Reduction of flurazepam convulsive threshold by Ro 15-1788

The influence of the benzodiazepine (BZ) receptor antagonist Ro 15-1788 on the convulsant properties of flurazepam (FLZ) was studied in rats. Animals were prepared with chronic epidural electrodes for EEG recording and respiratory rates were recorded via a rubber bulb connected to a pressure transducer. FLZ convulsive thresholds were determined by continuous IV infusion in the presence and absence of Ro 15-1788 pretreatment. All animals receiving the FLZ infusion experienced convulsions preceded by dose-dependent reduction of respiratory rate. Pretreatment with Ro 15-1788 substantially reduced the FLZ convulsive threshold, suggesting a blockade of the depressant (anticonvulsant) aspect of the FLZ effect, thus, augmenting a convulsant effect at a separate receptor. An alternate hypothesis is that Ro 15-1788 may increase the affinity or intrinsic activity of the receptor responsible for the convulsant aspect of FLZ. Ro 15-1788 did not appear to alter the respiratory rate depressant effect of FLZ, although early onset of convulsions in the pretreated animals precluded measurement of respiration at the higher dose of FLZ allowable in animals that were not pretreated with the antagonist.     

Dose-dependent reversal of chlordiazepoxide-induced discrimination impairment by Ro 15-1788

Chlordiazepoxide (10 mg/kg), administered on eight successive acquisition sessions, impaired a light-cued, successive discrimination in male Sprague-Dawley rats by increasing the number of incorrect responses. The benzodiazepine receptor antagonist Ro 15-1788 (5 and 10 mg/kg) reversed the discrimination impairment and reduced the number of incorrect responses in a generally dose-dependent manner when co-administered with chlordiazepoxide. These findings suggest that the impairment of successive discrimination by chlordiazepoxide is mediated by central benzodiazepine receptor sites. When administered alone, however, the 10 mg/kg dose of Ro 15-1788 (but not the 5 mg/kg dose) produced a mild benzodiazepine-like impairment in discrimination, which was accompanied by a small but significant increase in incorrect responses. These findings suggest that Ro 15-1788 may also have some intrinsic action of its own, which needs to be assessed independently of its use as a mediational research tool.Portions of these data were presented at the Annual Meeting of the Psychonomic Society, Seattle, Washington, 1987     

Changes in noradrenaline plasma levels and behavioural responses induced by benzodiazepine agonists with the benzodiazepine antagonist Ro 15-1788

Noradrenaline (NA) plasma levels were examined in 18 healthy volunteers on 2 consecutive days after a single treatment with either lormetazepam (0.06 mg/kg) (LMZ group), flunitrazepam (0.03 mg/kg) (FNZ group) or placebo (PLA group) in combination with the benzodiazepine (BZ) antagonist Ro 15-1788 (0.1 mg/kg). Behavioural responses (mood changes, anxiety) were also investigated in parallel. Both BZ decreased NA plasma levels to 50% of the basal values 10 min after the injection; administration of Ro 15-1788 15 min later reinstated NA plasma levels to basal values. A second administration of Ro 15-1788 (0.1 mg/kg) 24 h after BZ or PLA treatment increased NA plasma levels, estimated 10 min after the injection in both the LMZ- and the FNZ groups, but not in the PLA group. Behavioural responses measured under the same treatment also indicated minor anxiety responses followed by mood impairment. These data suggest that a stressful situation may be precipitated by the antagonist Ro 15-1788 24 h after a single BZ treatment, which resembles a withdrawal response, and increases NA plasma levels.     

Benzodiazepine antagonist Ro 15-1788: Neurological and behavioral effects

In neurological and behavioral studies in mice, rats, dogs and squirrel monkeys, the imidazobenzodiazepinone Ro 15-1788 acted as a potent benzodiazepine antagonist. The antagonistic activity was both preventive and curative and seen at doses at which no intrinsic effects were detected. It was highly selective in that it acted against CNS effects induced by benzodiazepines but not against those produced by other depressants, such as phenobarbitone, meprobamate, ethanol, and valproate. The onset of action was rapid even after oral administration. Depending on the animal species studied, the antagonistic effects lasted from a few hours to 1 day. The acute and subacute toxicity of Ro 15-1788 was found to be very low. Benzodiazepine-like effects were not seen.     

