Commentary on the mode of action of benzodiazepines

.I psyrhint.

Pergamon

Rcs., Pnnted

Vol.

21,

in Great

SupplI, pp.193-207,

1993 Elsevier Scicncc Ltd Britain. All rights reserved N22-3956/93 $6.00 + .OO

COMMENTARY ON THE MODE OF ACTION OF BENZODIAZEPINES B. E. LEONARD Pharmacology

Department,

University

College, Galway,

Ireland

Summary-Evidence is presented showing that the benzodiazepines produce their variety of pharmacological effects by activating GABA A receptors in the mammalian brain. Different classes of benzodiazepine receptor ligands have been developed which can cause or alleviate anxiety according to the nature of their interaction with the GABA A receptor. There is now evidence that natural ligands also exist in the brain which can modulate GABA A receptor function. The changes in the responsiveness of the GABA A receptor to chronic benzodiazepine treatment is discussed with reference to the phenomenon of tolerance dependence and withdrawal.

Introduction the late 1960s the symptoms of anxiety and insomnia were mainly treated with barbiturates. The barbiturates were known to cause dependence, and severe withdrawal effects were sometimes reported following the abrupt termination of their administration. Furthermore, their efficacy in the treatment of anxiety disorders was limited. The discovery of the benzodiazepine anxiolytic chlordiazepoxide (Librium) some 30 years ago, and the subsequent development of numerous analogues with an essentially similar pharmacological profile, rapidly led to the replacement of the barbiturates with a group of drugs that have been widely used for the treatment of anxiety disorders, insomnia, muscle spasm, epilepsy and as a pre-operative medication (Lader, 1989). The benzodiazepines have also been shown to have fewer side-effects than the barbiturates, to be relatively safe in overdose and to be less liable to produce dependence than the barbiturates. The benzodiazepines have now become the most widely used of all psychotropic drugs; during the last 25 years it has been estimated that over 500 million people world-wide have taken a course of benzodiazepine treatment. In recent years there has been growing concern among members of the public and the medical profession regarding the problem of dependence and possible abuse of the benzodiazepines (Uhlenhuth, 1988) and the recent decrease in the numbers of prescriptions of these drugs for the treatment of anxiety (Lader, 1989) reflects this concern. And yet, despite the decline in the short-term use of benzodiazepine drugs to treat anxiety, their use as hypnotic-sedatives is largely unchanged. Furthermore, their long-term use for the treatment of anxiety and/or insomnia continues; in the U.K. approximately 1.5% of the adult population have taken benzodiazepines continuously for 1 year or more, while nearly half of these have taken the drugs for at least 7 years (Lader & Pertursson, 1983). It has been variously estimated that approximately 0.25 million people have been taking benzodiazepines continuously for several years in the U.K. UNTIL

193

The purpose of this short review is to give anxiety so that the mechanism of action of understood. The review will conclude with a macological properties of chemical analogues currently undergoing development.

Chemical

an outline of the chemical pathogenesis of the benzodiazepines may be more readily brief discussion on the diversity of pharof the “classical” benzodiazepines that are

Pathogencsis

of Anxiety

Although different authors have ascribed dilrerent meanings to the term “anxiety”, and interchangeably. it is generally accepted have often used the terms “fear” and “anxiety” that anxiety is an unpleasant state accompanied by apprehension. worry, fear. nervousness and sometimes conflict. Arousal is usually heightened. An increase in the autonomic sympathetic nervous system is often associated with these psychological changes which may be manifest as an increase in blood pressure and heart rate. an erratic respiratory rate, decrease salivary tlow leading to dryness in the mouth and throat, and gastrointestinal disturbances. Whilst “physiological” anxiety is usually short-lived, often with a rapid onset and abrupt cessation once the aversive event has terminated. “pathological” anxiety occurs when the response of the individual to an anxiety-provoking event becomes excessive and affects the ability of the individual to lead a normal lift. It has been estimated that 2 4% of the population suffer from pathological anxiety and frequently no causative factor can be and non-bcnTodiazepine anxiolytics. such as buspironc. identified. The benzodiazepines. may be useful in alleviating the symptoms of pathological anxiety. The three neurotransmitters that appear to bc most directly involved in different aspects of anxiety are noradrenaline, serotonin and gamma-aminobutyric acid (GABA). No~&PIz&le is the ncurotransmitter most closely associated with the peripheral and central stress response. Thcrc is expcrimcntal evidence to show that drugs that block the noradrcnergic autoreceptors (for example. yohimbine). on cell bodies and nerve terminals cause fear and anxiety in both man and animals (Chnrney & Redmond, 1983). Conversely. drugs that stimulate these autoreceptors (as exemplified by clonidine) diminish the anxiety state because they reduce the release of noradrenaline (Keshavan & Crammer, 1985). BenzodiaLepines have been shown to inhibit the fear-motivated increase in the functional activity of noradrenaline in experimental animals. but it is now widely believed that the action of the ben/odiuzepines on the central noradrenergic system is only short term and may contribute to the sedative effects which most conventional benzodiazepines have. at least initially. Nevertheless, altered noradrenergic function may underlie certain forms 01 severe anxiety such as that seen in patients with panic attack or anxiety states associated with major depression. Such forms of anxiety generally respond to treatment with antidepressants or with benzodiazcpines that also have some mild antidepressant properties (e.g., alprazolam). Despite the clinical and experimental studies implicating changes in central noradrcnergic function in the effects of the benzodiazepines. there is cxperimcntal evidence to show that chronic bcnzodiazepine administration has little efEect on the density ofadrcncrgic rcccptors in the central nervous system (Stanford. Little, Nutt, & Taylor, 19X6) despite the evidence that the functional activity of these receptors changes following the abrupt withdrawal 01

