Novel molecular imaging ligands targeting matrix metalloproteinases 2 and 9 for imaging of unstable atherosclerotic plaques

Article abstract of DOI:10.1371/journal.pone.0187767

Molecular imaging of matrix metalloproteinases (MMPs) may allow detection of atherosclerotic lesions vulnerable to rupture. In this study, we develop a novel radiolabelled compound that can target gelatinase MMP subtypes (MMP2/9) with high selectivity and

Full text of DOI:10.1371/journal.pone.0187767

RESEARCH ARTICLE
Novel molecular imaging ligands targeting
matrix metalloproteinases 2 and 9 for imaging
of unstable atherosclerotic plaques
Nazanin Hakimzadeh1,2☯, Victorine A. Pinas3,4☯, Ger Molenaar5, Vivian de Waard6,
Esther Lutgens6,7, Berthe L. F. van Eck-Smit3, Kora de Bruin3, Jan J. Piek2, Jos L.
H. Eersels4, Jan Booij3, Hein J. Verberne3, Albert D. Windhorst4*
1 Department of Biomedical Engineering & Physics, Academic Medical Center, University of Amsterdam,
Amsterdam, The Netherlands, 2 Department of Cardiology, Academic Medical Center, Amsterdam, The
Netherlands, 3 Department of Radiology and Nuclear Medicine, Academic Medical Center, University of
Amsterdam, Amsterdam, The Netherlands, 4 Department of Radiology & Nuclear Medicine, VU University
Medical Center, Amsterdam, The Netherlands, 5 BV Cyclotron VU, Amsterdam, The Netherlands,
6 Department of Medical Biochemistry, Academic Medical Center, Amsterdam, The Netherlands, 7 Institute
for Cardiovascular Prevention (IPEK) Ludwig Maximilian’s University, Munich, Germany
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☯ These authors contributed equally to this work.
* ad.windhorst@vumc.nl
OPEN ACCESS
Abstract
Citation: Hakimzadeh N, Pinas VA, Molenaar G, de
Waard V, Lutgens E, van Eck-Smit BLF, et al.
(2017) Novel molecular imaging ligands targeting
matrix metalloproteinases 2 and 9 for imaging of
unstable atherosclerotic plaques. PLoS ONE 12
(11): e0187767. https://doi.org/10.1371/journal.
pone.0187767
Molecular imaging of matrix metalloproteinases (MMPs) may allow detection of atheroscle-
rotic lesions vulnerable to rupture. In this study, we develop a novel radiolabelled compound
that can target gelatinase MMP subtypes (MMP2/9) with high selectivity and inhibitory
potency. Inhibitory potencies of several halogenated analogues of MMP subtype-selective
inhibitors (N-benzenesulfonyliminodiacetyl monohydroxamates and N-halophenoxy-benze-
nesulfonyl iminodiacetyl monohydroxamates) were in the nanomolar range for MMP2/9.
The analogue with highest inhibitory potency and selectivity was radiolabelled with [123I],
resulting in moderate radiochemical yield, and high radiochemical purity. Biodistribution
studies in mice, revealed stabilization in blood 1 hour after intravenous bolus injection. Intra-
venous infusion of the radioligand and subsequent autoradiography of excised aortas
showed tracer uptake in atheroprone mice. Distribution of the radioligand showed co-locali-
zation with MMP2/9 immunohistochemical staining. In conclusion, we have developed a
novel selective radiolabeled MMP2/9 inhibitor, suitable for single photon emission computed
tomography (SPECT) imaging that effectively targets atherosclerotic lesions in mice.
Editor: Effie C. Tsilibary, National Center For
Scientific Research Demokritos, GREECE
Received: May 12, 2017
Accepted: October 25, 2017
Published: November 30, 2017
Copyright: © 2017 Hakimzadeh et al. This is an
open access article distributed under the terms of
the Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
files.
Introduction
Funding: This work was performed within the
framework of CTMM, the Center for Translational
Molecular Medicine (www.ctmm.nl), project
EMINENCE (grant 01C-204) and the EU FP7 project
INMIND, grant nr HEALTH-F2-2011-278850
(Molenaar, Windhorst). The financial contribution
of the Dutch Heart foundation is gratefully
Traditional diagnosis of atherosclerotic disease severity focused on the percentage of stenosis
detected by angiography. This simplistic view, that atherosclerosis is merely characterized by
pathological lipid deposition within arteries, is not inclusive of the complexities involving
active inflammation, a vast lipid core with a thin fibrous cap, expansive remodeling, intrapla-
que hemorrhage and intraplaque neovascularization of the vasa vasorum [1]. The latter are
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Radiolabeled MMP2/9 inhibitors for atherosclerotic plaque imaging
acknowledged. The funders had no role in study
design, data collection and analysis, decision to
publish, or preparation of the manuscript.
defined as microvessels sprouting from the adventitial layer via angiogenesis in response to
hypoxia and inflammation. This network of immature thin-walled vessels, are prone to leakage
due to their lack of endothelial gap junctions, and thereby serve a highly pathological role by
allowing infiltration of inflammatory cells, including macrophages and their respective proteo-
lytic enzymes, matrix metalloproteinases (MMP) [2]. Accumulation of MMP activity in
inflamed plaques has been suggested to mediate expansive vascular remodeling, following dis-
solution of extracellular matrix content in the fibrous cap [3]. These characteristics collectively
contribute to destabilization of atherosclerotic lesions and thereby lead to potentially fatal
events due to spontaneous plaque rupture (i.e. unstable plaques). Therefore, more advanced
diagnostic tools are required to detect unstable plaques. In this regard imaging of MMP activ-
ity is an accretive approach, that could provide critical insight on the extent of vascular insta-
bility and thereby allow targeted treatment and evaluation of therapeutic intervention efficacy.
