Comparative evaluation of 99mTc-MBIP-X/11[C] MBMP for visualization of 18 kDa translocator protein

Article abstract of DOI:10.1039/c9nj00180h

An elevated translocator protein (18 kDa, TSPO) density is observed during inflammation in the brain and peripheral organs making it a viable target for imaging. Recently, our group has explored a pharmacophore skeleton acetamidobenzoxazolone for positron emission tomography (PET) and single photon emission computed tomography (SPECT) applications to target TSPO. 2-(2-(5-Bromo/chloro benzoxazolone)acetamide)-3-(1H-indol-3-yl)propionate (MBIP-Br/Cl) were synthesized by using tryptophan methyl ester and compared with 2-[5-(4-methoxyphenyl)-2-oxo-1,3-benzoxazol-3(2H)-yl]-N-methyl-N-phenyl acetamide (MBMP) through tracer techniques. Computational docking showed similar results for MBIP-Br/Cl in comparison to MBMP. Their ex vivo and in vivo biodistributions were assessed in TSPO-rich organs as well as their release kinetics 0-120 min post injection. The ex vivo biodistribution showed a 7 fold higher uptake (5.16%ID per g vs. 0.72%ID per g) in the heart and a 2.5 fold higher uptake (12.91%ID per g vs. 4.69%ID per g) in the lungs for ^99mTc-MBIP-Cl compared to that of ^99mTc-MBIP-Br at 15 min. These findings demonstrated that ^99mTc-MBIP-Cl has improved pharmacokinetic properties compared to ^99mTc-MBIP-Br for SPECT application and is comparable to [¹¹C]MBMP.

Full text of DOI:10.1039/c9nj00180h

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Comparative evaluation of 99mTc-MBIP-X/11[C] MBMP for visualization of 18 kDa
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translocator protein
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Pooja Srivastava1,3, Neelam Kumari1, Dipti Kakkar1,Ankur Kaul1, Pravir Kumar3, Anjani K
Tiwari1,2, *
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1 Division of Cyclotron and Radiopharmaceutical Sciences, Institute of Nuclear Medicine and
Allied Sciences, Brig. S. K. Mazumdar Road, Delhi-110054, India
2Department of Chemistry, School of Physical & Decision Sciences (SPDS), Babasaheb
Bhimrao Ambedkar Central University, Lucknow-226025, UP, India
3 Molecular Neuroscience and Functional Genomics Laboratory, Department of
Biotechnoloy, Delhi Technological University, Delhi 110042, India
Abstract
An elevated translocator protein (18 kDa, TSPO) density is observed during inflammation in
brain and peripheral organs making it a viable target for imaging. Recently, our group has
explored a pharmacophore skeleton acetamidobenzoxazolone for positron emission
tomography (PET) and single photon emission computed tomography (SPECT) applications
to
yl)propionate (MBIP-Br/Cl) was synthesized by using tryptophan methyl ester and compared
with 2-[5-(4-methoxyphenyl)-2-oxo- 1,3-benzoxazol-3 (2H)-yl]-N-methyl-N-phenyl
acetamide (MBMP)
target
TSPO.
2-(2-(5-bromo/chloro
benzoxazolone)acetamide)-3-(1H-indol-3-
through tracer techniques. Computational docking showed similar results for MBIP- Br/Cl in
comparison to MBMP. Their ex-vivo and in-vivo biodistribution were assessed in TSPO-
rich organs and release kinetics 0-120 min post injection. The ex vivo biodistribution showed
a 7 fold higher uptake (5.16%ID/g vs 0.72%ID/g) in heart and 2.5 fold higher uptake
(12.91%ID/g vs 4.69%ID/g) in lung for 99mTc-MBIP-Cl compared to that of 99mTc-MBIP-Br
at 15 min. These findings demonstrated that 99mTc-MBIP-Cl has improved pharmacokinetic
properties than 99mTc-MBIP-Br for SPECT application which is comparable to [11C]MBMP.
