The multiplicity of actions of benzodiazepine receptor ligands.

The benzodiazepine receptor is an allosteric modulatory site present on most, if not all, gamma-aminobutyric acid A (GABAA) receptor channels (GABAA-R). The benzodiazepine receptor recognizes a large spectrum of compounds from different chemical classes that are grouped together as benzodiazepine receptor ligands--of benzodiazepine and non benzodiazepine structure. The GABAA-R is thought to be a heteropentameric protein complex composed of at least three different classes of subunits, with each subunit comprised of up to six structural variants. Binding of GABA to the extracellular domain of the receptor causes a conformational change that opens the channel pore to anions. A classical benzodiazepine achieves a positive allosteric modulation of the GABA channel gating function by increasing the affinity of the receptor for GABA and, possibly, by facilitating the conformational transition from the closed to the open form (benzodiazepine receptor agonists). Inverse agonists of benzodiazepine receptors cause negative allosteric modulation (a decrease in the GABA activity). Benzodiazepine receptor antagonists bind to the benzodiazepine receptor with little effect on GABAA-R functioning. The intrinsic efficacy of benzodiazepine receptor ligands determines the direction and magnitude of allosteric modulation. Benzodiazepine receptor agonists affect neuronal activity in all major neuronal networks. The classical pharmacological profile of benzodiazepine receptor agonists consists of anxiolytic, anticonvulsant, sedative, and myorelaxant activities. Partial agonists of benzodiazepine receptors conserve anxiolytic and anticonvulsant activity, with greatly reduced sedation and muscle relaxation. They promise to present therapeutic advantages, in particular for long term use. In initial studies. they have produced fewer side-effects and showed reduced tolerance development and physical dependence liability.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for a role of 5-HT1C receptors in the antiserotonergic properties of some antidepressant drugs.

A variety of antidepressants of different chemical classes were tested for their in vivo and in vitro activity at 5-HT1C receptors in the brain. Conventional tricyclic antidepressants (imipramine, desipramine, maprotiline, clomipramine, trimipramine, amitriptyline, nortriptyline, doxepin, amoxapine) as well as mianserin and trazodone were found to display high to low nanomolar affinity for 5-HT1C receptors. Antidepressants of other chemical classes and with other mechanisms of action (affecting amine uptake systems: fluoxetine, citalopram, sertraline, fluvoxamine, nomifensine, amineptine; or monoamine oxidase inhibitors: moclobemide, iproniazid) had negligible affinities (micromolar range) for 5-HT1C receptors, except fluoxetine. When tested in an in vivo rat model thought to reveal functional agonistic or antagonistic properties at 5-HT1C receptors, all antidepressants displaying high affinity for this receptor type (except clomipramine and trimipramine) were antagonists at 5-HT1C receptors. Antidepressants with a lower affinity for 5-HT1C receptors (except nomifensine) were inactive in this functional in vivo model. Taken together, these results suggest that antagonism at brain 5-HT1C receptors is a component of the antiserotonergic properties of a number of established antidepressants. In addition, the study confirmed that 5-HT1A receptors functionally interact with 5-HT1C receptors, which suggests that some degree of activity at 5-HT1A receptors may also be an important property for antidepressant activity.

Effects of repeated mild stress and two antidepressant treatments on the behavioral response to 5HT1C receptor activation in rats.

This study investigated the possible involvement of 5HT1C receptors in the development of depressive states and in the mode of action of antidepressants. The effects of repeated unpredictable mild stress (a regimen known to induce an anhedonic state in the rat) and of chronic administration of either of two recognized antidepressant treatments (sleep deprivation or inhibition of monoamine oxidase type A) in rats were studied on a 5HT1C receptor initiated response, i.e. mCPP-induced penile erection. A 3-week period of repeated, but unpredictable exposure to mild stressors induced a shift to the left of the dose-response curve for mCPP-induced penile erection. In contrast, 72-h REM sleep deprivation resulted in a shift to the right of the mCPP dose-response curve and 10-day administration of the monoamine oxidase type A inhibitor moclobemide (20 mg/kg IP bid) also resulted in a decreased number of mCPP-induced penile erections. These findings support the hypothesis that neuronal activities initiated via 5HT1C receptor stimulation may play a role in the pathophysiology and treatment of depression.

