Diazepam sensitive mice: differential sensitivity to the depressant and anticonvulsant effects of diazepam

A mouse line was developed by selecting for increased sensitivity to the hypnotic effect of diazepam. These "diazepam sensitive" mice showed a mean duration of loss of righting reflex (LORR) of approximately 150 min at a dose of 20 mg/kg diazepam, this dose failed to induce LORR in the control outbred mice. Rotarod treading times of the diazepam sensitive mice were significantly shorter than that of the control mice over the same dose range indicating that these mice are also more sensitive to the sedative/muscle relaxant effects of diazepam. On the contrary, the ability of diazepam to protect against pentylenetetrazole-induced convulsion was found to be the same in the sensitive and control mice. These observations strongly suggest that the heightened sensitivity to the sedative-hypnotic effects of diazepam in the sensitive mice is unlikely to be due entirely to changes in drug disposition.     

Role of the substantia nigra in antiaggressive and anticonvulsant effects of diazepam during pharmacological kindling

Having set up pharmacological kindling in rats by repeated injection of picrotoxin at a subthreshold dose i.p., a study was made of activity produced by injecting the trypsinized protein fragment, T5 — diazepam binding inhibitor (DBI) in man at a dose of 10 µg — into the reticular section of the substantia nigra (SN). Severity of convulsive effects increased in animals under the influence of DBI and the anticonvulsant action of diazepam declined. Intranigral injection of DBI did not affect the threshold triggering attacking behavior in rats when current was passed through the electrically-conducting floor, nor did the antiaggressive action of diazepam change under these conditions. Findings would indicate that the benzodiazepine receptors of the SN contribute to suppression of epileptic response during kindling and to the production of anticonvulsive (but not antiaggressive) diazepam action.N. I. Pirogov Medical Institute, Odessa. Translated from Neirofiziologiya, Vol. 22, No. 4, pp. 482–485, July–August, 1990.     

Identification of fungal metabolites of anticonvulsant drug carbamazepine

Carbamazepine, which has been used in the treatments of epilepsy, is often found in the environment. Although metabolism of carbamazepine by humans and rats has been characterized, the environmental fate of carbamazepine has not been studied. In this study, two model fungi Cunninghamella elegans ATCC 9245 and Umbelopsis ramanniana R-56, which have previously shown diverse metabolic activities, were tested for metabolism of carbamazepine. Both fungi produced three metabolites each (C1-C3 and M1-M3). All six metabolites showed [M + H](+) at m/z 253, suggesting addition of one oxygen to the parent compound. High-performance liquid chromatography and liquid chromatography-mass spectrometric analysis detected 10, 11-dihydro-10, 11-epoxycarbamazepine as a major product (C3 (47%) and M3 (85%)) and 3-hydroxycarbamazepine (C2 (15%) and M2 (7%)) from carbamazepine through mixed mono-oxidation reactions in both fungal strains. C. elegans was confirmed to produce 2-hydroxycarbamazepine (C1 (38%)) while U. ramanniana produced a yet unidentified ring-hydroxylated metabolite (M1 (8%)). The current study suggests that carbamazepine is likely to be subjected to initially diverse mono-oxygenation reactions by fungal metabolisms, resulting in the formation of the corresponding metabolites, which were similarly found in mammalian metabolisms.     

Inhibition of anticonvulsant action of carbamazepine by aminophylline and caffeine in rats

Interaction of two well known methyl xanthines, aminophylline--an antiasthmatic agent--and caffeine--commonly present in beverages, on the seizure protective ability of carbamazepine (CBZ) against electrically and chemically induced seizures in rats was investigated. Aminophylline (75 mg/kg, ip) did not alter the activity of CBZ (10 mg/kg, ip; ED100) on maximal electroshock seizures while dose dependent antagonism of CBZ efficacy was seen at 100 and 150 mg/kg, ip. Similar effects were observed with caffeine (200 and 250 mg/kg, ip). At the highest tolerated doses, aminophylline (150 mg/kg, ip) and caffeine (250 mg/kg, ip) produced antagonism of CBZ protection against pentylenetetrazole seizures. These observations support the possibility that the antagonism due to the interaction of these drugs could be related to their action at adenosine receptor sites in the brain.     

