Reversal of the antinociceptive effects of centrally-administered morphine by the benzodiazepine receptor antagonist Ro 15-1788

The effects of the benzodiazepine receptor antagonist, Ro 15-1788, were examined on analgesia induced by morphine after central (intracerebroventricular, i.c.v., or intrathecal, i.t.) and systemic administration. Analgesia was assessed in squirrel monkeys trained to respond under an electric shock tiltration procedure and in mice using the radiant heat tail-flick test. Central and systemic administration of morphine produced antinociceptive effects that were antagonized by 0.1 mg/kg of naloxone in both species. Ro 15-1788 antagonized the effects of morphine after central (i.c.v. or i.t.) administration but did not alter the effects of morphine given by the systemic route. This novel interaction suggests that Ro 15-1788 may be useful in pharmacologically separating neural substrates subserving opiate analgesia.     

The effect of a benzodiazepine antagonist, RO15-1788, in diazepam dependent rats

A benzodiazepine antagonist, RO15-1788, was administered intragastrically to diazepam-dependent gastric fistula rats and a precipitated abstinence syndrome was observed. The intensity of the RO15-1788 precipitated abstinence syndrome, calculated by the Precipitated Abstinence Scale, increased in intensity in a log-dose manner over a dose range of 1.0 to 15.0 mg/kg of RO15-1788 and plateaued at the 15.0 mg/kg dose. The RO15-1788 precipitated diazepam abstinence syndrome differed both qualitatively and quantitatively from the diazepam withdrawal syndrome.     

RO 15-1788 decreases hypnotic effects of sleep deprivation

The present study investigated the effects of 5 mg, 60 mg and 120 mg of the benzodiazepine antagonist RO 15-1788 on the ability to resist sleep and on mood of sleep deprived subjects. Repeated administration of 60 and 120 mg significantly increased subjects alertness in comparison with 5 mg and placebo. The 5 mg dose had a tendency to potentiate the hypnotic effects of sleep-deprivation. The higher levels of the drug also decreased positive mood and increased negative mood, and increased the density of sleep spindles during sleep. These results are interpreted to suggest a dose dependent effect of RO 15-1788 on arousal level.     

Behavioral effects and benzodiazepine antagonist activity of Ro 15-1788 (flumazepil) in pigeons

The selective benzodiazepine receptor antagonist, Ro 15-1788, produced behavioral effects in pigeons at doses at least 100 times lower than those previously reported to possess intrinsic pharmacological activity in mammals. In contrast to its effects in mammalian species, in pigeons, Ro 15-1788 does not exhibit partial agonist activity. Key-peck responses of pigeons were studied under a multiple fixed-interval 3-min, fixed-interval 3-min schedule in which the first response after 3-min produced food in the presence of red or white keylights. In addition, every 30th response during the red keylight produced a brief electric shock (punishment). Under control conditions, punished responding was suppressed to 30% of unpunished response levels. Ro 15-1788 (0.01 mg/kg, i.m.) increased unpunished response rates by 33% without affecting rates of punished responding. Doses of 0.1 to 1.0 mg/kg Ro 15-1788 produced dose-related decreases in both punished and unpunished responding. As is characteristic of other benzodiazepines, midazolam (0.1 and 0.3 mg/kg, i.m.) markedly increased punished responding but had little effect on rates of unpunished responding. Ro 15-1788 antagonized the increases in punished responding and also reversed the rate-decreasing effects of higher doses of midazolam. However, the effectiveness of Ro 15-1788 as a benzodiazepine antagonist was limited by its intrinsic activity: rate-decreasing doses of Ro 15-1788 were unable to completely reverse behavioral effects of midazolam. Midazolam was an effective antagonist of the behavioral effects of Ro 15-1788 (up to 0.1 mg/kg) but midazolam did not influence the rate-decreasing effects of 1.0 mg/kg Ro 15-1788 across a 100-fold dose range. In the pigeon, the behavioral effects of relatively low doses of Ro 15-1788 (0.01-0.1 mg/kg) appear to be related to benzodiazepine receptor mechanisms, whereas other systems appear to be involved in the effects of higher doses.     

Blood pressure response to Ro15-1788, a benzodiazepine antagonist

The effects of Ro15-1788, a benzodiazepine antagonist, on heart rate and blood pressure were studied in chloralose anesthetized cats. In previously untreated controls, Ro15-1788 lowered both systolic and diastolic arterial pressure about 15 mm Hg, and slightly decreased heart rate. In cats that had been given a single acute dose of diazepam or flurazepam, Ro15-1788 increased blood pressure about 40 mm Hg. A similar increase was measured in cats that were tolerant and physically dependent after 5 weeks of chronic flurazepam treatment. High spinal (C-1) section abolished all Ro15-1788 effects. It is suggested that the observed drug actions occur within the CNS rather than in the periphery, and that it might be useful to study further the cardiovascular actions of benzodiazepine agonists and antagonists.     

