Translocator Protein (18 kDa) as a Target for Novel Anxiolytics with a Favourable Side-Effect Profile

Journal of Neuroendocrinology From Molecular to Translational Neurobiology Journal of Neuroendocrinology 24, 82–92 ª 2011 The Authors. Journal of Neuroendocrinology ª 2011 Blackwell Publishing Ltd

REVIEW ARTICLE

Translocator Protein (18 kDa) as a Target for Novel Anxiolytics with a Favourable Side-Effect Profile C. Nothdurfter* , G. Rammes à, T. C. Baghai*, C. Schu¨le*, M. Schumacher§, V. Papadopoulos– and R. Rupprecht* ** *Department of Psychiatry and Psychotherapy, Ludwig-Maximilians-University Munich, Munich, Germany.  Max-Planck-Institute of Psychiatry, Munich, Germany. àDepartment of Anesthesiology, Technische Universita¨t Mu¨nchen, Munich, Germany. §UMR 788 INSERM and University Paris-Sud 11, Kremlin-Biceˆtre, France. –Departments of Medicine, Pharmacology and Therapeutics and Biochemistry, The Research Institute of the McGill University Health Centre, McGill University, Montreal, QC, Canada. **Department of Psychiatry and Psychotherapy, University of Regensburg, Regensburg, Germany.

Journal of Neuroendocrinology

Correspondence to: Caroline Nothdurfter, Department of Psychiatry and Psychotherapy, Ludwig-Maximilians-University Munich, Nußbaumstrasse 7, Munich 80336, Germany (e-mail: Caroline. [email protected]).

Anxiety disorders are frequent and highly disabling diseases with considerable socio-economic impact. In the treatment of anxiety disorders, benzodiazepines (BZDs) as direct modulators of the GABAA receptor are used as emergency medication because of their rapid onset of action. However, BZDs act also as sedatives and rather quickly induce tolerance and abuse liability associated with withdrawal symptoms. Antidepressants with anxiolytic properties are also applied as first line long-term treatment of anxiety disorders. However, the onset of action of antidepressants takes several weeks. Obviously, novel pharmacological approaches are needed that combine a rapid anxiolytic efficacy with the lack of tolerance induction, abuse liability and withdrawal symptoms. Neurosteroids are potent allosteric modulators of GABAA receptor function. The translocator protein (18 kDa) (TSPO) plays an important role for the synthesis of neurosteroids by promoting the transport of cholesterol from the outer to the inner mitochondrial membrane, which is the rate-limiting step in neurosteroidogenesis. Etifoxine not only exerts anxiolytic effects as a TSPO ligand by enhancing neurosteroidogenesis, but also acts as a weak direct GABAA receptor enhancer. The TSPO ligand XBD173 enhances GABAergic neurotransmission via the promotion of neurosteroidogenesis without direct effects at the GABAA receptor. XBD173 counteracts pharmacologically-induced panic in rodents in the absence of sedation and tolerance development. Also in humans, XBD173 displays antipanic activity and does not cause sedation and withdrawal symptoms after 7 days of treatment. XBD173 therefore appears to be a promising candidate for fast-acting anxiolytic drugs with less severe side-effects than BZDs. In this review, we focus on the pathophysiology of anxiety disorders and TSPO ligands as a novel pharmacological approach in the treatment of these disorders. Key words: TSPO, neurosteroid, anxiety disorder, GABAA receptor, benzodiazepine.

