Etienne Hanon

Neuroprotective properties of the novel antiepileptic drug levetiracetam in the rat middle cerebral artery occlusion model of focal cerebral ischemia

Levetiracetam (LEV) is a new antiepileptic drug with a promising preclinical profile involving both anticonvulsant and antiepileptogenic effects in kindling models. The latter stimulated the present study to compare its neuroprotective properties with the potent and selective, non-competitive NMDA antagonist, MK-801, in the rat middle cerebral artery occlusion model. Twenty-four hours after a transient occlusion of 90 minutes the animals were sacrificed and infarct volume and lesion distribution were determined from stained coronal sections. LEV was administered by intraperitoneal (i.p.) bolus injections of 5.5, 11, 22 and 44 mg x kg(-1), 30 minutes before occlusion followed by a continuous 24 hour i.p. infusion of 1.25, 2.6, 5.1 and 10.2 mg x kg(-1) per hour, respectively. LEV administration did not alter body temperature but reduced the infarct volume by 33% (P< 0.05) at the highest dose tested. An i.p. bolus injection of 0.04, 0.12 and 0.4 mg x kg(-1) of MK-801 followed by continuous i.p. infusion of 0.036, 0.108 and 0.36 mg x kg(-1) per hour, reduced the infarct volume by 49, 51 and 74% (P< 0.05), respectively. However, only the highest dose of MK-801 induced a significant reduction in the infarct volume (P< 0.05) and this was associated with hypothermia. These results suggest that LEV possesses neuroprotective properties which may be relevant for its antiepileptogenic action.

Proepileptic phenotype of SV2A-deficient mice is associated with reduced anticonvulsant efficacy of levetiracetam

Purpose:  Synaptic vesicle protein 2A (SV2A) constitutes a distinct binding site for an antiepileptic drug levetiracetam (Keppra). In the present study we characterized SV2A (+/−) heterozygous mice in several seizure models and tested if the anticonvulsant efficacy of levetiracetam is reduced in these mice.

The Use of Real-Time Cell Analyzer Technology in Drug Discovery Defining Optimal Cell Culture Conditions and Assay Reproducibility with Different Adherent Cellular Models

The use of impedance-based label-free technology applied to drug discovery is nowadays receiving more and more attention. Indeed, such a simple and noninvasive assay that interferes minimally with cell morphology and function allows one to perform kinetic measurements and to obtain information on proliferation, migration, cytotoxicity, and receptor-mediated signaling. The objective of the study was to further assess the usefulness of a real-time cell analyzer (RTCA) platform based on impedance in the context of quality control and data reproducibility. The data indicate that this technology is useful to determine the best coating and cellular density conditions for different adherent cellular models including hepatocytes, cardiomyocytes, fibroblasts, and hybrid neuroblastoma/neuronal cells. Based on 31 independent experiments, the reproducibility of cell index data generated from HepG2 cells exposed to DMSO and to Triton X-100 was satisfactory, with a coefficient of variation close to 10%. Cell index data were also well reproduced when cardiomyocytes and fibroblasts were exposed to 21 compounds three times (correlation >0.91, p < 0.0001). The data also show that a cell index decrease is not always associated with cytotoxicity effects and that there are some confounding factors that can affect the analysis. Finally, another drawback is that the correlation analysis between cellular impedance measurements and classical toxicity endpoints has been performed on a limited number of compounds. Overall, despite some limitations, the RTCA technology appears to be a powerful and reliable tool in drug discovery because of the reasonable throughput, rapid and efficient performance, technical optimization, and cell quality control.

Selection of cytotoxicity markers for the screening of new chemical entities in a pharmaceutical context: A preliminary study using a multiplexing approach

The present study was undertaken to validate a battery of cytotoxicity assays performed in a multiplex format to screen pharmaceutical compounds at an early stage of drug development. Two experiments were performed on HepG2 cells and the parameters were measured in 96-well plates. Biological and technical triplicates were performed to evaluate the reproducibility of the assay. In the first experiment, HepG2 cells were exposed to tamoxifen, staurosporine, phenobarbital and triton X-100 for 2 and 24h. The following nine cytotoxicity parameters were analyzed, cell viability, lactate dehydrogenase (LDH), adenosine triphosphate (ATP), caspase-3/7, aspartate aminotransferase (AST), glutamate dehydrogenase (GLDH), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and alpha-glutathione-S-transferase (alpha-GST). In the second experiment, HepG2 cells were exposed to doxorubicin, t-butyl hydroperoxide, ferrous sulfate and sulfamoxole for 2 and 24h. Based on the results of the first experiment, six cytotoxicity parameters were selected for further evaluation (cell viability, ATP, LDH, caspase, AST and GLDH). ALT (activity always below detection limit), ALP (no response to drug treatment) and alpha-GST (too labor intensive and not possible to multiplex) were eliminated. The analysis of the data revealed that the reproducibility of the assays was accurate according to principal component analysis. Our data also clearly indicated that the potential of this battery of selected assays measured in a multiplex format not only made it possible to rank and select the most promising drug candidates based on their cytotoxic potential, but also to gather information that may help to understand some of the toxic events occurring in the cells.