Other MMP molecular imaging tracers have been shown to target a large spectrum of
MMPs including gelatinases, interstitial collagenases as well as broad specificity stromelysins
[4, 5]. Collectively, the substrate repertoire of MMPs includes extracellular matrix (ECM) com-
ponents including fibrillary collagens, elastin, matrix proteoglycan core proteins, in addition
to non-matrix substrates [6]. The involvement of MMPs in ECM destabilization in atheroscle-
rotic plaques consequently leads to plaque rupture, and can thereby lead to myocardial and
cerebral infarction. Gelatinases (MMP2 and MMP9) have been shown to be the predominant
MMPs secreted by T lymphocytes and macrophages [7, 8]. In the setting of atherosclerotic pla-
que development, T lymphocytes and macrophages occupy plaques throughout all stages and
play a crucial role in modulating acute and chronic inflammatory responses [9, 10]. Smooth
muscle cells, fibroblasts and endothelial cells stimulated by cytokines released by pro-inflam-
matory cells, are also large producers of MMP2/9.
Competing interests: The authors have declared
that no competing interests exist.
Abbreviations: 3JHH, Vicinal proton-proton
coupling constant; δ, Chemical shifts; ACN,
Acetonitrile; APCI, Atmospheric pressure chemical
ionization; br s, Broad singlet; d, Doublet; DAB,
3,3’-diaminobenzidine; DCM, Dichloromethane;
DMF, Dimethylformamide; DMSO-d6, Dimethyl
sulfoxide-d6; ECF, Electrochemical fluorination;
ESI, Electrospray ionization; EtOAc, Ethyl acetate;
ECM, Extracellular matrix; HPLC, High performance
liquid chromatography; IC50, Half maximal
inhibitory concentration; IDA, Iminodiacetic acid;
LC-MS, Liquid chromatography-mass
spectrometry; m, Multiplet; MRI, Magnetic
resonance imaging; MMPs, Matrix
metalloproteinases; NMM, N-Methylmorpholine;
NMR, Nuclear magnetic resonance; PET, Positron
emission tomography; q, Quartet; Rf, Retardation
factor; s, Singlet; SD, Standard deviation; SEM,
Standard error of the mean; SPECT, Single photon
emission computed tomography; t, Triplet; TEA,
Triethanolamine; THF, Tetrahydrofuran; TLC, Thin
layer chromatography; UV, Ultraviolet.
Molecular-based single photon emission computed tomography (SPECT) and positron
emission tomography (PET) imaging modalities have been used to evaluate key molecular pro-
cesses involved in cardiovascular disease, including atherosclerosis, ventricular remodeling
post myocardial infarction and ischemia-induced angiogenesis [11]. Relative to magnetic reso-
nance imaging (MRI), nuclear imaging approaches are particularly well suited for in vivo
molecular imaging due to their high sensitivity, acceptable spatial resolution as well as wide
availability of instrumentation and molecular probes [12].
In this study we sought to develop a novel highly selective MMP radioligand suitable for
molecular imaging of atherosclerotic lesions. To do this, we have examined the influence of
halogen substitution on the change in affinity of previously described MMP inhibitors [13–
16]. Halogen substituted analogs (fluorine, bromine and iodine) would ultimately deem the
compounds suitable for radiolabeling and would thereby allow us to generate novel molecular
imaging agents targeting MMPs. We have focused on sulfonamide based hydroxamic acids for
halogenation, whereby the non-halogenated form of these compounds have previously been
shown to be potent MMP inhibitors. Hydroxamate based MMP inhibitors are the strongest
class of MMP inhibitors as they achieve bidentate binding to the Zinc ion region of MMPs,
resulting in a distorted geometry. In a key publication of Santos et al. [14], novel non-peptidic
hydroxamate-based MMP inhibitors capable of targeting the deep S1’ pocket of MMPs were
introduced. We have thereby synthesized a series of halogenated sulfonamide based com-
pounds based on the lead inhibitor presented by Santos et al. [14] as well as traditional sulfon-
amide based MMP inhibitors.
The inhibitory potency of these halogenated ligands towards MMP2 and MMP9 was deter-
mined, followed by radiolabeling of the iodine substituted ligand. Radiolabeling was con-
ducted with [123I], as this is a suitable radionuclide for SPECT imaging. Finally, we examined
the biodistribution of the selected radiolabeled ligand as well as conducted validation studies
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Radiolabeled MMP2/9 inhibitors for atherosclerotic plaque imaging
to confirm the efficacy of the tracer to selectively bind atherosclerotic lesions. Validation stud-
ies were conducted using ex vivo autoradiography and immunochemistry in an ApoE-/-
mouse model, known for the spontaneous development of unstable atherosclerotic plaques,
comprised of MMP2 and 9.