Aspects of benzodiazepine receptor structure and function with relevance to drug tolerance and dependence.

Potential development of tolerance to and dependence on benzodiazepine tranquilizers often limit their use for long-term treatment of epilepsy, anxiety and insomnia. Current developments in benzodiazepine receptor pharmacology, i.e. the advent of partial agonists and receptor subtype specific agonists (Fig. 1), however, might eventually overcome these limitations, thus greatly improving therapeutic prospects. The present study demonstrates that subchronic administration of alprazolam (a high-efficacy agonist) results in strong withdrawal reactions upon injection of a benzodiazepine receptor antagonist in mice and monkeys. The partial agonist bretazenil, as well as the benzodiazepine receptor type 1-preferring agonist zolpidem, however, are much less prone to producing such reactions. Neurochemical studies showed that subchronic infusion of lorazepam (a high-efficacy agonist), in contrast to betrazenil, led to benzodiazepine receptor downregulation in vivo and reduced potentiation of gamma-aminobutyric acid (GABA)-stimulated chloride flux by diazepam ex vivo. These findings indicate that partial and receptor subtype 1-selective agonists differ from full, non-selective agonists in their liability to induce drug dependence and tolerance upon chronic administration. It is hypothesized that the neurochemical basis of these adaptive phenomena may be receptor downregulation and/or reduced coupling between GABAA receptor/chloride channel gating and benzodiazepine receptor binding.

Pharmacological characterization of benzodiazepine receptor ligands with intrinsic efficacies ranging from high to zero.

Several benzodiazepine receptor ligands were pharmacologically characterized in a battery of functional tests after oral administration in mice, rats, and monkeys. Previous experiments have consistently demonstrated that diazepam exhibits high intrinsic efficacy, bretazenil exhibits intermediate intrinsic efficacy, Ro 42-8773 and Ro 41-7812 both show low intrinsic efficacy, and flumazenil exhibits virtually zero intrinsic efficacy. In the test battery used here it appears that nearly full intrinsic efficacy is required for clear anterograde amnesia or rotarod impairment. In contrast, full protection in the pentetetrazol test was achieved with intermediate-to-high intrinsic efficacy and nearly full protection with lower intrinsic efficacy. In the audiogenic seizure test full anticonvulsant effects were produced with intrinsic efficacy ranging from low to high. Clear inhibition of punished operant responding was observed for all test compounds except for Ro 41-7812 and flumazenil, which exhibit the lowest intrinsic efficacies. All of the test compounds enhanced palatable food consumption, with even those having low intrinsic efficacy producing maximum effects approximating that of diazepam. By additionally taking into consideration the degree of receptor occupancy required to obtain pharmacological activity in each of the tests in this battery it is possible to order the compounds with respect to intrinsic efficacy: diazepam > bretazenil > Ro 42-8773 > Ro 41-7812 > flumazenil. The latter four compounds all exhibited a maximum antagonistic activity in tests involving reversal of meclonazepam- or flunitrazepam-induced central nervous system depression. Thus, using these tests appears to permit the accurate ordinal classification of benzodiazepine receptor ligands for intrinsic efficacy.(ABSTRACT TRUNCATED AT 250 WORDS)

Behavioral profile of the 5HT1A receptor antagonist (S)-UH-301 in rodents and monkeys.

The effects of the new 5HT1A receptor antagonist (S)-UH-301 were investigated in several neurological and behavioral tests in rodents and monkeys. By itself, (S)-UH-301 was found to decrease palatable food consumption in rats, to exhibit anticonvulsant activity in mice, and anxiolytic-like properties in two rodent models of anxiety (light-dark test and elevated plus-maze test). (S)-UH-301 antagonized various symptoms and behaviors induced by the selective 5HT1A receptor agonist 8-OH-DPAT, such as lower lip retraction and flat body posture in rats, hyperphagia for palatable food in rats, and displacement activities (considered as indices of anxiety) in squirrel monkeys. These results further characterize (S)-UH-301 as an in vivo active 5HT1A receptor antagonist and suggest that this antagonistic activity might confer the compound with anxiolytic-like properties.