Brivaracetam Differentially Affects Voltage-Gated Sodium Currents Without Impairing Sustained Repetitive Firing in Neurons

Brivaracetam (BRV) is an antiepileptic drug in Phase III clinical development. BRV binds to synaptic vesicle 2A (SV2A) protein and is also suggested to inhibit voltage‐gated sodium channels (VGSCs). To evaluate whether the effect of BRV on VGSCs represents a relevant mechanism participating in its antiepileptic properties, we explored the pharmacology of BRV on VGSCs in different cell systems and tested its efficacy at reducing the sustained repetitive firing (SRF).

Repeated assessment of cardiovascular and respiratory functions using combined telemetry and whole-body plethysmography in the rat

INTRODUCTION As the currently recommended laboratory techniques for assessing cardiovascular and respiratory functions are telemetry and plethysmography, we therefore combined both in a single rodent model. The purpose of the present work was to assess the potential influence of body growth on the recorded parameters, to verify the sensitivity of the system to detect well known pharmacological effects of reference drugs, and to determine their reproducibility over time. METHODS Telemetry instrumented rats were enrolled in successive experiments over a total of 5 months. In each run, they were placed in individual plethysmography chambers for 6h, and received a single intraperitoneal injection of vehicle or test compound. Heart rate, blood pressure, body temperature and respiratory parameters were measured in real time. Six of these 16 rats were submitted 5 times at one month intervals to a vehicle injection, and six rats received theophylline (30 mg/kg) twice at 4 months interval. Nine other reference compounds were also tested at a single dose. RESULTS Analysis of baseline data mainly showed correlations between body weight or age and heart rate, as well as tidal volume. In the five successive runs, handling-induced perturbations were noted in all the parameters during 60 to 90 min. The effects of the different reference drugs were consistent with data published in animals and man. The response to theophylline was qualitatively similar at 4 months interval. DISCUSSION We established a combined model of telemetry and plethysmography in the conscious rat, allowing the reuse of the animals over several successive pharmacodynamic studies. Although a shift of some parameters, particularly heart rate and tidal volume, was noted with age and body weight, this can easily be managed by appropriate design measures. We showed that the combined system can detect negative or positive effects on both cardiovascular and respiratory functions with enough sensitivity.

Brivaracetam does not alter spatial learning and memory in both normal and amygdala-kindled rats

Several antiepileptic drugs (AEDs) may induce memory deficits when tested in preclinical models at doses that exert significant protection against seizures. Brivaracetam (BRV) is a novel high-affinity SV2A ligand also displaying inhibitory activity at neuronal voltage-gated sodium channels. In the present study we have investigated the effects of BRV, at doses that exerted marked anticonvulsant effects in kindled rats, upon cognitive functioning and memory in both normal and amygdala-kindled rats using place learning version of Morris water maze. In addition the effect of BRV on long-term potentiation (LTP) in rat hippocampal slices has been investigated. BRV (2.1, 6.8 or 21.0mg/kg i.p.) was injected daily, 60min before each session. Results indicated that in both normal and amygdala-kindled rats BRV did not alter the latency to find the hidden platform or swimming speed during the four consecutive days of learning. Similarly, the time spent in the target quadrant, used as a further independent index of spatial memory, was not modified by BRV treatment. Likewise, BRV did not affect the LTP induction in CA1 hippocampal region when tested at 3-30microM concentration range, which had been demonstrated to significantly reduce epileptiform activity in slice models. Based on the results of the present study it can be expected that BRV will not have detrimental effects on hippocampal-dependent cognitive functions in patients with epilepsy.

