Bidirectional effects of chronic treatment with agonists and inverse agonists at the benzodiazepine receptor

We have studied in rodents the effects of beta-carboline inverse agonists on chronic treatment and after repeated administration of benzodiazepine agonists. Chronically, the inverse agonist FG 7142 caused chemical kindling, i.e., a decrease in the threshold to the convulsive effects of the drug. This change was accompanied by decreases in the effects of beta-carboline but not benzodiazepine agonists. In addition the effects of GABA receptor agonists were decreased and the effects of GABA antagonists marginally increased. The GABA stimulated benzodiazepine binding was lower after FG 7142 kindling. Some evidence was found in mice to suggest that these changes were accompanied by behavioural alterations, but studies in rats did not show any changes. Repeated administration of benzodiazepine agonists, sufficient to cause tolerance to their pharmacological actions and to those of beta-carboline agonists, increased all of the effects of the partial inverse agonists and some of the actions of the full inverse agonists. We suggest that this is due not to precipitation of withdrawal but to a "withdrawal shift" in the coupling at the receptor inophore. This would increase the intrinsic properties of inverse agonists and decrease those of agonists. Evidence for this hypothesis is summarised.     

Enhanced sensitivity to β-carboline inverse agonists in rats chronically treated with FG 7142

The biochemical and behavioural effects of the chronic administration of the beta-carboline inverse agonist FG 7142 were studied in the rat. Repeated administration of FG 7142 (15 mg/kg IP, twice daily for 10 consecutive days) induced sensitization to the effects of this drug, which from proconvulsant became a full convulsant. Thus, myoclonic seizures were observed in 30% and 80% of the animals by the third and the eighth day of treatment, respectively. The sensitization to the convulsant effect of FG 7142 persisted for up to 50 days after withdrawal and was completely prevented by the concurrent administration of the benzodiazepine receptor antagonist Ro15-1788 (15 mg/kg IP, twice a day for 10 days). Moreover, four to twelve days after withdrawal from chronic treatment with FG 7142, an increased sensitivity to the proconvulsant beta CCE and to the convulsant DMCM was observed. In addition, convulsions induced by isoniazid (350 mg/kg, SC) were potentiated in rats chronically treated with FG 7142 at 5 and 20 days after withdrawal. These pharmacological effects were paralleled by a decrease in the density of low affinity GABA receptors in the cerebral cortex and cerebellum. These results are consistent with the view that repeated administration of FG 7142 induces a long-lasting down-regulation of the GABAergic function which results in an increased sensitivity to beta-carboline inverse agonists and isoniazid. The possibility that a concomitant decrease in the responsiveness to benzodiazepines and Ro15-1788 takes place after chronic treatment with FG 7142 is also discussed.     

Behavioral neurobiology of inverse agonist FG 7142 induced anxiety syndrome in rats

1. Neurobehavioral survey of inverse agonist FG 7142 was performed employing a novel anxiety paradigm namely FR-2 Two-Way crossover in a shuttle box. 2. FG syndrome was found to be similar to learned helplessness following shock treatment. Significant increase in mean latency to escape was observed from 0 to 25th trial. Effect of FG 7142 on the behavioral and neurological profile did not deviate significantly from controls. However, a general increase in arousal, darting and sideway movement (weaving) of the head were noted. 3. Drugs with specificity at benzodiazepine (BDZ) receptor site were employed as pretreatments in order to study their influence on FG 7142 induced anxiety syndrome. Diazepam and ZK 91296, significantly blocked the inverse agonist response. Head weaving as well as darting movements were completely abolished although rats demonstrated arousal and vigilance. 4. A dose dependent inhibition of FG response was observed with RO 15-1788, a specific BDZ receptor blocker. ZK 93423 at low doses (1 mg/kg) failed to reverse the escape deficit induced by FG. Higher doses of ZK 93423 could not be tested as significant sedation and ataxia were noted. 5. It is suggested that a careful combination of a BDZ agonist and inverse agonist may be beneficial in the treatment of generalised anxiety disorders.     

