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

REVIEW ARTICLE

Translocator Protein (18 kDa) as a Target for Novel Anxiolytics with aFavourable Side-Effect ProfileC. Nothdurfter*�, G. Rammes��, T. C. Baghai*, C. Schule*, 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.

�Department of Anesthesiology, Technische Universitat Munchen, Munich, Germany.

§UMR 788 INSERM and University Paris-Sud 11, Kremlin-Bicetre, 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.

Pharmacology of anxiety disorders

Anxiety disorders (according to the Diagnostic and Statistical Man-

ual 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

strategies in the treatment of anxiety disorders are psychopharma-

cological 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

Journal ofNeuroendocrinology

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 asso-

ciated 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 antidepres-

sants takes several weeks. Obviously, novel pharmacological approaches are needed that com-

bine 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 mitochon-

drial 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 neurotransmis-

sion 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.

doi: 10.1111/j.1365-2826.2011.02166.x

Journal of Neuroendocrinology 24, 82–92

ª 2011 The Authors. Journal of Neuroendocrinology ª 2011 Blackwell Publishing Ltd

Journal of NeuroendocrinologyFrom Molecular to Translational Neurobiology

neurotransmission (3,4). Although BZDs are very potent and fast-

acting anxiolytics, they are sedative drugs causing motor coordina-

tion 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 lia-

bility, 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 gener-

ally 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 sys-

tem. 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 hetero-

pentameric 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 modu-

lators. The BZD binding site is located at the interface of an a and

the c2 subunit (3) (Fig. 1).

Cl–

Cl–

Membranea

bab

g

Cytosol

BZD

GABAGABA

Neurosteroid

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 recep-

tor, although they occupy a different binding site than BZDs (13,14,104).

Cholesterol

P450scc

Pregnenolone

Deoxy-corticosterone

PregnenoloneProgesterone

3a-H

SD

3a-H

SD

5a-reductase

5a-reductase

3b-HSD Δ5-Δ4-isomerase

5a-DHDOC

Allopregnanolone3a,5a-THDOC

5a-dihydroprogesterone

HO

HO

HO

HO

HO

HO HO

O

HO

O

O O

O

H3C

H3C

H3CH3C

H3C

H3CH3C

H3C

H3CH3C

H3C

H3CH3C

H3C

H3CH3C

H3C

H3C

H3C

H3C

CH3

H3C

H3CH3C

H3C

H3CO

O

OO

O O

O

21b-hydroxlase

Fig. 2. Neurosteroid synthesis. The cholesterol side-chain-cleaving cytochrome-P450 enzyme (P450scc, CYP11A1) at the inner mitochondrial membrane con-

verts 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). Progester-

one 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-tetrahydrodeoxycorti-

costerone, 3a, 5a-THDOC) are formed (13,14,105).

TSPO as a target for anxiolytics 83

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

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 rel-

evant 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 pharmaco-

logical approaches in the treatment of anxiety disorders that

combine broad and high anxiolytic potency without BZD-like side-

effects, 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 proges-

terone and deoxycorticosterone are potent positive allosteric modu-

lators of GABAA receptor function, although they occupy a binding

site different from that of BZDs (13,14). During panic attacks in

patients suffering from panic disorder, 3a-reduced neurosteroid lev-

els are reduced (15). The translocator protein (18 kDa) (TSPO) plays

an important role for the synthesis of neurosteroids, thereby repre-

senting a putative novel pharmacological target for the develop-

ment of anxiolytic compounds.

Structure, expression and function of TSPO (18 kDa)

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 benzo-

diazepine 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 voltage-

dependent anion channel and the adenine nucleotide transporter,

which are associated with TSPO (19–21). Furthermore, interactions

Paracrine Paracrine

Paracrine

TSPO

TSPO

TSPO

Presynaptica2xx

a1,2,3,6b1,2,3gx

a1,4,5,6b2/3d/e

a5bg2/dab

Postsynaptic

Extrasynaptic

Auto

crin

e

Autocrine

without enzymesfor neurosteroid

synthesis

expressing5a-reductase

and3a-HSD

expressing5a-reductase and

3a-HSD

GlutamatergicGlutamatergicprincipal neuroneprincipal neurone

GlutamatergicGlutamatergicprincipal neuroneprincipal neurone

Glial cellGlial cell

GABAergicinterneuroneGABAergicinterneurone

GABAergicinterneuroneGABAergicinterneurone

GABAAreceptorGABAAreceptor

NeurosteroidsNeurosteroids

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 neuroster-

oids 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 differ-

ent types of GABAA receptors is depicted. Neuronal GABAA receptors are predominantly modulated by neurosteroids derived from glial and microglial cells, rep-

resenting 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 configu-

ration 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 sub-

unit 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-sub-

unit 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.

84 C. Nothdurfter et al.


of cytosolic proteins with TSPO (22) have been reported, suggesting

that TSPO serves as a mitochondrial anchor transducing intracellu-

lar signals to mitochondria (23).

TSPO is expressed in many organs, although the highest expres-

sion 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).

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, pro-

gesterone, 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 cho-

lesterol in glioma cells (36,37). TSPO-mediated translocation of cho-

lesterol 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 sub-

sequently revealed that TSPO ligands efficiently increase neuro-

steroidogenesis in rat brain (39–43).

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 ‘selec-

tive brain steroidogenic stimulants’ (44). They have been suggested

to interfere with neurosteroidogenic enzymes (e.g. the 3a-hydroxys-

teroid-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.

