Assessment of direct gating and allosteric modulatory effects of meprobamate in recombinant GABA(A) receptors.

Meprobamate is a schedule IV anxiolytic and the primary metabolite of the muscle relaxant carisoprodol. Meprobamate modulates GABAA (γ-aminobutyric acid Type A) receptors, and has barbiturate-like activity. To gain insight into its actions, we have conducted a series of studies using recombinant GABAA receptors. In αxßzγ2 GABAA receptors (where x=1-6 and z=1-3), the ability to enhance GABA-mediated current was evident for all α subunit isoforms, with the largest effect observed in α5-expressing receptors. Direct gating was present with all α subunits, although attenuated in α3-expressing receptors. Allosteric and direct effects were comparable in α1ß1γ2 and α1ß2γ2 receptors, whereas allosteric effects were enhanced in α1ß2 compared to α1ß2γ2 receptors. In "extrasynaptic" (α1ß3δ and α4ß3δ) receptors, meprobamate enhanced EC20 and saturating GABA currents, and directly activated these receptors. The barbiturate antagonist bemegride attenuated direct effects of meprobamate. Whereas pentobarbital directly gated homomeric ß3 receptors, meprobamate did not, and instead blocked the spontaneously open current present in these receptors. In wild type homomeric ρ1 receptors, pentobarbital and meprobamate were ineffective in direct gating; a mutation known to confer sensitivity to pentobarbital did not confer sensitivity to meprobamate. Our results provide insight into the actions of meprobamate and parent therapeutic agents such as carisoprodol. Whereas in general actions of meprobamate were comparable to those of carisoprodol, differential effects of meprobamate at some receptor subtypes suggest potential advantages of meprobamate may be exploited. A re-assessment of previously synthesized meprobamate-related carbamate molecules for myorelaxant and other therapeutic indications is warranted.

Electroencephalographic features of familial spontaneous epileptic cats.

A feline strain of familial spontaneous epileptic cats (FSECs) with typical limbic seizures was identified in 2010, and have been maintained as a novel animal model of genetic epilepsy. In this study, we characterized the electroencephalographic (EEG) features of FSECs. On scalp EEG under sedation, FSECs showed sporadic, but comparatively frequent interictal discharges dominantly in the uni- or bilateral temporal region. Bemegride activation was performed in order to evaluate the predisposition of epileptogenicity of FSECs. The threshold doses of the first paroxysmal discharge, clinical myoclonus and generalized convulsion in FSECs were significantly lower than those in control cats. Chronic video-intracranial EEG monitoring revealed subclinical or clinical focal seizures with secondarily generalization onset from the unilateral amygdala and/or hippocampus. Clinical generalized seizures were also recorded, but we were unable to detect the onset site. The results of the present study show that FSECs resemble not only feline kindling or the kainic acid model and El mouse, but also human familial or sporadic mesial temporal lobe epilepsy. In addition, our results indicate that FSECs are a natural and valuable model of mesial temporal lobe epilepsy.

Carisoprodol tolerance and precipitated withdrawal.

AIMS: Carisoprodol is a muscle relaxant that acts at the GABA(A) receptor. Concerns about the abuse liability of carisoprodol are increasing, but evidence that carisoprodol produces tolerance and a significant withdrawal syndrome has yet to be established. The purpose of the current study was to determine if repeated administration of carisoprodol produces tolerance and withdrawal signs in a mouse model. METHODS: Carisoprodol (0, 100, 200, 300, or 500 mg/kg bid, i.p.) was administered to Swiss-Webster mice for 4 days and loss-of-righting reflex was measured 20-30 min following each administration. On the fourth day, bemegride (20 mg/kg), flumazenil (20 mg/kg), or vehicle was administered following carisoprodol and withdrawal signs were measured. Separate groups of mice receiving the same treatment regimen and dose range were tested for spontaneous withdrawal at 6, 12 and 24 h after the last dose of carisoprodol. RESULTS: The righting reflex was dose-dependently impaired following the first administration of carisoprodol. A 75-100% decrease in the magnitude of the impairment occurred over the four days of exposure, indicating the development of tolerance to the carisoprodol-elicited loss-of-righting reflex. Withdrawal signs were not observed within 24h following spontaneous withdrawal; however, bemegride and flumazenil each precipitated withdrawal within 15-30 min of administration. CONCLUSIONS: Carisoprodol treatment resulted in tolerance and antagonist-precipitated withdrawal, suggesting it may have an addiction potential similar to that of other long-acting benzodiazepine or barbiturate compounds.

