The benzodiazepine recognition site inverse agonists Ro 15-4513 and FG 7142 both antagonize the EEG effects of ethanol in the rat

The aim of the present study was to compare the ability of Ro 15-4513 and FG 7142, two inverse agonists for benzodiazepine recognition sites, to antagonize the EEG effects of ethanol in freely moving rats. Ethanol (2.5 g/kg, p.o.) induced sedation and ataxia associated with a progressive suppression of the fast cortical activities and an enhancement of low frequencies in both cortical and hippocampal tracings. In contrast, Ro 15-4513 (2 mg/kg, i.p.) and FG 7142 (10 mg/kg, i.p.) both caused a state of alertness associated with desynchronized cortical activity and theta hippocampal rhythm as well as spiking activity which was predominantly observed in the cortical tracings. When rats were treated with FG 7142 or RO 15-4513 either before or after ethanol, a reciprocal antagonism of the behavioral and EEG effects of ethanol and of the partial inverse agonists was observed. These data support the view that the anti-ethanol effects of Ro 15-4513 may be related to its partial inverse agonist properties.     

Audiogenic seizures in DBA/2 mice discriminate sensitively between low efficacy benzodiazepine receptor agonists and inverse agonists

In experiments with audiogenic seizures in DBA/2 mice, we observed that several socalled benzodiazepine receptor antagonists exhibited either anticonvulsive (Ro 15-1788, PrCC) or proconvulsive (FG 7142, beta-CCE, CGS 8216) effects at high receptor occupancy (17-85%), as compared to benzodiazepines and DMCM which had anticonvulsive and proconvulsive actions, respectively, at very low receptor occupancy (less than 10%). Sensitive distinction between benzodiazepine receptor ligands with low anticonvulsive efficacy (partial agonists) and ligands with low proconvulsive, and maybe anxiogenic, efficacy (partial inverse agonists) can thus be obtained in sound seizure susceptible mice.     

Reversal of the effects of centrally-administered morphine on colonic motility in dogs by the benzodiazepine receptor antagonist RO 15-1788

The effects of intravenous (i.v.) and intracerebroventricular (i.c.v.) administration of morphine on jejunal and colonic motility were investigated in conscious dogs chronically prepared with strain gage transducers and compared to those of i.c.v. DAGO, a highly selective opiate mu agonist. Morphine i.v. (100 micrograms/kg) and i.c.v. (10 micrograms/kg) administered 3 hrs after a meal stimulated colonic motility for 3-5 hrs and induced a phase 3 on the jejunum, which appeared after a 15-60 min delay following i.c.v. administration. These effects were reproduced by DAGO administration at doses of 2 micrograms/kg i.v. and 0.2 micrograms/kg i.c.v. The effects of i.v., but not those of i.c.v., morphine and DAGO on jejunal and colonic motility were blocked by a previous administration of naloxone (100 micrograms/kg i.v.). The colonic stimulation but not the jejunal phase 3 induced by i.c.v. morphine and DAGO were blocked by RO 15-1788 (1 mg/kg i.v.), a selective benzodiazepine antagonist. The colonic stimulation induced by i.v. morphine or DAGO was not modify by i.v. RO 15-1788. It is concluded that i.c.v. administration of mu agonist involved benzodiazepine but not opiate receptors to stimulate colonic motility in dogs.     

Blockade of a putative GABA-mediated neurotransmission in the cerebellum by benzodiazepine receptor inverse agonists

An exponential relationship was observed between the firing rate of cerebellar Purkinje cells in urethane-anaesthetized rats and the duration of inhibition evoked in these cells by electrical stimulation of the nearby cortical surface. Benzodiazepines, administered i.v., decreased cell firing and increased the duration of the inhibitory response but did not alter the relationship between the two parameters. These effects of one benzodiazepine, RU 32007, were reversed by the benzodiazepine antagonist Ro15-1788 which had little effect alone. The benzodiazepine inverse agonists methyl- or ethyl-beta-carboline-3-carboxylate increased cell firing with the expected reductions in duration of inhibitory response in some cases. However, in 50% of recordings the inhibitory response disappeared, independent of the firing rate. All the effects of the beta-carboline esters were reversed by Ro15-1788 or the benzodiazepine, RU 32007. This action of the benzodiazepine receptor inverse agonists represents an in vivo blockade of an endogenous synaptic inhibition which is thought to be mediated by release of GABA.     