BENZODIAZEPINEMODE OF ACTION

195

the drugs from both animals (Rastogni, Lapierre, & Singal, 1978) and man (Nutt & Molyneux, 1987). One possible explanation for the apparent indirect effect of benzodiazepines on central adrenoceptors may involve corticotropin releasing factor (CRF). It has been shown that both CRF and adrenergic neurons activate the locus coeruleus (Al-Damluji et al., 1987) which suggests that a positive feedback loop exists between these stress inducing neurotransmitters. Roy et al. (1989) have reported that a positive correlation exists between the concentration of diazepam binding inhibitor and the concentration of CRF in the cerebrospinal fluid of depressed patients. Since GABA is known to exert an inhibitory effect on the release of CRF, the increase in the diazepam binding inhibitor may result in a reduced inhibitory effect of GABA on the release of CRF. From such studies, it may be concluded that the administration of a benzodiazepine receptor agonist to a patient with anxiety or panic disorder reduces central sympathetic hyperactivity by facilitating the inhibitory action of GABA on the release of CRF. Clearly this hypothesis remains to be further tested both experimentally and clinically. Several experimental studies have suggested that reduction in the activity of serofonin in the brain results in an anxiolytic effect and therefore the anxiolytic effects of the benzodiazepines may be at least partly mediated by a reduction in central serotonergic neurotransmission (Collinge, Pycock, & Taberner, 1983; File & Vellucci, 1978). Other studies have shown that benzodiazepines inhibit the firing of serotonergic neurons in the mid-brain raphe region, an area of the brain that contains serotonergic cell bodies that send projections to the limbic (emotional) and cortical regions of the brain (Laurent, Margold, Humbel, & Haefely, 1983). The link between the serotonergic pathways and the control of anxiety has been further strengthened by the introduction on non-benzodiazepine anxiolytics, such as buspirone, ipsapirone and gepirone, which decrease central serotonergic function by stimulating a subclass of serotonin receptors (5HT1 A) and result in a decrease in serotonergic function. Chopin and Briley (1987) have reviewed the actions of such drugs on the behaviour of animals in several anxiety-provoking situations. Despite the connection between the decreased functional activity of the serotonergic system and the anxiolytic effects of the benzodiazepines, it would appear that their effect on serotonergic transmission is indirect and probably mediated via a facilitation of GABA (Lista, Blier, & De Montigny, 1990; Nanopoulos, Belin, Matire, Vincendon, & Pujol, 1982; Soubrie et al., 1980). IJnlike the biogenic amines, noradrenaline and serotonin, GABA is one of the most widely distributed neurotransmitters in the mammalian brain occupying some 40% of all synapses. GABA is an inhibitory transmitter and therefore reduces the firing rate of excitatory neurons in which it is in contact. In various animal models of anxiety, the facilitation of GABAergic activity is associated with a reduction in anxiety (Ticku & Davis, which specifically block GABA receptors, 198 I). Conversely drugs such as bicuculline, precipitate the symptoms of anxiety. There is also experimental evidence to show that the anti-anxiety effects of the benzodiazepines may be inhibited by GABA receptor antagonists or by drugs that reduce the synthesis of GABA (Vellucci & Webster, 1984). From such studies it may be concluded that the primary action of “classical” benzodiazepines is to facilitate central GABAergic transmission but due to the modulatory effects of GABAergic neurons on other neurotransmitter systems in the brain, secondary changes occur in nor-

adrenergic and serotonergic these drugs.

pathways

which may contribute

I Iow do the BcnzodiaLepines

Augment

GABA

to the anxiolytic

efrects of

Function?

