Does RO 15-4513 reverse the anxiolytic effects of ethanol by its intrinsic properties?

In order to better understand the antagonistic effects of the partial inverse agonist of benzodiazepine receptors, RO 15-4513, against the disinhibitory action of ethanol, we examined the effects of RO 15-4513 at a dose (2.0 mg/kg) that did not alter locomotor activity, given alone or in combination with ethanol, on the behavior of mice confronted with the light/dark choice procedure and the staircase test. At this dose, RO 15-4513 given alone was found to have slight anxiogenic properties and when given in combination with ethanol, to completely reverse the disinhibitory effects of ethanol. Since we previously observed postictal depression after higher doses of RO 15-4513 given alone and antagonistic effects of these same doses on the action of ethanol, it can be suggested that the antagonistic effects of RO 15-4513 against ethanol are due to its anxiogenic or depressive properties depending on doses. However, this hypothesis can only be regarded as being in early stages of development at the present time since these results do not parallel with those of several other studies and the question whether the antagonistic action of RO 15-4513 against ethanol is additive or interactive remains open.     

Interaction of RO 15-4513 and ethanol on the behaviour of mice: antagonistic or additive effects?

Opposite effects were observed of ethanol on the behaviour of mice in the two chambered light/dark test. At a low dose, it had anxiogenic effects, while it produced anxiolytic effects at a higher dose. Selective antagonistic actions of RO 15-4513 against the behavioural effects of ethanol have been reported by others without intrinsic actions. In contrast, we found intrinsic depressive properties of RO 15-4513. This drug reduced locomotion in a running wheel test. We suggest that RO 15-4513 reversed certain effects of ethanol in an additive, rather than interactive, manner. In addition, RO 15-4513 did not block the sedation produced by a high dose of ethanol. Since RO 15-4513 revealed proconvulsant properties, it is proposed that the depressive effects of this drug could be related to its proconvulsive activity.     

The effects of FG 7142 and RO 15-1788 on the release of punished responding produced by chlordiazepoxide and ethanol in the rat

Previous results in our laboratory have shown that both chlordiazepoxide and ethanol will release punished responding in a rat operant conflict test using incremental shock. In the present study, a benzodiazepine antagonist and a benzodiazepine inverse agonist were used to explore the neurochemical basis for this behavioral action. N-methyl--carboline-3-carboxamide (FG 7142) at high doses (20 and 40 mg/kg) produced suppression of both punished and unpunished responding, and reversed the release of punished responding produced by both chlordiazepoxide and ethanol, but only at doses that produced an effect on its own. FG 7142 thus acted to oppose the actions of both ethanol and benzodiazepines but in an additive, not interactive, manner. In contrast, RO 15-1788 produced no changes when injected by itself in doses as high as 12 mg/kg and reversed chlordiazepoxide-induced but not ethanol-induced release of punished responding. RO 15-1788 also reversed the decrease in punished responding produced by FG 7142. Results suggest that ethanol does not interact directly with the benzodiazepine binding sites on the GABA/benzodiazepine ionophore complex to produce its anxiolytic action.     

Behavioural effects of the benzodiazepine receptor partial agonist RO 16-6028 in mice

The imidazo-diazepinone RO 16-6028 is a benzodiazepine receptor partial agonist which exhibits some anticonflict effects in the two-chambered light/dark test without significantly affecting the behaviour of mice confronted with the staircase test. In addition, this drug slightly reduced locomotion and more markedly rearing in a free exploration procedure. These results indicate that RO 16-6028 appears to produce some anxiolytic and sedative properties like full agonists, but with weaker magnitude. This could be related to the benzodiazepine partial agonistic profile of the compound.     

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.     

Behavioural effects of pentylenetetrazole reversed by chlordiazepoxide and enhanced by RO 15-1788

Summary The effects of low doses of pentylenetetrazole (PTZ) on exploratory behaviour and locomotor activity in the rat, were measured in the holeboard apparatus. PTZ (30 mg/kg) significantly reduced exploratory head-dipping, an effect that was significantly reversed by chlordiazepoxide (5 mg/kg) and enhanced by RO 15-1788 (20 mg/kg). RO 15-1788 (20 mg/kg) also significantly enhanced the reduction in locomotor activity produced by PTZ (30 mg/kg); although this reduction was not reversed by chlordiazepoxide (5 mg/kg) the reduction in locomotor activity with the drug combination was no greater than that with either drug alone. The results are discussed with respect to a coupling between the benzodiazepine receptors and the site at which PTZ acts.     

