Antagonism of ethanol effects by Ro 15-4513: an electrophysiological analysis

Ethanol (ETH) and general anesthetics have been reported to facilitate the chloride channel opening, possibly, or at least partly, through an interaction with the GABA-benzodiazepine (BZ) receptor-gated chloride ionophore "supramolecular complex". Recently Ro 15-4513, a novel BZ ligand, has been indicated as a potent and selective antagonist of various ETH-induced behavioral and biochemical effects. However, since its precise characterization is still a matter of debate, we have tested and compared the effect of Ro 15-4513, as well as its antagonism against ETH, in two objective electrophysiological parameters, i.e., the electroencephalograph (EEG) pattern in freely moving rats and single unit activity of reticulata neurons. Ro 15-4513 produced an EEG state of alertness and antagonized the behavioral impairment and the EEG deterioration by ETH. However, while its protective action was consistent against moderate doses (2 g/kg) of ETH, it was much less evident versus higher doses (4 and 8 g/kg). On reticulata cells, Ro 15-4513 potently stimulated their spontaneous firing and reversed the depression by both ETH and Na-pentobarbital. Moreover, the beta-carboline DMCM also had similar effects. The "pure" BZ antagonist Ro 15-1788 was completely inefective against ETH, yet fully cancelled the reversing actions of Ro 15-4513 and DMCM upon ETH or Na-pentobarbital effects. It is concluded that Ro 15-4513 behaves as a BZ inverse agonist, so that its opposition to ETH and Na-pentobarbital is probably the result of its "negative" coupling with the BZ recognition site that triggers the closing of chloride channels. It suggests that BZ inverse agonists might constitute, in the near future, a new class of analeptic drugs.     

The effect of the imidazodiazepine Ro 15-4513 on the anticonvulsant effects of diazepam, sodium pentobarbital and ethanol

The ability of the imidazodiazepine Ro 15-4513 to antagonize the anticonvulsant effects of diazepam, sodium pentobarbital and ethanol was investigated. Ro 15-4513 alone significantly lowered seizure threshold to bicuculline and this effect subtracted from the anticonvulsant effects of sodium pentobarbital and ethanol. In contrast, Ro 15-4513 completely reversed the anticonvulsant effects of diazepam, consistent with suggestions that it is a competitive ligand for benzodiazepine receptors.     

Characterization of a benzodiazepine receptor site with exceptionally high affinity for Ro 15-4513 in the rat CNS

The binding of [³H]Ro 15-4513, [³H]flunitrazepam and [³H[Fumazenil to rat CNS membranes was studied at 2°C, 22°C and 37°C using ligand concentrations ranging from ≈ 0.06 nM to 10 μM. Analysis of the binding saturation data suggested the existence of high-affinity sites (K_d < 10nM) and low-affinity sites (K_d > 100nM) for each ligand. When binding was performed using very low ligand concentrations a benzodiazepine site with an exceptionally high affinity for Ro 15-4513 (K_d ≈ 0.1nM) was evident in all major regions of the CNS except the cerebellum. This site was most prevalent in the hippocampus, medulla and spinal cord where it accounted for ≈ 70% of the specific binding when [³H]Ro 15-4513 was ≈ 0.06 nM. The selectivity of Ro 15-4513 for this site as compared to other high-affinity sites was 20- to 60-fold depending on the incubation temperature and CNS region. The affinity for the very high-affinity site was decreased ≈ 3-fold as temperature was increased from 2°C to 37°C (K_d ≈ 0.1nM and ≈ 0.3, respectively), which was similar to the effect of temperature on other high-affinity sites (K_d ≈ 2.6nM at 2°C and ≈ 8 nM at 37°C). Flumazenil, flunitrazepam, and diazepam did not differentiate the very high-affinity [³H]Ro 15-4513 site from other BZ sites, but alpidem exhibited a low affinity for it (IC₅₀ ≈ 5 μM). GABA at 100 μM had little effect on theK_d value for the very high-affinity site (GABA shift: ≈ 0.8 to 1.0), suggesting that Ro 15-4513 is a partial inverse agonist or an antagonist at this site. These findings provide further evidence for the pharmacologic diversity of BZ sites on different subtypes of GABA_A receptors.     

Pharmacokinetic studies on Ro 15-1788, a benzodiazepine receptor ligand, in the brain of the rat

Methods for determining Ro 15-1788 in brain tissue were developed using gas chromatography with nitrogen-phosphorus detection, and using reverse-phase high performance liquid chromatography. Application of the methods to pharmacokinetic studies in the rat found the elimination half-life of Ro 15-1788 from rat brain to be 16 min. Ro 15-1788 was undetectable in rat plasma at the time points studied. Concentrations of Ro 15-1788 in the brain were reduced if chlordiazepoxide was coadministered.     

