Characterization of Vixotrigine, a Broad-Spectrum Voltage-Gated Sodium Channel Blocker.

Voltage-gated sodium channels (Navs) are promising targets for analgesic and antiepileptic therapies. Although specificity between Nav subtypes may be desirable to target specific neural types, such as nociceptors in pain, many broadly acting Nav inhibitors are clinically beneficial in neuropathic pain and epilepsy. Here, we present the first systematic characterization of vixotrigine, a Nav blocker. Using recombinant systems, we find that vixotrigine potency is enhanced in a voltage- and use-dependent manner, consistent with a state-dependent block of Navs. Furthermore, we find that vixotrigine potently inhibits sodium currents produced by both peripheral and central nervous system Nav subtypes, with use-dependent IC50 values between 1.76 and 5.12 µM. Compared with carbamazepine, vixotrigine shows higher potency and more profound state-dependent inhibition but a similar broad spectrum of action distinct from Nav1.7- and Nav1.8-specific blockers. We find that vixotrigine rapidly inhibits Navs and prolongs recovery from the fast-inactivated state. In native rodent dorsal root ganglion sodium channels, we find that vixotrigine shifts steady-state inactivation curves. Based on these results, we conclude that vixotrigine is a broad-spectrum, state-dependent Nav blocker. SIGNIFICANCE STATEMENT: Vixotrigine blocks both peripheral and central voltage-gated sodium channel subtypes. Neurophysiological approaches in recombinant systems and sensory neurons suggest this block is state-dependent.

Neuroservice proconvulsive (NS-PC) set: A new platform of electrophysiology-based assays to determine the proconvulsive potential of lead compounds.

INTRODUCTION: Failures in drug development often result from the emergence of unexpected adverse drug reactions. It is clear that adverse drug reactions, including seizure liability, should be assessed earlier. The goal of the present work was to develop a new platform of in vitro assays, NS-PC set (for Neuroservice proconvulsive set), to determine the proconvulsive potential of compounds earlier in preclinical development. METHODS: Assays were based on electrophysiological recordings in acute hippocampal slices performed with multielectrode arrays. 4 reference proconvulsive/seizurogenic compounds (4-aminopyridine, bicuculline, kainate and carbachol) and 4 anti-epileptic drugs (AEDs; phenobarbital, carbamazepine, clonazepam and valproic acid) were evaluated on electrophysiological endpoints involved in seizure risk (neuronal excitability, balance of excitatory/inhibitory synaptic transmission, occurrence of neuronal synchronization mechanisms materialized by epileptiform discharges). RESULTS: The reference compounds increased the number and area under the curve of population spikes, triggered epileptiform discharges and enhanced the firing rate of CA1 neurons. The effects of the 4 antiepileptic drugs were assessed on these 3 parameters. They were able to partially of completely reverse the effects of proconvulsive compounds. DISCUSSION: The use of reference proconvulsive compounds and AEDs validated the electrophysiological parameters to detect proconvulsive risk. Systematic evaluation of compounds with the 3 complementary endpoints increase the probability to detect seizure liability in vitro. Depending on the compound mechanism of action, only one or two of the identified parameters might be modified.

Mechanistic characterization of S 38093, a novel inverse agonist at histamine H3 receptors.

