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 [3H]PK11195 with an IC50=18.3±1.2 μM, 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, 5α-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 5α-reductase that converts progesterone into its 5α-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 GABAA receptor function, may partially contribute to the anxiolytic-like effects of etifoxine.

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.

Anxiety disorders are often associated with autonomic symptoms, including heart palpitations, sweating, elevation of body temperature and alterations of gastrointestinal motility. Some of the alterations observed in animals exposed to stress are analogous to changes in a number of physiological and endocrine parameters observed in anxious patients. With the purpose to guide further clinical studies in subtypes of anxious patients, etifoxine, a nonbenzodiazepine anxiolytic compound, was evaluated in two rat models of anxiety with measures of physiological manifestations: stress-induced hyperthermia (SIH) and conditioned-fear-stress-induced freezing behavior and activation of colonic motility. The sequential handling of animals induced a rise in body temperature attenuated by etifoxine (50 mg/kg IP). The emotional stress induced by fear to receive electric foot shocks is accompanied by freezing behavior and an increase of the frequency of ceco-colonic spike bursts: both parameters were reduced by etifoxine (25–50 mg/kg IP), independently of changes in pain perception and memory-related processes. In response to a stressful event, the stimulation of the corticotropin-releasing hormone (CRH) system is probably involved in the observed modifications of body temperature and colonic motility. It is hypothesized that stress-induced CRH activation is attenuated by the enhancement of the inhibitory GABAergic system activity associated with etifoxine. These findings will guide future evaluation of etifoxine in the treatment of selected anxious patients with altered autonomic symptomatology.

The objective of this study was to resolve discrepancies regarding the possible antinociceptive synergy between morphine and nefopam in animal models of pain. Firstly, we have examined the antinociceptive activity of nefopam, a nonopioid antinociceptive compound that inhibits monoamine reuptake, in pain models of allodynia and hyperalgesia induced by carrageenan injection, or skin and muscle incision of the rat hind paw. Single subcutaneous administration of nefopam at 30 mg/kg blocked carrageenan- and incision-induced thermal hyperalgesia, and weakly but significantly diminished carrageenan-induced tactile allodynia. A weaker dose of nefopam (10 mg/kg) only reduced carrageenan-induced tactile allodynia and incision-induced thermal hyperalgesia. Secondly, we assessed the usefulness of the coadministration of nefopam with morphine. Combination of a nonanalgesic dose of nefopam (10 mg/kg) with a nonanalgesic dose of morphine (0.3 or 1.0 mg/kg) completely inhibited carrageenan- or incision-induced thermal hyperalgesia, respectively. In carrageenan-induced tactile allodynia, coadministration of weak analgesic doses of nefopam (10 and 30 mg/kg) with a nonanalgesic dose (1 mg/kg) or moderately analgesic dose (3 mg/kg) of morphine significantly reduced or reversed allodynia, respectively. In conclusion, coadministration of nefopam with morphine enhances the analgesic potency of morphine, indicating a morphine sparing effect of nefopam.

The present study explored the role of the histaminergic system in nefopam analgesia based on the structural relationship between nefopam and diphenhydramine. In vitro binding assays revealed that nefopam possesses moderate affinity for histamine H1 and H2 receptor subtypes, with IC50 of 0.8 and 6.9 microM, respectively, but no affinity for histamine H(3) receptor subtype until 100 microM. Subcutaneous nefopam administration dose-dependently inhibited pain in acetic acid-induced writhing (1-30 mg/kg) and formalin (1-10 mg/kg) tests in the mouse. Pretreatment with the histamine-depleting agent alpha-fluoromethylhistidine (alpha-FMH, 50 mg/kg), the histamine H1 receptor antagonist pyrilamine (3 or 10 mg/kg), or the histamine H2 receptor antagonists cimetidine (100 mg/kg) and zolantidine (10 or 30 mg/kg) did not significantly modify nefopam antinociception in both tests. The histamine H3 receptor agonist R(-)alpha-methylhistamine (RAMH, 10 mg/kg) did not significantly modify the nefopam analgesic activity in the writhing test. At 25 mg/kg, RAMH inhibited nefopam antinociception at 3 mg/kg, but not at 10 mg/kg in the formalin test. However, pretreatment with the histamine H3 receptor antagonist thioperamide (25 mg/kg) inhibited nefopam antinociception in the writhing test, but not in the formalin test. In conclusion, nefopam analgesic activity is not mediated by histamine H1 or H2 receptors, but can be slightly modulated by histamine H3 receptors in mouse pain tests.

