Benzodiazepine receptors: new vistas

seminars in THE NEUROSCIENCES, Vo13, 1991 : pp 191-203

Benzodiazepine

receptors:

new vistas *

Grayson Richards, Peter Schoch and Willy Haefely behaviour. The hopes raised by several second generation anxiolytic drugs depend upon clinical proof of this partial agonist concept. Recent research has provided evidence for a structural diversity of GABA* receptors, the targets for benzodiazepine receptor ligands in the CNS (see Tobin, this issue’). Since the composition and stoichiometry of the native receptor(s) are not yet known, the physiological role and the pharmacological implications of such a diversity (polymorphism) remain a matter of speculation. It is conceivable, however, that the pharmacological profiles of some partial agonists might be due to their interaction with target neurons differing not only in their receptor reserves but also their GABAA receptor variants. Here we describe: the role of GABA in the mechanism of benzodiazepine action; the identification of benzodiazepine receptors; the molecular neuroanatomy of GABA* receptors; the spectrum of benzodiazepine receptor ligands; and partial agonists

The therapeutic #e&s of benzodiazepinesare triggered by their interaction with allosteric sites (benzodiazepine recognition sites) on GABA* receptors in the CNS in a way that facilitates GABA- mediated inhibitory neurotransmission. The discovery of a spectrum of ligandr interacting with differing efficacies at benzodiazepine receptors, as well as the growing evidencefor a diversity of GABA* receptors, has created new opportunities for producing benzodiazepine receptor ligands with potentials un&e and evenunexpectedtherapeuticprojles. Key words: GABA receptors I receptor diversity I allosteric sites /partial agonists I intrinsic efficacy

BENZODIAZEPINES (prototype: diazepam) are widely prescribed anxiolytics, antiepileptics and hypnotics, the success of this class of therapeutic drugs being undoubtedly due to not only their efficacy and rapid onset of action but also to their extremely high safety. Research on their mechanism of action in the CNS has revealed a facilitatory effect on inhibitory neurotransmission mediated by the amino acid GABA (y-aminobutyric acid), This facilitation is achieved by the positive allosteric modulation of GABA* receptors, which regulate the chloride conductance of the subsynaptic membrane in target neurons through a convertible ion channel (Figure 1). The modulatory site, the benzodiazepine receptor, is in fact recognized by a spectrum of structurally different ligands (benzodiazepines and non-benzodiazepines) with differing allosteric modulatory activities, from full agonists (positive modulators) through antagonists to inverse agonists (negative modulators). Partial agonists acting at the benzodiazepine receptor are novel potential therapeutic agents (as efficacious anxiolytics and antiepileptics) because of their greatly reduced sedative and muscle relaxant activity and reduced liability to induce tolerance, physical dependence and drug-seeking

- -Figure 1. Simplified schematic view of a GABAergic synapse consisting of a nerve terminal with vesicular stores of GABA (0) and the subsynaptic membrane with GABA, receptors. The latter are depicted as pentameric structures (isofonns of cr, p, y subunits) of as yet unknown subunit composition and stoichiometry, which are thought to allow the conductance of chloride ions through the GABA-gated ion channel. A separate site binds benzodiazepines, which modify the action of GABA on the ion channel.

From the Pharma Division-Preclinical Research, F HojinannLa Roche Ltd, CH-4002 Basel, Switzerland *Dedicated to Dr Lowell 0. Randall on the occasion of his 80th birthday 01991 by W. B. Saunders Company 1044-5765/91/0303-0003$5.00/0

191

G. Richards et al

1 92 versus full agonists as novel potential therapeutic agents . We conclude with a discussion on future perspectives including unexpected potential therapeutic indications .