Bidirectional effects of beta-carbolines and benzodiazepines on cognitive processes

Experiments with benzodiazepine receptor ligands in two paradigms involving cognitive processing were performed in order to test whether the concept of bidirectional effects of benzodiazepine receptor ligands could also be applied to cognitive functions. Benzodiazepine receptor agonists like chlordiazepoxide, lorazepam, ZK 93423 and ZK 91296 induced amnesia in a passive avoidance paradigm. Mice treated with the benzodiazepine receptor antagonist, ZK 93426, reached a learning criterion after fewer foot-shocks than saline treated mice both in naive animals and in scopolamine pre-treated animals. Furthermore, ZK 93246, attenuated the amnesic effect of corneal electroshock. The inverse agonists FG 7142 and DMCM decreased the detrimental effect of scopolamine on retrieval. In a signal detection paradigm, chlordiazepoxide impaired signal detection. In aged rats ZK 93426, ZK 90886 and FG 7142 had no effect on signal detection but ZK 93426 and FG 7142 attenuated the impairment of signal detection induced by scopolamine. These effects of benzodiazepine receptor ligands may reflect changes in arousal/vigilance, suggesting that BZ inverse agonists may have useful properties in enhancing vigilance.     

Suppression of the immune response by benzodiazepine receptor inverse agonists

24 h after administration of a single dose of the benzodiazepine receptor inverse agonists N'-methyl-beta-carboline-3-carboxamide (FG 7142) and 3-carbomethoxy-4-ethyl-6,7-dimethoxy-beta-carboline (DMCM), a profound suppression of the immune response was observed in rodents. This immunosuppression was manifest as a decrease in phytohemagglutinin (PHA) and concanavalin-A (Con-A) stimulated T cell proliferation in rats and mice administered FG 7142 and a decrease in allogeneic cytotoxic T lymphocyte activity in mice administered either FG 7142 or DMCM. The effects of FG 7142 were antagonized by the prior administration of Ro 15-1788, a benzodiazepine receptor antagonist. These findings demonstrate that the neural pathways subserved by benzodiazepine receptors can modulate immune function, and suggest that these receptors may be involved in the stress-induced modulation of immune function.     

Kindling with the beta-carboline FG7142 suggests separation between changes in seizure threshold and anxiety-related behaviour

The aim of this paper was to determine whether the prolonged decrease in seizure threshold produced by chemical kindling was accompanied by behavioural changes in tests of anxiety and aggression. The responses of rats in five tests were examined after chronic treatment with the benzodiazepine inverse agonist FG7142. This treatment caused chemical kindling, so that the originally proconvulsant drug caused full seizures. This effect is very long-lasting, and our previous work with mice had suggested that it might be accompanied by an increase in anxiety-related behavior. In the present work no significant differences were found between the behaviour of FG7142-kindled rats and vehicle-treated controls in social interaction test, elevated plus maze, or the Vogel conflict test of anxiety or in tests of home cage aggression or startle responses. The results therefore show that prolonged changes in seizure threshold can occur without alterations in the apparent level of anxiety.     

Bidirectional changes in the consumption of food produced by β-carbolines

β-Carboline derivatives provide examples of benzodiazepine receptor ligands which span the range: full agonist-partial agonist-antagonist-partial inverse agonist-full inverse agonist. Taken together, the effects of these compounds illustrate two important principles: firstly, the bidirectionality of effects which can be achieved using benzodiazepine receptor ligands; secondly, the selectivity of effects which are produced by partial agonists. Applied to the study of feecling processes, these principles imply that both hyperphagic and anorectic effects can be generated by actions of selected ligands at benzodiazepine receptors. Furthermore, they suggest that a hyperphagic effect may occur in the absence of side-effects (e.g., sedation, muscle-relaxation), which are characteristic of classical benzodiazepines. Experimental data in support of these predictions are presented. A microstructural approach to feecling behaviour indicated that a benzodiazepine receptor agonist and an inverse agonist extend and abbreviate, respectively, the duration of individual bouts of eating. Preference for a saccharin solution was attenuated by the β-carboline inverse agonist, FG 7142, but rejection of a quinine solution was not increased. Adrenalectomy had no effect on the anorectic effect of inverse agonists.     