Key words: TSPO ligand, Molecular docking, Acetamidobenzoxazolone, TSPO,
SPECT/PET and Inflammation
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Introduction
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The 18 kDa translocator protein (TSPO), also known as peripheral benzodiazepine receptor
(PBR) and a highly conserved protein during evolution, has been studied for its importance in
various life essential functions.1-4 TSPO is mainly found on the outer surface of mitochondria
in central nervous system as well as in peripheral tissues. TSPO is an intracellular protein
which forms a complex with adenine nucleotide translocator (ANT, 30 kDa) and voltage
dependent anion channel (VCAD, 32 kDa) in mitochondria.
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The molecular imaging of neuroinflammation was started by PET studies with isoquinoline
carboxamide [11C]PK11195, the prototype ligand of translocator protein-18 kDa (TSPO),
besides that RoS-4864 and FGIN-127 which are also isoquinoline carboxamide, have been
evaluated. These three ligands have shown compatibility to tetrapyrrole protoporphyrin IX
(PPIX) structure, a well-known endogenous TSPO ligand.5-6
TSPO, a highly hydrophobic five transmembrane domain protein expressed in the outer
mitochondrial membrane has been investigated as biomarker for inflammatory conditions in
brain. lung, liver and kidney etc. Its role has also been studied in many neuro-disorders like
depression, anxiety and about all type of neurodegenerative disorders.7-12 Therefore, high
affinity TSPO probes were prime focus for non-invasive diagnosis of such conditions. 6,13-17
11C(R) PK11195 is the gold standard for TSPO related studies and being most widely studied
ligand from first generation in late eighties. The main limitation of this ligand was high
lipophilicity and low in vivo specific binding which were overcome by second generation
TSPO ligands withr improved imaging. Some of the TSPO ligands used in clinical human
6,12-16
studies are [11C]DAA, [18F]FEDAA, [11C]AC-5216, [11C]PBR28, and [18F]DPA714.
These ligands were able to solve most of the problems from first generation but later showed
problem of inter-subject variability due to single nucleotide polymorphism.17
Recently we have developed a new PET skeleton acetamidobenzoxazolone (ABO) which has
been derived from a ligand from first generation RoS-4864 by opening the diazepine ring and
reorganizing it.18-20
The first ligand which we explored in this category for diagnostic application through
positron emission tomography (PET) was 2-[5-(4-[11C]Methoxyphenyl)-2-oxo-1,3-
benzoxazol-3(2H)-yl]-N-methyl-N-phenylacetamide (11C-MBMP) which showed promising
result. Later two fluoro alkyl derivatives were also synthesized by us naming 2-[5-(4-[18F]
fluoroethoxy/propoxyphenyl)-2-oxo-1,3-benzoxazol-3(2H)-yl]-N-methyl-N-phenylacetamide
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(18F-FEBMP and F-FPBMP). The same skeleton was also analyzed through 99mTc-MBIP
for SPECT.20-25 In this report we are comparing 99mTc-MBIP-Br, 99mTc-MBIP-Cl alongwith
11C-MBMP in terms of ex vivo and in vivo properties in TSPO enriched organs.
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Materials and methods
Chemistry
2.1. Chemicals
All the chemicals and solvents were procured from Sigma-Aldrich and Merck. Procurement
of 99mTc was from Regional Centre for Radiopharmaceuticals, Board of Radiation and
Isotope Technology (BRIT), Department of Atomic Energy, India. Thin layer
chromatography (TLC), column chromatography and instant thin layer chromatography
(ITLC) were carried out during the tracer study. HPLC separation and analysis were
performed using the JASCO and Agilent HPLC system. Effluent radioactivity was monitored
using a NaI (Tl) scintillation detector system, and radioactivity measurement was performed
during synthesis and animal studies with a Curiemeter
2.2. Instrumentation
1
13
The synthesized compound were characterized by spectroscopic techniques like H and C
NMR spectroscopy (Bruker Avance III 600 MHz system) at 600 MHz and 150 MHz,
respectively. Agilent 6310 system mass spectroscopy was used in ESI mode. Siemens
Symbia Camera was used for imaging and γ-scintillation counter for radioactivity counting.
