GABAA receptor subtypes as targets for neuropsychiatric drug development

Pharmacology & Therapeutics 109 (2006) 12 – 32 www.elsevier.com/locate/pharmthera

Associate editor: A.L. Morrow

GABAA receptor subtypes as targets for neuropsychiatric drug development Esa R. Korpi *, Saku T. Sinkkonen Institute of Biomedicine, Pharmacology, Biomedicum Helsinki, P.O. Box 63 (Haartmaninkatu 8), FI-00014 University of Helsinki, Finland

Abstract The main inhibitory neurotransmitter system in the brain, the g-aminobutyric acid (GABA) system, is the target for many clinically used drugs to treat, for example, anxiety disorders and epilepsy and to induce sedation and anesthesia. These drugs facilitate the function of pentameric A-type GABA (GABAA) receptors that are extremely widespread in the brain and composed from the repertoire of 19 subunit variants. Modern genetic studies have found associations of various subunit gene polymorphisms with neuropsychiatric disorders, including alcoholism, schizophrenia, anxiety, and bipolar affective disorder, but these studies are still at their early phase because they still have failed to lead to validated drug development targets. Recent neurobiological studies on new animal models and receptor subunit mutations have revealed novel aspects of the GABAA receptors, which might allow selective targeting of the drug action in receptor subtype-selective fashion, either on the synaptic or extrasynaptic receptor populations. More precisely, the greatest advances have occurred in the clarification of the molecular and behavioral mechanisms of action of the GABAA receptor agonists already in the clinical use, such as benzodiazepines and anesthetics, rather than in the introduction of novel compounds to clinical practice. It is likely that these new developments will help to overcome the present problems of the chronic treatment with nonselective GABAA agonists, that is, the development of tolerance and dependence, and to focus the drug action on the neurobiologically and neuropathologically relevant substrates. D 2005 Elsevier Inc. All rights reserved. Keywords: GABAA receptor subtypes; Subtype selectivity; Intrinsic activity; Extrasynaptic receptors; Alcoholism; Anxiety Abbreviations: BPAD, bipolar affective disorder; COGA, The Collaborative Study on the Genetics of Alcoholism; GABA, g-aminobutyric acid; SNP, single nucleotide polymorphism; THIP, gaboxadol, 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3ol.

Contents 1. 2. 3. 4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Basic pharmacology and molecular biology of g-aminobutyric acid type A receptor Synaptic and extrasynaptic g-aminobutyric acid type A receptors as drug targets. . g-Aminobutyric acid type A receptor subtypes in psychiatric disorders . . . . . . . 4.1. The gene cluster a1-a6-h2-g2 . . . . . . . . . . . . . . . . . . . . . . . . 4.2. The gene cluster a2-a4-h1-g1 . . . . . . . . . . . . . . . . . . . . . . . . 4.3. The gene cluster a3-q-u. . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. The gene cluster a5-h3-g3 . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5. Conclusions on the genetic studies . . . . . . . . . . . . . . . . . . . . . .

* Corresponding author. Tel.: +358 9 191 25330; fax: +358 9 191 25364. E-mail address: [email protected] (E.R. Korpi). 0163-7258/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.pharmthera.2005.05.009

. . . system . . . . . . . . . . . . . . . . . . . . .

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Review and criticism of the current pharmacological attempts to modulate g-aminobutyric acid type A receptor activity in neuropsychiatric disorders . . . . . . . . . . . . . . . . 5.1. Benzodiazepine site inverse agonist compounds . . . . . . . . . . . . . . . . . . 5.2. g-Aminobutyric acid site compounds . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Neurosteroid compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Future possibilities in the development of drugs acting on g-aminobutyric acid type A receptors: subtype selectivity, minor subtypes, subcellular targeting . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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5.

1. Introduction In very simple terms, neuropharmacology and psychopharmacology are based on the fundamental balance between chemical excitation and inhibition. Both processes are indispensable in the networks of neurons, and all neurons have receptors for inhibitory and excitatory neurotransmitters in different domains of their cell plasma membranes. Although excitation is the primary way of information transfer between various brain regions, inhibition, especially that mediated by interneurons, is responsible in many networks for bursting and oscillatory activity. This bursting and oscillatory activity can be initially produced by intrinsic pacemaker-like membrane ion channel mechanisms or it can be elicited by afferent activity from input neurons. The g-aminobutyric acid (GABA) system is one of the mechanisms that take care of chemical inhibition in the brain, and it has been widely used for pharmacological modulation of brain functions. Most brain neurons express GABA receptors, the GABA type A (GABAA) receptors being considered as the most important for pharmacological modulation. These receptors have integral anion channels that, when activated, can pass chloride ions through the membrane along their electrochemical gradients, thus usually producing inhibition of depolarization or even hyperpolarization of the neuronal membrane. In the following, we briefly summarize the GABAA receptor system including the mechanisms of action of the present drugs acting on this receptor, give an overview of some of the compounds presently under development, and review the receptor subunit/psychiatric disease associations to illustrate the need for new approaches in relation to the advanced neurobiology of psychiatric diseases. It is very important to understand that, in most cases, we try to explain the effects of drugs, here those acting on GABAA receptors, solely by their intrinsic efficacy mode and/or selectivity on various receptor target populations, in the absence of real knowledge on the neurobiology or neuropathology of psychiatric illnesses. Therefore, we need to carry on innovative and active basic neuroscience research to develop new target-based drugs and selective tools. Also, it is important to note that most of the current developmental ideas originate from studies on experimental animals and animal models of disorders, and thus, the clinical applicability of these ideas is always subject to critical evaluation.

21 22 23 24 25 25

2. Basic pharmacology and molecular biology of ;-aminobutyric acid type A receptor system The first GABAA receptor subunit, the a1 subunit, was cloned almost 20 years ago (Schofield et al., 1987), and that was followed by a rapid progress in finding additional subunits with interesting features. There are several recent reviews on the topic (Wisden & Seeburg, 1992; Sieghart, 1995; McKernan & Whiting, 1996; Sigel & Buhr, 1997; Hevers & Lu¨ddens, 1998; Moss & Smart, 2001; Korpi et al., 2002a; Ernst et al., 2003; Lu¨scher & Keller, 2004; Olsen et al., 2004; Rudolph & Mohler, 2004; Steiger & Russek, 2004; Vicini & Ortinski, 2004), and here, only basic facts are summarized. There are 19 different human GABAA receptor subunits, all encoded by different genes. The molecular heterogeneity of the receptor subtypes stems mainly from sequence differences between the subunits, and on this basis, the subunits can be classified into a (1 –6), h (1– 3), g (1 – 3), y, q, k, u, and U (1 3) subunit classes. The function will then depend on the subunit combinations of the pentameric receptor complex, that is, on receptor subtypes. Various subunits have either widespread or very restricted expression profiles in various brain regions and cell types, making it possible to have different receptor subtypes to be modulated differently in different neuronal populations. The main problem still in relating receptor subtypes to drug actions is the lack of exact knowledge of the native receptor subunit combinations. It is evident from studies on native brain receptors that the number of receptor subtypes is restricted (see McKernan & Whiting, 1996), but we still cannot exclude the possibility that a minor subtype, located in a specific neuronal circuit, would be critical for certain behavior and therefore more important or useful as a drug target than a subtype that is abundant and widespread. The main subunit combination is the a1h2g2 receptor that accounts for about 40% of all GABAA receptors (McKernan & Whiting, 1996). It should be noted that the relative and absolute amounts of receptor subtypes are not precisely known. The receptor subtype issue is far more complicated due to significant assembly of more than 1 type of a subunit in a pentamer (Benke et al., 2004). The a1, h2, and g2 subunits are ubiquitous and often expressed in the same brain regions/neurons. When the a1 and h2 subunits are expressed in heterologous expression systems, they