Bemegride lengthens seizure duration during electric convulsive therapy in a schizophrenic patient with increased seizure threshold.

A 48-year-old man who had a history of schizophrenia for 30 years was treated with electroconvulsive therapies. Because of poor seizure even at maximum electrical dosage, aminophylline was administered just before initiating electroconvulsive therapy. Although aminophylline augmentation lengthened the seizure duration, tachycardia and hypertension were observed. Therefore, we switched to bemegride, an antagonist to barbiturate, and seizure length was improved without any side effects. The present case suggested that bemegride is one of the alternative measures in patients with poor seizure quality.

Changes of serum adrenocorticotropic hormone and cortisol levels during sleep seizures.

OBJECTIVE: Measuring the serum concentrations of adrenocorticotropic hormone (ACTH) and cortisol in epileptic seizures during sleep to investigate their link to the EEG changes. METHODS: Pre-surgical evaluation was performed by video-EEG monitoring using 24 channel recording. Thirty six epilepsy patients could be attributed to two groups: 28 patients had spontaneous seizures, and the other 8 patients whose seizures were induced by bemegride. Another 11 persons with confirmed psychogenic non-epileptic seizures (PNES) served as control group. Blood samples were obtained at five points: wake (08:00 a.m.), sleep (00:00 a.m.), and shortly before, during and after an epileptic seizure. The serum ACTH and cortisol were measured and analyzed by chemiluminescent immunoassay. RESULTS: The levels of ACTH and cortisol in serum underwent significant changes: declining below the average sleep-level shortly before seizures, increasing during seizures, and far above the average wake-level after seizures (P < 0.001). Such changes did not occur in the control group (P > 0.05). The ACTH and cortisol levels had no significant difference between spontaneous group and bemegride-induced group (P > 0.05). CONCLUSION: The serum concentrations of ACTH and cortisol during sleep seizures are linked with pre-ictal and ictal EEG changes in epilepsy patients.

Changes of serum adrenocorticotropic hormone and cortisol levels during sleep seizures / 神经科学通报·英文版

<p><b>OBJECTIVE</b>Measuring the serum concentrations of adrenocorticotropic hormone (ACTH) and cortisol in epileptic seizures during sleep to investigate their link to the EEG changes.</p><p><b>METHODS</b>Pre-surgical evaluation was performed by video-EEG monitoring using 24 channel recording. Thirty six epilepsy patients could be attributed to two groups: 28 patients had spontaneous seizures, and the other 8 patients whose seizures were induced by bemegride. Another 11 persons with confirmed psychogenic non-epileptic seizures (PNES) served as control group. Blood samples were obtained at five points: wake (08:00 a.m.), sleep (00:00 a.m.), and shortly before, during and after an epileptic seizure. The serum ACTH and cortisol were measured and analyzed by chemiluminescent immunoassay.</p><p><b>RESULTS</b>The levels of ACTH and cortisol in serum underwent significant changes: declining below the average sleep-level shortly before seizures, increasing during seizures, and far above the average wake-level after seizures (P < 0.001). Such changes did not occur in the control group (P > 0.05). The ACTH and cortisol levels had no significant difference between spontaneous group and bemegride-induced group (P > 0.05).</p><p><b>CONCLUSION</b>The serum concentrations of ACTH and cortisol during sleep seizures are linked with pre-ictal and ictal EEG changes in epilepsy patients.</p>

[Changes in energy metabolism in the brain of bemegride-kindled rabbits]. / Zminy enerhetychnoho metabolizmu v tkanynakh mozku kroliv pry bemehrydovomu kindlinhu.

