Flunitrazepam and lormetazepam do not affect the pharmacokinetics of the benzodiazepine antagonist Ro 15-1788.

Following a single intravenous dose of 0.1 mg/kg, the pharmacokinetics of Ro 15-1788, a specific benzodiazepine antagonist, have been investigated in 20 healthy male volunteers. In random order injection of Ro 15-1788 has been preceded (5 min) by intravenous dosing with 0.06 mg/kg of lormetazepam (n = 6), 0.03 mg/kg of flunitrazepam (n = 8) or placebo (n = 6). The rapid elimination of the antagonist could be characterized by an elimination half-life between 0.9 to 1.4 h and a total plasma clearance of 727 to 1440 ml/min. These single dose studies indicate that the disposition of Ro 15-1788 was not affected by the acute coadministration of both 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.     

An antagonist-induced benzodiazepine abstinence syndrome

Cats were treated for 35 days with flurazepam, 5 mg/kg per day. The drug was administered through a gastric fistula. Ro15-1788, a benzodiazepine antagonist, caused an abstinence syndrome when administered through the gastric fistula 24 h after the last dose of chronic treatment. Abstinence signs included increased muscle tone, tremor, piloerection, pupillary dilation, panting, and excessive salivation. Neither convulsions nor delirium was seen. Severity of abstinence was similar after Ro15-1788 doses of 2-100 mg/kg, but lasted longer (8-10 h) after the higher dose. Readministering Ro15-1788 a week after the end of chronic treatment precipitated an attenuated abstinence syndrome, but was inactive after 2 weeks.     

Pharmacodynamic interaction between midazolam and a specific benzodiazepine antagonist in humans

The pharmacodynamic interaction between midazolam and the specific benzodiazepine antagonist Ro 15-1788 has been investigated in six healthy male volunteers. Hypnotic steady-state concentrations of midazolam (55 +/- 11 ng/mL; mean +/- SD) have been achieved rapidly by an intravenous bolus of 0.07 mg/kg and maintained by an individual but constant infusion rate of 0.025 to 0.04 mg/kg/hr for eight hours. Following a two-hour control period, the antagonist (2.5 mg) or the solvent were injected double-blind in random order. Three hours later, the other medication was administered. Whereas plasma levels of midazolam remained constant throughout the complete eight-hour trial (Clearance = 670 +/- 96 mL/min) concentrations of Ro 15-1788 declined rapidly with an elimination half-life between 0.7 and 1.8 hours and a total plasma clearance of 702 +/- 235 mL/min. Concentrations of Ro 15-1788 approached the analytic limit of 2 ng/mL within three hours. The pharmacodynamic response to midazolam and the antagonist was assessed by a sedation index using visual analogue scales, reaction time (RT) measurements, and transformed Fourier analysis of the power spectrum of the recorded electroencephalogram (EEG). About 30 to 45 seconds following the injection of Ro 15-1788, hypnotic action of midazolam was completely reversed as visualized by return to alpha rhythm in the EEG, shortening of prolonged RT, and normalization of the elevated sedation index. The antagonistic action lasted for about two to three hours. The abrupt arousal from sleep was not associated with any unpleasant sensations, however, three subjects experienced a profound perspiration for about ten minutes following the injection of Ro 15-1788.(ABSTRACT TRUNCATED AT 250 WORDS)     

Periodic benzodiazepine antagonist administration prevents benzodiazepine withdrawal symptoms in primates

Daily administration of diazepam (1.5 or 6 mg/kg) in Rhesus monkeys results in the progressive development of physical dependence, as evidenced by Ro15-1788 (5 mg/kg i.m.) precipitated withdrawal symptoms including retching, vomiting, face and limb tremors. Every third day administration of the benzodiazepine antagonist, Ro15-1788, during a similar period of continuous diazepam exposure, significantly decreases withdrawal behaviors. During the course of diazepam exposure (with or without periodic Ro15-1788 administration) effects of chronic diazepam on spontaneously elicited sedative and active behaviors were not altered. It is postulated that physical dependence reverts to a drug naive state after each exposure to the benzodiazepine antagonist. This treatment may represent a possible therapeutic approach for preventing the (time dependent) development of physical dependence and the accompanying severe withdrawal symptoms.     

