Characterization of a novel acetamidobenzoxazolone-based PET ligand for translocator protein (18 kDa) imaging of neuroinflammation in the brain

Article abstract of DOI:10.1111/jnc.12670

We developed the novel positron emission tomography (PET) ligand 2-[5-(4-[¹¹C]methoxyphenyl)-2-oxo-1,3-benzoxazol-3(2H)-yl]-N-methyl- N-phenylacetamide ([¹¹C]MBMP) for translocator protein (18 kDa, TSPO) imaging and evaluated its efficacy in ischemic rat brains. [¹¹C]MBMP was synthesized by reacting desmethyl precursor (1) with [¹¹C] CH₃I in radiochemical purity of ≥ 98% and specific activity of 85 ± 30 GBq/μmol (n = 18) at the end of synthesis. Biodistribution study on mice showed high accumulation of radioactivity in the TSPO-rich organs, e.g., the lungs, heart, kidneys, and adrenal glands. The metabolite analysis in mice brain homogenate showed 80.1 ± 2.7% intact [¹¹C]MBMP at 60 min after injection. To determine the specific binding of [¹¹C]MBMP with TSPO in the brain, in vitro autoradiography and PET studies were performed in an ischemic rat model. In vitro autoradiography indicated significantly increased binding on the ipsilateral side compared with that on the contralateral side of ischemic rat brains. This result was supported firmly by the contrast of radioactivity between the ipsilateral and contralateral sides in PET images. Displacement experiments with unlabelled MBMP or PK11195 minimized the difference in uptake between the two sides. In summary, [¹¹C]MBMP is a potential PET imaging agent for TSPO and, consequently, for the up-regulation of microglia during neuroinflammation. We developed 2-[5-(4-[ ¹¹C]methoxyphenyl)-2-oxo-1,3-benzoxazol-3(2H)-yl]-N-methyl-N- phenylacetamide ([¹¹C]MBMP) as a novel positron emission tomography ligand for imaging of translocator protein (18 kDa, TSPO) in the brain. [ ¹¹C]MBMP exhibited high in vitro and in vivo specific binding with TSPO in the ischemic rat brain. We developed 2-[5-(4-[¹¹C] methoxyphenyl)-2-oxo-1,3-benzoxazol-3(2H)-yl]-N-methyl-N-phenylacetamide ([ ¹¹C]MBMP) as a novel positron emission tomography ligand for imaging of translocator protein (18 kDa, TSPO) in the brain. [¹¹C]MBMP exhibited high in vitro and in vivo specific binding with TSPO in the ischemic rat brain.

Full text of DOI:10.1111/jnc.12670

JOURNAL OF NEUROCHEMISTRY
|
2014
|
129
|
712–720
doi: 10.1111/jnc.12670
,
,
*Molecular Imaging Center, National Institute of Radiological Sciences, Chiba, Japan
†Division of Cyclotron and Radiopharmaceutical Sciences, Institute of Nuclear Medicine and Allied
Sciences, Delhi, India
‡SHI Accelerator Service Co. Ltd., Tokyo, Japan
Abstract
and PET studies were performed in an ischemic rat model. In
We developed the novel positron emission tomography (PET)
ligand 2-[5-(4-[11C]methoxyphenyl)-2-oxo-1,3-benzoxazol-3
(2H)-yl]-N-methyl-N-phenylacetamide ([11C]MBMP) for trans-
locator protein (18 kDa, TSPO) imaging and evaluated its
efficacy in ischemic rat brains. [11C]MBMP was synthesized
vitro autoradiography indicated significantly increased binding
on the ipsilateral side compared with that on the contralateral
side of ischemic rat brains. This result was supported firmly
by the contrast of radioactivity between the ipsilateral and
contralateral sides in PET images. Displacement experi-
ments with unlabelled MBMP or PK11195 minimized the
difference in uptake between the two sides. In summary,
by reacting desmethyl precursor (1) with
[
11C]CH3I in
radiochemical purity of ≥ 98% and specific activity of
85 Æ 30 GBq/lmol (n = 18) at the end of synthesis. Biodis-
tribution study on mice showed high accumulation of radio-
activity in the TSPO-rich organs, e.g., the lungs, heart,
kidneys, and adrenal glands. The metabolite analysis in mice
brain homogenate showed 80.1 Æ 2.7% intact [11C]MBMP at
60 min after injection. To determine the specific binding of
[
11C]MBMP is a potential PET imaging agent for TSPO and,
consequently, for the up-regulation of microglia during
neuroinflammation.
Keywords: [11C]MBMP, microglia activation, neuroinflam-
mation, positron emission tomography, translocator protein
(18 kDa).
[
11C]MBMP with TSPO in the brain, in vitro autoradiography
J. Neurochem. (2014) 129, 712–720.
Microglia activation is the most predominant cellular
response to inflammation within the central nervous system
(CNS). This process is also characterized by a sharp change
in the phenotype of these cells and by the over-expression of
peripheral benzodiazepine receptor or, as recently proposed,
translocator protein (18 kDa, TSPO) on the outer mitochon-
drial membranes of TSPO binding site (Papadopoulos et al.
2006a; Chauveau et al. 2008).
Over the past two decades, several researchers have
demonstrated that the imaging of activated microglia in the
CNS can be performed by positron emission tomography
(PET) using TSPO ligands (Venneti et al. 2008; Batarseh
and Papadopoulos 2010; Winkeler et al. 2010; Trapani et al.
