ZCDD083: A PFKFB3-Targeted PET Tracer for Atherosclerotic Plaque Imaging

Article abstract of DOI:10.1021/acsmedchemlett.9b00677

PFKFB3, a glycolysis-related enzyme upregulated in inflammatory conditions and angiogenesis, is an emerging target for diagnosis and therapy of atherosclerosis. The fluorinated phenoxindazole [18F]ZCDD083 was synthesized, radiolabeled in 17 ± 5percent rad

Full text of DOI:10.1021/acsmedchemlett.9b00677

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Letter
[18F]ZCDD083: a PFKFB3-targeted PET
tracer for atherosclerotic plaque imaging
Carlo De Dominicis, Paola Perrotta, Sergio Dall'Angelo, Leonie
Wyffels, Steven Staelens, G. R. Y. de Meyer, and Matteo Zanda
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00677 • Publication Date (Web): 19 Feb 2020
Downloaded from pubs.acs.org on February 20, 2020
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[18F]ZCDD083: a PFKFB3-targeted PET tracer for atherosclerotic
plaque imaging
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Carlo De Dominicis,1 Paola Perrotta,2 Sergio Dall’Angelo,1 Leonie Wyffels,3 Steven Staelens,3,*
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G.R.Y. De Meyer,2,* Matteo Zanda,1,4,†,*
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1Kosterlitz Centre for Therapeutics, University of Aberdeen, AB25 2ZD Foresterhill, Aberdeen (UK)
2Laboratory of Physiopharmacology, University of Antwerp (Belgium)
3Molecular Imaging Center Antwerp, University of Antwerp (Belgium)
4CNR-ICRM, via Mancinelli 7, 20131 Milan (Italy)
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ABSTRACT: PFKFB3, a glycolysis-related enzyme upregulated in inflammatory conditions and angiogenesis, is an emerging
target for diagnosis and therapy of atherosclerosis. The fluorinated phenoxindazole [18F]ZCDD083 was synthesised,
radiolabelled in 17 ±5% radiochemical yield and >99% radiochemical purity, and formulated for pre-clinical PET/CT imaging
in mice. In vivo stability analysis showed no significant metabolite formation. Biodistribution studies showed high blood pool
activity and slow hepatobiliary clearance. Significant activity was detected in the lung 2 h post-injection (pi) (11.0 ±1.5
%ID/g), while at 6 h pi no pulmonary background was observed. Ex vivo autoradiography at 6 h pi showed significant high
uptake of [18F]ZCDD083 in the arch region and brachiocephalic artery of atherosclerotic mice, and no uptake in control mice,
matching plaques distribution seen by lipid staining along with PFKFB3 expression seen by immunofluorescent staining. In
vivo PET scans showed higher aortic region uptake of [18F]ZCDD083 in atherosclerotic ApoE-/-Fbn1C1039G+/- than in control
mice (0.78 ± 0.05 vs 0.44 ± 0.09 %ID/g). [18F]ZCDD083 was detected in aortic arch and brachiocephalic artery of ApoE-/-
(with moderate atherosclerosis) and ApoE-/-Fbn1C1039G+/- (with severe, advanced atherosclerosis) mice, suggesting this tracer
may be useful for the non-invasive detection of atherosclerotic plaques in vivo.
KEYWORDS: Atherosclerosis, Plaque, Fluorine, PET
imaging, glycolysis, preclinical.
hypoxic conditions.8,9 Several studies have shown
a
correlation between PFKFB3 and angiogenesis, which is a
feature of vulnerable atherosclerotic plaques.10,11,12,13 Here
we describe the development of the 18F-radiolabelled
INTRODUCTION
Atherosclerosis is a pathological process characterised by
progressive accumulation of lipids, inflammatory cells and
connective tissue in the arterial wall.1 This process, leading
to the formation of the so-called “atherosclerotic plaques”,
is predominantly asymptomatic. However, the progression
of the disease can cause progressive stenosis of arterial
lumen, which can eventually result in ischaemic heart pain.
Acute clinical events, such as myocardial infarction, stroke
and unstable angina, are mainly associated with instability
of plaques and formation of occlusive thrombi.2
The progression of atherosclerotic plaques is initially
asymptomatic, characterised by the slow growth of “silent”
stable plaques. However, plaque rupture is associated with
unstable angina, acute myocardial infarction, and sudden
cardiac death. Similarly, rupture of carotid artery plaque is
associated with cerebral ischemic events.3 The risk of
thrombotic complications of atherosclerosis is mostly
related to the instability of an atheroma rather than the size
of the plaque.
Currently, cardiovascular medicine aims principally to
identify atherosclerotic plaques prone to rupture, improve
the risk stratification, monitor the disease progression,
assess new anti-atherosclerotic therapies and promptly
evaluate the efficacy of therapeutic treatment. PET imaging
is non-invasive and has a great potential in this context, but
there is a clear need for new targets and radiotracers for
imaging unstable atherosclerotic plaque.4,5
phenoxindazole compound ZCDD083 as a PFKFB3-
targeted PET radiotracer for imaging the atherosclerotic
plaque in vivo.14 ZCDD083 is a close structural mimic of the
potent inhibitor AZ68 (IC50= 4nM), whose radiofluorination
was deemed to be chemically viable and unlikely to affect
the binding to PFKFB3.