Na, K-ATPase activity of the meninges in electroshock and the action of the anticonvulsant agent, diazepam]

It was shown that the phenomenon of inactivation of Na, K-ATPase of the non-purified fraction of the rat cortical synaptosomes under electroshock may be related to "modification" of the potassium active center of the enzyme. The anticonvulsant diazepam injected intramuscularly also inhibits Na, K-ATPase of the cerebral membranes. However, in subsequent electrical stimulation of the brain the drug activates Na, K-ATPase as compared to controls. Diazepam also abolishes clonic convulsions induced by electrical stimulation of the brain. At the same time it does not eliminate compensatory shifts in the activity of acetyl-cholinesterase of the rat cerebral and spinal synaptosomes, characteristic of electroshock. The results are discussed from the standpoint that inhibition of the activity of Na, K-ATPase of the nerve endings membranes may underlie the pathogenetic mechanism of the convulsive activity.     

Brain anticonvulsant protection of mice given chronic carbamazepine under various fatty acid and magnesium diet conditions

The anticonvulsant and mood stabilizer drug carbamazepine (CBZ) was evaluated for anti-seizure activity after drug pretreatment of young weaning mice given various oil-based diets. These diets had various mono-(MUFA) and poly-(PUFA) unsaturated fatty acid contents, were associated or not with magnesium deprivation, and were given over the entire experimental period (34 days). The diets included a commercial and three purified synthetic diets (n-6 PUFA, n-3 PUFA and MUFA-based chows containing 5% corn/sunflower oils 1:3, 5% rapeseed oil and 5% high oleic acid sunflower oil/sunflower oil 7:3, respectively). A 10-days CBZ treatment (50mg/kg/day fragmented in two daily intraperitoneal injections of 25mg/kg) was given 20 days after initiating diet administration and evaluations of mice was performed 4 days after arrest of CBZ in various seizure tests. In these conditions, CBZ pretreatment still exhibited anticonvulsant protection especially in magnesium-deficient animals. Ethosuximide (ESM)-like profiles under MUFA and n-3 PUFA diets and unusual GABA(A)ergic profile under n-6 PUFA diet in magnesium-deficiency dependent audiogenic seizures (MDDAS) test as well as protection against NMDA-induced seizures in all lipid (n-3 PUFA>MUFA and n-6 PUFA) diet conditions were observed in CBZ-pretreated mice. By highlighting ESM-like and anti-NMDA mechanisms previously induced by an n-3 PUFA diet, present CBZ anticonvulsant properties suggest brain protective targets common to CBZ and n-3 PUFAs.     

Inhibition of slow TTX-insensitive inward current by the anticonvulsant carbamazepine in an identified neuron of Lymnaea stagnalis

1. Pentylenetetrazol (PTZ), induces tonic depolarization and bursting activity in an identified neuron, B1, of Lymnaea stagnalis. This is due in part to activation of a slow, tetrodotoxin-insensitive inward sodium current.2. Carbamazepine (CBZ) reversed the effect of PTZ on both membrane potential and inward current, after a delay of up to 5 min. CBZ alone had no effect on voltage or current responses.3. These results suggest that CBZ blocks the slow sodium current, possibly via a decrease in intracellularly stored calcium ions.     

Teratogenic effects of diazepam in the hamster.

Pregnant hamsters were treated with different doses of oral and intravenous diazepam during the period of organogenesis. Teratogenic effects of diazepam were observed following oral treatment on days 8 and 10 and following intravenous treatment on day 11 of gestation. Types of malformations included cleft palate, exencephaly, limb anomalies, and hemorrhage. A dose-effect relationship was not observed. Comparison with reported literature seems to indicate that diazepam may be a mild teratogen in some species.     

