Effects Of Benzodiazepine Receptor Ligands Research Articles

Partial and full agonists/inverse agonists affect [ 35S]TBPS binding at different occupancies of central benzodiazepine receptors

Concentration-dependent effects of benzodiazepine receptor ligands were examined on nonequilibrium binding of t-butylbicyclophosphoro[ 35S]thionate (TBPS, 20 min of incubation at 25°C) to synaptosomal membranes of rat cerebral cortex. Benzodiazepine receptor occupancies were calculated from the displacing potencies of the ligands determined for [ 3H]flumazenil binding under identical conditions. Greater maximal enhancing (i.e. accelerating) effects of the full agonists diazepam and flunitrazepam on [ 35S]TBPS binding were reached at lower occupancies of benzodiazepine receptors than the smaller enhancing effects of the partial agonists bretazenil and the β-carboline ZK 91296. Similarly, the maximal decreasing effect of the full inverse agonist methyl 6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM) on TBPS binding was reached at lower occupancy than that of the partial inverse agonist FG 7142. Half-maximal effects on TBPS binding corresponded to about 20–30% occupancies for the full agonists and DMCM, while for partial agonists and FG 7142 they exceeded 60–80% occupancies. Different (concave versus convex) shapes of the occupancy-effect curves can also differentiate partial from full agonists and inverse agonists. The results suggest that different pharamcological efficacies of benzodiazepine receptor ligands are associated with differences in coupling between benzodazepine and convulsant binding sites to modulate the chloride ionophores.

Abecarnil: a novel anxiolytic with mixed full agonist/partial agonist properties in animal models of anxiety and sedation.

Although the benzodiazepines are without doubt the most effective agents currently available for the treatment of anxiety disorders, they possess a number of properties which limit their use or which give rise to problems on withdrawal. Thus, currently available benzodiazepine anxiolytics are sedative and muscle relaxant, induce memory impairment, increase the intoxicating potency of alcohol, may be subject to nontherapeutic use (abuse), and, following chronic use, may give rise to problems of dependence. For these reasons, it has been the aim of medicinal chemistry to synthesize compounds with the therapeutic strengths of the benzodiazepines, but avoiding their unwanted properties. Given the proven efficacy of anxiolytics acting at benzodiazepine receptors, an obvious approach is to seek compounds acting at this site, but which have been modified so that the unwanted properties of the benzodiazepines have been eliminated. In recent years, two approaches have been used to achieve a better dissociation of the effects of benzodiazepine-receptor ligands; the first entails the use of compounds acting at partial agonists at benzodiazepine receptors (Haefely et al. 1990), but with the discovery of a number of subtypes of y-amino- butyric acid type A (GABAA) receptors, a second approach has become feasible. Although pharmacological evidence for benzodiazepine-receptor heterogeneity has existed for some time (Corda et al. 1988; Squires et al. 1979), it is only recently that the application of molecular biological techniques to the understanding of GABAA receptors has allowed us a fuller understanding of their complexity (see Luddens, this volume).

Strain differences in sensitivity to the hypothermic effects of benzodiazepine receptor ligands in mice

The hypothermic effects of intraperitoneal (IP) administration of the full benzodiazepine agonist loprazolam (1, 10 mg/kg); the partial agonist Ro 17-1812 (1, 10 mg/kg); the benzodiazepine receptor antagonist flumazenil (10, 20 mg/kg); the benzodiazepine inverse agonists Ro 15-4513 (1, 3, 10 mg/kg) and Ro 19-4603 (0.03, 0.1, 0.3 mg/kg) and the beta-carboline inverse agonists FG 7142 (10, 30 mg/kg) and DMCM (1, 3, 10 mg/kg) were investigated in three strains of mice. TO mice were less sensitive than CBA/cA and DBA/2 mice, since only loprazolam and the partial and full beta-carboline inverse agonists FG 7142 and DMCM lowered body temperature in these animals. CBA/cA mice were particularly sensitive to the hypothermic effects of loprazolam and Ro 17-1812, and also responded to the beta-carboline but not the benzo diazepine inverse agonists. In contrast, DBA/2 mice responded with moderate hypothermia to loprazolam, Ro 17-1812, and to the partial inverse agonist Ro 15-4513, and exhibited marked hypothermia in response to the more efficacious benzodiazepine inverse agonist Ro 19-4603 and to FG 7142 and DMCM. Flumazenil did not alter body temperature. DBA/2 mice were also more sensitive to the convulsant activity of inverse agonists than TO mice. CBA/cA mice exhibited enhanced sensitivity to the convulsant, but not the hypothermic, effects of Ro 19-4603, showing dissociation of these responses. The mechanisms underlying the genetic differences in sensitivity of mice to the hypothermic and convulsant action of the different ligands are unknown and warrant further investigation.

