Benzodiazepine antagonist, CGS-8216, in diazepam- or pentobarbital-dependent and non-dependent rats

CGS-8216, a benzodiazepine antagonist, was administered to rats acutely dosed with diazepam, and to rats chronically dosed with diazepam or pentobarbital. The effects of an acute dose of diazepam were antagonized by CGS-8216 but signs of precipitated abstinence were not observed. An apparent arousing effect was seen in non-dependent rats when CGS-8216 was administered after placebo, but no arousal was observed when Ro15-1788 was administered after placebo in non-dependent rats. A precipitated abstinence syndrome was elicited with CGS-8216 in rats chronically dosed with diazepam and was very similar to the abstinence syndrome precipitated by Ro15-1788 in diazepam-dependent rats. Like Ro15-1788, CGS-8216 elevated Precipitated Abstinence Scale (PAS) scores in a dose-related manner until a plateau was reached with 5 mg/kg. No signs of precipitated abstinence were observed when CGS-8216 was administered to rats dependent on phenobarbital.     

Differential actions of RO 15-1788 and diazepam on poikilothermia, motor impairment and sleep produced by ethanol

In adult male Sprague-Dawley rats kept at an ambient temperature of 23-25 degrees C, ethanol was injected intraperitoneally in a dose of 4.0 g/kg to produce a clear-cut impairment of autonomic and motorial functions. Following the injection of ethanol, motor coordination, measured on a rotorod, behavioral sleep, righting reflex and colonic temperature were monitored at predetermined intervals for 5.0-7.0 hr. In the first experiment, either 1.0 mg/kg RO 15-1788 (flumazenil), a benzodiazepine (BZ), receptor antagonist, or 1.0-5.0 mg/kg diazepam, a classical benzodiazepine receptor agonist, were injected intraperitoneally either alone or concurrently with ethanol's administration. In the second study, either RO 15-1788 (1.0 or 2.0 mg/kg) or diazepam (1.0 or 5.0 mg/kg) was injected at the nadir of the fall of body temperature induced by ethanol. Although RO 15-1788 alone failed to affect the rats' temperature, it did not prevent the characteristic ethanol-induced hypothermia but rather potentiated it in a dose-dependent manner. Further, this BZ receptor antagonist exacerbated motor incoordination and other behavioral effects when given either simultaneously with ethanol or at the nadir in the animals' core temperature. Although diazepam evoked a dose-dependent hypothermia, it did not enhance ethanol-induced hypothermia when both drugs were administered simultaneously. However, diazepam augmented motor incoordination and other effects and served to delay their recovery. When given to the rats at the nadir of ethanol hypothermia, diazepam did not potentiate ethanol's thermolysis but retarded the recovery from hypothermia; it caused also a dose-dependent delay in the recovery of motor coordination and other responses.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for a unique interaction of loreclezole with the GABA receptor complex

The interaction of loreclezole (a structurally novel non-benzodiazepine anticonvulsant), diazepam, phenobarbital sodium, and ethosuximide with the benzodiazepine antagonists β-CCE, Ro 15-1788, and CGS 8216 and the purine antagonist aminophylline were examined in the rat i.v. metrazol seizure test. The anticonvulsant activity of loreclezole was inhibited by β-CCE and CGS 8216 but not by Ro 15-1788 or aminophylline. The inhibition produced by CGS 8216 was dose dependent (ID₅₀ = 0.04 mg/kg i.p. CGS 8216) and surmountable. When the interactions of the various antagonists with loreclezole were compared to those of diazepam, phenobarbital sodium, and ethosuximide, a clear differentiation of activity was evident. β-CCE blocked the anticonvulsant activity of all four compounds whereas Ro 15-1788 blocked only the anticonvulsant effects of diazepam. CGS 8216 blocked the anticonvulsant effects of diazepam and loreclezole in a similar manner while being much less effective against the anticonvulsant effects of ethosuximide and ineffective against phenobarbital sodium. Aminophylline blocked the anticonvulsant effects of only ethosuximide. Based on these data and the anticonvulsant profile of loreclezole, we suggest that loreclezole represents a new class of agents which modulate GABAergic transmission via a unique interaction with the GABA receptor complex.     