The benzodiazepine receptor ligands RO 5-4864 and RO 15-1788 do not block the inhibition of PAF-induced platelet aggregation seen with the hetrazepine WEB2086

The hypothesis was tested that the hetrazepine WEB 2086 acts as an inhibitor of PAF-induced platelet aggregation via interaction with the platelet benzodiazepine receptor(BDZR). WEB 2086 is a potent inhibitor of rabbit platelet aggregation and ATP secretion induced by 370 nM PAF. The two BDZR ligands RO 5-4864 and RO 15-1788 (7-96 microM) are inactive as PAF antagonists. When platelets were pretreated with either BDZR ligand, and then exposed to various concentrations of WEB 2086, there was no alteration of the dose-response relationship of the hetrazepine on PAF-induced aggregation, as reflected by threshold concentration, ED50, or maximum inhibition seen with WEB 2086. Pretreatment of platelets with the BDZR ligands also failed to block the inhibitory action of WEB 2086 on PAF-induced ATP release. The data are consistent with the notion that WEB 2086 acts as a PAF antagonist through its action at a specific PAF receptor, and is dissociated from, and independent of, interaction with the benzodiazepine receptor.     

Convulsions induced by submaximal dose of pentylenetetrazol in mice are antagonized by the benzodiazepine antagonist Ro 15-1788

The effects of benzodiazepine antagonist Ro 15–1788, alone or with diazepam, were studied in mice on convulsions induced by pentylenetetrazol (PTZ). We found that Ro 15–1788 (1 mg/kg) was able to antagonize the anticonvulsive effects of diazepam (1 mg/kg), but also had, with submaximal doses of PTZ (65 mg/kg), its own anti-convulsive action. At very low doses (0.1 mg/kg), it even potentiated the anticonvulsive effects of diazepam (0.05 mg/kg). This dual action provides evidence for partial agonist properties of the antagonist Ro 15–1788.     

Effect of RO 15-1788 and RO 5-4684 on the evoked activity of neurons in hippocampal sections

The action of specific benzodiazepine (BD) antagonist R015-1788 and peripheral benzodiazepine receptor (BDR) ligand R05-4864 on the evoked activity of hippocampal neurons was studied using brain slice method. The extracellular activity was registered in CAI area upon single and paired pulse stimulation of Schaffer collaterals. R015-1788 application (5 microM, for 3-6 min) reduced paired pulse inhibition (PPI). More prolonged application produced a depression of the population spike (PS). R015-1788 (5 microM) blocked diazepam (2 microM), hexobarbital (10 microM) and GABA (40 microM) potentiation of PPI. Interaction of R015-1788 with endogenous BD-like ligand as a possible explanation for the effects under study is discussed. R05-4864 (10 microM) reduced PPI and decreased PS evoked by single pulse stimulation. Frequency stimulation revealed the generation of additional PS after drug application. The data presented suggest that suppression of hippocampal inhibitory circuits may be a general feature of anxiogenic BDR ligands.     

The benzodiazepine receptor inverse agonists beta-CCM and RO 15-3505 both reverse the anxiolytic effects of ethanol in mice

The antagonistic effects of the benzodiazepine receptor inverse agonist beta-CCM (1 mg/kg) and of the partial inverse agonist RO 15-3505 (3 mg/kg) on the anxiolytic properties of ethanol (1 g/kg) in mice confronted with a light/dark choice procedure and with the staircase test were investigated. Both drugs reversed the effects of ethanol on some of the behavioral parameters, but beta-CCM alone elicited anxiogenic intrinsic effects. RO 15-3505 induced seizures in mice treated with a subconvulsant dose of pentylenetetrazole, the most efficient doses being 3 and 6 mg/kg. These data indicate that beta-CCM and RO 15-3505 can reverse some of the anxiolytic effects of ethanol, acting probably to oppose GABA function via the benzodiazepine receptor.     

