Development and Evaluation of an 18F-Radiolabeled Monocyclam Derivative for Imaging CXCR4 Expression

Article abstract of DOI:10.1021/acs.molpharmaceut.9b00069

In humans, C-X-C chemokine receptor type 4 (CXCR4) is a protein that is encoded by the CXCR4 gene and binds the ligand CXCL12 (also known as SDF-1). The CXCR4-CXCL12 interaction in cancer elicits biological activities that result in tumor progression and

Full text of DOI:10.1021/acs.molpharmaceut.9b00069

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Article
Development and Evaluation of an 18F-Radiolabeled
Monocyclam Derivative for Imaging CXCR4 Expression
Diana Brickute, Marta Braga, Maciej A. Kaliszczak, Chris Barnes, Doreen Lau, Laurence Carroll,
Elizabeth Stevens, Sebastian Trousil, Israt S. Alam, Quang-De Nguyen, and Eric O. Aboagye
Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/
acs.molpharmaceut.9b00069 • Publication Date (Web): 18 Mar 2019
Downloaded from http://pubs.acs.org on March 19, 2019
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Development and Evaluation of an 18F-
Radiolabeled Monocyclam Derivative for
Imaging CXCR4 Expression
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Diana Brickute,a,† Marta Braga,a,† Maciej A. Kaliszczak,a Chris Barnes,a Doreen
Lau,a Laurence Carroll,a Elizabeth Stevens,a Sebastian Trousil,a Israt S. Alam,a
Quang-Dé Nguyen a and Eric O. Aboagyea*
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a Cancer Imaging Centre, Department of Surgery and Cancer, Imperial College
London, Faculty of Medicine, Hammersmith Hospital Campus, Du Cane Road,
London W12 0NN, United Kingdom.
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† These authors contributed equally to this work.
Keywords: CXCR4, Cyclam, [18F]MCFB, AMD3465
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ABSTRACT
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C-X-C chemokine receptor type 4 (CXCR4) is a protein that in humans is encoded by
the CXCR4 gene and binds the ligand CXCL12 (also known as SDF-1). The CXCR4-
CXCL12 interaction in cancer elicits biological activities that result in tumor
progression and has accordingly been the subject of significant investigation for
detection and treatment of disease. Peptidic antagonists have been labeled with a variety
of radioisotopes for detection of CXCR4, but methodology utilizing small molecules
have predominantly used radiometals. We report here the development of a 18F-
radiolabeled cyclam-based small molecule radioprobe, [18F]MCFB, for imaging
CXCR4 expression. The IC50 of [19F]MCFB for CXCR4 was similar to that of
AMD3465 (111.3 and 89.9 nM, respectively). In vitro binding assays show that the
tracer depicted differential CXCR4 expression, which was blocked in the presence of
AMD3465, demonstrating specificity of [18F]MCFB. Positron emission tomography
(PET) imaging studies showed distinct uptake of radioprobe in lymphoma and breast
cancer xenografts. High liver and kidney uptake were seen with [18F]MCFB leading us
to further examine the basis of its pharmacokinetics in relation to the tracer’s cationic
nature, thus, the role of organic cation transporters (OCTs). Substrate competition
following the intravenous injection of metformin led to a marked decrease in urinary
excretion of [18F]MCFB, with moderate changes observed in other organs, including
the liver. Our results suggest involvement of OCTs in renal elimination of the tracer. In
conclusion, the 18F radiolabeled monocyclam, [18F]MCFB, has potential to detect tumor
CXCR4 in non-hepatic tissue.
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INTRODUCTION
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C-X-C chemokine receptor type 4 (CXCR4) is a seven-transmembrane domain G
protein-coupled receptor that in humans is encoded by the CXCR4 gene. Binding of
CXCR4 to its cognate ligand, CXCL12 (also known as SDF-1), leads to the activation
of several downstream signaling pathways that control cellular responses including
proliferation, survival, chemotaxis and transcription.1,2 These CXCR4-regulated
pathways exert critical functions in development and normal physiology, as well as in
disease processes. In fact, CXCR4 upregulation has been reported in over 20 types of
malignant cancers and is correlated with poor prognosis and an aggressive phenotype.