Pharmacology of anxiety disorders Anxiety disorders (according to the Diagnostic and Statistical Manual of Mental Disorders, 4th edition: generalised anxiety disorder, panic disorder with and without agoraphobia, specific phobias, social phobia, obsessive–compulsive disorder and post-traumatic stress disorder) belong to the most frequent mental disorders. Their estimated life-time prevalence reaches almost 30% (1). Therapeutic

doi: 10.1111/j.1365-2826.2011.02166.x

strategies in the treatment of anxiety disorders are psychopharmacological compounds or cognitive–behavioral psychotherapy, or even a combination of both (2). Especially psychopharmacological treatment remains a challenge because ‘the optimum anxiolytic compound’ has not yet been developed. Emergency ⁄ short-term medication versus long-term medication can be differentiated, with both exhibiting specific disadvantages. Benzodiazepines (BZDs) (e.g. diazepam) quickly exert anxiolytic effects by enhancing GABAergic

TSPO as a target for anxiolytics

neurotransmission (3,4). Although BZDs are very potent and fastacting anxiolytics, they are sedative drugs causing motor coordination deficits and memory impairment, and their continuous use rather quickly induces tolerance effects and abuse liability (4). By contrast, antidepressants lack tolerance development and abuse liability, which makes them more suitable for the long-term treatment of anxiety disorders. Selective serotonin reuptake inhibitors (SSRI) and serotonin-norepinephrine reuptake inhibitors (SNRI) are generally considered as the first-line treatment option as a result of their broad anxiolytic efficacy and rather good tolerability (5). However, agitation and insomnia as initial adverse effects may occur under SSRI ⁄ SNRI treatment, which may have a negative impact on patients’ compliance. Moreover, the onset of anxiolytic efficacy of antidepressants usually takes several weeks (6). So far, one of the most important pharmacological targets for the development of anxiolytic compounds is the GABAergic system. GABA is the major inhibitory neurotransmitter of the central nervous system (CNS) (7) and plays an outstanding role in the pathogenesis of anxiety disorders (8). GABAA receptors are heteropentameric ligand-gated ion channels that represent anion selective chloride channels, which open upon GABA binding (8–10). The receptor subunit composition 2*a1 ⁄ 2*b2 ⁄ 1*c2 is most abundant (7,8). The a2 subunit and, presumably to a lesser extent, the a3 subunit are assumed to play a key role in anxiety ⁄ anxiolysis (3,11). GABAA receptors contain multiple binding sites for different modulators. The BZD binding site is located at the interface of an a and the c2 subunit (3) (Fig. 1).

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Cl– Fig. 1. GABAA receptor binding sites. GABAA receptors are heteropentameric ligand-gated chloride channels (8–10). Most abundantly, the receptor is composed of the subunits 2*a1 ⁄ 2*b2 ⁄ 1*c2 (7,8). GABAA receptors contain multiple binding sites for different modulators. The benzodiazepine (BZD) binding site is located at the interface of an a and the c2 subunit (3). Also neurosteroids are potent positive allosteric modulators of the GABAA receptor, although they occupy a different binding site than BZDs (13,14,104).

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Fig. 2. Neurosteroid synthesis. The cholesterol side-chain-cleaving cytochrome-P450 enzyme (P450scc, CYP11A1) at the inner mitochondrial membrane converts cholesterol to pregnenolone (13,14,16,38). In the cytoplasm (diffusion marked by dark grey arrow), progesterone is formed from pregnenolone by the microsomal 3b-dehydrogenase ⁄ D5–D4 isomerase (13,14). Progesterone is then metabolised to deoxycorticosterone by the 21-hydroxylase (CYP21B). Progesterone and deoxycorticosterone are reduced by the 5a-reductase to 5a-dihydroprogesterone and 5a-dihydrocorticosterone (5a-DHDOC). By further reduction through 3a-hydroxysteroid dehydrogenase (3a-HSD), the neurosteroids allopregnanolone and allotetrahydrodeoxycorticosterone (3a, 5a-tetrahydrodeoxycorticosterone, 3a, 5a-THDOC) are formed (13,14,105). ª 2011 The Authors. Journal of Neuroendocrinology ª 2011 Blackwell Publishing Ltd, Journal of Neuroendocrinology, 24, 82–92