Effects of benzodiazepine receptor ligands with different intrinsic activities on seizures induced by inhibition of GAD.

Effects of benzodiazepine receptor ligands with different intrinsic activities on seizures induced by inhibition of GAD.

In vivo bidirectional modulatory effect of benzodiazepine receptor ligands on GABAergic transmission evaluated by positron emission tomography in non-human primates

The central type benzodiazepine receptor (BDZr), an allosteric modulatory site of the GABA A receptor-anion channel, has been shown in vitro to respond to drugs with positive efficacy (agonists), zero efficacy (competitive antagonists) and drugs with negative efficacy (inverse agonists). However, this general concept of the function of BDZr drugs has rarely been assessed in intact living brain. We report here in on a non-invasive in vivo assessment of the intrinsic efficacies of BDZr drugs in the brain of non-human primates. We have performed an in vivo simultaneous determination of fractional BDZr occupancy and the resulting pharmacological efficacies of the full agonist diazepam, the partial agonist bretazenil, the antagonist flumazenil (Ro15-1788), the partial inverse agonist Ro15-4513 and the full inverse agonist methyl β-carboline-3-carboxylate (β-CCM). Positron emission tomography (PET) was used to estimate fractional BDZr occupancy measured as the in vivo displacement in the brain of the positron emitter radioligand, [ 11C]flumazenil. Simultaneously, the proconvulsant or anticonvulsant efficacies of the BDZr drugs were measured as their abilities to facilitate or counteract the central effects of an infusion of pentylenetetrazol, a non-competitive GABA antagonist acting on the picrotoxin site of the receptor complex. This was measured using electroencephalographic recording (EEG). Our results show that, in vivo, the fractional receptor occupancy by a given drug is perfectly correlated with its resulting graded pharmacological effects, as predicted from the competitive drug receptor interaction theory. Furthermore, the slope of the relationship between fractional receptor occupancies and the resulting pharmacological effects (an index of intrinsic efficacy) strictly depends on the BDZr ligand considered. Diazepam displayed a strong positive intrinsic efficacy and, in contrast, β-CCM a marked negative one. Between these two extremes, the partially active drugs bretazenil and Ro15-4513, which required a large fractional receptor occupancy to produce significant anti- or proconvulsant effects, respectively, displayed only a weak intrinsic efficacy. Flumazenil did not produce any significant pharmacological effect. We observed that the in vivo intrinsic efficacies of diazepam, flumazenil and β-CCM correlate with their intrinsic efficacies as measured by their modulatory effects on the GABA-dependent membrane chloride conductance in vitro. Thus, the intrinsic efficacies measured using PET and EEG are likely to reflect the different in vivo abilities of BDZr drugs to induce or stabilize the GABA A-benzodiazepine chloride channel in a given conformation. Our results confirm the general concept that, in vivo, the bidirectional effects (anti- to proconvulsant) of BDZr drugs are mainly the result of their different intrinsic efficacies in modulating the effects of GABA on the chloride channel.

Opposite effects of agonist and inverse agonist ligands of benzodiazepine receptor on self-defensive and submissive postures in the rat.

