Results from a Joined Prospective Study to Evaluate the Sensitivity of the In Vivo Dog QT Assay in Line with the ICH E14/S7B Q&A Best Practices.

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.

Differential Effects of Aripiprazole on Electroencephalography-Recorded Gamma-Band Auditory Steady-State Response, Spontaneous Gamma Oscillations and Behavior in a Schizophrenia Rat Model.

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.

Ocular Administration of Palonosetron in the Prevention of Cisplatin-Induced Nausea and Vomiting.

Cancer treatments are frequently associated with nausea and vomiting despite greatly improved preventive medication. Administration of antinausea agents as eye drops might provide easy and rapid access to the systemic circulation for prevention of nausea and vomiting and for the treatment of breakthrough nausea, but the ocular administration route has rarely been evaluated. Palonosetron is a second-generation 5-hydroxytryptamine 3 receptor antagonist approved for prevention and treatment of chemotherapy-induced nausea and vomiting. We compared ocular administration of palonosetron to non-active vehicle eye drops and to intravenous palonosetron in the prevention of cisplatin-induced nausea and vomiting in beagle dogs. Palonosetron ocular drops at the dose of 30 µg/kg reduced cumulative nausea over time as measured with the area under the visual analog scale curve by 98% compared with the vehicle and reduced nausea-associated dog behavior by 95%. Vomiting was completely prevented with repeated palonosetron ocular dosing. Hydroxypropyl-ß-cyclodextrin (HP-ß-CD) palonosetron formulation was well tolerated locally at the palonosetron concentration of 3 mg/ml. Absorption of palonosetron from eye drops was fast. Ten minutes after ocular administration, palonosetron plasma concentrations were similar compared with intravenous administration, and remained similar for six hours. We conclude that palonosetron is rapidly absorbed into the systemic circulation from eye drops. Ocularly administered palonosetron was well tolerated in the HP-ß-CD formulation and was highly effective in the prevention of cisplatin-induced nausea and vomiting. Evaluation of the safety and efficacy of ocular administration of palonosetron is warranted in the prevention and treatment of chemotherapy-induced nausea and vomiting in clinical trials. SIGNIFICANCE STATEMENT: Palonosetron, an effective and well-tolerated antiemetic drug was rapidly absorbed into the systemic blood circulation when administered as eye drops. The achieved palonosetron blood concentrations prevented cisplatin-induced nausea and vomiting in beagle dogs. Palonosetron eye drops might provide an easy and quick method for administering palonosetron when parenteral administration is desired and intravenous administration is not feasible.

In vitrolong term differentiation and functionality of three-dimensional bioprinted primary human hepatocytes: application forin vivoengraftment.

In recent decades, 3Din vitrocultures of primary human hepatocytes (PHHs) have been increasingly developed to establish models capable of faithfully mimicking main liver functions. The use of 3D bioprinting, capable of recreating structures composed of cells embedded in matrix with controlled microarchitectures, is an emergent key feature for tissue engineering. In this work, we used an extrusion-based system to print PHH in a methacrylated gelatin (GelMa) matrix. PHH bioprinted in GelMa rapidly organized into polarized hollow spheroids and were viable for at least 28 d of culture. These PHH were highly differentiated with maintenance of liver differentiation genes over time, as demonstrated by transcriptomic analysis and functional approaches. The cells were polarized with localization of apico/canalicular regions, and displayed activities of phase I and II biotransformation enzymes that could be regulated by inducers. Furthermore, the implantation of the bioprinted structures in mice demonstrated their capability to vascularize, and their ability to maintain human hepatic specific functions for at least 28 d was illustrated by albumin secretion and debrisoquine metabolism. This model could hold great promise for human liver tissue generation and its use in future biotechnological developments.

Mismatch negativity as EEG biomarker supporting CNS drug development: a transnosographic and translational study.