Changes in the energetic metabolism in the cortex were examined in bemegride-kindled rabbits. A reliable decrease in the ATP and an increase in the AMP contents in the cortical tissues as well as low ATP/AMP and energetic charge coefficients have been found after kindling the rabbits for 14 and 30-days as compared to the control groups of animals. Considerable disorders in the oxidative phosphorilation in the mitochondria of the cortical tissues have been determined after the succinate and glutamate oxidation: a decrease in the oxygen utilization rate at V3 active metabolic state, lowering the respiratory control coefficient and the disscciation between phosphorilation and oxidation. Those disorders were more expressed after glutamate oxidation as compared to a succinate one. In addition, it has been determined that oxygen utilization at V2 state was reliably decreased; both coefficient of energetic production and ATP-ase reserve activity lowered.

Clinico-ECoG correlate of megimide-induced epileptic seizures in epilepsy-prone El and its mother strain nonepileptic ddY mice.

Megimide-induced epileptic seizures were recorded in nine male El and six male ddY mice with the chronic implantation technique for monopolar recording of electrocorticogram (ECoG). ECoG were recorded from preictal period until 30 min after general tonic clonic convulsion (GTC). In total, 14 GTC in ddY and 15 in El were recorded. There were three periods as for clinico-ECoG correlate in both strains: a twitching period, GTC, and a postictal period. The GTC had three phases: a tonic phase, a tonic phase with clonic movements, and a clonic phase. Twitching movements (twitching period) consisted of rapid backward jerks of the neck, associated with different electrocortical phenotypes: focal spikes, generalized spikes, or generalized spike and wave complex. During tonic phase of GTC, clonic movement of the limbs was the most frequently observed clinical phenotype, whereas twitchings were the predominant manifestation during clonic phase of GTC in both strains. The ictal ECoG phenotype was strain specific: generalized spikes predominated in ddY and occipital spikes in El mice. There were postictal spikes without specificity for localization in both strains.

Pharmacological studies on a new dihydrothienopyridine calcium antagonist, S-312-d. 5th communication: anticonvulsant effects in mice.

S-312, S-312-d, but not S-312-l, L-type calcium channel antagonists, showed anticonvulsant effects on the audiogenic tonic convulsions in DBA/2 mice; and their ED50 values were 18.4 (12.8-27.1) mg/kg, p.o. and 15.0 (10.2-23.7) mg/kg, p.o., respectively, while that of flunarizine was 34.0 (26.0-44.8) mg/kg, p.o. Although moderate anticonvulsant effects of S-312-d in higher doses were observed against the clonic convulsions induced by pentylenetetrazole (85 mg/kg, s.c.) or bemegride (40 mg/kg, s.c.), no effects were observed in convulsions induced by N-methyl-D-aspartate, picrotoxin, or electroshock in Slc:ddY mice. S-312-d may be useful in the therapy of certain types of human epilepsy.

[The role of lipid peroxidation in the pathogenesis of neuropathological syndromes and in experimental therapy]. / Rol' perekisnogo okisleniia lipidov v patogeneze nekotorykh neiropatologicheskikh sindromov i pri éksperimental'noi terapii.

Activated lipid peroxidation observed in brain homogenates in experimental choreoid hyperkinesis and bemegride epilepsy can be arrested by antioxidant pretreatment. The same is true for parkinsonian and "malignant locomotion" syndromes provoked by intrastriatal pro-oxidant (oxidized oleinic acid) microinjection and systemic administration of haloperidol. Experimental therapy of choreoid hyperkinesis with GABA-positive and dopamine-blocking drugs alone proved ineffective. The effect was achieved at combined application of antioxidant and GABA-positive drugs.