Ro 19-8022, a nonbenzodiazepine partial agonist at benzodiazepine receptors: neuropharmacological profile of a potential anxiolytic.

The partial agonist at benzodiazepine receptors, Ro 19-8022, has been characterized as a putative anxiolytic drug with an improved side effect profile. This orally active compound is a representative of a quinolizinone structure class and shows potent anticonflict activity in mice and rats. It protects rodents from convulsions induced by pentylenetetrazol, N-methyl-D-aspartic acid and maximal electroshock, as well as against audiogenic seizures, with an efficacy comparable to that of the full agonist alprazolam. No appreciable sedative or motor-impairing effects could be detected up to a very high dose (100 mg/kg) in the horizontal wire test or the rotarod performance test in mice and rats and in spontaneous behavior in monkeys. Consistent with its characterization as a partial agonist, Ro 19-8022 antagonized the motor impairment induced by the full agonists diazepam or meclonazepam measured in horizontal wire and rotarod tests in rodents, and reduced flunitrazepam-induced effects in squirrel monkeys, with an efficacy comparable to that of the benzodiazepine receptor antagonist flumazenil. After subchronic administration of Ro 19-8022 to mice, antagonist-precipitated withdrawal syndrome was dramatically weaker than after alprazolam treatment, which is indicative of a lower physical dependence liability of Ro 19-8022. Pharmacodynamic effects recorded in convulsion and reversal of motor impairment tests after i.v. administration suggest a long duration of action of this compound. Taken together, such preclinical data suggest that benzodiazepine receptor partial agonists with a neurological and behavioral profile such as that of Ro 19-8022 may offer an innovative therapeutic approach to the treatment of anxiety disorders.

Relationship between benzodiazepine receptor occupancy and functional effects in vivo of four ligands of differing intrinsic efficacies.

Agonists at the benzodiazepine receptor (BZR) produce their effects through potentiation of the inhibitory alpha-aminobutyric acid-mediated neurotransmission in the central nervous system via positive allosteric modulation of the gamma-aminobutyric acidA receptor. Agonists with high intrinsic efficacy are anticonvulsant, anxiolytic, muscle relaxant and sedative, whereas agonists with low intrinsic efficacy (partial agonists) are predominantly anticonvulsant and anxiolytic, but antagonize muscle relaxant and sedative effects of full agonists. The four BZR ligands triazolam, diazepam, Ro 19-8022 (a benzoquinolizinone) and bretazenil (Ro 16-6028, an imidazobenzodiazepinone) were pharmacologically characterized in various neurological and behavioral paradigms in mice: two anticonvulsant tests (prevention of audiogenic and pentylenetetrazol-induced seizures), a conflict test which reveals both anxiolytic and sedative properties and two tests which mainly measure motor impairment (rotarod and horizontal wire test). Although triazolam and diazepam elicited an effect in all tests, Ro 19-8022 and bretazenil exhibited anticonvulsant and anxiolytic properties, but virtually failed to induce motor impairment and severe sedation. In separate experiments, fractional BZR occupancy in vivo was assessed by inhibition of [3H]flumazenil binding and correlated with the pharmacological effects. Although diazepam and triazolam produced effects beginning at low to intermediate fractional BZR occupancy, Ro 19-8022 and bretazenil required a higher BZR occupancy to do so, in accordance with their partial agonistic character. With the two full agonists, anticonvulsant and anticonflict activities were elicited at a lower fractional BZR occupancy than muscle relaxant and sedative effects.(ABSTRACT TRUNCATED AT 250 WORDS)

Relationship between benzodiazepine receptor occupancy and potentiation of gamma-aminobutyric acid-stimulated chloride flux in vitro of four ligands of differing intrinsic efficacies.