Modification of the anticonvulsant efficacy of diazepam by Ro-15-1788 in the kindled amygdaloid seizure model

The effects of various doses of diazepam and the new central benzodiazepine antagonist Ro-15-1788 were investigated in fully amygdaloid kindled rats. Diazepam had a pronounced dose-dependent anticonvulsant effect in this model. Ro-15-1788 dose-dependently reduced the behavioral ranks of the elicited kindled seizures to a maximum of 60% of control without consistently modifying the afterdischarge duration. No prestimulation convulsant effects were seen with Ro-15-1788. When 2 mg/kg i.p. of Ro-15-1788 was given after various doses of diazepam, the prestimulation sedation and ataxia anticonvulsant effects of diazepam (0.5-2.0 mg/kg) were attenuated by treatment with 2 mg/kg dose of Ro-15-1788. At the low dose of diazepam (0.25 mg/kg), increased reduction of behavioral rank and after discharge duration was seen after the 2 mg/kg dose of Ro-15-1788. Thus, Ro-15-1788 appears not to have proconvulsant properties in the kindled amygdaloid seizure model. Further, Ro-15-1788 appears to have some anticonvulsant properties of its own. Mixed agonist and antagonist effects were seen with Ro-15-1788 when given after various doses of diazepam in this model.     

Effect of diazepam, carbamazepine, sodium valproate and their combinations with vitamin preparations on epileptic activity

The anticonvulsive action of diazepam, carbamazepine, sodium valproate and their combinations with pyridoxal-5-phosphate, nicotinamide, and alpha-tocopherol were investigated in acute experiments on mice with corazole-induced seizures. Diazepam (0.5 mg/kg), carbamazepine (50 mg/kg) and sodium valproate (200 mg/kg) were shown to reduce convulsive intensity and lethality. Vitamins nicotinamide (250 mg/kg), pyridoxal-5-phosphate (10 mg/kg) and alpha-tocopherol (100 mg/kg) potentiated anticonvulsive action of the above antiepileptic drugs. The results of the investigation suggest the efficacy of pathogenetic therapy and give new evidence of the advisability of using vitamins in combination with synthetic anticonvulsive drugs.     

Pineal mediation of the thermoregulatory and behavioral activating effects of beta-endorphin

Intraventricular administration of the opioid peptide, beta-endorphin to goldfish altered their body temperatures and activity levels. Low doses (0.5-5.0 pg g-1 body weight) of beta-endorphin significantly increased behaviorally selected body temperatures while higher doses (15 pg g-1) decreased the preferred temperatures selected in horizontal thermal gradients. There was a significant day-night rhythm in the extent of these effects. These thermoregulatory effects could be blocked and reversed by systemic administration of the opiate antagonist, naloxone, supporting mediation of the thermoregulatory effects at opioid receptors. In addition, administration of naloxone by itself significantly decreased preferred temperature. Removal of the pineal gland significantly increased the preferred temperatures selected by goldfish and eliminated the thermoregulatory effects of beta-endorphin administration in both the day and the night. The behavioral activity effects of beta-endorphin were dependent on the thermal conditions. In fish held at a constant temperature (20 degrees C) beta-endorphin caused a dose-dependent increase in activity, while in individuals held in thermal gradients administration of beta-endorphin had no effects on activity. In both situations naloxone caused a decrease in activity levels. Pinealectomy also eliminated the behavioral activating effects of beta-endorphin, though it had no apparent effects on the actions of naloxone. These results indicate that the pineal gland is involved in the mediation of the thermoregulatory and behavioral activating effects of beta-endorphin. Speculations are made as to the possible mechanisms of action of the pineal gland in mediating the effects of opioid neuropeptides.     

Caffeine antagonizes several central effects of diazepam

In spinal cats, caffeine (3–30 mg·kg^?1 i.v.) reduced the increase of dorsal root potentials (DRPs) caused by diazepam (0.1–1 mg·kg^?1 i.v.) without affecting the prolongation of DRPs evoked by phenobarbitone (10–20 mg·kg^?1 i.v.). Caffeine antagonized the depression by diazepam, but not that by phenobarbitone, of the ventral root-evoked Renshaw cell discharge. In unrestrained cats, 50 mg·kg^?1 caffeine i.p. abolished the elevation induced by 1 mg·kg^?1 diazepam i.p. of the threshold for eliciting a rage reaction by stimulation of the lateral hypothalamus, but was ineffective against the threshold increase caused by 20 mg·kg^?1 phenobarbitine i.p. In the horizontal wire test in mice, caffeine was more potent in reversing the depression of performance induced by diazepam that that by phenobarbitone (ED50 1.8 mg·kg^?1 and 139 mg·kg^?1 p.o., respectively). The reduction of skeletal muscle tone in mice produced by diazepam was antagonized by low doses of caffeine (ED50 0.53 mg·kg^?1 p.o.). While caffeine at low doses (0.3-3 mg·kg^?1 p.o.) abolished the anticonflict effect of diazepam in rats, high doses (ED50 160 mg·kg^?1 p.o.) were necessary to antagonize the anticonvulsant effect of diazepam on pentylene-tetrazole-induced seizures in mice. The interaction between caffeine and diazepam is not due to a competition at the benzodiazepine receptors but may involve purinergic mechanisms.     