The effects of benzodiazepine receptor ligands on different types of defensive behaviours were examined in intruder male rats confronted with offensive residents. Chronic administration, via a subcutaneous silastic pellet, of a full agonist (diazepam) for 15 days increased self-defensive postures as well as social and non-social behaviour whereas submissive postures and flight were reduced. Acute administration of a partial agonist (ZK 91296) resulted in a similar increase in self-defensive postures and a decrease of submission and non-social elements. Acute administration of a partial inverse agonist (FG 7142) reduced defensive postures and social behaviour whereas submissive postures were increased. These results show that activation of benzodiazepine receptors by full or partial agonists increased self-defensive responses to attacks by a conspecific, while decreasing submissive postures. On the contrary, "inverse activation" of these receptors by an inverse agonist increased submissive postures while decreasing self-defensive responses. These data suggest that benzodiazepine receptors are involved in the control of the animal's strategy to respond to an attack of another rat.

Behavioral analysis of the effects of benzodiazepine receptor ligands in the conditioned burying paradigm

In the conditioned burying paradigm, 10 mutually exclusive behavioral sequences were encoded in order to characterize the effects of various benzodiazepine receptor ligands. It was found that each ligand produced a specific behavioral pattern, the elements of which were consistent with results obtained in other experimental situations. Whereas anxiolytic (diazepam, Ro 16-6028) and ‘anxiogenic’ ligands (FG 7142) both decreased the mean duration of burying, these two classes of drugs largely differed when approaches to the prod and escape movements away from the prod were considered. The benzodiazepine receptor antagonist Ro 15-1788 was devoid of effects on all of the behavioral sequences considered. The main characteristics of these behavioral profiles persisted during an extinction test, although the animals were no longer treated. It is proposed that additional behavioral measures, or better, an ethological analysis of the behavior may increase the potency of the conditioned defensive burying paradigm.

Behavioral differences in an elevated plus-maze: correlation between anxiety and decreased number of GABA and benzodiazepine receptors in mouse cerebral cortex.

In an elevated plus-maze model of anxiety mice treated with the benzodiazepine inverse agonist DMCM (0.5-1.5 mg/kg i.p.) spent significantly less time on the open arms and showed the decreased number of open arm entries. The opposite i.e. increased time spent on the open arms and the higher number of open arm entries was registered after diazepam (1.5 mg/kg). The results are consistent with the results obtained in the other animal tests and support the idea that this procedure is suitable for detecting anxiolytic/anxiogenic effects of benzodiazepine receptor ligands. After testing of 84 mice in an elevated plus-maze substantial differences were detected between the individuals. According to the behavioral response two subgroups of animals with DMCM or diazepam like exploratory activity (as compared to the whole group data) termed as "anxious" or "non-anxious", respectively, were chosen for further binding studies. "Anxious" animals had significantly lower numbers of 3H-flunitrazepam and 3H-muscimol binding sites as compared to "non-anxious" animals in cerebral cortex but not in cerebellum. No differences in the affinity were found between the two groups studied. The results indicate that behavioral anxiety in mice is in correlation with the decreased number of GABA and benzodiazepine receptors in cerebral cortex.

Bidirectional effects of β-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 93426, 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.

Lorazepam and FG 7142 Induce Tolerance to the DMCM Antagonistic Effect of Benzodiazepine Receptor Ligands

Mice were given chronic treatment with lorazepam 10 mg/kg PO or FG 7142 40 mg/kg IP once a day for 14 days. The pretreatments with lorazepam and FG 7142 did not change the sensitivity of the mice to the convulsant effect of DMCM. Lorazepam pretreated mice showed a significantly lower sensitivity to the anticonvulsant effects of the benzodiazepine (BZ) receptor ligands lorazepam, ZK 93423, ZK 91296, Ro 15-1788 and ZK 93426 administered acutely by the IP route when challenged with DMCM 24 hr after the last dose of lorazepam. FG 7142 pretreated mice showed a significantly lower sensitivity to the anticonvulsant effect of the two agonists lorazepam and ZK 93423 and to the antagonist Ro 15-1788, whereas the effects of ZK 91296 and ZK 93426 were left unchanged. The reduced DMCM antagonistic effects of the BZ receptor ligands may indicate that these ligands may either have lost some of their affinity to those BZ receptors being responsible for the DMCM-induced seizures or they may have lost some efficacy in allosterically inhibiting DMCM binding or as a third possibility may have lost efficacy at a BZ receptor site downstream to the seizure-inducing center in the brain.