Intrinsic efficacy is the inherent ability of a ligand to induce the conformational change of its receptor that is required to transduce the event of signal recognition into a physiologically relevant response. Relating fractional receptor occupancy to fractional effect is an indirect but reliable way to assess relative intrinsic efficacy. The receptor studied was the benzodiazepine receptor (BZR), a modulatory site on the gamma-aminobutyric acidA (GABAA) receptor-chloride channel. The relationship between fractional BZR occupancy, as assessed by inhibition of [3H]flumazenil binding, and potentiation of GABA-stimulated 36Cl- influx into membrane vesicles of rat cerebral cortex was evaluated for four ligands under identical experimental conditions. Triazolam and the quinolizinone Ro 19-8022 potentiated the effect of GABA maximally by nearly 50%, diazepam by about 40% and bretazenil by approximately 20%. Potentiation of GABA-stimulated 36Cl- flux by 25% was observed at about 35% BZR occupancy for diazepam, about 45% for triazolam and about 95% for Ro 19-8022. Bretazenil did not produce 25% potentiation even at receptor saturation. Although the curves relating fractional BZR occupancy to GABA potentiation were hyperbolic and nearly superimposable for triazolam and diazepam, those for Ro 19-8022 and bretazenil displayed parabolic characteristics by inducing an effect only at very high BZR occupancy, reflecting the partial agonistic profile of the latter two compounds. The rank order of relative intrinsic efficacy determined in this study was: triazolam congruent to diazepam much greater than Ro 19-8022 greater than bretazenil.

Antidepressant treatment prevents chronic unpredictable mild stress-induced anhedonia as assessed by ventral tegmentum self-stimulation behavior in rats.

The effect of chronic unpredictable mild stress on sensitivity to reward was evaluated using the brain self-stimulation procedure. Rats were allowed to electrically self-stimulate the ventral tegmental area, one of the main cerebral structures subserving positive reinforcement. Stimulation thresholds (frequency of stimuli) for self-stimulation responses were determined prior to, during, and following a 19-day period of exposure to a variety of mild unpredictable stressors. Stimulation threshold was increased in stressed rats, suggesting a decrease in the rewarding properties of brain stimulation. This deficit became evident after about 1 week of mild stress, lasted throughout the stress period, and progressively diminished following termination of the stress regime. In stressed rats concomitantly treated with the tricyclic antidepressant desipramine (5 mg/kg b.i.d.), no stress-induced increase in self-stimulation threshold was observed. However, desipramine did not modify self-stimulation threshold in non-stressed animals. Thus, the increased threshold for brain self-stimulation produced by a period of chronic unpredictable mild stress can be completely prevented by concomitant antidepressant treatment and may provide an heuristic animal model of depression.

Aniracetam improves radial maze performance in rats.

The memory enhancing effect of the pyrrolidinone derivative aniracetam was investigated in rats trained in a delayed-response task in an 8-arm radial maze. Oral administration of aniracetam (100, 200, 400, or 800 mg kg-1) 16 h and again 1 h prior to a first trial of exposure to a given configuration of 4 baited arms resulted in a significant improvement in performance during a second trial in the maze given 3 h later in which there was access to all 8 arms but only the other 4 arms were baited. The pattern of baited arms was varied daily. The performance enhancement was greatest for the highest doses. These results extend the demonstration of the cognition enhancing effects of aniracetam to a spatial memory task in rats.

Physical dependence induced in DBA/2J mice by benzodiazepine receptor full agonists, but not by the partial agonist Ro 16-6028.

Continuous administration of triazolam, alprazolam or diazepam for a 7-day period by means of minipumps or chronic (17 days) p.o. treatment with alprazolam induced clear physical dependence in DBA/2J mice as assessed by precipitation of a withdrawal syndrome with an i.v. injection of the benzodiazepine receptor partial inverse agonist Ro 15-3505. In contrast, no precipitated withdrawal signs were observed following chronic exposure to high doses of the benzodiazepine receptor partial agonist Ro 16-6028. The use of minipumps and precipitation with a benzodiazepine receptor antagonist permits a simple and rapid evaluation of the physical dependence liability of potent compounds acting at the benzodiazepine receptor. Furthermore, these results support the hypothesis that benzodiazepine receptor partial agonists are less likely to induce physical dependence than full agonists.