Role of GABA in the mechanism of benzodiazepine action

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effects of benzodiazepines is such that they are now the best understood of the major neuropsychotropic drugs, creating a more rational basis for improve-

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ments in their therapeutic profiles . GABA is the most abundant and functionally most important inhibitory neurotransmitter in the

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mammalian CNS . Two distinct classes of receptors for GABA exist : GABAA and GABAB receptors, characterized by their affinity for specific agonists and antagonists, the effector systems to which they

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After little more than 15 years of intense research on the role of GABA in the mechanism of action of benzodiazepines (reviewed in ref 2), our knowledge of the molecular events underlying the therapeutic

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Figure 2 . Benzodiazepine receptor ligands of different chemical classes : diazepam, triazolam, alprazolam (1, 4-benzodiazepines), zopiclone (cyclopyrrolone), zolpidem (imidazopyridine) . All compounds are full agonists .

are coupled and the presence or not of allosteric modulatory sites . Whereas GABA A receptors 3 are convertible ion channels regulating the chloride

main reasons for the extremely high safety of these drugs .

conductance of the subsynaptic membrane, GABA B receptors 4 are coupled to GTP-binding protein(s) regulating adenylate cyclase, as well as calcium and potassium channels . Although GABA is the natural

The first clue of the mechanism by which benzodiazepines produce their effects was provided by the observation that diazepam enhanced segmental dorsal root potentials (for review, ref 2) . The relevance of

agonist for both receptors, there exist selective agonists and antagonists : e .g . muscimol and bicuculline for GABAA receptors and baclofen and phaclofen for GABAB receptors (see articles by Macdonald and Twyman, Johnston, and Bowery and Maguire, this

these findings became clear when this so-called presynaptic inhibition was shown to be only one of many GABA-mediated responses which are potentiated by diazepam . Subsequent electrophysiological, biochemical and behavioural studies have led

issues -7 ) . Allosteric sites on neuronal GABAA receptors are the targets through which benzodiazepines (and non-benzodiazepines) (Figure 2) modulate

to the currently held belief that benzodiazepines act as positive allosteric modulators to enhance submaximal GABAergic synaptic transmission . 2 The benzodiazepines have been shown, electrophysio-

GABAA receptor function ; other allosteric sites include those for barbiturates, convulsants (such as picrotoxin, TBPS), and general anaesthetics8 (such as halothane, propofol, steroids and perhaps ethanol) (Figure 3) (see Ticku, this issue9) . A unique feature of the allosteric site to which

logically, to have GABA-potentiating properties in various parts of the mammalian CNS, where they modulate the effectiveness of either GABAergic principal output neurons, e .g . cerebellar Purkinje cells and neurons of the GABAergic pallido- and striato-nigral pathways, or GABAergic intrinsic cells, i .e . interneurons such as those mediating presynaptic inhibition in the spinal cord and dorsal column nuclei, cerebellar Golgi, basket and stellate cells, hippocampal basket cells and intrinsic neurons of the cerebral cortex . Potentiation of exogenous GABA

benzodiazepines bind is its ability to mediate opposite pharmacological effects by facilitating or impeding GABA receptor function, depending on the type of receptor ligand (see below) . These modulatory effects are of limited maximal intensity, i .e . the maximum response to the physiological transmitter GABA is not exceeded . This property is probably one of the

(or a GABA mimetic, such as isoguvacine) in cultured neurons or in slice preparations in vitro

Bentodiazepine

receptors: new vistas

193

GABAsite GABA muscimol isoguvacine ;rgyl$

Barbiturate site barbiturates etomidate etazolate

Benzodiazepine site

agonists antagonists 3 inverse agonists membrane

benzodiazepines non-benzodiazepines

General , anesthetics

Subsynaptic

propofol steroids halothane ethanol

Picrotoxinin site bicyclophosphates TBPS tetrazoles

3. Functional binding sites on ion channel GABA* receptors. Binding sites for the natural transmitter GABA and its agonists and antagonists, as well as allosteric drug recognition sites for a variety of ligands, are depicted schematically.

Figure

is routinely used for electrophysiological gations10-i2 of the GABA-potentiating benzodiazepines.

