Stiripentol inhibits spike-and-wave discharges in animal models of absence seizures: A new mechanism of action involving T-type calcium channels.

OBJECTIVE: Stiripentol (STP; Diacomit®) is an antiepileptic drug indicated for Dravet syndrome that has been identified as a γ-aminobutyric acid (GABAergic) positive allosteric modulator. Dravet syndrome is characterized by multiple seizure types: generalized tonic-clonic, focal, myoclonic, and absence seizures. In addition to its antiepileptic effects on tonic-clonic seizures, STP has also been reported to reduce the frequency of atypical absence seizures in patients. Our study focused on STP potential effects on absence seizures, to better characterize its full spectrum of mechanisms of action. METHODS: STP effects on absence seizures were quantified by electroencephalographic recording in two animal models: rats treated with a low dose of pentylenetetrazol (20 mg/kg ip) and rats from the WAG/Rij strain. In addition, we characterized STP effects on T-type calcium channel activity. Peak currents were recorded with manual patch clamp on cells transfected with cDNA encoding for the human isoform for Cav 3.1, Cav 3.2, and Cav 3.3. RESULTS: STP administered before pentylenetetrazol almost completely abolished the generation of spike-and-wave discharges (SWDs) at the dose of 300 mg/kg. At this dose, STP also statistically significantly decreased SWD cumulated duration and number in WAG/Rij rats. Its antiepileptic effect was maintained in WAG/Rij rats, whose seizures were aggravated by the GABA agonist THIP (gaboxadol hydrochloride). Furthermore, electrophysiological recordings showed that STP inhibits T-type calcium channel peak activity, with a higher specificity for the Cav 3.3 subtype. SIGNIFICANCE: In addition to its previously characterized anticonvulsive properties, these data highlight a new mechanism of action of STP on abnormal thalamocortical activity. This strong antiabsence effect on seizures is correlated with an inhibition of T-type calcium channels. This new mechanism of action could be implicated in the specificity of STP therapeutic effects in Dravet syndrome.

Involvement of the GABAA receptor α subunit in the mode of action of etifoxine.

Etifoxine (EFX) is a non-benzodiazepine psychoactive drug which exhibits anxiolytic effects through a dual mechanism, by directly binding to GABAA receptors (GABAARs) and to the mitochondrial 18-kDa translocator protein, resulting in the potentiation of the GABAergic function. The ß subunit subtype plays a key role in the EFX-GABAAR interaction, however this does not explain the anxiolytic effects of this drug. Here, we combined behavioral and electrophysiological experiments to challenge the role of the GABAAR α subunit in the EFX mode of action. After single administrations of anxiolytic doses (25-50 mg/kg, intraperitoneal), EFX did not induce any neurological nor locomotor impairments, unlike the benzodiazepine bromazepam (0.5-1 mg/kg, intraperitoneal). We established the EFX pharmacological profile on heteropentameric GABAARs constructed with α1 to α6 subunit expressed in Xenopus oocyte. Unlike what is known for benzodiazepines, neither the γ nor δ subunits influenced EFX-mediated potentiation of GABA-evoked currents. EFX acted first as a partial agonist on α2ß3γ2S, α3ß3γ2S, α6ß3γ2S and α6ß3δ GABAARs, but not on α1ß3γ2S, α4ß3γ2S, α4ß3δ nor α5ß3γ2S GABAARs. Moreover, EFX exhibited much higher positive allosteric modulation towards α2ß3γ2S, α3ß3γ2S and α6ß3γ2S than for α1ß3γ2S, α4ß3γ2S and α5ß3γ2S GABAARs. At 20 µM, corresponding to brain concentration at anxiolytic doses, EFX increased GABA potency to the highest extent for α3ß3γ2S GABAARs. We built a docking model of EFX on α3ß3γ2S GABAARs, which is consistent with a binding site located between α and ß subunits in the extracellular domain. In conclusion, EFX preferentially potentiates α2ß3γ2S and α3ß3γ2S GABAARs, which might support its advantageous anxiolytic/sedative balance.