Histaminergic H3 inverse agonists, by stimulating central histamine release, represent attractive drug candidates to treat cognitive disorders. The present studies aimed to describe the mechanistic profile of S 38093 a novel H3 receptors inverse agonist. S 38093 displays a moderate affinity for rat, mouse and human H3 receptors (Ki=8.8, 1.44 and 1.2µM, respectively) with no affinity for other histaminergic receptors. In cellular models, the compound was able to antagonize mice H3 receptors (KB=0.65µM) and to suppress cAMP decrease induced by an H3 agonist via human H3 receptors (KB=0.11µM). The antagonism properties of the compound were confirmed by electrophysiological studies on rat hippocampal slices (from 0.1µM). In cells expressing a high H3 density, S 38093 behaved as a moderate inverse agonist at rat and human H3 receptors (EC50=9 and 1.7µM, respectively). S 38093 was rapidly absorbed in mouse and rat (Tmax=0.25-0.5h), slowly in monkey (2h), with a bioavailability ranging from 20% to 60% and t1/2 ranging from 1.5 to 7.4h. The compound was widely distributed with a moderate volume of distribution and low protein binding. The brain distribution of S 38093 was rapid and high. In mice, S 38093 significantly increased ex vivo N-tele-Methylhistamine cerebral levels from 3mg/kg p.o. and antagonized R-α-Methylhistamine-induced dipsogenia from 10mg/kg i.p. Taken together, these data suggest that S 38093, a novel H3 inverse agonist, is a good candidate for further in vivo evaluations, in particular in animal models of cognition.

Phosphodiesterase 10A Inhibition Improves Cortico-Basal Ganglia Function in Huntington's Disease Models.

Huntington's disease (HD) symptoms are driven to a large extent by dysfunction of the basal ganglia circuitry. HD patients exhibit reduced striatal phoshodiesterase 10 (PDE10) levels. Using HD mouse models that exhibit reduced PDE10, we demonstrate the benefit of pharmacologic PDE10 inhibition to acutely correct basal ganglia circuitry deficits. PDE10 inhibition restored corticostriatal input and boosted cortically driven indirect pathway activity. Cyclic nucleotide signaling is impaired in HD models, and PDE10 loss may represent a homeostatic adaptation to maintain signaling. Elevation of both cAMP and cGMP by PDE10 inhibition was required for rescue. Phosphoproteomic profiling of striatum in response to PDE10 inhibition highlighted plausible neural substrates responsible for the improvement. Early chronic PDE10 inhibition in Q175 mice showed improvements beyond those seen with acute administration after symptom onset, including partial reversal of striatal deregulated transcripts and the prevention of the emergence of HD neurophysiological deficits. VIDEO ABSTRACT.

MPX-004 and MPX-007: New Pharmacological Tools to Study the Physiology of NMDA Receptors Containing the GluN2A Subunit.

GluN2A is the most abundant of the GluN2 NMDA receptor subunits in the mammalian CNS. Physiological and genetic evidence implicate GluN2A-containing receptors in susceptibility to autism, schizophrenia, childhood epilepsy and neurodevelopmental disorders such as Rett Syndrome. However, GluN2A-selective pharmacological probes to explore the therapeutic potential of targeting these receptors have been lacking. Here we disclose a novel series of pyrazine-containing GluN2A antagonists exemplified by MPX-004 (5-(((3-chloro-4-fluorophenyl)sulfonamido)methyl)-N-((2-methylthiazol-5-yl)methyl)pyrazine-2-carboxamide) and MPX-007 (5-(((3-fluoro-4-fluorophenyl)sulfonamido)methyl)-N-((2-methylthiazol-5-yl)methyl)methylpyrazine-2-carboxamide). MPX-004 and MPX-007 inhibit GluN2A-containing NMDA receptors expressed in HEK cells with IC50s of 79 nM and 27 nM, respectively. In contrast, at concentrations that completely inhibited GluN2A activity these compounds have no inhibitory effect on GluN2B or GluN2D receptor-mediated responses in similar HEK cell-based assays. Potency and selectivity were confirmed in electrophysiology assays in Xenopus oocytes expressing GluN2A-D receptor subtypes. Maximal concentrations of MPX-004 and MPX-007 inhibited ~30% of the whole-cell current in rat pyramidal neurons in primary culture and MPX-004 inhibited ~60% of the total NMDA receptor-mediated EPSP in rat hippocampal slices. GluN2A-selectivity at native receptors was confirmed by the finding that MPX-004 had no inhibitory effect on NMDA receptor mediated synaptic currents in cortical slices from GRIN2A knock out mice. Thus, MPX-004 and MPX-007 offer highly selective pharmacological tools to probe GluN2A physiology and involvement in neuropsychiatric and developmental disorders.