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Because of the manifold side-effects of BZDs, many efforts have been made to develop subtype-selective GABAA receptor modulators that specifically target the subunits a2 and a3, which are most relevant for anxiolysis. Furthermore, some drugs are available that potentiate GABAergic neurotransmission by different mechanisms than BZDs. Although most of these compounds are used in the treatment of convulsive disorders, the anxiolytic potential of some of these drugs could be demonstrated more or less successfully. Obviously, there is a need for the development of novel pharmacological approaches in the treatment of anxiety disorders that combine broad and high anxiolytic potency without BZD-like sideeffects, such as sedation, tolerance induction and abuse liability, which are associated with withdrawal symptoms. Neurosteroids are endogenous modulators of the GABAA receptor (12–14). They are derived from cholesterol and synthesised in the brain (13). Especially 3a-reduced metabolites of the steroids progesterone and deoxycorticosterone are potent positive allosteric modulators of GABAA receptor function, although they occupy a binding Glutamatergic principal neurone expressing 5a-reductase and 3a-HSD

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TSPO is an ubiquitous protein, which is primarily localised in the outer mitochondrial membrane (16,17). Because the BZD diazepam was shown to bind to TSPO, the protein was formerly called the ‘peripheral-type benzodiazepine receptor’ or ‘mitochondrial benzodiazepine receptor’. TSPO consists of a 169-amino acid sequence that is arranged as a five transmembrane helix structure (18). Moreover, specific mitochondrial proteins exist, namely the voltagedependent anion channel and the adenine nucleotide transporter, which are associated with TSPO (19–21). Furthermore, interactions

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site different from that of BZDs (13,14). During panic attacks in patients suffering from panic disorder, 3a-reduced neurosteroid levels are reduced (15). The translocator protein (18 kDa) (TSPO) plays an important role for the synthesis of neurosteroids, thereby representing a putative novel pharmacological target for the development of anxiolytic compounds.

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Fig. 3. Neuronal networks targeted by translocator protein (18 kDa) (TSPO) ligand-induced neurosteroid signalling; modified according to Rupprecht et al. (98). 3a-pregnane-reduced neurosteroids in general are very efficient positive allosteric modulators of GABAA receptor function, whereas other neurosteroids (i.e. pregnenolone sulphate and dehydroepiandrosterone sulphate) are negative modulators (106). However, the modulation of GABAA receptors by neurosteroids is complex and may differ between neurones and between distinct GABAA receptors (e.g. synaptic and extrasynaptic receptors) on the same neurone. An example of the formation of neurosteroids via TSPO in glial cells and glutamatergic principal output neurones and the positive allosteric modulation of different types of GABAA receptors is depicted. Neuronal GABAA receptors are predominantly modulated by neurosteroids derived from glial and microglial cells, representing a paracrine mechanism. As an autocrine mechanism, neurosteroids from principal neurones may modulate the different subtypes of GABAA receptors located at the same synapse. Neurosteroids may also modulate GABAA receptors located at distal neurones in a paracrine fashion. The known subunit configuration of different GABAA receptor subtypes is indicated in the figure. ‘X’ represents unknown subunits. Extrasynaptic GABAA receptors contain subunits a1,4,5b2,3,d in the dentate gyrus granular cells of the hippocampus, a4,5b2d in the ventrobasal nucleus of the thalamus, a6bc2 ⁄ d in cerebellar granule cells (57–66), and a5bc2 ⁄ d in the CA1 region of the hippocampus (60). GABA is released from GABAergic interneurones and targets presynaptic (a2-containing) (60,67), postsynaptic (a1,2,3,6b2,3c2-containing) (60, 68) and extrasynaptic receptors at glutamatergic principal output neurones. Depending on the receptor subunit composition, allopregnanolone and 3a, 5a-THDOC differentially modulate the overall charge transfer of chloride through GABAA receptors (107–109). GABA-evoked currents mediated by a1b1c2 or a3b1c2 receptors, for example, are enhanced by rather low concentrations of allopregnanolone, whereas a2, a4-, a5- or a6-subunit-containing receptors require three- to ten-fold higher concentrations for equal potentiation effects (110). Receptors containing the c1-subunit are less sensitive to allopregnanolone than receptors expressing c2- or c3-subunits (109,110). Moreover, 3a, 5a-THDOC efficiently enhances GABA-evoked currents through a1b3d-containing receptors in contrast to a1b3c2L-containing receptors (108). 3a-HSD, 3a-hydroxysteroid dehydrogenase. ª 2011 The Authors. Journal of Neuroendocrinology ª 2011 Blackwell Publishing Ltd, Journal of Neuroendocrinology, 24, 82–92