Corticotropin releasing factor and amphetamine exaggerate partial agonist properties of benzodiazepine antagonist Ro 15-1788 in the conflict test

The central nervous system stimulants corticotropin releasing factor (CRF) and amphetamine were administered in combination with the benzodiazepine ligands Ro 15-1788 and FG 7142 in order to assess their benzodiazepine agonist and antagonist receptor properties in an operant conflict test in rats. Ro 15-1788, which was without behavioral activity in this test when given alone, reversed the suppression of punished responding produced by CRF and amphetamine in a dose-dependent manner. Chlordiazepoxide, which produced a release of punished responding by itself, also reversed the suppression of punished responding produced by CRF but not that of amphetamine. The benzodiazepine inverse agonist FG 7142, in contrast, enhanced the rate suppressing actions of both CRF and amphetamine. In a locomotor activity test, Ro 15-1788 failed to block the locomotor activation observed with CRF and amphetamine. The results suggest that anxiety or stress-enhancing compounds may enhance the partial agonist properties of Ro 15-1788 in certain test situations.     

RO 15-1788 does not influence postpartum aggression in lactating female rats

Recently, Hansen et al. (1985) suggested behavioural similarities between lactating rats and non-maternal rats treated with benzodiazepines (BDZ), indicating that lactation may be associated with an increased activity state at the GABA/BDZ receptor complex similar to BDZ treatment. A logical prediction of this hypothesis is that BDZ antagonists should decrease typical maternal behaviours involved, such as aggression.We tested this hypothesis by measuring the behavioural effects of the BDZ antagonist RO 15-1788 (1.25–10 mg/kg IP) on aggressive behaviour of lactating female rats confronted with male intruders. We could not support the hypothesis; no consistent behavioural effects of RO 15-1788 on aggression were found. The implications of this finding for the proposed hypothesis are discussed.     

Recovery from lorazepam tolerance and the effects of a benzodiazepine antagonist (RO 15-1788) on the development of tolerance

After 3 days of dosing rats with lorazepam (0.25 mg/kg), tolerance developed to its sedative effects. Recovery from this tolerance was rapid. No differences could be detected in undrugged behaviour 24 h after the last dose and no differences in response to a probe injection could be found when 2 drug-free days intervened between the chronic treatment and test dose. RO 15-1788 (1–4 mg/kg) antagonised the sedative effects of acute lorazepam (0.5 and 0.25 mg/kg), but chronic treatment with these doses concomitantly with lorazepam did not prevent the development of tolerance. However, 4 mg/kg RO 15-1788 administered for 5 days at the same time as lorazepam (0.5 mg/kg) and again 45 min later attenuated the development of tolerance. Plasma concentrations after acute and chronic treatment did not differ for 0.25 mg/kg lorazepam, but they were lower following chronic treatment with 0.5 mg/kg. Therefore the development of behavioural tolerance in rats to the sedative effects of benzodiazepines probably involves changes in benzodiazepine receptors, in addition to a pharmacokinetic contribution after treatment with high doses.Wellcome Trust Senior Lecturer     

Effects of RO 15-1788 on a running response rewarded on continuous or partial reinforcement schedules

Two experiments were run in which rats were rewarded with food for running in a straight alley at one trial a day, followed by extinction of the running response. During acquisition of the response, reward was delivered either on a continuous reinforcement (CRF) or on a quasirandom 50% partial reinforcement (PRF) schedule. The groups given PRF were more resistant to extinction than those given CRF, the well-known partial reinforcement extinction effect. In Experiment 1 different groups of rats were injected during acquisition only with 1, 5 or 10 mg/kg of the benzodiazepine antagonist, RO 15-1788, or with placebo. In Experiment 2, 5 mg/kg RO 15-1788 or placebo were administered in a full cross-over design during acquisition, extinction or both. At the end of Experiment 2 only [³H]-flunitrazepam binding was measured in either the presence or absence of added -aminobutyrate (GABA) in homogenates of hippocampi dissected from the animals that had received behavioural training. The drug affected running speeds during both acquisition and extinction in different ways depending upon the schedule of reinforcement (CRF or PRF) and also gave rise to enhanced GABA stimulation of [³H]-flunitrazepam binding. The results are discussed in relation to the hypothesis that the neurochemical pathways by which reinforcement schedules modify behaviour include a step influenced by benzodiazepine receptors.     