Bidirectional changes in the consumption of food produced by beta-carbolines

beta-Carboline derivatives provide examples of benzodiazepine receptor ligands which span the range: full agonist-partial agonist-antagonist-partial inverse agonist-full inverse agonist. Taken together, the effects of these compounds illustrate two important principles: firstly, the bidirectionality of effects which can be achieved using benzodiazepine receptor ligands; secondly, the selectivity of effects which are produced by partial agonists. Applied to the study of feeding processes, these principles imply that both hyperphagic and anorectic effects can be generated by actions of selected ligands at benzodiazepine receptors. Furthermore, they suggest that a hyperphagic effect may occur in the absence of side-effects (e.g., sedation, muscle-relaxation), which are characteristic of classical benzodiazepines. Experimental data in support of these predictions are presented. A microstructural approach to feeding behavior indicated that a benzodiazepine receptor agonist and an inverse agonist extend and abbreviate, respectively, the duration of individual bouts of eating. Preference for a saccharin solution was attenuated by the beta-carboline inverse agonist, FG 7142, but rejection of a quinine solution was not increased. Adrenalectomy had no effect on the anorectic effect of inverse agonists.     

Low and high doses of benzodiazepine receptor inverse agonists respectively improve and impair performance in passive avoidance but do not affect habituation

The benzodiazepine receptor inverse agonists, methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) and N-methyl-beta-carboline-3-carboxamide (FG 7142), were given to rats at various stages of a passive avoidance task. When the drugs were given before trial 1, low doses enhanced, and high doses impaired, performance as assessed 24 h later. A group given drugs on both trials showed that the impairment was not due to state-dependent effects. When the drugs were given immediately after trial 1, or before trial 2, they were without effect, except for the low dose of DMCM which impaired consolidation. It is discussed whether the changes in passive avoidance performance are due to direct or indirect effects. Between-trial habituation of exploratory head-dipping was measured in a holeboard. When FG 7142 was given before trial 1, the high dose impaired between-trial response decrement; but this was because it decreased the level of head-dipping on trial 1. When FG 7142 was given immediately after trial 1, or before trial 2, it was without effect on between-trial habituation.     

Stimulation of cortical acetylcholine efflux by FG 7142 measured with repeated microdialysis sampling

The effects of the benzodiazepine receptor partial inverse agonist β-carboline FG 7142 on cortical ACh efflux were determined using in vivo microdialysis in freely-moving rats. Additionally, a within-subjects, repeated-dialysis experimental design (four microdialysis sessions; removable dialysis probe) was evaluated as a method for measuring changes in basal and FG 7142-stimulated ACh efflux in the frontoparietal cortex. FG 7142 (4.0, 8.0, and 16.0 mg/kg) produced a 150–470% increase in cortical ACh efflux, with a dose-dependent effect on the duration of the increase in efflux. Basal cortical ACh efflux was lower in session 4 than in session 1. However, the ability of FG 7142 to stimulate efflux was unchanged by repeated dialysis testing. The ability of tetrodotoxin (1.0 μM) to suppress both basal and FG 7142-stimulated ACh efflux was also unaffected by repeated dialysis testing. These results demonstrate that systemically administered benzodiazepine receptor inverse agonists stimulate cortical ACh efflux, and that repeated-measures experimental designs can be valid for determining certain changes in cortical ACh efflux with in vivo microdialysis. © 1995 Wiley-Liss, Inc.     