Organ-specific metastatic spread, for instance, has been attributed in part to CXCR4-
CXCL12 interaction,3,4 wherein CXCR4 expressing tumor cells migrate to CXCL12-
rich environments such as lymph nodes, lung, liver, and bone marrow. Along with
metastatic spread, the CXCR4-CXCL12 axis impact other aspects of tumor
progression, including provision of a protective niche for tumor cells in the bone
marrow and supporting hypoxia-driven angiogenesis through recruitment of CXCR4-
positive pro-angiogenic cells.5,6
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Increased understanding of the pivotal role of CXCR4 in tumor biology has resulted
in the development of imaging probes that can provide sensitive and rapid detection of
CXCR4 expression. Positron emission tomography (PET) is a sensitive nuclear
functional imaging modality used frequently in oncology for diagnosis, staging and
monitoring treatment response. The development of novel PET imaging agents to target
receptors that are characteristic of malignant phenotype is an important area of research
and strives towards personalized medicine. CXCR4 is an attractive target for
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developing PET radioligands because of its role and overexpression in some cancers
and other diseases. One of the most studied classes of CXCR4 imaging agents comprise
of peptidic radioprobes; screening of molecules against HIV infection led to the
discovery of T140, a 14-residue peptide with a disulphide bridge that has been
functionalized with various chelators/radionuclides for CXCR4 detection.7–9
Cyclopentapeptides have also been used for the development of new imaging tracers.
An important milestone in CXCR4 imaging was achieved by Demmer et al, in
developing a 68Ga-labeled cyclopentapeptide, [68Ga]CPCR4-2 ([68Ga]Pentixafor), that
has reached clinical trials due to its persistent tumor accumulation and favorable
pharmacokinetics.10,11
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NH
NH
N
NH
NH
N
H
H
N
N
HN
N
HN
N
H
F
Target molecule
AMD3465
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Figure 1. Chemical structures of the target molecule (MCFB or compound 5) and
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AMD3465.
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An extensive array of small molecule CXCR4 antagonists covering over 20 different
chemical classes of compounds have been developed and discussed;12 however, their
application to imaging has been limited. The cyclam AMD3465 (Figure 1) and
bicyclam AMD3100 currently represent the most studied class of non-peptide CXCR4
inhibitors for nuclear imaging, predominantly PET. The metal-chelating properties of
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cyclams have been used to radiolabel both AMD3100 and AMD3465 with Cu.13–16
While both compounds gave significant tumor uptake in a CXCR4 expressing
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transgenic cell line, [64Cu]AMD3465 was more hydrophilic and showed superior
pharmacokinetic properties in comparison to [64Cu]AMD3100, resulting in higher
tumor imaging contrast. Both compounds, however, exhibited considerable uptake in
the liver and kidneys; the liver uptake has been hypothesized to be partly due to
transchelation of 64Cu to liver. Other strategies have been evaluated, whereby
AMD3100 and AMD3465 were radiolabeled with 68Ga17 and 11C,18 respectively;
however, these copper-free approaches showed no improvements in biodistribution in
vivo. Notably the high liver uptake,14,18,19 independent of use of radiometal or
transchelation, has been assigned in part to the previously reported cytoplasmic and
cell-surface expression of CXCR4 protein in liver tissue.20 Less studied is, however,
the role of non-specific transporters - in particular organic cationic transporters (OCTs),
given the cationic nature of the cyclam probes - in the liver uptake of the tracers.