The lack of translation from basic research into new medicines is a major challenge in CNS drug development. The need to use novel approaches relying on (i) patient clustering based on neurobiology irrespective to symptomatology and (ii) quantitative biomarkers focusing on evolutionarily preserved neurobiological systems allowing back-translation from clinical to nonclinical research has been highlighted. Here we sought to evaluate the mismatch negativity (MMN) response in schizophrenic (SZ) patients, Alzheimer's disease (AD) patients, and age-matched healthy controls. To evaluate back-translation of the MMN response, we developed EEG-based procedures allowing the measurement of MMN-like responses in a rat model of schizophrenia and a mouse model of AD. Our results indicate a significant MMN attenuation in SZ but not in AD patients. Consistently with the clinical findings, we observed a significant attenuation of deviance detection (~104.7%) in rats subchronically exposed to phencyclidine, while no change was observed in APP/PS1 transgenic mice when compared to wild type. This study provides new insight into the cross-disease evaluation of the MMN response. Our findings suggest further investigations to support the identification of neurobehavioral subtypes that may help patients clustering for precision medicine intervention. Furthermore, we provide evidence that MMN could be used as a quantitative/objective efficacy biomarker during both preclinical and clinical stages of SZ drug development.

Implication of nigral dopaminergic lesion and repeated L-dopa exposure in neuropsychiatric symptoms of Parkinson's disease.

This study aims to investigate the contribution of nigral dopaminergic (DA) cell loss, repeated exposure to DA medication and the combination of both to the development of neuropsychiatric symptoms observed in Parkinson's disease (PD). A bilateral 6-OHDA lesion of the substantia nigra pars compacta (SNc) was performed in rats. A set of animals was repeatedly administered with L-dopa (20 mg/kg/day) and benserazide (5 mg/kg/day) over 10 days starting from day 11 post-lesion. Behavioural testing was performed in week 3 post-lesion: novel object recognition (NOR), elevated plus maze (EPM) social interaction (SI) tests, and amphetamine-induced hyperlocomotion (AIH). Immunohistochemical analysis revealed a significant partial lesion (48%) in 6-OHDA versus sham rats. This lesion was not associated with motor impairment. However, lesioned rats displayed a significant deficit in the NOR, which was reversed by acute treatment with l-dopa/benserazide (12.5 mg/kg and 15 mg/kg respectively). Lesioned rats also displayed a deficit in the EPM which was not reversed by acute treatment with l-dopa. No difference was observed in the SI test or in the AIH assay. In all assays, no effect of chronic l-dopa exposure was observed. This study provides new insights into the neuropathophysiology associated with neuropsychiatric symptoms of PD. Our data strongly emphasises a not previously clearly identified critical role in cognition for the SNc. The results suggest that DA pathways were less directly involved in lesion-induced anxiety-like behaviour. We did not report any effect of chronic l-dopa exposure in the context of partial nigral cell loss.

Validation of a New Scoring Scale for Behavioral Assessment of L-Dopa-Induced Dyskinesia in the Rat: A New Tool for Early Decision-Making in Drug Development.

The 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated nonhuman primate (NHP) has been described as the most translatable model for experimental reproduction of L-dopa-induced dyskinesia (LID). However, from a drug discovery perspective, the risk associated with investment in this type of model is high due to the time and cost. The 6-hydroxydopamine (6-OHDA) rat dyskinesia model is recommended for testing compounds but relies on onerous, and nonstandard behavioral rating scales. We sought to develop a simplified and sensitive method aiming at assessing LID in the rat. The purpose was to validate a reliable tool providing earlier insight into the antidyskinetic potential of compounds in a time/cost-effective manner before further investigation in NHP models. Unilaterally 6-OHDA-lesioned rats were administered L-dopa (20 mg/kg) and benserazide (5 mg/kg) daily for 3 weeks starting 4 weeks postlesion, then coadministered with amantadine (20-30-40 mg/kg). An adapted rating scale was used to score LID frequency and a severity coefficient was applied depending on the features of the observed behavior. A gradual increase (about 3-fold) in LID score was observed over the 3 weeks of L-dopa treatment. The rating scale was sensitive enough to highlight a dose-dependent amantadine-mediated decrease (about 2.2-fold) in LID score. We validated a simplified method, able to reflect different levels of severity in the assessment of LID and, thus, provide a reliable tool for drug discovery.

Memory facilitating effects of agomelatine in the novel object recognition memory paradigm in the rat.

The aim of the present study was to evaluate the effects of agomelatine, an antidepressant with melatonergic agonist and 5-HT(2C) antagonist properties, in the rat novel object recognition (NOR) task, a model of short-term episodic memory. To assess the potential involvement of its chronobiotic activity, single intraperitoneal administration of agomelatine and NOR testing were performed either in the evening or in the morning. In both conditions, using a 24h retention interval, vehicle-treated rats did not discriminate between the novel and the familiar object (recognition index was not different from chance performance) while object memory performance of rats treated with agomelatine either in the evening (10 and 40mg/kg) or in the morning (2.5, 10, and 40mg/kg) was significantly improved. Moreover, the selective 5-HT(2C) antagonist SB 242,084 (0.63, 2.5, and 10mg/kg) and melatonin (2.5, 10, and 40mg/kg) displayed also memory facilitating effects in both administration conditions. Finally, thioperamide used as positive reference compound to validate the experimental conditions, demonstrated a memory facilitating effect. In conclusion, agomelatine was shown to possess memory facilitating effects in the rat NOR task and both melatonergic agonist and 5-HT(2C) antagonist properties could be involved in these effects.