The benzodiazepine antagonist Ro 15-1788 does not antagonize the ethanol withdrawal syndrome

It has been suggested that the signs and symptoms of the ethanol withdrawal syndrome may be due to the increased production of an "inverse agonist" that binds to the central benzodiazepine (BZ) recognition site in the brain. Ro 15-1788 (a potent antagonist at the central BZ recognition site), diazepam, and Ro 15-1788 plus diazepam were administered to groups of rats undergoing overt ethanol withdrawal. Ro 15-1788 did not alter the severity of the ethanol withdrawal reactions, but antagonized the ameliorative effect of diazepam. The results of our studies suggest that (1) the ethanol withdrawal syndrome is not produced by an endogenous ligand acting on the central BZ recognition site, and (2) diazepam decreases the severity of the ethanol withdrawal syndrome, at least in part, by its action at the central BZ recognition site.     

In vivo labeling of central benzodiazepine receptors with the partial inverse agonist [3H]Ro 15-4513

Ro 15-4513 is an imidazobenzodiazepine and a partial inverse agonist at the central benzodiazepine receptors (BZDr). It has been shown to antagonize behavioral and biochemical effects of ethanol. In vivo binding of [3H]Ro 15-4513 was evaluated in mouse brain. After intravenous injection [3H]Ro 15-4513 was readily taken up by the brain and distributed to brain areas enriched in benzodiazepine receptors. Binding was specific for central BZDr, saturable and reversible. A high degree of specific binding, relative to non-specific binding, was achieved. Analysis of dissociation kinetics revealed that [3H]Ro 15-4513 was retained significantly longer in hippocampus compared to other brain regions. In view of the known distribution of benzodiazepine receptor subtypes, this suggests that, in vivo, [3H]Ro 15-4513 has a higher affinity for benzodiazepine receptors type II and may explain quantitative differences in the regional distribution of this ligand compared to the antagonist [3H]Ro 15-1788. We conclude from these studies that Ro 15-4513 is a suitable ligand for in vivo studies of benzodiazepine receptors. Labeled with a positron-emitting isotope, it could be used with positron emission tomography to study BZDr in man under a variety of conditions.     

Visualization of alpha5 subunit of GABAA/benzodiazepine receptor by 11C Ro15-4513 using positron emission tomography

Although [(11)C]Ro15-4513 and [(11)C]flumazenil both bind to the central benzodiazepine (BZ) receptors, the distributions of the two ligands are not identical in vivo. Moreover, the in vivo pharmacological properties of [(11)C]Ro15-4513 have not been thoroughly examined. In the present study, we examined the pharmacological profile of [(11)C]Ro15-4513 binding in the monkey brain using positron emission tomography (PET). [(11)C]Ro15-4513 showed relatively high accumulation in the anterior cingulate cortex, hippocampus, and insular cortex, with the lowest uptake being observed in the pons. Accumulation in the cerebral cortex was significantly diminished by the BZ antagonist flumazenil (0.1 mg/kg, i.v.), but not that in the pons. Using the pons as a reference region, the specific binding of [(11)C]Ro15-4513 in most of the cerebral cortex including the limbic regions clearly revealed two different affinity sites. On the other hand, specific binding in the occipital cortex and cerebellum showed only a low affinity site. Zolpidem with affinity for alpha1, alpha2, and alpha3 subunits of GABA(A)/BZ receptor fully inhibited [(11)C]Ro15-4513 binding in the occipital cortex and cerebellum, while only about 23% of the binding was blocked in the anterior cingulate cortex. Diazepam with affinity for alpha1, alpha2, alpha3, and alpha5 subunits inhibited the binding in all brain regions. Since Ro15-4513 has relatively high affinity for the alpha5 subunit in vitro, these in vivo bindings of [(11)C]Ro15-4513 can be interpreted as the relatively high accumulation in the fronto-temporal limbic regions representing binding to the GABA(A)/BZ receptor alpha5 subunit.     