RESULTS
Phenol intermediate 2 (Scheme 1) was synthesised from
N-Boc-proline and O-Bn-4-aminophenol, which were
coupled to afford compound 1 that was debenzylated by
hydrogenation over Pd/C.
Boc
O
N
HATU,
DIPEA
H
N
H
N
OH
H2
OH
(83%)
OBn
N
Boc
O
N
Boc
O
Pd/C
DMA
2
1
(88%)
H2N
OBn
Scheme 1. Synthesis of phenol intermediate 2.
Indazole 4 (Scheme 2) was synthesised by NIS-promoted
iodination of (2-amino)phenyl-acetonitrile, followed by
indazole ring formation via Sandmeyer reaction and
intramolecular ring closure of the diazonium salt formed
from 3.
The PFKFB3 enzyme is emerging as a new target for the
in vivo assessment of atherosclerotic plaques.6,7 PFKFB3 is a
glycolysis-related enzyme upregulated in inflammatory and
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N
N
N
N
O
1
2
3
4
5
O
O
N
I
K222
/
18F-
O
O
N
N
N
N
I
N
N
1) HCl, NaNO2
2) NH4OH
O
ACN
CN
I
H
H
CN
NH2
N
N
°
100
5 min.
Boc
Boc
NH2
(89%)
N
TsO
18F
7
10
H
3
4 (54%)
6
7
Scheme 2. Synthesis of indazole 4.
90 °C
1 N HCl
10 min.
8
9
The radiofluorination precursor 7 (Scheme 3) was
synthesized next. Commercial 2-methyl-1,3-propanediol
was treated with tosyl chloride to give the mono tosylated
derivative 5, which was reacted with the indazole 4 via
Mitsunobu reaction to give compound 6. Ullmann-type
coupling with phenol 2 afforded the target tosylated
precursor 7.
N
N
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O
O
N
N
H
NH
18F
[
18F]ZCDD083
N
Scheme 5. Radiosynthesis of [18F]ZCDD083.
[18F]ZCDD083 was obtained with
I
OH OH
p-TsCl
OH OTs
4
N
N
a
17 ±5%
Py, DCM
DIAD, PPh3
radiochemical yield (decay-corrected to EOB) and the
activity was in the range of 4.7-7.2 GBq (n = 7). The average
time taken for synthesis, purification and formulation was
65 minutes. [18F]ZCDD083 was obtained with
radiochemical purity >99%, specific activity >130
GBq/μmol, no impurities were detected in the UV
chromatogram (Fig. 1) and no residual solvents other than
ethanol (<10%) were detected (see Fig. S4, Supporting
Information).
TsO
5
(73%)
6 (59%)
N
N
O
2
O
N
N
H
CuI
N
CyDMEDA
K2CO3
Boc
TsO
7
(25%)
Scheme 3. Synthesis of radiofluorination precursor 7.
The ‘cold’ (e.g. non-radioactive) tracer ZCDD083
(Scheme 4) was prepared from the indazole tosylate 6,
which was treated with TBAF to give the fluorinated
compound 8. The latter was subjected to the Ullmann
coupling with 2 affording 9, which was N-Boc-deprotected
to afford the target molecule.
CN
CN
N
I
Figure 1. Chemical and radiochemical purity of
[18F]ZCDD083 assessed by analytical RP-HPLC. No impurities
were detected in the UV (top) and radioactive (bottom)
chromatograms. Column: Phenomenex Luna C18 column (5
µm, 100 Å, 250 × 4.6 mm). Mobile phase: solvent A = H2O +
0.1% TFA, solvent B = CH3CN + 0.1% TFA; Isocratic method
40% B.
I
2
TBAF
N
N
N
CuI
CyDMEDA
K2CO3
TsO
F
6
8
(70%)
CN
CN
N
O
O
O
O
N
N
HCl/dioxane
N
N
N
H
H
N
A preliminary metabolite study was performed in healthy
C57BL/6J mice (6 weeks old, female) to evaluate the in vivo
plasma stability of [18F]ZCDD083. The radio-HPLC analysis
showed no significant metabolite formation within 6 h. The
eluate from HPLC was collected in fractions and the
radioactivity in each fraction was measured in a γ-counter.
At 6 h pi, 78.9% ±1.2 of [18F]ZCDD083 was intact (see Table
S1 and Fig. S5, Supporting Information).
NH
Boc
F
F
ZCDD083 (89%)
9
(45%)
Scheme 4. Synthesis of ‘cold’ ZCDD083.
Starting from the tosylated precursor 7, [18F]ZCDD083
was radiosynthesised in a two-step synthesis (Scheme 5)
using standard 18F-fluoride chemistry, producing first the
protected tracer 10 which was then treated with HCl in
order to remove the N-Boc-protection.