A PET study following treatment with a pharmacological stressor,FG7142, in conscious rhesus monkeys

FG7142 is a benzodiazepine partial inverse agonist, which is known as a pharmacological stressor. Several reports demonstrated that FG7142 produced anxiety in humans, non-human primates, and rodents, and impaired working memory in non-human primates and rodents. In this study, we examined the effect of FG7142 on cerebral blood flow and glucose metabolism using positron emission tomography (PET) in conscious rhesus monkeys. Male rhesus monkeys were intramuscularly treated with FG7142 (0.2 or 1.0 mg/kg, n=5, respectively), and regional cerebral blood flow (rCBF) and regional cerebral metabolic rate of glucose (rCMRglc) were measured by PET 20 min and 40 min after treatment, respectively. During PET measurement, physiological parameters and plasma cortisol levels were monitored. FG7142 significantly decreased rCBF in the thalamus and rCMRglc in all brain regions examined in a dose-dependent manner without changes in physiological parameters. FG7142 also significantly increased plasma cortisol levels. The present study may provide an important insight into the understanding of the pathophysiology of anxiety and stress-related disorders in humans, and strongly suggesting that prevention of anxiety or stress is important when measuring conscious brain function.     

Beta-carbolines characterized as benzodiazepine receptor agonists and inverse agonists produce bi-directional changes in palatable food consumption

Drugs which bind to specific benzodiazepine recognition sites fall into three categories: agonists, antagonists, and inverse agonists. A set of biochemical parameters is available which distinguishes between the three. In addition, actions of the drugs result in physiological and behavioural effects which are distinguishable. beta-Carboline derivatives provide a group of compounds which show high affinity for the benzodiazepine sites, and which contains examples belonging to each of the three categories. Evidence is reviewed which shows that beta-carboline benzodiazepine receptor agonists (ZK 93423, ZK 91296) produce increases in the consumption of a palatable diet by non-deprived rats, that beta-carboline inverse agonists (FG 7142, DMCM) produce an anorectic effect, and that the beta-carboline ZK 93426 acts as a benzodiazepine receptor antagonist. The results support the proposal of bi-directional control of feeding responses through the action of drugs at a common benzodiazepine receptor. Furthermore, benzodiazepine receptor inverse agonists provide a novel class of anorectic agents. Evidence is also reviewed which is suggestive of the modulation of food-related reward by drug actions at benzodiazepine receptors.     

Chronic treatment with diazepam or the inverse benzodiazepine receptor agonist FG 7142 causes differential changes in the effects of GABA receptor stimulation

Daily treatment of mice with diazepam leads to the development of tolerance to the anxiolytic and anticonvulsant effect of the benzodiazepine, while daily treatment with the proconvulsant benzodiazepine receptor inverse agonist FG 7142 produces sensitization to its effects in that seizures develop (chemical kindling). In the present study, the effects of GABA receptor stimulation were studied 2 days after termination of 13 days treatment with diazepam, 20 mg/kg i.p./day, and FG 7142, 40 mg/kg i.p./day. For GABA receptor stimulation, the GABA agonist progabide was chosen because among several GABA receptor stimulants tested it was the only compound that induced increases in seizure threshold in non-toxic doses. Using the threshold for maximal (tonic extension) electroconvulsions as a measure for anticonvulsant efficacy, the anticonvulsant effect of progabide (100 mg/kg i.p.) was unchanged after chronic treatment with diazepam but was lost in FG 7142 kindled animals. Conversely, the hypothermic effect of progabide was reduced after treatment with diazepam but not with FG 7142. Baseline seizure threshold was unchanged 2 days after chronic administration of diazepam but increased in the FG 7142 pretreated mice. The data indicate that tolerance to benzodiazepines and kindling by FG 7142 are associated with different changes in GABA receptor function.     

Bidirectional effects of beta-carbolines in rats with spontaneous petit mal-like scizures