Naltrexone potentiates both amnestic and anxiolytic effects of chlordiazepoxide in mice

Studies have suggested that opioid antagonists potentiate the anxiolytic effect but not the amnestic action of chlordiazepoxide (CDZ). We investigated the effects of naltrexone (NAL) on the anxiolytic and amnestic effects of CDZ in mice tested in the plus-maze discriminative avoidance task (DAT). Mice are conditioned to choose between two enclosed arms (one of which aversive) while avoiding the two open arms of the apparatus. This task measures memory (time spent in the aversive vs. time in the non-aversive enclosed arms) and anxiety (time spent in the open arms). Mice treated with saline (SAL) or 5 mg/kg NAL, and SAL or 2.5 mg/kg CDZ were submitted to DAT training. The test was performed 24 h later, without aversive stimuli. In the training, NAL + CDZ group showed higher percent time spent in the open arms than all the other groups. In the test, NAL + CDZ (but not SAL + CDZ) group showed higher percent time spent in the aversive enclosed arm than SAL + SAL and NAL + SAL groups. The data suggest that NAL potentiates the small decreases in anxiety and retention induced by a subeffective dose of CDZ.     

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.     

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.     

Differences in the mechanism of the antihypoxic action of benzodiazepine receptor agonists and muscimol

Neuropharmacological analysis of previously revealed antihypoxic activity of benzodiazepines (BDZ) has been performed in experiments on mice exposed to hypoxia. Antihypoxic effect of diazepam is shown to be antagonized by the central BDZ receptor blocker, Ro 15-1788. A certain degree of antihypoxic activity also abolished by Ro 15-1788 is exhibited by hypothetical ligands of BDZ receptors: inosin, nicotinamide, ethyl-beta-carboline-3-carboxylate. The effect of dipyridamole, a drug with high affinity for BDZ receptors of the peripheral type is not antagonized by Ro 15-1788, another evidence of Ro 15-1788 affinity precisely to the central BDZ receptors. GABA-mimetics (muscimol and GABA cetyl ester) were also found to have marked antihypoxic activity. Unlike BDZ receptor agonists, this effect is reduced by bicuculline and not by Ro 15-1788. The data obtained suggest that antihypoxic activity of BDZ is caused by their direct interaction with the central BDZ receptors, probably with the type which is not modulated by GABAA receptors.     

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.     

Effects of the imidazobenzodiazepine RO15-4513 on the stimulant and depressant actions of ethanol on spontaneous locomotor activity

The purpose of this study was to investigate the effects of the imidazobenzodiazepine RO15-4513, a partial inverse agonist at benzodiazepine (BDZ) receptors, on the stimulant and depressant actions of ethanol in mice. For comparative purposes, another BDZ inverse agonist, FG-7142, was examined as well. Neither RO15-4513 nor FG-7142 influenced the low-dose excitatory effects of ethanol on spontaneous locomotor activity. However, both RO15-4513 and FG-7142 significantly antagonized the depressant effects of ethanol, and this antagonism was completely reversed by pretreatment with the BDZ receptor antagonist, RO15-1788. These data suggest that RO15-4513 is capable of antagonizing only some of the behavioral effects of ethanol, and in particular, those responses to ethanol that are mediated by modulation of the GABA/BDZ-chloride channel receptor complex.     

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.     