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 for-

mation. 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 oli-

godendrocytes (50–52) and principal output neurones (glutamater-

gic pyramidal, GABAergic reticulothalamic, striatal and Purkinje

neurones), whereas these enzymes are almost absent in telence-

phalic or hippocampal GABAergic interneurones (53).

Neurosteroids synthesised in cortical glutamatergic principal neu-

rones may act at GABAA receptors in an autocrine (i.e. at postsyn-

aptic 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 neu-

rosteroids, the most likely mechanism appears to be the paracrine

Mitochondrion

TSPO ligand

ANT

VDAC

COOH

TSPOTranslocator

protein (18 kDa)

Fig. 4. Translocator protein (18 kDa) (TSPO) associated proteins and ligand

binding; modified according to Rupprecht et al. (98). TSPO is primarily local-

ised in the outer mitochondrial membrane (16,17) and consists of a 169-

amino acid sequence arranged as a five transmembrane helix structure (18).

Specific mitochondrial proteins are associated with TSPO, namely the volt-

age-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 (cho-

lesterol recognition amino acid consensus) domain (82,83); all other drug

ligands bind to a region within the amino-terminus (82,84,85).

Cl

(A)CH3

CH3

CH3

O

H

N

N

(B)CH3

CH3

O

O

NN

N N

N

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-2-

phenyl-purin-9-yl)acetamide (98).



release of neurosteroids from glial and microglial cells (54). Never-

theless, 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 inhibi-

tion. 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 inter-

neurones 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)-3-

isoquinolinecarboxamide (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 deter-

mined 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 suici-

dality (77), a reduction of TSPO expression could be demon-

strated.

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

120

100200 * *

**

150

100

50

0

200

150

100

50

0

80

60

40

20

0

80

60

40

20

0

0 3

Am

plitu

de (p

A)

Cha

nge

%C

hang

e %

Am

plitu

de (p

A)

6 9 12 32 35

Time (min)

XBD173 (5 µM)

XBD173

Finasteride + XBD173

Finasteride(10 µM)

Finasteride (10 µM)+ XBD173 (5 µM)

XBD173(1 µM)

XBD173 (5 µM)

XBD173(5 µM)

Amplitude Charge

Amplitude Charge

Control

Control

20 pA

5 pA

50 ms

50 ms

Time (min)

38 41 44

0 3 6 9 12 32 35 38 41 44

(A)

(B)

Fig. 6. Effect of XBD173 on GABAergic neurotransmission; according to Rupprecht et al. (103). The effect of XBD173 was monitored with whole-cell record-

ings 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 experi-

ments 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.



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).

TSPO (18 kDa) ligands

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-affin-

ity 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 pre-

cursor, 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-con-

taining proteins (ACBD) and renamed ACBD1 (90). Within the CNS,

DBI is primarily expressed in glial cells. Interestingly, b-amyloid

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 carboxa-

mide PK 11195 and the BZD 7-chloro-5-(4-chlorophenyl)-1,3-dihy-

dro-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 char-

acterisation, 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

1200

800

API

-AU

C

400

0Placebo

PlaceboBaseline

Day 7*

*

**

XBD173 XBD173 XBD173 Alprazolam(10 mg) (30 mg) (90 mg) (2 mg)

XBD173 XBD173 XBD173 Alprazolam(10 mg)

0

–10

–30

–20

–40

–50

–60

Ratio

del

ta A

UC

/bas

elin

e A

UC

+ S

EM (%

)

(30 mg) (90 mg) (2 mg)(A) (B)

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% per-

centile. (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).



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 eti-

foxine (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 anx-

iolytic properties in animal models and in humans by enhancing

neurosteroidogenesis in brain slices, thereby potentiating the ampli-

tude and duration of GABA-mediated inhibitory postsynaptic cur-

rents, 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 (express-

ing rat a1c2 and human b3 GABAA receptor subunits), thereby dem-

onstrating 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 compa-

rable 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 treat-

ment 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 dif-

ference 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 anx-

iolytic properties and fewer side-effects for XBD173 compared to

BZDs in humans, which makes this compound a promising candi-

date for a novel class of anxiolytics.

Conclusions and prospects

The concept of direct modulators of the GABAergic system as anx-

iolytic 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 obvi-

ous.

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 psychiat-

ric disorders, such as anxiety disorders, which may constitute a

novel class of compounds related to the pathophysiology of these

TSPOexpression

Anxiety

Anxiety

Neuroprotection

Neurodegeneration

Stroke

Parkinson’s disease

Alzheimer’s disease

Glioma

Morbus Niemann Pick

peripheral nerve lesionsM

icro

glia

act

ivat

ionDepression ?

Depression?

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 activa-

tion 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).



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 pro-

file of TSPO ligands under prolonged application has to be investi-

gated in view of the high expression of TSPO in peripheral tissues,

which might also reduce drug specificity. In addition, drug specific-

ity is further challenged by the fact that TSPO ligands do not selec-

tively 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 exist-

ing treatment options remains to be clarified. These questions can

only be answered by systematic, clinical studies involving prolonged

administration and safety monitoring. Moreover, further investiga-

tions on the underlying molecular mechanisms of these compounds

that may contribute toward explaining the putative favourable side-

effect 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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Translocator Protein (18 kDa) as a Target for Novel Anxiolytics with a Favourable Side‐Effect...

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