Chronic pentobarbital administration alters γ-aminobutyric acidA receptor α6-subunit mRNA levels and diazepam-insensitive [3H]Ro15-4513 binding

In order to study the chronic effects of pentobarbital, a positive GABA_A receptor modulator, on the inverse agonist binding of the benzodiazepine site, binding of [³H]Ro15-4513 and levels of GABA_A receptor α₆-subunit mRNA were investigated in the brains of pentobarbital-tolerant/dependent animals, using receptor autoradiography and in situ hybridization histochemistry in consecutive brain sections. Pentobarbital was administered to rats either 60 mg/kg, i.p., once, for acute treatment, or 300 μg/10μl/h i.c.v. continuously for 6 days via osmotic minipumps to render rats tolerant to pentobarbital. Rats assigned to the dependent group were sacrificed 24 h after discontinuance of pentobarbital infusion, while those assigned to the tolerant group were sacrificed at the end of infusion. The α₆ subunit mRNA was increased in the tolerant group only. Diazepam-insensitive [³H]Ro15-4513 binding was increased in the cerebellar granule layer of pentobarbital-tolerant and -dependent rats. No alterations in these parameters were observed in acutely treated animals. These data suggest that chronic pentobarbital treatment induced expression of α₆-subunit mRNA. This was in contrast to α₁- and γ₂-subunit mRNA, which in tolerant animals are unchanged, but for which withdrawal triggers a surge in levels. Because the α₆-subunit is a major component of the diazepam-insensitive [³H]Ro15-4513 binding site, the increased diazepam-insensitive [³H]Ro15-4513 binding implied de novo synthesis of the receptor subunit protein. © 1996 Wiley-Liss, Inc.     

A benzodiazepine antagonist, Ro 15-1788, can block the phase-shifting effects of triazolam on the mammalian circadian clock

A single injection of the short acting benzodiazepine, triazolam, can induce permanent phase advances as well as phase delays in the onset of the circadian rhythm of wheel running behavior in hamsters free-running under constant environmental conditions. If the phase shifting effects of triazolam on the circadian system are mediated through the benzodiazepine-GABA receptor complex, then it should be possible to block these effects with RO 15-1788, a selective benzodiazepine antagonist, which acts at the benzodiazepine-GABA receptor level. To test this hypothesis, hamsters free running in constant light received an intraperitoneal injection of various doses of Ro 15-1788 15 min before a single i.p. injection of 0.5 mg of triazolam. This dose of triazolam is known to induce maximal phase shifts in the circadian rhythm of wheel running behavior in hamster. Treatment with Ro 15-1788 totally blocked both the phase advancing and phase delaying effects of triazolam, while the administration of Ro 15-1788 alone did not phase shift the activity rhythm. These results support the hypothesis that the phase shifting effects of triazolam are mediated through the benzodiazepine-GABA receptor complex. The absence of any phase shifting effects of Ro 15-1788 when delivered alone suggests that Ro 15-1788 has no partial agonist properties in this experimental paradigm.     

Ro 15-1788 suppresses the development of kindling through the benzodiazepine receptor

beta-Carboline (norharman) has been shown to produce kindled seizures when given systemically for long periods of time. The expression of the kindled seizure activity can be blocked by ligands of the benzodiazepine receptor suggesting the receptor as a site of vulnerability in the kindling mechanism. The present data show that Ro 15-1788, a receptor antagonist, suppresses the development of kindled seizures as demonstrated by the delayed appearance of each behavioral stage and the decreased severity of symptoms within each stage. Animals treated with Ro 15-1788 still expressed lower behavioral stages when Ro 15-1788 was eliminated from the trials indicating that this compound suppresses the very process of kindling itself and not just the expression of the kindled seizures. Ro 15-1788 given with norharman causes an increase in Bmax of [3H]flunitrazepam binding to the benzodiazepine receptor in cortex. It is not known if this increase is instrumental in lowering the kindling rate.     

Effect of ethanol on memory consolidation in mice: antagonism by the imidazobenzodiazepine Ro 15-4513 and decrement by familiarization with the environment

Three sets of experiments were carried out with CD1 mice tested in a one-trial inhibitory avoidance task. In a first set of experiments the posttraining administration of ethanol (1 or 2 g/kg) impaired, while that of the imidazobenzodiazepine Ro 15-4513 (5 or 10, but not 2.5 mg/kg) improved the retention performance of the animals. In a second set of experiments a by itself ineffective dose of Ro 15-4513 (2.5 mg/kg) antagonized the effect of ethanol (1 and 2 kg/kg). These results are discussed on the basis of the interaction of these drugs with the GABAergic system. In a third set of experiments, in which the performances of mice familiarized with the apparatus were compared with those of non-familiarized mice, ethanol was less effective in impairing memory processes of the experienced subjects. These results are discussed in terms of attenuation of emotionality, resulting in impaired retention, following posttraining ethanol administration.     