Effects of Ro 15-1788 and CGS 8216 in diazepam-dependent baboons

Baboons received continuous intragastric infusions of diazepam (20 mg/kg per day) for one or more months. While diazepam treatment continued, baboons received intragastric doses of Ro 15-1788 (0.032-32.0 mg/kg) or CGS 8216 (1.0-100.0 mg/kg) at intervals of two or more weeks. Baboons were observed following administration of these antagonists for the presence of precipitated withdrawal signs. The following results were obtained: (1) both Ro 15-1788 and CGS 8216 produced signs of precipitated withdrawal in the baboon; (2) a more severe overall withdrawal syndrome was precipitated with Ro 15-1788 than with CGS 8216 at testable doses; (3) Ro 15-1788 produced dose-related increases in the overall severity of withdrawal, while CGS 8216 did not produce a clear dose-related increase in the overall severity of withdrawal; (4) dose-effect curves for Ro 15-1788 for certain signs (e.g. limb-tremor) were monotonicly increasing, while for other signs dose-effect curves plateaued at lower doses of Ro 15-1788 (e.g. retching and vomiting) or were an inverted U-shape (e.g. scratching). CGS 8216 precipitated withdrawal signs were less clearly dose-dependent; (5) onset of Ro 15-1788 precipitated withdrawal signs were rapid (5-15 min) and reliable, while the onset of CGS 8216 precipitated withdrawal signs were generally slower (approximately 30 min) and more variable; (6) at doses of Ro 15-1788 and CGS 8216 that produced equal levels of vomiting and retching, Ro 15-1788 produced more limb-tremor than CGS 8216. These studies indicate that Ro 15-1788 and CGS 8216 may produce quantitatively and qualitatively different precipitated withdrawal syndromes.     

Specific antagonism by RO 15-1788 of benzodiazepine-induced increases in food intake in rhesus monkeys

Food intake of rhesus monkeys was limited to a single daily 2 hr session of banana flavored pellet availability, seven days a week. Following stabilization of intake, the effects of intragastric bromazepam, diazepam and pentobarbital, when given alone, were determined by delivering a dose twice weekly, 60 min before the session. Dose-dependent increases in food intake were observed with the following descending order of potencies: bromazepam, diazepam and pentobarbital. RO 15-1788 (0.5-1.0 mg/kg, IM), when given alone, five min before the session had no effect on food intake. When given in combination with bromazepam and diazepam, RO 15-1788 completely blocked the increases in food intake observed when the benzodiazepines were given alone. The specificity of this antagonism was shown by the failure of RO 15-1788 to alter the food intake increases induced by pentobarbital. These results confirm and extend previous reports of the specific antagonism of benzodiazepine behavioral effects by RO 15-1788 to an additional species and another behavioral effect of benzodiazepines.     

The effects of GABA and benzodiazepine receptor antagonists on the anti-conflict actions of diazepam or ethanol

The effects of picrotoxin, bicuculline or RO 15-1788 on the anti-conflict action(s) of diazepam or ethanol were studied in rats using a modified Vogel's conflict test procedure. RO 15-1788 antagonized the anti-punishment effects of diazepam (2.5 mg/kg, IP), whereas various doses of bicuculline or picrotoxin did not interfere with diazepam's anti-conflict effect in this test situation. The anti-conflict action of ethanol (2 g/kg, IP) was antagonized by picrotoxin (1.0 mg/kg, IP), whereas both bicuculline and RO 15-1788 were without effect on the increased punishment response produced by ethanol. These data suggest that the anti-conflict properties of ethanol are at least partially mediated through an enhancement of central GABAergic activity.     

3-((+-)-2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) and phencyclidine produce a deficit of passive avoidance retention in rats

3-((+-)-2-Carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP), phencyclidine (PCP) and diazepam were evaluated for their ability to produce a deficit for a single trial step-through passive avoidance response in rats. Pretraining administration with CPP at doses ranging from 2.0 to 10.0 mg/kg s.c. significantly decreased retention latencies 24 h after passive avoidance training. Similar effects were found with PCP at doses ranging from 0.5 to 1.7 mg/kg s.c. and diazepam at doses between 5.0-18.0 mg/kg s.c. Pretraining administration with the benzodiazepine antagonist, RO15-1788 at doses between 0.1-15 mg/kg s.c., did not alter retention latencies. Co-administration of RO15-1788 (0.01-15.0 mg/kg s.c.) with CPP (6.0 mg/kg s.c.) or PCP (1.0 mg/kg s.c.) failed to block decreases in latencies. However, when RO15-1788 was co-administered with diazepam (9.0 mg/kg s.c.) a dose-related antagonism of diazepam's effects were found. These results suggest that the behavioral actions of CPP and PCP on passive avoidance retention are not mediated via the benzodiazepine receptor complex.     