Methods
Expanded methods section provided in S1 Appendix. ARRIVE Guidelines checklist reporting
in vivo experiments provided in S2 Appendix.
Chemical synthesis
All non-aqueous reactions were carried out under nitrogen atmosphere. Reagents and solvents
were obtained from commercial sources and were used without further purification. Yields
refer to purified products and are not optimized. Analytical thin layer chromatography (TLC)
was performed on Merck silica gel 60 F254 aluminum-backed plates. Compounds were visual-
ized by ultraviolet (UV) light (254 nm). Flash column chromatography was performed with
kiesel gel 60 F254 (Baker). Nuclear Magnetic Resonance (NMR) spectra were recorded a Bruker
Advance 400 MHz spectrometer (400 MHz for 1H, 100 MHz for 13C). Chemical shifts (δ) are
expressed in parts per million (ppm) with the solvent signal as reference, 2.50 for DMSO-d6,
7.27 for CDCl3. Splitting patterns are designated as s (singlet), d (doublet), t (triplet), q (quar-
tet), m (multiplet), br s (broad singlet). LC-MS (SHIMAZU, LCMS-2020 combined with LC
(UFLC) UFLCXR) analyses were performed on either Acquity C-18 column (2.1 mm x 100
mm, 1.7 μm) using electrospray ionization (ESI) or on a Gemini C-18 column (4.6 mm x 50
mm, 5 μm) using atmospheric pressure chemical ionization (APCI). The purity of all tested
compounds was determined by liquid chromatography-mass spectrometry (LC-MS) and ele-
mental analyses, resulting in a purity < 96% for all tested compounds. All commercially
obtained chemicals were used without prior purification.
In vitro MMP inhibition & selective binding capacity assays
The inhibitory potency and selective binding of MMP-ligands was investigated by examining
their capacity to block MMP1, MMP2 and MMP9 activity, using in vitro fluorometric assays
(SensoLyteTM 520 MMP-1, SensoLyteTM 520 MMP-2, SensoLyteTM 520 MMP-9; AnaSpec,
San Jose, USA). Assay buffer was warmed to 37˚C and used to dilute the reference inhibitor N-
isobutyl-N-(4-methoxyphenylsulfonyl)glycyl hydoxamic acid (NNGH 1:100), calibration stan-
dard (1:50), substrate and enzyme (1:60). Components were loaded into a 96-well plate. MMP
ligands 10a-d were also aliquoted to the respective 96-well plate at varying concentrations
(10pM, 100pM, 1nM, 10nM, 100nM, 1μM, 10 μM and 100 μM), followed by incubation at
37˚C for 30 minutes. Reactions were initiated by addition of the substrate to the respective
wells, after which fluorescence generated was measured using a microplate reader (Synergy
HT Multi-detection Microplate Reader, BioTek, 310–405nM) continuously for 30 minutes at
60 second intervals. Data analysis was conducted using Sigma Plot and Microsoft Excel.
In vivo biodistribution
All experiments were approved by the institutional committee for animal experiments at the
Academic Medical Center. C57BL/6 female mice (14–16 week) were purchased from Harlan
and housed in the animal care facility at the Academic Medical Center. Animals were anesthe-
tized using 126 mg ketamine, 100 μg dexdomitor, 500 μg atropine, 7.5 mL NaCl 0.9% of which
0.015 mL/g body weight was administered by intraperitoneal injection. In order to investigate
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Radiolabeled MMP2/9 inhibitors for atherosclerotic plaque imaging
the in vivo biodistribution of MMP ligand [123I]10c, 4 MBq was administered intravenously in
the tail vein and allowed to circulate for either 5, 15, 30, 60 minutes or 2, 3, 4, 6 and 24 hours
(n = 3–4 animals for each time point). At the end of each circulation time, animals were sacri-
ficed by bleeding through phosphate buffered saline solution (PBS) perfusion. After the ani-
mals were sacrificed, whole blood was collected by cardiac puncture, and selected organs were
excised (heart, lungs, liver and kidneys). The organs were weighed and radioactivity was mea-
sured using a scintillation counter (TRT-CARB 2000CA, Perkin Elmer 1987). The measured
radioactivity was expressed as the percentage of the injected dose per gram of tissue per gram
of body weight (% ID/g/g).