Myotonic dystrophy CTG expansion affects synaptic vesicle proteins, neurotransmission and mouse behaviour.

Myotonic dystrophy type 1 is a complex multisystemic inherited disorder, which displays multiple debilitating neurological manifestations. Despite recent progress in the understanding of the molecular pathogenesis of myotonic dystrophy type 1 in skeletal muscle and heart, the pathways affected in the central nervous system are largely unknown. To address this question, we studied the only transgenic mouse line expressing CTG trinucleotide repeats in the central nervous system. These mice recreate molecular features of RNA toxicity, such as RNA foci accumulation and missplicing. They exhibit relevant behavioural and cognitive phenotypes, deficits in short-term synaptic plasticity, as well as changes in neurochemical levels. In the search for disease intermediates affected by disease mutation, a global proteomics approach revealed RAB3A upregulation and synapsin I hyperphosphorylation in the central nervous system of transgenic mice, transfected cells and post-mortem brains of patients with myotonic dystrophy type 1. These protein defects were associated with electrophysiological and behavioural deficits in mice and altered spontaneous neurosecretion in cell culture. Taking advantage of a relevant transgenic mouse of a complex human disease, we found a novel connection between physiological phenotypes and synaptic protein dysregulation, indicative of synaptic dysfunction in myotonic dystrophy type 1 brain pathology.

Easy-to-fabricate conducting polymer microelectrode arrays.

A simple and versatile fabrication process is used to define conducting polymer microelectrode arrays (MEAs), patterning at the same time the recording electrodes as well as the insulating layer. Thanks to the low impedance of poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) (PEDOT:PSS) electrodes, these MEAs allow in vitro recording of action potentials from rat hippocampus slices.

Characterization of neurophysiological and behavioral changes, MRI brain volumetry and 1H MRS in zQ175 knock-in mouse model of Huntington's disease.

Huntington's disease (HD) is an autosomal neurodegenerative disorder, characterized by severe behavioral, cognitive, and motor deficits. Since the discovery of the huntingtin gene (HTT) mutation that causes the disease, several mouse lines have been developed using different gene constructs of Htt. Recently, a new model, the zQ175 knock-in (KI) mouse, was developed (see description by Menalled et al, [1]) in an attempt to have the Htt gene in a context and causing a phenotype that more closely mimics HD in humans. Here we confirm the behavioral phenotypes reported by Menalled et al [1], and extend the characterization to include brain volumetry, striatal metabolite concentration, and early neurophysiological changes. The overall reproducibility of the behavioral phenotype across the two independent laboratories demonstrates the utility of this new model. Further, important features reminiscent of human HD pathology are observed in zQ175 mice: compared to wild-type neurons, electrophysiological recordings from acute brain slices reveal that medium spiny neurons from zQ175 mice display a progressive hyperexcitability; glutamatergic transmission in the striatum is severely attenuated; decreased striatal and cortical volumes from 3 and 4 months of age in homo- and heterozygous mice, respectively, with whole brain volumes only decreased in homozygotes. MR spectroscopy reveals decreased concentrations of N-acetylaspartate and increased concentrations of glutamine, taurine and creatine + phosphocreatine in the striatum of 12-month old homozygotes, the latter also measured in 12-month-old heterozygotes. Motor, behavioral, and cognitive deficits in homozygotes occur concurrently with the structural and metabolic changes observed. In sum, the zQ175 KI model has robust behavioral, electrophysiological, and histopathological features that may be valuable in both furthering our understanding of HD-like pathophyisology and the evaluation of potential therapeutic strategies to slow the progression of disease.

TRO40303, a new cardioprotective compound, inhibits mitochondrial permeability transition.