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.

The anxiolytic compound etifoxine (2-ethylamino-6-chloro-4-methyl-4-phenyl-4H-3,1-benzoxazine hydrochloride) potentiates GABAA 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 GABAA receptor subunit composition on the effects of etifoxine, using recombinant murine GABAA receptors expressed in Xenopus oocytes. Basal chloride currents mediated by homomeric β receptors were reduced by micromolar concentrations of etifoxine, showing that β subunits possess a binding site for this modulator. In oocytes expressing α1βx GABAA 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 α2βx, βxγ2s or α1βxγ2s combinations. The extent of potentiation was clearly β-subunit-dependent, being more pronounced at receptors containing a β2 or a β3 subunit than at receptors incorporating a β1 subunit. The mutation of Asn 289 in the channel domain of β2 to a serine (the homologous residue in β1) did not significantly depress the effects of etifoxine at α1β2 receptors. This specific pattern of inhibition/potentiation was compared with that of other known modulators of GABAA receptor function like benzodiazepines, neurosteroids, barbiturates or loreclezole.

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.

The activity of nefopam, a centrally acting compound, not structurally related to other analgesics, was examined in acute and postoperative thermal pain models in the rat. Its antinociceptive potency was evaluated using heat noxious stimuli either in intact or in injured animals after skin and muscular incisions. In the hot plate and in the plantar tests, nefopam after acute administration by different routes exhibited a dose-dependent attenuation of the nociceptive responses at 10-30 mg x kg(-1) by intraperitoneal or subcutaneous administration, at 60 mg x kg(-1) by oral dosing, and from 3 mg x kg(-1) after intravenous injection. In the postoperative pain model, at 30 mg x kg(-1) nefopam augmented the endpoint to thermal threshold, 60 and 90 minutes after administration compared to the threshold recorded after the incision. In the same conditions, morphine and tramadol displayed antinociceptive activities. As the plantar test provides a good index of nociception in humans, these results point out the usefulness of nefopam for attenuating moderate to severe pain, and for postoperative analgesia. In conclusion, nefopam has shown potent properties to reduce thermal hypersensitivity after acute or postoperative pain in rats.

We looked for an interaction between etifoxine and the neurosteroid allopregnanolone at central gamma-aminobutyric acid (GABA(A)) receptors. Etifoxine (2 microM) did not affect the affinity of allopregnanolone (IC(50)=108 nM) for its site in preparations of Sprague-Dawley rat cerebral cortex membranes, as determined by the inhibition of [(35)S] t-butylbicyclophosphorothionate binding, a specific ligand of the GABA(A) receptor chloride channel site. Etifoxine and allopregnanolone were anticonvulsants, blocking the clonic convulsions induced by bicuculline (an antagonist of the GABA(A) receptor) in CD1 mice. A combination of subactive doses of the two compounds showed additive anticonvulsant effects. These results suggest that etifoxine and allopregnanolone bind to distinct putative recognition sites at or near the chloride channel site. Functionally, their binding may have an additive effect by enhancing GABA(A) inhibitory transmission.

To justify the inclusion of phenazone, independently of its anti-inflammatory properties, in combination with a local anaesthetic, such as lidocaine, in some ear drop medications, we have studied the effect of this compound on the local anaesthetic activity of lidocaine in an animal model, that of sciatic nerve blockade in mice. Lidocaine and phenazone were tested alone and in combination at various concentrations. The local anaesthetic activity was estimated as the loss of motor activity of the hindlimb after topical injection of the drugs in the region of the sciatic nerve. Lidocaine, at concentrations ranging from 0.03 to 0.25%, induced a concentration-dependent anaesthetic effect. Phenazone alone had no effect at 0.25-1%. When combined, the two compounds acted synergistically. The local anaesthesia induced by lidocaine plus phenazone was significantly more intense and longer lasting than that induced by lidocaine alone. Phenazone enhanced the potency of lidocaine in this animal model. It is suggested that the potentiated local anaesthetic effect of the combination may be partly due to enhanced local bioavailability of lidocaine.