Despite the superior properties of 18F-radioisotope, such as low positron energy and
adequate half-life (109.7 min), strategies employing this radionuclide for small
molecule labeling are limited. A few pyrimidine-based derivatives were labeled with
18F. The small molecule and CXCR4 modulator, MSX-122, was shown to inhibit tumor
metastasis and inflammation in vitro; however, no in vivo imaging evaluation was
carried out.21 Another pyrimidine-pyridine derivative showed no probe accumulation
in CXCR4-expressing tumors due to rapid metabolism of the radioligand.22 Work by
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Poty et al. used AMD3100 analogues as precursors for F labeling, but no in vivo
validation was performed.23 A structurally similar tracer to AMD3465, [18F]RPS-544,
has been evaluated recently.24 The radioligand showed a moderate uptake in PC3-
CXCR4 tumor model and, in addition to substantial uptake in the liver and kidneys,
[18F]RPS-544 also accumulated in small and large intestines, differing from the
excretion profile of other labeled cyclams.
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In this study, we aimed at developing and evaluating a new CXCR4-targeting
radioligand that would capitalize on the advantages of 18F, whilst preserving the ability
to sensitively detect CXCR4 expression in tumors. Due to the potent binding affinity
and selectivity of AMD3465 towards CXCR4, this molecule was chosen as a reference
for the development of the tracer, [18F]MCFB.
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The most important aspect of the tracer design was high in vivo metabolic stability.
Pyridines can generally be reacted with [18F]fluoride in the 2 or 4 position to obtain the
corresponding labeled fluoropyridines.25 However, their in vivo stability is
unpredictable, often resulting in defluorination, with associated uptake of radioactivity
in bone.22,24 Therefore, to facilitate 18F labeling, the 2-pyridylmethylamine moiety was
substituted with 1-aminomethyl-4-fluorobenzene. The former was linked to the original
1,4-phenylenebismethylene linker by N-substituted ethylene chain. The introduction of
18F isotope was achieved by using the easily accessible prosthetic group,
[18F]fluorobenzaldehyde, which can be further converted into [18F]fluorobenzylamine
through reductive amination.26
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The applicability of [18F]MCFB for sensitive and specific imaging of CXCR4 was
evaluated, as well as its in vivo pharmacokinetics and biodistribution in relevant tumor
models.
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MATERIALS AND METHODS
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Materials
Unless otherwise indicated, reagents and solvents were purchased from Sigma-
Aldrich (Haverhill, UK) and used without further purification. AMD3465 was
purchased from Tocris Bioscience (R&D Systems, Abingdon, UK).
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4-N,N,N-trimethylammonium benzaldehyde (triflate salt) was synthesized according
to a method published in the literature.27 Flash column chromatography was carried
out on silica gel (Aldrich silica gel, pore size 60 Å, particle size 230-400 mesh). Thin
layer chromatography (TLC) was performed on Merck Silica 60 F254 plates. 1H NMRs,
13C NMRs and 19F NMRs were recorded on a Bruker AV-400 spectrometer at a
frequency of 400, 101 and 376 MHz, respectively. Chemical shifts (δ) are given in parts
per million (ppm) as referenced to the appropriate residual solvent peaks. Signals are
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assigned as s, d, m and br for singlet, doublet, multiplet and broad, respectively. C
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NMRs are complicated by existence of C-F coupling (long range including) and
conformers. Mass spectra (m/z) were obtained in a positive electrospray ionization on
a Waters LCT Premier (ES-ToF). Crude reaction mixtures were analyzed by TLC and
HPLC.