Schmidt and colleagues in 1967 (see Sternbach. 1973) were the first to show that diazcpam could potentiate the inhibitory effect of GABA on the cat spinal cord. Later it was shown that the effect of diazepam could bc abolished if the endogenous GABA content was depleted, thus establishing that dia7cpam, and related benzodiazepincs. did not act directly on GABA receptors but in some way modulated inhibitory transmission via GABA (Pole & Haefely, 1977). Two groups of investigators were responsible for explaining how the benzodiazepines modulate GABAergic transmission at the molecular level (Costa. Guidotti. Mao. & Suria, 1975; Haefcly et al., 1975). These investigators showed that the benzodiazcpines bind with high afinity and specificity to neuronal elements in the mammalian brain and that there was an excellent correlation between the atfinity of the bcn7odiazepines for their specific binding sites and their pharmacological potencies in alleviating anxiety in both man and animals (Braestrup & Squires, 197X). The binding of a benzodiazepincs to this receptor site is enhanced in the presence of GABA or ;I GABA agonist, thcrcby suggesting that ;I functional. but independent. relationship exists between the GABA receptor and the benzodiarepine receptor. Two distinct classes of receptors for GABA exist in the mammalian brain, GABA A and CBA B receptors. These are characterized by their afinity for specific agonists and antagonists. the efrect or systems to which they arc coupled and the prcscnce or absence of allosteric modulatory sites. Whereas, the GABA A receptors arc ion channels that regulate the chloride conductance of the subsynaptic membrane (Olsen & Venter, 1986). GABA B receptors are coupled to GTP binding proteins that regulate adenylate cyclase in addition to potassium and calcium channels (Bowery, 1989). GABA is the natural agonist for both types of receptors but selective agonists and antagonists exist which enable these receptor types to be distinguished. Thus muscimol and baclofen are agonists. while bicuculline and phaclofcn are antagonists of the GABA A and GABA B receptors respectively (Bowel-y, Maguire. & Pratt, 1991; .lohnston. 1991). Allosteric sites on ncuronal GABA A receptors are targets through which benzodiazepines. and ben/odiaLepine ligands such as the cyclopyrrolone zopiclone and the imida/opyridine-rolpidem. modulate GABA A rcccptor function. Other allostcric sites on the GABA A receptor include those for the barbiturates. convulsants (such as picrotoxin) and general anaesthetics as exemplified by halothane. propofol, some steroids and ethanol (Ticku. 1991). A unique feature of the GABA A receptor is its ability to mediate opposite pharmacological efrects by facilitating or impeding GABA rcccptor function depending on the nature of the receptor ligand that binds to the allosteric sites. These modulatory effects are limited in their intensity such that the maximal response to the physiological effects of GABA is not cxcccdcd. This property of the GABA A rcccptor is probably one of the primary reasons why agonists of the ben/odiazepine receptor have a high therapeutic index. Figure I is a diagrammatic representation of the different allosteric sites which have been identified on the GABA A rcccptor.

197

A = GABA site (muscimol, isoguvacine, bicuculline) B = Benzodiazepine site (diazepam, flumazenil, RO 19-4603 C = Barbiturate site (phenobarbitone; etomidate; etazolate) D = General anaesthetic site (halothane; ethanol; steroids; propofol) E = Picrotoxinin site (tetrazoles, picrotoxin, bicyclophosphates) F = Storage site for GABA G = GABA auto receptor (valproate, carbamazepine)