In vivo binding to atherosclerotic plaques
To investigate selective binding of [123I]10c to MMP2/9 found in atherosclerotic lesions, we
examined the distribution of the [123I]10c in 8–10 week old female ApoE-/- mice (n = 5,
Charles River) that were fed a high fat Western diet (15% fat, 0.25% cholesterol; Arie Blok
Diervoeding, Netherlands) for 4 weeks. These animals subsequently developed atherosclerotic
plaques. 40 MBq of [123I]10c were administered by intravenous injection in the tail vein and
allowed to circulate for 1 hour. Animals were euthanized by bleeding via PBS infusion. The
aortic arch was excised and 10 μm serial sections were made. Aortic arch sections were placed
on a phosphor imaging plate (Fuji) and the amount of radioactivity was measured using a stor-
age phosphor imaging apparatus (Typhoon Trio; GE Healthcare Life Sciences). Sections were
then stained with hematoxylin/eosin staining, or used for MMP2 and MMP9 detection. Prior
to incubation with monoclonal goat-anti-mouse MMP2 (R&D Systems) and rabbit-anti-
mouse MMP9 (Abcam), the sections were heated to improve antigen retrieval. Rabbit-anti-
goat secondary antibodies (DAKO) were utilized on the MMP2 sections. Thereafter, all sec-
tions were probed with the PowerVision anti-rabbit polymer, conjugated to horseradish per-
oxidase (Immunologic), to reveal positive primary antibody binding by means of chromogen
conversion of 3,3’-diaminobenzidine (DAB), followed by nuclear counterstaining with hema-
toxylin. Histological sections were imaged with a bright-field microscope. The respective sec-
tion used for MMP9 staining was also subsequently stained for smooth muscle cells. After the
MMP9 staining, the section was incubated with anti-alpha-smooth muscle actin antibody
(clone 1A4; DAKO) in Tris buffered saline (TBS) with 1% normal goat serum for 1h at room
temperature. Thereafter, the section was washed in TBS and incubated with BrightVision poly
alkaline phosphatase (AP)-anti Mouse IgG (ImmunoLogic) for 30 min, followed by TBS wash-
ing and incubation with Vector Blue AP substrate (SK-5300; Vector Laboratories Inc) for 1h.
Similar to the other histological sections, imaging was conducted with a bright-field
microscope.
Results
We synthesized and examined two series of halogenated sulfonamide hydroxamate based
compounds for MMP inhibition, 4a-e and 10a-d (Fig 1, Table 1). Our goal was to investigate
the effects of halogenation on these compounds such that they could be ultimately utilized for
radiosynthesis.
Chemistry
The commercially available compounds 1a-e were converted into the intermediate tertiary
butyl protected iminodiacetic acid derivatives 2a-e in the presence of triethylamine in dichlor-
omethane by reaction with di-tert-butyl iminodiacetate (Fig 2). Deprotection was achieved by
stirring 2a-e overnight in formic acid at room temperature to give the dicarboxylic acid
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Radiolabeled MMP2/9 inhibitors for atherosclerotic plaque imaging
Fig 1. Hydroxamate target compounds. Schematic depicting halogenated forms of two sulfonamide
hydroxamate based compounds examined (4a-e and 10a-d).
https://doi.org/10.1371/journal.pone.0187767.g001
compounds 3a-e. After selective activation of one carboxylic acid group of the N-substituted
iminodiacetic acid derivatives with ethylchloroformate in the presence of N-methylmorpho-
line and subsequent reaction with hydroxylamine in dichloromethane the target compounds
4a-e were obtained in moderate overall yields.
The arylsulfonyl chlorides 7a-d were prepared from the commercially available compounds
5a-d, introduction of the sulphonic acid moiety was achieved with chlorosulphonic acid lead-
ing to 6a-d which were treated without intermediate purification with thionyl chloride to yield
7a-d (S1 Fig). Subsequent synthesis of 9a-d in a heterogeneous THF/H2O system (Schotten-
Baumann conditions) as described previously [14] was unsuccessful. Alternatively, reaction of
di-tert-butyl protected iminodiacetic acid with 7a-d in the presence of triethylamine, followed
by deprotection with formic acid yielded 9a-d. Selective activation of one carboxylic acid
group and condensation was performed using the same strategy as described for compounds
4a-e and provided the target compounds 10a-d (Fig 3). The purity of all tested compounds
was found to be > 96% and was determined with liquid chromatography-mass spectrometry
and elemental analysis.
Inhibitory potency of candidate ligands towards MMP2 and 9
To determine the capacity of each candidate ligand to inhibit the activity of MMP2 and
MMP9, as well as to examine their selectivity for these MMP subtypes, in vitro inhibitory
potency assays were conducted with the compounds 4a-e and 10a-d. As depicted in Table 2,
all compounds demonstrated inhibitory potency in the nanomolar range. Relative to
Table 1. List of halogenated forms of two sulfonamide hydroxamate based compounds examined.
Compound
Symbol
Compound name
4a
4b
2-(N-(2(hydroxyamino)-2-oxoethyl)phenylsulfonamido) acetic acid
2-(4-fluoro-N-(2-hydroxyamino)-2oxoethyl)phenylsufonamido acetic acid
2-(4-Iodo-N-(2-hydroxyamino)-2-oxoethyl) phenylsufonamido acetic acid
2-(4-bromo-N-(2-hydroxyamino)-2-oxoethyl) phenylsufonamido acetic acid
2-(4-chloro-N-(2-hydroxyamino)-2-oxoethyl)phenylsufonamido acetic acid
([4-Phenoxy-benzenesulphonyl]-hydroxycarbamoylmethyl-amino) acetic acid
4c
4d
4e
10a
10b
[4-(4-Fluoro-phenoxy)-benzenesulphonyl]-hydroxycarbamoylmethyl-amino) acetic
acid
10c
10d
([4-(4-Iodo-phenoxy)-benzenesulphonyl]-hydroxycarbamoylmethyl-amino) acetic
acid
([4-(4-Bromo-phenoxy)-benzenesulphonyl]-hydroxycarbamoylmethyl-amino) acetic
acid
https://doi.org/10.1371/journal.pone.0187767.t001
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Radiolabeled MMP2/9 inhibitors for atherosclerotic plaque imaging
Fig 2. Synthesis of hydroxamate target compound 4a-e from 1a-e. (i): HN(CH2CO2tbu)2,TEA, DCM, rt, o/n, yield 39–75%; (ii): HCOOH, rt, o/n,
yield 30–93%; (iii): ECF, NMM, THF, NH2OH x HCl, MeOH, 0˚C, 2 h, yield 63–97%.