Interactions between the benzodiazepine receptor antagonist Ro 15-1788 (flumazepil) and the inverse agonist beta-CCE: behavioral studies with squirrel monkeys

The effects of Ro 15-1788 and ethyl-beta-carboline-3-carboxylate (beta-CCE) were studied alone and in combination on the behavioral performances of squirrel monkeys. Under one procedure, performances maintained by food were suppressed by electric shock presentation (punishment or "conflict" procedure). Under a second procedure, responding was maintained either by food or electric shock delivery under a 5-min fixed-interval schedule. Doses of beta-CCE between 0.1 and 3.0 mg/kg, i.m., produced graded decreases in punished responding which were reversed by pretreatment with Ro 15-1788 (1.0 - 10.0 mg/kg, i.m.). Low doses of beta-CCE (0.03 - 0.3 mg/kg, i.m.) increased responding of monkeys maintained by shock presentation, but did not affect food-maintained responding; higher doses of beta-CCE decreased responding under both schedules. These effects of beta-CCE are opposite those produced by the benzodiazepines under this procedure. Ro 15-1788 (1.0 mg/kg i.m.) antagonized the effects of beta-CCE, producing a shift to the right in the dose-response curves. These findings provide further support for the view that beta-CCE and Ro 15-1788 produce effects mediated by the same benzodiazepine receptor recognition site.     

Effect of an imidazobenzodiazepine (RO 15-1788) on aggressive behavior in mice

Imidazobenzodiazepine (Ro 15-1788, 5 mg/kg) similarly to a lose dose of apomorphine (0.1 mg/kg) decreased the intensity of footshock aggression in male rats. Ro 15-1788 significantly potentiated the antiaggressive action of apomorphine. Pirenperone (0.01 mg/kg) potentiated the effect of both drugs, whereas haloperidol (0.01 mg/kg) had an opposite action. After long-term treatment with apomorphine and Ro 15-1788 the tolerance to their antiaggressive action developed. This change was in agreement with increased serotonin metabolism in the forebrain. Unlike the action on aggressive behavior, Ro 15-1788 similarly to haloperidol (0.05 mg/kg) decreased the motor depressant effect of apomorphine (0.01 mg/kg) in mice. This effect correlated with the lowered serotonin metabolism after Ro 15-1788 administration. Unlike apomorphine, Ro 15-1788 reversed catalepsy induced by haloperidol (0.25 mg/kg). Administration of pirenperone (0.03 mg/kg) and destruction of serotoninergic terminals by p-chloroamphetamine (2 X 15 mg/kg) significantly potentiated the sedative action of apomorphine. It appears that different action of Ro 15-1788 on behavioral effects of apomorphine is related to different influence of Ro-1788 on serotoninergic processes in the striatum and limbic structures.     

RO 15-1788 selectively reverses antagonism of pentylenetetrazol-induced discriminative stimuli by benzodiazepines but not by barbiturates

The specificity of ethyl 8-fluro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo (1,5-a) (1,4) benzodiazepine-3-carboxylate (RO 15-1788) in reversing the effectiveness of diazepam and des-methylclobazam, but not of pentobarbital, in antagonizing discriminative stimuli produced by pentylenetetrazol is described. Male hooded rats were trained to discriminate pentylenetetrazol-induced interoceptive discriminative-stimuli (IDS) in a two-lever choice paradigm on an FR10 schedule of food reinforcement. These IDS pharmacologically model verbal report of anxiogenic activity in humans. Diazepam (1,4 benzodiazepine), des-methylclobazam (1,5 benzo-diazepine), and pentobarbital antagonized pentylenetetrazol-IDS. RO 15-1788 neither generalized to nor antagonized pentylenetetrazol-IDS. It also did not cause convulsions in pentylenetetrazol sensitized rats at doses up to 40 mg/kg. It did, however, antagonize the action of diazepam (10 mg/kg) as well as that of des-methylclobazam (160 mg/kg) but not that of pentobarbital. These data suggest that RO 15-1788 is not an anxiomimetic, anxiolytic or a convulsant drug, but it is a specific and effective antagonist of anxiolytic action of benzodiazepines.     

Antagonism of RO 15-1788 with benzodiazepines in the effect on motivated aggression and the action of analgesics

The influence of Ro 15-1788 and bicuculline on the action of GABA-positive drugs (muscimol), GABA cethyl ester, piracetam and depakine and benzodiazepine tranquilizers (diazepam, phenazepam) on motivated aggression has been studied. It has been shown that Ro 15-1788 which has a weak antiaggressive effect selectively antagonizes the anti-aggressive effect of tranquilizers but not that of GABA-positive drugs. Bicuculline antagonizes antiaggressive activity of the drugs of both types. The action of these antagonists on the effect of the drugs under study as regards the analgetic activity of morphine was also studied. It has been shown that Ro 15-1788 antagonizes the potentiation of morphine analgesia caused by diazepam. At the same time Ro 15-1788 does not influence morphine analgesia potentiated by muscimol. Bicuculline removes the potentiation of morphine analgesia caused both by diazepam and muscimol it is concluded that bicuculline-sensitive GABA receptors modulate the antiaggressive effect of benzodiazepines and their influence on the analgetic action of opiates.     