The barbiturates, and to some extent alcohol, also seem to produce their anxiolytic and sedative effects by facilitating GABAergic transmission. This action of chemically unrelated compounds (i.e. alcohol, barbiturates and benzodiazepines) can be explained by their abilities to stimulate specific sites on the GABA receptor complex, the most marked effect being due to the benzodiazepines when they activate their specific receptor site (Tallman & Gallagher, 1985). Thus benzodiazepines bind with high affinity to the benzodiazepine receptor and, as a result, change the structural conformation of the GABA receptor so that the action of GABA on its receptor is enhanced. This enables GABA to produce a stronger inhibition of the post-synaptic neuron than would occur in the absence of the benzodiazepine, the anxiolytic effect being produced by an allosteric enhancement of the action of GABA. The relationship between the various components of the GABA receptor and the GABA nerve terminal, and the actions of various benzodiazepine ligands on the GABAbenzodiazepine receptor complex, has been discussed and illustrated by Haefely (1990). The inhibitory effect of GABA is mediated by chloride ion channels. When the GABA receptor is occupied by GABA, or a drug acting as an agonist. such as muscimol, the ion channels open and chloride ions together with potassium ions diffuse into the cell. Thus an

H. E. LEONAKI)

198

inhibitory transmitter like GABA is thereby hyperpolarizing the cell. The chloride ion channel contains at least two binding sites. One of these sites is activated by barbiturates that have weak anxiolytic and hypnotic properties (for example, pentobarbitone and phenobarbitone). Such drugs facilitate inhibitory transmission by increasing the duration of opening of the chloride ion channel. Another class of experimental anxiolytic agents that are not structurally related to the benzodiazepines (the pyrazolopyridines, ofwhich etazolate is a clinically active example), also act at a specific site within the chloride ion channel and enhance GABAergic function by increasing the frequency of channel opening (Collins. Sakalis, & Minn, 1976). Thus, it may be concluded that the “classical” benzodiazepines such as diazepam, act as anxiolytics by activating a specific benzodiazepine receptor which facilitates inhibitory GABAergic transmission. Other drugs with anxiolytic properties, such as some of the barbiturates and alcohol, also facilitate GABAergic transmission by acting on sites associated more directly with the chloride ion channel. Diversity

of Drugs Acting

on the Benzodiazepine

Receptor

Until about 1980, it was widely accepted that the benzodiazepinc structure was a prerequisite for the anxiolytic profile and for the recognition and binding to the benzodiazepinc unrelated drug. the cyclopyrrolone receptor. More recently, however, a chemically zopiclone, which has a benzodiazepine-like profile, has been shown to be a useful sedative hypnotic. Other chemical classes of drugs that are also structurally dissimilar to the benzodiazepines (for example the triazolopyridazines) have also been developed and shown to have anxiolytic activity in man; these non-benzodiazepines also act via the bcnzodiazepinc receptor (Haefcly, Kyburz, Gcrecke, & Moehler, 1985). Thus the term “bcnzodiazepinc receptor ligand” has been introduced to describe all drugs, irrespective of their chemical structure, that act on benzodiazepine receptors and thereby modulate inhibitory transmission in the brain. Over the last decade there has been an increase in our knowledge of the relationship between the structure of the benzodiazepine receptor ligand and its pharmacological properties. This has led to the development of potent receptor agonists, that stimulate the receptor and produce pharmacological effects qualitatively similar to diazepam and related “classical” benzodiazepines, antagonists, which block the effects of the agonists without having any effects themselves, and a group of drugs that have a mixture of agonists and antagonist (so called partial-agonist) properties. In addition, an intriguing group of compounds has been developed that have the opposite effects on the benzodiazepine receptor to the pure agonists. These are known as inverse agonists. The pharmacological properties of these different types of benzodiazepine receptor ligands are summarized in Scheme I. At the molecular level, the differences between the agonist and antagonist benzodiazepine are ascribed to the ability of the drug to induce a conformational change in the fine structure of the receptor molecule that produces functional consequences in terms of the cellular changes. The partial agonists have intrinsic activity that lies between the full agonists and the antagonists. When administered, they have qualitatively similar etfects to full agonists but may not be quite as potent: when given with full agonists they reduce the potency of

BENZODIAZEPINE MODE OF ACTION

199

Scheme 1 Properties

of Benzodiazepine

Receptor

Ligand.7

Full agonists (e.g., Diazepam. Midazolam) Partial agonists (e.g., RO 16-6028)

Full inverse agonist (e.g., RO 19-4603) Partial inverse agonist (e.g., RO 15-4513) Antagonist (e.g., Flumazenil)

I -anxiolytic -anticonvulsant -myrorelaxant -amnesic --facilitutes GABA

I -little direcl effect on receptor -Hocks eflects qf agonists and inverse agonists transmission