Ex vivo biodistribution of [18F]ZCDD083 was initially
studied in healthy female C57BL/6J mice (Table S2, Supp
Information). Measurement of radioactivity by γ-counter
showed a high overall accumulation of [18F]ZCDD083 in
tissues and organs within 2 h pi, as shown in Fig. 2. More
specifically, high blood radioactivity was observed (7.2 ±2.2
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%ID/g at 15 min pi, 5.5±1.6 %ID/g at 1 h pi, and 2.1±0.4
%ID/g at pi), revealing slow clearance of
Ex vivo biodistribution evaluation at 6 h pi was carried out
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2
h
a
also in atherosclerotic ApoE-/- mice (Table S3, Supp
Information). A similar [18F]ZCDD083 biodistribution
profile was observed in atherosclerotic ApoE-/- and control
C57BL/6J mice. Pharmacokinetic studies in ApoE-/-
atherosclerotic mice confirmed the plasma half-life of
[18F]ZCDD083 (1.4 ±0.2 %ID/g in blood). Residual high
radioactivity accumulation was observed in lungs (8.6 ± 1.9
%ID/g), whereas the maximal uptake was found in small
and large intestine (40.6 ± 45.8 %ID/g and 35.6 ± 2.3 %ID/g
respectively).
[18F]ZCDD083 from blood pool. Also, high pulmonary
uptake was found (34.7 ± 6.0 %ID/g at 15 min) with a slow
washout from lungs (11.0 ± 1.5 %ID/g at 2 h. On the
contrary, at 6 h pi the radioactivity in lungs and blood was
significantly lower (5.5±3.9 %ID/g and 1.5±1.1 %ID/g
respectively). Instead, at the late time point [18F]ZCDD083
had mostly accumulated in the excretory organs (i.e. 25.5 ±
3.8 %ID/g in small intestine and 47.9 ± 6.4 %ID/g in large
intestine). Level of uptake in the liver and the intestines
were significantly higher compared to the kidneys.
Therefore, it can be concluded that [18F]ZCDD083 is
predominantly cleared via the hepatobiliary system into the
intestines and that the renal clearance is less significant.
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15 min
30 min
1 h
60
40
20
2 h
6 h
0
t
s
s
s
er
ine
st
Fat
Skin
Liver
Bone
Brain
Urine
Hear
Blood
Lung
te
testine
Spleen
Muscle
Bladd
Kidney
Stomach
Pancrea
Small In
Large In
Figure 2. Ex vivo biodistribution of [18F]ZCDD083. Slow blood clearance and high tracer uptake in the lungs were observed at
earlier time points within 2 h pi. At 6 h pi [18F]ZCDD083 was mostly accumulated in the intestines.
[18F]ZCDD083 PET imaging scans were performed using
two different disease models (ApoE-/-, with moderate
atherosclerosis, and ApoE−/−Fbn1C1039G+/−
, with severe
advanced atherosclerosis, both female) and aged-matched
wild type C57BL/6J female mice as controls. Representative
PET/CT scans of ApoE-/-Fbn1C1039G+/- mice are shown in
Fig. 3. Spherical ROIs were drawn manually over each aortic
arch and ascending aorta using the axial view of CT images.
The [18F]ZCDD083 signal was then quantified as SUVmean
(mean Standardized Uptake Values). Tracer uptake was
different in ApoE-/-Fbn1C1039G+/- (SUV 0.78 ± 0.05), ApoE-/-
(SUV 0.55 ± 0.06) and C57BL/6J mice (SUV 0.44 ± 0.09),
with significant difference between first and latter series, as
shown in Fig. 4.
Figure 3. Representative axial, sagittal and coronal views of
PET/CT images of ApoE-/-Fbn1C1039G+/-acquired at 6 h
after injection of [18F]ZCDD083. Radioactive signal in aorta
(white arrows) was observed.
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(***p = 0.0006, one-way ANOVA followed by Brown-Forsythe
test).
1.0
0.8
0.6
0.4
0.2
0.0
***
1
2
3
4
5
6
7
8
C57BL/6J
ApoE-/-
ApoE-/-Fbn1C1039G+/-
C57BL/6J
ApoE-/-
ApoE-/-Fbn1C1039G+/-
n=2-6; *** p<0.05
Figure 4. Comparison of SUV measurements in C57BL/6J (n
= 6), ApoE-/- (n = 6) and ApoE−/−Fbn1C1039G+/− mice (n =
2). All values are given as mean ± SD. One-way ANOVA was
performed to test for difference between groups. One
ApoE−/−Fbn1C1039G+/− mouse was excluded from the
analysis because of a PET image acquisition technicality.