3,5-Seco-4-nor-cholestan-5-one oxime-3-ol (TRO40303) is a new cardioprotective compound coming from a chemical series identified initially for neuroprotective properties. TRO40303 binds specifically to the mitochondrial translocator protein 18 kDa (TSPO) at the cholesterol site. After intravenous administration, TRO40303 tissue distribution was comparable to that of TSPO, and, in particular, the drug accumulated rapidly in the heart. In a model of 35 min of myocardial ischemia/24 h of reperfusion in rats, TRO40303 (2.5 mg/kg) reduced infarct size by 38% (p < 0.01 versus control), when administered 10 min before reperfusion, which was correlated with reduced release of apoptosis-inducing factor from mitochondria to the cytoplasm in the ischemic area at risk. Although TRO40303 had no effect on the calcium retention capacity of isolated mitochondria, unlike cyclosporine A, the drug delayed mitochondrial permeability transition pore (mPTP) opening and cell death in isolated adult rat cardiomyocytes subjected to 2 h of hypoxia followed by 2 h of reoxygenation and inhibited mPTP opening in neonatal rat cardiomyocytes treated with hydrogen peroxide. The effects of TRO40303 on mPTP in cell models of oxidative stress are correlated with a significant reduction in reactive oxygen species production and subsequent calcium overload. TRO40303 is a new mitochondrial-targeted drug and inhibits mPTP triggered by oxidative stress. Its mode of action differs from that of other mPTP inhibitors such as cyclosporine A, thus providing a new pharmacological approach to study mPTP regulation. Its efficacy in an animal model of myocardial infarctions makes TRO40303 a promising new drug for the reduction of cardiac ischemia-reperfusion injury.

Specific antinociceptive activity of cholest-4-en-3-one, oxime (TRO19622) in experimental models of painful diabetic and chemotherapy-induced neuropathy.

Diabetes and cancer chemotherapies are often associated with painful neuropathy. The mechanisms underlying neuropathic pain remain poorly understood, and the current therapies have limited efficacy and are associated with dose-limiting side effects. We recently described the pharmacological characterization of cholest-4-en-3-one, oxime (TRO19622), a cholesterol-like compound, that significantly reduced axonal degeneration and accelerated recovery of motor nerve conduction in a model of peripheral neuropathy induced by crushing the sciatic nerve. These results triggered investigation of efficacy in other preclinical models of peripheral neuropathy. Here, we report evidence that daily oral administration of TRO19622, while similarly improving motor nerve conduction impaired in streptozotocin-induced diabetic rats, also reversed neuropathic pain behavior as early as the first administration. Further exploration of these acute antinociceptive effects demonstrated that TRO19622 was also able to reverse tactile allodynia in vincristine-treated rats, a model of chemotherapy-induced neuropathic pain. It is interesting to note that TRO19622 did not have analgesic activity in animal models of pain produced by formalin injection, noxious thermal or mechanical stimulation, or chronic constriction injury of the sciatic nerve, indicating that painful diabetic or chemotherapy-induced neuropathies share a common mechanism that is distinct from acute, inflammationdriven, or lesion-induced neuropathic pain. These results support the potential use of TRO19622 to treat painful diabetic and chemotherapy-induced neuropathies.

Identification and characterization of cholest-4-en-3-one, oxime (TRO19622), a novel drug candidate for amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by progressive death of cortical and spinal motor neurons, for which there is no effective treatment. Using a cell-based assay for compounds capable of preventing motor neuron cell death in vitro, a collection of approximately 40,000 low-molecular-weight compounds was screened to identify potential small-molecule therapeutics. We report the identification of cholest-4-en-3-one, oxime (TRO19622) as a potential drug candidate for the treatment of ALS. In vitro, TRO19622 promoted motor neuron survival in the absence of trophic support in a dose-dependent manner. In vivo, TRO19622 rescued motor neurons from axotomy-induced cell death in neonatal rats and promoted nerve regeneration following sciatic nerve crush in mice. In SOD1(G93A) transgenic mice, a model of familial ALS, TRO19622 treatment improved motor performance, delayed the onset of the clinical disease, and extended survival. TRO19622 bound directly to two components of the mitochondrial permeability transition pore: the voltage-dependent anion channel and the translocator protein 18 kDa (or peripheral benzodiazepine receptor), suggesting a potential mechanism for its neuroprotective activity. TRO19622 may have therapeutic potential for ALS and other motor neuron and neurodegenerative diseases.