Seven benzodiazepine-receptor ligands of the beta-carbolines' group were administered IP in Wistar rats from (1) a strain displaying spontaneous petit mal-like seizures (PMLS) characterized by spike and wave discharges (SWD) and, (2) a strain where no seizure is ever observed (NS). Five different types of effects were observed. (1) Injection of a full agonist (ZK 93 423) suppressed SWD in PMLS rats, in a dose-dependent manner, and induced marked sedation with alteration of EEG background activity; (2) Injection of partial agonists (ZK 95 962, ZK 91 296) suppressed SWD in PMLS rats without sedation; (3) Injection of low doses of a full inverse agonist (DMCM) significantly increased the total duration of SWD in PMLS rats and induced SWD in NS rats. Higher doses of DMCM induced convulsions in both strains; (4) Injection of partial inverse agonists (FG 7142, ZK 90 886) aggravated SWD in PMLS and induced SWD in NS rats. FG 7142 induced convulsions only in PMLS animals, whereas no convulsions were ever observed with ZK 90 886; (5) Injection of an antagonist (ZK 93 426) did not significantly modify SWD in PMLS rats. However, this compound was able to reverse both antiepileptic effects of agonists and epileptogenic effects of inverse agonists. These results suggest the involvement of the benzodiazepine-GABA receptor complex in the control of petit mal-like seizures in rats.     

The anorectic effect of FG 7142, a partial inverse agonist at benzodiazepine recognition sites, is reversed by CGS 8216 and clonazepam but not by food deprivation

The benzodiazepine partial inverse agonist N′-methyl-β-carboline-3-carboxamide (FG 7142; 5.0 and 10.0 mg/kg, i.p.) produced a dose-dependent reduction in the consumption of a familiar, highly palatable diet by non-food-deprived male rats. At dose levels which exhibited no significant intrinsic effects, the benzodiazepine receptor antagonist 2-phenylpyrazolo-[4,3-c]-quinoline-3(5H)-one (CGS 8216; 1.25–5.0 mg/kg, i.p.) reversed the anorectic effect of FG 7142. When clonazepam and FG 7142 were given in combination, mutual cancelling of their opposite effects occurred. These results are consistent with an action of FG 7142 at benzodiazepine recognition sites to reduce the level of palatable food consumption, and imply that a bidirectional control of food intake via benzodiazepine recognition sites can be achieved. The anorectic effect of FG 7142 was not reversed by 24-h food deprivation, indicating a possible separation from the effects of hunger mechanisms.     

Suppression of natural killer cell activity by FG 7142, a benzodiazepine receptor "inverse agonist"

A dose-dependent (5-50 mg/kg) suppression of natural killer (NK) cell activity was observed 2 h after administration of the benzodiazepine receptor "inverse agonist" FG 7142 (N-methyl-beta-carboline-3-carboxamide), and was still manifest 24 h later. Addition of FG 7142 (1-1000 nM) to the 4 h 51 Cr release assay did not affect NK cell activity. Pretreatment of mice with the benzodiazepine receptor antagonist Ro 15-1788 (10 mg/kg) blocked FG 7142-induced suppression of NK cell activity, but had no effect when administered alone. The suppression of NK cell activity by FG 7142, a compound which produces a syndrome resembling stress or anxiety in both animals and man, provides further evidence that the central nervous system pathways subserved by the benzodiazepine/GABA receptor chloride channel complex ("supramolecular complex") may play a role in the modulation of immune function.     

β-Carbolines characterized as benzodiazepine receptor agonists and inverse agonists produce bi-directional changes in palatable food consumption

Drugs which bind to specific benzodiazepine recognition sites fall into three categories: agonists, antagonists and inverse agonists. A set of biochemical parameters is available which distinguishes between the three. In addition, actions of the drugs result in physiological and behavioural effects which are distinguishable. β-Carboline derivatives provide a group of compounds which show high affinity for the benzodiazepine sites and which contains examples belonging to each of the three categories. Evidence is reviewed which shows that β-carboline benzodiazepine receptor agonists (ZK 93423, ZK 91296) produce increases in the consumption of a palatable diet by non-deprived rats, that β-carboline inverse agonists (FG 7142, DMCM) produce an anorectic effect and that the β-carboline ZK 93426 acts as a benzodiazepine receptor antagonist. The resuks support the proposal of bi-directional control of feeding responses through the action of drugs at a common benzodiazepine receptor. Furthermore, benzodiazepine receptor inverse agonists provide a novel class of anorectic agents. Evidence is also reviewed which is suggestive of the modulation of food-related reward by drug actions at benzodiazepine receptors.     