The identification receptors

investieffects of

of benzodiazepine

Although the molecular target mediating the pharmacological effects of benzodiazepines was first described in 1977, the term benzodiazepine receptor was already used in 197 1 to indicate specific drug recognition sites through which benzodiazepine responses were triggered (reviewed in ref 3). With the knowledge of the essential role of GABA in the mechanism of action of benzodiazepines, we prepared 3H-diazepam of high specific activity with the aim of visualizing, radioautographically, the benzodiazepine bound to its presumed receptor in GABAergic synapses in the CNS. Our attempt failed because of the rapid diffusion of the ligand in the standard tissue preparation in use at the time. (Procedures developed by other research groups have since overcome these initial diffculties.r3) In the meantime, as a result of discussions with Dr R.F. Squires we decided to study 3H-diazepam binding to brain homogenates and in 1977 investigations performed independently in Copenhagen,i4

New Y ork15 and Roche, Baseli6 revealed the presence of specific high-affinity and saturable binding sites for 3H-diazepam in brain membranes. The binding sites were restricted to grey matter and the B,, for various brain regions differed markedly. Although most of the criteria for identifying these drug recognition sites with pharmacological receptors (reviewed in ref 2) could be satisfied at that time (high affinity, saturable, pharmacologically specific, interaction with GABA, restricted to CNS, phylogenetic and ontogenetic development, correlation between receptor occupancy and pharmacological effects, distribution in CNS regions with high densities of GABAA receptors), definite proof was provided only by the discoveryi7,18 of pharmacologically inert ligands that competitively blocked the effects of benzodiazepines by occluding these binding sites. The benzodiazepine receptor is now known to be recognized by not only benzodiazepines but also drugs of other chemical classes, ligands displaying differences in both affinity and intrinsic efficacy (see below: Spectrum of benzodiazepine receptor ligands). Although diazepam and related benzodiazepines (probably of dietary origin or from gut flora) have been measured in rat and human brains (using antibodies to benzodiazepines as well as mass spectroscopic and radiometric techniques), it remains to be demonstrated whether benzodiazepine receptors are

194 primarily the molecular targets for endogenous neurotransmitter modulators or for xenobiotic ligands (for reviews, refs 2,19). Regions of the mammalian CNS highly innervated by GABAergic neurons contain a high density of GABA* receptors and its associated binding sites for various allosteric modulators including benzodiazepines (Figures 4-6). Quantitative receptor radioautography (for reviews, refs 20,21) has revealed the distribution and abundance of selective binding sites for the following components of the receptor complex: high-affinity GABAA receptors

G. Richards

revealed by “H-muscimol (a GABAA receptor agonist); low affinity GABAA receptors revealed by 3H-bicuculline and 3H-SR 95531 (GABAA receptor ;22 high-affinity benzodiazepine receptors antagonists) revealed by 3H-flunitrazepam, 3H-clonazepam, 3Hflumazenil and 3H-Ro 15-45 13 (benzodiazepine agonists, antagonist and partial inverse agonist, respectively);13~20~23 and high-affinity binding sites thought to be located on or close to the pore of the GABA* receptor channel complex revealed by 3H-TBPS (t-butyl-bicyclophosphorothionate, a cage convulsant).24 According to Olsen et ~1,~~the differential capacity of various regions of the receptor channel complex to bind the above radioligands (see also ref 25) indicates the existence of at least four subtypes of receptors. Monoclonal antibodies to the GABA* receptor crl-subunit and fi2-, /33-subunits have been used to reveal the regional, cellular and subcellular localization of the receptors in the mammalian CNS.26-28 Receptors are not confined to synapses but have also been found in extrasynaptic membranes. In vivo binding investigations with “H-flumazenil (reviewed in ref 20) have demonstrated the feasibility of non-invasive computer tomographic (PET and SPECT) analyses of human brain using l ‘C-flumazenil and 1231-Ro 16-0154 (an iodinated derivative of flumazenil) 2g for studying receptor changes (as cause or effect) in neurological disorders such as chronic anxiety, epilepsies and Huntington’s chorea.

Molecular receptors

Figures 4-6. Radioautographic distribution of benzodiazepine receptors (Figures 4,5) and high-affinity binding sites for the GABA, receptor-associated chloride ionophore (Figure 6) in parasagittal sections of rat (Figures 4, 6) and mouse (Figure 5) brain: White areas indicate the brain regions with an abundance of receptors revealed by [ 3H] flumazenil binding in vitro (Figure 4) and in vivo (Figure 5) and 35S-TBPS binding in vitro (Figure 6). Calibration bars = 2 mm.