Clonazepam-induced hyperphagia in nondeprived rats: tests of pharmacological specificity with Ro5-4864, Ro5-3663, Ro15-1788 and CGS 9896

Nondeprived male rats were familiarised with daily 60 min access to a highly palatable diet, consisting of powdered rat diet, sweetened condensed milk and water. Clonazepam (0.625-5.0 mg/kg, IP) produced a substantial increase in food consumption within the first 30 min of access. The increase was similar across all dose conditions, suggesting that a maximal effect may have been achieved with a dose as small as 0.625 mg/kg. The hyperphagia induced by clonazepam was reversed by the benzodiazepine receptor antagonist, Ro15-1788 (5.0-20.0 mg/kg), indicating that the effect was benzodiazepine receptor-mediated. Treatments with the peripheral-type benzodiazepine agonist, Ro5-4864, did not induce a hyperphagic response. Instead, food consumption was significantly depressed following the administration of Ro5-4864 at 20 and 40 mg/kg, IP. A comparison of the clonazepam and Ro5-4864 data suggests that benzodiazepine-induced hyperphagia is mediated by central-type benzodiazepine binding sites. The pyrazoloquinoline, CGS 9896, binds with high affinity to benzodiazepine sites and has recently been described as a nonsedating anxiolytic. CGS 9896 (2.5-20.0 mg/kg, administered either IP or PO) did not affect consumption of the highly palatable diet. In consequence, anxiolytic and hyperphagic effects of drug actions at benzodiazepine receptors may be dissociated in the case of this compound. The atypical 1,4-benzodiazepine, Ro5-3663, a GABA antagonist which may act at the picrotoxinin site, produced a dose-related reduction in food consumption. Comparison with the results for Ro5-4864 rules out an interpretation for the anorexia in terms of anxiogenic effects.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antagonism by CGS 8216 and Ro 15-1788, benzodiazepines antagonists, of the action of chlordiazepoxide on a timing behavior in rats

Rats were trained to respond under a differential reinforcement of low rate (DRL) schedule of reinforcement. Pretreatment with relatively low doses of chlordiazepoxide (1-10 mg/kg) produced increases in total DRL responses and decreases in the numbers of reinforced responses. Chlordiazepoxide produced a shift in the interresponse time (IRT) distribution of DRL responses. Low doses of chlordiazepoxide shifted the IRT distribution of DRL responses. Low doses of chlordiazepoxide shifted the IRT distribution from the reinforced to the non-reinforced bins. In addition there was marked increase in the number of responses that occurred in the earliest IRT bin (0-3.75 sec). The highest dose of chlordiazepoxide (32 mg/kg) produced a decrease in total DRL responses and resulted in an even IRT distribution of responses. Both CGS 8216 and Ro 15-1788 had minimal effect on DRL responding when given alone. Ro 15-1788 had no effect at either 10 or 32 mg/kg, while CGS 8216 produced decreases in DRL responding at 32 and 100 mg/kg. Both Ro 15-1788 and CGS 8216 antagonized the effects of high and low chlordiazepoxide doses on total DRL responding and on the IRT distribution of responding.     

Midazolam cue in rats: Effects of Ro 15-1788 and picrotoxin

The discriminative stimulus effect of midazolam, a short-acting benzodiazepine, was used for testing the effects of drugs thought to act as antagonists at different sites in the proposed benzodiazepine receptor complex. Rats were trained in a standard two-bar operant conditioning procedure with food reinforcers delivered on a tandem schedule. The 0.4 mg/kg dose of midazolam used for training was well discriminated, typically yielding at least 95% correct responding. The benzodiazepine receptor antagonist Ro 15-1788 blocked the discriminative effect of midazolam but did not influence generalization to pentobarbitone (7.5 mg/kg). The indirect GABA antagonist picrotoxin attenuated both generalization to pentobarbitone and its response rate-reducing effect. Picrotoxin had no effect on the discriminative effect of midazolam at 0.4 mg/kg but it blocked the effect of 01 mg/kg. Even in doses which reduced overall response rates, nicotine did not block discrimination of midazolam (0.4 mg/kg). The results are consistent with models which postulate a GABA-linked ion channel which is a site of action for barbiturates and which is downstream of the benzodiazepine receptor itself.     

Ro 15-1788 and beta-CCE selectively eliminate diazepam-induced feeding in the rabbit

Food intake was monitored in three female and one male adult rabbits following the administration of three drugs known to result in feeding increases in other species. The drugs, diazepam (1.0 mg/kg), cyproheptadine (0.03 mg/kg) and chlorpromazine (1.0 mg/kg) all produced large increases in food intake; of these, only the effect of diazepam, a benzodiazepine, was reversed by doses of the benzodiazepine antagonists Ro 15-1788 (0.3 mg/kg) and Ethyl beta-carboxylate (beta-CCE) (1.0 mg/kg) which, when given alone, did not affect feeding. The results support evidence suggesting that Ro 15-1788 and beta-CCE are specific antagonists of the benzodiazepine receptor and of their effects on a wide range of behaviors.     