Agonistic action of a benzodiazepine antagonist: effects of Ro 15-1788 and midazolam on hypertonic NaCl intake

Rats adapted to a 23-hr water deprivation regimen were allowed a daily 1-hr water rehydration session. On test days the session drinking fluid was 1.5% NaCl solution rather than water. One group was injected (SC) with the benzodiazepine antagonist Ro 15-1788 (2.5-10 mg/kg) and another group with the agonist agent midazolam (0.13-2.0 mg/kg) every 5-6 days at 10 and 15 min before a test session, respectively. Both Ro 15-1788 and midazolam increased 1.5% NaCl solution intake in a dose-related manner. In this study and in previous research, benzodiazepines and barbiturates were shown to increase the intake of hypertonic NaCl solutions. The present results reveal a similar effect for Ro 15-1788, indicating an agonistic dimension to the spectrum of action of this specific receptor antagonist.     

beta-Carboline FG 7142-reduced aggression in male rats: reversed by the benzodiazepine receptor antagonist, Ro15-1788

The beta-carboline FG 7142 decreases conspecific aggression in male hooded rats. The purpose of this study was to examine the effects of pretreatment with Ro15-1788 or chlordiazepoxide (CDP) in this paradigm. The six groups (n = 8) were saline, FG 7142 (5 mg/kg, immediate, IP), CDP (5 mg/kg, -10 min, IP), CDP (5 mg/kg, -10 min) plus FG 7142 (5 mg/kg, immediate), Ro15-1788 (10 mg/kg, -10 min, IP), and Ro15-1788 (10 mg/kg, -10 min) plus FG 7142 (5 mg/kg, immediate). Following injection of the more aggressive member of a pair of isolation-housed rats, the pair was observed in a living cage over four 6-min trials interpolated over a 40 min session. In the first 20 min after the injection FG 7142 decreased aggression, decreased the pinning of the other animal, and increased avoiding behavior. These effects were the opposite of those seen in the Ro15-1788-injected rats and Ro15-1788 pretreatment reversed the effects of FG 7142. CDP alone caused prolonged aggressive behavior but as a pretreatment only partially reversed the effects of FG 7142.     

Increased sensitivity to benzodiazepine antagonists in rats following chronic treatment with a low dose of diazepam

The effects of benzodiazepine (BZ) antagonists on operant behavior were examined in rats chronically administered a low dose of diazepam (DZ). The low maintenance dose of DZ (5 mg/kg twice daily) was selected as more closely associated with its anxiolytic effects than the higher treatment doses previously used to study BZ dependence. Food-restricted rats were trained to press a lever for food reinforcement under a FR20 schedule of reinforcement prior to the start of DZ administration. Acute administration of DZ caused a dose-dependent reduction of response rates, with 5 mg/kg causing a 50% decrease. Rats treated chronically with DZ became tolerant to its rate-suppressant effects as shown by a 5-fold increase in the dose of DZ required to reduce FR20 response rates by 50%. The BZ antagonist flumazenil (formerly Ro 15-1788; 10–56 mg/kg) suppressed rates of responding in rats treated chronically with DZ. The suppression of operant responding was obtained when flumazenil was given up to 3 h, but not 18 h, after the last treatment with DZ. In contrast, only the highest dose of flumazenil (56 mg/kg) caused reductions of operant responding when given to rats treated with saline. The BZ antagonist CGS 8216 (3.3–33 mg/kg IP), given 10 min prior to the session, was similarly more potent and effective at suppressing operant responding in rats treated chronically with DZ than saline. This procedure may provide a model for the clinical problem of physical dependence to chronically-administered low, anxiolytic doses of BZ tranquilizers.     