Received October 7, 2013; revised manuscript received January 11,
2014; accepted January 29, 2014.
Address correspondence and reprint requests to Ming-Rong Zhang,
Molecular Probe Program, Molecular Imaging Center, National Institute
of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555,
Japan. E-mail: zhang@nirs.go.jp
Abbreviations used: % ID/g, percentage of the injected dose per gram
of wet tissue; [11C]MBMP, 2-[5-(4-[11C]methoxyphenyl)-2-oxo-1,3-
benzoxazol-3(2H)-yl]-N-methyl-N-phenylacetamide; BBB, blood–brain
barrier; BPND
chromatography;
,
binding potential; HPLC, high-performance liquid
MBMP, 2-[5-(4-methoxyphenyl)-2-oxo-1,3-ben-
zoxazol-3(2H)-yl]-N-methyl-N-phenylacetamide; PET, positron emis-
sion tomography; SRTM, simplified reference-tissue model; SUV,
standardized uptake value; TAC, time-activity curve; tR, retention time;
TSPO, translocator protein (18 kDa).
712
© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 129, 712--720

[
11C]MBMP for TSPO in brain
713
Effluent radioactivity was monitored using a NaI (Tl) scintillation
detector system, and radioactivity measurement was performed during
synthesis and animal studies with a Curiemeter (Aloka, Tokyo, Japan).
2013). Radiolabelling of the isoquinoline-3-carboxamide
derivative PK11195 with 11C in the early 1980s paved way
for the current concepts of PET imaging of neuroinflamma-
tion through TSPO ligands (Camsonne et al. 1984; Dolle
et al. 2009). A number of PET ligands are being employed,
such as benzodiazepine derivative [11C]Ro5-4864 (Watkins
et al. 1988); phenoxyphenylacetamide derivatives [11C]
DAA1106, [18F]FEDAA1106, and [11C]PBR28 (Zhang
et al. 2003, 2004; Maeda et al. 2004; Hines et al. 2013);
pyrimidineacetamide derivative [11C]DPA-713, [18F]DPA-
714, and [11C]CLINME (Boutin et al. 2007; Van Camp
et al. 2009, 2010; Arlicot et al. 2012); indoleacetamide
derivative [11C]SSR180575 (Chauveau et al. 2011); and 8-
oxopurine derivatives [11C]AC-5216 and [18F]FEDAC
(Zhang et al. 2007; Yui et al. 2010). Although these PET
ligands are promising and parts of them have been used to
visualize TSPO in human brains, several issues need to be
resolved before TSPO radioligands can be routinely used in
clinical studies. It is necessary to find the optimal ligand
showing requisite amounts of sensitivity and specificity and
understand how TSPO polymorphisms can alter uptake of
the PET ligands (Venneti et al. 2008; Owen et al. 2012;
Kreisl et al. 2013; Venneti et al. 2013).
Chemical synthesis
2-[5-(4-Methoxyphenyl)-2-oxo-1,3-benzoxazol-3(2H)-yl]-N-methyl-
N-phenylacetamide (MBMP)
A
mixture of 2-(5-bromo-2-oxo-1,3-benzoxazol-3(2H)-yl)-N-
methyl-N-phenylacetamide (Fukaya et al. 2012) (1.44 g, 4.0 mmol)
and 4-methoxyphenylboronic acid (729 mg, 4.8 mmol), K2CO3
(829 mg, 6.0 mmol) and tetrakis(triphenylphosphine)palladium(0)
(Pd(PPh3)4; 462 mg, 0.4 mmol) in 1,4-dioxane/water (3/1, 20 mL)
was heated at 100°C under nitrogen atmosphere. The reaction
mixture was stirred for 4 h and cooled to 25°C. Subsequently, the
mixture was extracted with EtOAc. The organic layer was washed
with brine and dried over sodium sulfate. After the solvent was
removed in vacuo, and the residue was purified by silica gel column
chromatography using hexane/EtOAc (2/1, v/v) as the eluent to give
MBMP (1.23 g, 79.9% yield): white solid; mp 168–170°C (Fukaya
et al. 2012). Chemical purity: > 99% by HPLC: Capcell Pack UG80
C18 column (Shiseido, Tokyo, Japan), 4.6 mm i.d. 9 250 mm;
acetonitrile (MeCN)/H2O/Et3N, 6/4/0.01 (v/v/v); flow rate, 1.0 mL/
1
min; kuv, 254 nm); retention time (tR), 10.1 min. H NMR (CDCl3,
d) 3.32 (3H, s), 3.86 (3H, s), 4.35 (2H, s), 6.96–7.00 (3H, m), 7.19–
7.23 (2H, m), 7.34 (2H, d, J = 7.7 Hz), 7.41–7.54 (5H, m). high
resolution mass spectrometer (HRMS) (FAB) calculated for
C23H21O4N2, 389.1501; Observed:, 389.1511.
Following the development of [11C]DAA1106 (Zhang
et al. 2003) and [11C]AC-5216 (Zhang et al. 2007), we are
developing a new PET ligand for TSPO imaging. In this
paper we have introduced a novel acetamidobenzoxazol-
one skeleton 2-[5-(4-methoxyphenyl)-2-oxo-1,3-benzoxazol-
3(2H)-yl]-N-methyl-N-phenylacetamide (MBMP, Fig. 1) as
a candidate ligand for TSPO. Recently, a group of research-
ers have prepared a library of acetamidobenzoxazolone
derivatives as TSPO ligands (Fukaya et al. 2012). The
selection of our candidate ligand, MBMP, was based on the
pharmacophore model with high TSPO binding affinity
(Ki = 0.29 nM) and appropriate lipophilicity (computed
LogD = 3.5). The aim of our present study was to inves-
tigate the potential use of [11C]MBMP (Fig. 1) as a new PET
ligand for neuroinflammation, and to demonstrate that this
radioligand binds to TSPO sites, while having improved
imaging properties over (R)[11C]PK11195.