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produce functional ah receptors that have high sensitivity to GABA (for a review, see Hevers & Lu¨ddens, 1998) but a low channel conductance. When the g2 subunit is expressed together with the a1 and h2 subunits, the receptors have a lower GABA sensitivity but higher conductance, which corresponds to the properties of main native GABAA receptor populations. The smaller conductance of the a1h channels might also explain why the knockout mice without g2 subunits are not viable (Gu¨nther et al., 1995). However, the g2 subunits have been shown to regulate the targeting of GABAA receptors to synapses via interaction with gephyrin, a protein also needed for clustering of inhibitory glycine receptors (Kneussel & Betz, 2000). Without the g2 subunits, receptors are more localized on extrasynaptic membranes (Nusser et al., 1998; Mangan et al., 2005), where they may also mediate important regulatory function by sensing low concentrations of GABA spilled over from neighbouring synapses (Mody, 2001). The g subunit, especially the most abundant g2 variant, is obligatory for high-affinity benzodiazepine binding and efficacy (Pritchett et al., 1989). GABAA receptor forms an integral anion-selective channel, which normally mediates fast chloride ion influx opposing depolarization or producing hyperpolarization of the neuronal plasma membrane and thereby reducing any firing activity of the postsynaptic neuron. This action may explain the sedative and hypnotic effects of GABAA receptor agonists, such as benzodiazepines, neurosteroids, barbiturates, alcohol, anesthetics, and GABA-site agonists, although the detailed mechanisms of the various agonists may differ. Physiological effects mediating inhibition (GABAA receptor function) require some gradient of Cl from outside the cell to in, which is produced by the chloride pumping action of various ion transporters. An interesting example is the KCC2 transporter, whose expression is developmentally regulated in many neurons (Rivera et al., 1999). Early in the development, the KCC2 transporter is not expressed and GABA responses of the GABAA receptors are depolarizing due to lack of adult-like electrochemical Cl gradients. Although various GABAA receptor subtypes differ in kinetics of the channel function (Hevers & Lu¨ddens, 1998), it is not known how these differential properties would make a contribution in physiology. At least in some neurons, the subcellular location of the receptor subtypes in the neurons (Nusser et al., 1998; Nusser & Mody, 2002) and their activation by presynaptic neurons or spillover GABA may be as important for physiology as any channel property conferred by different receptor subunits. The subunit combination, though, is decisive in subcellular targeting of the receptor subtypes (Moss & Smart, 2001). Other neurochemical mechanisms to affect GABAA receptor function, such as post-translational modifications, have been recently reviewed (Lu¨scher & Keller, 2004). A simple classification of GABAA/benzodiazepine receptors can be made on the basis of benzodiazepine affinities. In terms of clinical pharmacology, it is useful to

make the classification on the basis of diazepam and zolpidem affinities with the help of 1 experimental ligand, Ro 15-4513. The high-affinity binding of flumazenilsensitive, tritium-labelled Ro 15-4513 is very little affected by natural molecular heterogeneity of ahg2 receptors, and therefore, it has been used as the ligand for receptor subtyping (Lu¨ddens et al., 1995). For example, the classical benzodiazepine diazepam displaces [3H]Ro 15-4513 from all other benzodiazepine receptors (diazepam-sensitive receptors), except for the ones having a4 or a6 as the a subunit (diazepam-insensitive receptors). Thus, diazepam acts as an agonist (increases allosterically the affinity for GABA) on a1/2/3/5 subunit-containing ahg2 receptors, but fails to act on a4 and a6 subunit-containing receptors. The a1 subunit has a histidine residue at the position of about 100 in the extracellular domain, and this residue is obligatory for classical benzodiazepine actions (Wieland et al., 1992). Homologous histidine is also found in other benzodiazepine-sensitive receptor subtypes having the a2, a3, or a5 subunits, but the insensitive subunits a4 and a6 harbor arginine in the same position. This residue is critical for the benzodiazepine action as a domain of the binding site and/or a domain for regulating allosteric interactions (Dunn et al., 1999). Recent molecular modelling studies have indicated that this histidine/arginine residue is close to the interface between a and g2 subunits (Ernst et al., 2003), and experiments with concatenated subunits have clarified the subunit order within the pentameric complex to allow or prevent diazepam sensitivity in a1a6h2g2 receptors (Minier & Sigel, 2004). The 100th residue has also been important in constructing several novel mouse models, which have helped to reveal the roles of the a subunits in behavior and physiology (see, for reviews, Rudolph et al., 2001; Korpi et al., 2002a; Vicini & Ortinski, 2004). In particular, these studies have revealed that the a1 subunits make a large contribution to the sedative, amnestic, and anticonvulsant components of the benzodiazepine action (Rudolph et al., 1999; McKernan et al., 2000), while the a2 subunits seem to be responsible for the anxiolytic actions of benzodiazepines (Low et al., 2000). These preclinical results have a major impact on the future development of subtypeselective compounds for specific disorders and clinical indications. Further details and suggestions of pharmacological significance of the receptor subunits and subtypes are compiled in Table 1. In addition, there are also many other residues that are pharmacologically important in various receptor subunits, which make it possible to look for receptor subtype-selective new compounds, especially for the benzodiazepine site. For example, the hypnotic imidazopyridine zolpidem, but not classical benzodiazepine flurazepam, loses its behavioral and GABA-potentiating activity in mice with a F77I point mutation in the g2 subunit (Cope et al., 2004). In addition to benzodiazepines, many other drugs act by facilitating the action of GABAA receptors. Barbiturates and neurosteroids seem to have rather little subtype specificity

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15

Table 1 Properties of GABAA receptors containing specific subunits Subunit

Preferred subunit combinations (McKernan & Whiting, 1996)

Suggested main subcellular localization (Mody, 2001; Brunig et al., 2002)

Pharmacology (Sieghart, 1995; Korpi et al., 2002a)

Putative neuropsychiatric indications or effects of selective agonists (A) or antagonists (AN) as predicted from animal model data (Korpi et al., 2002a; Rudolph & Mohler, 2004), human genetic correlation studies (Table 2), or pharmacological properties (this table)

a1

a1h2g2

Synaptic

DZ-S, Zol-S, Niflu agon (Sinkkonen et al., 2003), La3+ agon

a2

a2h2/3g2, a2hXg1

Synaptic

DZ-S

a3

a3hXg2/3

Synaptic/extrasynaptic

DZ-S, low GABA-S

a4

a4h2/3, a4h2/3y, a4h2/3g2

Extrasynaptic

a5

a5h3g2/3

Extrasynaptic

DZ-IS, high THIP-S (Brown et al., 2002), Furo-S, high EtOH-S (Wallner et al., 2003), high NS-S (Brown et al., 2002) DZ-S, L655,708-S

Sedation (A) (Rudolph et al., 1999; McKernan et al., 2000), sleep disorders (A), agitation (A), anterograde amnesia (A), mood disorders (A?/AN?), epilepsy (A?/AN?) Anxiety (A) (Low et al., 2000), myorelaxation (A), alcoholism (A?/AN?) Myorelaxation (A), mood disorders (A?/AN?), alcoholism (A?/AN?) Anxiety (A), amnesia (A), alcoholism (A?/AN?). Hypnosis (A)?

a6

a6h2/3g2, a6h2/3y

Extrasynaptic

h1

a3h1g2/3 (suggested by localization) a1h2g2, a2h2g1/2, a4h2, a4h2y, a4h2g2, a6h2g2, a6h2y a2h3g2

Synaptic

DZ-IS, high GABA-S, high EtOH-S (Wallner et al., 2003), Furo-S, Niflu antagon (Sinkkonen et al., 2003), high Zn2+-S, La3+ antagon SCS-S (Thompson et al., 2004)

Synaptic/extrasynaptic

Lore-S, Furo-S, Etom-S, Prop-S

Synaptic/extrasynaptic

Lore-S, Furo-S, Etom-S, Prop-S, high EtOH-S (Wallner et al., 2003)

h2

h3

g1

a2h3g1 (suggested by localization)

?

BZ-S

g2

aXhXg2

Synaptic

g3 y

a3hXg3, a5h3g3 a4h2/3y, a6h2/3y

? Extrasynaptic

q

a3hu/q (suggested by localization; Luque et al., 1994; Sinkkonen et al., 2000; Moragues et al., 2002)

?

BZ-S, Niflu agon (Sinkkonen et al., 2003), TCZ agon (Thompson et al., 2002) BZ-S BZ-IS, high GABA-S, high THIP-S (Brown et al., 2002), high EtOH-S (Wallner et al., 2003), high NS-S (Brown et al., 2002), high Zn2+-S BZ-IS, NS-IS, TCZ antagon (Thompson et al., 2002)

u

a3hu/q (suggested by localization; Luque et al., 1994; Sinkkonen et al., 2000; Moragues et al., 2002)

?