Pharmacology of the benzodiazepine receptor.

The benzodiazepine receptor (BZR) is an intrinsic allosteric modulatory site of the GABAA-receptor-chloride channel complex of neuronal membranes mediating the main action of the major inhibitory neurotransmitter GABA. The BZR is unique in recognizing three classes of ligands, two of them producing opposite modulatory effects on the GABAA receptor function in an allosteric fashion (agonists and inverse agonists) and the third acting as antagonists of the two others. Agonists and partial agonists of the BZR (belonging to various chemical classes) have therapeutic applications as broad-spectrum tranquilizers and specific anxiolytics- anticonvulsants, respectively. The BZR antagonist flumazenil, recently introduced in therapy, increases the versatility of agonists in therapy and greatly simplifies the treatment of agonist overdosing. Inverse agonists are interesting probes to analyse the biological basis of anxiety-related emotional disorders.

Preclinical profiles of the novel reversible MAO-A inhibitors, moclobemide and brofaromine, in comparison with irreversible MAO inhibitors.

Acceptance into clinical practice of monoamine oxidase (MAO) antidepressants requires unequivocal evidence that novel, non-hepatotoxic and reversible MAO-A inhibitors carry little or no risk of inducing severe hypertensive crises (cheese effect). This study summarizes the most relevant preclinical aspects which differentiate the novel reversible MAO-A inhibitors moclobemide and brofaromine, from previous irreversible MAO inhibitors of the old generation, particularly phenelzine and tranylcypromine. Moclobemide and brofaromine bear no chemical relation to irreversible inhibitors such as hydrazine derivatives (phenelzine) or aminocyclopropyl derivatives (tranylcypromine). Experiments in rats show that moclobemide and brofaromine increase the level of serotonin (5-hydroxytryptamine) and decrease that of 3,4-dihydroxyphenylacetic acid for only 16-24 hours. In vitro, moclobemide and brofaromine behave as mechanism-based, enzyme-activated inhibitors since their intrinsic inhibitory activity increases with the duration of their interaction with the enzyme in tissue homogenates. In contrast to irreversible monoamine oxidase inhibitors, which are much more potent in vitro than in vivo, moclobemide has the characteristic to be virtually equipotent in vitro and in vivo. MAO-A inhibition induced by moclobemide in the rat in vivo was rapidly reversed by simply incubating liver homogenates at 37 degrees C in the absence of the inhibitor, indicating a rapid metabolic inactivation of moclobemide in vitro. This reversibility is a distinctive feature of moclobemide, when compared with brofaromine or irreversible MAO inhibitors. Hepatotoxicity is not an inherent property of MAO inhibitors indeed, moclobemide or brofaromine, due to their chemical structures, cannot be converted into isopropyl hydrazine, the hepatotoxic metabolite of iproniazid suspected to induce liver necrosis. Results from preclinical and clinical investigations demonstrate that moclobemide and brofaromine, in contrast to tranylcypromine and phenelzine, very weakly potentiate the pressor effects of orally administered tyramine. In conclusion, the reversible MAO-A inhibitors moclobemide and brofaromine, due to their well-documented safety characteristics, to their lack of anticholinergic-effects and to their good tolerability, will provide innovative tools for clarifying the role of MAO-A inhibitors in the treatment of endogenous and atypical depressive states.

Neurochemical profile of moclobemide, a short-acting and reversible inhibitor of monoamine oxidase type A.