2.3. Animal models
Institutional Animal Ethics Committee (INM/DASQA/IAEC/09/015) has approved the
protocols for studies. BALB/c mice (22-28 g) were used for biodistribution studies and
imaging.
Sprague-Dawley (SD) rats (male, 8−9 weeks old, 240−330 g) were used. The animals were
housed under a 12/12-h dark/ light cycle under optimal conditions.
A lung inflammation model was prepared in balb/c mice by intratracheal delivery of
lipopolysaccharide (LPS) as per our previous work.24 Saturation studies were performed in
these animal models by pre administering 10 mg/kg unlabelled PK11195, 10 min prior to
tracer injection.
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2.4 Synthesis
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R
NH2
OH
O
H2C
CH
NH2
NH
C
O
a
b
CH2
O
R
NH
NH
C
O
CH
C
O
O
NH
O
O
3
1) where R=Cl
2) where R=Br
c
O
O
O
NH
R
N
O
O
HN
4) where R=Cl
5) where R=Br
Scheme 1: Synthesis of 2-(2-(5-bromo/chloro benzoxazolone) acetamido)-3-(1H-indol-3-yl)
propanoate (MBIP-Br/Cl)
Reagents and conditions: a) CDI, THF, Reflux 2 h; b) Chloroacetyl chloride, Et3N,
H2O/DCM, 0oC-rt; c) K2CO3, DMF, 60o-70oC, 3h.
2.4.1 Synthesis of methyl 2-(2-chloroacetamido)-3-(1H-indol-3-yl)propanoate:
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To a solution of L-tryptophan methyl ester (500 mg, 2.32 mmol) and deionizeDdOIw: 10a.1t0e3r9/(C190NJ00180H
ml), triethyl amine (355 µl, 2.55 mmol) was added with constant stirring under nitrogen
atmosphere on ice bath for 5 min. To the mixture, chloroacetyl chloride (203 µl, 2.55 mmol)
in dichloromethane (10 ml) was added drop wise over 1 h and the reaction was carried out for
4 h. Thereafter, the mixture was extracted with dichloromethane (3X). The organic layer was
washed twice by deionized water and brine solution. After washing, the organic layer was
dried over anhydrous sodium sulphate. After filtration, the solvent was removed in vacuo,
and purified by silica gel column chromatography using 100% CHCl3 as eluent (Yield=75%).
1H-NMR (CDCl3, 600 MHz): δ(ppm) 3.3-3.4 (d, 2H), 3.7 (s, 3H), 4.0 (s, 2H), 4.9-5.0 (m,
1H), 7.0–7.6 (m, 5H), 8.5 (br s, 1H)
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13C-NMR (CDCl3, 150 MHz): δ(ppm) 27.5, 42.5, 52.6, 53.3, 109.3-136.3 (8 peaks), 166.0,
171.8
MS (ESI) m/z: 293.1 [M-H+], calculated m/z: 293.1 [M-H+]
2.4.2 Synthesis of 5-Chloro benzoxazol-2(3H)-one
To a solution of 4-chloro-2-aminophenol (5 g, 34.83 mmol) in 150 ml THF, 1,1’-
carbonyldiimidazole (5.65 g, 34.83 mmol) was added. The mixture was refluxed with stirring
for 2 h. The reaction mixture was quenched by adding 2 M HCl solution after cooling down
to room temperature. EtOAc was used for extraction which was washed with brine, dried
over anhydrous sodium sulphate and filtered. Thereafter, solvent was removed in vacuo to get
the product as a white solid (yield= 91%).