I -anxiogenic -convulsant or pro-convulsant -arousing --spasmogenic -promnesic ~-depresses GABA fransmission

the full agonist. Some 10 years ago, the Danish investigators Braestup and Nielsen (1983) found that a group of non-benzodiazepine compounds, the beta-carbolines, not only antagonized the actions of the full agonists but also had intrinsic activity themselves. Such compounds were clearly not pure antagonists which lack intrinsic activity but were found to be inverse agonists because they had the exact opposite biological effects to the pure agonists, i.e., they caused anxiety, convulsions and were promnestic. Regions of the mammalian brain that are innervated by GABAergic neurons contain a high density of GABA A receptors and its associated binding sites. The application of quantitative receptor autoradiographic methods has led to the identification of selective binding sites. These include high and low affinity sites for GABA, as well as the sites for benzodiazepine ligands and the modulatory sites associated directly with the chloride ion channel. The identification of these different sites, has led to the conclusion that there are at least four main subtypes of the GABA A receptor (Richards, Schock, & Haefely, 1991). Encoded by a family of at least fifteen genes, GABA A receptor subtypes are assembled from several subunits termed alpha 1 and alpha 6, beta 1, beta 4 and gamma 1, gamma 3, delta and pi. Immuno precipitation studies with subunit specific antibodies have shown that the most prevalent subunits (alpha 2, beta 2, beta 3 and gamma 2) are frequently coassembled to form a basic structure for most of the GABA A receptors (Benke, Mertens, Trzeciak, Gillessen, & Mohler, 1991). It is now known that different populations of neurons may be distinguished by specific GABA A receptor subtypes. For example. the alpha 1 subunit is frequently localized in GABAergic neurons, whereas monoaminergic and cholinergic neurons, which are modulated by GABAergic neurons, selectively express the alpha 3 subunit (Mohler & Fritschy, 1992). Based on the differences in the basic structure of the GABA A receptor, four classes of receptors have been identified due to their interactions with different types of benzodiazepine receptor ligands. Type I receptors, with the structure of alpha 1, beta, gamma 2, show a high affinity for the imidazopyridine zolpidem. Type II receptors, with the subunit structure alpha 2 (3) B, gamma 2, have an intermediate affinity for zolpidem, while Type III receptors (alpha 5, beta, gamma 2) have a very low affinity for zolpidem. By contrast, Type IV receptors (alpha 6, beta, gamma 2) have a low affinity for the 1,4_benzodiazepine receptor agonist, but a high affinity for the inverse agonist Ro 15-45 13. It would appear that the type of gamma subunit strongly influences the affinities of the receptor for antagonists and inverse agonists (Richards et al., 1991).

200

H. E.

LLONAKI)

At the subcellular level, it has been postulated that the GABA receptor might spontaneously oscillate between states ofhigh and low affinity for GABA. It would appear that agonists and inverse agonists stabilize the receptor in the high and low affinity conformation respectively. Conversely. competitive antagonists might bc unable to distinguish between these two conformational states but act by preventing the access of either the positive or negative allosteric modulators. Partial agonists, by contrast, product a shift in the equilibrium between the low and high affinity state that is less marked than the full agonist because they cannot so readily distinguish between the two conformations. The interaction between benzodiazepine receptor ligands and their allosteric sites on the GABA A receptor complex is determined by its binding affinity and its intrinsic efficacy. Full agonists have positive intrinsic efficacy, invcrsc agonists have negative intrinsic efficacy, while between these extremes, competitive antagonists have zero efficacy. The intrinsic efficacy of a benzodiazepine receptor ligand may be quantitied by measuring the GABA stimulated chloride flux; such changes arc directly reflected in the pharmacological profile of the ligand. For example, the I ,4_benzodiazepines like diazcpam are anxiolytic, sedative, muscle relaxant and anticonvulsant. whcrcas the full inverse agonists such as the bctacarboline DMCM have the opposite effects. An agonist or inverse agonist that has a low capacity to activate the receptor is described as a partial agonist or partial inverse agonist. Thus benzodiazepine receptor ligands show a continuous spectrum of intrinsic efficacies in modulating GABA response from potent convulsants. such as DMCM that inhibit GABA responses, to potent central depressants. such as diazepam, that potentiate such responses. The partial agonists, partial inverse agonists and antagonists occur in between these extremes. The benzodiazepine receptor is so far unique in that it has a bidirectional function. This discovery could be of major importance in designing drugs in which the adverse effects ol the “classical” benzodiazepines could bc reduced but their beneficial effects maintained. Although the I ,6benzodiazepinc anxiolytics are effective and safe drugs with a rapid onset of action. there is a practical need for drugs which arc less sedative. with a lower dependence potential and a lack of interaction with alcohol. Similarly, the benzodiazcpine hypnotics arc liable to cause hangover effects. show a liability to tolerance development following prolonged use, cause amnesia and have an abuse potential (see Lopez et al.. 1990). It is possible that partial agonists may offer therapeutic advantages over the conventional full agonists; the partial agonist brctazcnil has pharmacological propertics which appear to support this hypothesis. Thus, it has been shown to product only a mild degree of sedation at doses that are far higher than those which are anticonvulsant and which have marked anticonflict effects. It shows little interaction with ethanol but antagonizes the sedative and motor-impairing etfccts of high doses of diazepam. In animal studies. there is little evidence of physical dependence (Haefcly, Martin, & Schoch, 1990). Preliminary clinical studies also suggest that bretazenil is an effective anxiolytic with a lower propensity to cause sedation, muscle relaxation and amnesia than diazepam. The abuse potential also appears to be low (Delini-Stula. 1992). While these preliminary clinical studies suggest that bretazcnil is not entirely devoid of undesirable properties, it dots show a higher therapeutic index than the “classical” bcnzodiazepincs. Thus the development of partial agonists may be particularly important in the production OF anxiolytics that lack the sedative and amnestic properties or the full agonists.

