Inhibition of hypothermic effect of cholecystokinin by benzodiazepines and a benzodiazepine antagonist, Ro 15-1788, in mice

Intracisternally administered cholecystokinin produced long lasting hypothermia in mice, and the hypothermic effect was significantly antagonized by benzodiazepines like chlordiazepoxide and diazepam and by a benzodiazepine antagonist, Ro 15-1788, that were administered intraperitoneally. Proglumide, a cholecystokinin antagonist, failed to prevent the hypothermia induced by cholecystokinin at a dose of 200 mg/kg. Benzodiazepines and Ro 15-1788 revealed a considerably strong hypothermic effect, but they still prevented the hypothermic effect of cholecystokinin. The precise mechanism of the antagonism between cholecystokinin and benzodiazepines remains unclear at present.     

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.     

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     

Conformational changes in benzodiazepine receptors induced by the antagonist Ro 15-1788

The binding kinetics of [3H]Ro 15-1788, a selective benzodiazepine receptor antagonist, to synaptosomal membranes of rat cerebral cortices was studied. [3H]Ro 15-1788 binds with high affinity (dissociation constant, 0.53 nM) to a single class of binding sites (maximal binding capacity, 1.97 pmoles/mg of protein). Equilibrium binding was not affected by gamma-aminobutyric acid (GABA), NaCl, pentobarbital, or pretreatment of the membranes at 37 degrees. Association at 0 degrees was identical whether measured in the absence or presence of GABA or bicuculline methiodide or after preincubation of the membranes at 37 degrees. The association rate under pseudo-first order conditions was curvilinear and consisted of a fast component and a slow component. Dissociation at 0 degrees with 1 X 10(-5)M clonazepam was also curvilinear and could best be fitted by two linear exponential components. The dissociation rate was not altered by GABA, NaCl, pentobarbital, or pretreatment of membranes at 37 degrees. The dissociation rate was similar for 0.1, 1, and 10 nM [3H]Ro 15-1788. The ratio of slow to fast dissociation component for 10 nM [3H]Ro 15-1788 was larger than that for 0.1 and 1 nM [3H]Ro 15-1788. In contrast, the dissociation rate for 20 nM [3H]flunitrazepam ( [3H]FNP) was much greater than that for 2 nM [3H]FNP. Using ligand concentrations occupying the same fraction of receptors, the ratio of slow to fast dissociation components was invariably greater for [3H]Ro 15-1788 than that for [3H]FNP. The rate of dissociation for [3H]Ro 15-1788 was faster under pre-equilibrium conditions than under equilibrium conditions. These results, discussed in terms of the cyclic model of interaction between receptors and benzodiazepines, suggest that [3H]Ro 15-1788 is a powerful ligand in inducing conformational changes in the initial, more labile, binary complex. They also suggest that different conformational states deduced from studies of in vitro binding kinetics may not correspond to the distinct pharmacological actions of benzodiazepines. It is speculated that intrinsic activities of benzodiazepines probably are determined by the step beyond the complex formation and conformational changes suggested to occur by these studies of binding kinetics.     

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.     

Naloxone-induced analgesia: effects of the benzodiazepine antagonist Ro 15-1788

Repeated exposure to pain under the influence of the opiate antagonists naloxone and naltrexone leads to the recruitment of substantial analgesia as measured by paw-lick latency on the hot-plate test (4,11). One hypothesis to explain this naloxone-induced analgesia (NIA) is that nociceptive stimulation in the face of opiate blockade becomes stressful enough to activate an analgesic adaptation that otherwise would not occur. This hypothesis was examined in two experiments by the administration of a benzodiazepine antagonist with anxiogenic properties (Ro 15-1788, in a dose of 10 mg/kg) in conjunction with repeated administrations of naloxone (5 mg/kg). One experiment incorporated defecation as a relatively direct measure of stress. Ro 15-1788 reliably augmented NIA. Defecation was increased by naloxone alone and in combination with Ro 15-1788. Overall, the results were most consistent with the hypothesis that NIA is a form of stress-induced analgesia that is at least partly nonopiate in nature.     

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.     