1H-NMR (DMSO, 600 MHz): δ (ppm) 7.1-7.4 (m, 3H)
13C-NMR (DMSO, 150 MHz): δ (ppm) 110.3, 111.3, 122.0, 128.2, 132.2, 142.6, 154.7
MS (ESI) m/z: 167.8 [M-H+], Calculated m/z: 168.0 [M-H+]
2.4.3 Synthesis of 2-(2-(5-Chloro benzoxazolone)acetamido)-3-(1H-indol-3-yl)
propanoate (MBIP-Cl)
To a solution of 5-chloro benzoxazol-2(3H)-one (250 mg, 1.48 mmol) and K2CO3 (306 mg,
2.22 mmol) in DMF (5 ml), methyl-2-(2-chloroacetamido)-3-(1H-indol-3-yl)propionate (435
mg, 1.48 mmol) was added with cooling on ice bath. The reaction mixture was stirred at 60-
700C for 3 h. Thereafter, the reaction mixture was cooled to room temperature before adding
water. A mixture of toluene and ethyl acetate, in 1:1 ratio, was used as extraction solvent.
Water and brine solution were used for washing the organic layer and anhydrous sodium
sulphate for drying. Solvent was removed in vacuo after filtration and was purified by silica
gel column chromatography using CHCl3/EtOAc (4:1, v/v) as eluent (yield= 70%).
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1H-NMR (CDCl3, 600 MHz): δ (ppm) 3.5-3.7 (m, 2H), 3.8 (s, 3H), 3.9-4.0 (m, 2H), 5.0-5.1
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(m, 1H), 6.7-7.5 (m, 8H), 8.1 (br s, 1H)
13C-NMR (CDCl3, 150 MHz): 24.1, 51.2, 53.1, 54.3, 110.4- 148.8 (14 peaks), 155.5, 168.2,
168.8
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MS (ESI) m/z: 426.3 [M-H+], Calculated m/z: 426.1 [M-H+]
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2.4.4 Synthesis of 2-(2-(5-bromo benzoxazolone)acetamido)-3-(1H-indol-3-yl)
propanoate (MBIP-Br)
MBIP-Br was synthesized by the procedure mentioned in scheme 1 as per our previous
paper.24
1H-NMR (DMSO, 400 MHz): δ (ppm) 3.1–3.2 (m, 2H), 3.6 (s, 3H), 4.4–4.6 (m, 3H), 6.9–7.5
(m, 8H)
13C-NMR (DMSO, 100 MHz): δ (ppm) 28.7, 44.4, 52.4, 53.9, 111.0– 141.5 (14 C), 154.2,
166.3, 172.3.
MS (ESI) m/z: 470.2 [M-H+], calculated: 470.0 [M-H+]
Scheme 2: Synthesis of [5-(4-Hydroxyphenyl)- 2-oxo-1,3-benzoxazol -3(2H)-yl]-N-
methyl -N-phenylacetamide and Radiosynthesis of [11C ]MBMP
Reagents and conditions: (I) Pd(PPh ) , K CO , 4-(4,4,5,5-tetramethyl-1,3,2- dioxaborolan-
3 4
2
3
2-yl) phenol (when R = OH) / 4-methoxyphenylboronic acid (when R = OCH3) , 1,4-
dioxane:water (v/v -3/1) reflux at 1000C under nitrogen atmosphere for 4 - 4.5 h (II)
[11C]CH I, NaOH at 700C for 5 min in DMF
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2.4.5 Synthesis of 2-[5-(4-Methoxyphenyl)-2-oxo-1,3-benzoxazol-3(2H)-yl]-N-methyl-N-
phenylacetamide (MBMP)
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It was synthesised as per scheme 2. Briefly, a mixture of 2-(5-bromo-2-oxo-1,3-DbOeIn: 1z0o.10x3a9z/Co9lN-J00180H
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3(2H)-yl)-N-methyl-N-phenylacetamide (0.72 g, 2.0 mmol) and 4-methoxyphenylboronic
acid
(365
mg,
2.4
mmol),
K2CO3
(415
mg,
3.0
mmol)
and
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tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4; 230 mg, 0.2 mmol) in 1,4-
dioxane/water (3/1, 4.0 mL) was heated at 100°C under nitrogen atmosphere. The reaction
mixture was stirred for 4 h and cooled to room temperature. Subsequently, the mixture was
extracted with EtOAc. The organic layer was washed with brine and dried over anhydrous
sodium sulphate. After filtration, 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)
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1H NMR (CDCl3, δ) 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).