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Figure 5. [18F]ZCDD083 aortic uptake vs plaque distribution. Ex vivo autoradiography (left part of the insets) and fat staining
using Oil-red O (right part of the insets) of longitudinally opened aortas at 6h post injection of [18F]ZCDD083 in C57BL/6J control
mice (left) compared to atherosclerotic mice (ApoE-/- and ApoE-/-Fbn1C1039G+/-, centre and right respectively, as indicated by the
white arrows. [18F]ZCDD083 uptake colocalises with atherosclerotic plaques. Scale bar = 1 cm.
At the end of the PET imaging scans, three mice of
C57BL/6J, ApoE-/- and ApoE−/−Fbn1C1039G+/− were sacrificed
and the aorta was harvested for Red Oil O staining and
autoradiography (ARG). The ex vivo distribution of
atherosclerotic plaques within the aorta was assessed by
the tracer accumulation within the aorta matched exactly
with the distribution of atherosclerotic lesions. More
specifically, the radioactivity was accumulated mostly in the
aortic arch and in the branches. In these regions,
[18F]ZCDD083 uptake was significantly higher in ApoE−/−
(0.022 ± 0.004 %ID/µL) than control mice (0.011 ± 0.003
%ID/µL) (p = 0.0378, unpaired t test).
Red Oil
O staining, whereas the accumulation of
[18F]ZCDD083 was assessed by autoradiography of the in
vivo injected [18F]ZCDD083. This aforementioned Red Oil O
staining and ex vivo autoradiography were performed on
the same aortic specimens in order to visually compare fat-
stained areas and tracer distribution within the aorta (Fig.
5).
The correlation between plaques and tracer uptake was
also consistent using ApoE−/−Fbn1C1039G+/− mice with higher
plaque load than the ApoE−/− strain.27 The comparison of
corresponding en face lipid staining and ARG signals
demonstrated a high correlation of fat-stained areas and
radioactivity distribution. ApoE−/−Fbn1C1039G+/− mice
showed more extended atherosclerotic lesions in the arch
region, brachiocephalic artery along with thoracic
descending aorta. [18F]ZCDD083 was, indeed, detected in
the same areas along the aorta.
Aortas from C57BL/6J control mice did not show focal
lipid stained areas and radioactivity signal. Conversely,
atherosclerotic plaques were found in ApoE-/- mice,
especially in the aortic arch, proximal aorta and
brachiocephalic aorta. The Oil Red O staining showed also a
low plaque load in the thoracic descending aorta of the
ApoE−/− strain. The ARG of the same specimen showed that
Aortic specimens of C57BL/6J and ApoE-/- (n = 3/ each
group) were used for further histological analysis after PET
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imaging scans. Longitudinal sections of aortic arch and right ultrasound (IVUS), optical coherence tomography (OCT)
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common carotid artery as well as cross sections of
brachiocephalic aorta and proximal aorta were stained with
haematoxylin and eosin (H&E) to visualise atherosclerotic
plaques and lesion morphology. Aortas of healthy mice did
not show atherosclerosis, whereas ApoE-/- mice showed
and near-infrared spectroscopy (NIRS), can provide only a
limited plaque characterisation but, unfortunately, they are
invasive and thus not useful for patient diagnosis and follow
up.16,17,18 Therefore, new non-invasive approaches to detect
potentially unstable plaque are urgently needed.
fibrous/atheromatous
plaques
and
some
small
In this context, PET imaging has great potential, since -
compared to other molecular imaging modalities - it shows
higher sensitivity allowing better visualisation of biological
and biochemical processes involved in the development of
atherosclerotic plaques.19 [18F]FDG (inflammation,
approved for clinical use) and [18F]NaF (micro-calcification)
have been investigated in a clinical setting for detection and
characterisation of atherosclerotic plaques in the major
coronary arteries.19,20 However, new targets and
radiotracers are currently being investigated preclinically
for imaging atherosclerotic plaque.21 Clinical studies
showed that 18F-FDG is useful to assess carotid artery
stenosis in asymptomatic patients.22,23 However, carotid
artery imaging using 18F-FDG PET is challenging due to (1)
the low spatial resolution of PET (≈ 3 mm in human PET and
1.2 mm in rodent PET, using our setting), (2) cardiac motion
and (3) myocardial spill over.24 To address the latter
limitation, new imaging targets and radiotracers having
lower unspecific myocardial uptake are currently under
investigation.25
calcifications. The H&E staining confirmed atherosclerotic
disease especially in brachiocephalic artery, proximal aorta
and aortic arch. The immunohistochemistry of ApoE-/-
aortic arch sections, revealed that the enzyme target
PFKFB3 is overexpressed inside atherosclerotic plaques
(see Fig. S6, Supporting Information).
The comparison among brachiocephalic arteries of
atherosclerotic and control mice showed that PFKFB3 was
highly expressed within atherosclerotic plaques, whereas in
normal vessels PFKFB3 is present just in a basal level along
the arterial wall (Fig. 6).
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The results above validate the PFKFB3 enzyme as an
imaging target of atherosclerosis in two mouse models.26,27
Firstly, we observed – via PET/ARG – an increased target
engagement in cross sections of diseased aortas of
atherosclerotic mice compared to those of healthy mice.