The benzodiazepine receptor inverse agonists FG 7142 and RO 15-4513 both reverse some of the behavioral effects of ethanol in a holeboard test

The intrinsic effect of the benzodiazepine receptor inverse agonists RO 15-4513 and FG 7142 on the behavior of mice in a holeboard were investigated. Both drugs caused dose-related decreases in exploratory head-dipping. The highest dose of FG 7142 (40 mg/kg) also reduced locomotor activity. RO 15-4513 (1.5 and 3.0 mg/kg) and FG 7142 (10 and 20 mg/kg) reversed the reductions in the number of head-dips caused by ethanol (2 g/kg). The higher doses of these two drugs also partially reversed the locomotor stimulant action of ethanol. Animals that received ethanol in combination with either inverse agonist spent less time head-dipping than vehicle-treated controls. These data indicate that FG 7142 and RO 15-4513 can reverse, at least in part, some of the behavioral effects of ethanol. Neither drug significantly altered blood alcohol concentrations suggesting that the antagonism does not result from pharmacokinetic changes.     

Involvement of different mechanisms in the stimulatory effects of cholecystokinin octapeptide on gastrointestinal and colonic motility in dogs

The effects of an intravenous infusion of cholecystokinin octapeptide (CCK-8, 1 microgram.kg-1.h-1) were investigated in conscious fasted dogs chronically fitted with strain-gauge transducers on the antrum, the jejunum, and the colon. Attempts to antagonize the increase of motility appearing at the three levels during CCK infusion were made using different blockers to elucidate the mechanisms involved. Asperlicin (a specific CCK antagonist) blocked the effects of CCK-8 at the three levels, while atropine and somatostatin were only effective in the jejunum and colon. Methyl-levallorphan (a mu-opiate antagonist that poorly crosses the blood-brain barrier) antagonized the CCK-induced colonic stimulation when intracerebroventricularly administered. Serotonin, histamine, substance P, and K-antagonists as well as a benzodiazepine did not modify the CCK-8 induced stimulation. It was concluded that the stimulatory effect of CCK-8 resulted from (a) a direct stimulation of the smooth muscle cells at gastric level, (b) a cholinergic activation of the jejunum and the colon, and (c) the involvement of a mu-opioid central component in the colonic response only.     

Benzodiazepine receptor binding in vivo with [3H]-Ro 15-1788

In vivo benzodiazepine receptor binding has generally been studied by "ex vivo" techniques. In this investigation, we identify the conditions where [3H]-Ro 15-1788 labels benzodiazepine receptors by true "in vivo" binding, i.e. where workable specific to nonspecific ratios are obtained in intact tissues without homogenization or washing. [3H]-Flunitrazepam and [3H]-clonazepam did not exhibit useful in vivo receptor binding.     

Neurochemical effects of benzodiazepine and morphine on freshwater mussels

The purpose of this study was to examine the neurochemical effects of morphine, diazepam, a common benzodiazepine, and an effluent concentrate on the endemic freshwater mussel Elliptio complanata. Mussels were exposed to the drugs and to the solid-phase concentrate of a municipal effluent and left to stand at 15 °C for 48 h. Neurochemical effects were determined by monitoring changes in dopamine, serotonin, glutamate and γ-aminobutyric acid (GABA) levels in the visceral mass (containing the nerve ganglia) of mussels. The activities of acetylcholinesterase (AChE), dopamine and serotonin-dependent adenylyl cyclase (ADC) were also determined in the mussels. Oxidative stress was determined by tracking changes in lipid peroxidation (LPO) in the mitochondrial and post-mitochondrial fractions. The results revealed that the drugs and the effluent extract were biologically active in mussels. Morphine reduced serotonin and increased dopamine in mussel tissues while reducing AChE activity and increasing GABA levels. This suggests the induction of a relaxation state in mussels. Diazepam also reduced serotonin levels but produced no change in dopamine levels. However, dopamine-sensitive ADC activity was readily activated, indicating the potential effect on opiate signaling. Diazepam increased glutamate levels slightly, but AChE remained stable. The increase in both dopamine ADC activity and glutamate concentrations was also associated with greater oxidative stress on the mitochondrial and post-mitochondrial fractions in cells. A comparison of the global response pattern of these drugs with those of the effluent extract revealed only a relative proximity to morphine. In conclusion, the data warrant more studies on the analysis of opiates and benzodiazepines in municipal effluents to better address the potential environmental hazard of these neuroactive drug classes to aquatic organisms.     