BZ-IS, NS-IS

Memory and learning (AN) (Collinson et al., 2002; Crestani et al., 2002b), myorelaxation (A), mood disorders (A?/AN?) Anxiety (A), amnesia (A), myorelaxation (A), alcoholism (A?/AN?), mood disorders (A?/AN?) Alcoholism (A?/AN?) Sedation (A) (Reynolds et al., 2003), alcoholism (A?/AN?), schizophrenia (A?/AN?) Immobilization (A) (Jurd et al., 2003), hyperactivity and hyperreactivity (A) (Homanics et al., 1997), memory and learning (AN), alcoholism (A?/AN?), autism (A?/AN?), Angelman’s syndrome (A), epilepsy (A?/AN?) Localization in the hypothalamus, septum, and amygdala: role in neuroendocine functions? Anxiety (A) (Crestani et al., 1999), schizophrenia (A?/AN?) Alcoholism (A?/AN?), autism (A?/AN?) Anxiety (A), amnesia (A), hypnosis (A), alcoholism (A?/AN?)

Opiate withdrawal (A) (Heikkila et al., 2001). Cholinergic and monoaminergic localization (Sinkkonen et al., 2000; Moragues et al., 2002): role in anxiety, arousal, attention, panic and drug withdrawal, learning? Monoaminergic localization (Sinkkonen et al., 2000; Moragues et al., 2002): role in anxiety, attention, panic, and drug withdrawal?

In Pharmacology column: agon, agonistic or positive modulation; antagon, antagonistic or negative modulation; S, sensitive; IS, insensitive; BZ, benzodiazepine; DZ, diazepam; EtOH, ethanol; Etom, etomidate; Furo, furosemide; L655,708, a5-selective BZ-site inverse agonist; Lore, loreclezole; Niflu, niflumate; NS, neurosteroid agonist; Prop, propofol; SCS, salicylidene salicylhydrazide; TCZ, tracazolate; Zol, zolpidem.

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and their actions are not dependent on the g2 subunits. A general intravenous anesthetic etomidate requires the h2 or h3 subunits for its efficacy, and it has been pointed out that the residues in the 2nd transmembrane region have a critical role either for the binding or for the allosteric interactions of this drug (Belelli et al., 1997). Homologous or closely located amino acid residues of various other GABAA receptor subunits have also shown to be important for the high-concentration actions of ethanol and volatile anesthetics (Mihic et al., 1997). Some of these residues may form hydrophobic binding pockets for the drugs in the transmembrane domains (Jenkins et al., 2001). The h subunits might contribute to the selectivity of certain compounds by other unspecified domains, too (Hamon et al., 2003). Whatever is the exact mechanism on how the h subunits affect drug responses remains to be established, but recent animal models have revealed specific contributions of the h2 and h3 subunits to different components of anesthesia: The h2 subunit-containing receptors mediate the sedative component of the etomidate anesthesia (Reynolds et al., 2003), whereas the h3 subunit-containing receptors mediate the deeper anesthesia, including immobility and analgesia (Jurd et al., 2003). These important results, showing the GABAA receptor subtype distinction between sedation and anesthetic immobility and analgesia, were possible with the point-mutated mouse lines, one with etomidate-insensitive h2(N265S) subunits (Reynolds et al., 2003) and the other with etomidate and propofol-insensitive h3(N265M) subunits (Jurd et al., 2003). More selective intravenous anesthetics might be produced by selective targeting of the h3 subunit-containing GABAA receptors. The inhalation anesthetics enflurane and halothane act almost normally also in h3(N265M) knockin mice (Jurd et al., 2003), and generally, the volatile anesthetics act less specifically via the GABAA receptor system (Sonner et al., 2003). For example, in the absence of the a1 subunit, only the supraspinal amnestic actions of isoflurane are reduced (Sonner et al., 2005). Furthermore, a point mutation in the critical amino acid for the volatile anesthetic action (Nishikawa et al., 2002; Nishikawa & Harrison, 2003) in the a1 subunit of a1(S270H) knockin mice produces strong alterations in GABAA receptor function itself and in baseline behavioral phenotype (Homanics et al., 2005), so far preventing the study of the molecular mechanism of volatile anesthetic action in vivo in a similar fashion as what has been achieved with benzodiazepines and intravenous anesthetics. Anyway, all this detailed information from molecules to behaving animals should start promoting our possibilities to rationally modulate the inhibitory circuits in the brain in various neuropsychiatric disorders. The GABA binding site has been also resolved in the middle of the extracellular domain of the GABAA receptor subunits. Like the benzodiazepine binding site, it is apparently also located in the interface between 2 subunits, now between the a and h subunits. There are many different residues that have been found to affect the efficacy and

binding of GABA and antagonists in various a and h subunit variants (see for a review, Korpi et al., 2002a), and the amino acid domains responsible for the functional diversity between the receptor subtypes have started to be revealed (Wagner & Czajkowski, 2001; Bo¨hme et al., 2004). There seems to be some brain regional variation in the potency and efficacy of various GABA site agonists and antagonists (Rabe et al., 2000), suggesting that different receptor subtypes would be differentially affected by the compounds. Muscimol is a prototypic specific agonist for GABAA receptors. The regional distribution of [3H]muscimol binding suggests that high-affinity interaction is limited to a6, a4, and y subunit-containing receptors, especially in the thalamic and cortical regions and in the cerebellar granule cell layer (Korpi et al., 2002b). The y subunit does not support benzodiazepine sites. The expression of a4 and y subunits is high, for example, in the thalamus, where a1h2g2 receptor is not as abundant as in many other regions (Wisden et al., 1992; Pirker et al., 2000). Therefore, it is possible that some direct GABA agonists might offer profiles of receptor subtype stimulations clearly different from those of benzodiazepines.

3. Synaptic and extrasynaptic ;-aminobutyric acid type A receptors as drug targets Many basic physiological characteristics of GABAAergic tonic inhibition are still unclear (Mody, 2001), but the most interesting question at the moment, at least to pharmacologists and the pharmacological industry, concerns drug therapy: Where are actually the primary targets of GABAAergic drugs, in the postsynaptic membranes or on peri- and extrasynaptic locations? GABAAergic drugs usually exert their actions by enhancing the actions of GABA. It has been estimated that peak GABA concentrations in the synaptic cleft reach 0.3 – 3 mM (Mozrzymas et al., 1999; Perrais & Ropert, 1999), but most receptor subtypes have an EC50 for GABA below 50 AM (Hevers & Lu¨ddens, 1998). The discrepancy between the receptors’ affinity for GABA and the apparently overwhelming agonist concentrations in the synaptic cleft during transmission makes it tempting to speculate that the primary targets for GABAAergic drugs are the extrasynaptic receptors. In these receptors, positive modulators would have powerful effects on inhibitory tone in the presence of low ambient GABA. In fact, their effects would be largely independent of synaptic activity. The y subunit-containing receptors are addressed to nonsynaptic membranes in the cerebellum, hippocampus, and thalamus (Nusser et al., 1998; Nusser & Mody, 2002; Wei et al., 2003), being primarily assembled in the forebrain with the a4 subunits (Korpi et al., 2002b; Peng et al., 2002) and in the cerebellum with the a6 subunits (Jones et al., 1997). The y subunit-containing receptors may thus form the major extrasynaptic receptor subtype, and their proper-