https://doi.org/10.1371/journal.pone.0187767.g002
compounds 4a-e, ligands 10a-d showed higher inhibitory potency towards MMP2/9, with less
binding towards MMP1, and thus showed the desired selectivity.
Fig 3. Synthesis of 10 a-d. (i): HN(CH2COOH, THF/H2O, rt, o/n, yield 3–35%; (ii): HN(CH2CO2tbu)2, TEA, DCM, rt, o/n, 78–99%; (iii): HCOOH, rt,
o/n, yield 75–94%; (iv): ECF, NMM, THF, NH2OH x HCl, MeOH, 0˚C, 2 h, yield 52–91%.
https://doi.org/10.1371/journal.pone.0187767.g003
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Radiolabeled MMP2/9 inhibitors for atherosclerotic plaque imaging
Table 2. MMP inhibitory potencies of compounds 4a-e and 10a-d. IC50: half maximal inhibitory concentration. Values expressed in nM are presented as
mean ± SD of three independent experiments in duplicate. Selectivity was calculated as the ratio of IC50 values; MMP1 IC50 for each respective compound
was divided by the respective MMP2 or MMP9 IC50. The higher the selectivity value, the greater the selectivity towards either MMP2 or MMP9 relative to
MMP1 for each respective compound.
IC50 (nM)
MMP2
Selectivity
Compound
MMP1
51 ± 0.10
75 ± 0.15
59 ± 0.17
63 ± 0.13
61 ± 0.11
1670 ± 980
4690 ± 2220
910 ± 560
1320 ± 790
MMP9
MMP1/MMP2
1.31
MMP1/MMP9
3.64
4a
4b
39 ± 0.16
12 ± 0.17
27 ± 1.00
19 ± 0.19
41 ± 0.19
1.89 ± 1.11
0.13 ± 0.03
0.87 ± 0.40
7.64 ± 1.43
14 ± 1.05
21 ± 0.19
11 ± 1.14
23 ± 0.21
18 ±0.16
6.25
3.57
4c
2.19
5.36
4d
3.32
2.74
4e
1.49
3.39
10a
10b
10c
10d
4.31 ± 2.06
0.27 ± 0.15
1.95 ±1.10
1.61 ±1.41
884
387
1.74 x 104
3.60 x 104
1.05 x 103
173
467
820
https://doi.org/10.1371/journal.pone.0187767.t002
Radiosynthesis of 123I-N-arylsulfonyl iminodiacetyl monohydroxamate
([123I]10c)
Radiolabelling was considered only for compounds 10a-d. Furthermore, although compound
10b showed the highest affinity for MMP2/9 and selectivity towards MMP1, compound 10c
was selected as the most suitable candidate for targeting MMP2/9 since its affinity for MMP2/
9 was high as well was its selectivity. More importantly, the chemical structure offers the
opportunity for radiolabeling with Iodine-123. This radioisotope is suitable for SPECT imag-
ing which could in turn be used for SPECT imaging in small animals. In addition, the relatively
long half-life of Iodine-123 (13.2 hours) infers that a dedicated on-site radionuclide production
facility is not required for utilization of compounds labelled with this radionuclide. Finally,
SPECT scanners are more readily available than PET scanners.
Several routes for the radiosynthesis of 123I-N-arylsulfonyl iminodiacetyl monohydroxa-
mate [123I]10c were explored. In a first attempt electrophilic iodination was investigated with
the stannylated precursor 11, which was synthesized starting from 10c (S2 Fig). Unfortunately
only starting material was recovered after the radiolabeling and no radiolabeled product was
obtained. Protection of the hydroxymate function with a t-butyl moiety also did not generate
radiolabeled product. The labeling of the stannylated di-t-butyl protected analogue of 8c (S3
Fig) was however successful, [123I]8c was obtained in > 90% radiochemical yield. Compound
[
123I]8c was subsequently deprotected under acidic conditions into [123I]9c. The target com-
pound, [123I]10c was finally obtained from [123I]9c after evaporation of the acidic reagents, by
hydroxylamine condensation in basic conditions with a radiochemical purity of > 98% (S3
Fig). Drawback of this 3-step radiosynthesis route, besides the non-favorable multistep reac-
tion sequence with radiolabeled compounds, was the deprotection step to form [123I]9c using
2 M hydrochloric acid in diethylether. This reaction gave rise to several side products, resulting
is a low overall radiochemical yield of 4 1% (corrected for decay).