Etifoxine improves sensorimotor deficits and reduces glial activation, neuronal degeneration, and neuroinflammation in a rat model of traumatic brain injury.

BACKGROUND: Traumatic brain injury (TBI) results in important neurological impairments which occur through a cascade of deleterious physiological events over time. There are currently no effective treatments to prevent these consequences. TBI is followed not only by an inflammatory response but also by a profound reorganization of the GABAergic system and a dysregulation of translocator protein 18 kDa (TSPO). Etifoxine is an anxiolytic compound that belongs to the benzoxazine family. It potentiates GABAergic neurotransmission, either through a positive allosteric effect or indirectly, involving the activation of TSPO that leads to an increase in neurosteroids synthesis. In several models of peripheral nerve injury, etifoxine has been demonstrated to display potent regenerative and anti-inflammatory properties and to promote functional recovery. Prior study also showed etifoxine efficacy in reducing brain edema in rats. In light of these positive results, we used a rat model of TBI to explore etifoxine treatment effects in a central nervous system injury, from functional outcomes to the underlying mechanisms. METHODS: Male Sprague-Dawley rats received contusion (n = 18) or sham (n = 19) injuries centered laterally to bregma over the left sensorimotor cortex. They were treated with etifoxine (50 mg/kg, i.p.) or its vehicle 30 min following injury and every day during 7 days. Rats underwent behavioral testing to assess sensorimotor function. In another experiment, injured rats (n = 10) or sham rats (n = 10) received etifoxine (EFX) (50 mg/kg, i.p.) or its vehicle 30 min post-surgery. Brains were then dissected for analysis of neuroinflammation markers, glial activation, and neuronal degeneration. RESULTS: Brain-injured rats exhibited significant sensorimotor function deficits compared to sham-injured rats in the bilateral tactile adhesive removal test, the beam walking test, and the limb-use asymmetry test. After 2 days of etifoxine treatment, behavioral impairments were significantly reduced. Etifoxine treatment reduced pro-inflammatory cytokines levels without affecting anti-inflammatory cytokines levels in injured rats, reduced macrophages and glial activation, and reduced neuronal degeneration. CONCLUSIONS: Our results showed that post-injury treatment with etifoxine improved functional recovery and reduced neuroinflammation in a rat model of TBI. These findings suggest that etifoxine may have a therapeutic potential in the treatment of TBI.

Neuroprotective activity of stiripentol with a possible involvement of voltage-dependent calcium and sodium channels.

A growing body of data has shown that recurrent epileptic seizures may be caused by an excessive release of the excitatory neurotransmitter glutamate in the brain. Glutamatergic overstimulation results in massive neuronal influxes of calcium and sodium through N-methyl-D-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, and kainic acid glutamate subtype receptors and also through voltage-gated calcium and sodium channels. These persistent and abnormal sodium and calcium entry points have deleterious consequences (neurotoxicity) for neuronal function. The therapeutic value of an antiepileptic drug would include not only control of seizure activity but also protection of neuronal tissue. The present study examines the in vitro neuroprotective effects of stiripentol, an antiepileptic compound with γ-aminobutyric acidergic properties, on neuronal-astroglial cultures from rat cerebral cortex exposed to oxygen-glucose deprivation (OGD) or to glutamate (40 µM for 20 min), two in vitro models of brain injury. In addition, the affinity of stiripentol for the different glutamate receptor subtypes and the interaction with the cell influx of Na(+) and of Ca(2+) enhanced by veratridine and NMDA, respectively, are assessed. Stiripentol (10-100 µM) included in the culture medium during OGD or with glutamate significantly increased the number of surviving neurons relative to controls. Stiripentol displayed no binding affinity for different subtypes of glutamate receptors (IC50 >100 µM) but significantly blocked the entry of Na(+) and Ca(2+) activated by veratridine and NMDA, respectively. These results suggest that Na(+) and Ca(2+) channels could contribute to the neuroprotective properties of sitiripentol.

Nefopam analgesia and its role in multimodal analgesia: A review of preclinical and clinical studies.