Central and peripheral type benzodiazepine ligands displace [3H][3-ME-HIS2]TRH from its binding sites in the brain and the anterior pituitary and antagonize the effect of TRH in the rat duodenum.

The effects of central (clonazepam, an agonist, and FG 7142, an inverse agonist), mixed (diazepam) or peripheral type (Ro 5-4864) benzodiazepine receptor ligands on the action of TRH on the transmurally stimulated rat duodenum and binding of [3H][3-Me-His2] TRH in the rat anterior pituitary, hypothalamus, cortex and brainstem have been studied. TRH dose-dependently inhibited the contractions of transmurally stimulated rate duodenum. Clonazepam (5 x 10(-6) M), diazepam (10(-5) M), Ro 5-4864 (10(-5) M) or FG 7142 (10(-5) M) attenuated the response of TRH in the rat duodenum. The action of these compounds was antagonized neither by the central type benzodiazepine antagonist flumazenil nor by peripheral type antagonist PK 11195 but instead PK 11195 itself counteracted TRH. TRH displaced [3H][3-Me-His2]TRH with Ki-values ranging 0.08 to 0.31 microM. Ki-values for clonazepam diazepam, Ro 5-4864, PK 11195 and FG 7142 ranged 6-117 microM, 3-23 microM, 20-67 microM, 20-40 microM and 260-420 microM, respectively, demonstrating fairly weak affinity to TRH-receptors. In saturation experiments, clonazepam and PK 11195 significantly increased KD but not Bmax of the labelled ligand while Ro 5-4864 increased both KD and Bmax. This indicates that all these compounds competitively inhibit the binding of [3H][3-Me-His2]TRH in the CNS which may also be the mechanism for their antagonism of the effect of TRH in the rat duodenum.     

Bidirectional effects of benzodiazepine receptor ligands against picrotoxin- and pentylenetetrazol-induced seizures

Summary The dose response curves of picrotoxin-induced seizures and pentylenetetrazol-induced seizures were shifted to the right by the benzodiazepine (BZ) receptor agonist lorazepam, and to the left by the inverse agonists, DMCM, ZK 90886, FG 7142 and CGS 8216. The BZ receptor antagonists ZK 93426 and Ro 15-1788 had no effect on the dose response curves. The anticonvulsive action of lorazepam and the proconvulsive action of DMCM against picrotoxin-induced seizures and against pentylenetetrazol-induced seizures was inhibited by low doses of ZK 93426 and Ro 15-1788.These results indicate that the bidirectional effects of benzodiazepine receptor ligands on picrotoxin and pentylenetetrazol induced seizures is actually mediated through benzodiazepine receptors.     

Differential sensitivity to inverse agonists of GABA(A)/benzodiazepine receptors in rats with genetic absence-epilepsy.

A strain of Wistar rats, genetic absence epilepsy rats from Strasbourg (GAERS), was selected and inbred over 40 generations for occurrence of spontaneous spike-wave discharges characteristic of absence seizures, simultaneously with a strain of non-epileptic rats (NER). GAERS demonstrate an excessive sensitivity to antagonists of the GABA(A) receptor. The sensitivity to convulsions induced by various inverse agonists of the GABA(A)/benzodiazepine receptor was compared in GAERS and NERs. The beta-carbolines FG 7142 and DMCM, and the imidazobenzodiazepines RO 19-4603 and the alpha 5-selective RY 024 were several times more convulsant in GAERS than in NERs. The largest differences were found with the non-selective RO 19-4603- and FG 7142. The proconvulsant imidazobenzodiazepine RO 15-4513, binding also to diazepam-insensitive receptors, had low efficacy. The high affinity binding of GABA(A)/BZD receptors with (3H) RO 15-1788 in the brain of naive rats and after administration of FG 7142 did not differ in GAERS and NERs. The data indicate that the hypersensitivity of GAERS to various inverse agonists of the GABA(A)/benzodiazepine receptor involves cortical GABA(A) receptors and is not related to differential activity of a subunit-selective receptor.