TSPO as a target for anxiolytics

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Moreover, certain antidepressants can enhance the synthesis of neurosteroids (especially the 3a-reduced steroids), which may play a role for their antidepressant and anxiolytic properties (44–46). SSRIs such as fluoxetine have therefore been referred to as ‘selective brain steroidogenic stimulants’ (44). They have been suggested to interfere with neurosteroidogenic enzymes (e.g. the 3a-hydroxysteroid-dehydrogenase) (47). Neurosteroids can alter the release of neurotransmitters or the activity of neurotransmitter receptors (48), thereby acting as inhibitors or enhancers of neuronal excitability (49). In view of their modulatory potency at the GABAA receptor (Fig. 1), neurosteroids play an important role in the pathophysiology of anxiety disorders.

Mitochondrion Fig. 4. Translocator protein (18 kDa) (TSPO) associated proteins and ligand binding; modified according to Rupprecht et al. (98). TSPO is primarily localised in the outer mitochondrial membrane (16,17) and consists of a 169amino acid sequence arranged as a five transmembrane helix structure (18). Specific mitochondrial proteins are associated with TSPO, namely the voltage-dependent anion channel (VDAC) and the adenine nucleotide transporter (ANT) (19–21). With regard to the binding sites of TSPO ligands, cholesterol binds to the cytosolic carboxy-terminus containing a conserved CRAC (cholesterol recognition amino acid consensus) domain (82,83); all other drug ligands bind to a region within the amino-terminus (82,84,85).

of cytosolic proteins with TSPO (22) have been reported, suggesting that TSPO serves as a mitochondrial anchor transducing intracellular signals to mitochondria (23). TSPO is expressed in many organs, although the highest expression levels are found in tissues that contain steroid-synthesising cells (e.g. adrenal, gonad and brain cells) (16,24). Within the CNS, TSPO is expressed in glia and microglia (25,26) and in reactive astrocytes (27, 28). Nonetheless, TSPO has also been detected in some neuronal cell types (e.g. neurones of the mammalian olfactory bulb) (29,30). TSPO is assumed to mediate various mitochondrial functions, including cholesterol transport and steroid hormone synthesis, mitochondrial respiration, mitochondrial permeability transition pore opening, apoptosis and cell proliferation (24–26,31–33).

Cell-specific neurosteroid signalling The synthesis of neurosteroids is brain region and neurone-specific and depends on the relative amount of TSPO, as well as on the expression of the neurosteroidogenic enzymes mediating their formation. 5a-reductase and 3a-hydroxysteroid dehydrogenase, for example, which catalyse the synthesis of allopregnanolone and 3a, 5a-THDOC (both positive allosteric modulators of GABAA receptor function), can be detected in type 1 and type 2 astrocytes and oligodendrocytes (50–52) and principal output neurones (glutamatergic pyramidal, GABAergic reticulothalamic, striatal and Purkinje neurones), whereas these enzymes are almost absent in telencephalic or hippocampal GABAergic interneurones (53). Neurosteroids synthesised in cortical glutamatergic principal neurones may act at GABAA receptors in an autocrine (i.e. at postsynaptic receptors of the same neurone) and ⁄ or in a paracrine (i.e. at receptors located at distal neurones) fashion (Fig. 3). Regarding the neuronal networks involved in GABAA receptor modulation by neurosteroids, the most likely mechanism appears to be the paracrine