Nefopam is a non-opioid, non-steroidal, centrally acting analgesic drug used to prevent postoperative pain, primarily in the context of multimodal analgesia. This paper reviews preclinical and clinical studies in which nefopam has been combined with opioids, non-steroidal anti-inflammatory compounds, and paracetamol. This report focuses on the literature during the last decade and discusses the translational efforts between animal and clinical studies in the context of multimodal or balanced analgesia. In preclinical rodent models of acute and inflammatory pain, nefopam combinations including opioids revealed a synergistic interaction or enhanced morphine analgesia in six out of seven studies. Nefopam combinations including non-steroidal anti-inflammatory drugs (NSAIDs) (aspirin, ketoprofen or nimesulide) or paracetamol likewise showed enhanced analgesic effects for the associated compound in all instances. Clinical studies have been performed in various types of surgeries involving different pain intensities. Nefopam combinations including opioids resulted in a reduction in morphine consumption in 8 out of 10 studies of severe or moderate pain. Nefopam combinations including NSAIDs (ketoprofen or tenoxicam) or paracetamol also demonstrated a synergic interaction or an enhancement of the analgesic effect of the associated compound. In conclusion, this review of nefopam combinations including various analgesic drugs (opioids, NSAIDs and paracetamol) reveals that enhanced analgesia was demonstrated in most preclinical and clinical studies, suggesting a role for nefopam in multimodal analgesia based on its distinct characteristics as an analgesic. Further clinical studies are needed to evaluate the analgesic effects of nefopam combinations including NSAIDs or paracetamol.

Anti-hypercholesterolemic effect of Saccharomyces boulardii in the hamster.

BACKGROUND/AIMS: Hypercholesterolemia is a major risk factor for coronary artery disease and probiotics have been suggested as tools to manage elevated cholesterol levels. METHODS: The present study investigated the ability of the biotherapeutic agent Saccharomyces boulardii (Sb-Biocodex) to reduce the hypercholesterolemia induced by a 0.1% cholesterol-enriched diet in the hamster. RESULTS: In a first experiment, chronic oral treatment with S. boulardii at 12 × 10(10) CFU/kg (3 g/kg) twice a day was started from the beginning of the cholesterol diet and continued for 14 days ('preventive protocol'). In the second experiment, S. boulardii was given 14 days after the beginning of the cholesterol diet when hypercholesterolemia had developed and continued for an additional 14 days ('curative protocol'). In the preventive protocol, administration of the yeast significantly reduced hypercholesterolemia (14%) induced by the cholesterol-enriched diet compared to the group receiving only the cholesterol diet. In the curative protocol, S. boulardii significantly reduced hypercholesterolemia (12%) induced by the cholesterol-enriched diet, too. Moreover, the yeast significantly decreased the serum triglyceride increase by 39%. CONCLUSION: S. boulardii possesses anti-hypercholesterolemic properties in the hamster worthy of further evaluation in clinical studies.

An Update on Stiripentol Mechanisms of Action: A Narrative Review.

Stiripentol (Diacomit®) (STP) is an orally active antiseizure medication (ASM) indicated as adjunctive therapy, for the treatment of seizures associated with Dravet syndrome (DS), a severe form of childhood epilepsy, in conjunction with clobazam and, in some regions valproic acid. Since the discovery of STP, several mechanisms of action (MoA) have been described that may explain its specific effect on seizures associated with DS. STP is mainly considered as a potentiator of gamma-aminobutyric acid (GABA) neurotransmission: (i) via uptake blockade, (ii) inhibition of degradation, but also (iii) as a positive allosteric modulator of GABAA receptors, especially those containing α3 and δ subunits. Blockade of voltage-gated sodium and T-type calcium channels, which is classically associated with anticonvulsant and neuroprotective properties, has also been demonstrated for STP. Finally, several studies indicate that STP could regulate glucose energy metabolism and inhibit lactate dehydrogenase. STP is also an inhibitor of several cytochrome P450 enzymes involved in the metabolism of other ASMs, contributing to boost their anticonvulsant efficacy as add-on therapy. These different MoAs involved in treatment of DS and recent data suggest a potential for STP to treat other neurological or non-neurological diseases.