Secondly, longitudinal sections of aortic arch specimens of
ApoE-/- mice showed higher expression level of PFKFB3
enzyme within atherosclerotic plaques compared to normal
vessel wall. Hence, all these results demonstrated the
appropriateness and the specificity of the PFKFB3 target for
the detection of atherosclerotic plaques.
The new PFKFB3-targeted tracer, [18F]ZCDD083 could be
obtained in high radiochemical yields, activity
concentrations and quality parameters. [18F]ZCDD083
demonstrated high in vivo metabolic stability and long
circulation time in the blood. The slow clearance from the
blood may be explained by the high hydrophobicity of
[18F]ZCDD083. Indeed, a LogD7.4 of 3.6 was reported for the
parent compound AZ68.14 The correlation of high
hydrophobicity with high binding to plasma proteins and
prolonged circulation time in the blood is well known.28
High binding to plasma proteins is documented for the
compound AZ2614 (chemically similar to AZ68) and might
be valid also for our tracer [18F]ZCDD083. This may likely
explain the slow tracer clearance. Also, the excretion route
is usually related to lipophilicity, as lipophilic compounds
are generally cleared via the hepatobiliary system. This is in
accordance with our ex vivo biodistribution results which
showed a predominant excretion from the liver to the
intestines. We also noticed a high uptake in the lung.
However, it is not clear whether this high uptake in the lung
is due to nonspecific binding (due to the high [18F]ZCDD083
lipophilicity) or somehow correlated with PFKFB3 tissue
distribution. In 2003, Minchenko et al. studied the in vivo
expression of the PFKFB enzyme family and its response to
hypoxia, disclosing that lungs exhibit high basal level of
Figure 6. Immunohistochemistry of murine brachiocephalic
arteries. Upper panels = immunofluorescent staining for von
Willebrand factor to detect endothelial cells (green) and
PFKFB3 (red). Counter-staining with DAPI to detect nuclei
(blue). Lower panels = PFKFB3 staining (red). ApoE-/- mice
exhibit high expression level of PFKFB3 enzyme in contrast to
C57BL/6J controls. Scale bar = 50 µm.
DISCUSSION
Atherosclerosis is responsible for the majority of acute
cardiovascular
events.
Specifically,
rupture
of
atherosclerotic plaques and consequent thrombosis
remains the largest underlying cause of cardio- and
cerebrovascular death. Despite extraordinary advances in
the understanding of the pathophysiology of
atherosclerosis, both the diagnosis and treatment of the
disease still present limitations. Currently, the main
challenge in cardiovascular medicine is to identify patients
who are at risk of coronary plaque rupture and subsequent
heart attack or stroke originating from carotid plaque
depositions.15 Among the cardiovascular imaging
modalities used, coronary angiography has a wide clinical
application but provides information only on vessel
stenosis. Other imaging techniques, such as intravascular
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autoradiography and histology. Copies of the 1H, 19F and 13C
NMR spectra of all the new compounds.
PFKFB3 mRNA.29 Furthermore, it was recently shown that
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4
lung endothelial cells express high levels of glucose
metabolic enzymes, such as PFKFB3.30 Finally, the proteome
analysis reported in the Human Protein Atlas shows that
lung, stomach and placenta have the highest PFKFB3
expression among all tissues considered.31
The Supporting Information is available free of charge on the
ACS Publications website.
5
Our PET imaging studies showed significantly increased
signals in the aorta of ApoE-/- (+25%) and
ApoE−/−Fbn1C1039G+/− (+79%) compared to C57BL/6J mice.
This is in accordance with the higher plaque burden in
ApoE−/−Fbn1C1039G+/− mice compared to ApoE-/- mice.27
Moreover, these results were supported by aortic
autoradiography which showed a 2-fold increased uptake in
the aortic arch of ApoE-/- compared to WT mice. The
autoradiographic signal in ApoE−/−Fbn1C1039G+/− was
considerably higher too. The increment in uptake is clearly
correlated with the increment in plaque load, as confirmed
by histological evaluation. Indeed, the highest uptake was
found in ApoE−/−Fbn1C1039G+/− at an advanced stage of the
disease and high level of plaques in the aortic arch along
with the thoracic aorta.
Unfortunately, despite the higher uptake, the
[18F]ZCDD083 signal in these small sized atherosclerotic
plaques was affected by partial volume effect and, due to the
limited spatial resolution of PET,32 no “hotspots” could be
detected in the in vivo imaging studies. Indeed, the vessel
wall in mouse aorta is usually 30-80 µm, whereas the
resolution in rodent PET imaging is ~1,2 mm.
The specificity of the tracer accumulation at the target
structure (i.e. atherosclerotic plaque) was demonstrated by
the combination of ex vivo autoradiography with en face Oil
Red O staining of the same aortic specimens. Indeed, we
observed co-localisation of [18F]ZCDD083 signal with
plaque distribution along the aorta. These cross studies,
involving C57BL/6J, ApoE-/- and ApoE−/−Fbn1C1039G+/−
demonstrated high sensitivity of [18F]ZCDD083 to detect
atherosclerotic plaques, whereas little signal was detected
in normal vessel or outside atherosclerotic lesions. These
stained areas exhibited a higher tracer accumulation.