Ro 15-1788 antagonizes the discriminative stimulus effects of diazepam in rats but not similar effects of pentobarbital

The benzodiazepine antagonist properties of Ro 15-1788 were evaluated in rats trained to discriminate between saline and either 1.0 mg/kg of diazepam or 10 mg/kg of pentobarbital in a two-choice discrete-trial shock avoidance procedure. When administered alone, 1.0 mg/kg of diazepam and 10 mg/kg of pentobarbital produced comparable amounts of drug-appropriate responding (> 84%), whether rats were trained to discriminate between diazepam or pentobarbital and saline. Ro 15-1788 (3–32 mg/kg, p.o.), administered 10 min before diazepam or pentobarbital, produced a dose-related blockade of the discriminative effects of diazepam in both groups of rats, but was completely ineffective in blocking the discriminative effects of pentobarbital. The dose-effect curve for the discriminative effects of diazepam was shifted to the right in a parallel fashion 3- and 13-fold by 10 and 32 mg/kg of Ro 15-1788, respectively, indicating that Ro 15-1788 acts as a surmountable, competitive antagonist of diazepam. When administered alone, Ro 15-1788 (32–100 mg/kg, p.o.) produced primarily saline-appropriate responding, although 100 mg/kg of Ro 15-1788 produced drug-appropriate responding in one out of eight rats. When administered orally 30 min after diazepam, Ro 15-1788 (32 mg/kg) completely reversed within 10 min the discriminative effects of diazepam. The blockade of diazepam's discriminative effects by 32 mg/kg of Ro 15-1788 appeared to last at least as long (approximately 2 hr) as the effects of diazepam alone.     

The benzodiazepine receptor antagonist, flumazenil does not block clinical effects of delta-9-tetrahydrocannabinol

Fifteen normal volunteer men were given intravenous doses of 2 mg of delta-9-tetrahydrocannabinol (THC) and 3 were given doses of 0.5 mg. Five, 15 and 30 minutes later, they were given intravenous doses of the benzodiazepine receptor antagonist, flumazenil. Doses of this compound ranged between 0.1 and 3.2 mg for single doses and 0.7 and 6.4 mg for total doses, being increased progressively with each successive subject, until dose-ranging was completed in 10 subjects. After that 5 subjects were given doses of 2 mg of THC on two occasions, followed by either doses of 3.2 or 6.4 mg flumazenil or placebo, administered under blind conditions. Three subjects were treated with 0.5 mg doses of THC followed by 3.2 mg of flumazenil or placebo under similar conditions. Despite doses of the antagonist which would have been adequate to reverse the effects of substantial doses of benzodiazepines, little ameliorative action was observed on the level of intoxication or the degree of conjunctival injection, two quite reliable clinical indicators of THC action. The benzodiazepine receptor does not seem to play any significant role in the psychoactive actions and conjunctival injection produced by THC.     

Role of calcitonin receptor-like receptor in colonic motility and inflammation

Calcitonin gene-related peptide (CGRP) mediates neurogenic inflammation and modulates intestinal motility. The CGRP receptor is a heterodimer of calcitonin receptor-like receptor (CLR) and receptor-associated modifying protein 1. We used RNA interference to elucidate the specific role of CLR in colonic motility and inflammation. Intramural injection of double-stranded RNA (dsRNA) against CLR (dsCLR) into the colonic wall at two sites caused the spatial and temporal downregulation of CLR in the colon within 1 day of dsRNA injection. Knockdown of CLR persisted for 7-9 days, and the effect of knockdown spread to approximately 2 cm proximal and distal to the injection sites, whereas control dsRNA injection did not affect CLR expression. Measurement of isometric contractions of isolated colonic muscle segments revealed that in control dsRNA-injected rats, CGRP abrogated contractions entirely and decreased resting muscular tone, whereas in dsCLR-injected rats, CGRP decreased muscle tone but slow-wave contractions of varying amplitude persisted. In trinitrobenzene sulfonic acid-induced colitis, rats with knockdown of CLR displayed a significantly greater degree of edema and necrosis than saline- or control dsRNA-injected rats. Levels of the proinflammatory cytokines TNF-alpha and IL-6 were markedly upregulated by trinitrobenzene sulfonic acid treatment. TNF-alpha mRNA levels were further increased in CLR knockdown rats, whereas levels of IL-6 were unaltered. Thus this study demonstrates that CLR is a functional receptor for CGRP.