et al

neuroanatomy

of GABA*

GABA* receptors belong to the superfamily of ligand-gated ion channels30 and assemble from several homologous subunits to form a GABA-gated, chloride-conducting pore, a convertible ion channel. The receptor is an integral part of one or several subunits of a transmembrane hetero-oligomeric glycoprotein complex (see Tobin, this issue’). Biochemical investigations (for reviews, refs 3,31,32), that is, photoaffinity labelling with [ 3H] flunitrazepam and [ 3H ] Ro 15-4513, affinity purification and immunoprecipitation studies, initially suggested that the GABAA receptor consists of CY-and p-subunits with molecular sizes of 50 kDa and 55 kDa. Molecular size determination of the intact receptor suggested that it is composed of four subunits although, to ac%ommodate an estimated open pore diameter of 5.6 A, a pentamerit structure was proposed. The high-affinity

195

Benzodiazepine receptors . new vistas

binding sites for GABA and benzodiazepines were originally considered to be located on the ß- and a-subunits respectively . More recent evidence (reviewed in ref 33), however, indicates the presence of GABA and benzodiazepine binding sites on both subunits or on the interfaces between them . Whereas benzodiazepine tranquilizers increase the current flowing through activated GABA A receptor channels, presumably by increasing the frequency of opening, barbiturates (via different allosteric sites) do so by increasing the channel open time (for review, ref 34 ; see also Macdonald and Twyman, this issue5) . It has been postulated that the receptor fright spontaneously oscillate between states with high and low affinity for GABA . Agonists and inverse agonists at the benzodiazepine receptor could then stabilize the receptor in the high- and lowaffinity conformation, respectively, i .e . they might shift the conformational equilibrium to the respective affinity state through their selective affinity for either the high- or low-affinity states . Competitive antagonists might not be able to distinguish between the two conformation states but would prevent access of either positive or negative allosteric modulators . Partial agonists, on the other hand, would produce a shift of the equilibrium between low and high affinity GABA A receptor states that is less marked than full agonists because they are less able to distinguish the two conformations . Molecular cloning studies 30,35-41 (reviewed in refs 33,42,43) have now revealed the primary structures of the subunits proposed to constitute the supramolecular complex of the GABAA receptor channel. As detailed in the article by Tobin (this issuer), cloning has revealed four subunits, a, ß, y and 6 with less than 50% sequence homology, and variants thereof with >,70% identity, al-6, ß1-3, yl,2 and 6 . Electrophysiological studies on oocytes injected with subunit-specific mRNAs 44 or on transiently transfected mammalian cells expressing simultaneously various subunit combinations have revealed that receptors composed of a and ß subunits form GABA-gated chloride channels which can be blocked by bicuculline and picrotoxin, and facilitated by barbiturates (see articles by Macdonald and Twyman ; Johnston, and Ticku, this issue 5,6'9) . For consistent positive or negative allosteric modulation by benzodiazepines, however, a -y subunit is also required, although the type of a-subunit influences not only the sensitivity of the receptor to GABA but also the affinity of different ligands for the benzodiazepine recognition site . 35-39,45 Four classes of

receptors have been proposed : I, alßy2 (high affinity for zolpidem) ; II, a2(3)ßy2 (intermediate affinity for zolpidem) ; III, a5ßy2 (extremely low affinity for zolpidem) ; IV, a6ßy2 (low affinity for 1,4benzodiazepine agonists but high affinity for Ro 15-4513, an inverse agonist) . N .B . 0 follows the nomenclature of Pritchett and Seeburg ;37 the same sequence was published earlier and called a4 by Khrestchatisky et al. 33 Whereas the antagonist flumazenil has a high affinity for receptor classes IIII, it has only a moderate to low affinity for class IV . The type of -y-subunit, on the other hand, strongly influences the affinities for antagonists and inverse agonists . 39 The variety of neuronal responses to GABA resulting from this receptor diversity increases plasticity (see ref 46) . Using polyclonal antibodies to al- and -y2-specific synthetic peptides, it has been recently demonstrated 47 that these two subunit isoforms are integral components of the GABAA receptor and that the al subunit has the allosteric benzodiazepine binding site which can be photolabelled . The y2 subunit probably induces a conformational change in the receptor complex, favoring the binding of benzodiazepines to the a subunit . These findings explain different but interrelated roles of these subunits in receptor modulation by benzodiazepines . Distribution of GABAA receptor subunit isoforms Despite rapid advances in our knowledge of the structural diversity of subunit components of the GABAA receptor, its native composition and stoichiometry are not yet known . The prerequisites for identifying the composition of native receptors include the evidence for : (i) the cellular co-localization of subunit isoforms from in situ hybridization histochemistry (mRNA) and immunohistochemistry (protein) ; (ii) the unique physiology and pharmacology of co-localized subunits in injected oocytes and transfected cells, also in native receptors of the identified CNS regions by single-channel recordings on brain slices from pertinent regions ; and (iii) the presence of individual subunits in isolated receptor preparations demonstrated by antibodies using e .g. sequential immunoprecipitation assays (see ref 42) . As antibodies to oligopeptides specific for subunit isomers have not been used extensively for immunohistochemistry, most of the available information on the co-localization of receptor subunits has come from in situ hybridization histochemistry (Figure 7) .