Since this radiolabeling procedure was complicated and the yield not adequate to perform
experiments with the radiolabeled product, the radiolabeling starting from the bromine pre-
cursor via a Cu+ assisted nucleophilic aromatic substitution was also investigated (Fig 4). This
approach is advantageous since it can be performed in one reaction step in contrast to the
complex multistep procedure. The elevated reaction temperature however, is a serious disad-
vantage since the risk for the formation of side products under these reaction conditions is
increased. In addition, performing a nucleophilic aromatic substitution reaction is technically
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Radiolabeled MMP2/9 inhibitors for atherosclerotic plaque imaging
Fig 4. Radiosynthesis of [123I]10c from 10d by nucleophilic radioiodination. (i): Na[123I]I, ethanol, citric acid, 2,5 dihydroxybenzoic acid,
CuSO4, SnSO4, 130˚C, 40 min, yield 51%.
https://doi.org/10.1371/journal.pone.0187767.g004
more complicated compared to a electrophilic reaction. Nonetheless, by means of nucleophilic
aromatic substitution reaction conditions, radiolabeling of the target compound [123I]10c,
resulted the desired product in a decay-corrected radiochemical yield and purity 38–63%
and > 98%, respectively.
In vivo biodistribution of [123I]10c
The biodistribution of [123I]10c was investigated following intravenous infusion of 4 MBq in
healthy C57BL/6 mice. Following the bolus injection, the compound was allowed to circulate
for 5, 15, 30, 60 minutes, 2, 3, 4, 6 and 24 hours. As shown in Fig 5, radioactivity was highest in
the liver and blood, while accumulation in fat was the lowest. In the blood, stabilization of the
compound was reached after at least 1 hour of circulation.
Uptake of [123I]10c in atherosclerotic lesions
To validate the efficacy of [123I]10c as an appropriate tracer for detection of atherosclerotic pla-
ques, we examined the in vivo uptake of this ligand in ApoE-/- mice that were fed a high-fat
diet. ApoE-/- mice develop atherosclerotic lesions spontaneously due to lack of ApoE expres-
sion, while the 6 week high fat diet accelerates lesion formation [17]. 40 MBq of [123I]10c was
administered intravenously and allowed to circulate for 3 hours, after which the animals were
Fig 5. Biodistribution of [123I]10c following intravenous infusion in healthy mice (n = 3–4 /group).
Injected dose (ID) of radioactivity was measured in fat (A), muscle (B), liver (C), kidney (D), spleen (E), blood
(F), heart (G) and lung (H) at varying time-points after injection. Values shown are mean ± SEM.
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Radiolabeled MMP2/9 inhibitors for atherosclerotic plaque imaging
euthanized and the aortic arch was excised for measuring radioactivity by storage phosphor
imaging [18].
As depicted in Fig 6A, the uptake of [123I]10c was detectable in atherosclerotic lesions
found in the aortic arch of ApoE-/- mice. The tracer uptake was relatively high in the diseased
regions of the vessel wall. Fig 6B and 6C shows nuclear and cytoplasm staining of the respec-
tive histological section shown in Fig 6A, depicting the presence of anatherosclerotic lesion.
To elucidate the selectivity of this radioligand towards MMP2 and MMP9, immunohistochem-
ical staining revealed positive areas for MMP2 and MMP9 in the lesions co-localizing with
uptake of [123I]10c that was detected by storage phosphor imaging (Fig 6D and 6E). This
murine atherosclerotic lesion showed especially high MMP9 staining in the plaque (foam cell)
macrophages, and not in the adjacent medial layer which consists of smooth muscle cells (Fig
6F). In conclusion, in this proof of concept study, the [123I]10c MMP2/9 tracer effectively tar-
geted atherosclerotic lesions.
Discussion
In this study we have developed a novel radiolabeled inhibitor of MMP2/9, which is able to tar-
get atherosclerotic lesions. The ligand we have derived in this study demonstrated an inhibi-
tory potency in the desired nanomolar range, with a high selectivity in vitro for MMP2/9 over
MMP1. We also demonstrated successful radiolabeling of the MMP inhibitor with [123I] using
a single step approach. Thus, we have developed a novel highly potent molecular imaging
agent for visualization of MMP2/9 activity by SPECT imaging, and thereby allowing for the
identification of atherosclerotic lesions with high proteolytic MMP activity, which are consid-
ered vulnerable to rupture.