Moclobemide belongs to a new generation of short-acting, reversible, monoamine oxidase (MAO) inhibitors. In vitro (rat brain homogenates) moclobemide inhibits MAO-A selectively with lower potency than many of the reference MAO inhibitors. However, when measured ex vivo in the rat, the potency of moclobemide is similar to that of reference compounds. In vivo the drug induces a dose-dependent, short-lasting (8-16 hr) and preferential inhibition of MAO-A in the brain and both MAO-A and MAO-B inhibition in extracerebral organs (liver, small intestine and kidney). In the extracerebral tissues of the rat moclobemide induces marked peripheral MAO-B inhibition due to rapid and extensive biotransformation of its morpholine ring. The active molecular species is probably the metabolite Ro 16-6491. The moderate MAO-B inhibition measured after moclobemide intake in human platelets indicates that only minor amounts of Ro 16-6491 are formed in humans. Virtually all metabolites of moclobemide so far identified have been tested in vitro and ex vivo in the rat and proved to be either equipotent or, mostly, less effective than moclobemide as MAO-A inhibitors. In liver homogenates of moclobemide-treated rats MAO-A activity recovers during dialysis or simple incubation at 37 degrees C, suggesting a biodegradation of moclobemide and/or the moclobemide-derived active metabolite(s) by MAO itself or a slow dissociation of the active inhibitory species from the enzyme. Similar to other MAO-A inhibitors, moclobemide induces an increase in the rat brain levels of 5-hydroxytryptamine, norepinephrine and dopamine and a concomitant decrease of their deaminated metabolites. These effects are of short duration (8-16 hr) and parallel the time course of MAO-A inhibition. Moclobemide administered subchronically down-regulates beta adrenoceptors as shown by binding experiments with brain cortical membranes using dihydroalprenolol as ligand. In vitro MAO inhibition by moclobemide is specific in that the compound does not affect other amine oxidases or monoamine uptake mechanisms; furthermore, it does not interact with various neurotransmitter or drug receptor sites. In conclusion, a large body of preclinical evidence characterizes moclobemide as a short-acting and reversible MAO-inhibitor. The neurochemical profile of moclobemide indicates clearly that this nonhydrazine nonhepatotoxic MAO-A inhibitor represents a novel and safe drug for treatment of affective disorders.

Dopamine D2 receptor stimulation inhibits inositol phosphate generating system in rat striatal slices.

Previous studies on the transduction mechanisms triggered by dopamine receptor stimulation have established that both D1 and D2 subtypes of dopamine receptors are linked to the adenylate cyclase system, the former in a stimulatory and the latter in an inhibitory manner. The present report provides the first evidence that stimulation of D2 receptors in rat brain tissue affects the turnover of polyphosphoinositides, as revealed by changes of the content of inositol phosphates. We found that the basal level of [3H]inositol trisphosphate, [3H]inositol bisphosphate and [3H]inositol monophosphate decreased following the stimulation of the D2 receptor. The rank order of potency was quinpirole (IC50 5 nM) greater than lisuride (IC50 8 nM) greater than RU 24213 (IC50 50 nM) greater than dopamine (IC50 200 nM). In contrast, selective D1 receptor stimulation by fenoldopam did not alter the inositol monophosphate, inositol bisphosphate and inositol trisphosphate content. The quinpirole effect was prevented by selective D2 antagonists, such as domperidone and L-sulpiride (both 5 microM) while it was unaffected by the selective D1 antagonist SCH 23390 (100 nM) and by the pharmacologically inactive D-isomer of sulpiride. Our data indicate that the activation of striatal D2 receptors leads to the inhibition of inositol phosphate production.

On tyramine, food, beverages and the reversible MAO inhibitor moclobemide.

The pathways for the biosynthesis and metabolism of tyramine are described as a basis for the discussion of the interaction between MAO inhibitors and tyramine. While a role of endogenous tyramine in the antidepressant action of MAO inhibitors remains purely hypothetical at this time, the mechanisms leading to the potentiation of the tyramine pressor effect ("cheese effect") are well known. Experiments in animals and man have provided concordant quantitative information on the effect of irreversible and some novel reversible MAO inhibitors on the presystemic disposition of orally ingested tyramine and on the noradrenaline-releasing action of tyramine in noradrenergic nerve terminals. There is a profound difference in the magnitude of tyramine potentiation between the irreversible inhibitor tranylcypromine and the reversible inhibitor moclobemide. A systematic analysis of the tyramine content of current European food and beverage is reported and serves as a rational basis for providing advice to patients on moclobemide. Most of the food and beverages analyzed contain less tyramine than previously reported and a few rules concerning rare cheeses with high tyramine content are sufficient to eliminate the risk of hypertensive crises.