196

G. Richards

et al

Figure 7. Radioautographic distribution of GABA, receptor mRNA in a parasagittal section of rat brain. White areas indicate the brain regions with an abundance of mRNA revealed bv in silu hvbridization with 35S-labelled synthetic oligonucleotide specific for the o 1-subunit. Calibration bar = 2 mm. These studies33,40,48-54 have revealed that in rat brain crl , fl2 and y2 subunits are the most ubiquitously distributed and abundant of those investigated to date (Persohn et al, in preparation). They co-localize in several regions (olfactory bulb mitral cells, cerebral cortex layers II-VI, pallidum, hippocampal formation, thalamus, substantia nigra reticular, colliculi, cerebellar Purkinje and granule cells, deep cerebellar and brainstem nucleiinterpositus, vestibular, facial, motor trigeminal). Other subunit mRNAs, in contrast, have a more restricted and unique distribution-olfactory bulb granule cells: 63 > (r2 > (~3, ~5, ~2, 6; caudate putamen: p3> >(r2, 72, 6>(r3, fil, 02; hippocampus: a3 poorly expressed, (r6 and 6 absent; septum, hypothalamus and medial amygdala: yl; dentate gyrus: (r3 poorly expressed, (r6 absent; thalamus: a2, ~5, ~6, 03 absent; substantia nigra compacta: (r3; cerebellar Bergmann glia: CY~,01, yl ; cerebellar Golgi cells: (r3; deep cerebellar and brainstem nuclei: (x5, a6, 01 and 6 absent. Lastly, of all the subunit isoforms studied to date (r6 is the most restricted (to cerebellar granule cells). We also found evidence for receptor diversity in the spinal cord,50 where the subunit isoforms a2, /33 and y2 co-localize in motor neurons of Rexed layer IX whereas in the spinal ganglia the expression of various subunit mRNAs provides further evidence for the axo-axonal inhibition of primary sensory afferents by GABAA receptors.

Spectrum

of benzodiazepine

receptor

ligands

The interaction between benzodiazepine receptor ligands and their allosteric sites on the GABA*

receptor complex (Figure 8) is determined by two intrinsic properties of the ligand: its binding affinity and its intrinsic efficacy; full agonists have a positive intrinsic efficacy and inverse agonists a negative intrinsic efficacy (for reviews, refs 2,55). Between these two extremes are competitive antagonists whose intrinsic efIicacies are zero, i.e. they are unable to activate the receptor to which they bind. The modulation of GABA responses by benzodiazepine receptor ligands can be easily quantified by measuring GABA-stimulated chloride flux, which is the primary effect of GABA receptor activation. Figure 9 illustrates the concentration-response curve for GABA and how it is shifted along the abscissa under the influence of an agonist (diazepam), antagonist (flumazenil) and inverse agonist (the fl-carboline DMCM) of the benzodiazepine receptor. In correspondence to the pharmacological profile, diazepam potentiates GABA whereas DMCM reduces its apparent potency. Flumazenil as an antagonist has no measurable intrinsic efficacy in this test. If an agonist or inverse agonist has a low capacity to activate the receptor (positively or negatively) it is described as a partial agonist or a partial inverse agonist. Comparing different benzodiazepine receptor ligands with varying pharmacological profiles including agonists, inverse agonists and antagonists in the chloride flux assay shows that indeed there exists a continuous spectrum of intrinsic efficacies in modulating GABA responses. Strong convulsants, e.g. DMCM, inhibit and strong central depressants, e.g. diazepam, potentiate the GABA response. In between are the partial (inverse) agonists with reduced and antagonists with zero intrinsic efficacy (Figure 10).