Glycine potentiates the anticonvulsant action of diazepam and phenobarbital in kindled amygdaloid seizures of rats

The effect of glycine on the anticonvulsant activity of diazepam and phenobarbital in fully developed kindled amygdaloid seizures in rats was determined. Glycine alone had no significant effect on the seizure response, either when administered orally 1 hr prior to the seizure test or when given chronically in a 0.5 M solution as the source of water.

Administration of glycine (10 mmol/kg, oral) together with diazepam produced a significant reduction in both cortical epileptiform afterdischarge and the severity of seizures at doses of diazepam which had no significant effect on the seizures when administered alone. Glycine potentiated the effects of phenobarbital on the cortical afterdischarge but not the severity of the seizures. The observed potentiation of the effects of diazepam and phenobarbital suggests a glycinergic mechanism in the anticonvulsant action of these drugs which may be mediated in part by the inhibitory γ-aminobutyric acid (GABA) systems.     

The effect of a glycine derivative (CP 1552-S) on kindled seizures in rats

The effects of the glycine derivative, CP 1552-S (2-N-pentylaminoacetamide hydrochloride) were evaluated for potential anticonvulsant activity in rats which were cortically- or amygdaloid-kindled. Large doses (300-600 mg/kg, i.p.) of CP 1552 given 30 min before stimulation resulted in significant reductions in duration of afterdischarge after both partially-developed and fully-developed cortically-kindled seizures. The largest dose tested (600 mg/kg, i.p.) markedly reduced the duration of the elicited afterdischarge and the severity of seizure. This dose was associated with prestimulation sedation and a 50% incidence of post-afterdischarge spontaneous, electrical seizure activity. Against kindled amygdaloid seizures, CP 1552-S significantly reduced the duration of afterdischarge at 300 mg/kg (i.p.) without modifying the seizure and without prestimulation behavioral or electrical effects. The largest dose tested (600 mg/kg, i.p.) resulted in a significant reduction of the elicited duration of afterdischarge but was associated with a 25% incidence of prestimulation spontaneous electrical seizure activity and a 45% incidence of post-afterdischarge electrical seizure activity. When CP 1552-S (30-300 mg/kg, i.p.) was administered daily, prior to the amygdaloid kindling stimulus, no difference was noted in the rate of acquisition of the kindled amygdaloid response. It is concluded that the glycine derivative CP 1552-S, has little anticonvulsant activity against the acquisition or development of kindled amygdaloid seizures. It appears to have significant anticonvulsant effects against both cortically- and amygdaloid-kindled afterdischarges with little effect on the behavioral severity of the seizure. Further, large doses of CP 1552-S appeared to result in paradoxical post-afterdischarge and possibly prestimulation electrical seizure activity.     

Attenuation of the biphasic effects of ethanol on avoidance extinction by RO 15-4513 in rats

Ethanol had biphasic effects on jump-up avoidance extinction with low doses (1 g/kg) increasing, and high doses (2.5 g/kg) decreasing number of trials to extinction criterion. In Experiment 1 these doses of ethanol were studied alone, and in combination with RO 15-4513 (0.3, 3 or 6 mg/kg). The stimulation of responding produced by low ethanol doses was reversed by 3 and 6 mg/kg doses of RO 15-4513 which had intrinsic suppressive effects, but the depressed responding produced by higher ethanol doses was not attenuated by RO 15-4513. Experiment 2 analysed the interaction between ethanol and benzodiazepine antagonists RO 15-1788 and CGS 8216. RO 15-1788 did not have intrinsic action and did not interact with ethanol. CGS 8216 showed an intrinsic suppressive action much like RO 15-4513, and also reversed the stimulation produced by low dose ethanol, but not the effects of the high dose. Experiment 3 showed that the benzodiazepine agonist, chlordiazepoxide, had effects much like low dose ethanol which were reversed by CGS 8216 and RO 15-4513. The major conclusions were that RO 15-4513 and CGS 8216 possess inverse agonist properties which may cancel out the effects of alcohol under certain circumstances.     