A behavioral examination of convulsant benzodiazepine and GABA antagonist, Ro 5-3663, and benzodiazepine-receptor antagonist Ro 15-1788

The potential "anxiogenic" effects of convulsant benzodiazepine and GABA-antagonist, Ro 5-3663 and specific antagonist of benzodiazepine receptors, Ro 15-1788 were compared in the Geller-Seifter conflict paradigm. Chlordiazepoxide (CDP) (5 mg/kg) was used as a "positive" control. Both Ro 5-3663 (1 mg/kg) and Ro 15-1788 (10 mg/kg) antagonized the anticonflict effect of CDP. However, while Ro 15-1788 had a modest anticonflict potency. Ro 5-3663 had an anxiogenic effect in its own right.     

Amnestic effects of lormetazepam and their reversal by the benzodiazepine antagonist Ro 15-1788

A combined visual (pictures) and auditory (word lists) memory test developed to trace the course of information processing under pharmacological or other influences was validated in a group of 20 subjects (control group) and then applied to determine the amnesic effects of lormetazepam and the reversal of these effects by the benzodiazepine antagonist Ro 15-1788. Three groups of n=10 subjects received either 0.02 mg/kg IV lormetazepam (groups B and C) or placebo (group A) followed 14 min later by 0.03 mg/kg IV Ro 15-1788 (groups A and C) or placebo (group B). The time course of memory performance in the three groups was investigated and compared across three consecutive 14-min phases: before (phase 1) and after (phase 2) the first intravenous administration, and after the second treatment (phase 3). Results were also compared with those of 20 subjects from a drug-free control group in order to verify the memory test. Lormetazepam clearly impaired immediate and delayed free recall as well as recognition in both visual and auditory tasks. These effects were completely reversed by Ro 15-1788, which alone had no clear effect on memory performance in this study. Psychometric scales indicated concomitant sedation and impaired concentration after lormetazepam alone. Interestingly, lormetazepam retrogradely enhanced performance in the visual test of delayed free recall. The impaired acquisition of new information after the administration of lormetazepam may be associated with an improvement of the consolidation process of information acquired before drug treatment.This article is part of the doctoral thesis of Dagmar Berenberg     

Electrophysiological aspects of benzodiazepine antagonists, Ro 15-1788 and Ro 15-3505.

The comparative action of two specific benzodiazepine antagonists, Ro 15-1788 and Ro 15-3505 was examined in six healthy volunteers. Medication was given i.v. in a double-blind cross over pattern, and EEG was recorded throughout each experimental session. Ten minutes after the injection of one of the antagonists or placebo, midazolam was injected in incremental doses until first signs of drowsiness appeared in the EEG. The EEG was computer analyzed, using adaptive segmentation and time-dependent clustering. Continuous power profiles for various frequency bands, as well as power ratios for the physiological frequency bands (e.g. sigma/alpha power ratio) were generated. It has been found that sigma/alpha power ratio was the most sensitive parameter detecting early effects of midazolam on the EEG signal, thus enabling a semi-quantitative titration of the antagonists by midazolam. Ro 15-1788 in doses of 5 mg i.v. was counteracted on average by 7.3 mg midazolam. From the EEG analysis it has been found that Ro 15-3505 was at least 4-5 times more potent than Ro 15-1788.     

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.     

Does the benzodiazepine antagonist Ro 15-1788 antagonize the action of ethanol?

Ethanol aggravates benzodiazepine-induced central nervous depression by pharmacokinetic and/or pharmacodynamic interactions and Ro 15-1788 reverses promptly the hypnotic effects of benzodiazepines. We therefore studied the acute effects of Ro 15-1788 on the ethanol-induced sedation in six healthy male subjects. Subsequently to an oral loading dose (0.54 g ethanol kg-1) ethanol was infused for 4 h (0.15 g ethanol kg-1 h-1) and steady state blood levels between 0.9 to 1.2 g l-1 were reached within 2 h. At steady state and during the elimination phase of ethanol an intravenous bolus of 0.5 mg Ro 15-1788 or placebo was administered in a randomized, double-blind crossover fashion. The marked sedative effects of ethanol as assessed by visual analogue scales (2 to 6 fold increase in the sedation index), and choice reaction time (25 to 40% prolongation) were not affected by Ro 15-1788. However, the pharmaco-EEG indicated that Ro 15-1788 seems to reverse transiently the ethanol-induced changes in total alpha, delta, and slow alpha bands. There was no pharmacokinetic interaction between both agents since elimination of Ro 15-1788 (t1/2 = 1.2 +/- 0.7 h) and of ethanol (0.17 +/- 0.02 g l-1 h-1) were in good agreement with control values. Thus, it could be concluded that Ro 15-1788 might affect for a short while the action of ethanol by interfering with the benzodiazepine receptors.     