Different functional effect of Ro 15-4513 and Ro 19-4603 on synaptic responses of Purkinje cells in the rat cerebellar slice

Modulation of GABA-mediated neurotransmissions by Ro 15-4513 in cerebellar slices was assessed following stimulation of the parallel fibre input, which, in this preparation, preferentially activates the inhibitory interneurones innervating Purkinje cells. Peristimulus-time histogram analysis of inhibitory responses of spontaneously-active Purkinje cells showed only a decrease in the duration of inhibition induced by Ro 19-4603. This is consistent with inverse agonism on the BZ₁ receptors associated with postsynaptic GABAa receptors on Purkinje cells. 1 μM Ro 15-4513 induced a similar response but 100 nM Ro 15-4513 induced a biphasic response, with an increase in duration of inhibition preceding the decrease during continued perfusion of the compound. At lower concentrations of Ro 15-4513 the increase in inhibition predominated, the minimal effective concentration being 10 pM. 1 μM flumazenil blocked both components of this response to 100 nM Ro 15-4513, but at 100 nM flumazenil only blocked the decrease in inhibition. The ability of Ro 15-4513 but not Ro 19-4603 to enhance inhibition and its relative insensitivity to 100 nM flumazenil, parallel the affinities of these compounds for diazepam-insensitive (DI) binding sites in the cerebellum. These data suggest that the enhancement of inhibition induced by Ro 15-4513 results from its inverse agonist activity on DI receptors causing disinhibition of both granule cells and their parallel fibres and increased sensitivity to the electrical stimuli inducing activation of the inhibitory interneurones innervating Purkinje cells.     

First clinical investigation of the benzodiazepine antagonist Ro 15-1788 in comatose patients

In medical practice a number of antagonists which are capable of abolishing the effect of endogenous substances are available. The discovery of the benzodiazepine antagonist Ro 15-1788 has opened up new possibilities in the treatment and diagnosis of comas of different aetiology. This compound makes it possible to shorten the duration of unconsciousness and to eliminate selectively the effect of the benzodiazepine in an organism which has been intoxicated by different substances. The benzodiazepine antagonist Ro 15-1788 occupies the benzodiazepine receptors in the brain and, without influencing the receptor itself, prevents an active benzodiazepine from binding to this site and from exercising its pharmacological effect. On the basis of 9 clinical examples in special situations, the efficacy of treatment using the benzodiazepine antagonist is presented and discussed.     

Effects of the imidazobenzodiazepine Ro 15-4513 on saccharin choice and acceptance, and on food intake, in the rat

The general aim of the present series of experiments was to investigate the effects of the imidazobenzodiazepine, Ro 15-4513, on ingestional behavior in the rat. The more specific aims were to test its effects on preference for sweet taste, to determine if it acts as a benzodiazepine-receptor inverse agonist, and if it selectively reduces sweetness preference. The results indicated that Ro 15-4513 (1.0-10 mg/kg, i.p.) abolished the preference for a 0.05% sodium saccharin solution in a two-choice test. Water intake in the same test was unaffected. Second, at 10 mg/kg, it suppressed saccharin ingestion in an acceptance test; this effect was completely reversed by the selective benzodiazepine antagonist, Ro 15-1788 (20 mg/kg). Third, Ro 15-4513 (1.0-10 mg/kg) reduced palatable food consumption in non-deprived rats, an effect which was also antagonized by Ro 15-1788. The results are consistent the bidirectional modulation of ingestional responses to palatable taste stimuli as a consequence of drug actions at benzodiazepine receptors. Furthermore, they emphasize that any reduction in consummatory responses produced by Ro 15-4513 is likely to reflect inverse agonist characteristics, as distinct from any putative ethanol antagonist property.     

GABAA/Benzodiazepine receptor binding in patients with schizophrenia using [11C]Ro15-4513, a radioligand with relatively high affinity for alpha5 subunit

Dysfunction of the GABA system is considered to play a role in the pathology of schizophrenia. Individual subunits of GABA(A)/Benzodiazepine (BZ) receptor complex have been revealed to have different functional properties. alpha5 subunit was reported to be related to learning and memory. Changes of alpha5 subunit in schizophrenia were reported in postmortem studies, but the results were inconsistent. In this study, we examined GABA(A)/BZ receptor using [(11)C]Ro15-4513, which has relatively high affinity for alpha5 subunit, and its relation to clinical symptoms in patients with schizophrenia. [(11)C]Ro15-4513 bindings of 11 patients with schizophrenia (6 drug-na?ve and 5 drug-free) were compared with those of 12 age-matched healthy control subjects using positron emission tomography. Symptoms were assessed using the Positive and Negative Syndrome Scale. [(11)C]Ro15-4513 binding was quantified by binding potential (BP) obtained by the reference tissue model. [(11)C]Ro15-4513 binding in the prefrontal cortex and hippocampus was negatively correlated with negative symptom scores in patients with schizophrenia, although there was no significant difference in BP between patients and controls. GABA(A)/BZ receptor including alpha5 subunit in the prefrontal cortex and hippocampus might be involved in the pathophysiology of negative symptoms of schizophrenia.     