Fig 6. Binding of [123I]10c to atherosclerotic lesions containing MMP2 and MMP9. A) Storage phosphor image depicting binding of [123I]10c in a
cross-section of the aortic arch of an ApoE-/- mouse. The region with highest apparent radioactivity has been outlined. B) Hematoxylin and eosin staining
of the respective aortic arch cross-section shown in (A). The respective outlined region in (A) coincides with an atherosclerotic lesion at the base of a
branching artery, as outlined in (B). C) Enlargement of the outlined region in (B). D) Cross-section of the atherosclerotic plaque in a consecutive section,
displaying MMP2 (brown) distribution mainly in the smooth muscle cell-rich medial area of the aorta, and nuclei (blue). E) Cross-section of the
atherosclerotic lesion in a consecutive section, depicting MMP9 (brown) distribution mainly in foam cell macrophages of the atherosclerotic plaque, and
nuclei (blue). F) Respective section in (E) stained with anti-alpha-smooth muscle actin depicting smooth muscle rich layer (purple) in the aorta vessel
wall, adjacent to the plaque core where apparent foam cells are present.
https://doi.org/10.1371/journal.pone.0187767.g006
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Radiolabeled MMP2/9 inhibitors for atherosclerotic plaque imaging
[
123I]10c shows selectivity towards gelatinase MMP subtypes (MMP2 and MMP9) over
MMP1. This is an important revelation, as previous broad spectrum inhibitors targeting
MMP1 have led to adverse side effects due to interference with normal remodeling processes.
We have demonstrated co-localization of [123I]10c in atherosclerotic lesions containing
MMP2/9 in mice. This further highlights the feasibility to target MMP2/9-rich regions with
the tracer and indicates that its accumulation in lesions is not due to vascular leakage.
The MMP inhibitor we have developed in this study displays highly potent inhibitory
capacity in the range of 0.87 to 1.95 nM at MMP2/9. Although other existing MMP inhibitors
display an even higher inhibitory potency [4, 14], these inhibitors did not show the high selec-
tivity we have observed. Since MMP2/9 are typically expressed in areas under stress, such as
atherosclerotic plaque lesions, the high selectivity for MMP2/9 will allow preferential targeting
of atherosclerotic lesions.
We have selected iodine-123 as a suitable radionuclide to radiolabel compound 10c as this
would infer the compound appropriate for SPECT imaging. We selected a SPECT tracer due
to the wider availability of SPECT machines, along with the convenient physical half-life of
13.2 hrs of the radionuclide. In addition, while PET scanners have superior sensitivity relative
to SPECT imaging, SPECT cameras can detect different energies and thereby allow for simul-
taneous imaging of multiple radioisotopes [19, 20].
Radiolabeling of 10c with iodine-123 was achieved successfully in a single step approach.
This is advantageous to obtain the product with maximum radioactivity. In addition, stabiliza-
tion of the compound in blood was noted approximately one hour after intravenous adminis-
tration. Thus, the optimum time for imaging whereby signal-to-noise ratio is high is well
within the limits of the half-life of iodine -123.
Other applications of [123I]10c
As a result of the ECM remodeling capacity of MMPs and their participation in regulatory
signaling in chronic inflammatory responses, these enzymes are involved in numerous
pathologies, including cancer, arthritis in addition to atherosclerosis [21, 22]. The initial
interest in MMP imaging, evolved from their role in cancer progression [12]. MMPs, partic-
ularly MMP2 and MMP9 modulate the degradation of the basal membrane, and thereby
facilitate the migration of metastatic cells into proximal and distal tissues [23]. Thus, it is
plausible that the MMP2 and MMP9 inhibitor developed in this study may have farther-
reaching applications than only atherosclerotic lesion visualization and quantification.
Nonetheless, future work must examine the efficacy of the ligand to also target metastatic
tumors and allow visualization.
Study limitations
Accumulation of [123I]10c in atherosclerotic lesions is not direct evidence of tracer binding to
MMP2/9. However, taking into account that [123I]10c uptake co-localizes with regions of
MMP2/9 immunostaining as well as the specific inhibitory potency towards MMP2/9, this
accumulation of [123I]10c is suggestive of MMP2/9 binding. In addition, [123I]10c uptake has
been visualized using phosphor imaging of excised tissue, rather than live in vivo SPECT imag-
ing. Nevertheless, when combined with immunohistochemistry, this proof of concept method
effectively demonstrates [123I]10c uptake in atherosclerotic lesions. Furthermore, although we
have examined the inhibitory potency of our compound in relation to MMP2, 9 and 1, we can-
not comment on the inhibitory potency of these compounds towards other MMP subtypes.
We chose to examine the inhibitory potency of these compounds towards MMP1 in particular
as previous broad spectrum MMP inhibitors have demonstrated severe side effects due to
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Radiolabeled MMP2/9 inhibitors for atherosclerotic plaque imaging
impairment of normal tissue remodeling that is regulated by MMP1. Nonetheless, as our com-
pound 10a is comparable to compound B3 of Santos et al. we can speculate that it has similar
inhibitory potencies for the other MMP subtypes. Santos et al. demonstrated that this com-
pound shows highest inhibitory potency towards MMP2, MMP13 and MMP9 [14]. We are
limited by a lack of metabolite assessment. In vivo biodistribution results of the tracer over
time demonstrated a multi-phasic response in some tissues, suggesting the possibility of
metabolite generation. Future studies should include examining the validation of this tracer in
larger animal models, using animal SPECT imaging as well as examining the metabolic stabil-
ity of the tracer. In addition, although we have examined the biodistribution of the tracer over
time in blood, we did not examine its stability in plasma.