Nefopam (NEF) is a potent analgesic compound administered as a racemic mixture. Previous in vitro and in vivo studies with nefopam enantiomers have shown that (+)nefopam [(+)NEF] is substantially more potent than (-)nefopam [(-)NEF]. Differences between enantiomers have also been suggested in metabolic studies in vitro. The impact of these differences in vivo is not known because there is little or no information on the relative plasma concentrations of the enantiomers or on their kinetics. In this study, individual enantiomers of nefopam were synthesized and examined for acute toxicity in male and female rats and mice. Pharmacologic properties of enantiomers were examined using in vitro binding assays and antinociceptive tests in rats and mice. Additionally, a pharmacokinetic study was conducted in human volunteers. Subjects were administered 20 mg nefopam as Acupan(R) either as a 5- or 20-min intravenous infusion. In a control phase, subjects were administered only vehicle. Blood samples were collected through the following 24 h. Plasma samples were analyzed for individual enantiomers using a chiral assay developed for this purpose. The pharmacologic differences of previous studies were confirmed in receptor binding assays and in the hot plate and the formalin tests in mice. Neither enantiomer demonstrated substantial activity in the tail flick test in rats. No significant differences were revealed between LD(50) values of nefopam enantiomers after oral or intravenous administration in male and female rats or mice. There were no significant differences in AUC(0-infinity), C(max), or half-life between enantiomers following intravenous administration. Based on these findings, there is currently no compelling rationale to justify administering or monitoring individual enantiomers.

We have investigated the effects of 2-ethylamino-6-chloro-4-methyl-4-phenyl-4H-3,1-benzoxazine hydrochloride (etifoxine) on GABAA receptor function. Etifoxine displaced [35S]TBPS (t-butylbicyclophosphorothionate) from GABAA receptors of rat cortical membranes with an IC50 of 6.7±0.8 μM and [3H]PK11195 from peripheral (mitochondrial)-type benzodiazepine receptors (PBRs) of rat heart homogenates with an IC50 of 27.3±1.0 μM. Etifoxine displayed anxiolytic properties in an anticonflict test in rats, and potentiated GABAA receptor-mediated membrane currents elicited by submaximal (5–10 μM) but not saturating (0.5 mM) concentrations of GABA in cultured rat hypothalamic and spinal cord dorsal horn neurones. In hypothalamic cultures, etifoxine induced a dose-dependent inward current for concentrations >1 μM which reflected the post-synaptic potentiation of a small (∼20 pA) tonic and bicuculline-sensitive GABAA receptor-gated Cl− current. Etifoxine also increased the frequency of spontaneous and miniature GABAergic inhibitory post-synaptic currents without changing their amplitude and kinetic characteristics. Both effects of etifoxine were insensitive to flumazenil (10 μM), an antagonist of central-type benzodiazepine sites present at GABAA receptors, but were partly inhibited by PK11195 (10 μM) an antagonist of PBRs which control the synthesis of neurosteroids. Our results indicate that etifoxine potentiates GABAA receptor-function by a direct allosteric effect and by an indirect mechanism involving the activation of PBRs.

This study examined the nature of the interactions of etifoxine, an anxiolytic and anticonvulsant compound, with the GABA(A) receptor/chloride channel complex. In membrane preparations of Sprague-Dawley rat cerebral cortex, etifoxine competitively inhibited the binding of [35S]t-butylbicyclophosphoro-thionate (TBPS), a specific ligand of the GABA(A) receptor chloride channel site. In vivo studies demonstrated an anticonvulsant effect of etifoxine (50 and 75 mg/kg, i.p.) against the clonic convulsions induced by TBPS in CD1 mice. Flumazenil (10 and 40 mg/kg, i.p.), an antagonist of benzodiazepine sites at GABA(A) receptors, had no effect on the action of etifoxine. These findings suggest that etifoxine exerts its effect by interacting with the Cl- channel of GABA(A) receptors and probably by facilitating GABAergic inhibition.