Pharmacokinetics of the selective benzodiazepine antagonist Ro 15-1788 in man

Summary The pharmacokinetics of the selective benzodiazepine antagonist Ro 15-1788 has been studied in 6 healthy male volunteers following a single intravenous dose of 2.5 mg. The drug was only slightly bound to plasma proteins (40±8%, mean±SD). A negligible amount (<0.2% of the dose) of unchanged drug was recovered in urine. Hepatic elimination was rapid, as shown by a short t_1/2 of 0.9±0.2 h, and high total plasma and blood clearances of 691±216 ml/min and 716±199 ml/min, respectively. The fast decline of plasma levels from about 60 to 2 ng/ml accounts for the short-lasting reversal of benzodiazepine-induced sedation by Ro 15-1788.     

Ethanol self-administration in freely feeding and drinking rats: effects of Ro15-4513 alone,and in combination with Ro15-1788 (flumazenil)

Recent work in our laboratory demonstrated that Ro15-4513, a partial inverse benzodiazepine (BDZ) agonist, decreases ethanol (ETOH) self-administration in rodents under fluid deprivation conditions. The present study further examined the effects of Ro15-4513 (2.5 and 5.0 mg/kg) alone and in combination with Ro15-1788, (flumazenil) (8.0 and 16.0 mg/kg), a BDZ receptor antagonist on ETOH self-administration in freely feeding and drinking rats. Animals were trained to consume ETOH (11% v/v) using a limited access procedure. Measurements were taken at 10- and 60-min intervals. Ro15-4513 (2.5 and 5.0 mg/kg) markedly attenuated ETOH consumption at both intervals. The antagonistic actions of Ro15-4513 were completely blocked by the higher dose of flumazenil at both intervals; the lower dose failed to antagonize the Ro15-4513-induced reduction of ETOH intake. When flumazenil was given alone, both doses reduced ETOH self-administration at 60 min; although the magnitude of the antagonism was comparable to that of Ro15-4513 only with the highest does of flumazenil (16.0 mg/kg). Neither Ro15-4513 nor flumazenil alone or in combination significantly altered water intake at any of the tested doses. Rats pretreated with Ro15-4513 showed a substantial reduction in blood ethanol concentration (BEC) compared with the Tween-80 vehicle condition at the 10-min interval. However, the BEC of animals given Ro15-4513 in combination with flumazenil were similar to rats given Tween-80 vehicle. The present study extends our previous research by demonstrating that Ro15-4513 and flumazenil attenuate ETOH self-administration in non-food or water deprived rats. These studies suggest that the suppressant effects of Ro15-4513 and flumazenil on ETOH self-administration are associated with actions at the BDZ site of the GABA_A receptor complex. These data are discussed in relation to the possible mechanism(s) by which Ro-15-4513 and flumazenil exert their antagonism on ETOH self-administration.Portions of this work were presented at the Annual Meeting of the Research Society on Alcoholism under the symposium entitled GABA/BDZ Receptor Update, Marco Island, FL, June 10, 1991.We would like to dedicate this paper to the late Dr. Richard G. Lister, a friend, teacher, colleague and exceptional scientist who contributed substantially to the area of alcohol abuse and alcoholism. Over the past years, Dr. Lister investigated the interactions of alcohol with inverse benzodiazepine agonists and benzodiazepine receptor antagonists. His seminal and more recent papers played an important role in delineating the GABA benzodiazepine systems in the neurobehavioral actions of alcohol. Richard, we will miss you.     

Evaluation of visual function in healthy subjects after administration of Ro 15-1788

Summary Ro 15-1788 is a specific benzodiazepine antagonist, which has also been shown to have some agonist properties. Since benzodiazepine receptors are involved in the physiological mechanisms of vision, a possible intrinsic effect of Ro 15-1788 was sought in 6 healthy volunteers by study of psychophysical flicker thresholds, including critical fusion frequency and low frequency modulation threshold, and pattern reversal visual evoked response, using double blind cross-over methodology. In each session 2 tablets of Ro 15-1788 30 mg were administered. Using a two factorial univariate analysis of variancé, no change was detected in any of the parameters studied.     