Conclusions and future perspectives
We have developed a novel radioligand (N-[123I]iodophenylsulfonyl iminodiacetyl monohy-
droxamate) with high selectivity for MMP2/9 binding over MMP1. Nonetheless, as it is been
published recently by Newby et al. [24] that other MMP subtypes also play a role in plaque
rupture, the affinity to a larger panel of MMPs should be investigated in future studies. We
have also demonstrated the efficacy of this tracer to target atherosclerotic lesions consisting of
MMP2/9 in mice. Radiolabeling of 10c was achieved using a single step approach, resulting in
moderate radiochemical yield and high radiochemical purity. Biodistribution studies further
revealed fast stabilization of the compound in blood. Nonetheless, our study is limited by a
lack of metabolite detection. Future efforts will focus on further validation studies by in vivo
SPECT imaging of mice prone to atherosclerotic lesion development along with the assess-
ment for metabolite formation. Furthermore, it is imperative to examine the efficacy of these
ligands in other species, with the ultimate realization of efficacy to target human MMP2/9.
Development of tracers for visualization of atherosclerotic lesions prone to rupture, is an
important step in the realization that atherosclerotic plaque complexity is an important indica-
tor of the true risk of plaque rupture, rather than only angiographic assessment of atheroscle-
rotic narrowing.
Supporting information
S1 Appendix. Supplemental methods describing chemical synthesis of analogues.
(DOCX)
S2 Appendix. ARRIVE guidelines checklist.
(PDF)
S1 Fig. Synthesis of the arylsulfonylchlorides 7a-d from 5a-d (i): ClSO3H, CH2Cl2, 0˚C, 2h,
(ii): SOCl2, DMF (cat), reflux, 6h, yield 80–97%.
(TIF)
S2 Fig. Attempted radiosynthesis of [123I]10c (i): (n-Bu3Sn)2, Pd(PPh3)4, toluene, reflux, o/
n, yield 72%; (ii): Na[123]I, H2O2, CH3COOH, rt, 20 min, yield 0%.
(TIF)
S3 Fig. Radiosynthesis of [123I]10c from 8c via electrophilic radioiodination. (i): (n-
Bu3Sn)2, Pd(PPh3)4, toluene, reflux, o/n, yield 76%; (ii): NaI[123], H2O2, CH3COOH, 25 min.;
(iii): 2M HCl in Et2O, rt, 30 min.; (iv): a) ethylchloroformate, NMM, THF, 15 min, b) NH2OH
x HCl, MeOH, 0˚C, 15 min. Overall (steps ii to iv) decay corrected radiochemical yield of
[
(TIF)
123I]10c was 4%.
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Radiolabeled MMP2/9 inhibitors for atherosclerotic plaque imaging
Acknowledgments
This work was performed within the framework of CTMM, the Center for Translational
Molecular Medicine (www.ctmm.nl), project EMINENCE (grant 01C-204) and the EU FP7
project INMIND, grant nr HEALTH-F2-2011-278850 (Molenaar, Windhorst). The financial
contribution of the Dutch Heart foundation is gratefully acknowledged. The funders had no
role in study design, data collection and analysis, decision to publish, or preparation of the
manuscript.
Author Contributions
Conceptualization: Vivian de Waard, Berthe L. F. van Eck-Smit, Jan Booij, Hein J. Verberne,
Albert D. Windhorst.
Data curation: Victorine A. Pinas, Vivian de Waard, Esther Lutgens, Kora de Bruin.
Formal analysis: Nazanin Hakimzadeh, Victorine A. Pinas, Vivian de Waard, Kora de Bruin,
Jos L. H. Eersels.
Funding acquisition: Berthe L. F. van Eck-Smit, Jan J. Piek, Jan Booij, Hein J. Verberne,
Albert D. Windhorst.
Investigation: Nazanin Hakimzadeh, Victorine A. Pinas, Ger Molenaar, Vivian de Waard,
Berthe L. F. van Eck-Smit, Kora de Bruin, Jan Booij, Hein J. Verberne, Albert D.
Windhorst.
Methodology: Nazanin Hakimzadeh, Victorine A. Pinas, Vivian de Waard, Kora de Bruin, Jos
L. H. Eersels.
Resources: Nazanin Hakimzadeh, Victorine A. Pinas, Ger Molenaar, Vivian de Waard, Esther
Lutgens, Berthe L. F. van Eck-Smit, Kora de Bruin, Jos L. H. Eersels, Jan Booij, Albert D.
Windhorst.
Supervision: Vivian de Waard, Berthe L. F. van Eck-Smit, Jan J. Piek, Jan Booij, Hein J. Ver-
berne, Albert D. Windhorst.
Validation: Nazanin Hakimzadeh, Victorine A. Pinas, Vivian de Waard, Esther Lutgens, Kora
de Bruin.
Visualization: Nazanin Hakimzadeh, Victorine A. Pinas, Vivian de Waard, Esther Lutgens,
Kora de Bruin.
Writing – original draft: Nazanin Hakimzadeh, Victorine A. Pinas.
Writing – review & editing: Nazanin Hakimzadeh, Victorine A. Pinas, Ger Molenaar, Vivian
de Waard, Esther Lutgens, Berthe L. F. van Eck-Smit, Kora de Bruin, Jan J. Piek, Jos L. H.
Eersels, Jan Booij, Hein J. Verberne, Albert D. Windhorst.
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