2-[5-(4-Hydroxyphenyl)-2-oxo-1,3-benzoxazol-3(2H)-yl]-N-methyl-
N-phenylacetamide (1)
Compound 1, as a desmethyl precursor for radiosynthesis, was
prepared from 2-(5-bromo-2-oxo-1,3-benzoxazol-3(2H)-yl)-N-
methyl-N-phenylacetamide (718 mg, 2.0 mmol) and 4-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (528 mg, 2.4 mmol) in
a manner similar to that described for MBMP, as a white solid
(284 mg, 75.7% yield) after purification by silica gel column
chromatography using hexane/EtOAc (1/1, v/v); mp: 202–205°C.
Chemical purity: > 99% by HPLC: Capcell Pack UG80 C18 column,
4.6 mm i.d. 9 250 mm; MeCN/H2O/Et3N, 6/4/0.01 (v/v/v); flow
rate, 1.0 mL/min; kuv, 254 nm; tR, 3.0 min. 1H NMR (DMSO-d6,
d): 3.22 (3H, s), 4.42 (2H, s), 6.86 (2H, d, J = 8.4 Hz), 7.29–7.57
(10H, m), 9.56 (1H, s). HRMS (FAB) calculated for C22H19O4N2,
375.1345; observed, 375.1308.
Materials and methods
2-[5-(4-[11C]Methoxyphenyl)-2-oxo-1,3-benzoxazol-3(2H)-yl]-N-
methyl-N-phenylacetamide ([11C]MBMP)
Chemicals and instrumentation
Cyclotron-produced [11C]CO2 was bubbled into 0.4 M LiAlH4 in
anhydrous tetrahydrofuran (0.3 mL). After the evaporation of
tetrahydrofuran, the remaining complex was treated with 57%
hydroiodic acid (0.3 mL) to give [11C]CH3I, which was distilled
with heating and transferred under N2 gas flow to a solution of 1
(1 mg) and NaOH (5 lL, 0.5 M) in N,N-dimethylformamide
(DMF) (0.3 mL) at À15°C. After trapping was completed, this
reaction mixture was heated at 70°C for 5 min. HPLC separation
was performed on a Capcell Pack UG80 C18 column (10 mm
i.d. 9 250 mm) using MeCN/H2O/Et3N (6/4/0.01, v/v/v) at
5.0 mL/min. The radioactive fraction corresponding to [11C]MBMP
All chemicals were purchased from (Wako Pure Chemical Industries
[Osaka, Japan], Tokyo Chemical Industries [Tokyo, Japan] and
Sigma-Aldrich [Tokyo, Japan]), and used as received. Melting
points were measured by
a micro melting point apparatus
(MP-500P). 1H NMR (300 MHz) spectra were recorded on a
JEOL-AL-300 NMR spectrometer (JEOL, Tokyo, Japan) with
tetramethylsilane as an internal standard. Mass peaks were calcu-
lated by high-resolution fast atom bombardment (FAB) mass
spectra, which were obtained and recorded on a JEOL JMS-Sx
102A spectrometer (JEOL). HPLC separation and analysis were
performed using the JASCO HPLC system (JASCO, Tokyo, Japan).
© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 129, 712--720

714
A. K Tiwari et al.
Fig. 1 Chemical synthesis and radiosynthesis. Reagents and condi-
1,4-dioxane/water (3/1), reflux, 4–5 h. b) [11C]CH3I, NaOH, DMF,
70°C, 5 min.
tions: a) Pd(PPh3)4, K2CO3, 4-substituted arylboron derivative,
(tR = 9.8 min) was collected in a flask in which Tween 80
(0.075 mL) and ethanol (0.3 mL) was before the radiosynthesis.
The fraction was evaporated to dryness, redissolved in 3 mL of
sterile normal saline, and passed through a 0.22 lm Millipore filter
(Billerica, MA, USA). The identity of [11C]MBMP was confirmed
by co-injection with unlabelled MBMP on a reverse phasedÀana-
lytical HPLC on a Capcell Pack UG80 C18 column (4.6 mm
i.d. 9 250 mm) using MeCN/H2O/Et3N (6/4/0.01, v/v/v) at
1.0 mL/min (tR = 10.2 min). In the final product solutions, their
radiochemical purity was higher than 99% at the end of synthesis
(EOS). No other significant peak was found on the HPLC chart. The
specific activity was measured and calculated by comparing the
assayed radioactivity with the mass measured at UV (254 nm). To
determine the radiochemical stability of [11C]MBMP, after the
formulated product solution was maintained for 30, 60, and 90 min
at 25°C, analytic sample was taken from the solution to measure the
radiochemical purity of [11C]MBMP with HPLC.
(1480 Wizard 3, Perkin-Elmer, Waltham, MA, USA) and expressed
as percentages of the injected dose per gram of wet tissue (% ID/g).
All radioactivity measurements were corrected for decay.