The adult rat hippocampal slice revisited with multi-electrode arrays.

The multi-electrode arrays (MEA) technology for the recording of brain slices is available for more than 10 years. However, despite its relative simplicity, this recording technique is not widely used in academic or pharmaceutical research laboratories. We illustrate here that MEA provide multiple possibilities to investigate some network physiological properties as well as to evaluate the pharmacological effects of compounds. We first document that MEA allow to trigger and to record conventional FP which are inhibited by the block of action potential propagation (with 500 nM TTX). FP recorded with MEA are sensitive to ionic substitutions, to ionotropic glutamate receptor antagonists (CNQX or NBQX) and to energetic failure. Second, we illustrate that different "classical" protocols (paired-pulse, LTP, chemical LTD), revealing synaptic plasticity mechanisms, could be performed. Third, we document that MEA allow spatial and temporal discriminations for the effects of known pharmacological compounds such as competitive antagonist (gabazine, bicuculline) and allosteric modulators (steroids) of GABA(A) receptors. In conclusion, we illustrate that MEA recordings of adult rat hippocampal slices constitute a powerful and sensitive system to evaluate the effect of molecules on basic synaptic propagation/transmission and on synaptic plasticity processes.

Allosteric modulation of ligand-gated ion channels.

Ligand-gated ion channels (LGICs) are cell surface proteins that play an important role in fast synaptic transmission and in the modulation of cellular activity. Due to their intrinsic properties, LGICs respond to neurotransmitters and other effectors (e.g. pH) and transduce the binding of a ligand into an electrical current on a microsecond timescale. Following activation, LGICs open allowing an ion flux across the cell membrane. Depending upon the charge and concentration of ions, the flux can cause a depolarization or hyperpolarization, thus modulating excitability of the cell. While our understanding of LGICs has significantly progressed during the past decade, many properties of these proteins are still poorly understood, in particular their modulation by allosteric effectors. LGICs are often thought as a simple on-off switches. However, a closer look at these receptors reveals a complex behavior and a wide repertoire of subtle modulation by intrinsic and extrinsic factors. From a physiological point of view, this modulation can be seen as an additional level of complexity in the cell signaling process. Here we review the allosteric modulation of LGICs in light of the latest findings and discuss the suitability of this approach to the design of new therapeutic molecules.

Chronic nicotine exposure upregulates nicotinic receptors by a novel mechanism.

Nicotine addiction is initiated by its binding to high-affinity nicotinic receptors in brain composed primarily of alpha4 and beta2 subunits. For nicotinic receptors expressed in vivo or heterologously, nicotine exposure over hours to days increases or "upregulates" high-affinity nicotine binding to receptors through a posttranslational mechanism thought to increase receptor numbers. Using heterologous expression, we find nicotine exposure causes a fourfold to sixfold higher binding to alpha4beta2 receptors that does not correspond with any significant change in the number of surface receptors or a change in the assembly, trafficking, or cell-surface turnover of the receptors. However, upregulation does alter the functional state of the receptor, slowing desensitization and enhancing sensitivity to acetylcholine. Based on these findings, we propose an alternative mechanism to explain nicotine-induced upregulation in which nicotine exposure slowly stabilizes alpha4beta2 receptors in a high-affinity state that is more easily activated, thereby providing a memory for nicotine exposure.

Nicotine addiction: the possible role of functional upregulation.

