2. Benzodiazepine
• A benzodiazepine (sometimes colloquially "benzo"; often abbreviated
"BZD") is a psychoactive drug whose core chemical structure is the fusion
of a benzene ring and a diazepine ring.
• Benzodiazepines enhance the effect of the neurotransmitter gamma-
aminobutyric acid (GABA) at the GABAA receptor, resulting
in sedative, hypnotic, anxiolytic, anticonvulsant, and muscle
relaxant properties.
3. CBR and PBR
Central benzodiazepine
receptor
GABAA
receptor
CNS
α-subunit
binding
Inhibitory
effect
Peripheral benzodiazepine
receptor
TSPO
Peripheral
tissues and
glial cells
Heteromeric
complex
Cholesterol
transport
• BZDs also bind to other receptors, located mainly in peripheral
tissues and glial cells in the brain.
4. TSPO
The translocator protein (18 kDa) (TSPO) is a five transmembrane
domain protein that is localized primarily in the outer
mitochondrial membrane.
5. PBR distribution
• The PBR is widely expressed throughout the body, with high
densities found in steroid-producing tissues.
Christopher J. Endres et. al.
6. Functions
• Cholesterol transport: TSPO binds with high affinity to cholesterol and transports it
across the mitochondrial membrane where it is used in steroid synthesis.
• Immunomodulation: Expression of TSPO has been linked to inflammatory
responses that occur after ischemia-reperfusion injury, following brain injury, and in
some neurodegenerative diseases.
• Apoptosis: Ligands of TSPO have been shown to induce apoptosis in human colorectal
cancer cells.
• Cell proliferation
• An indicator of the importance of TSPO is that disruption of the gene in mice results
in embryonic death.
7. Structure of TSPO, docking with cholesterol and
mitochondrial localization
Rupprecht R et.al. Nat Rev Drug Discov. 2010 Dec;9(12):971-88. doi: 10.1038/nrd3295.
8.
9. Therapeutic Applications
• TSPO has been shown to be involved in a
number of processes such
as inflammation,and TSPO ligands may be
useful anti-cancer drugs. Activation of
TSPO is also required for
steroidogenesis to take place, and this is
particularly important for the production
of neuroactive steroids such
as allopregnanolone in the brain. This
makes some TSPO ligands such as
emapunil (XBD-173) useful as
potential anxiolytics which may have less
side effects than traditional
benzodiazepine-type drugs.
10. Emapunil (XBD-173) - potential anxiolytic
XBD173 (AC-5216, emapunil) is a phenylpurine with high and rather selective affinity to TSPO,
which has recently been investigated for the treatment of anxiety disorders. XBD173 enhances
neurosteroidogenesis in the brain, thereby exerting anxiolytic properties in animal models and in
humans.
Eur Arch Psychiatry Clin Neurosci (2012) 262 (Suppl 2):S107–S112
score
11. Ligands
• The benzodiazepine Ro5-4864 and the isoquinoline carboxamide PK11195 exhibit
nanomolar affinity for the PBR, and are the archetypic pharmacological tools for
characterizing the receptor and its function.
• PK11195 [1-(2-chlorophenyl)-N-methyl-N-(1-methylprolyl)-3-isoquinoline
carboxamide,] is currently the most widely used PBR ligand.
12. Effect of PBR ligands on cellular
activity in different cell types
Gavish et. al. 1999
13. A translocator protein ligand PK11195 shows antigrowth
activity in human choriocarcinoma cells
• BeWo choriocarcinoma cells showed significant sensitivity to PK11195 with a
concentration of 8.0×10−5M which caused a 50 % inhibition (ED50) in their growth. On
the other hand, choriocarcinoma cells showed little sensitivity to diazepam from a
concentration of 1×10−7 to 1×10−4 M.
• PK11195 led to a dramatic increase in the sub-G0/G1 apoptotic cell population and
G0/G1 phases of the cell cycle compared to treatment with vehicle alone, with a
concomitant decrease in the proportion of cells in the S phase.
Tumor Biol. (2012) 33:1505–1510
14. Alterations in PBR binding characteristics in various
hyperplastic and cancer tissues
Noninvasive molecular imaging of TSPO
expression could form the basis of a predictive
cancer imaging biomarker.
Gavish et. al. 1999
15. PK or not PK
Historically, the TSPO ligand used most commonly with PET is 11C-R-PK11195. Although it has
been used successfully for numerous clinical studies, 11C-R-PK11195 has pharmacokinetic
properties, such as high nonspecific binding, that are suboptimal for imaging.