Cognition-enhancing properties of Dimebon in a rat novel object recognition task are unlikely to be associated with acetylcholinesterase inhibition or N-methyl-D-aspartate receptor antagonism.

Dimebon (dimebolin) treatment enhances cognition in patients with Alzheimer's disease (AD) or Huntington's disease. Although Dimebon was originally thought to improve cognition and memory through inhibition of acetylcholinesterase (AChE) and the N-methyl-d-aspartate (NMDA) receptor, the low in vitro affinity for these targets suggests that these mechanisms may not contribute to its clinical effects. To test this hypothesis, we assessed whether Dimebon enhances cognition in rats and if such an action is related to either mechanism or additional candidate mechanisms. Acute oral administration of Dimebon to rats (0.05, 0.5, and 5 mg/kg) enhanced cognition in a novel object recognition task and produced Dimebon brain concentrations of 1.7 +/- 0.43, 14 +/- 5.1, and 172 +/- 94 nM, respectively. At these concentrations, Dimebon did not alter the activity of recombinant human or rat brain AChE. Unlike the AChE inhibitors donepezil and galantamine, Dimebon did not change acetylcholine levels in the hippocampus or prefrontal cortex of freely moving rats. Dimebon displays affinity for the NMDA receptor (K(i) = 105 +/- 18 microM) that is considerably higher than brain concentrations associated with cognition enhancement in the novel object recognition task and 200-fold weaker than that of memantine (K(i) = 0.54 +/- 0.05 microM). Dimebon did not block NMDA-induced calcium influx in primary neuronal cells (IC(50) > 50 microM), consistent with a lack of significant effect on this pathway. The cognition-enhancing effects of Dimebon are unlikely to be mediated by AChE inhibition or NMDA receptor antagonism, and its mechanism of action appears to be distinct from currently approved medications for AD.

Arenicola marina extracellular hemoglobin: a new promising blood substitute.

The need to develop a blood substitute is now urgent because of the increasing concern over Europe's BSE outbreak and the worldwide HIV/AIDS epidemic, which have cut blood supplies. Extracellular soluble hemoglobin has long been studied for its possible use as a safe and effective alternative to blood transfusion, but this has met with little success. Clinical trials have revealed undesirable side effects-oxidative damage and vasoconstriction-that hamper the application of cell-free hemoglobin as a blood substitute. We have addressed these problems and have found a new promising extracellular blood substitute: the natural giant extracellular polymeric hemoglobin of the polychaete annelid Arenicola marina. Here we show that it is less likely to cause immunogenic response; its functional and structural properties should prevent the side effects often associated with the administration of extracellular hemoglobin. Moreover, its intrinsic properties are of interest for other therapeutic applications often associated with hemorrhagic shock (ischemia reperfusion, treatment of septic shock and for organ preservation prior to transplantation). Moreover, using natural hemoglobin is particularly useful since recombinant DNA techniques could be used to express the protein in large quantities.

Alzheimer's disease: the pharmacological pathway.

The current pharmacological treatment of Alzheimer's disease (AD) comes down to four marketed drugs (tacrine, donepezil, rivastigmine and galantamine) all of which are cholinesterase inhibitors, conforming to the cholinergic hypothesis. The future is clearly directed at new biological targets closely linked to the pathophysiology of the disease and more precisely, the pathological hallmark of AD which includes widespread neuronal degeneration, neuritic plaques containing beta-amyloid and tau-rich neurofibrillary tangles. For clinicians, this means that new curative drugs will have to be prescribed early in the course of the disease. This review describes the main entry pathways for drug discovery in AD: (1) supplementation therapy, (2) anti-apoptotic compounds, (3) substances with a mitochondrial impact, (4) anti-amyloid substances, (5) anti-protein aggregation and (6) lipid-lowering drugs. The rapidity at which these compounds will be at our disposal is highly dependent on the policy of the pharmaceutical companies.