The effect of diazepam on ventricular automaticity induced by a local injury. Evidence of involvement of "peripheral type" benzodiazepine receptors

The effect of diazepam on ectopic cardiac automaticity has been examined in rats. We also investigated whether "central" or "peripheral type" benzodiazepine receptors are involved, as well as the role of calcium, on the possible effect of diazepam, by studying the interaction of this drug with either GABA, picrotoxin, RO 15-1788, PK 11195, diltiazem or Bay K 8644. A local injury of the rat isolated right ventricle produced a sustained abnormal rhythm which was completely abolished by diazepam (30-50 microM). This effect was not modified by the presence of either GABA (100 microM) picrotoxin (2 microM) or RO 15-1788 (5 microM) but it was reduced by the antagonist of "peripheral type" benzodiazepine receptors PK 11195 (0.1 microM). On the other hand the calcium channel blocker diltiazem (5 microM) and the calcium channel activator Bay K 8644 (3 nM), respectively, potentiated and reduced the effect of diazepam. It is concluded that diazepam effectively reduces ectopic cardiac automaticity in the rat. The "central type" benzodiazepine receptors are not involved in this effect, but it seems to be, at least, partially mediated by "peripheral type" receptors and is a calcium-dependent phenomenon.     

Spontaneous and RO 15-1788-induced reversal of subsensitivity to GABA following chronic benzodiazepines

Chronic daily injections of diazepam (5 mg/kg i.p.) for 21-30 days have previously been shown to cause a selective subsensitivity to microiontophoretically applied GABA in serotonergic dorsal raphe neurons in the rat. Following termination of chronic benzodiazepine treatment, GABAergic sensitivity remained depressed for up to 96 h even though pharmacologically active concentrations of diazepam and its active metabolites could no longer be detected in brain tissue. In contrast, a single injection of the specific benzodiazepine antagonist, RO 15-1788, given 22 h prior to electrophysiological recording, restored GABAergic sensitivity to the control range. Sensitivity of dorsal raphe neurons to serotonin was not altered by either chronic treatment with or withdrawal from diazepam. These results indicate that (1) RO 15-1788 can accelerate the time course of withdrawal and (2) administration of this benzodiazepine antagonist can induce a persistent change in GABAergic responsiveness.     

Chronic administration of diazepam downregulates adenosine receptors in the rat brain

Following chronic administration (10 or 20 days) of diazepam (5 mg/kg/day, subcutaneous pellets) or RO 15-1788 (5 mg/kg/day, intraperitoneally), adenosine and benzodiazepine receptors in different rat brain areas were assessed by radioligand binding studies using [3H]R-PIA for A1 receptors, [3H]NECA and [3H]R-PIA for A2 receptors and [3H]FNZ for benzodiazepine receptors. Chronic administration of diazepam for 10, but not for 20 days, decreased A2 receptors in the striatum by 46% (p less than 0.05) and A1 receptors in the hippocampus by 13% (p less than 0.05). Administration of diazepam for 10 days and 20 days failed to alter [3H]FNZ binding in all brain areas studied. However, 20 days of diazepam administration decreased the magnitude of GABA enhancement of [3H]FNZ binding in the cortex by 25% (p less than 0.05). In contrast, chronic administration of RO 15-1788 failed to alter [3H]R-PIA, [3H]NECA and [3H]FNZ binding in all brain areas. These results suggest that adenosine receptors may play a role in the CNS actions of benzodiazepines.     

Is Ro15-1788 a partial agonist at benzodiazepine receptors?