A search for quantum mechanical methods suitable for the conformational analysis of models related to some inverse agonists of the benzodiazepine receptor

The target of this work consists in a search, among several methods of quantum mechanical calculations, for the most suitable one (both with regard to accuracy and speed) for doing conformational analysis on models related to molecules belonging to a group of indol-3-yl glyoxylyl derivatives, many of which behave as ligands of the benzodiazepine receptor (BzR). Among the wide variety of ligands of the BzR, -carboline derivatives are included, many of which show a pharmacological profile similar to the one exhibited by compounds of interest to us. A structure analogy between our compounds and -carboline derivatives could be hypothesized, depending upon the preferred arrangement of the investigated molecules with respect to the dihedral angles of the indol glyoxylyl moiety. Therefore a potential energy surface scan has been carried out on selected models either by the quantum mechanical semiempirical methods MNDO, AM1, PM3 (by using the programs included in themopac package) or by a quantum mechanical SCFab initio method (by using thegaussian-80 program), both in order to find support for the choice of the more suitable semiempirical method, and in order to verify a structural analogy between our compounds and the -carboline ring. The results showed that AM1 and PM3, unlike MNDO, seem to be suitable semiempirical methods for doing potential energy surface scans on this kind of molecule. The preferred arrangement showed by the models with respect to the indol glyoxylyl moiety appeared to support the hypothesis about the structure analogy between the investigated ligands and -carboline derivatives.     

Androgens with activity at estrogen receptor beta have anxiolytic and cognitive-enhancing effects in male rats and mice

Testosterone (T) and its metabolites may underlie some beneficial effects for anxiety and cognition, but the mechanisms for these effects are unclear. T is reduced to dihydrotestosterone (DHT), which can be converted to 5α-androstane,3α,17β-diol (3α-diol) and/or 5α-androstane-3β,17β-diol (3β-diol). Additionally, T can be converted to androstenedione, and then to androsterone. These metabolites bind with varying affinity to androgen receptors (ARs; T and DHT), estrogen receptors (ERβ; 3α-diol, 3β-diol), or GABA_A/benzodiazepine receptors (GBRs; 3α-diol, androsterone). Three experiments were performed to investigate the hypothesis that reduced anxiety-like and enhanced cognitive performance may be due in part to actions of T metabolites at ERβ. Experiment 1: Gonadectomized (GDX) wildtype and ERβ knockout mice (βERKO) were subcutaneously (SC) administered 3α-diol, 3β-diol, androsterone, or oil vehicle at weekly intervals, and tested in anxiety tasks (open field, elevated plus maze, light–dark transition) or for cognitive performance in the object recognition task. Experiment 2: GDX rats were administered SC 3α-diol, 3β-diol, androsterone, or oil vehicle, and tested in the same tasks. Experiment 3: GDX rats were androsterone- or vehicle-primed and administered an antagonist of ARs (flutamide), ERs (tamoxifen), or GBRs (flumazenil), or vehicle and then tested in the elevated plus maze. Both rats and wildtype mice, but not βERKO mice, consistently had reduced anxiety and improved performance in the object recognition task. Androsterone was only effective at reducing anxiety-like behavior in the elevated plus maze and this effect was modestly reduced by flumazenil administration. Thus, actions at ERβ may be required for T's anxiety-reducing and cognitive-enhancing effects.     

Androgens with activity at estrogen receptor beta have anxiolytic and cognitive-enhancing effects in male rats and mice