Hence, the [18F]ZCDD083 uptake was clearly correlated
with plaque distribution as well as with the progression of
the disease.
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AUTHOR INFORMATION
Corresponding Author
*Phone: +44 01509564129. E-mail: m.zanda@lboro.ac.uk
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*Phone : +3232652737. E-mail:
guido.demeyer@uantwerpen.be
*Phone : +3232652820. E-mail:
steven.staelens@uantwerpen.be
Present Addresses
†Centre for Sensing and Imaging Science, Loughborough
University, LE11 3TU Loughborough (UK)
Author Contributions
CDD performed and co-designed all the experiments,
contributed to writing the manuscript. SDA, and LW designed
and performed radiochemistry experiments, reviewed the
manuscript. SS designed in vivo experiments and reviewed
the manuscript. GDM designed biology experiments and
reviewed the manuscript. MZ designed the experiments and
wrote the manuscript.
Funding Sources
We thank the European Union's Horizon 2020 research and
innovation programme under the Marie Skłodowska-Curie
ITN-European Joint Doctorate MOGLYNET (grant agreement
No. 675527).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
We are grateful to Rita Van Den Bossche, University of
Antwerp, for performing the immunohistochemical stainings.
ABBREVIATIONS
CONCLUSION
CCR2, CC chemokine receptor 2; CCL2, CC chemokine ligand 2;
CCR5, CC chemokine receptor 5; TLC, thin layer
chromatography.
In summary, we efficiently radio-synthesized the novel
PET
tracer
[18F]ZCDD083
which
targets
the
PFKFB3enzyme. Using in vivo and ex vivo studies, we
characterized and validated [18F]ZCDD083, demonstrating
its ability to detect atherosclerotic plaques. This tracer may
represent a promising tool for the non-invasive diagnosis
and follow-up of atherosclerotic plaques prone to rupture,
which in turn could help improving risk stratification and
evaluation of the efficacy of anti-atherosclerotic therapies.
REFERENCES
(1)
Lusis, A. J. Atherosclerosis. Nature 2000, 407 (6801), 233–
241. https://doi.org/10.1038/35025203.
(2)
Ross, R. Atherosclerosis — An Inflammatory Disease. N.
Engl.
J.
Med.
1999,
340
(2),
115–126.
https://doi.org/10.1056/NEJM199901143400207.
Spagnoli, L. G.; Bonanno, E.; Sangiorgi, G.; Mauriello, A.
Role of Inflammation in Atherosclerosis. J. Nucl. Med.
(3)
(4)
(5)
2007,
48
(11),
1800–1815.
https://doi.org/10.2967/jnumed.107.038661.
Tawakol, A.; Finn, A. V. Imaging Inflammatory Changes in
Atherosclerosis. JACC Cardiovasc. Imaging 2011, 4 (10),
1119–1122.
https://doi.org/10.1016/j.jcmg.2011.09.001.
Rudd James H.F.; Hyafil Fabien; Fayad Zahi A.
Inflammation Imaging in Atherosclerosis. Arterioscler.
ASSOCIATED CONTENT
Supporting Information
Experimental procedures for the synthesis of all the
compounds described in this paper, radiochemistry
procedures for
metabolite analysis, ex vivo biodistribution, PET/CT imaging,
[
18F]ZCDD083, animal models, in vivo
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 8 of 21
Thromb. Vasc. Biol. 2009, 29 (7), 1009–1016.
(19)
Hammad, B.; Evans, N. R.; Rudd, J. H. F.; Tawakol, A.
Molecular Imaging of Atherosclerosis with Integrated PET
Imaging. J. Nucl. Cardiol. 2017, 24 (3), 938–943.
https://doi.org/10.1007/s12350-016-0766-y.
Moghbel, M.; Al-Zaghal, A.; Werner, T. J.; Constantinescu, C.
M.; Høilund-Carlsen, P. F.; Alavi, A. The Role of PET in
Evaluating Atherosclerosis: A Critical Review. Semin. Nucl.
https://doi.org/10.1161/ATVBAHA.108.165563.
Van Schaftingen, E.; Jett, M. F.; Hue, L.; Hers, H. G. Control
of Liver 6-Phosphofructokinase by Fructose 2,6-
Bisphosphate and Other Effectors. Proc. Natl. Acad. Sci. U.
S. A. 1981, 78 (6), 3483–3486.
Okar, D. A.; Lange, A. J. Fructose-2,6-Bisphosphate and
Control of Carbohydrate Metabolism in Eukaryotes.
1
2
3
4
(6)
(7)
(20)
5
Med.
2018,
48
(6),
488–497.
6
7
8
9
BioFactors
1999,
10
(1),
1–14.
https://doi.org/10.1053/j.semnuclmed.2018.07.001.