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Role of TSPO (18 kDa) in neurosteroidogenesis The steroid biosynthetic pathway results in the formation of an array of steroid hormones and neurosteroids (13,34,35), including oestradiol, testosterone, pregnenolone, pregnenolone sulphate, progesterone, allopregnanolone, allotetrahydrodeoxycorticosterone (3a, 5a-THDOC), dehydroepiandrosterone and dehydroepiandrosterone sulphate (Fig. 2). In this context, TSPO ligands were initially shown to stimulate the synthesis of pregnenolone from endogenous cholesterol in glioma cells (36,37). TSPO-mediated translocation of cholesterol from the outer to the inner mitochondrial membrane is the rate-limiting step in the synthesis of pregnenolone, which is the precursor of all other neurosteroids (16,24,38). In vivo studies subsequently revealed that TSPO ligands efficiently increase neurosteroidogenesis in rat brain (39–43).

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N O Fig. 5. Chemical structures of translocator protein (18 kDa) ligands for the treatment of anxiety disorders. (A) Etifoxine: benzoxazine, 4-(3-chlorophenyl)N-ethyl-4,6-dimethyl-3,1-benzoxazin-2-amine (98). (B) XBD173 ( ⁄ AC-5216 ⁄ Emapunil): phenylpurine acetamide, N-benzyl-N-ethyl-2-(7-methyl-8-oxo-2phenyl-purin-9-yl)acetamide (98).

ª 2011 The Authors. Journal of Neuroendocrinology ª 2011 Blackwell Publishing Ltd, Journal of Neuroendocrinology, 24, 82–92

C. Nothdurfter et al.

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Fig. 6. Effect of XBD173 on GABAergic neurotransmission; according to Rupprecht et al. (103). The effect of XBD173 was monitored with whole-cell recordings and minimal stimulation in slices of mouse medial prefrontal cortex. The mean amplitude of all inhibitory postsynaptic currents (IPSCs) in the absence of compounds was 26.0  2.7 pA (decay time constant s: 27.8  2.8 ms), the mean charge was 1.5  0.7 pC (mean  SEM of n = 54). Electrophysiological data were analysed by the t-test for paired samples. *P < 0.05 compared to control experiments. The diagrams on the left show the individual response amplitudes during the course of one representative experiment. The diagrams in the middle show the average traces from all consecutive IPSCs for the control experiments and in the presence of 5 lM XBD173 or 10 lM finasteride ⁄ 5 lM XBD173. The diagrams on the right show the average data of all experiments (mean  SEM of n = 6–8). (A) XBD173 increases the amplitude and charge of IPSCs. (B) Antagonism of the effects of XBD173 by finasteride.

release of neurosteroids from glial and microglial cells (54). Nevertheless, there are in vitro studies suggesting that neurones can also express TSPO (27,28,55,56), which could not yet be confirmed in vivo. The question of whether GABAergic interneurones express TSPO remains to be clarified. The respective subunit composition of synaptic and extrasynaptic receptors plays an important role for the sensitivity of GABAA receptors to the modulation by neurosteroids (49). TSPO drug ligand-induced formation of neurosteroids may therefore result in a brain region-specific enhancement of GABAergic neuronal inhibition. Extrasynaptic GABAA receptors contain subunits a1,4,5b2,3,d in the dentate gyrus granular cells of the hippocampus, a4,5b2d in the ventrobasal nucleus of the thalamus, and a6bc2 ⁄ d in cerebellar granule cells (57–66). Subunits a5bc2 ⁄ d are found in the CA1 region of the hippocampus (60). GABA released by GABAergic interneurones targets presynaptic (a2-containing) (60,67), postsynaptic (a1,2,3,6b2,3c2-containing) (60,68) and extrasynaptic receptors at glutamatergic principal output neurones.