Caffeine and theophylline counteract diazepam effects in man

A total of 237 healthy subjects were studied in four placebo-controlled double-blind trials with parallel treatment groups. The subjects ingested a capsule (diazepam or placebo) and decaffeinated coffee with or without added caffeine or theophylline. Diazepam (10 and 20 mg) impaired dose dependently cognitive skills as measured by digit symbol substitution and letter cancellation, the balance of extraocular muscles, flicker fusion, and tapping speed. With diazepam 10 mg statistically significant effects were seen on digit symbols and exophoria only. Theophylline (10 mg/kg) increased tapping speed and heart rate, whereas other objective measurements were negative for the effects of theophylline or caffeine (250 and 500 mg) alone. Subjectively they reduced calmness, and caffeine also increased alertness. Caffeine 250 mg counteracted diazepam induced (10 mg) impairment of cognitive skills and relaxation of extraocular muscles whereas caffeine 500 mg counteracted the same effects of diazepam 20 mg, respectively. Theophylline antagonized diazepam-induced impairment in digit symbol substitution and tapping speed tests. Subjectively, caffeine and theophylline counteracted diazepam induced drowsiness and mental slowness. The results suggest, therefore, that the ample use of methylxanthines compensates various side-effects of benzodiazepines in man. It may also increase the need of sedation and thus the consumption of benzodiazepines.     

Effect of GABA-negative preparations on the anxiolytic and sedative effects of diazepam

Experiments on rats have shown that bicuculline (2 mg/kg) and picrotoxin (2 mg/kg) abolish the anxiolytic action of diazepam (2.5 mg/kg). Bicuculline (2 and 4 mg/kg) decreases while picrotoxin transforms the sedative effect of diazepam to the anxiolytic one. Picrotoxin (2 mg/kg) reduces the sedative action of gamma-acetylenic GABA (100 mg/kg) but does not favour the manifestation of its anxiolytic effect. It is suggested that the GABA-ergic mechanisms play an important role in the sedative effect of diazepam.     

Dopaminergic mediation of the diuretic and natriuretic effects of ANF in the rat

Atrial natriuretic factor (ANF) increases sodium (Na+) and water excretion 8-10 fold on repeated administration to anesthetized rats. SCH-23390 (80 micrograms/kg i.v.) and R-sulpiride (80 micrograms/kg i.v.), selective antagonists of dopamine receptors in the renal vasculature, inhibited diuresis and natriuresis induced by AP III and dopamine. These findings suggest that ANF exerts its effects on renal Na+ and water handling via a dopaminergic mechanism; however, changes in intrarenal hemodynamics secondary to dopamine receptor blockade may attenuate the actions of ANF.     

Gender-related differences in diazepam effects on performance

In order to investigate the possible differences of diazepam effects in the two sexes, two placebo-controlled double-blind studies were conducted on healthy volunteer students. In one study the subjects received diazepam 10 mg alone or combined with 0.5 g/kg of alcohol in a parallel group design; in the other 0.2 mg/kg of diazepam or placebo were given in a cross-over manner. In both trials diazepam impaired the psychomotor skills of women more than men. The difference was similar in tasks measuring cognitive (digit symbol substitution), motor (balance of extraocular muscles) and sensory (critical flicker fusion) performances. Tapping speed was affected to a similar degree in both genders. Diazepam 10 mg did not cause impairment of body balance, a parameter sensitive to alcohol. The combined effect of diazepam and alcohol was of similar magnitude in both sexes in all objective tests. Subjectively the women felt themselves clumsier than did the men. The calming effect was similar in both groups. The results suggest that while the performance of women may be more vulnerable than men to impairment by diazepam they also are aware of it. The difference of effects is of such magnitude that it may cause bias in experiments unless carefully balanced groups are used.     

ortho Substituent effects on the anticonvulsant properties of 4-hydroxy-trifluoroethyl phenols