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.     

Chronic RO 15-1788 treatment increases the number of benzodiazepine receptors in rat cerebral cortex and hippocampus

Administration of 4 mg/kg per day of diazepam in the drinking water for 14 days produced a small, but not significant decrease of benzodiazepine binding sites for [3H]flunitrazepam and [3H]ethyl-beta-carboline-3-carboxylate. Similar treatment with the benzodiazepine antagonist RO 15-1788 resulted in a marked increase in the density of sites for both ligands in the synaptosomal membranes of cerebral cortex and hippocampal formation.     

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.     

Effects of valproate on hyponeophagia in rats: Competitive antagonism with picrotoxin and non-competitive antagonism with RO 15-1788

The effects of valproate (30–500 mg/kg), alone and in combination with picrotoxin (1.5 mg/kg) or RO 15-1788 (10 mg/kg) were studied in two experiments on hyponeophagia in rats. Valproate reduced eating latency and incrased eating time and amount eaten of novel food, except at 500 mg/kg which reduced feeding. Picrotoxin induced generally opposite actions alone and shifted valproate dose/response curves to the right. RO 15-1788 had no detectable intrinsic action, but prevented both the behavioural facilitation and inhibition produced by valproate. These findings are discussed in the context of the GABA hypothesis of benzodiazepine action, with the conclusions that they provide behavioural support for the hypothesis of a receptor complex with GABA and benzodiazepine binding sites, and that an optimal and submaximal level of activity at the benzodiazepine site is a necessary condition for anxiolytic actions of valproate.     

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.     

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.     

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.     

The effects of the benzodiazepine antagonist Ro 15-1788 on psychophysiological performance and subjective measures in normal subjects

Ro 15-1788 is an imidazodiazepine which was initially described as a pure benzodiazepine antagonist lacking in intrinsic actions. Although recent animal work has shown the drug to have differing intrinsic actions depending on the dose, the majority of studies on human subjects conclude that it is a pure antagonist of benzodiazepines. Two oral doses of Ro 15-1788 (30 mg and 100 mg) were compared with 5 mg diazepam and placebo in their intrinsic effects on a range of psychophysiological, performance and subjective measures in 12 healthy adult subjects. At both these doses Ro 15-1788 showed a mixture of agonist (benzodiazepine-like) effects and other non-benzodiazepine-like effects on the variables measured. Although there was no clear-cut dose-response relationship, the results suggested a predominance of benzodiazepine-like effects at the higher dose on physiological measures whilst the lower dose was observed to have greater effects on a number of behavioural and subjective dimensions. The subjective changes were the opposite of those normally found for benzodiazepines.     

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.     

Effects of diazepam,FG 7142, and RO 15-1788 on schedule-induced polydipsia and the temporal control of behavior

Although benzodiazepine agonists and inverse agonists have opposite effects on drinking elicited by water deprivation, there is much less information about the effects of these drugs on nonhomeostatic drinking. In this experiment the effects of diazepam (0.3–5.0 mg/kg), a benzodiazepine receptor agonist, and FG 7142 (1.0–9.0 mg/kg), an inverse agonist, were determined on drinking elicited by a FT-60 schedule of food delivery (SIP). Both diazepam and FG 7142 dose-dependently reduced SIP, measured as either licking or volume consumed. In addition, diazepam reduced panel pressing for food, decreased locomotor activity, and changed the time course of each behavior. In contrast, FG 7142 reduced schedule-induced drinking without significantly altering other behaviors. The antagonist RO 15-1788, when given in combination with these drugs, only partially restored the reductions in licking produced by diazepam, but was much more effective in reversing the effects of FG 7142 at doses of the antagonist that failed by themselves to affect responding. The opposite pattern of effects was seen on the volume of water consumed. These effects are discussed in terms of the behavioral and pharmacological specificity of these drugs.