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ties, such as very high sensitivity and very slow desensitisation (Saxena & Macdonald, 1994; Hevers et al., 2000), fit well to monitor low extracellular agonist concentrations and to regulate neuronal excitability on that basis (Mody, 2001). The details of extrasynaptic GABAA receptor action are poorly known, and several other receptor subtypes may be involved in specific neurons. For example, the diazepamsensitive a5 subunit-containing receptors are extrasynaptic in the hippocampus (Brunig et al., 2002; Lindquist et al., 2003), and novel diazepam-insensitive q and u subunitcontaining receptors in various aminergic nuclei might be extrasynaptic, as they should lack the g2 subunits (Sinkkonen et al., 2000; Moragues et al., 2002; Sergeeva et al., 2005), and some of these extrasynaptic non-y receptors might also constitute targets for drug development. But is it possible to selectively modulate 1 modality of the GABAAergic inhibition without affecting the other? It has been demonstrated that the tonic conductance in CA1 pyramidal neurons of rat hippocampal slices is potentiated by midazolam and propofol more than the phasic conductance (Bai et al., 2001). Vigabatrin, an antiepileptic drug that increases brain GABA levels, reduces phasic but increases tonic GABAAergic inhibition (Overstreet & Westbrook, 2001; Wu et al., 2003). More recently, the y subunitcontaining receptors have been suggested to mediate the main actions of neurosteroids and, perhaps, the low concentration effects of ethanol (Mihalek et al., 1999; Belelli et al., 2002; Sundstrom-Poromaa et al., 2002; Spigelman et al., 2003; Wallner et al., 2003), and, interestingly, there seems to be a significant involvement of neurosteroids in the tolerance and dependence processes to ethanol (reviewed in Kumar et al., 2004). Neurosteroid modulation is reduced in the hippocampal dentate gyrus of y subunit-deficient mice in conjunction with deletion of extrasynaptic receptors (Stell et al., 2003). Volatile anesthetics might target a5 subunit-containing hippocampal extrasynaptic receptors (Caraiscos et al., 2004). Taking these recent findings together, and accepting the fact that the subunit composition of the receptor determines its subcellular localization and pharmacological properties, it should be possible to introduce novel compounds acting selectively on phasic or tonic GABAAergic conductance. With these compounds, better and safer treatment could be feasible. Considering only some of the brain regions where tonic conductance could be selectively modulated, novel treatments affecting, for example, memory performance and seizure threshold (hippocampus), sleep and anesthesia (among others thalamus), and motor coordination (among others cerebellum) could be envisaged. One possible drawback in the venture for selective modulation of phasic or tonic inhibition might arise from the apparent delicate balance between the 2 forms of GABAAergic inhibition. In the 2 animal models, where tonic inhibition is increased due to ectopic expression of a6 subunit (Wisden et al., 2002) or knockout of GABA transporter-1 (Jensen et al., 2003), there is an yet unidenti-

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fied compensatory mechanism that balances the increased tonic inhibition with reduction in phasic inhibition. In the ectopic a6 mouse model, this compensation seems to be associated with enhanced propensity to convulse in response to GABAA receptor blockade (Sinkkonen et al., 2004). The GABA uptake inhibitor tiagabine, causing increased ambient GABA levels and enhanced tonic inhibition (Wisden et al., 2002), interestingly is more potent but less efficient in preventing seizures in the mutant than wild-type mice (Sinkkonen et al., 2004). This would argue for the correct balance between synaptic and extrasynaptic inhibition. If similar balance prevails during the course of chronic drug therapy, as suggested by the effects of vigabatrin (Overstreet & Westbrook, 2001; Wu et al., 2003), the applicability of selective ligands is obviously more complex. Still, the possibility of finding novel drug targets, indications, and safety profiles by studying the properties of extrasynaptic GABAA receptors is currently 1 path not to be forgotten in the drug development of nonbenzodiazepine-site compounds focusing especially on a4, a6, or y subunitcontaining receptors. 4. ;-Aminobutyric acid type A receptor subtypes in psychiatric disorders It is difficult to demonstrate a causative role for disturbed GABAA receptor function in the pathophysiology of any disorder, but a connection between these two may be surmised on the basis of the following findings: (1) a genetic linkage between disorder incidence and, for example, subunit mutation or polymorphism in humans, (2) altered GABAA receptor function (due to changes in receptor subunit composition or subunit expression) in patients with the disorder, (3) mouse models with selective GABAA receptor alterations displaying similarities to human disorder, and (4) good clinical efficacy of GABAAergic drugs in the treatment of the disorder. Based on these criteria, a role for the GABAAergic system may be proposed in a variety of psychiatric disorders. Disorders linked with an altered GABAAergic system, or which can be efficiently treated with GABAAergic drugs, include at least alcoholism, Angelman’s syndrome, anxiety disorders, autism, depression, mania, premenstrual syndrome, schizophrenia, and sleep disorders (Smith et al., 1998; Benes, 1999; DeLorey & Olsen, 1999; Lancel, 1999; Buxbaum et al., 2002; Brambilla et al., 2003; Davies, 2003). Diverse molecular pharmacology of the GABAA receptors is a result of different subunit combinations, which makes the individual subunits the key elements for rational drug design. To get some idea of the possible roles of different GABAA receptor subtypes in the pathogenesis of neuropsychiatric diseases and their possible value as drug targets, we have gathered information about the studies addressing genetic associations between GABAA receptor subunits and neuropsychiatric disorders in human (Table 2).

18

Subunit

Disorder

The gene cluster a1-a6-b2-c2 a1 Bipolar mood disorder

a1

a6

Depression, both sexes; Bipolar mood disorder, only females Depression, only females; Bipolar mood disorder, only females Neuroticism; suggestive for depression Alcoholism and low response to alcohol Alcoholism

a6

Alcoholism

a6 a6

Altered salivary cortisol levels Altered stress response

h2 h2

Alcoholism Schizophrenia

g2

None

a6

a6 a6

The gene cluster a2-a4-b1-c1 a2 Alcoholism

a2 a4

Alcoholism None

Genetic marker

Ethnicity

Sample

Reference

Haplotype (GT repeat polymorphism in intron 2 and SNP T156C in exon 4) SNP in intron 10

Japanese and USA (multicentric; NIMH Initiative Bipolar Pedigrees) Japanese

125 bipolar cases and 191 controls and 88 multiplex pedigrees with 480 subjects 203 mood disorder cases and 202 controls

Horiuchi et al., 2004 Yamada et al., 2003

SNP C to A at cDNA nucleotide 1497

Japanese

203 mood disorder cases and 202 controls

Yamada et al., 2003

Pro385Ser variant

Caucasian

384 healthy subjects

Sen et al., 2004

Pro385Ser variant

Caucasian

41 children of alcoholics

SNP T1519C in 3Vuntranslated region Pro385Ser variant

Scottish

108 alcoholics and 54 controls

Schuckit et al., 1999 Loh et al., 1999

Southwestern Native-Americans and Finnish Swedish

419 sib pairs, 110 alcoholics, 277 relatives and 124 controls

Radel et al., 2005

284 healthy male subjects

USA

56 healthy subjects

Rosmond et al., 2002 Uhart et al., 2004

Scottish Han Chinese

108 alcoholics and 54 controls ¨120 schizophrenics and ¨120 controls

Loh et al., 1999 Lo et al., 2004

SNPs (mostly in introns, exonic did not cause amino acid changes) and their haplotypes

USA (multicentric)

Edenberg et al., 2004

10 SNPs and their haplotypes

European Americans

The Collaborative Study on the Genetics of Alcoholism (COGA) sample: Families with at least 3 alcoholic members 446 alcoholics and 334 controls

SNP T1519C in 3Vuntranslated region SNP T1519C in 3Vuntranslated region Silent exonic SNP T1412C 5 SNPs (in introns 7 and 8) and their haplotypes

Covault et al., 2004

E.R. Korpi, S.T. Sinkkonen / Pharmacology & Therapeutics 109 (2006) 12 – 32

Table 2 Positive genetic associations between GABAA receptor subunits (in gene clusters) and human neuropsychiatric disorders

h1

Alcoholism

Tetranucleotide repeat polymorphism in intron 8 Tetranucleotide repeat polymorphism in intron 2

USA

133 alcoholics and 89 controls

h1

Alcoholism

g1

None

USA (multicentric; COGA)

363 trios families

Dinucleotide (CA) repeat polymorphism

Caucasian

314 alcoholics and 174 controls

Dinucleotide (CA) repeat polymorphism in intron 8

European (multicentric)

185 bipolar cases with 370 matched controls

The gene cluster a5-b3-c3 a5 Autism

Individual SNPs and their haplotypes

Caucasian

123 multiplex autistic families

a5

Bipolar mood disorder

Dinucleotide (CA) repeat polymorphism

Greek

48 bipolar cases and 50 controls

h3

Autism

USA, mostly Caucasian

h3

Autism

h3

Autism Alcoholism

European Caucasian

138 families (125 trios, 13 parent – child pairs) 80 families (59 multiplex and 21 trios) 108 families (76 multiplex, 32 singleton) 171 alcoholics and 45 controls