Radiolabelled metabolites analysis
The ddY mice were intravenously injected with [11C]MBMP
(37–54 MBq/0.4À0.7 nmol, 61 Æ 19 GBq/lmol in 200À300 lL
per mouse) through the tail vein. The mice were killed by cervical
dislocation at 5, 15, 30 or 60 min (n = 4 for each point). Blood
(0.7À1.0 mL) and whole brain samples were obtained and treated as
reported previously (Zhang et al. 2003, 2004). The supernatant of
plasma and brain homogenate was analyzed according to the
following conditions: Capcell Pack UG80C18 column, 4.6 mm
i.d. 9 250 mm; MeCN/H2O/Et3N, 7/3/0.01 (v/v/v); flow rate,
1.0 mL/min. Percentages of the unchanged [11C]MBMP were
calculated with correction of decay. At the same time, radioactivity
fractions in the HPLC waste solution were measured using a auto-
gamma counter.
(R)[11C]PK11195
The R enantiomer of [11C]PK11195, the standard PET ligand for
TSPO, was synthesized as reported in the literature (Shah et al.
1994). The radiochemical purity of (R)[11C]PK11195 was 99% and
the specific activity was calculated as 74 Æ 8 GBq/lmol (n = 3) at
EOS.
Ischemic model of rat brain
Mild focal ischemia was produced by intraluminal occlusion of the
middle cerebral artery for 30 min using an intraluminal thread
model (Yui et al. 2013). Briefly, a SD rat was anesthetized with
4% (v/v) isoflurane and maintained with 1.8% isoflurane. A
ligation was conducted with the right internal carotid artery. A 4.0-
monofilament nylon suture coated with silicon was inserted
(16–18 mm) into the internal carotid artery up to the level of
middle cerebral artery branches. The neck incision was closed with a
silk suture. After regaining consciousness from anesthesia, the rats
were reanesthetized after 30 min, and the filament was carefully
removed for reperfusion. Throughout the surgery, the body
temperature was monitored and maintained at an optimal level.
All rats were employed for experiments 7 days after the surgery.
Animals
All animals were maintained and handled in accordance with the
recommendations of the National Institute of Health and institu-
tional guidelines of the National Institute of Radiological Sciences
(NIRS). The experiments conducted in the NIRS were approved by
the Animal Ethics Committee of the NIRS. Male ddY mice (male,
8 weeks old, 34À36 g) and Sprague–Dawley (SD) rats (male,
8À9 weeks old, 240À330 g) were purchased from Japan SLC
(Shizuoka, Japan). The animals were housed under a 12/12-h dark/
light cycle under optimal conditions. The ARRIVE guidelines were
complied with.
In vitro autoradiography
Brain sagittal sections (10 lm) were prepared from frozen rat brains
using a cryostat (HM560, Carl Zeiss, Oberkochen, Germany). The
brain sections were pre-incubated for 20 min in 50 mM Tris-HCl
buffer (pH 7.4) at 25°C followed by incubation in the same buffer
containing [11C]MBMP (18 MBq, 0.2 nmol, 1 nM) at 25°C for
30 min. For the inhibition studies, unlabelled MBMP (10 lM,
prepared from 10 mM MBMP solution in distilled water containing
15% ethanol and 10% Tween 80) or PK11195 (10 lM, prepared
from 10 mM PK11195 solution in distilled water containing 15%
ethanol and 10% Tween 80) was co-incubated with [11C]MBMP.
After incubation, the brain sections were treated as reported
previously (Yui et al. 2013). Autoradiograms were acquired using
Biodistribution study
A saline solution of [11C]MBMP (5.6 MBq/0.08 nmol in 100 lL
per mouse) was injected into the ddY mice through the tail vein, and
the mice were killed at five time intervals (1, 5, 15, 30, and 60 min).
All ddY mice for each group (n = 4) were killed by cervical
dislocation after injection. Blood samples were acquired, and the
cerebellum, cerebral cortex, other brain structures, liver, lungs,
heart, kidneys, spleen, adrenal glands, small intestine, large
intestine, and testes were quickly removed and weighed. The
radioactivity in each tissue was measured using a gamma counter
© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 129, 712--720

[
11C]MBMP for TSPO in brain
715
a Bio-Imaging analyzer system (BAS-5000, Fujifilm, Tokyo, Japan).
The radioactivity level on the brain regions was measured by the
Multi Gauge analysis software version 2.3 (Fujifilm) and expressed
as photo-stimulated luminescence per unit area (PSL/mm2).
Redmond, WA, USA). All data were expressed as the mean Æ SD.
For autoradiography, the changes in values were compared using
Student’s paired t-test. PET data were examined by one-way
repeated-measure ANOVA. p-values less than 0.05 were considered
statistically significant.
PET imaging
PET scans were conducted using a small-animal PET scanner
(Inveon; Siemens Medical Solutions, Knoxville, TN, USA). Seven
days after ischemic surgery, a rat was anesthetized with 1–2%
isoflurane during the scan and its body temperature was maintained
with a 40°C water circulation system (T/Pump TP401, Gaymar
Industries, Orchard Park, NY, USA). An emission scan was
immediately acquired for 60 min after injection of [11C]MBMP
(37 MBq/0.4–0.6 nmol, 70 Æ 21 GBq/lmol in 100 lL) or (R)
Results
Chemistry
Figure 1 shows the synthetic pathway for the synthesis of
[11C]MBMP and its precursor 1 along with unlabelled
MBMP. The desmethyl precursor 1 for radiosynthesis was
synthesized by Suzuki’s coupling reaction between the
bromo compound and 4-(4,4,5,5-tetramethyl-1,3,2-dioxa-
borolan-2-yl)phenol in the presence of Pd(PPh3)4). For the
synthesis of MBMP, 4-methoxyphenylboronic acid was used
for coupling of the methoxyphenyl ring. The yield of both
the reactions was 75.7% for 1 and 79.9% for MBMP. Proton
NMR and high-resolution MS spectra confirmed the pro-
posed stoichiometry and structure for the novel acet-
amidobenzoxazolone derivatives.