BE~~ZODIAZEPINE Mom OF ACTION

Are These Natural

Ligands

for the Benzodiazepine

201

Receptor

on the Brain?

The presence of benzodiazepine receptors in the brain would suggest that there are natural ligands present which modulate these receptors. To date, a specific compound(s) has not been unequivocally identified but a number of candidates have been isolated that show agonist or inverse agonist activity. Some of these candidates are listed in Table 1. Table

I

Nicotinamide Inosine and hypoxanthine Ethyl beta-carbolinc-3 carboxylate Tribulin Nephentin Diazepam displacing activity in human Diazepam binding inhibitor (DBI)

cerebrospinal

fluid

Of the putative ligands for the benzodiazepines receptor that are listed in Table I, diazepam binding inhibitor (DBI), nephentin and tribulin appear to be particularly interesting. DBI is a polypeptide that has been isolated, and its structure elucidated, from mammalian and human brain. It is called diazepam binding inhibitor because it can inhibit the binding of tritiated diazepam to the benzodiazepine receptor; recently, it has also been shown to inhibit the binding of antagonists and inverse agonists to the benzodiazepine receptor. Pharmacological studies show that DBI has anxiogenic properties and its concentration in the brain appears to be sufficiently high to block benzodiazepine receptors under appropriate conditions. It is only present in trace amount in tissues other than the brain. A detailed account of properties of DBI has been published by Barbaccia, Berkovich, Guarneri and Slobadyansky (1990). Tribulin is a relatively low molecular weight compound with acidic or neutral properties that has been isolated from human urine (Sandler, 1983). The presence of this compound increases following stress and it has been found to inhibit the binding of benzodiazepines to their receptor site. Sandler (1983) has suggested that tribulin might be related to the endogenous anxiogenic factor and be structurally related to the beta-carbolines. Nephentin is also a large polypeptide that has been shown to have a relatively high affinity for the benzodiazepine receptor and does not have any effect upon other neurotransmitter receptors (Woolf & Nixon, 1981). Unlike DBI, however, the concentration of nephentin is much higher in non-nervous peripheral tissues such as the bile duct than it is in the brain. Furthermore, the distribution of nephentin in the brain does not coincide with that of the benzodiazepine receptors. It is possible, nevertheless, that nephentin is a precursor of a lower molecular weight peptide that can block the benzodiazepine receptor. Less progress has been made in the detection of natural compounds that may act as agonists on the benzodiazepine receptor (Mueller. 1987). Three non-peptides (nicotinamide, inosine and hypoxanthine) have been shown to have low affinities for the benzodiazepine receptor, and there is some experimental evidence suggesting that they have mixed agonistantagonist properties. Nevertheless, the concensus of opinion would appear to suggest that these substances are not the endogenous ligands for the benzodiazepine receptor. It is