[The effect of hormonal systems on the activity of convulsant agents]. / Vliianie gormonal'nykh sistem na aktivnost' sudorozhnykh sredtsv.

The dynamics of convulsive action exerted by Bemegride, 15 mg/kg, and Corasol (Pentamethylenetetrasol, 50 mg/kg, was examined in rat experiments during extirpation of endocrine glands (pituitary, thyroid, adrenals and gonads). Their effects were found to be unequal in various series of experiments: thyroidectomy intensified the nature of convulsive reactions while gonadectomy attenuated them; sex-specific differences in the effects of the agents were insignificant. It has been suggested that the hormone-mediator interaction is essential in the mechanisms of convulsive action of Bemegride and Corasol.

Paradoxical enhancement of hypoperfusion single photon emission CT images in epileptic focus with bemegride activation. Observation from subtraction of 99mTc hexamethyl-propyleneamine oxime brain single photon emission CT images.

Changes of brain images in single photon emission CTs (SPECTs) before and after intravenous injection of bemegride were examined with simultaneous EEG recording in a patient with occipital lobe epilepsy and in a hysteric patient, using a new method, i.e. subtraction of the SPECT images with 99mTc hexamethyl-propyleneamine oxime. The bemegride injection in the epileptic patient paradoxically intensified the hypoperfusion images as epileptic foci in SPECTs, associated with enhanced interictal epileptic discharges, whereas the hysteric showed no such effects. These findings suggest that the hypoperfusion images closely correlate to the severity of epileptic activity, reflecting a functional rather than a morphological deficit.

[The anticonvulsant activity of digoxin in rats of different ages and in mice]. / Protivosudorozhnaia aktivnost' digoksina u krys raznogo vozrasta i myshei.

The anticonvulsant activity of digoxin was studied on rats of different ages and mice. Cramps were simulated by using electroshock or a subcutaneous administration of bemegrid or corasol. The effect of digoxin (in the doses amounting to 1/10 or 1/2 of LD50) on the time of the onset of cramps, the duration of tonic and clonic phases as well as the number of the deceased animals was evaluated. The detected anticonvulsant effect of digoxin is explained by its influence on the activity of the membrane Na+, K+, ATP-ase and K+ conductivity.

Posttetanic and frequency potentiation in slices of rat olfactory cortex.

The electrical tetanization of the lateral olfactory tract at a frequency of 30/sec for 15 sec elicited the development of posttetanic potentiation of populational EPSP and IPSP in surviving slices of rat olfactory cortex. The stimulation of the lateral olfactory tract by series of stimuli at a constant frequency of 10/sec and with intervals of 4-5 sec between series facilitates the emergence of the phenomenon of frequency potentiation. The data obtained indicate that such forms of functional plasticity as posttetanic and frequency potentiation are manifested in the pyriform cortex.

Reversed-phase high-performance liquid chromatography method for the determination of bemegride in serum and brain tissue: pharmacokinetics and brain distribution of an intraperitoneal subconvulsive dose in rats.

A simple and rapid HPLC method has been developed for the quantification of bemegride in serum and brain tissue, using p-methylphenobarbital as an internal standard. Serum and brain tissue homogenate samples were extracted with ethyl acetate and the evaporated and redissolved extracts injected into a reversed-phase column. The compounds were eluted with an acetonitrile-phosphate buffer mixture and monitored at 200 nm. A linear response was obtained in the range 1-40 micrograms ml-1 for serum and 1-40 micrograms g-1 for brain tissue. Within-day and between-day precisions were less than 5% and the analytical recovery greater than 76.4%. This method has been used to investigate the kinetic profiles of the drug in serum and discrete areas of rat brain after intraperitoneal administration of a subconvulsive dose of bemegride (10 mg kg-1). Peak concentrations occurred in the brain and serum at the same time (30 min), followed by a biphasic decay. The results also indicated the accumulation of the drug in the brain, with no significant differences (p greater than 0.05) in the impregnation of the different brain areas investigated.