[
11C]PK11195 (37 MBq/0.5–0.7 nmol, 66 Æ 10 GBq/lmol in
100 lL) through the tail vein. For the displacement experiments,
respective unlabelled TSPO ligands (MBMP (1 mg/kg) or
PK11195 (3 mg/kg) in 300 lL of saline containing 15% ethanol
and 10% Tween 80) were injected 20 min after the injection of
[
11C]MBMP. Each experiment was performed in a group of three
animals, and only one PET scan was performed with one animal.
[11C]MBMP was synthesized by reacting precursor 1 with
[11C]CH3I in the presence of NaOH in good radiochemical
yields. After the reaction, separation, and formulation, 0.9–
2.3 GBq (1.5 Æ 0.6 GBq, n = 18) of [11C]MBMP was
obtained after EOS, which started at 15.5–22.2 GBq
(18.5 Æ 2.1 GBq) of cyclotron-produced [11C]CO2. The
specific activity was 50–130 GBq/lmol (85 Æ 30 GBq/
lmol, n = 18) at the end of the 32–36 min synthesis. The
radiochemical purity of [11C]MBMP was higher than 99%
and remained radiochemically stable for 1.5 h at 25°C. These
analytical results were in compliance with our in-house
quality control/assurance specifications.
Image analysis
PET data modelling was performed into three-dimensional sino-
grams, which were changed into two-dimensional sinograms
(frames 9 min: 4 9 1, 8 9 2, 8 95) by Fourier rebinning. Filtered
back-projection using Hanning’s filter with a Nyquist cut-off
frequency of 0.5 cycle/pixel was used for dynamic image recon-
struction. PET images were analyzed using ASIPro VMTM (Analysis
Tools and System Setup/Diagnostics Tool; Siemens Medical
Solutions) with reference to the magnetic resonance (MR) imaging
template. A region of interest (ROI) with fixed size was manually
positioned at the center of the striatum on the ipsilateral side defined
on a summation image for each experiment, which coincided to the
ROI used in autoradiography. The ipsilateral ROI was copied and
symmetrically pasted into the contralateral striatum on the same slice
to yield a contralateral ROI of identical volume and shape. Time-
activity curves (TACs) for ipsilateral and contralateral striatum were
generated. Decay correction was performed for the brain uptake of
radioactivity with reference to the injection time and expressed as the
standardized uptake value (SUV), which was normalized for the
injected radioactivity and body weight. The SUV was calculated
according to the following formula: SUV = (radioactivity per cubic
centimetre tissue/injected radioactivity) 9 grams body weight.
The binding potential (BPND) was calculated as a quantitative
value, which represented the receptor binding with the radioligand,
using PMOD software (PMOD Technologies, Zurich, Switzerland).
The BPND was estimated using a simplified reference-tissue model
(SRTM) by taking the TAC of the contralateral side as the reference
region instead of the plasma input function. The SRTM analysis in
the ipsilateral side of the striatum was performed using the TAC in
the contralateral side as a reference region (Lammertsma and Hume
1996). R1 (k1/k1’), which represented the ratio of tracer delivery, was
estimated instead of input function.
Biodistribution
Table 1 shows the biodistribution of radioactivity at the five
time points after the injection of [11C]MBMP into the mice
(n = 4 for each point). At 1 min, a high uptake of
radioactivity (% ID/g) was shown immediately to reach a
peak in the blood, heart, lung, and brain. In the lung, the
radioactivity reached 160.8 Æ 23.8% ID/g, which decreased
very rapidly within 5 min to 60.3 Æ 2.3% ID/g. The
radioactivity in the adrenal glands increased until 60 min,
whereas in other organs like the spleen and kidney it reached
maximum on 15 min and then decreased. The initial brain
uptake was higher than 2.0% ID/g at 1 min. The maximum
amount of radioactivity was found in cerebellum. The uptake
in the brain might be because of the lipophilic property of
[11C]MBMP (cLogP = 3.5).
Radiolabelled metabolites analysis
Up to 60 min after the injection of [11C]MBMP, only one
radioactive metabolite peak was recovered from the plasma
and brain tissues on radio-HPLC, in addition to intact [11C]
Statistical analysis
Statistical analyses were performed using SPSS software (IBM,
Armonk, NY, USA) and Microsoft Office Excel 2007 (Microsoft,
© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 129, 712--720