HRMS (FAB) calcd for C23H21O4N2, 389.4320; found, 389.1511.
2.7 Radiopharmaceutical efficacy of 99mTc-MBIP-Br/Cl:
The compounds were labeled with 99mTc using procedure from literature with slight
modifications. 26-29 Briefly, 100 µL of sterile sodium pertechnetate (approximately ７５–110
MBq of 99mTcO4) was reduced with 50 µL of stannous chloride solution (1x10-2 M in 10%
glacial acetic acid) and pH was adjusted to 6.5 using 0.5 M sodium bicarbonate. A solution of
MBIP-Cl and MBIP-Br were added to this mixture separately and after thorough mixing,
incubated for 15 mins at room temperature (25°C). ITLC-SG strips as stationary phase and
acetone and PAW (pyridine, acetic acid and water in 3:5:1.5 ratio) as mobile phase were used
to determine complexation of MBIP-Cl and MBIP-Br with 99mTc and purity of the labelled
compound.
In vitro human serum stability of the radiolabeled compounds was evaluated using our
previous approach with some modifications.28 Blood collected from healthy volunteers was
clotted in humidified incubator for 1h under 5% CO2 and 95% air and used to prepare serum.
Sample was centrifuged at 400 rpm and filtered through 0.22 µm filter into sterile plastic
culture tubes. 99mTc-MBIP-Cl and 99mTc-MBIP-Br were incubated with freshly prepared
serum at 37oC. Samples were analyzed at different time points using ITLC-SG strips.
Acetone was used as mobile phase for measurement to find out percentage dissociation of the
complex in serum with a function of time.
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Balb/c mice were used for the initial in vivo assessment of 99mTc-MBIP-Cl and 99DmOTI: c10-.M103VB9ie/CIwP9ANr-tJic0le0O18nl0inHe
Br24. Radioactivity uptake was measured in specific organs of the mice at different time
points i.e. 15 min, 30 min, 60 min and 120 min post injection of 100 µCi of both radioligands
via a tail vein and results were expressed as percentage of injected dose per gram (%ID/g) of
tissue (n = 3). Total blood volume was calculated as 7% of the total body weight.
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2.8 Radiolabeling of 2-[5-(4-[11C]Methoxyphenyl)-2-oxo-1,3-benzoxazol-3(2H)-yl]-N-
methyl-N-phenylacetamide ([11C]MBMP
Cyclotron-produced [11C]CO2 was bubbled into 0.4 M LiAlH4 in anhydrous tetrahydrofuran
(THF, 0.3 mL). After the evaporation of THF, the remaining complex was treated with 56%
hydroiodic acid (0.3 mL) to give [11C]CH3I, which was distilled with heating and transferred
under N2 gas flow to a solution of desmethyl MBMP (1 mg) and NaOH (5 L, 0.5 M) in
DMF (0.3 mL) at -15°C. After trapping was completed, this reaction mixture was heated at
80°C for 3 min. HPLC separation was performed on a Capcell Pack UG80 C18 column (10
mm i.d. × 250 mm) using MeCN/H2O/Et3N (6/4/0.01, v/v/v) at 5.0 mL/min.
3.0 Results and Discussion
Encouraged by the outcome of our recently developed TSPO ligands [11C]MBMP and
[18F]FEBMP, [18F]FPBMP and 99mTc-MBIP-Br having ki in nanomolar range20-22, we
directed our efforts towards improvement of acetamidobenzoxazolone(ABO) skelton for
99mTc labeling to target 18 kDa TSPO. The new ligands conjugated to ABO to tryptophan
methyl ester. 99mTc-MBIP-Cl showed fast clearance from the TSPO enriched organs as well
as from circulating blood. The 99mTc-MBIP-Cl exhibited reasonable uptake in heart, lungs,
kidney and spleen which are known to express TSPO.