Upregulation of binding to nicotinic acetylcholine receptors (nAChRs) is observed in the brains of both smokers and animals chronically exposed to nicotine, although whether this in vivo change is accompanied by an increase in receptor function is unknown. In vitro recordings indicate that alpha4beta2- and alpha7-subtypes of nAChRs, which are the most abundant subtypes in the brain, are functionally upregulated following prolonged exposure to nicotine. The possible consequences of functional upregulation for nicotine addiction are discussed. Moreover, we propose a new paradigm that describes the unusual behavior of these neuronal nAChRs and helps to explain the effects of nicotine in the CNS and the diffuse effects of ACh.

Potentiation of human alpha4beta2 neuronal nicotinic acetylcholine receptor by estradiol.

The modulation of neurotransmitter receptors by various substances can reflect important physiological mechanisms involved in the regulation of neural function. Furthermore, such substances, in particular specific allosteric modulators, can reveal promising therapeutic targets for diseases of the nervous system. From this perspective, we investigated the effects of the steroid hormone estradiol on human neuronal nicotinic acetylcholine receptors expressed either in Xenopus laevis oocytes or human embryonic kidney cells. Acetylcholine-evoked currents were potentiated both by pre- and coapplications of estradiol in alpha4beta2 and alpha4beta4 receptors, but not in alpha3beta2 or alpha3beta4 receptors. The reversible potentiation of alpha4-containing receptors could be induced within seconds in X. laevis oocytes and at micromolar concentrations of estradiol. The potentiation was greatest for responses evoked by low concentrations of acetylcholine, resulting in an apparent increase of receptor affinity. At the single channel level, estradiol potentiation resulted from an increase in opening probability. Finally, the use of functional chimeric or truncated alpha4 subunits demonstrated that a site at the C-terminal tail of the alpha4 subunit is required for estradiol potentiation. These results suggest the presence of a specific site at the human nicotinic acetylcholine receptor alpha4 subunit through which estradiol can cause an allosteric potentiation of acetylcholine-evoked responses.

Effect of Glutamate and Ionomycin on the Release of Arachidonic Acid, Prostaglandins and HETEs from Cultured Neurons and Astrocytes.

The release of arachidonic acid (ArA) metabolites from mouse neurons and astrocytes in primary culture has been studied in response to ionomycin or glutamate stimulation. Cells were preincubated with [3H]ArA for 24 h and the radioactivity released was examined by HPLC. In striatal, cortical and hippocampal neurons, glutamate and ionomycin strongly stimulated the release of ArA, but neither prostaglandins (PGs) nor hydroxyeicosatetraenoic acids (HETEs) could be detected. If they were released, these latter compounds represented < 0.02% of the amount of ArA. In contrast, in astrocyte cultures, ionomycin (but not glutamate) strongly stimulated the release of PGs and HETEs as well as ArA. Reversed- and straight-phase HPLC analysis revealed the presence of PGD2, PGE2, PGF2alpha, 12-hydroxyheptadeca-5,8,10-trienoic acid (HHT) and HETEs (15-HETE, 11-HETE and 5-HETE). Indomethacin inhibited the release of PGs and HHT, but also that of 11- and 15-HETE, indicating that these two HETEs may be produced through the cyclooxygenase pathway. Metabolism of [3H]ArA was also examined in cellular homogenates. Although > 50% of the [3H]ArA was metabolized to PGF2alpha, PGE2, PGD2, HHT, 15- and 11-HETE in cultured astrocyte homogenates, no [3H]ArA metabolism could be detected in cultured striatal neuron homogenates. Moreover, neuronal homogenates did not inhibit the metabolism of [3H]ArA observed in either astrocyte or platelet homogenates. These results indicate that central neurons in primary culture possess very low lipoxygenase and cyclooxygenase activities. They emphasize the need to identify the cellular source of ArA metabolites in the brain, particularly when considering the multiple new messenger roles proposed for these molecules, such as that of retrograde messengers involved in synaptic plasticity phenomena.