716
A. K Tiwari et al.
Table 1 Radioactivity distribution in % ID/g of mice tissue (n = 4) during 1–60 min time intervals
Time after the injection (min)
Organ/tissue
1
5
15
0.49 Æ 0.20
30
0.48 Æ 0.02
60
0.47 Æ 0.02
Blood
1.98 Æ 0.93
1.01 Æ 0.12
Lung
160.80 Æ 23.75
17.21 Æ 2.84
0.99 Æ 0.15
3.12 Æ 0.96
4.95 Æ 2.11
8.27 Æ 0.50
1.41 Æ 0.31
0.79 Æ 0.15
0.41 Æ 0.20
2.43 Æ 0.40
2.53 Æ 0.58
2.26 Æ 0.21
60.31 Æ 2.29
15.53 Æ 0.91
2.92 Æ 0.27
9.05 Æ 0.39
12.95 Æ 3.05
13.55 Æ 0.87
2.76 Æ 0.22
1.51 Æ 0.22
0.72 Æ 0.03
1.84 Æ 0.18
2.41 Æ 0.18
1.92 Æ 0.12
34.17 Æ 1.82
13.80 Æ 0.66
3.09 Æ 0.34
12.06 Æ 2.34
16.88 Æ 4.55
15.66 Æ 1.88
2.62 Æ 0.21
1.75 Æ 0.20
0.82 Æ 0.07
1.12 Æ 0.28
1.48 Æ 0.43
1.16 Æ 0.25
23.52 Æ 2.76
8.19 Æ 1.36
3.05 Æ 0.56
10.30 Æ 1.13
18.26 Æ 6.77
14.94 Æ 1.13
2.53 Æ 0.34
1.64 Æ 0.26
0.97 Æ 0.09
0.58 Æ 0.07
1.15 Æ 0.08
0.72 Æ 0.08
16.96 Æ 2.33
5.91 Æ 0.54
2.81 Æ 0.48
9.82 Æ 0.76
21.78 Æ 7.27
13.51 Æ 0.95
2.73 Æ 0.29
1.65 Æ 0.07
1.23 Æ 0.13
0.53 Æ 0.09
0.80 Æ 0.17
0.53 Æ 0.03
Heart
Liver
Spleen
Adrenal gland
Kidney
S. Intestine
L. Intestine
Testis
Cerebral cortex
Cerebellum
Other brain structures
Table 2 Metabolite analysis of [11C]MBMP in the plasma and brain of
mice (n = 4 at each time point)
(a)
(d)
Time after the
injection (min)
Percentage (%) in
the plasma
Percentage (%) in
the brain
(b)
(c)
5
64.9 Æ 4.6
38.0 Æ 5.3
24.1 Æ 2.2
21.0 Æ 1.9
98.6 Æ 0.5
94.3 Æ 0.5
83.4 Æ 1.8
65.7 Æ 2.7
15
30
60
MBMP (tR = 6.6 min). Table 2 shows the percentages of
intact [11C]MBMP in the plasma and brain. The fraction
corresponding to intact [11C]MBMP in the plasma gradually
decreased to 21.0 Æ 1.9% at 60 min after the injection.
However, in the brain, intact [11C]MBMP (98.6 Æ 0.5%)
was detected with one polar metabolite (tR = 2.9 min) at
5 min. After 60 min the intactness of [11C]MBMP in the
brain was 65.7 Æ 2.7%. The recovery of radioactivity from
HPLC analysis for all samples was greater than 95%.
Fig. 2 In vitro autoradiographic results of 2-[5-(4-[11C]methoxyphe
nyl)-2-oxo-1,3-benzoxazol-3(2H)-yl]-N-methyl-N-phenylacetamide ([11C]
MBMP) in ischemic rat brains (n = 4). Arrows indicate ischemic areas.
Representative autoradiograms of
[
11C]MBMP alone (a) or with
unlabelled MBMP (b) or with PK11195 (c) were depicted in identical
scales. Quantitative effects of unlabelled MBMP or PK11195 on the in
vitro binding of [
11C]MBMP on brain sections (d). Radioactivity
In vitro autoradiography
concentrations determined in the brain regions are expressed as
photo-stimulated luminescence (PSL)/mm2. *p < 0.05.
Figure 2 shows the in vitro autoradiographic results of [11C]
MBMP on the ischemic rat brain. In the control section (a),
the ipsilateral side (right) showed high radioactive signal
levels compared with the contralateral side (left). However,
the difference in radioactivity levels between the contralateral
and ipsilateral sides was diminished by co-incubation with an
excess of unlabelled MBMP (b) or PK11195 (c). In the
control sections, the ratio of radioactive signals in the
ipsilateral side compared with the contralateral side was
approximately 4 : 1. In the brain sections treated with
unlabelled MBMP or PK11195, the ratio of radioactive
signals in the ipsilateral side compared with the contralateral
side was approximately 1. Compared with the control, the
treatment with unlabelled MBMP or PK11195 indicated a
large reduction of ipsilateral binding (Fig. 2, À97.4% for
unlabelled MBMP and À94.7% for PK11195).
PET
Figure 3 shows representative PET/MR images and TACs in
the brain of ischemic rats treated with [11C]MBMP (a, b) and
(R)[11C]PK11195 (c, d), respectively. The PET study showed
that [11C]MBMP and (R)[11C]PK11195 had higher uptakes
in the ipsilateral side than in the contralateral side. Initially,
the maximum uptake was 1.35 Æ 0.12 SUV for [11C]MBMP
© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 129, 712--720

[
11C]MBMP for TSPO in brain
717
sides. These data indicate that displacement with unlabelled
MBMP or PK11195 significantly reduced [11C]MBMP
binding in the ipsilateral side.
(b)
(a)
Discussion
In this study, we have synthesized [11C]MBMP, which is the
first PET ligand derivatized from a novel skeleton, acet-
amidobenzoxazolone, for TSPO imaging. The in vivo studies
demonstrated that [11C]MBMP crosses the blood–brain
barrier (BBB) with observable brain uptake within a few
min and interacts selectively with TSPO without significant
interference from radiolabelled metabolites.
