronotropic changes without inotropic changes. MFI assay presented sufficient specificity (83%) and sensitivity (88%), and RNA-sequence analysis provided an insight into the relationship between occurrence of the false compounds and target gene expression. DISCUSSION: We demonstrated the utility of MFI assay using hiPSC-CMs to assess drug-induced contractile function. These results will facilitate the de-risking of compounds during early drug development.

Exposure-response analysis of drug-induced QT interval prolongation in telemetered monkeys for translational prediction to human.

INTRODUCTION: The preclinical in vivo assay for QT prolongation is critical for predicting torsadogenic risk, but still difficult to extrapolate to humans. This study ran preclinical tests in cynomolgus monkeys on seven QT reference drugs containing the drugs used in the IQ-CSRC clinical trial and applied exposure-response (ER) analysis to the data to investigate the potential for translational information on the QT effect. METHODS: In each of six participating facilities in the J-ICET project, telemetered monkeys were monitored for 24 h following administration of vehicle or 3 doses of test drugs, and pharmacokinetic profiles at the same doses were evaluated separately. An individual rate-corrected QT interval (QTca) was derived and the vehicle-adjusted change in QTca from baseline (∆∆QTca) was calculated. Then the relationship of concentration to QT effect was evaluated by ER analysis. RESULTS: For QT-positive drugs in the IQ-CSRC study (dofetilide, dolasetron, moxifloxacin, ondansetron, and quinine) and levofloxacin, the slope of the total concentration-QTca effect was significantly positive, and the QT-prolonging effect, taken as the upper bound of the confidence interval for predicted ∆∆QTca, was confirmed to exceed 10 ms. The ER slope of the negative drug levocetirizine was not significantly positive and the QTca effect was below 10 ms at observed peak exposure. DISCUSSION: Preclinical QT assessment in cynomolgus monkeys combined with ER analysis could identify the small QT effect induced by several QT drugs consistently with the outcomes in humans. Thus, the ER method should be regarded as useful for translational prediction of QT effects in humans.

In vivo binding of protoporphyrin IX to rat translocator protein imaged with positron emission tomography.

In vitro experiments have shown that protoporphyrin IX (PPIX) binds to the translocator protein 18 kDa (TSPO), which transports cholesterol across the outer mitochondrial membrane. The purpose of this study was to examine whether binding of PPIX to TSPO can also be detected in vivo using positron emission tomography and [(11)C]PBR28, a radioligand that binds with high affinity and selectivity to TSPO. Rats were injected with a high dose of 5-aminolevulinic acid (ALA, 200 mg/kg i.v.), which is a precursor for PPIX. ALA-pretreatment significantly decreased the uptake of [(11)C]PBR28 in TSPO-rich organs such as heart, kidneys, lungs, parotid glands, and spleen by 57-80%. As a control experiment, injection of a receptor saturating does of PK 11195, which is selective for TSPO, produced a pattern of displacement similar to that after ALA but with greater magnitude (88-97%). This study provides the first evidence that PPIX binds in vivo to TSPO. Although PPIX at physiological concentrations would likely occupy an insignificant percentage of TSPOs, it does reach high-enough concentrations in porphyria to occupy and have pharmacological effects via this target.