Systematic evaluation of the nefopam-paracetamol combination in rodent models of antinociception.

1. The aim of the present study was to explore the concept of multimodal anaesthesia using a combination of two non-opioid analgesics, namely nefopam, a centrally acting non-opioid that inhibits monoamine reuptake, and paracetamol, an inhibitor of central cyclo-oxygenases. The antinociceptive characteristics of the combination were evaluated using four different animal models of pain. 2. In the mouse writhing test, antinociceptive properties were observed with ED50 values of 1.5 ± 0.2 and 120.9 ± 14.8 mg/kg for nefopam and paracetamol, respectively. In the mouse formalin test, both compounds significantly inhibited the licking time of the injected hind paw, with ED50 values in the early phase of 4.5 ± 1.1 and 330.7 ± 80.3 mg/kg for nefopam and paracetamol, respectively, compared with 4.3 ± 0.2 and 206.1 ± 45.1 mg/kg for nefopam and paracetamol, respectively, in the inflammatory phase. Isobolographic analysis revealed that this drug combination was synergistic in the writhing test and additive in the formalin test. 3. In a rat incision model of postoperative thermal hyperalgesia, coadministration of nefopam at a non-analgesic dose (3 mg/kg) with paracetamol at a low analgesic dose (300 mg/kg) showed the appearance of a strong antihyperalgesic effect, maintained for at least 3 h. In rat carrageenan-induced tactile allodynia, the combination of low analgesic doses of nefopam (10 or 30 mg/kg) with a non-analgesic dose of paracetamol (30 mg/kg), significantly blocked allodynia with a longer duration of efficacy. 4. In conclusion, coadministration of nefopam with paracetamol is worthy of clinical evaluation.

Etifoxine improves peripheral nerve regeneration and functional recovery.

Peripheral nerves show spontaneous regenerative responses, but recovery after injury or peripheral neuropathies (toxic, diabetic, or chronic inflammatory demyelinating polyneuropathy syndromes) is slow and often incomplete, and at present no efficient treatment is available. Using well-defined peripheral nerve lesion paradigms, we assessed the therapeutic usefulness of etifoxine, recently identified as a ligand of the translocator protein (18 kDa) (TSPO), to promote axonal regeneration, modulate inflammatory responses, and improve functional recovery. We found by histologic analysis that etifoxine therapy promoted the regeneration of axons in and downstream of the lesion after freeze injury and increased axonal growth into a silicone guide tube by a factor of 2 after nerve transection. Etifoxine also stimulated neurite outgrowth in PC12 cells, and the effect was even stronger than for specific TSPO ligands. Etifoxine treatment caused a marked reduction in the number of macrophages after cryolesion within the nerve stumps, which was rapid in the proximal and delayed in the distal nerve stumps. Functional tests revealed accelerated and improved recovery of locomotion, motor coordination, and sensory functions in response to etifoxine. This work demonstrates that etifoxine, a clinically approved drug already used for the treatment of anxiety disorders, is remarkably efficient in promoting acceleration of peripheral nerve regeneration and functional recovery. Its possible mechanism of action is discussed, with reference to the neurosteroid concept. This molecule, which easily enters nerve tissues and regulates multiple functions in a concerted manner, offers promise for the treatment of peripheral nerve injuries and axonal neuropathies.

In Vitro and In Vivo Neuroprotective Effects of Etifoxine in ß-Amyloidinduced Toxicity Models.