Benzodiazepine

receptors: new vistas

197 Partial Agonists

Agonists

Partial Inverse Agonists

Antagonists

inverse Agonista

+

Figure 8. A simplified schematic view of the three basic types of ligand-receptor interactions and their different modes of coupling to the GABA, receptor. Whereas agonists reduce the levels of anxiety, muscle tension, convulsions, vigilance and memory, inverse agonists have diametrically opposite effects. Competitive antagonists, while having no intrinsic activity per se, prevent or abolish the effects of agonists and inverse agonists by excluding access to the benzodiazepine receptor. 500-

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Figure 9. GABA-stimulated 36chloride flux in a membrane preparation from rat cerebral cortex. Before the stimulation of chloride flux by GABA (duration 3 s at 30 “C) and filtration, the membranes were preincubated for 10 min in the presence of the benzodiazepine receptor ligands at the receptor saturating concentration of 1 FM. The typical modulation of the GABA-response by the benzodiazepine receptor agonist diazepam and inverse agonist DMCM are shown by the lower part of the curves. The intersection of the curves at higher GABA concentrations is probably due to different desensitization kinetics of the GABA response in the presence of the respective ligands. Discussion of this effect is beyond the scope of this review.

Ligands with differing intrinsic efficacies (Figure 11) produce different maximal pharmacological effects in neuronal populations which differ in receptor density or efficiency of signal transduction. The fractional receptor occupancies required for the same intensity of pharmacological effect is usually much higher for partial agonists than for full agonists. With full agonists a maximal pharmacological response is frequently obtained well before all receptors are activated. For such ligands the target cells are said to have a functional receptor reserve. When partial agonists interact with the receptor, only a small proportion of the ligand-receptor complexes results in receptor activation. Consequently, partial agonists require a higher fractional receptor occupancy than full agonists for an effect of identical intensity so that in target cells with little or no functional receptor reserve, a maximal effect may not be achieved even at receptor saturation (Table 1).

Full

agonists:

ideal

clinical

profile?

Benzodiazepines are the most widely used drugs for the treatment of acute and chronic anxiety, for sleep disturbances, conscious sedation, induction and potentiation of anaesthesia, for muscle spasticity and



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Figure 10 . The comparison of benzodiazepine receptor ligands with differing pharmacological profiles reveals a continuous spectrum of intrinsic efficacies with regard to their modulation of GABA-induced chloride fluxes in a membrane preparation from rat cerebral cortex . Agonists enhance whereas inverse agonists reduce GABA-response (positive and negative modulation of the GABA receptor) . Full agonists show maximal, antagonists show zero intrinsic efficacy . The intrinsic efficacies of partial agonists lie between those of full agonists and antagonists .

seizures . One would think that this broad therapeutic spectrum, coupled with the potency and acknowledged safety of these drugs would not require improvement

indications . Of potential therapeutic interest are competitive antagonists (prototype ; flumazenil) and partial inverse agonists (prototype : sarmazenil) . 55

but it is now widely accepted that the ideal clinical profile of second generation drugs should include several characteristics that are lacking in currently marketed compounds .

Pure competitive antagonists have no intrinsic activity

Although as anxiolytics, benzodiazepines have good efficacy, rapid onset of action and undisputed safety, there is a need for drugs with less sedation and

experiments and clinical studies, the competitive antagonist flumazenil (Anexate ® , the imidazobenzodiazepinone Ro 15-1788) has been shownl 7 to

dependence potential as well as less interaction with ethanol . The ideal profile of future anxiolytics should include : good efficacy and anxioselectivity ; rapid onset and long duration of action ; excellent tolerability ; minimal physical dependence, abuse liability, ethanol interaction and muscle relaxation ; and no or only

antagonize the behavioural, neurological and physiological effects of benzodiazepine receptor ligands . It is currently used in several clinical indications, for example in intensive care medicine, in anaesthesiology (for general anaesthesia, dentistry and bronchial