Effects of the benzodiazepine receptor ligands midazolam, Ro 15-1788, and Ro 5-4864, alone and in combinations, on platelet serotonin uptake

The benzodiazepine (BZ) agonist midazolam, the BZ antagonist Ro 15-1788, and the peripheral BZ binding site ligand Ro 5-4864 have been assessed, separately and in combinations, as to their effect on the active uptake of serotonin (5HT) in human blood platelets in vitro, in an artificial, protein-free medium. Midazolam had a moderate noncompetitive (or mixed competitive/noncompetitive) inhibitory effect on the uptake. This effect was not influenced by Ro 15-1788, which in itself had only a very weak inhibitory effect. Ro 4-5864 showed in several independent experiments (but not always) a biphasic inhibitory effect, with a moderate but significant inhibition in the concentration range of about 10(-8) to 10(-6) M, and a stronger, noncompetitive inhibitory effect above 10(-6) M. When Ro 5-4864 was tested in the presence of a fixed concentration of midazolam (4 X 10(-6) M), the inhibitory effect of the former was markedly reduced (at higher concentrations), eliminated (at intermediate concentrations), or even reversed into a relative stimulatory effect (at low concentrations). Thus, low concentrations of Ro 5-4864 reduced the inhibitory effect of midazolam. A possible explanation of this interaction is that low concentrations of Ro 5-4864 have both an inhibitory and a stimulatory effect on platelet 5HT uptake (often seen to vary in relative strength between experiments), and that the stimulatory component is unmasked in the presence of a drug that already has an inhibitory effect, probably mediated by the same site.(ABSTRACT TRUNCATED AT 250 WORDS)     

The benzodiazepine antagonist, Ro 15-1788, increases REM and slow wave sleep in the dog

The benzodiazepine antagonist, Ro 15-1788, increases REM and deep slow-wave in dogs at a dose of 10 mg/kg orally. These actions are opposite to that of diazepam. This suggests either that Ro 15-1788 possesses hitherto undescribed intrinsic partial inverse agonist activity or that it is antagonizing an endogenous benzodiazepine-like substance which is involved in modulating sleep-wakefulness.     

Interactions between benzodiazepine antagonists, inverse agonists, and acute behavioral effects of ethanol in mice

The behavioral manifestations of acute ethanol intoxication resemble those of benzodiazepines, barbiturates and general anesthetics. This has led to speculation that these drugs share common mechanisms or sites of actions within the brain. The discovery of a specific benzodiazepine receptor site, and the subsequent development of selective receptor antagonist and inverse agonist drugs, provides a framework to test the involvement of the benzodiazepine receptor complex in mediating ethanol's behavioral effects. The partial inverse agonist Ro15-4513, an analog of the benzodiazepine receptor antagonist Ro15-1788 (flumazenil), has been reported to block or reduce some of ethanol's acute effects in rodents by a benzodiazepine receptor-mediated action. There has been some controversy over whether the "antialcohol" effect of Ro15-4513 is a unique property of this compound or is shared by other benzodiazepine antagonists with inverse agonist activity. We have studied the effects of Ro15-4513 and other benzodiazepine receptor antagonists on acute ethanol intoxication in mice and have obtained evidence that 1) Ro15-4513 differentially affects acute effects of ethanol, 2) an "antialcohol" property is not a general feature of all benzodiazepine antagonists and inverse agonists, and 3) "antialcohol" activity may not be unique to Ro15-4513.     

Chronic treatment with ethanol alters the physiological action of nicotine.

1. Ethanol decreases the release of acetylcholine through effects on presynaptic neurons at both muscarinic and nicotinic junctions. 2. Blockade of the release of acetylcholine should produce denervation supersensitivity at both muscarinic and nicotinic cholinergic junctions. 3. Chronic but not acute treatment with ethanol produces supersensitivity to the hypothermic effects of a muscarinic agonist in the rat. 4. The authors now report that chronic treatment with orally administered ethanol blunts (rather than enhances) the hypothermic response to nicotine in the rat. 5. This could have major public health implications. 6. Smoking and the use of ethanol containing beverages positively covary. 7. Ethanol induced reduction in sensitivity to nicotine suggests that the heavy consumption of ethanol may necessitate that one drink more than otherwise in order to obtain the desired effects of nicotine.