AIM: The aim of this study is to examine the effect of etifoxine on ß-amyloid-induced toxicity models. BACKGROUND: Etifoxine is an anxiolytic compound with a dual mechanism of action; it is a positive allosteric modulator of GABAergic receptors as well as a ligand for the 18 kDa mitochondrial Translocator Protein (TSPO). TSPO has recently raised interest in Alzheimer's Disease (AD), and experimental studies have shown that some TSPO ligands could induce neuroprotective effects in animal models. OBJECTIVE: In this study, we examined the potential protective effect of etifoxine in an in vitro and an in vivo model of amyloid beta (Aß)-induced toxicity in its oligomeric form, which is a crucial factor in AD pathologic mechanisms. METHODS: Neuronal cultures were intoxicated with Aß1-42, and the effects of etifoxine on oxidative stress, Tau-hyperphosphorylation and synaptic loss were quantified. In a mice model, behavioral deficits induced by intracerebroventricular administration of Aß25-35 were measured in a spatial memory test, the spontaneous alternation and in a contextual memory test, the passive avoidance test. RESULTS: In neuronal cultures intoxicated with Aß1-42, etifoxine dose-dependently decreased oxidative stress (methionine sulfoxide positive neurons), tau-hyperphosphorylation and synaptic loss (ratio PSD95/synaptophysin). In a mice model, memory impairments were fully alleviated by etifoxine administered at anxiolytic doses (12.5-50mg/kg). In addition, markers of oxidative stress and apoptosis were decreased in the hippocampus of these animals. CONCLUSION: Our results have shown that in these two models, etifoxine could fully prevent neurotoxicity and pathological changes induced by Aß. These results confirm that TSPO ligands could offer an interesting therapeutic approach to Alzheimer's disease.

Contribution of transient receptor potential vanilloid subtype 1 to the analgesic and antihyperalgesic activity of nefopam in rodents.

In order to further elucidate the mechanism(s) of action of analgesic and antihyperalgesic nefopam, its interactions with the transient receptor potential vanilloid subtype 1 (TRPV1) were investigated. In sensory neurons of rat embryos, dorsal root ganglion (DRG) in culture, nefopam (3-30 mumol/l) and capsazepine (TRPV1 antagonist, 10 mumol/l) prevented intracellular calcium elevation and calcitonin gene-related peptide release induced by vanilloid agonist capsaicin. Unlike nefopam, capsazepine failed to inhibit these same responses induced by KCl excess. In vivo, nefopam (0.5 and 2 mg/kg, i.v.) and capsazepine (40 mg/kg, i.p.) reduced the licking response due to intraplantar injection of capsaicin in mice. These findings suggest that nefopam exerts its analgesic and antihyperalgesic effects through multiple mechanisms including blockade of TRPV1 in addition to voltage-dependent calcium channels in the DRG.

Analgesic and anti-edemic properties of etifoxine in models of inflammatory sensitization.

Inflammatory processes are critical promoting factors of chronic pain states, mostly by inducing peripheral and central sensitization of the nociceptive system. These processes are associated with a massive increase in glutamatergic transmission, sometimes facilitated by spinal disinhibition. In this study, we used etifoxine, a non-benzodiazepine anxiolytic known to amplify inhibition mediated by gamma-aminobutyric acid type A (GABAA) receptors in pain processing regions, either directly (through allosteric modulation) or indirectly (through the synthesis of endogenous neurosteroids). We used different models of local inflammation to evaluate the possible direct action of etifoxine on analgesia and edema. Pain symptom and edema measurements were performed after intraplantar carrageenan injection or after topical ear inflammation. We found that etifoxine treatment was associated with reduced plantar surface temperature 24 h after intraplantar carrageenan injection. In this model, etifoxine also alleviated thermal hot and mechanical hyperalgesia. A similar finding was observed while analyzing pain symptoms in the late phase of the formalin test. In a model of ear inflammation, etifoxine appeared to have a moderate anti-edemic effect after topical application. This slight action of etifoxine on the limitation of inflammatory processes could be mediated in part by cyclo-oxygenase 1 activity inhibition. Etifoxine appears as a promising therapeutic tool contributing to the limitation of inflammatory pain symptoms. Since etifoxine is already prescribed as an anxiolytic in several countries, it could be a good candidate for the prevention of inflammatory-driven edema and hyperalgesia, although the precise mechanism of action relative to its anti-inflammatory potential remains to be elucidated.

Investigation of the anticonvulsive effect of acute immobilization stress in anxious Balb/cByJ mice using GABA A-related mechanistic probes.