possible that purinergic mechanism are activated by inosine and hypoxanthinc and that the modulation of benzodiazepine receptor function is a secondary consequence of this (Snyder, 1985). The detection of compounds with a benzodiazepine structure in human brain and breast milk raises the possibility that such compounds could act as endogenous agonists at benzodiazepine receptor sites (see de Bias et al., 1985). There is no evidence that such compounds are synthesized in vivo and may be the products of bacterial activity in the gastrointestinal tract and/or from plant products in the diet. However. evidence is now accumulating that benzodiazepines are synthesized in the mammalian brain and may play a physiological rote in brain function (see Iquierdo & Medina. 1993). It may be concluded that there is some evidence to suggest that anxiety arises as ~1 consequence tither of a deficiency of an endogcnous agonist or to the prcscnce of cndogenous inverse agonist acting on the benzodiazepine ~GABA receptor complex. Thus, one possible approach to drug design in the future may be in the development of drugs that either facilitate the synthesis of endogenous agonists, or reduce the synthesis of inverse agonists, at the benzodiazepines receptor sites. Changes

in BenLodiaTepine

Receptor Function Following Bena~dia7epines

Chronic

Administration

ol

It is a well-established biological phenomenon that receptors adapt to the prolonged presence or absence of an agonist by changing their sensitivity, thereby attempting to return their function to normal levels. Such changes, sometimes termed “up”or “down” regulation, tnay develop slowly or rapidly, the former being due to changes in the synthesis of the receptor, while the tatter probably rcflccts the movement of receptors into, or out of, the neuronal membrane. This area has been reviewed by Enna (1984) and Schwcitjer. Bonnet and FricdhofT (1984). Experimental studies in rodents have clearly demonstrated that high doses of”classica1” benzodiazepines such as diazepam, lorazepam and flurazepam cause a decrease in benLodiazcpine receptors in the cortex of the brain (Braestrup, Nielsen, & Squires, 1978; Chiu & Rosenberg, 1978) but the number of receptors rapidly returns to normal (approximately 5 days) following the abrupt cessation of drug treatment. There is also electrophysioiogical evidence to show that the functional activity of the GABA receptors that are linked to the benzodiazepines receptors is also decreased following prolonged treatment with chlordiazcpoxide (Gallagher, Kakoski, Consalves, & Rauch, 1984). even though the actual number of GABA receptors is increased (Mueller, 1987). In vitro cvidcnce also suggests that chronic benzodiazepine treatment results in an uncoupling of the benzodiazepine receptor from the GABA receptor complex (Rota, Rozenberg, Farranti. & Farb, 1990). Functional tolerance following chronic treatment with the benzodiazepines is well documented in animals and man, and represents a pharmacodynamic rather than pharmacokinetic phenomenon. Tolerance appears to occur more rapidly with the sedative and anticonvulsant than the anxiolytic properties of the “classical” bcnzodiazepincs. However, since clinically relevant tolerance develops with therapeutic doses, but changes in receptor tolerance only occur with very high doses of the drugs that are usually far in excess of those used clinically, little experimental evidence exists at present whereby the functional tolerance

BENZODIAZEPINEMow OF ACTION

203

to benzodiazepines can be explained on the basis of benzodiazepine receptor “down” regulation (Rosenberg & Chiu, 1982; Tietz, Rosenberg, & Chiu, 1986). However, one must be cautious in extrapolating the results of animal experiments to the patient with an anxiety disorder who is being treated with a benzodiazepine. The benzodiazepine receptor complex shows marked plasticity in the animal brain, but relatively few changes have been noted in their receptor system in samples obtained from post-mortem human brain, even when the patients suffered from epilepsy at the time of death (Sherwin, Matthew, Blain, & Guevremont, 1986; Wusterman, Reynolds, & Martin, 1985). This suggest that the regulation or plasticity of the benzodiazepine receptor in the human brain differs considerably from that of the brain of the experimental animal although the molecular properties of the benzodiazepine receptor appear to be similar! Benzodiazepine