The partial benzodiazepine agonist properties of Ro 15-1788 in pentylenetetrazol-induced seizures in cats

The effects of Ro 15-1788, a specific benzodiazepine antagonist, were studied on pentylenetetrazol-induced seizures in cats. Ro 15-1788 decreased the number of myoclonic jerks induced by a subconvulsive dose of pentylenetetrazol (12.5 mg/kg, i.m.). Ro 15-1788 suppressed generalized convulsive seizures induced by a minimal convulsive dose of pentylenetetrazol (25-35 mg/kg), but did not block the effects of higher doses (35-45 mg/kg). These results indicate that Ro 15-1788 is not a pure benzodiazepine antagonist, but has partial agonistic properties.     

Relationship of the effects of the benzodiazepine derivative clorazepate on corticosterone secretion with its behavioural actions. Antagonism by Ro 15-1788

The effectiveness of Ro 15-1788, a benzodiazepine receptor antagonist, in modifying the effects of the benzodiazepine derivative clorazepate on schedule-controlled behaviour and pituitary-adrenal activity of rats was investigated. At low doses (5 mg/kg) clorazepate increased punished responding and slightly decreased basal plasma corticosterone levels. At high doses (40 mg/kg), clorazepate suppressed fixed ratio responding, raised basal plasma levels of corticosterone and reduced the stress response produced by exposure to a novel environment. Pretreatment with Ro 15-1788 blocked the behavioural and neuroendocrine effects of the high dose of clorazepate. These results suggest that both types of effects of clorazepate share a common mechanism which involves brain benzodiazepine receptors.     

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.     

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.     

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     

Benzodiazepine-like effects of inosine on punished behavior of pigeons

Behavioral effects of the putative endogenous benzodiazepine receptor ligand, inosine, were studied alone and in combination with the benzodiazepine antagonist Ro 15-1788. Keypeck responses were maintained by food under a multiple fixed-interval 3-min, fixed-interval 3-min schedule of food delivery. Under the multiple schedule, the first response after 3 min produced food in the presence of either white (no punishment) or red keylights and, in addition, each 30th response produced a brief electric shock (punishment) when the keylight was red. Inosine increased the low rates of punished responding (10-100 mg/kg IM) and the higher rates of unpunished responding (30 mg/kg). The benzodiazepine antagonist Ro 15-1788 (0.03 mg/kg, IM) antagonized the rate-increasing effects of inosine but had no effect when given alone. Combinations of inosine (30 mg/kg) with higher doses of Ro 15-1788 (0.1-1 mg/kg) decreased responding much like Ro 15-1788 alone. The marked rate-decreasing effects of 1000 mg/kg inosine were not affected by concurrent administration of Ro 15-1788 (0.01-1 mg/kg). The behavioral effects of inosine alone resembled effects of benzodiazepines but not those of benzodiazepine antagonists. The response rate-increasing effects of inosine may be due to its benzodiazepine receptor binding properties, whereas the rate decreases produced by higher doses of inosine appear to be unrelated to benzodiazepine receptors.     

The benzodiazepine antagonist Ro 15-1788 reverses the effect of methyl-beta-carboline-3-carboxylate but not of harmaline on cerebellar cGMP and motor performance in mice

The cerebellar cGMP level in mice was decreased in a dose-dependent manner 30 min after diazepam (ED50 = 2 mg/kg p.o.). This effect was reversed by the specific benzodiazepine antagonist Ro 15-1788. Methyl-beta-carboline-3-carboxylate (beta-CCM) and harmaline increased cGMP. Ro 15-1788 dose dependently counteracted the beta-CCM- but not the harmaline-induced increase in cGMP. In the horizontal wire test Ro 15-1788 antagonized the impairment of motor performance induced by beta-CCM, but not that induced by harmaline. These findings further support the view that harmaline in contrast to beta-carboline-3-carboxylates does not act through benzodiazepine receptors, and that Ro 15-1788 antagonizes only those convulsants and stimulants that act through specific benzodiazepine receptors.     

