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Vol. 30, No. 1 (2018)
A Novel F Labelled Imidazo-oxazolopyridine Derivative as β-Amyloid Imaging Agent 185
chromatography (DCM/MeOH) yielding 0.084 g (92 %) of an
orange product. δ (400 MHz; DMSO; Me Si): 9.53 (1H, s, CH),
.53 (1H, s, CH), 8.50 (1H, d, J = 4.4 Hz, CH), 8.20 (1H, d, J =
.4 Hz, CH), 7.97 (1H, q, J = 1.6 Hz, CH), 7.91 (2H, d, J = 8.4
Radiometabolite analysis: The SD rats were intravenously
18
H
4
injected with [ F]FPIPOP (7.5 MBq per rat) through the tail
vein. The animals were sacrificed by cardiac puncture at 5,
15, 30 and 60 min (n = 3 for each point). Blood (0.8-1.0 mL)
and the entire brain samples were obtained and treated as
reported previously. The supernatant of the brain homogenate
and plasma was analyzed under the following conditions: Capcell
8
8
Hz, CH), 7.74 (1H, d, J = 10 Hz, CH), 7.42 (1H, J = 3.2 Hz,
1
H), 7.19 (2H, d, J = 8.4 Hz, CH); δ
C
(100 MHz; DMSO; Me
4
Si):
163.75 (CH), 130.62 (2CH), 129.06 (CH), 128.33 (C), 128.25
(
1
C), 123.37 (CH), 121.33 (CH), 120.37 (2CH), 119.48 (CH),
Pack UG80 C18 column, 4.6 mm i.d. × 250 mm; MeCN-H
2
O-
+
17.62 (CH); ESI-MS(+): m/z [M+H ] calc (found): 331 (331).
Et N, 6/4/0.01(v/v/v); flow rate, 1.0 mL/min. the percentages
3
1
8
18
Radiosynthesis of 2-[2-(4- F-fluorophenyl)imidazo[1,2-
of the intact [ F]FPIPOP were calculated, simultaneously, the
radioactivity fractions in the HPLC waste solution were measured
using a 1480 Wizard 3" auto-gamma counter.
1
α]pyridine-6-yl]oxazolo[4,5-b]pyridine ([ F]FPIPOP])
8
18
18
18
18
[
F]4: [ O]H
2
O (95 %) was used for F production. [ F]HF
was extracted and separated from the cyclotron target by elution
PET study and image analysis: A small-animal PET
scanner from Siemens Medical Solutions was used for imaging.
Normal SD rats were anaesthetized during the scan and the
animals’body temperature was maintained at 40 °C using water
circulation system (T/Pump TP401, Gaymar Industries). The
emission scans were acquired at different time intervals post
injection of [ F]FPIPOP (5.18 ± 0.29 MBq/0.02-0.04 nmol)
through the tail vein. All image frames were summed at 0-30
min. The time activity curves (TACs) for the striatum, hippo-
campus and cerebellum were generated from the PET data
in order to parameterize the radioactivity uptake, clearance
and distribution in healthy rat. The radioactivity (% ID/g) was
estimated as the ratio of the regional activity concentration
normalized by the injected dose {SUV = [(MBq/g of tissue)/
(MBq injected/patient body in g)]} and the weight of the animal
to give the PET-generated biodistribution pattern over different
parts of brain.
18
18
2
with [ O]H O, respectively. H[ F]F was concentrated on a short
QMA column followed by elution with 400 µL of a solution
®
with aqueous K
MeCN (8 mL). Aqueous K[ F]F was transferred to a reaction
vial and evaporated to remove H O and MeCN at 110 °C for
5 min. after 3 (2 mg) in 1,2-dichlorobenzene (150 µL) was
2
CO
3
(10 mg/8 mL), Kryptofix 222 (30 mg) in
18
2
18
1
-1
added to the vial, the reaction gas (10 mL min ) for 2 min and
trapped in a solution of DMSO (300 µL).After the radioactivity
plateaued, the reaction mixture was heated at 120 °C for 15
min. HPLC purification was carried out using a mobile phase
of MeCN-H
2
O-Et N (6.0/4.0/0.01, v/v/v) and flow rate was
3
-1
maintained at 5.0 mL min . Desired radiolabelled product was
collected in a sterile flask followed by evaporated to dryness
in vacuo. Further, redissolved in 3 mL of sterile normal saline
and passed through a 0.22 µm Millipore filter. The retention
18
time of [ F]FPIPOP] was 9.7 min for purification and 9.6 min
for analysis on HPLC [30].