The ability of the imidazodiazepine derivative Ro15-1788, a potent and specific displacer of benzodiazepine binding, to induce and reduce benzodiazepine-like activity has been evaluated against the convulsant activity evoked by leptazol, bicuculline and the convulsant benzodiazepine Ro5-3663, in mice. Ro15-1788 (10-50 mg/kg) produced a dose dependent reduction in the activity of all 3 convulsants (significant at 50 mg/kg), when they were given at just maximal convulsant doses, but this effect was lost with supramaximal doses of the convulsants, as used in other reported experiments. In addition to this weak benzodiazepine-like activity, Ro15-1788 also reduced the anticonvulsant activity of diazepam and a triazolopyridazine derivative but not that of phenobarbitone. This effect was more marked at 10 than 25 or 50 mg/kg Ro15-1788. It is concluded that although Ro15-1788 has a specific effect on drugs acting at the benzodiazepine receptor it should be classified as a weak partial agonist rather than an antagonist at this site.     

Sound vibration,a non-invasive stress: antagonism by diazepam

Rats, subjected to sound-vibration stress, showed an abrupt increase in plasma corticosterone (CS). This stimulation was reliably produced using a Burgess brand vibro-graver, a standard tool used for engraving. With the tool set at 8 or coarse, the barrel of the tool was placed on the animal's flank and the point held against the side of the metal cage for 15 s. Plasma CS increased to 29.3±4.7 µg/dl at 15 min and 15.7±1.8 µg/dl at 30 min. These levels were significantly higher than animals pretreated with diazepam, 5 mg/kg IV, 2 h prior to stimulation (9.2±2.0 and 7.4±1.5 µg/dl, respectively). Animals which were pretreated with CGS-8216 (a mixed agonist/antagonist at the benzodiazepine receptor), 2 mg/kg IV, 30 min prior to diazepam had the protective effects of diazepam abolished. Sound/vibration produced a significant elevation in plasma CS in animals given CGS-8216 alone; but, this elevation was significantly lower than in vehicle-treated controls. This comparatively lower plasma CS level suggests a partial-agonist, diazepam-like effect by CGS-8216. Experiments were done in conscious unrestrained male Sprague-Dawley rats with chronic IV catheters. Except for 15 s stimulation exposure, all animals remained isolated in sound-attenuated one-way vision boxes for the duration of the serial blood sampling. Control stimulation exposure involved similar handling without turning on the engraving tool. These results demonstrate: 1) the usefulness of this tool to provide a repeatable stress stimulus; 2) the ability of diazepam to abolish the stress response; 3) that CGS-8216 can antagonize the action of diazepam; and 4) a demonstration of the partial agonist effects of CGS-8216.     

Using Ro 15-1788 to investigate the benzodiazepine receptor in vivo: Studies on the anticonvulsant and sedative effect of melatonin and the convulsant effect of the benzodiazepine Ro 05-3663

Both the anticonvulsant and sedative effects of diazepam (5 mg/kg) were reversed by subsequent administration of the suggested specific benzodiazepine antagonist Ro 15-1788. In contrast neither the seizure threshold raising or sedative effect of melatonin (200 mg/kg) was reversed by Ro 15-1788. Ro 15-1788 had no effect on the convulsant action of the benzodiazepine Ro 05-3663. These data therefore argue against the suggestion that melatonin produces its sedative and anticonvulsant effects in vivo by interacting with the benzodiazepine receptor, and also strengthens the suggestion that Ro 05-3663 does not act at this site. The use of Ro 15-1788 in demonstrating whether a drug acts in vivo at the benzodiazepine site to produce a pharmacological response is discussed.     

Effect of aminophylline on amygdaloid-kindled postictal inhibition

Previous studies have shown that methylxanthines such as aminophylline increase the clinical severity and length of electrically elicited limbic afterdischarges in naive and kindled rats without lowering seizure threshold. When fully amygdaloid-kindled rats are electrically stimulated at intertrial stimulation intervals of less than 60 minutes, significant residual inhibition can be demonstrated. The present study examines the effect of three doses of aminophylline (25, 50 and 100 mg/kg) on repeated daily stimulations of fully amygdaloid-kindled rats. After 100 mg/kg aminophylline, the first elicited amygdaloid-kindled seizure afterdischarge was doubled in length compared to saline controls. The second elicited seizure 15 minutes later resulted in status epilepticus and hindlimb extension in the majority of the aminophylline-treated animals with death occurring in 28%. When 25 or 50 mg/kg of aminophylline was given daily for five days before the first of five daily stimulation trials, each separated by 15 minutes, no significant reduction in postictal inhibition was demonstrated compared to saline controls. The 50 mg/kg aminophylline dose consistently and significantly lengthened only the first afterdischarge of each day without affecting the postictal inhibition seen with repeated stimulations. The neural substrate that governs immediate postictal inhibition of amygdaloid-kindled seizures appears to be resistant to modification by aminophylline at low doses. At high doses of aminophylline (100 mg/kg), sustained epileptical activity occurred. The sustained seizure activity seen at the high dose of aminophylline may be secondary to blockade of the processes which normally terminate seizure activity, or it may represent actual inhibition of the immediate postictal inhibitory processes.     