RATIONALE: A disordered regulation of neuroactive steroids release in response to acute stress could induce GABAergic dysfunctions underlying anxiety disorders. OBJECTIVES: First, we conducted studies indicating that a short immobilization stress in anxious Balb/cByJ mice produced an anticonvulsive effect. Second, the effects of different positive allosteric modulators (etifoxine, progesterone, clonazepam, and allopregnanolone) of GABA A receptors were compared in a mouse model mimicking the disruption of the acute stress-induced neuroactive steroids release with finasteride (types I and II 5alpha-reductase inhibitor). RESULTS: The acute stress-induced anticonvulsive effect, expressed by the threshold dose of t-butylbicyclophosphorothionate-producing clonic seizures, was time-dependent. The extent of the enhancement of acute stress-induced anticonvulsive effect was lowered in the presence of finasteride. The same effect was observed with PK11195, which behaves as an antagonist of the peripheral benzodiazepine receptor in the dose range used in this study. Picrotoxin reduced the acute stress anticonvulsive effect, proving that this effect operates through the GABA A receptor. Contrary to progesterone (up to 30 mg/kg), etifoxine (50 mg/kg), allopregnanolone (10 mg/kg), and clonazepam (10 microg/kg) inhibited the finasteride effect in stressed animals. The effect of etifoxine was blocked in the presence of finasteride and picrotoxin combined in stressed animals. CONCLUSIONS: These findings support the hypothesis suggesting an involvement of neuroactive steroids in the anticonvulsive effect of restraint stress. The dual and complementary mechanisms of action of etifoxine (directly on the GABA A receptor and indirectly via the neuroactive steroids) may represent a therapeutic benefit in the treatment of various anxiety disorders with abnormal production of neuroactive steroids.

Moclobemide attenuates anoxia and glutamate-induced neuronal damage in vitro independently of interaction with glutamate receptor subtypes.

Recent data suggested the existence of a bidirectional relation between depression and neurodegenerative diseases resulting from cerebral ischemia injury. Glutamate, a major excitatory neurotransmitter, has long been recognised to play a key role in the pathophysiology of anoxia or ischemia, due to its excessive accumulation in the extracellular space and the subsequent activation of its receptors. A characteristic response to glutamate is the increase in cytosolic Na(+) and Ca(2+) levels which is due mainly to influx from the extracellular space, with a consequent cell swelling and oxidative metabolism dysfunction. The present study examined the in vitro effects of the antidepressant and type-A monoamine oxidase inhibitor, moclobemide, in neuronal-astroglial cultures from rat cerebral cortex exposed to anoxia (for 5 and 7 h) or to glutamate (2 mM for 6 h), two in vitro models of brain ischemia. In addition, the affinity of moclobemide for the different glutamate receptor subtypes and an interaction with the cell influx of Na(+) and of Ca(2+) enhanced by veratridine and K(+) excess, respectively, were evaluated. Moclobemide (10-100 microM) included in the culture medium during anoxia or with glutamate significantly increased in a concentration-dependent manner the amount of surviving neurons compared to controls. Moclobemide displayed no binding affinity for the different glutamate receptor subtypes (IC(50)>100 microM) and did not block up to 300 microM the entry of Na(+) and of Ca(2+) activated by veratridine and K(+), respectively. These results suggest that the neuroprotective properties of moclobemide imply neither the glutamate neurotransmission nor the Na(+) and Ca(2+) channels.

Lack of interaction between etifoxine and CRF1 and CRF2 receptors in rodents.