In vitro binding assay withAβ42 and tau aggregates: The
18
Determination of lipophilicity: The experimental determi-
K
i
and K values of [ F]FPIPOP binding to the tau aggregates
d
18
nation of partition coefficients of [ F]FPIPOP and was performed
in 1-octanol and 0.02 M phosphate buffer at a pH of 7.4. Both
the phases were pre-saturated with each other. 1-Octanol (3
mL) and phosphate buffer (3 mL) were taken into a test tube
and syntheticAβ42 aggregates purchased from SigmaAldrich
were determined utilizing methods previously reported [31].
The values of K and Bmax were determined using GraphPad
d
Prism 7 XML software.
18
containing 0.38 MBq of [ F]FPIPOP. The test tube was vortexed
for 10 s and centrifuged (5 min, 4000 rpm). Aliquots of 500
µL from the 1-octanol and buffer phases were transferred into
a new test tube. 1-Octanol (2 mL) and phosphate buffer (3 mL)
were taken into the same test tube. The centrifuging, vortexing
and counting were repeated. The amount of radioactivity was
measured in each tube with a γ counter and corrected for decay.
The partition coefficient was calculated using eqn. 1: (counts/
µL in 1-octanol)/(counts/µL in buffer) = r.
Animals: Sprague-Dawley (SD) rats (male, 8-9 weeks old,
240-330 g) were taken for studies. Animals were kept under
optimal conditions with a 12/12 h dark/light cycle and animals
were handled as per recommendations of ethical committee.
RESULTS AND DISCUSSION
Computational analysis of FPIPOP: In order to design
and synthesize Aβ plaques imaging agent with optimum lipo-
philicity, good binding affinity and higher specificity, we began
with docking study (Schrödinger Maestro 9.4) of newly designed
ligand with the 3D structure ofAβ42 was obtained from protein
data bank (PDB: 2BEG) [25].After the optimization of model,
as the exact binding site was unknown, we zeroed in on three
major binding sites (Fig. 2). Two of them were within the fibril
whereas one was on the surface of the two sites of Met35.
Site 1 is constituted by terminal residues (Leu17,Val18, Phe19,
Gly38 andVal40) and is in the proximity of the solution environ-
ment, which is favourable for ligand access in this peptide. At
this site, the ligand showed hydrophobic interactions. Solvent
interactions are also exhibited by the backbone of FPIPOP.
Site 2 is deeply buried inside the fibril and thus has a highly
hydrophobic environment. It is constituted by side chains of
Phe19, Ala21 and Val36 and the backbone atoms of Phe20
All assays were performed in triplicate.
Ex vivo biodistribution study: Biodistribution study was
18
carried out in the SD rat. [ F]FPIPOP (1.85 MBq, 10 pmol)
was injected through the tail vein and animals were sacrificed
at six time intervals (2, 5, 15, 30 and 60 min). Blood samples
were collected at each time point from each group (n = 4) and
the adrenal gland, brain, heart, kidneys, liver, lungs, spleen,
small intestine and testis were quickly removed and weighed.
Radioactivity counts in each tissue/organ, at each time point
was done by a 1480 Wizard 3" autogamma counter (Perkin
Elmer, Waltham, MA, USA). It is expressed as the percentage
of the injected dose per gram of the wet tissue (% ID/g). The
decay correction was taken into consideration during the radio-
activity counts.