Anticonvulsant actions of the putative gamma-aminobutyric acid (GABA)-mimetic, ethylenediamine.

1 Ethylenediamine, 31.6-1000 mg/kg intraperitoneally, inhibited the convulsive effects of pentylenetetrazol, 100 mg/kg (i.p.) in mice. 2 Ethylenediamine, 100-1000 mg/kg (i.p.) increased the convulsion threshold to the intravenous infusion of three convulsants in the order pentylenetetrazol greater than bicuculline greater than strychnine. 3 The benzodiazepine antagonist R0 15-1788, 10 mg/kg (i.p.), significantly inhibited the anticonvulsant action of diazepam, 50 micrograms/kg, but not ethylenediamine, 1000 mg/kg. 4 These results clearly indicate that ethylenediamine has anticonvulsant properties and are consistent with the hypothesis that ethylenediamine is a gamma-aminobutyric acid (GABA)-mimetic.     

Distinctive behavioral effects of the pyrazoloquinoline CGS 8216 in squirrel monkeys

The behavioral effects of the pyrazoloquinoline CGS 8216 were studied in squirrel monkeys trained to respond under a fixed-interval (FI) schedule of food presentation. Dose-effect curves were determined by administering cumulative doses IV during timeout periods that preceded sequential components of the FI schedule. CGS 8216 (0.1-3.0 mg/kg) produced dose-related decreases in the rate of FI responding. In comparison, diazepam (0.1-3.0 mg/kg) had biphasic effects under identical conditions: intermediate doses increased the rate, whereas high doses decreased the rate of FI responding. Pretreatment with the benzodiazepine antagonist Ro 15-1788 (3.0 or 5.6 mg/kg) attenuated the decreases in response rate normally produced by high doses of CGS 8216. The behavioral effects of CGS 8216 were not altered systematically by pretreatment with either diazepam (0.3-3.0 mg/kg) or the alpha 2-adrenergic agonist clonidine (0.01-0.03 mg/kg). The results suggest that CGS 8216 has benzodiazepine inverse agonist effects on schedule-controlled behavior of squirrel monkeys. CGS 8216 can, however, be distinguished from inverse agonists of the beta-carboline type on the basis of its effects in the presence of diazepam or clonidine.     

The sedative effects of CL 218,872, like those of chlordiazepoxide,are reversed by benzodiazepine antagonists

The effects of CL 218,872, initially classified as a non-sedative anxiolytic, were investigated and compared with those of chlordiazepoxide in the holeboard. The ability of two drugs that antagonise the effects of benzodiazepines, CGS 8216 and Ro 15-1788, to reverse the effects of CL 218,872 and chlordiazepoxide were also investigated, to see whether their effects might be mediated via benzodiazepine receptors. CL 218,872 (10 mg/kg) was found to be significantly sedative in both mice and rats (i.e., both locomotor activity and head-dipping were significantly decreased). In mice, the effects of CL 218,872 and of chlordiazepoxide were very similar over a range of doses, except that the stimulatory effect seen with low doses of chlordiazepoxide on head-dipping just failed to reach significance with CL 218,872. This study is in agreement with recently published results from different tests showing that sedative effects can be obtained with doses of CL 218,872 that are low and not much higher than those leading to anxiolysis. The sedative effects of both CL 218,872 (10 mg/kg) and chlordiazepoxide (20 mg/kg) were significantly reversed by RO 15-1788 (10 and 20 mg/kg) and CGS 8216 (10 mg/kg), suggesting that their effects are mediated via benzodiazepine receptors. The increase in head-dipping seen with chlordiazepoxide (2.5 mg/kg) was also reversed by RO 15-1788 and CGS 8216.