Meester, E. J.; Krenning, B. J.; de Swart, J.; Segbers, M.;
Barrett, H. E.; Bernsen, M. R.; Van der Heiden, K.; de Jong,
M. Perspectives on Small Animal Radionuclide Imaging;
Considerations and Advances in Atherosclerosis. Front.
Med. 2019, 6. https://doi.org/10.3389/fmed.2019.00039.
Skagen, K.; Johnsrud, K.; Evensen, K.; Scott, H.; Krohg-
Sørensen, K.; Reier-Nilsen, F.; Revheim, M.-E.; Fjeld, J. G.;
Skjelland, M.; Russell, D. Carotid Plaque Inflammation
Assessed with 18F-FDG PET/CT Is Higher in Symptomatic
Compared with Asymptomatic Patients. Int. J. Stroke
https://doi.org/10.1002/biof.5520100101.
(21)
(22)
(8)
(9)
Pober, J. S.; Sessa, W. C. Evolving Functions of Endothelial
Cells in Inflammation. Nat. Rev. Immunol. 2007, 7 (10),
803–815. https://doi.org/10.1038/nri2171.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Zhang, R.; Li, R.; Liu, Y.; Li, L.; Tang, Y. The Glycolytic
Enzyme PFKFB3 Controls TNF-α-Induced Endothelial
Proinflammatory Responses. Inflammation 2019, 42 (1),
146–155. https://doi.org/10.1007/s10753-018-0880-x.
Tawakol, A.; Singh, P.; Mojena, M.; Pimentel-Santillana, M.;
Emami, H.; MacNabb, M.; Rudd, J. H. F.; Narula, J.; Enriquez,
J. A.; Través, P. G.; et al. HIF-1α and PFKFB3 Mediate a
Tight Relationship between pro-Inflammatory Activation
(10)
2015,
10
(5),
730–736.
https://doi.org/10.1111/ijs.12430.
and
Macrophages. Arterioscler. Thromb. Vasc. Biol. 2015, 35
(6), 1463–1471.
Anaerobic
Metabolism
in
Atherosclerotic
(23)
(24)
Tawakol, A.; Migrino, R. Q.; Bashian, G. G.; Bedri, S.;
Vermylen, D.; Cury, R. C.; Yates, D.; LaMuraglia, G. M.; Furie,
K.; Houser, S.; et al. In Vivo 18F-Fluorodeoxyglucose
https://doi.org/10.1161/ATVBAHA.115.305551.
Positron Emission Tomography Imaging Provides a
(11)
(12)
(13)
Parathath, S.; Yang, Y.; Mick, S.; Fisher, E. A. Hypoxia in
Murine Atherosclerotic Plaques and Its Adverse Effects on
Macrophages. Trends Cardiovasc. Med. 2013, 23 (3), 80–
84. https://doi.org/10.1016/j.tcm.2012.09.004.
Sluimer, J. C.; Daemen, M. J. Novel Concepts in
Atherogenesis:
Atherosclerosis. J. Pathol. 2009, 218 (1), 7–29.
https://doi.org/10.1002/path.2518.
Perrotta, P.; Emini Veseli, B.; Van der Veken, B.; Roth, L.;
Martinet, W.; De Meyer, G. R. Y. Pharmacological Strategies
to Inhibit Intra-Plaque Angiogenesis in Atherosclerosis.
Noninvasive Measure of Carotid Plaque Inflammation in
Patients. J. Am. Coll. Cardiol. 2006, 48 (9), 1818–1824.
https://doi.org/10.1016/j.jacc.2006.05.076.
Joshi, N. V.; Vesey, A. T.; Williams, M. C.; Shah, A. S. V.;
Calvert, P. A.; Craighead, F. H. M.; Yeoh, S. E.; Wallace, W.;
Salter, D.; Fletcher, A. M.; et al. 18F-Fluoride Positron
Emission Tomography for Identification of Ruptured and
Angiogenesis
and
Hypoxia
in
High-Risk Coronary Atherosclerotic Plaques:
A
Prospective Clinical Trial. The Lancet 2014, 383 (9918),
705–713.
https://doi.org/10.1016/S0140-
6736(13)61754-7.
Vascul.
Pharmacol.
2019,
112,
72–78.
(25)
Rudd, J. H. F.; Narula, J.; Strauss, H. W.; Virmani, R.; Machac,
J.; Klimas, M.; Tahara, N.; Fuster, V.; Warburton, E. A.;
Fayad, Z. A.; et al. Imaging Atherosclerotic Plaque
Inflammation by Fluorodeoxyglucose With Positron
Emission Tomography. J. Am. Coll. Cardiol. 2010, 55 (23),
2527–2535. https://doi.org/10.1016/j.jacc.2009.12.061.
Emini Veseli, B.; Perrotta, P.; De Meyer, G. R. A.; Roth, L.;
Van der Donckt, C.; Martinet, W.; De Meyer, G. R. Y. Animal
Models of Atherosclerosis. Eur. J. Pharmacol.
https://doi.org/10.1016/j.ejphar.2017.05.010.