Testosterone (T) and its metabolites may underlie some beneficial effects for anxiety and cognition, but the mechanisms for these effects are unclear. T is reduced to dihydrotestosterone (DHT), which can be converted to 5α-androstane,3α,17β-diol (3α-diol) and/or 5α-androstane-3β,17β-diol (3β-diol). Additionally, T can be converted to androstenedione, and then to androsterone. These metabolites bind with varying affinity to androgen receptors (ARs; T and DHT), estrogen receptors (ERβ; 3α-diol, 3β-diol), or GABA_A/benzodiazepine receptors (GBRs; 3α-diol, androsterone). Three experiments were performed to investigate the hypothesis that reduced anxiety-like and enhanced cognitive performance may be due in part to actions of T metabolites at ERβ. Experiment 1: Gonadectomized (GDX) wildtype and ERβ knockout mice (βERKO) were subcutaneously (SC) administered 3α-diol, 3β-diol, androsterone, or oil vehicle at weekly intervals, and tested in anxiety tasks (open field, elevated plus maze, light–dark transition) or for cognitive performance in the object recognition task. Experiment 2: GDX rats were administered SC 3α-diol, 3β-diol, androsterone, or oil vehicle, and tested in the same tasks. Experiment 3: GDX rats were androsterone- or vehicle-primed and administered an antagonist of ARs (flutamide), ERs (tamoxifen), or GBRs (flumazenil), or vehicle and then tested in the elevated plus maze. Both rats and wildtype mice, but not βERKO mice, consistently had reduced anxiety and improved performance in the object recognition task. Androsterone was only effective at reducing anxiety-like behavior in the elevated plus maze and this effect was modestly reduced by flumazenil administration. Thus, actions at ERβ may be required for T's anxiety-reducing and cognitive-enhancing effects.     

Cross-dependence to opioid and alpha 2-adrenergic receptor agonists in NG108-15 cells

Clonidine, a partial alpha 2-agonist, has been used empirically to alleviate opiate withdrawal symptoms, but the mechanism of its effects is not completely understood. We studied the interactions of opioid and adrenergic receptor agonists in the NG108-15 cells, which are a model of opiate dependence. We determined that in these cells the adenylate cyclase (AC) [EC 4.6.1.1; ATP pyrophosphate-lyase (cyclizing) overshoot response to opioid or alpha 2-agonist withdrawal can be significantly attenuated or suppressed by the other agonist. Subsequently, the AC overshoot response can be triggered with the antagonist to the second agonist to which the cells were not dependent. These results demonstrate that convergent dependence to morphine and alpha 2 agonists can occur in a homogeneous cell population without neuronal loops. Therefore, the basic mechanisms that can account for convergent dependence in this model take place at the level of intracellular regulatory pathways that do not require neuronal networks.     

A 5-HT4 receptor transmembrane network implicated in the activity of inverse agonists but not agonists

Activation of G protein-coupled receptors is thought to involve disruption of intramolecular interactions that stabilize their inactive conformation. Such disruptions are induced by agonists or by constitutively active mutations. In the present study, novel potent inverse agonists are described to inhibit the constitutive activity of 5-HT(4) receptors. Using these compounds and specific receptor mutations, we investigated the mechanisms by which inverse agonists may reverse the disruption of intramolecular interactions that causes constitutive activation. Two mutations (D100(3.32)A in transmembrane domain (TMD)-III and F275(6.51)A in TMD-VI) were found to completely block inverse agonist effects without impairing their binding properties nor the molecular activation switches induced by agonists. Based on the rhodopsin model, we propose that these mutated receptors are in equilibrium between two states R and R* but are unable to reach a third "silent" state stabilized by inverse agonists. We also found another mutation in TMD-VI (W272(6.48)A) that stabilized this silent state. This mutant remained fully activated by agonists. Molecular modeling indicated that Asp-100, Phe-275, and Trp-272 might constitute a network required for stabilization of the silent state by the described inverse agonists. However, this network is not necessary for agonist activity.     

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.     

Relating the structure,activity, and physical properties of ultrashort-acting benzodiazepine receptor agonists

The ultrashort-acting benzodiazepine (USA BZD) agonists reported previously have been structurally modified to improve aqueous solubility. Lactam-to-amidine modifications, replacement of the C5-haloaryl ring, and annulation of heterocycles are presented. These analogues retain BZD receptor potency and full agonism profiles.