Differential effects of benzodiazepine receptor ligands on isotonic saline and water consumption in water-deprived rats

Water-deprived male rats were adapted to a 30 min test of water or saline drinking in a single-bottle acceptance test. The potent benzodiazepine agonist, clonazepam, produced significant increases in both water and saline consumption. Increases in the consumption of both were also obtained with the non-benzodiazepine agonist, zopiclone (a cyclopyrrolone), but not with the pyrazoloquinoline agonist, CGS 9896. Hence, some but not all, benzodiazepine receptor agonists enhance drinking responses. The benzodiazepine receptor antagonists, Ro15-1788 and CGS 8216, had no significant effect on the intake of either isotonic saline or water. In contrast, the β-carboline FG 7142, which has been described as an inverse agonist acting at benzodiazepine receptors, reduced both saline and water drinking at 10 and 20 mg/kg. Although the baseline level of saline drinking was considerably higher than that of water, there was no general indication that any drug effect on consumption interacted with the type of fluid in the drinking test. However, in the case of agonist-induced increases in consumption, peak effects occurred at different doses; they were lower saline- than for water-drinking.

Selective changes in the in vivo effects of benzodiazepine receptor ligands after chemical kindling with FG 7142

It has recently been demonstrated that kindling occurs with repeated administration of the benzodiazepine “inverse agonist” FG 7142. The present study was an investigation of the effects of other ligands for the benzodiazepine receptor in mice kindled with FG 7142. It was shown that over a range of doses the lowering effects of FG 7142 on the seizure threshold were greater in kindled animals than in control. In contrast, the hypothermie effect of FG 7142 was unaltered. The effects of the partial inverse agonist CGS 8216 were unaltered. The effects of the full inverse agonist DMCM were unchanged except for an enhancement of its convulsant effect when infused at a concentration of 100 μgm 1 −1. Studies with the full agonist benzodiazepine, flurazepam and the full agonist β-carboline, ZK 93423, showed small but significant reductions in their hypothermic effects. The sedative and anticonvulsant effects of flurazepam were unaltered, whereas the anticonvulsant effects of ZK 93423 were decreased in animals kindled with FG 7142. There was a pronounced reduction in the anticonvulsant and hypothermic effects of the partial agonist β-carboline, ZK 91296. These data do not fit any simple explanation of kindling being due to a change in the function of benzodiazepine receptors, although they may offer some support for the idea that kindling with FG 7142 produces a change in the effects of all β-carboline compounds which act at the benzodiazepine receptor.

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

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.

Benzodiazepine antagonist Ro 15-1788: binding characteristics and interaction with drug-induced changes in dopamine turnover and cerebellar cGMP levels.

The recently discovered benzodiazepine antagonist Ro 15-1788 was characterized in binding studies, and its potency and selectivity were determined in vivo by interaction with drug-induced changes in dopamine turnover and cerebellar cGMP level. Ro 15-1788 reduced [3H]flunitrazepam binding in the brain in vivo with a potency similar to that of diazepam and effectively inhibited [3H]diazepam binding in vitro (IC50 = 2.3 +/- 0.6 nmol/liter). [3H]Ro 15-1788 bound to tissue fractions of rat cerebral cortex with an apparent dissociation (KD) of 1.0 +/- 0.1 nmol/liter. The in vitro potency of various benzodiazepines in displacing [3H]Ro 15-1788 from its binding site was of the same rank order as found previously in [3H]diazepam binding. Autoradiograms of [3H]Ro 15-1788 binding in sections of rat cerebellum showed the same distribution of radioactivity as with [3H]flunitrazepam. The attenuating effect of diazepam on the chlorpromazine- or stress-induced elevation of homovanillic acid in rat brain was antagonized by Ro 15-1788. Among a series of compounds which either decreased or increased the rat cerebellar cGMP level, only the effect of benzodiazepine receptor ligands (diazepam, zopiclone, CL 218 872) was antagonized by Ro 15-1788. Thus, Ro 15-1788 is a selective benzodiazepine antagonist acting at the level of the benzodiazepine receptor in the central nervous system. Peripheral benzodiazepine binding sites in kidney and schistosomes were not affected by Ro 15-1788.