Status epilepticus induced by pentylenetetrazole modulates in vivo [11C]Ro 15-1788 binding to benzodiazepine receptors. Effects of ligands acting at the supramolecular receptor complex

Positron emission tomography (PET) was used to investigate, in the living baboon, the in vivo modulation of [11C]Ro 15-1788 binding to benzodiazepine receptors in brain and the changes with ligands acting at the supramolecular complex during status epilepticus induced by pentylenetetrazole. The central type benzodiazepine receptors were labelled in vivo by intravenous injection of [11C]Ro 15-1788. Simultaneous positron emission tomography and electroencephalographic activity recording evidenced a modulation of the brain binding of [11C]Ro 15-1788 during pentylenetetrazole-induced status epilepticus. We investigated the changes in the modulation of radioligand kinetics and in seizure activity after intravenous administration of a benzodiazepine agonist (diazepam, 1.5 mg/kg), a benzodiazepine antagonist (Ro 15-1788, 2 mg/kg), a GABA agonist (progabide, 50 mg/kg) and a ligand of the picrotoxin/barbiturate binding sites (LY81067, 3.5 mg/kg). The results showed that there is an in vivo competitive interaction of pentylenetetrazole with the benzodiazepine receptors, as reflected by the low displacement of [11C]Ro 15-1788 in the first 10 min of the status epilepticus. However, in contrast to diazepam, progabide and LY81067, a dose (2 mg/kg) of Ro 15-1788 that saturates the benzodiazepine receptors was unable to block the seizures induced by pentylenetetrazole. This indicates that the benzodiazepine receptors play only a minor role in the status epilepticus induced by pentylenetetrazole. The contribution of other binding sites within the supramolecular complex is assessed.     

Reversal of antinociceptive effect of cholecystokinin by benzodiazepines and a benzodiazepine antagonist, Ro 15-1788

Intraperitoneally administered benzodiazepines, chlordiazepoxide (2-5 mg/kg), diazepam (1 mg/kg), flurazepam (1 mg/kg) and a benzodiazepine antagonist, Ro 15-1788 (0.5 mg/kg), reversed the antinociceptive effect in mice which was induced by intracisternal administration of 1 microgram of sulfated cholecystokinin octapeptide. The antinociceptive effect of cholecystokinin was reversed by naloxone, suggesting that the antinociceptive action involves endogenous opioid peptides in its production. On the other hand, morphine-induced analgesia was not reversed by diazepam and Ro 15-1788. These facts rule out opioid receptors as the site of the antagonism between the benzodiazepines or Ro 15-1788 and cholecystokinin on the antinociceptive effect. Benzodiazepines and Ro 15-1788 seem to inhibit the release of opioid peptides induced by cholecystokinin.     

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.     

Antagonism of the effects of the atypical benzodiazepine, Ro 5-4864 on intracranial self-stimulation in the rat

The effects of Ro 5-4864 (chlordiazepam) were examined on responding for self-stimulation of the mid-lateral hypothalamus. Rewarding stimuli were delivered according to a 10-sec variable interval schedule of reinforcement. Ro 5-4864 (10-30 mg/kg, subconsulsive doses in these rats) decreased responding. This effect was antagonized by chlordiazepoxide (5-10 mg/kg) and phenobarbitone (35 mg/kg) but not by the benzodiazepine receptor antagonist Ro 15-1788 (10-20 mg/kg), the ligand for peripheral benzodiazepine receptors PK 11195 (60 mg/kg) or phenytoin (60 mg/kg). The pattern of interactions of Ro 5-4864 with these compounds differs from the pattern obtained with other procedures, and suggests that Ro 5-4864 has effects on systems unrelated to anxiety, convulsive activity or sedation.     

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.