Role of TSPO (18 kDa) in psychiatric disorders So far, relatively few studies have investigated TSPO expression with regard to psychiatric disorders. These studies examined either TSPO

mRNA expression (69,70) in peripheral mononuclear cells or the binding characteristics of the high-affinity TSPO ligand isoquinoline carboxamide 1-(2-chlorophenyl)-N-methyl-N-(1-methyl-propyl)-3isoquinolinecarboxamide (PK 11195) on platelet membranes (71– 79). Regarding depression and anxiety, neurosteroids have been shown to act as modulators of these disorders (14,15,45). The expression of TSPO on peripheral blood cells and platelets was reduced in anxious subjects (69,74), which remains to be determined for distinct brain areas. Reduced TSPO expression has also been found in lymphocytes and platelets of patients suffering from generalised anxiety disorder (70), social anxiety disorder (73), post-traumatic stress disorder (72) and panic disorder in the presence of adult separation anxiety disorder (75). Depression has not been associated with reduced TSPO expression levels (78). However, in patients suffering from depression or bipolar disorder with comorbid adult separation anxiety (71,79) or suicidality (77), a reduction of TSPO expression could be demonstrated. In schizophrenia, an association of reduced TSPO expression with anxiety, distress and aggression has been reported (76). Moreover, a genetic polymorphism in exon 4 of the TSPO gene appears to increase the susceptibility to panic disorder (80). Furthermore, a

ª 2011 The Authors. Journal of Neuroendocrinology ª 2011 Blackwell Publishing Ltd, Journal of Neuroendocrinology, 24, 82–92

TSPO as a target for anxiolytics

peptide, which is assumed to be involved in the pathophysiology of Alzheimer’s disease, has been shown to stimulate the synthesis of endozepines in astrocytes (91). In this context, elevated levels of endozepines have been detected in the cerebrospinal fluid of patients suffering from Alzheimer’s disease (92). Classical synthetic ligands of TSPO are the isoquinoline carboxamide PK 11195 and the BZD 7-chloro-5-(4-chlorophenyl)-1,3-dihydro-1-methyl-2H-1,4-benzodiazepin-2-one (Ro5-4864). PK 11195 binds exclusively to TSPO, whereas the Ro5-4864 also requires other mitochondrial protein components to display full binding capacity. Isoquinolines have become important diagnostic tools for the characterisation, expression and function of TSPO. Over the past two decades, various other TSPO ligands have been developed. The imidazopyridine alpidem (93), for example, was approved for the treatment of anxiety in France in 1991. However, alpidem was withdrawn in 1994 as a result of the occurrence of severe liver dysfunction (94–96). Further synthetic TSPO ligands were developed primarily as neuroimaging agents and diagnostic tools for brain inflammation (97). However, some TSPO ligands might also have therapeutic potential. Interestingly, some classical clinically relevant benzodiazepines, such as clonazepam and diazepam, which primarily act as allosteric modulators of the GABAA receptor, are TSPO ligands and thus may promote the synthesis of neurosteroids (26,98). Recently, a type of dual mechanism of action has been postulated for midazolam in that this compound inhibits long-term potentiation and learning

recent PET study showed a positive correlation between the binding of the TSPO ligand [11C]DAA1106, positive symptoms and duration of illness in patients with schizophrenia, which suggests the involvement of a glial reaction in the pathophysiology of positive symptoms (81).

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With regard to the binding sites of TSPO ligands, cholesterol binds to the cytosolic carboxy-terminus containing a conserved CRAC (cholesterol recognition amino acid consensus) domain (82,83). All other drug ligands bind to a region within the amino-terminus (82,84,85) (Fig. 4). Nonetheless, other overlapping binding sites for BZDs have also been reported. Cholesterol and porphyrins are important endogenous high-affinity ligands of TSPO (82,86). Further endogenous TSPO ligands are endozepines, a family of neuropeptides that can displace BZDs from their binding site at the GABAA receptor (87). Endozepines are derived from proteolytic processing of a common polypeptide precursor, the diazepam-binding inhibitor (DBI). DBI is encoded by a single gene widely expressed in the nervous system (88) and has been shown to bind long-chain (C12–C22) acyl-CoA esters; therefore, it is also known as acyl-CoA-binding protein (89). Recently, DBI was classified as a member of the acyl-CoA-binding domain-containing proteins (ACBD) and renamed ACBD1 (90). Within the CNS, DBI is primarily expressed in glial cells. Interestingly, b-amyloid