Hyperactivity of the corticotropin-releasing factor (CRF) system occurs in some patients with anxiety disorders and depression. Blockade of CRF1 and CRF2 receptors can underlie the anxiolytic effects of drugs. In the present investigation, in vivo and in vitro studies were designed to determine whether the anxiolytic drug etifoxine, known to enhance GABAergic synaptic transmission, behaves also as a CRF1 and CRF2 receptor antagonist. A drug exerting multiple actions may be of clinical interest in the treatment of various different forms of mood disorders. Using two animal models, it was found that etifoxine reversed the excess CRF-induced grooming but not the hypo-locomotion of the rat placed in an open field. Etifoxine attenuated the CRF-induced gastric emptying delay in the mouse. On the other hand, in vitro, binding of etifoxine to CRF1 and CRF2 receptors on rat brain membranes was negligible and functionally, etifoxine did not block the CRF1 and CRF2 activation-induced cAMP production in presence of CRF in human neuroblastoma SH-SY5Y cells. The selective anxiolytic properties of etifoxine appear unrelated to an antagonist activity at the CRF1 and CRF2 receptors. The decrease in CRF activity produced by etifoxine may be related to its GABAergic properties.

The anxiolytic etifoxine activates the peripheral benzodiazepine receptor and increases the neurosteroid levels in rat brain.

The peripheral benzodiazepine receptors (PBR) might be involved in certain pathophysiological events, such as anxiety, by stimulating the production of neuroactive steroids in the brain. A recent electrophysiological study has revealed an interaction between PK11195, a PBR ligand and the anxiolytic compound etifoxine at micromolar concentrations. The present work was aimed at further characterizing the etifoxine-PBR interaction. In membrane preparations from intact male rat forebrain, etifoxine uncompetitively inhibited the binding of [(3)H]PK11195 with an IC(50) = 18.3 +/- 1.2 microM, a value consistent with etifoxine plasma and brain concentrations measured after an anxiolytic-like dose (50 mg/kg). In vivo, that etifoxine dose was associated with increased concentrations of pregnenolone, progesterone, 5alpha-dihydroprogesterone and allopregnanolone in plasma and brain of sham-operated animals. In adrenalectomized and castrated rats, etifoxine enhanced the brain levels of these steroids, suggesting a stimulation of their local synthesis and/or a decrease of their disappearance rate, independently of peripheral sources. Finasteride, an inhibitor of 5alpha-reductase that converts progesterone into its 5alpha-reduced metabolites like allopregnanolone, attenuated the anti-conflict effect of etifoxine even though brain allopregnanolone contents were drastically reduced. These results indicate that following activation of the PBR in the brain, an increased cerebral production of allopregnanolone, a potent positive modulator of the GABA(A) receptor function, may partially contribute to the anxiolytic-like effects of etifoxine.

The modulatory effects of the anxiolytic etifoxine on GABA(A) receptors are mediated by the beta subunit.

The anxiolytic compound etifoxine (2-ethylamino-6-chloro-4-methyl-4-phenyl-4H-3,1-benzoxazine hydrochloride) potentiates GABA(A) receptor function in cultured neurons (Neuropharmacology 39 (2000) 1523). However, the molecular mechanisms underlying these effects are not known. In this study, we have determined the influence of GABA(A) receptor subunit composition on the effects of etifoxine, using recombinant murine GABA(A) receptors expressed in Xenopus oocytes. Basal chloride currents mediated by homomeric beta receptors were reduced by micromolar concentrations of etifoxine, showing that beta subunits possess a binding site for this modulator. In oocytes expressing alpha(1)beta(x) GABA(A) receptors (x=1, 2 or 3), etifoxine evoked a chloride current in the absence of GABA and enhanced GABA (EC10)-activated currents, in a dose-dependent manner. Potentiating effects were also observed with alpha(2)beta(x), beta(x)gamma(2s) or alpha(1)beta(x)gamma(2s) combinations. The extent of potentiation was clearly beta-subunit-dependent, being more pronounced at receptors containing a beta(2) or a beta(3) subunit than at receptors incorporating a beta(1) subunit. The mutation of Asn 289 in the channel domain of beta(2) to a serine (the homologous residue in beta(1)) did not significantly depress the effects of etifoxine at alpha(1)beta(2) receptors. This specific pattern of inhibition/potentiation was compared with that of other known modulators of GABA(A) receptor function like benzodiazepines, neurosteroids, barbiturates or loreclezole.

Effects of etifoxine on ligand binding to GABA(A) receptors in rodents.