Buxbaum et al., 2002 Shao et al., 2003

h3 h3

European Caucasian

86 male PTSD cases

Feusner et al., 2001

g3 g3

Severity of posttraumatic stress disorder (PTSD) Alcoholism Autism

Dinucleotide (CA) repeat polymorphism in intron 3 Dinucleotide (CA) repeat polymorphism in intron 3 Dinucleotide (CA) repeat polymorphism in 3Vuntranslated region Dinucleotide (CA) repeat polymorphism in 3Vuntranslated region Dinucleotide (CA) repeat polymorphism in 3Vuntranslated region 7 SNPs and their haplotypes SNP T539C in exon 5 and SNP in intron 5

USA (multicentric; COGA) USA

262 multiplex families with 2282 subjects 226 autistic trios families

Dick et al., 2004 Menold et al., 2001

y

None

The gene cluster a3-e-h a3 Alcoholism Bipolar mood disorder

q u

None None

USA, mostly Caucasian USA

Parsian & Cloninger, 1997 Massat et al., 2002

McCauley et al., 2004 Papadimitriou et al., 1998 Cook et al., 1998

Noble et al., 1998

E.R. Korpi, S.T. Sinkkonen / Pharmacology & Therapeutics 109 (2006) 12 – 32

a3

Parsian & Zhang, 1999 Song et al., 2003

19

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The table is by no means comprehensive, since research activity in this field is presently enormous. Here, we briefly describe some of the positive findings using the subunit gene clusters to illustrate the disorder/gene polymorphism associations. The receptor subunit gene clustering might not mean much physiologically, some of the genes being regulated rather independently, but others may join common regulatory elements (Steiger & Russek, 2004). 4.1. The gene cluster a1-a6-b2-c2 The genes for a1, a6, h2, and g2 subunits form a cluster in human chromosome 5q34– q35 (Russek, 1999). This chromosomal area is suggested to be linked to mood disorders (Rice et al., 1997). Associations between a1 subunit haplotypes and mood disorders were detected in a study on Japanese population and in another one of the National Institute of Mental Health Initiative Bipolar Pedigrees (Yamada et al., 2003; Horiuchi et al., 2004). No evidence on the haplotype effects on a1 subunit function or pathophysiology of the disorder was obtained. A study with bipolar patients also suggested a possible linkage between the a1 subunit gene and the disorder (De Bruyn et al., 1996). Associations between an a6 subunit gene haplotype and mood disorders in female patients was detected in the Japanese population (De Bruyn et al., 1996). No information about the effects of this haplotype on a6 subunit function or pathophysiology of the disorder was obtained. In conjunction, the a6 subunit gene variant (Pro385Ser) was found to be associated with higher neuroticism scores in a sample of non-Hispanic Caucasians, suggestive for a possible role in depression (Sen et al., 2004). The same Pro385Ser variant of a6 gene has previously been shown to affect benzodiazepine sensitivity (Iwata et al., 1999), but the mechanism is unclear. In addition, Pro385Ser was associated with low response to alcohol and alcoholism in a pilot sample of children of Caucasian alcoholics (Schuckit et al., 1999). Pro385Ser was recently associated with alcoholism in independent Southwestern Native-Americans and Finnish populations (Radel et al., 2005). Another link between a6 subunit gene polymorphism and alcoholism was provided by a study where a single nucleotide polymorphism (SNP) T1519C in the 3V untranslated gene region was associated with alcohol dependence in a Scottish population (Loh et al., 1999). Interestingly, the same SNP was shown to be associated with higher salivary cortisol levels in homozygotes for T allele in comparison to heterozygotes in a Swedish population (Rosmond et al., 2002). In that population, also a link between obesity on the T/T haplotype was suggested. Another link between alcoholism and GABAA receptor polymorphism was provided by a study where a silent exonic SNP (T1412C) in h2 subunit gene was associated with alcohol dependence in Scottish population (Loh et al., 1999). Another polymorphism in h2 gene has been

linked with schizophrenia in Chinese population (Lo et al., 2004). Thus far, no genetic associations between the g2 subunit gene and psychiatric disorders have been published. 4.2. The gene cluster a2-a4-b1-c1 In order to reveal the genes affecting the risk for alcoholism, the Collaborative Study on the Genetics of Alcoholism (COGA) performed a whole-genome survey on families of alcoholics and found linkage between chromosome 4p and alcohol dependence (Reich et al., 1998). GABAA receptor g1, a2, a4, and h1 subunit genes are clustered in this region (McLean et al., 1995), and Edenberg et al. (2004) performed a linkage disequilibrium analysis of SNPs of these genes. Only the SNPs in a2 subunit gene were significantly associated with alcoholism. Since the haplotypes derived from SNPs did not result in amino acid coding changes of the a2 gene, the authors suggested that the effect would be mediated through gene regulation. Furthermore, the a2 subunit gene polymorphism was associated with brain oscillations at beta frequency, indicating that the a2 subunit-containing GABAA receptors provide with strong effects on cortical neuronal networks. Similarly, in another study Covault et al. (2004) found that, in European American subjects with alcohol dependence, there was an association of SNP haplotypes of the a2 gene and alcoholism. Actual pharmacological effects of alcohol seem to be affected by the a2 subunit gene polymorphisms (Pierucci-Lagha et al., 2005), and interestingly, finasteride, a 5a-steroid reductase inhibitor used in the treatment of prostate hypertophy, attenuates many subjective effects of alcohol in an a2 gene-allele specific manner, perhaps by reducing the activity of progesterone-derived neurosteroids. Song et al. (2003) found linkage disequilibrium between h1 subunit polymorphism and alcoholism in COGA subjects with multiplex alcoholic pedigrees. Another study with alcoholics and controls also found a correlation between h1 gene polymorphism and alcoholism (Parsian & Zhang, 1999). 4.3. The gene cluster a3-e-h Several genetic linkage studies have suggested the chromosome region Xq26 – 28 to harbor genes associated with bipolar affective disorder (BPAD; for references, see Massat et al., 2002). This region contains the GABAA receptor a3 subunit gene. Massat et al. (2002) searched for a possible association between a dinucleotide repeat (CA) polymorphism of the a3 gene (Hicks et al., 1991) and BPAD in a European multicentric case-control sample. They found a correlation between the polymorphism and disorder, particularly in females. Since the polymorphism is located near the 3Vend of the gene in intron 8, it does not affect the properties of the receptors containing the a3 subunit, but may affect BPAD phenotype by altered gene expression

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levels. Parsian and Cloninger (1997) found association a3 subunit gene polymorphism and alcoholism in Caucasian population. Thus far, no genetic associations between the other members of the gene cluster, q and u, and psychiatric disorders have been found. 4.4. The gene cluster a5-b3-c3 Based on the GABAergic hypothesis of mood disorders, genetic association studies have been performed between GABAA receptor a5 subunit gene polymorphism and mood disorder patients. In a study with Greek subjects and matched controls, a correlation of a a5 gene dinucleotide repeat (CA) polymorphism with bipolar disorder, but not with unipolar disorder, was found (Papadimitriou et al., 1998). No information about the significance of gene polymorphisms in the receptor function was provided. Chromosome 15q11– q13 is a candidate region for autistic disorder. This region harbors gene cluster for GABAA receptor subunits a5, h3, and g3 (Sinnett et al., 1993; Russek, 1999). Using SNPs as markers, McCauley et al. (2004) found evidence for allelic association between a5 and h3 subunit genes and autism in the population of 123 multiplex autistic families. Also, an association of g3 subunit SNPs with autism has been reported (Menold et al., 2001). Various other studies have also found evidence for h3 and autism association (Table 2). Noble et al. (1998) and Dick et al. (2004) have suggested an association of h3 and g3 polymorphisms and alcoholism, respectively. Feusner et al. (2001) has proposed a link between severity of posttraumatic stress disorder and h3 subunit gene polymorphism.