mild sedative effect . Anti-epileptic benzodiazepines produce sedation and tolerance, and have a restricted efficacy in treating partial complex seizures . A longacting drug is required that is effective in treating all forms of epilepsies without developing tolerance . A second generation benzodiazepine hypnotic should be one lacking hangover effects, tolerance liability, ethanol interaction, behavioural disinhibition, amnesia and dependence and abuse potential . These ideal profiles could be satisfied by benzodiazepine receptor ligands acting as partial agonists (see below) . Receptor ligands with different pharmacological profiles might be envisaged for other therapeutic

but nevertheless have extremely useful pharmacological properties as antidotes to benzodiazepine receptor ligands in cases of overdose . In both animal

endoscopy) together with midazolam, 56 and in mixed intoxications involving benzodiazepine receptor ligands where it has diagnostic utility in coma of unknown etiology . Recent preclinical and clinical findings indicate a potential therapeutic use of flumazenil also for hepatic encephalopathy 57 and for re-setting the receptor in cases of benzodiazepine tolerance . 58 The partial inverse agonist sarmazenil (for review, ref 55), the imidazobenzodiazepinone derivative Ro 15-3505, is a congener of flumazenil and a potent antagonist of classical benzodiazepine tranquilizers, with a very low inverse (negative) intrinsic efficacy at the benzodiazepine receptor . The compound has

199

Benzodiazepine receptors : new vistas

1

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convulsions, from its stimulation of brain EEG activity and from its consistent cognition-improving effects in several behavioural paradigms . Compounds like sarmazeni1 59,60 could have therapeutic value for

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Figure 11 . Benzodiazepine receptor ligands of different intrinsic activities : midazolam is a full agonist (positive allosteric modulator) ; bretazenil, abecarnil, alpidem, RU 32698 and FG 8205 are partial agonists (weak positive allosteric modulators) ; clonazepam is a partial agonist with a relatively high intrinsic efficacy ; flumazenil is a competitive antagonist (virtually no intrinsic efficacy) ; sarmazenil, Ro 15-4513 and Ro 19-4603 are partial inverse agonists (weak negative allosteric modulators) ; DMCM is an inverse agonist (negative allosteric modulator) . a mild central stimulant activity, as is evident from its facilitation of chemically and physically induced

anxiolytics, antiepileptics and hypnotics, without the aforementioned characteristics, could be designed using the most rational exploitation of the present knowledge of the benzodiazepine receptor as the modulatory component of the GABAA receptor complex . The separation of their desirable and undesirable effects can be probably achieved by using the partial agonist concept, 55 for example to produce compounds which are anxiolytic and antiepileptic but with markedly reduced sedative and muscle relaxant effects and reduced liability to induce tolerance, physical dependence and drug-seeking behaviour (Figure 12) . 62-67 Different neuronal systems that are preferentially involved in the various effects of benzodiazepines vary in their GABAA receptor density and reserve .

Table 1 . Fractional receptor occupancies (in %) of diazepam and bretazenil at equieffective doses for different pharmacological effects in mice

Diazepam Bretazenil

Protection from audiogenic seizures in DBA/2J mice

Anticonflict activity in the Geller-Seifter test

Protection from pentetrazoleinduced convulsions

Impaired motor performance in the rotating rod test

15 65

30 95

40 100

50 inactive

The fractional receptor occupancies were measured by an in vivo competition assay using intravenous 3 H-flumazenil as radioligand . The doses of the test compounds corresponded to the oral ED 50 with the exception of the anticonflict test where the minimal effective dose was given ."



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Neurons mediating anticonvulsant and antianxiety effects seem to have the highest receptor reserve whereas a smaller reserve or lack of it is characteristic of neurons involved, e .g . in arousal control . Bretazenil (Ro 16-6028), as a partial agonist, should be able sufficiently to enhance GABA activity in the former systems to reach the same maximal CNS depression as full agonists but would be insufficient in the latter systems . Preclinical studies with bretazenil (for review, ref 55) have compared its pharmacological properties with those of the full agonist diazepam . Bretazenil has approximately a 10-times higher affinity for the benzodiazepine receptor than diazepam and produces anticonflict effects at much lower doses and over a much wider dose range . The following observations identify bretazenil as a benzodiazepine receptor partial agonist : it has a smaller, regionally-specific GABAshift profile in sections of rat brain and cat spinal cord in vitro (Figure 13) ; its fractional receptor