Actions of steroids and bemegride on the GABAA receptor of mouse spinal neurones in culture.

The effects of a synthetic and an endogenous steroid were studied on the GABAA receptors of isolated mouse spinal neurones, maintained in culture. Low doses of alphaxalone reversibly increased GABA-evoked whole-cell currents. Alphaxalone at higher doses (10-50 microM), when pressure ejected onto spinal neurones, also directly evoked a membrane chloride current. Such currents were reversibly suppressed by bicuculline (a GABAA antagonist) and enhanced by phenobarbitone. 5 beta-Pregnan-3 alpha-ol-20-one, a progesterone metabolite, dose-dependently potentiated the amplitude of GABA-evoked whole-cell currents. The mechanism of potentiation was examined at the single-channel level using outside-out patches from spinal neurones. The main action of the steroid on the GABAA receptor appears to be similar to that found for barbiturates, in that they prolonged GABA-activated bursts of channel openings. Bemegride had an antagonistic action on the GABAA receptor, suppressing both GABA- and pentobarbitone-evoked whole-cell currents to similar extents.

Protective effect of cinnarizine against convulsive elevation of nonesterified fatty acids in CNS.

In the brain tissue there exists a highly active "pool" of nonesterified fatty acids (NEFA). The greatest part of NEFA comes from the fraction of nerve endings. It was evidenced that the model convulsive activity provoked by Bemegrid increased NEFA in the brain cortex, hypothalamus and brain stem. Cinnarizine (StugeronR) markedly decreases the convulsive increase of NEFA. The greatest inhibitory effect was observed in the brain cortex. Cinnarizine has an objective stabilizing and anticonvulsive effect on the brain tissue.

[Effector modeling of the action of ligands of GABA-receptor complex. Functional interaction of the hypothetic alcohol receptor with other subunits of the complex]. / Effektornoe modelirovanie deistviia ligandov GAMK-retseptornogo kompleksa. Funktsional'noe vzaimodeistvie gipoteticheskogo retseptora spirtov s drugimi sub"edinitsami kompleksa.

The types of the interaction of the pharmacological effects of ethanol and barbiturate antagonists--picrotoxin, bemegride and corasol--were determined. The effect of ethanol was determined as competitive--for the convulsant effects of bicuculline, and non-competitive--for the effects of thiosemicarbazide. The indices of the anticonvulsant effects of n-aliphatic alcohols were compared. It is suggested that n-aliphatic alcohols alter the functional status of the supramolecular GABA-receptor channel ensemble. The pharmacological properties and the elements of the structural similarity of picrotoxin and n-propanol (the presumptive ligand of the GABA-receptor channel ensemble) are discussed.

[Effector modelling of the action of ligands of the GABA-receptor complex, cooperativity of the processes and the type of modification of the complex by convulsants and their reversible agonists]. / Effektornoe modelirovanie deistviia ligandov GAMK-retseptornogo kompleksa, kooperativnost' protsessov i tip modifikatsii kompleksa sudorozhnymi agentami i ikh obratnymi agonistami.

The expression of cooperativity of pharmacological effects of convulsants--exogenic ligands of supramolecular GABA-receptor-channel ensemble (GABA-RC)--bicuculline, picrotoxin, pentylenetetrazole, bemegride at intravenous infusion to intact animals and against a background of administration of barbital-Na and phenazepam is determined. The supposed mechanism of bemegride effect is discussed. Analysis of principles of GABA-RC functioning in vivo on the base of pharmacological data (cooperativity coefficients and types of modulation of GABA-RC functions at the interaction of convulsants and their reverse agonists) suggests the formation of biosystem response at modification of structures adequate to one of four subunits of "quartet" (tetrameric) model of GABA-RC.