21

ward, how polymorphisms might affect GABAAergic system without interfering the properties of the subunit proteins per se. Still, these are only possible mechanisms, and none of them have been actually demonstrated to take place in the brain. Considering these factors, it seems impossible, on the basis of the genetic linkage studies between subunit gene polymorphism and disorder incidence carried out so far, to speculate whether an agonist or antagonist of the given subunit would be suitable for the treatment of the disorder. With the above precautions in mind, one may get excited about the emerging roles of a2 and a6 subunit genes in alcoholism. While the a6 subunit gene polymorphism may not be important for all types of alcoholics (Dick et al., 2005), it seems to be a significant factor in some populations of alcoholics (Radel et al., 2005). The linkage between the a2 subunit gene and alcoholism (Edenberg et al., 2004) appears to hold more generally in various populations (Covault et al., 2004; Lappalainen et al., 2005), and some evidence for altered pharmacology keeps coming: Pierucci-Lagha et al. (2005) have found that the alcoholism-associated allele of the a2 subunit gene reduces the subjective effects of ingested alcohol. It clearly remains to be assessed how the brain GABAA receptor subtypes are functioning in individuals with differing a2 subunit gene alleles, before we may rationally formulate a hypothesis how to treat alcohol dependence by GABAA receptor modulation.

5. Review and criticism of the current pharmacological attempts to modulate ;-aminobutyric acid type A receptor activity in neuropsychiatric disorders

4.5. Conclusions on the genetic studies To our knowledge, none of the genetic polymorphisms presented in Table 2 causes such changes in receptor subunit protein structure or function that would provide an obvious pathophysiological mechanism for the disorder. In fact, most of the polymorphisms are found in introns or other untranslated regions. This is common in multigenetic diseases. What might then be the relevance of the findings? The genetic associations presented in the Table 2 may represent polymorphisms that are in linkage disequilibrium with a functional polymorphism or mutation in the open reading frame of the gene. Alternatively, the polymorphisms in the uncoding region may affect gene transcription and, for example, stability of the transcripted mRNA. These changes, in turn, may affect subunit protein levels and, thus, receptor subunit composition. Since alterations in the gene regulation of 1 GABAA receptor subunit may alter the regulation of the other genes at the cluster level (UusiOukari et al., 2000), changes in the level of 1 subunit may affect transcription levels of the others in the same cluster. Together, there are several ways, although not straightfor-

Historically, the GABAA receptor has been the target of many drug treatments. The earliest compounds were ions like bromide, then came barbiturates, and finally, from 1960s onwards, a number of benzodiazepines. The benzodiazepines were considered, at the time of their introduction, as very efficient and safe ‘‘minor tranquillizers’’, but more recently, their use has been criticized because of the dependence-producing effects. This concerns the prolonged use of especially the long-acting anxiolytic compounds rather than the short-acting sedative and sleep-inducing ones. This is also accompanied by clear tolerance development that has limited their use, for example, in epilepsy. Anyway, we have several efficient benzodiazepines in use, and a clinician can select a benzodiazepine agonist in relation to its length of action, dosage form, metabolic interactions, and other drug safety features. We do not see any strong need to develop further standard nonselective benzodiazepine-site agonist drugs, since there is a good selection of effective compounds for insomnia, anxiety, and sedation. In addition, there is a selection of a1 subunitpreferring hypnotics (zolpidem, zopiclone, and zaleplon),

22

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and this category might not need any more members for the clinical use. Therefore, we are not going to deal much with nonselective benzodiazepine agonist compounds that might still be under development, and we do so under full understanding that very minor differences between drug properties may make an unpredictably large impact on drug treatment in practice. 5.1. Benzodiazepine site inverse agonist compounds The main target domain in the drug development has been the allosteric benzodiazepine site, and most present drug development is still focussed on that site. This review picks up only some examples of these compounds and makes no further evaluation on each and every compound. Atack (2003) has recently reviewed the development status of partial agonists, such as imidazobenzodiazepines bretazenil, imidazenil, and FG 8025 (L-663581), the h-carboline mixed agonist/partial agonist abecarnil, the pyrazolopyrimidine ocinaplon (CL 273,547), the cyclopyrrolone pagoclone (RP 59037), the pyridobenzimidazole RWJ-51204, and (S)desmethylzopiclone, and receptor subtype-selective compounds L-838417, NGD 91-3, and SL651498. His review and the references therein should be consulted for details on these development projects. It should be noted that acutely benzodiazepines are safe drugs as compared, for example, to barbiturates. There have been rather few adverse effects (see for paradoxical actions, Mancuso et al., 2004), in addition to tolerance (loss of efficacy during repeated administrations) and dependence (withdrawal symptoms upon discontinuation) issues during chronic treatment (Bateson, 2002). Benzodiazepines do affect cognitive functions, for example, by inducing sedation, decreasing attention, and producing anterograde amnesia, only the 2 first effects apparently showing tolerance (Buffett-Jerrott & Stewart, 2002). The chronic use of benzodiazepines might thus affect the outcome of cognitive behavioral therapy (Westra & Stewart, 1998). The long-term cognitive effects would still need further research, but it should be noted that at least 1 case-control study found significantly less Alzheimer’s disease in elderly subjects with a history of prolonged use of benzodiazepines, especially in those using the short-acting ones (Fastbom et al., 1998). It may be speculated that the neuroprotective/ hypothermic effects of benzodiazepines (Schwartz-Bloom et al., 2000; Kuhmonen et al., 2002) could be involved. Benzodiazepine agonists have been tried in the treatment of alcoholism in the 1960s and 1970s, but the efficacy was clearly lacking. Presently, benzodiazepines are the treatment of choice only in the withdrawal phase to reduce the likelihood of seizures. This treatment is carried out as ‘‘loading’’ treatment, in which the withdrawing alcoholic is treated, for example, with a standard 20 mg dose of diazepam (or other long-acting full agonists) every 1– 2 hr depending on symptoms (Sellers et al., 1983; Saitz et al., 1994).

Benzodiazepine agonists, if anything, increase alcohol drinking in animal models (Wegelius et al., 1994; June et al., 1996), and as alcohol and benzodiazepines are often coabused, the agonists cannot act antialcoholic in humans, either. On the other hand, in preclinical experiments, it has been clearly shown that GABAA receptor antagonists, such as picrotoxin and bicuculline, can antagonize alcohol intoxication and drinking behavior (Kulonen, 1983; Hellevuo et al., 1989; Boyle et al., 1993). The same efficacy has also been detected with benzodiazepine-site inverse agonists, the prime example being Ro 15-4513 that was initially widely reported as an efficient and specific alcohol antagonist (Suzdak et al., 1986; Bonetti et al., 1988). However, soon thereafter, this compound was found to rather be a physiological antagonist, via its inverse agonism of the GABAA receptors (Hellevuo & Korpi, 1988; Hiltunen & Jarbe, 1988; June & Lewis, 1989). More recently, the inverse agonist compounds have shown efficacy in reducing alcohol drinking in rodents under different paradigms (June et al., 1991, 1996, 1998; Rassnick et al., 1993; Wegelius et al., 1994), and this is a property that is still under drug development. GABAA receptor ligands seem to affect bidirectionally alcohol drinking behavior similarly to consumption of palatable food (Cooper, 1986). Newer GABAA receptor benzodiazepine site partial inverse agonists RY-023 (t-butyl 8-(trimethylsilyl) acetylene-5,6-dihydro-5-methyl6-oxo-4H-imidazo [1,5a] [1,4] benzodiazepine-3-carboxylate), RY-80 (ethyl-8-acetylene-5,6-dihydro-5-methyl-6oxo-4H-imidazo [1,5a][1,4] benzodiazepine-3-carboxylate; showing also selectivity to a5 subunit-containing receptors; Liu et al., 1996; Strakhova et al., 2000), h-CCT (hcarboline-3-carboxylate-t-butyl ester), and 3-PBC (6-(propyloxy)-4-(methoxymethyl)-h-carboline-3-carboxylic acid ethyl ester) have all shown efficacy to reduce alcohol consumption in rats (Cox et al., 1998; June et al., 2001, 2003; McKay et al., 2004). To our knowledge, no clinical experiments have been started yet, and further experiments, for example, on monkeys, might be necessary before human trials (Shelton & Grant, 2001). One important thing to remember when administering benzodiazepine-site inverse agonists to people is that they most likely provoke anxiety (Dorow et al., 1983; Cole et al., 1995), and are proconvulsants or convulsants at higher doses (Lister & Karanian, 1987). Here, the future might be in GABAA receptor subtypeselective antagonism. However, if the a1 subunit-containing receptors need to be inhibited for producing the ‘‘antialcoholic’’ effect (see June et al., 2003; Tauber et al., 2003), then the precautions in human subjects have to be taken seriously. The a1 subunit-containing receptors are involved in proconvulsive and convulsive effects of the inverse agonists (Crestani et al., 2002a). This is even more serious after chronic alcohol administration, since then, at least in rats, the efficacy of inverse agonists is increased (Buck & Harris, 1990). There are likely several different alcoholic subpopulations, for example, certain alcoholic populations