Citrulline malate is known to improve performance in weakened muscles. The present experiment was designed to test the hypothesis that citrulline malate can limit the effect of endotoxins on muscle fatigability. Endotoxemia was induced in rats by injection of lipopolysaccharides from Klebsiella pneumoniae. Resistance to fatigue was quantified by measuring tension production during repetitive electrical stimulation of the isolated epitrochlearis muscle. Oral treatment by citrulline malate was found to increase resistance to fatigue in infected rats, whereas twitch tension was not modified. This demonstrates the efficacy of citrulline malate for limiting an increase in muscle fatigue elicited with bacterial endotoxins.

Disposition and metabolism of the new anticonvulsant 2,6-dimethylbenzamide N-(5-methyl-3-isoxazolyl) (D2916) was studied in male and female rats after oral administration of 14C-labeled material. D2916 was well absorbed in both sexes and distributed to all tissues, with maximal drug concentrations found in elimination and metabolization organs, as well as in fatty tissues. Striking differences in pharmacokinetic parameters of total radioactivity were observed between males and females; females had higher brain concentrations and longer blood and tissue half-lives. The study of blood, bile, urine, and brain metabolites showed that D2916 follows two degradation pathways related to hydroxylation of methyl groups. Males prefer to hydroxylate one of the methyl groups of the phenyl ring, and females prefer to hydroxylate the methyl of the isoxazolyl ring forming the active metabolite D3187. These findings suggest a sex difference in the location of the hydroxylation of the D2916 molecule and can explain the longer anticonvulsant effect observed in the female rat that is related both to an orientation of the metabolism toward the formation of the active metabolite and to a better ability to this metabolite to cross the blood-brain barrier, compared with the unchanged drug.

Objective: The spectrum of cytochrome P450 inhibition of stiripentol, a new anticonvulsant, was characterized in vitro and in vivo.Methods: Stiripentol was incubated in vitro with (R)-warfarin, coumarin, (S)-warfarin, (S)-mephenytoin, bufuralol, p-nitrophenol, and carbamazepine as probes for CYPs 1A2, 2A6, 2C9, 2C19, 2D6, 2E1, and 3A4, respectively. Caffeine demethylation and the 6 beta-hydroxycoaisol/cortisol ratio were monitored in vivo before and after 14 days of treatment with stiripentol as measures of CYP1A2 and CYP3A4 activity, and dextromethorphan O- and N-demethylation were used to measure CYP2D6 and CYP3A4 activity, respectively. In vivo inhibition constants for CYP3A4 were calculated with use of data that previously documented the interaction between stiripentol and carbamazepine.Results: In vitro, stiripentol inhibited CYPs 1A2, 2C9, 2C19, 2D6, and 3A4, with inhibition constant values at or slightly higher than therapeutic (total) concentrations of stiripentol, but it did not inhibit CYPs 2A6 and 2E1 even at tenfold therapeutic concentrations. In vivo inhibition of caffeine demethylation and dextromethorphan N-demethylation were consistent with inhibition of CYP1A2 and CYP3A4, respectively. The 6 beta-hydroxycortisol/cortisol ratio did not provide a reliable index of CYP3A4 inhibition. Inhibition of CYP2D6-mediated O-demethylation was not observed in vivo. With use of carbamazepine, in vivo inhibition constants for CYP3A4 ranged between 12 and 35 mu mol/L, whereas the corresponding in vitro value was 80 mu mol/L.Conclusions: Stiripentol appears to inhibit several CYP450 enzymes in vitro and in vivo. In vivo inhibition constants show that stiripentol inhibition of CYP3A4 is linearly related to plasma concentration in patients with epilepsy.

Some unsymmetrical N-exocyclic and N-endocyclic derivatives from benzoylation of 3- and 5-aminopyrazole were prepared with the aim of comparing their anticonvulsant activity towards the MES and scMET tests. Unambiguous proof of their structure was obtained from heteronuclear long-range correlation spectroscopy and NOE difference spectra, Only the N-exo-pyrazole benzamides showed good protection with respect to these tests.