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Fig. 7. Effects of XBD173 or alprazolam on cholecystokinin tetrapeptide (CCK-4) induced panic; according to Rupprecht et al. (103)]. (A) Area under the time curve (AUC) of the Acute Panic Inventory (API) score of healthy male volunteers during a first and a second CCK-4 challenge (7 days after the first challenge). Box plots represent the median equivalent to the 50% percentile (line within the boxes), the range containing all individual values above the 25% and below the 75% percentile (boxes) and the range of individual values within 150% above or below the difference between the 75% and the 25% percentile (error bars). Open circles depict outliers located more than 150% and asterisks depict extreme values located more than 300% of the box height above the 75% percentile. (B) Decrease in CCK-4 induced panic (delta API-AUC) after treatment with different dosages of XBD173, alprazolam or placebo in relation to baseline AUC (mean  SEM). Asterisks indicate a significant difference against placebo (ANCOVA: 90 mg XBD173, P < 0.036; alprazolam, P < 0.019). ª 2011 The Authors. Journal of Neuroendocrinology ª 2011 Blackwell Publishing Ltd, Journal of Neuroendocrinology, 24, 82–92

C. Nothdurfter et al.

through TSPO-mediated neurosteroidogenesis (99). In view of these data, the classification of certain benzodiazepines as ‘pure’ GABAA receptor modulators has to be questioned.

Etifoxine The benzoxazine etifoxine was the first TSPO ligand that revealed anxiolytic effects in a clinical trial (100) (Fig. 5A). Besides fewer side-effects, the anxiolytic efficacy of etifoxine was found to be comparable with lorazepam in patients suffering from adjustment disorders with anxiety (100). Etifoxine enhanced tonic inhibition in hypothalamic neurones mediated by extrasynaptic GABAA receptors (101). This effect could partially be inhibited by the 5a-reductase inhibitor finasteride (101). This observation was in line with an elevation of plasma and brain levels of pregnenolone, progesterone, 5a-dihydroprogesterone and allopregnanolone after the administration of etifoxine independently from the adrenal glands. These data suggest that an enhancement of neurosteroidogenesis contributes to the anxiolytic effects of etifoxine (43). However, etifoxine is not only a TSPO ligand, but also a weak direct GABAA receptor enhancer (101).

XBD173 XBD173 (AC-5216, Emapunil) is a novel selective phenylpurine high-affinity TSPO ligand that has very recently been investigated for the treatment of anxiety (102,103) (Fig. 5B). XBD173 exerts anxiolytic properties in animal models and in humans by enhancing neurosteroidogenesis in brain slices, thereby potentiating the amplitude and duration of GABA-mediated inhibitory postsynaptic currents, as shown in mouse prefrontal cortical neurones (Fig. 6A) (103). This potentiating effect on GABAergic neurotransmission could be prevented by the 5a-reductase inhibitor finasteride (Fig. 6B) (103). Furthermore, in contrast to BZDs, XBD173 did not enhance GABAA receptor-mediated chloride currents of WSS-1 cells (expressing rat a1c2 and human b3 GABAA receptor subunits), thereby demonstrating that XBD173 does not reveal direct modulatory effects at the GABAA receptor (103). Thus, the enhancement of GABAergic neurotransmission by XBD173 appears to be mediated indirectly through generation of neurosteroids. In vivo, XBD173 counteracted pharmacologically-induced panic attacks in rodents without exerting sedative effects (103). Also in humans, XBD173 revealed rapid-onset antipanic effects. In healthy male volunteers, the antipanic effectiveness of XBD173 was comparable to the BZD alprazolam during pharmacologically-induced panic by cholecystokinin tetrapeptide (CCK-4) (Fig. 7) (103). In this placebo-controlled parallel group proof of concept study on the efficacy and tolerability of XBD173, subjects with a sufficient panic response after CCK-4 application entered one of five treatment arms. Seventy-one healthy volunteers were randomised to treatment for 7 days with placebo, 10, 30 or 90 mg ⁄ day XBD173, or 2 mg ⁄ day alprazolam before undergoing a second CCK-4 challenge. The difference in the Acute Panic Inventory (area under the time curve) between the first and the second CCK-4 challenge relative to the effects of placebo was defined as an efficacy parameter for the