(c)
(d)
Neuroinflammation changes microglial morphology and
results in the over-expression of TSPO. The number of
cases of neuroinflammation and neurodegenerative disor-
ders has multiplied many fold in the last two decades,
which requires more non-invasive monitoring techniques
and novel diagnostic markers (Dolle et al. 2009). TSPO is
expressed at low levels on astrocytes and resting microg-
Fig. 3 Coronal positron emission tomography (PET) images and time-
activity curves for 2-[5-(4-[11C]methoxyphenyl)-2-oxo-1,3-benzoxazol-
3(2H)-yl]-N-methyl-N-phenylacetamide ([11C]MBMP) (a, b) and (R)
[
11C]PK11195 (c, d) in ischemic rat brains (n = 3). PET images (a, c)
were generated by summation of whole scans (0–60 min) and were
overlaid on magnetic resonance images as anatomical templates of
normal rat brain. Arrows indicate ischemic areas. Curves (b, d) were
plotted based on the PET images of the ipsilateral and contralateral
striatum. Significant difference (p < 0.05) was observed between two
sides.
ꢀ
lial cells in a healthy brain (Gulyas et al. 2011). However,
its levels are markedly increased during neuroinflammation
and, subsequently, in neurodegeneration. Moreover, TSPO
may be important in modulating neuronal damage and,
hence, may support accelerated microglial proliferation
(Papadopoulos et al. 2006b). Therefore, PET imaging of
TSPO can provide excellent information as a diagnostic
tool in the early stages of neurodegenerative disorders
(Casellas et al. 2002; Chen and Guilarte 2008; Takano
et al. 2010; Lavisse et al. 2012). The reference radioligand
(R)[11C]PK11195 has not gained wide acceptance as a
PET imaging agent because of persistent problems with its
quantification in vivo. To date, nearly 75–80 TSPO
radioligands, which can be classified into 7–8 chemical
classes, have been developed (Watkins et al. 1988; Maeda
et al. 2004; Zhang et al. 2004, 2007; Van Camp et al.
2009, 2010).
Several new radioligands have been assessed in human
subjects, including [11C]DAA1106, [18F]FEDAA1106, [11C]
PBR28, [11C]vinpocetine, [11C]AC-5216, [11C]DPA713, and
[18F]DPA714 (Van Camp et al. 2009; Yui et al. 2010;
Arlicot et al. 2012). However, the human studies of these
clinically used TSPO PET ligands are at their preliminary
stages with relatively small databases. Of these, [11C]
DAA1106 and [11C]PBR28 have shown binding capability
in diseased human living brain in Alzheimer and
schizophrenic conditions (Venneti et al., 2009; Takano et al.
2010; Kreisl et al. 2013).
(B) and 1.00 SUV Æ 0.42 (p < 0.05) for (R)[11C]PK11195
(d). In the contralateral side at 60 min after injection, lower
uptake of [11C]MBMP (0.23 Æ 0.03 SUV) was observed in
comparison with that of (R)[11C]PK11195 (0.33 Æ 0.05
SUV, p < 0.1). A high ipsilateral to contralateral ratio was
observed for [11C]MBMP, which was significantly higher
than (R)[11C]PK11195 (2.77 Æ 0.42 vs. 1.80 Æ 0.18,
p < 0.01).
In addition, the PET quantitative value of the ligand bound
to the receptor was presented as the BPND, which were non-
invasively obtained by SRTM analysis using the contralateral
side as the reference region. The BPND values in the
ipsilateral sides were 2.03 Æ 0.24 for [11C]MBMP and
1.24 Æ 0.39 (p < 0.05) for (R)[11C]PK11195. The R1 values
instead of the input function were 1.14 Æ 0.05 for [11C]
MBMP and 1.12 Æ 0.08 (p = 0.82) for (R)[11C]PK11195.
The displacement studies are shown in Fig. 4. The
treatment with unlabelled MBMP or PK11195 at 20 min
after injection of [11C]MBMP minimized the heterogeneous
radioactive signals between the ipsilateral and contralateral
sides in PET images, respectively (b, e). When unlabelled
MBMP or PK11195 was injected 20 min after the injection
of [11C]MBMP, as shown in their TACs (c, f), the uptake of
radioactivity in the ipsilateral side was lowered to a level
similar to that in the contralateral side. From 30 min to the
end of the PET scan, there was no difference in the
radioactivity uptake between the ipsilateral and contralateral
Our candidate molecule, MBMP, shows a similar binding
affinity for TSPO (Ki: 0.29 nM) (Fukaya et al. 2012) to other
well-established TSPO ligands of second generation. To
develop the optimal ligand for human clinical application to
be accepted widely, we synthesized [11C]MBMP to demon-
strate that [11C]MBMP binds to TSPO sites in vitro and in
© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 129, 712--720

718
A. K Tiwari et al.
(c)
(a)
(b)
Fig. 4 Coronal positron emission tomography
(PET) images and time-activity curves for 2-[5-(4-
[
11C]methoxyphenyl)-2-oxo-1,3-benzoxazol-3(2H)-
yl]-N-methyl-N-phenylacetamide ([11C]MBMP) with
unlabelled MBMP (1 mg/kg; n = 3) or PK11195
(3 mg/kg; n = 3) were determined in ischemic rat
brains. Arrows indicate ischemic areas. PET
images were generated by summation of the
early phase (0–20 min) before treatment with
unlabelled MBMP (a) or PK11195 (d) and the late
phase (30–60 min) after treatment with unlabelled
MBMP (b) or PK11195 (e). Curves (c, f) were
plotted based on the PET images of the ipsilateral
and contralateral striatum.