2,6-Dialkylphenols with isopropyl and sec-butyl substituent are well known anesthetic compounds. The 4-substitution with 1-hydroxy-2,2,2-trifluoroethyl (4-HTFE) group in these compounds led to the discovery of compounds with anticonvulsant activity in the 6Hz (32mA) model of partial epilepsy. In the present study a series of 2-alkyl-4-HTFE phenols with the 6-position being replaced with either hydrogen and bromine were designed, synthesized and tested to evaluate the effect of ortho-substitution on the anticonvulsant property. The studies show that 2-substituted branched alkyl chain (iso-propyl and sec-butyl) is necessary for the anti-seizure effect. Phenols with 2-substituted linear alkyl groups (methyl, ethyl and n-propyl) having no substitution at 6-position were found to be devoid of antiseizure effects. The 6-substitution with bromine moderately reduces the anticonvulsant effect in the compounds with branched alkyl chains, but led to enhanced anticonvulsant effect in the compound with a linear alkyl chain. This study shows that 4-HTFE phenols having isopropyl or sec-butyl ortho groups produce good antiseizure protection in the 6Hz therapy-resistant mouse model.     

Prostaglandin mediation of potassium effects on renin release

The effects of varying potassium concentrations on renin release from rabbit renal cortical slices were investigated. High potassium concentrations inhibited and low concentrations stimulated renin release, although the magnitude of the effect was greater with reduced concentrations. The fatty acid cyclooxygenase inhibitor Δ^5,8,11,14-eicosatetraynoic acid abolished the stimulatory effects of low potassium, suggesting a prostaglandin dependent effect of potassium on renin release.     

Abdominal vagal mediation of the satiety effects of CCK in rats

CCK type 1 (CCK1) receptor antagonists differing in blood-brain barrier permeability were used to test the hypothesis that satiety is mediated in part by CCK action at CCK1 receptors on vagal sensory nerves innervating the small intestine. Devazepide penetrates the blood-brain barrier; A-70104, the dicyclohexylammonium salt of N alpha-3-quinolinoyl-D-Glu-N,N-dipentylamide, does not. At dark onset, non-food-deprived control rats and rats with subdiaphragmatic vagotomies received a bolus injection of devazepide (2.5 micromol/kg i.v.) or a 3-h infusion of A-70104 (3 micromol.kg(-1).h(-1) i.v.) either alone or coadministered with a 2-h intragastric infusion of peptone (0.75 or 1 g/h). Food intake was determined from continuous computer recordings of changes in food bowl weight. In control rats both antagonists stimulated food intake and attenuated the anorexic response to intragastric infusion of peptone. In contrast, only devazepide was effective in stimulating food intake in vagotomized rats. Thus endogenous CCK appears to act both at CCK1 receptors beyond the blood-brain barrier and by a CCK1 receptor-mediated mechanism involving abdominal vagal nerves to inhibit food intake.     

Comparative study of the teratogenic effects of chlordiazepoxide and diazepam in the fetal hamster

A single, intraperitoneal injection of either chlordiazepoxide (280–3100 mg/kg) or diazepam (120–980 mg/kg) was administered to pregnant hamsters on day 8 of gestation. These dosages produced a dose-dependent sedation of pregnant animals lasting up to 36 hours, and a combined maternal mortality rate of 21% (chlordiazepoxide) and 5% (diazepam). Animals were sacrificed on gestation day 12 and the fetuses were examined for gross malformations. Although there was a dose-related increase in the frequency of malformed fetuses (3.4%–54.7% for chlordiazepoxide, and 0–58.7% for diazepam), the lowest teratogenic dose was noted to be 280 mg/kg, i.e., almost 500 times and 1000 times the average daily recommended therapeutic doses for chlordiazepoxide and diazepam, respectively. The majority of fetal malformations observed with both compounds were either exencephaly or cranioschisis. The results of this study should be interpreted with caution in so far as their relevance to humans is concerned. The terata were observed at exceedingly high doses (700–7000 times the average human use dose), and the malformations noted by us have not been associated with the use of minor tranquilizers in human pregnancy. Also, although we found no gross anomalies in the litters of dams deprived of food and water for up to 72 hours, the confounding role of prolonged periods of sedation and resulting inactivity, and possible hypoxia and hypothermia needs to be explored further.