anxiolytic potential of the respective compound. A significant difference from placebo was demonstrated for both alprazolam and the highest dose of XBD173. By contrast to the alprazolam group, the XBD173 groups did not suffer from sedation and withdrawal symptoms after 7 days of treatment, thereby indicating a better side-effect profile. These results suggest both rapid and potent anxiolytic properties and fewer side-effects for XBD173 compared to BZDs in humans, which makes this compound a promising candidate for a novel class of anxiolytics.

Conclusions and prospects The concept of direct modulators of the GABAergic system as anxiolytic compounds derives from clinical experiences with BZDs, which, however, have an unfavourable side-effect profile as a result of tolerance development and abuse liability. Therefore, the need for alternative pharmacological approaches thereby becomes obvious. TSPO mediates a broad spectrum of biological functions in the CNS, making TSPO ligands useful as diagnostic tools for monitoring physiological and pathophysiological processes (Fig. 8). Furthermore, TSPO ligands are under development for the treatment of psychiatric disorders, such as anxiety disorders, which may constitute a novel class of compounds related to the pathophysiology of these

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peripheral nerve lesions Fig. 8. Translocator protein (18 kDa) (TSPO) expression in the context of neuropsychiatric disorders. The expression of TSPO may be altered in response to different neuropsychiatric pathological processes (98). In response to injury, TSPO expression is up-regulated in the peripheral nervous system (111–113) and returns to baseline levels upon nerve regeneration, which suggests a key role of TSPO in repair processes (113). TSPO expression in the brain was originally considered to be specific for activated microglial cells and infiltrating macrophages (114). However, it is now suggested that reactive astrocytes (27,28) and certain central nervous system neurones (55,115) also express TSPO. TSPO up-regulation in microglia and astrocytes in response to lesions is directly associated with the degree of damage (116,117). The timing of TSPO expression reflects not only glial cell activation upon injury, but also during regeneration (118). TSPO was found to be strongly up-regulated at the sites of degenerative changes. A recent study associated dominant microglial TSPO expression with substantial neuronal loss, whereas less neuronal insult was associated with TSPO expression mainly in astrocytes (119). In animal models, TSPO levels remained elevated during recovery from disease and myelin repair, suggesting a possible role for TSPO in regenerative processes (116,120).

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disorders. Although initial clinical trials are promising, several issues remain to be addressed in this context: the medium- and long-term efficacy of TSPO ligands has to be determined. The side-effect profile of TSPO ligands under prolonged application has to be investigated in view of the high expression of TSPO in peripheral tissues, which might also reduce drug specificity. In addition, drug specificity is further challenged by the fact that TSPO ligands do not selectively enhance neurosteroids relevant for anxiety disorders. Therefore, TSPO ligand-associated side-effects as a result of the overall enhancement of neurosteroid synthesis still need to be determined. Initial experiences with etifoxine, which has been approved in France for the treatment of anxiety disorders since 1982, and the more recently developed compound XBD173, are promising (100,103). However, whether there is really an increased benefit along with an improved side-effect profile relative to existing treatment options remains to be clarified. These questions can only be answered by systematic, clinical studies involving prolonged administration and safety monitoring. Moreover, further investigations on the underlying molecular mechanisms of these compounds that may contribute toward explaining the putative favourable sideeffect profile are needed.

Conflict of interest R. R. has been on Novartis advisory boards. The clinical study on XBD173 (104) was sponsored by Novartis, Switzerland.

Received 2 March 2011, revised 29 April 2011, accepted 22 May 2011

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