I F-1> 300 H > 50

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Figure 12 . Profiles of activity of the full agonist diazepam (o) and the partial agonist bretazenil (u) in a range of behavioural and functional tests . Values are ED50 values or minimal effective doses (anticonflict activity) . PTZ, pentetrazole (leptazol) . DIAZEPAM 13





CLONAZEPAM 26.2 46

39

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Figure 13 . IC 50 profiles and GABA-shift profiles of diazepam, clonazepam and bretazenil in rat brain . IC 50 values (in nM, •) with 95% confidence limits do not differ dramatically from one brain region to another for all three compounds . GABA-shift profiles (thick bold bars, highly significant) differ markedly, reflecting the rank order of intrinsic efficacies (diazepam > clonazepam > bretazenil) . GABA-shift index = IC 50 - GABA/IC550 + GABA .

201

Benzodiazepine receptors : new vistas

Figure 14 . Modulation of GABA-stimulated 36chloride flux in a membrane preparation from rat cerebral cortex by a benzodiazepine receptor full agonist (diazepam), partial agonist (bretazenil) and full inverse agonist (DMCM) . GABA was added at the half-maximally effective concentration of 10µM . Bretazenil, a prototype partial agonist, exerts a positive modulatory influence albeit at reduced efficacy (intrinsic activity approx 26 of that of diazepam under the present test conditions) . For other assay conditions see Figure 9 .

occupancy is higher for the same magnitudes of pharmacological effects (Table 1), 66 it has lower efficacy in facilitating GABA-induced chloride flux in brain synaptoneurosomes (Figure 14) and lower efficacy in potentiating isoguvacine in hippocampal slices . 12 It has higher potency but lower efficacy in reducing cerebellar cGMP content (an index of Purkinje cell activity) ; low efficacy in producing mild sedation at doses far higher than those producing anticonvulsant and anticonflict effects (Figure 12) ; much less pronounced potentiation of ethanolinduced sedation ; and it antagonizes the sedating and motor performance-impairing effects of diazepam . Behavioural effects on chronic administration are sustained, i .e . there is little or no desensitization of GABAA receptors and virtually no physical dependence . 64,65 Lastly, in monkeys there is lack of induction and maintenance of intravenous self-administration . Clinical proof of the partial agonist concept is now required . To date, clinical studies have demonstrated the high potency of bretazenil as a therapeutic agent for generalized anxiety disorder and panic disorders, with minimal sedation in comparison to equi-anxiolytic doses of diazepam . Other partial agonists currently under clinical evaluation include FG 8205 (MS&D), 68 imidazo [ 1,2-a] pyrimidines e .g . RU 32698 (RousselUclaf), the ,ß-carboline abecarnil (Schering AG)69

and alpidem (Synthélabo) . 7° FG 8205 and abecarnil appear to have preclinical pharmacological profiles similar to that of bretazenil . Further clinical evaluation of bretazenil and its competitors is eagerly awaited .

Future perspectives Provided long-term clinical studies with partial agonists confirm the animal data (showing a virtual absence of profound sedation, tolerance, dependence liability and ethanol potentiation), benzodiazepine receptor ligands may well remain ideal therapeutic drugs . As yet undiscovered partial agonists with preferential affinities for certain receptor subunit variants 71 might also have unique pharmacological profiles with therapeutic utility . Recently, unexpected potential therapeutic indications for benzodiazepines have been revealed in clinical studies : the partial agonist bretazenil appears to have therapeutic potential for schizophrenia (see ref 2) and the antagonist flumazenil has been reported 58 to reverse tolerance to the antiepileptic effects of clonazepam, possibly by resetting the receptor to its naive state . The latter finding, if confirmed, will open up new avenues for the use of the respective antagonist for general treatment of tolerance and dependence to benzodiazepines and other therapeutic drugs .

202 Acknowledgement We thank our colleagues Drs P . Malherbe, J . Martin and E . Persohn as well as Miss M . Facklam for allowing us to quote unpublished findings and Mrs Martine Wdonwicki for secretarial assistance .

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