(f)
(d)
(e)
vivo, while having improved imaging properties over (R)
[11C]PK11195.
The in vitro autoradiography indicated that [11C]MBMP
bound with TSPO-rich lesions and enhanced radioligand
binding by more than 3.9-fold in the ipsilateral area compared
with the contralateral side (Fig. 2). The contrast between the
ipsilateral and contralateral sides disappeared in the presence
of an excess of MBMP or PK 11195, which demonstrated the
specificity of the in vitro binding with TSPO.
The biodistribution of radioligand is an important charac-
teristic to study because it demonstrates how the radioligand
will distribute in vivo and how it excretes. The present
biodistribution study revealed that the majority of the
radioactivity was distributed in prominent TSPO-expressing
organs (Table 1). Low radioactivity uptake was found in low
TSPO expressing major organs, such as the liver (≤ 3.0%
ID/g). The radioactivity distribution in mouse brains showed
fast penetration across the BBB into all brain regions at
1 min after injection, which was highest in the cerebellum,
the region with relatively high TSPO expression in normal
rodent brain (Zhang et al. 2003; Maeda et al. 2004).
After the injection of [11C]MBMP, only one radiolabelled
metabolite was detected by radio-HPLC in the plasma and
brain homogenates of the mice. As indicated by its tR of
2.9 min, the metabolite was more polar than [11C]MBMP
(tR = 6.6 min). The metabolite in the brain showed the same
tR as the metabolite found in the plasma. This result
confirmed the slow metabolism of [11C]MBMP in the brain
or, in general, a limited level (< 35% at 60 min after
injection) of radiolabelled metabolite originating from the
plasma routed to the brain. Therefore, this result certified that
the brain uptake was mainly because of [11C]MBMP itself.
Because of the low TSPO expression in normal brain, we
used an ischemic rat model in which TSPO expression is
elevated by neuroinflammation with microglial activation.
Our previous study had indicated that the ischemic surgery
did not cause widespread disruption and serious breakdown
of BBB (Yui et al. 2010). In fact, R1, the tracer delivery ratio
between the ipsilateral and contralateral sides, was close to
unity both for [11C]MBMP and (R)[11C]PK11195. This
result suggests that the brain uptake of [11C]MBMP and (R)[
11C]PK11195 determined in this study was mostly because
of specific uptake and minimally affected by a potential
disruption of BBB.
The PET study with [11C]MBMP confirmed that uptake
was significantly higher in the ipsilateral side than in the
contralateral side, which allowed clear visualization of the
lesion site on the PET images (Fig. 3). [11C]MBMP was
completely displaced from the ipsilateral area by an excess of
the corresponding MBMP or PK11195 and, within minutes,
reached the level of uptake in the contralateral area (Fig. 4).
Moreover, [11C]MBMP showed a higher ratio of ipsilateral
to contralateral uptakes and higher BPND than (R)[11C]
PK11195 under the same experimental conditions, suggest-
ing higher in vivo specific binding of [11C]MBMP for TSPO
in the brain.
In the present study, although [11C]MBMP showed higher
BPND than [11C]PK11195 in the same ischemic model, this
new tracer may not have significant advantage over second
generation TSPO radioligands. [11C]MBMP has the similar
in vitro binding affinity for TSPO with those TSPO
radioligands. More importantly, approximately 35% radiola-
belled metabolite of [11C]MBMP was found in the mouse
brain at 60 min after the ligand injection, whereas only less
than 10% radiolabelled metabolite of those TSPO radioli-
gands was in the rodent brains (Zhang et al. 2003, 2004;
Boutin et al. 2007; Van Camp et al. 2009, 2010; Arlicot
et al. 2012). This property is likely to hamper the clinical
application of [11C]MBMP. Therefore, we will not perform
further evaluation for [11C]MBMP, including comparison
with the TSPO radioligands of second generation and
determination for the selectivity of [11C]MBMP for low
and high affinity TSPO binding sites described with those
ligands (Owen et al. 2011).
© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 129, 712--720

[
11C]MBMP for TSPO in brain
719
In conclusion, [11C]MBMP was easily labelled by using
11C with high radiolabelling efficiency and showed favorable
biodistribution pattern, and high in vitro and in vivo specific
binding for TSPO. Optimization for [11C]MBMP is in
progress.
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[
Acknowledgments and conflict of interest
disclosure
Lammertsma A. A. and Hume S. P. (1996) Simplified reference tissue
model for PET receptor studies. Neuroimage 4, 153–158.
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Lavisse S., Guillermier M., Herard A. S. et al. (2012) Reactive
The authors would like to thank the staff at the National Institute of
Radiological Sciences for their support with the cyclotron operation,
radioisotope production, radiosynthesis, and animal experiments.
This study was supported by under NIRS-IAEA program 2013. The
first author is also grateful to Dr R. P. Tripathi, Director, Institute of
Nuclear Medicine and Allied Sciences (INMAS), Delhi for their
support and assistance. All experiments were conducted in compli-
ance with the ARRIVE guidelines. The authors have no conflict of
interest to declare.
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