The peripheral benzodiazepine receptor ligand Ro 5-4864 induces supraspinal convulsions in rabbits. Reversal by the central benzodiazepine antagonist Ro 15-1788

The peripheral BDZ receptor ligand Ro 5-4864 was administered to rabbits in doses ranging from 0.2 to 7 mg/kg IV. Changes in electrocortical activity appeared within 1 min after administration, characterized by trains of slow waves in the posterior sensorimotor and optic cortices (0.6–2 mg/kg) and by grand mal seizures (2–10 mg/kg). The low doses also induced alterations in the basic rhythms both of the hippocampus (reduced amplitude and spike-like waves) and of the nucleus ventralis of thalamus (trains of slow waves), not associated with observable behavioural changes. The paroxysmal EEG activity observed at higher doses of the drug was first recorded in the cortical areas and then spread to the subcortical structures. No change in electrical activity could be observed in the spinal cord. The paroxysmal activity was associated with tonic-clonic convulsions and scialorrea.The EEG and behavioural manifestations were inhibited by administration of Ro 15-1788. This drug at doses of 0.6 and 6 mg/kg antagonized the effects of Ro 5-4864 at doses of 0.6–5 mg/kg and 6–7 mg/kg, respectively. This effect began 1–3 min after administration of the antagonist, and led to EEG synchronization.These data suggest that in rabbits the convulsant effect of Ro 5-4864 is due to interference of the drug at the GABA-BDZ-picrotoxin receptor oligomeric complex. Such an effect seems to be mediated at least in part by central BDZ receptors. Offprint requests to: M. Massotti     

The action of benzodiazepine antagonist Ro 15-1788 on the effects of GABA-ergic drugs

Summary The interaction of Ro 15-1788 (5 mg kg^–1 i.p.), a benzodiazepine antagonist, with GABA-ergic drugs muscimol (1.4 mg kg^–1 i.p.), fenibut (100 mg kg^–1 i.p.) and baclofen (5 mg kg^–1 i.p.) was examined in behavioural and biochemical studies in rats. All the above-mentioned GABA-ergic drugs produced motor depression and with the exception of muscimol, where anti-aggressive effect was evident, fenibut and baclofen showed only slight antiagressive properties. Ro 15-1788 attenuated the motor depression produced by these compounds but potentiated their antiaggressive effect. Moreover, it was found that Ro 15-1788 itself possessed dose-related antiagressive properties. Fenibut increased, whereas muscimol and Ro 15-1788 decreased, the GABA content in the rat striatum. Ro 15-1788 and all the studied GABA-ergic compounds increased the level of the dopaminc metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) in the striatum. In spite of this no further increase of DOPAC was observed after concomitant use of Ro 15-1788 with GABA-ergic drugs. The action of investigated GABA-ergic drugs via GABA receptors linked to benzodiazepine receptors is suggested.     

Stimulus control induced by benzodiazepine antagonist Ro 15-1788 in the rat

The imidazodiazepine Ro 15-1788 is a proposed benzodiazepine receptor antagonist. Recently however, behavioural effects of Ro 15-1788 have been demonstrated. In the present study, rats (n=12) were trained to discriminate Ro 15-1788 (10 mg/kg, IP, t=15 min) from vehicle in a two-lever food-reinforced procedure. All rats showed a reliable discrimination (mean injection-appropriate lever responding >85%) after about 60 daily training sessions. Drug stimulus control was evidenced by an orderly generalization gradient obtained with 0.01–30 mg/kg Ro 15-1788 (ED₅₀ for stimulus generalization: 0.12 mg/kg). Since even low doses of Ro 15-1788 have discriminative effects in the rat, it is concluded that Ro 15-1788 may have potent behavioural activity.     

Scopolamine and benzodiazepine models of dementia: cross-reversals by Ro 15-1788 and physostigmine

The muscarinic antagonist scopolamine and the benzodiazepine lorazepam both produce transient impairments in memory and attention in normal volunteers. These impairments can be reversed by appropriate agents such as the cholinesterase inhibitor physostigmine in the case of scopolamine or the benzodiazepine antagonist Ro 15-1788 in the case of lorazepam. In this paper we investigated the pharmacological specificity of these reversals by examining the interactions of scopolamine and Ro 15-1788 and of lorazepam and physostigmine. There was no evidence that the effects of scopolamine and lorazepam on cognitive function could be attenuated, by Ro 15-1788 and physostigmine, respectively. The results are discussed in terms of pharmacological models of Alzheimer's disease.     