The GABA(A) receptor/chloride ionophore is allosterically modulated by several classes of anxiolytic and anticonvulsant agents, including benzodiazepines, barbiturates and neurosteroids. Etifoxine, an anxiolytic and anticonvulsant compound competitively inhibited the binding of [(35)S]t-butylbicyclophosphoro-thionate (TBPS), a specific ligand of the GABA(A) receptor chloride channel site. To investigate the etifoxine modulatory effects on the different binding sites of the GABA(A) receptor complex, we have examined the effects of etifoxine on binding of the receptor agonist [(3)H]muscimol and the benzodiazepine modulator [(3)H]flunitrazepam in rat brain membrane preparations. The anticonvulsant properties of etifoxine combined with muscimol and flunitrazepam were performed in mice with picrotoxin-induced clonic seizures. Etifoxine modestly enhanced binding of [(3)H]muscimol and of [(3)H]flunitrazepam by increasing the number of binding sites without changing the binding affinity of [(3)H]flunitrazepam. In contrast, the compound decreased the affinity of muscimol for its binding site. In vivo, the combination of subactive doses of etifoxine with muscimol or flunitrazepam produced an anticonvulsant additive effect against the picrotoxin-induced clonic seizures in mice. These results suggest that the interaction of etifoxine on the GABA(A) receptor complex would allosterically modify different binding sites due to conformational changes. Functionally, the resulting facilitation of GABA transmission underlies the pharmacological properties of etifoxine.

Nefopam blocks voltage-sensitive sodium channels and modulates glutamatergic transmission in rodents.

In order to specify the nature of interactions between the analgesic compound nefopam and the glutamatergic system, we examined the effects of nefopam on binding of specific ligands on the three main subtypes ionotropic glutamate receptors: N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), or quisqualic acid (QA) and kainic acid (KA) in rat brain membrane preparations. Functionally, we investigated the effects of nefopam against the seizures induced by agonists of these excitatory glutamate receptors in mice. Since the synaptic release of glutamate mainly depends upon the activation of membrane voltage-sensitive sodium channels (VSSCs), the nature of interactions between nefopam and these ionic channels was studied by evaluating the effects of nefopam on binding of 3H-batrachotoxinin, a specific ligand of the VSSCs in rat brain membrane preparations. The functional counterpart of the binding of nefopam on VSSCs was evaluated by its effects on the 22Na uptake-stimulated by veratridine on human neuroblastoma cells and in the maximal electroshock test in mice. Nefopam showed no affinity for the subtypes of ionotropic glutamate receptors up to 100 microM. On the other hand, nefopam was effective against NMDA, QA and KA induced clonic seizures in mice. Nefopam displaced 3H-batrachotoxinin and inhibited the uptake of 22Na in the micromolar range and it protected mice against electroshock induced seizures. Nefopam may block the VSSCs activity: consequently, at the presynaptic level, this effect led to a reduction of glutamate release and at the postsynaptic level, it led to a decrease of the neuronal excitability following activation of the glutamate receptors.

Effects of stress and etifoxine on pentobarbital-induced loss of righting reflex in Balb/cByJ and C57BL/6J mice.

We hypothesized that functional changes in the GABAergic system induced by stress would differ between two inbred mouse strains BALB/cByJ and C57BL/6J. We compared the effects of restraint stress and of the anxiolytic drug etifoxine (EFX) on the duration of pentobarbital-induced loss of righting reflex (hypnotic effect) in the two strains. Naive BALB/cByJ mice were less sensitive than naive C57BL/6J mice to the hypnotic effect of pentobarbital. C57BL/6J mice exhibited a shortening in the duration of pentobarbital-induced hypnosis following stress whereas stress had no effect in BALB/cByJ mice. EFX reversed the shortening of pentobarbital-induced hypnosis elicited by stress in C57BL/6J and shortened the duration of pentobarbital-induced hypnosis after stress in BALB/cByJ mice. Alterations in the GABAergic function in BALB/cByJ mice could be corrected by EFX, an enhancer of GABAergic transmission.