The two SPECT ligand MBIP-Cl/Br were synthesized by the procedure mentioned in scheme
1 and MBMP and its carbon -11 derivative was synthesized as per our previous work. 22,24
The synthetic of MBIP-Cl/Br was performed by synthesizing two important intermediate
namely,
methyl-2-(2-chloroacetamido)-3-(1H-indol-3-yl)propanoate
and
5-chloro
benzoxazol-2(3H)-one, which were combined in the final step. The synthesized MBIP-Cl had
good yield (70%). The synthetic scheme followed for MBIP-Cl is better than reported
scheme for its bromo analog in terms of yield and duration of overall synthesis.24
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Subsequently MBIP -Br/Cl analogs were radiolabeled with 99mTc and studiDeOsI: 1f0o.1r03t9h/Ce9iNrJ00180H
stability in saline as well as in serum (n=5). The purified compounds MBIP -Br and MBIP -
Cl were radiolabeled with >96% radiochemical yields. Both the radiolabeled compound
showed >94% stability in saline after 24 h indicating stable nature of radiocomlex. In vitro
serum stability clearly indicates the stable nature of radio complexes without any
transchelation to serum protein like albumin which was observed to have >91% and >89%
intactness for 99mTc- MBIP -Br and 99mTc- MBIP -Cl, respectively after 24 h. (Fig. 1)
Stability studies showed requisite stability of the complex to act as a SPECT agent.
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Fig1: Stability profile of 99mTc MBIP-Cl/Br
The blood clearance was studied on normal rabbits of the weight in the range of 2-2.5 kg. 300
μL of the complex having 4.3 MBq activity was administered intravenously through the
dorsal vein of ear. Both the trypophan methyl ester analogs demonstrated a rapid
radioactivity clearance from blood circulation. 99mTc-ABTO-Br and 99mTc-ABTO-Cl activity
clearance were >62% and >66% in 1 h; and >92% and >84% in 3 h. This reflects the fast
release kinetics which is desirable for diagnostic imaging.
Time activity curve (TAC, n=3) has been drawn for 99mTc-MBIP-Br (fig 2) 99mTc-MBIP-Cl
(fig 3) and [11C] MBMP (fig 4). All three showed uptakes in TSPO enriched organs which
was maximum in liver for 99mTc-MBIP-Br, kidney for 99mTc-MBIP-Cl and lungs for [11C]
MBMP.
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All three showed rapid clearance in lungs where initial uptake was high. Blood clDeOaIr:a10n.1c0e3V9iwe/Cwa9ANsrtJic0le0O18nl0inHe
fast in 99mTc-MBIP-Cl and [11C] MBMP. The main difference in SPECT and PET ligand was
uptake in liver which was 28.86 %ID/g and 19.74%ID/g in 99mTc-MBIP-Br 99mTc-MBIP-Cl
while in [11C]MBMP it was just 0.56%ID/g. Maximum quantity of radioactivity in liver
showed that the clearance of the SPECT radioligand is through hepatobiliary route.
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In brain, small uptake was observed which decreased with time point of observation. The
small %ID in brain could be attributed to moderate expression of TSPO in the healthy brain.
Fig2: Time activity curve (TAC) of 99mTc-MBIP-Br
TAC of 99mTc MBIP-Cl
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blood
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liver
spleen
kidney
intestine
brain
Fig3: Time activity curve (TAC) of 99mTc-MBIP-Cl
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TAC of 11[C] MBMP
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Fig4: Time activity curve (TAC) of [11C] MBMP
Comparative ex-vivo biodistribution of 99mTc-MBIP-Br , 99mTc-MBIP-Cl and [11C] MBMP
has been shown in figure 5 which demonstrate that at 30 and 60 min the maximum
radioactivity reaches in lungs, spleen and heart which are supposed to be tspo organs. Only
one unexpected outcome was found for 99mTc-MBIP-Cl in terms of kidney which was
relatedly very high and further investigation in direction may help to get the possible reason
for it.