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might have genetic susceptibility associated to GABAA a2 and others to a6 subunit genes (see above). Once the effects of these gene polymorphisms are known at the functional level of the receptors and neuronal networks, we may be able to develop selective ligands to stimulate or inhibit the required receptor subtypes. From the drug development point of view, this is a real possibility, since especially for the a6 subunit-containing receptors, there are several prototypic compounds known that have selective (though not specific) actions on them (e.g., furosemide and niflumic acid; Korpi et al., 1995; Sinkkonen et al., 2003), which could lead the way to more specific compounds. In addition to alcoholism, inverse agonism at the GABAA receptor benzodiazepine site might be beneficial in improving cognition (Duka et al., 1988), such as memory impairment in dementia patients (Maubach, 2003). None of the inverse agonists developed thus far, for example, for alcoholism treatment, have been tried for treating dementia patients. But, for example, in a series of naphthyridine derivatives, 5-(3-methoxyphenyl)-3-(5-methyl-1,2,4-oxadiazol-3-yl)-2-oxo-1,2-dihydro-1,6-naphthyridine (AC-3933) has shown promise in preclinical in vitro and in vivo models for improving memory, and it has entered the phase II proof-of-concept trials in Alzheimer’s disease-related dementia. Similarly, an arylimidazoquinoline 2-(3-isoxazolyl)-3,6,7,9-tetrahydroimidazo[4,5-d]pyrano[4,3-b]pyridine (S-8510) is also a partial inverse agonist, highly efficient in preclinical models in vitro and in vivo (Kawasaki et al., 1996; Abe et al., 1998). It has reached the phase II trials, but no information is available on its efficacy in Alzheimer’s disease. The authors do not know whether any compounds with inverse agonist activity have proven efficient in dementia patients. It remains to be estimated whether general inverse agonism of the GABAA receptors can become a part of the treatment of dementia, especially as many of the patients are agitated and have difficulties in sleeping. Therefore, they often need additional benzodiazepine agonist medication, which, by definition, should counteract any beneficial effects of the inverse agonists (but see Fastbom et al., 1998). Again, subtype-selectivity might turn out to be helpful, for example, compounds like RY-80 and L-655,708 (ethyl (S)-11,12,13,13a-tetrahydro-7-methoxy-9-oxo-9H-imidazo[1,5-a]pyrrolo[2,1-c][1,4]benzodiazepine-1-carboxylate), being partial inverse agonists at a5 subunit-containing receptors, should make a rational approach. The preclinical studies both on a5 subunit knockout and knockin mouse models have shown either enhancement or impairment of certain hippocampal memory-related performances (Collinson et al., 2002; Crestani et al., 2002b). Indeed, the development of several series of novel compounds with various efficacies at receptor subtypes has recently been reported. The most interesting compounds for improving cognition may show relatively selective inverse agonism at a5 subunit-containing receptors, and they include derivatives from the following structural classes: Certain benzo-

23

thiophene (Chambers et al., 2003), triazolophthalazine (Sternfeld et al., 2004; Street et al., 2004), and pyrazolotriazine (Chambers et al., 2004) derivatives show selective inverse agonism at a5 subunit-containing receptors, while having weak agonistic or inverse agonistic effects on a1, a2, and a3 subunit-containing GABAA receptors, and strongly reduced affinity to a4 and a6 receptors. Since there is presently little information on their possible clinical safety, dose ranges or efficacy in treating cognitive decline in dementia patients. In animal models, the selective inverse agonism at a5 subunit-containing receptors by these orally active compounds does not seem to be associated with sedative, motor-impairing, proconvulsive, convulsive or anxiogenic actions (Chambers et al., 2003, 2004; Sternfeld et al., 2004; but see for L-655,708, Navarro et al., 2002). 5.2. c-Aminobutyric acid site compounds Gaboxadol (THIP; 4,5,6,7-tetrahydroisoxazolo[5,4c]pyridin-3ol; Braestrup et al., 1979) is a GABA-site ligand that mainly acts as a partial agonist on GABAA receptors (Krogsgaard-Larsen et al., 2004). It was earlier tested for various indications, such as anxiety, pain, tardive dyskinesia, and epilepsy in clinical studies, but its use was associated with strong side effects, especially sedation, preventing efficient actions on indication symptoms (Korsgaard et al., 1982; Hoehn-Saric, 1983; Kjaer & Nielsen, 1983; Lindeburg et al., 1983; Petersen et al., 1983; Valentin & Bank-Mikkelsen, 1983; Thaker et al., 1987; see also Soares et al., 2004). These studies were stopped, but gaboxadol got a new start, when Lancel and coworkers studied the sedative and sleep-inducing actions of this compound in animal models and patient populations (Lancel & Faulhaber, 1996; Faulhaber et al., 1997; Lancel, 1997; Lancel & Langebartels, 2000; Lancel et al., 2001; Mathias et al., 2001, 2005), finding it efficient in inducing and maintaining sleep, with promotion of slow wave sleep and little action on REM sleep. Preclinical experiments suggest no cross-tolerance to benzodiazepines, at least in actions on motor functions (Voss et al., 2003). The effects on sleep parameters by gaboxadol seem to be also different from those produced by benzodiazepines (Lancel & Faulhaber, 1996). Now, this compound is in a randomized, doubleblind phase III trial in multiple centers, and the results of this study will be essential for further development of gaboxadol as a hypnotic drug. Even if gaboxadol has been long classified as a partial agonist, more recently, gaboxadol has been tested for efficacy on various subunit combinations using recombinant receptors: It acts efficiently on a2, a5, and a6 subunitcontaining receptors, and less efficiently on a1 and a3 receptors (Ebert et al., 1997). But maybe, more importantly, it shows a higher efficacy than GABA at special populations of GABAA receptors (Brown et al., 2002). These receptor populations, containing the a4 subunits with the y subunit instead of g2, are not targeted to synapses but remain

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extrasynaptically. The extrasynaptic y subunit-containing receptors are highly sensitive to GABA, not at all sensitive to benzodiazepines, and desensitize slowly, if at all (Mody, 2001; Liang et al., 2004; Kullmann et al., 2005). Their function is believed to focus on the regulation of resting membrane potential, especially in cerebellar and thalamic neurons (Brickley et al., 1999; Hamann et al., 2002; Nusser & Mody, 2002; Porcello et al., 2003; Chadderton et al., 2004). Gaboxadol has a significantly higher affinity and efficacy on extrasynaptic receptors than on prototypical synaptic a1hg2 receptors (Ebert et al., 1994, 1997; Brown et al., 2002), which might make it relatively selective for increasing tonic inhibition in vivo. At least, its clinically effective concentration (¨ 1 AM; Madsen et al., 1983; Faulhaber et al., 1997) is far closer to the affinity of the extrasynaptic receptors, and 2 AM gaboxadol has been shown to activate extrasynaptic GABAA receptors in CA1 pyramidal neurons of hippocampal slices (Lindquist et al., 2003; Liang et al., 2004). It is thus possible that gaboxadol might become an alternative to some of the indications of benzodiazepine-site ligands, but its efficacy and safety, for example, in human populations with insomnia, should first be demonstrated. Furthermore, gaboxadol will be an interesting tool to understand the role of extrasynaptic GABAA receptors in brain function. Further development of sleeping medications related to the GABAA receptors should also take into account the specific GABAergic hypothalamic nuclei and pathways from ventrolateral preoptic nuclei to other sleep centers in the brain (Mignot et al., 2002). There is still a need for basic neurobiology to establish rational working models and possible new selective target sites. 5.3. Neurosteroid compounds GABAA receptors are known to harbor also binding site(s) for neurosteroids and neuroactive steroids (Lambert et al., 2003; Schumacher et al., 2003) and mediate the fast nongenomic effects of these compounds either by enhancing or inhibiting receptor activity (Majewska, 1992). Some of the compounds, such as progesterone metabolites allopregnanolone (5a-pregnan-3a-ol-20-one) and pregnanolone (5h, 3a) and the corticosterone metabolite 5a,3a-tetrahydrodeoxycorticosterone, may be endogenous ligands in the brain, and changes in their levels may be involved, for example, in the menstruation-related anxiety, painfulness, and epilepsy in females and stress responses and anxiety in general (Backstrom et al., 2003; Schumacher et al., 2003). Steroid metabolism also produces the glucocorticoid dehydroepiandrosterone (DHEA), which is used as a precursor for both androgenic and estrogenic steroids. Its deficiency might be involved in the symptoms of putative andropause. DHEA and its sulphate form (DHEAS) are also neurotrophic/protective in preclinical models (Lapchak & Araujo, 2001). Neurosteroids and synthetic neuroactive steroids have profound effects on brain function at higher concen-