Effect of the benzodiazepine antagonist Ro 15-1788 on flunitrazepam-induced sleep changes.

1 The modifications of human sleep induced by benzodiazepines, and particularly by flunitrazepam, are complex. Stage 4 and paradoxical sleep are both decreased; however, these two effects have a different evolution during and after single or short-term drug administration. 2 The benzodiazepine antagonist Ro 15-1788 also tends to depress stage 4, but with immediate recovery in the post-drug night, and does not modify paradoxical sleep. 3 In combined administration, this drug totally reverses the hypnogenic effect of flunitrazepam, as well as its effect on paradoxical sleep but not the decrease of slow wave sleep. 4 Some of the benzodiazepine-induced alterations of sleep may be related to receptors different from central benzodiazepine receptors, or to mechanisms not directly connected to this type of receptors.     

The benzodiazepine antagonist, Ro 15-1788 does not decrease ethanol withdrawal convulsions in rats

The effects of the benzodiazepine antagonist, Ro 15-1788 on ethanol withdrawal convulsions were investigated in rats. The study originated from recent reports of benzodiazepine binding activity in urine of alcoholics during withdrawal. No alleviation of convulsions was found with Ro 15-1788. This suggested that this component of the withdrawal syndrome is not due to endogenous production of a benzodiazepine inverse agonist (contragonist), as Ro 15-1788 prevents the action of this type of compound.     

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.     

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.     

Evidence for intrinsic behavioural activity of the benzodiazepine antagonist, Ro15-1788, in male mice

It would be predicted that putative benzodiazepine should be released under anxiety-provoking conditions and that behavioural changes should be observed following pretreatment with selective antagonists of the benzodiazepine receptor. To test this hypothesis, adult male albino mice were briefly exposed to a novel, brightly-illuminated arena during the dark phase of their LD cycle. Under these test conditions, Ro15-1788 (10 mg/kg) enhanced total rearing whilst, a 5-10 mg/kg, it significantly altered the normal pattern of rearing over the test session. However, at the highest dose tested (20 mg/kg), such behavioural changes were no longer apparent. A similar, though non-significant, trend was observed for locomotor activity. These data, the first to demonstrate-low-dose intrinsic activity of Ro15-1788 in mice, suggest that benzodiazepine antagonists may prove to be powerful tools in the study of the behavioural significance of the benzodiazepine receptor.     

Antagonism of dermorphin-induced catalepsy with naloxone, TRH-analog CG3703 and the benzodiazepine antagonist, Ro 15-1788

Intracerebroventricular (i.c.v.) administration of the highly selective opiate mu-receptor agonist, dermorphin, produced dose-dependent catalepsy in conscious rats. The cataleptic effect of dermorphin was abolished by pretreatment (intraperitoneal, i.p.) with the opiate-antagonist naloxone (5 mg/kg), thyrotropin-releasing hormone analog CG3703 (1 mg/kg) or the benzodiazepine-antagonist Ro 15-1788 (5 mg/kg) whereas pretreatment with the benzodiazepine alprazolam (1 mg/kg) potentiated the cataleptic effect of dermorphin. When given to cataleptic rats, naloxone and CG3703, but not Ro 15-1788, reversed the dermorphin-induced catalepsy. The data suggest an involvement of benzodiazepine receptors in the induction of catalepsy mediated by opioid mu-receptors. Other opioid-modulating neuronal systems, antagonized by CG3703, may be involved in maintaining the dermorphin-induced cataleptic state.