Dependence

Tolerance, dependence and withdrawal have been observed following the chronic administration of all types of benzodiazepine receptor agonists to animals and man. The development of tolerance, defined as a reduced pharmacological response with repeated use, varies according to the pharmacological response being measured. Thus tolerance develops to the sedative and ataxic effects and, lastly, the anxiolytic effects (Gent & Haigh, 1983; File, 1983). Experimental studies show that there are clearly differences in the rate of tolerance development both within and between species (File, 1983). Withdrawal effects are known to result following the abrupt discontinuation of treatment following the chronic administration of benzodiazepine receptor agonists. Such effects are indicated by the behavioural and psychological changes that are the opposite to those seen during the acute administration of the agonists. Thus, in man, severe dysphoria, anxiety and even seizures were initially demonstrated following the chronic administration of large doses of chlordiazepoxide (Hollister, Motzenbecker, & Degan, 1961) but withdrawal reactions from therapeutic doses of the benzodiazepines were only convincingly demonstrated more recently (see Petursson & Lader, 1981; Tyrer, Owen, & Dawling, 1983). Whereas some of the symptoms that occur following benzodiazepine withdrawal may reflect the emergence of the underlying symptoms of anxiety, the presence of symptoms that were not present in the previously untreated state (for example, muscle cramps, confusion, paranoid ideation and changes in visual perception) suggest that true withdrawal effects can occur even following the long-term administration of therapeutic doses of bcnzodiazepine receptor agonists. Benzodiazepine dependence can be inferred from the occurrence of the withdrawal effects in both animals (Eisenberg, 1987) and man (Woods, Katz, & Winger, 1987). Psychological dependence also occurs and undoubtedly may contribute to benzodiazepine use and abuse in some individuals. However, it should be emphasized that the benzodiazepines have a low abuse potential in comparison with other drugs including the barbiturates (Griffiths, Bigelow, Liebin, & Kaliszak, 1980; Woods et al., 1987). The phenomenon of tolerance, dependence and withdrawal is dependent on benzodiazepine receptor occupation as evidenced by the observation that co-administration of a benzodiazepine receptor antagonist, such as flumazenil, can prevent the occurrence of

204

B. E.

LEONAKU

tolerance (File, Lister, & Nutt, 1982). Clearly pharmacokinetic characteristics of the benzodiazepines also contribute to the severity of the withdrawal symptoms. Thus drugs with a relatively short half-life, such as lorazepam, that have a rapid clearance, produce more severe withdrawal symptoms than those with a slow clearance (e.g., diazepam). As withdrawal symptoms can be precipitated by the administration of antagonist to an individual to whom benzodiazepincs had been chronically administered (Wilson & Gallagher, 19X8), it is possible to use this procedure to evaluate the degree of benzodiazepine dependence. Factors such as the rate of entry of the benzodiazepine into the brain, and its rate of clearance, are also of importance in determining the abuse potential of these drugs (Griffiths, McLeod, Bigelow, Liebson, & Roache, 19X4). At the cellular level, benzodiazepine dependence has been explained by changes that occur within the GABA-benzodiazepine receptor complex and also in the intra-cellular events which occur as a consequence of the changes in benzodiazepine rcccptor function. Nutt and co-workers, in their rodent studies of the chronic effects of lorazepam in the presence and absence of flumazenil. have suggested that a benzodiazepine receptor shift in the direction of the inverse agonist state accounts for many of the features of benzodiazepine tolerance and withdrawal (Little, Nutt, & Taylor. 1987). This shift in the sensitivity of the benzodiazepine receptor has been termed a “withdrawal shift” and is shown by an enhanced erect (seizures) of a partial inverse agonist (FG 7 142). Such changes would not appear to be due to other components of the GABA receptor complex. Presumably these changes result from a shift in the coupling mechanism between the bcnzodiazepine receptors and the GABA receptor chloride ionophorc from optimal agonist efficacy to increased inverse agonist cfhcacy (Mele, Sagratella. & Massotti, 1984). As such effects have been shown to occur relatively rapidly (hours), it may be concluded that other adaptive changes proximal to the GABA-benzodiazepine receptor complex arc also involved in the development of tolerance and dependence following the long-term administration of such drugs. Conclusion In this short review, evidence is presented to show that the benzodiazepines produce their variety of pharmacological effects by activating specific receptors that form part of the main inhibitory neurotransmitter receptor system, the GABA receptor, in the mammalian brain. Different classes of benzodiazepine receptors ligand have been developed which can alleviate anxiety or produce anxiety according to the fine structural changes that occur when the drugs interact with the benzodiazepine receptor. There is some evidence that natural substances occur in the human brain that can cause either an increase or a reduction in the anxiety state by acting on the benzodiazepine receptor. The unique nature of the benzodiazepine receptor, and the disparate propcrtics of the drugs that act on this receptor, should allow plenty of scope for the development of novel compounds with selective anxiolytic and other properties in the future. Lastly, despite the evidence from animal studies that benzodiazepine receptor function changes in response to chronic drug treatment, there is little evidence from human brain studies that such changes are relevant to the phenomenon of tolerance, dependence and withdrawal effects that have been the recent cause for public concern.

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