trations, suggesting that they might be useful in producing global effects such as anesthesia or to be useful in conditions in which there might be abnormalities in steroid metabolism and activity. There have been several attempts to develop neurosteroid compounds as anesthetics, since they have been long known by the pioneering studies of Hans Selye to possess powerful, rapidly acting, and quickly residing (quick metabolism) effects. For example, alphaxalone (combined with alphadolone in Althesin) was rather extensively used/studied in the clinics, but finally, its use was met with unacceptable problems especially in repeated usage due to allergic hypersensitivity (Clarke, 1981). This was apparently due to its vehicle Chemophor EL. Later, the endogenous agonist 5h-pregnan-3a-ol-20-one (pregnanolone, eltanolone) was studied with a nonantigenic vehicle Intralipid. However, even eltanolone or the water-soluble steroid minaxolone has not been clinically successful, because they seem to have prolonged recovery from anesthesia as compared with thiopental or propofol (Sear & Prys-Roberts, 1981; Kallela et al., 1994; Eriksson et al., 1995; Tang et al., 1997). A synthetic steroid ganaxolone (3a-hydroxy-3h-methyl5a-pregnan-20-one) is a potent GABAA receptor modulator without efficacy on nuclear hormone receptors (Carter et al., 1997). It has been developed for several years and holds promise in some forms of epilepsies, such as infantile spasms and catamenial epilepsy (Rogawski & Reddy, 2002). Its oral formulations (Monaghan et al., 1997) have been tested in treating infantile spasms in an add-on open label trial of intractable cases (Kerrigan et al., 2000), and it benefited 30 – 60% of the patients with reductions in seizure frequency and caused only mild adverse effects. Ganaxolone has been also tested in a double-blind monotherapy trial in which the patients, with a history of complex partial seizures, poorly responding to antiepileptics, were randomized to ganaxolone or placebo groups during washout of the antiepileptic medication for presurgical assessment (Laxer et al., 2000). Intent-to-treat survival analyses suggested better efficacy of ganaxolone over placebo. Further larger studies are needed to verify the efficacy in epileptic patients. Theoretically, catamenial epilepsy, which is associated with increased seizure frequency just before or during menstruation coinciding with the falling progesterone levels, seems to provide an important target disease for neurosteroid agonists. Ganaxolone showed increased efficacy and potency in an animal model of perimenstrual epilepsy (Reddy & Rogawski, 2000), and clinical testing might be warranted for this indication. Also, other orally active neurostroid agonists have been developed, at least one of them, 3a,21-dihydroxy-3h-trifluoromethyl-19-nor-5h-pregnan-20-one, showing efficacy in preclinical tests for anxiolytic activity (Vanover et al., 2000). DHEA has been recently tried in a small randomized double-blind study on schizophrenic patients and found, especially in females, after 3 weeks of treatment to improve negative symptoms and also to help anxiety and depression

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symptoms (Strous et al., 2003). No effects were observed on positive symptoms. DHEA and DHEAS have both antagonistic action on GABAA receptors (Majewska, 1992), and since there seems to be a structural and functional impairment (e.g., reduced nonpyramidal cell numbers, increased GABA binding sites, and reduced axo-axonic synaptic contacts between interneurons and pyramidal cells) in the cortical GABA system in schizophrenia (Benes & Berretta, 2001), further studies and theories are needed to verify and understand the treatment effects and their actions at receptor and neuronal network levels. DHEA was tried for Alzheimer’s disease in a randomized, double-blind, placebo-controlled study, but it had only a small transient efficacy in the usual outcome measures (Wolkowitz et al., 2003), which have, in other studies, been partially normalized by acetylcholinesterase inhibitors and the NMDA receptor antagonist memantine (Evans et al., 2004). The neurosteroids provide an interesting group for drug development, not least because they might preferentially target the extrasynaptic y subunit-containing receptors (Mihalek et al., 1999; Mody, 2001; Belelli et al., 2002) and/or act via GABAA receptor after their specific modifications by the intracellular enzymes modifying the phosphorylation status of the receptor subunits (Brussaard & Koksma, 2003). It may well turn out to be difficult to successfully develop them for similar indications as, for example, propofol and short-acting barbiturates. However, there may be other indications, such as premenstrual dysphoric disorder and menstruation-cycle related epilepsies, where they might be better suited at lower doses and where there might be an actual shortage of endogenous agonists (Rogawski & Reddy, 2002; Smith, 2002). For these indications, especially the orally active formulations should be studied in larger clinical experiments. It should be also noted that the induction of panic attacks has been shown to cause a quick dramatic decline in blood concentrations of endogenous agonistic neurosteroids in a small group of panic disorder patients (Strohle et al., 2003), but not in normal controls (Zwanzger et al., 2004), which would justify a clinical efficacy study of neurosteroid agonists in the treatment of panic anxiety patients.

6. Future possibilities in the development of drugs acting on ;-aminobutyric acid type A receptors: subtype selectivity, minor subtypes, subcellular targeting Recent neurobiological experiments on the roles of various receptor subtypes in behavior have strongly supported the view that it will be possible to produce significant drug effects by affecting only a minor population of the synaptic or extrasynaptic GABAA receptors in the brain (Mohler et al., 2001; Rudolph et al., 2001; Bateson, 2004; Vicini & Ortinski, 2004; Rowlett et al., 2005). We are on this road now, and receptor

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subtype-selective compounds will hopefully be in clinical tests in a couple of years. The neurobiology has also told us that we might also benefit from using inverse agonisms of the GABAA receptors to treat alcoholism (and eating disorders/obesity?) and cognitive impairment of dementia. In this context, it seems to be clear that the new compounds should have subtype-selectivity rather than being general nonselective inverse agonists. Nonselective benzodiazepine-site inverse agonists are known to be strongly anxiogenic and proconvulsant or convulsant, and in chronic treatment, these actions might get sensitized and abolish any initial treatment effect. Subtype-selective inverse agonists might be safer in this regard, but it remains to be fully established. Furthermore, the modern clinical genetic studies are providing an impetus to assess the roles of the GABAA receptors in psychiatric disorders, and indeed, we need much more neurobiology and neuropathology to understand what kind of treatment via the very complicated GABA system might be beneficial in pharmacotherapy. Previous development of GABAA receptor active drugs has mainly dealt with the main receptor subtypes, especially the a1 subunit-containing receptors, but one should not forget the minor ones, for example, q and u subunits that are highly enriched in certain monoaminergic nuclei (Table 1) and might serve as selective targets for nonbenzodiazepine site compounds to regulate neuronal activity in various disorders. They are most likely assembled with the a3 subunits, but no reports assessing the pharmacology and functional activity of these receptor subtypes have been published. Finally, it should be remembered that the GABAA receptor is a large molecular complex having many different binding sites. Therefore, it will be clear that the future brings us more compounds having novel sites of interaction at the receptor complex or at the receptorassociated proteins, which might then provide different pharmacological profiles and effects in human. Soon, we will be in the position to really use rational drug design to look for novel target domains, as the structure of the GABAA receptor is being modelled at higher precision and validity (Ernst et al., 2003). In any case, all new compounds must provide a benefit over the existing drugs, such as benzodiazepine-site ligands, volatile and intravenous anesthetics, in the indications for which they are being developed.

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