Intrinsic actions of the benzodiazepine receptor antagonist Ro 15-1788

The imidazodiazepine Ro 15-1788 is a benzodiazepine receptor antagonist that was initially reported to be lacking in intrinsic activity in a variety of test situations in which benzodiazepine-like effects can be identified. However, many recent studies have shown that this compound does indeed have intrinsic activity in a variety of behavioural, neurological, electrophysiological and biochemical preparations in both animals and man. The purpose of the present review is firstly to describe these intrinsic actions, and secondly to consider to what extent these intrinsic actions of Ro 15-1788 have implications for current concepts of the functioning of the benzodiazepine receptor.     

Absence of central effects in man of the benzodiazepine antagonist Ro 15-1788

The benzodiazepines are typified by a profile of side effects which includes drowsiness, ataxia and incoordination. Ro 15-1788, an imidazodiazepine derivative, exhibits marked antagonism of the behavioural and biochemical effects of the benzodiazepines in animals and man. It is devoid of any behavioural activity in animals, except at very high doses. In the present study the effects of single rising oral doses of Ro 15-1788 on cognitive, psychomotor and subjective function in man have been assessed using a battery of psychometric tests designed to identify the sedative action of the benzodiazepines. At all doses up to 600 mg, Ro 15-1788 demonstrated none of the classical behavioural effects of the benzodiazepines.     

Enhancement of saline consumption by chlordiazepoxide in thirsty rats: Antagonism by Ro15-1788

Water-deprived rats were given access to either water or a highly preferred 0.9% NaCl solution in a 30 min drinking test. The animals consumed substantially more saline than water. Chlordiazepoxide (2.5–20.0 mg/kg) was administered IP before the drinking test. Analysis of the results revealed a significant drug treatment × fluid condition interaction. Chlordiazepoxide produced a preferential enhancement of saline intake, achieving a peak effect at 5.0 mg/kg. Consideration of the time-course of drinking showed some complexity in the way in which chlordiazepoxide affected the saline drinking. During the first 6 min of the drinking period, the drug treatments tended to depress consumption, reaching a significant effect at 20.0 mg/kg. However, in the following 16 min interval of the drinking test, chlordiazepoxide significantly elevated saline consumption. The mechanism for this second effect may have been a retardation in the development of satiety. Finally, at the end of the drinking test, when saline consumption had become satiated, chlordiazepoxide exerted no discernible effect. The enhancement of saline consumption by chlordiazepoxide appears to have been benzodiazepine-receptor mediated, since the effect was reversed by treatment with Ro15-1788, a benzodiazepine receptor antagonist. The implications of a benzodiazepine-induced increase in salt intake are briefly considered. The overconsumption of salt is contraindicated in certain clinical conditions. Both stress and hypertension are associated with elevated salt appetite. Treatment of these conditions using benzodiazepines may require due consideration of the possible stimulant effect of these drugs on salt appetite.     

Differential interactions of Ro 15-4513 with benzodiazepines, ethanol and pentobarbital

The effects of the imidazobenzodiazepine Ro 15-4513 in combination with three CNS depressants (ethanol, benzodiazepine agonists and pentobarbital) were examined in three different experiments. Full antagonism of classical benzodiazepines by Ro 15-4513 was seen in all three situations. Partial antagonism of ethanol occured in the pull up test of muscle relaxation in rats, but not in the inhibition of ultrasounds produced in rat pups by mild stress. The depressant effect of ethanol on twitching of the urethane-anaesthetised rat suprahyoid muscles was reversed. No attenuation of the effects of pentobarbital was seen in any test.     

Biphasic effects of Ro 15-1788 on spontaneous petit mal-like seizures in rats

The effects of various doses of the potent and specific benzodiazepine antagonist Ro 15-1788 were investigated in rats with spontaneous non convulsive, petit mal-like seizures. In preliminary experiments, Ro 15-1788, 2 mg/kg i.p., completely but transiently antagonized the antiepileptic action of diazepam, 2 mg/kg i.p. Ro 15-1788, 2 mg/kg, given alone, exhibited no intrinsic activity. At 10-80 mg/kg, it acted as an antiepileptic; this dose-dependent suppressant effect developed slowly over 20-40 min after injection and was never total even at 80 mg/kg. At the highest dose, Ro 15-1788 also had a transient epileptogenic effect immediately following the injection. These results confirm that Ro 15-1788 is not a pure benzodiazepine antagonist but also has partial 'agonist' and 'inverse agonist' properties.