Lack of synaptic vesicle protein SV2B protects against amyloid-β25–35-induced oxidative stress, cholinergic deficit and cognitive impairment in mice

SV2B is a synaptic protein widely distributed throughout the brain, which is part of the complex vesicle protein machinery involved in the regulation of synaptic vesicle endocytosis and exocytosis, and therefore in neurotransmitters release. The aims of the present work were twofold: (1) phenotype SV2B knockout mice (SV2B KO) in a battery of cognitive tests; and (2) examine their vulnerability to amyloid-β25-35 (Aβ25-35) peptide-induced toxicity. SV2B KO mice showed normal learning and memory abilities in absence of Aβ25-35 injection. SV2B KO mice were protected against the learning deficits induced after icv injection of an oligomeric preparation of amyloid-β25-35 peptide, as compared to wild-type littermates (SV2B WT). These mice failed to show Aβ25-35-induced impairments in a number of cognitive domains: working memory measured by a spontaneous alternation procedure, recognition memory measured by a novel object recognition task, spatial reference memory assessed in a Morris water-maze, and long-term contextual memory assessed in a inhibitory avoidance task. In addition, SV2B KO mice were protected against Aβ25-35-induced oxidative stress and decrease in ChAT activity in the hippocampus. These data suggest that SV2B could be a key modulator of amyloid toxicity at the synaptic site.

Comparison of a genomic and a multiplex cell imaging approach for the detection of phospholipidosis

Phospholipidosis (PLD) is a topic of concern in drug development because it may be associated with toxicological consequences. The aim of the study was to determine the best method to screen proprietary compounds with regard to their potential to induce PLD. Two in vitro approaches, a genomic method previously evaluated in our laboratory and a fluorescent cell based approach to detect PLD were compared using HepG2 cells. The same set of reference compounds (15 PLD inducing, 7 non-PLD inducing and 4 additional compounds) were used to compare both approaches. The same sensitivity (15/15) and similar specificity (7/7 versus, 6/7 for the genomic approach) were obtained. In addition, 11 proprietary compounds were tested in 4-day exploratory rat toxicity studies as well as in both in vitro approaches. Two of the 11 compounds induced alveolar foamy macrophages and lung vacuolization in vivo and were considered as PLD inducers. Sensitivity (2/2) and specificity (7/9) were better with the fluorescent method than with the genomic approach (1/2 and 3/9, respectively). In conclusion, compared to the genomic approach, the fluorescent method is the test of choice for screening compounds at an early stage of drug development. Indeed, the fluorescent method is more adapted to medium throughput, detects the positive reference compounds at lower (8/15) or equal (7/15) concentrations, allows multiplexing and is associated with higher sensitivity and specificity to predict lung foamy macrophages and vacuolization in vivo. Nevertheless, to confirm our conclusion, it would be necessary to compare the predictivity of both in vitro approaches by using a wide range of reference and proprietary compounds, with information on their potential to induce PLD under in vivo conditions.

Paving the Route to Plasma miR-208a-3p as an Acute Cardiac Injury Biomarker: Preclinical Rat Data Supports Its Use in Drug Safety Assessment

Drug-induced cardiac injury (DICI) detection remains a major safety issue in drug development. While circulating microRNAs (miRs) have emerged as promising translational biomarkers, novel early detection biomarkers of cardiotoxicity are needed. This work aims at evaluating whether a panel of putative cardiac injury plasma miRs could serve as early DICI biomarkers in a 4-day rat preclinical model. Out of a panel of 68 selected targets, we identified plasma miR-208a-3p as being significantly upregulated after single administration with either isoproterenol (ISO) or allylamine (AAM). This provides the first evidence of miR-208a-3p detection after AAM administration. Moreover, similarly to cardiac troponins (cTn), plasma miR-208a-3p expression profile appears to be compound-specific with most significant early changes occurring in ISO-treated rats. Overall, miR-208a-3p performance in detecting the severity of myocardial injury, as well as the magnitude of miR-208a-3p increase after ISO or AAM administration, were comparable to that of cTn. Our results highlight the importance of assessing the whole time-dependent profiles of miR expression. Hence, time course evaluation revealed plasma miR candidates whose expression was not stable across the duration of the study in the vehicle group, restricting their utility as cardiac injury-specific biomarkers. In light of these findings, miR-208a-3p has a potential to complement the existing biomarkers of cardiac injury specifically in the context of evaluating toxicity in a time-dependant manner. Assessment of miR-208a-3p in other DICI settings would strengthen its robustness as an early detection biomarker leading to a warranted extensive and rigorous validation.