Fig5: Comparative ex-vivo biodistribution of 99mTc-MBIP-Br , 99mTc-MBIP-Cl and [11C] MBMP
Data are derived from tissues taken from animal tissue (n=3) at different time points after intravenous
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injection via tail vein in each mouse. Radioactivity uptake in different organs were displayed as mean
of % injected dose per gram ±SD
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Further, 99mTc-MBIP-Cl/Br were compared with the ligands reported in literature. As
compared to 99mTc-labeled 2-quinolinecarboxamide, the uptake of 99mTc-MBIP-Cl, was found
to be higher in heart (2.96%ID/g vs negligible uptake) and lung (6.16%ID/g vs 1.19 %ID/g)
and comparable in spleen (2.18%ID/g vs 2.07%ID/g) at 15 min as reported in CD-1 mice.31
Uptake of [123I]-CLINME, an iodine based SPECT ligand, in heart, lungs, and spleen of SD
rat were 3.53%ID/g, 3.53%ID/g, 2.65%ID/g at 15 min. This was lower/comparable to the
uptake of 99mTc-MBIP-Cl/Br in respective organs30. The liver uptake was lowest for 99mTc-
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MBIP-Cl in comparison with 99mTc-labeled 2-quinolinecarboxamide and 99mTc-MBIP-Br24, 31
This accounts for better image visualization (target organ to liver ratio) for 99mTc-MBIP-Cl.
.
In addition, the release kinetic rates between 15-60 min for 18F-FEBMP20, 11C-MBMP22, 18F-
2-(5-(6-Fluoropyridin-3-yl)-2-oxobenzo[d]oxazol-3(2H)-yl)-N-methyl-N-phenylacetamide32
and 99mTc-MBIP-Cl are 0.175%ID/g/min, 0.150%ID/g/min, 0.070%ID/g/min and
0.085%ID/g/min in heart, this ratio became more comparable as 0.382%ID/g/min,
0.375%ID/g/min, 0.155%ID/g/min and 0.231%ID/g/min in lungs. In liver and kidney, release
kinetic rates were approximately 3-32 folds faster in 99mTc-MBIP-Cl which makes it
interesting for further evaluation. In vivo specificity of MBIP-Cl was analyzed by saturation
study by PK11195 in inflammation models. SPECT images 40 min post injection in normal
and inflamed mice confirms uptake in tspo enriched organs (Fig. 6 and 7).
a(i)
a(ii)
b(i)
b(ii)
Fig 6: In vivo SPECT imaging of 99mTc-MBIP-Cl a(i) without and a(ii) with PK11195
preadministration and 99mTc- MBIP-Cl b(i) without and b(ii) with PK11195 preadminstration
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at 40 min post injection of radioligand
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(A to H are the points of higher uptake of radioactivity taken as randomly by operator)
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(a)
(b)
Fig. 7: In vivo SPECT image showing activity distribution of 99mTc-MBIP-Cl in a) normal
and b) inflamed mouse without and with blocking at 40 min post injection
As per comparative evaluation all the three ligands 99mTc-MBIP-Br, 99mTc-MBIP-Cl and
[11C] MBMP were found suitable for TSPO targeting. In SPECT ligands 99mTc-MBIP-Cl was
found to be better than 99mTc-MBIP-Br in terms of in vivo uptake of the compound in tissues
expressing TSPO but has a limiting factor of sustained activity in kidney. Further
Investigation in other specific models may give a warrant for human clinical studies.
Conclusion
The cost of production of these synthetic TSPO ligand and easy chemistry makes them
potential candidates for further exploration as imaging agents. Though few models have been
used for validation, other TSPO expressing pathological conditions can also be used for
validation and quantification. These skeletons can also be modified for other metal-based
PET and SPECT agents.
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Acknowledgement:
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We extend special thanks to Director INMAS and Dr A. K. Mishra, Head, Division of
Cyclotron and Radiopharmaceutical Science, INMAS, Delhi, for providing us the facility to
carry out experiments. Besides that we also thank Dr Sonia Gandhi, and Mr Sunil Pal,
INMAS for their technical support. This work was supported by INMAS, Delhi under Task
Project ST/15-16/INM-03.
There is no conflict of interest among authors of this manuscript.
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Cover Figure:
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