Van der Donckt, C.; Van Herck, J. L.; Schrijvers, D. M.;
Vanhoutte, G.; Verhoye, M.; Blockx, I.; Van Der Linden, A.;
Bauters, D.; Lijnen, H. R.; Sluimer, J. C.; et al. Elastin
Fragmentation in Atherosclerotic Mice Leads to
Intraplaque Neovascularization, Plaque Rupture,
Myocardial Infarction, Stroke, and Sudden Death. Eur.
https://doi.org/10.1016/j.vph.2018.06.014.
(14)
Boyd, S.; Brookfield, J. L.; Critchlow, S. E.; Cumming, I. A.;
Curtis, N. J.; Debreczeni, J.; Degorce, S. L.; Donald, C.; Evans,
N. J.; Groombridge, S.; et al. Structure-Based Design of
Potent and Selective Inhibitors of the Metabolic Kinase
PFKFB3. J. Med. Chem. 2015, 58 (8), 3611–3625.
https://doi.org/10.1021/acs.jmedchem.5b00352.
Qureshi, W. T.; Rana, J. S.; Yeboah, J.; bin Nasir, U.; Al-
Mallah, M. H. Risk Stratification for Primary Prevention of
Coronary Artery Disease: Roles of C-Reactive Protein and
Coronary Artery Calcium. Curr. Cardiol. Rep. 2015, 17
(12), 110. https://doi.org/10.1007/s11886-015-0666-9.
Vigne, J.; Thackeray, J.; Essers, J.; Makowski, M.; Varasteh,
Z.; Curaj, A.; Karlas, A.; Canet-Soulas, E.; Mulder, W.;
Kiessling, F.; et al. Current and Emerging Preclinical
Approaches for Imaging-Based Characterization of
Atherosclerosis. Mol. Imaging Biol. 2018, 20 (6), 869–887.
https://doi.org/10.1007/s11307-018-1264-1.
Rathod, K. S.; Hamshere, S. M.; Jones, D. A.; Mathur, A.
Intravascular Ultrasound Versus Optical Coherence
Tomography for Coronary Artery Imaging – Apples and
Oranges? Interv. Cardiol. Rev. 2015, 10 (1), 8–15.
https://doi.org/10.15420/icr.2015.10.1.8.
de Boer, S. P. M.; Brugaletta, S.; Garcia-Garcia, H. M.;
Simsek, C.; Heo, J. H.; Lenzen, M. J.; Schultz, C.; Regar, E.;
Zijlstra, F.; Boersma, E.; et al. Determinants of High
Cardiovascular Risk in Relation to Plaque-Composition of
a Non-Culprit Coronary Segment Visualized by near-
Infrared Spectroscopy in Patients Undergoing
Percutaneous Coronary Intervention. Eur. Heart J. 2014,
(26)
(27)
(15)
(16)
Heart
J.
2015,
36
(17),
1049–1058.
https://doi.org/10.1093/eurheartj/ehu041.
(28)
(29)
(30)
Gee, A. D.; Bongarzone, S.; Wilson, A. A. Small Molecules as
Radiopharmaceutical Vectors. In Radiopharmaceutical
Chemistry; Lewis, J. S., Windhorst, A. D., Zeglis, B. M., Eds.;
Springer International Publishing: Cham, 2019; pp 119–
136. https://doi.org/10.1007/978-3-319-98947-1_7.
Minchenko, O.; Opentanova, I.; Caro, J. Hypoxic Regulation
(17)
(18)
of
the
6-Phosphofructo-2-Kinase/Fructose-2,6-
Bisphosphatase Gene Family (PFKFB-1–4) Expression in
Vivo. FEBS Lett. 2003, 554 (3), 264–270.
https://doi.org/10.1016/S0014-5793(03)01179-7.
Cao, Y.; Zhang, X.; Wang, L.; Yang, Q.; Ma, Q.; Xu, J.; Wang, J.;
Kovacs, L.; Ayon, R. J.; Liu, Z.; et al. PFKFB3-Mediated
Endothelial
Glycolysis
Promotes
Pulmonary
35
(5),
282–289.
Hypertension. Proc. Natl. Acad. Sci. 2019, 116 (27),
https://doi.org/10.1093/eurheartj/eht378.
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ACS Medicinal Chemistry Letters
13394–13403.
(32)
Position paper of the Cardiovascular Committee of the
European Association of Nuclear Medicine (EANM) on
PET imaging of atherosclerosis SpringerLink
https://doi.org/10.1073/pnas.1821401116.
Tissue expression of PFKFB3 - Summary - The Human
(31)
|
Protein
Atlas
https://link.springer.com/article/10.1007%2Fs00259-
015-3259-3 (accessed Feb 5, 2019).
https://www.proteinatlas.org/ENSG00000170525-
PFKFB3/tissue (accessed Feb 18, 2020).
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