Alex Sik Chung Ching et.al. Insights Imaging(2012)3:111–119
Binding of benzodiazepines to this receptor complex promotes binding of GABA, which in turn increases the total conduction of chloride ions across the neuronal cell membrane. This increased chloride ion influx hyperpolarizes the neuron's membrane potential. As a result, the difference between resting potential and threshold potential is increased and firing is less likely
Based on its pharmacological profile, the PBR was initially
considered a subtYpe of CBR
The translocator protein (TSPO) was originally discovered and named the peripheral-type benzodiazepine receptor (PBR) in 1977 when investigators found a second binding site for the benzodi-azepine diazepam in renal tissue distinct from GABAAreceptorwithin the central nervous system
Cytosolic loops 1and 2 are responsible for the binding of small molecule ligands, such as benzodiazepines or pyrazolopyrimidines. The cytosolic C-terminal region binds cholesterol molecules.
Summed coronal 2-dimensional projections of tracer
activity after bolus injection of 11C-DPA-713. Seven whole-body
passes were performed as described. Images from left to right are
from passes 1, 3, and 5 and show tracer biodistribution from 1 to 3,
from 10 to 16, and from 28 to 42 min after injection, respectively.
Images are displayed to same maximum. Lung uptake is prominent
immediately after injection. Substantial tracer uptake is also observed
in kidneys, spleen, liver, and brain.
The highest residence times
were obtained in the lungs, liver, red marrow, brain, kidney,
and small intestine. The highest absorbed doses were
obtained in the lungs, spleen, kidney, pancreas, heart, and
liver.
TSPO expression
may constitute a biomarker for brain inflammation that could
be monitored by using TSPO tracers as neuroimaging agents.
The PBR appears to be a heteromeric complex of at
least three different subunits, including an isoquinoline
binding subunit (18 kDa), a VDAC (32 kDa), and an
adenine nucleotide carrier
PAP7 is a functional element of the hormone-induced signal transduction cascade leading to steroidogenesis
Fig. 3 Modified according to [46]. Effect of XBD173 on CCK-4 induced panic. a Area under the time curve (AUC) of the acute panic inventory (API) score of healthy male volunteers during a first (before treatment) and a second (after 7 days of treatment) CCK-4 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
represent outliers located more than 150 %, and asterisks represent extreme values located more than 300 % of the box height above the
75 % percentile. b Decrease in CCK-4 induced panic (delta APIAUC) after treatment with different dosages of XBD173, the BZD
alprazolam or placebo in relation to baseline AUC (mean ± SEM). Asterisks indicate a significant difference from placebo (ANCOVA:
90 mg XBD173 P\0.036, alprazolam P\0.019)
In contrast to BZDs, XBD173 did not directly enhance GABAA receptor-mediated chloride currents [46].
XBD173 counteracted pharmacologically induced panic attacks in rodents without exerting sedative effects [46]. In
healthy male volunteers, the antipanic efficacy of XBD173 was comparable to the BZD alprazolam during pharmacologically
induced panic by cholecystokinin tetrapeptide (CCK-4) [46] (Fig. 3). This placebo–controlled, parallel
group-proof of concept study included subjects with a sufficient panic response after CCK-4 application. Seventyone
subjects were randomized to a 7-day treatment with placebo, 10, 30 or 90 mg/day XBD173 or 2 mg/day
alprazolam. At the end of the study, subjects underwent a second CCK-4 challenge. The difference in the acute panic
inventory (API) (area under the time curve; AUC) between the first and the second CCK-4 challenge relative to the
effects of placebo was defined as readout for anxiolytic efficacy. For both alprazolam and the highest dose of
XBD173, a significant difference from placebo could be demonstrated. Interestingly, the XBD173 groups did not
suffer from sedation or withdrawal symptoms after the 7-day treatment in contrast to alprazolam treated
subjects.
positron emission tomography
(PET)
PET principle: imaging agents used for PET are radioactive
probes emitting positively charged anti-electrons (positrons). The
positron travels a short distance (∼1–3 mm) before it annihilates with
any electron to give two photons of 511 keV travelling under a mutual
angle of 180°. It is this pair of coincident high energy photons that
underly the PET principle allowing in vivo quantitative 3D imaging
through coincident detection using a gantry of multiple detectors
around the patient
(A)T2-weightedMRimageofC6glioma–bearingratinrightbrainhemisphere.(B)CoronalPETimageobtainedfromdynamic
scanof
18F-DPA-714PET(summeddynamicscan,0–90min).(C)
18F-DPA-714time–activitycurvesfortumor(blue),contralateralbrain
(green),andplasma(red).(D)TransversePETimageobtainedfromdynamicscanof
18F-DPA-714PET(summeddynamicscan,0–90min).
(E)Standard hematoxylin and eosin staining of serial tissue section.(F)ImmunohistochemistryanalysisofTSPOexpressionintypicalC6
glioma.Tumorlocationisindicatedwitharrowsinimages.%ID/cc 5 percentageinjecteddosepercubiccentimeter.