HPLC Methods (a) Purity analysis of nonradioactive and radioactive compounds
was carried out on a Shimadzu Prominence HPLC system equipped with a LC-10Ai
pump, a SPD-20A UV detector (λ= 254 nm), a Flow-RAM sodium iodide/PMT gamma
detector and a Laura 4.1.1.3 software (LabLogic); Phenomenex Gemini 5µ C18 150 x
4.6 mm and Luna 5µ Phenyl-Hexyl 150 x 4.6 mm columns were used and a mobile
phase comprising of Water (0.1%TFA)/MeCN with a gradient of 5% organic for 1 min,
5→95% in 16 min, 95% organic for 2 min, 95→5% organic in 2 min, delivered at a
flow rate of 1 mL/min. (b) Semi-preparative radio-HPLC was carried out using a
Shimadzu LC-20AT pump, a Bioscan Flowcount FC3200 sodium iodide/PMT gamma
detector (LabLogic) and a Knauer UV detector (λ= 254 nm). Sample injection, product
isolation and data collection were performed using an in-house build Multi-stream
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HPLC system and bespoke software package (Hammersmith Imanet Ltd., UK) and an
Agilent 5µ Exlipse XDB-C18 250 × 9.4 mm column with an isocratic mobile phase of
Water (0.1%TFA)/MeCN (62:38 v/v) at a flow rate of 3mL/min; injection loop 10 mL.
(c) Metabolite analysis was carried out using an Agilent 1100 series G1312A pump and
a LabLogic Laura 3 software equipped with a linear G1314A UV detector (λ=254 nm),
a γ-RAM model 3 detector and a Phenomenex Luna 5µ Phenyl-Hexyl 250 × 10 mm
column with an isocratic mobile phase of Water (0.1%TFA)/MeCN (80:20 v/v) at a
flow rate of 3mL/min; injection loop 1 mL.
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Synthesis of precursor and reference compound
1,1',1''-(1,4,8,11-tetraazacyclotetradecane-1,4,8-triyl)tris(2,2,2-trifluoroethan-
1-one) (1)
To a stirred solution of cyclam (1 g, 4.99 mmol) and triethylamine (0.69 mL, 4.99
mmol) in methanol (10 mL) was added dropwise ethyl trifluoroacetate (2.36 mL, 19.9
mmol). The homogeneous reaction mixture was cooled with an ice-water bath to control
the mild exothermic reaction. Stirring was continued under N2 overnight. Volatiles were
removed under reduced pressure. The residue was dissolved in EtOAc and passed
through the plug of silica. The solvent was concentrated to give the product as a white
foam (2.6 g, 93%). HRMS (ESI) = 489.1549 (M + H)+. Calc. for C16 H22 N4 O3 F9:
489.1548. 1H NMR (400 MHz, CDCl3): δ 3.91-3.34 (m, 12H), 3.07-2.84 (m, 2H), 2.81-
2.50 (m, 2H), 2.40-2.07 (m, 2H), 1.98-1.67 (m, 2H), 1.17-0.60 (bs, 1H). 13C NMR
(101 MHz, CDCl3): δ 160.06-154.85 (m, C=O,), 121.77-111.81 (q, CF3, J=288.6 Hz),
52.55-43.85 (m, CH2-N), 31.74-25.94 (m, CH2). 19F NMR (376 MHz, CDCl3): δ -63.56
– -72.90 (m).
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1,1',1''-(11-(4-(bromomethyl)benzyl)-1,4,8,11-tetraazacyclotetradecane-1,4,8-
triyl)tris(2,2,2-trifluoroethan-1-one) (2)
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To a stirred solution of 1 (1.95 g, 3.99 mmol) and K2CO3 (717 mg, 5.2 mmol) in
CH3CN (70 mL) was added α,α’-dibromo-p-xylene (5.25 g, 19.9 mmol) and the
reaction mixture stirred at 70oC for 1 h. The solution was cooled to room temperature
and the solvent removed under reduced pressure. The residue was partitioned between
brine (50 mL) and CH2Cl2 (100 mL). The organic phase was separated, dried (Na2SO4)
and silica added, the mixture was evaporated to dryness. The compound impregnated
on silica was purified by flash chromatography (first with CH2Cl2, followed by
CH2Cl2/Ether, 9:1) to give the desired compound as a white solid (1.8 g, 67%). HRMS
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(ESI) = 671.1273 (M + H)+. Calc. for C24 H29 N4 O3 Br F9: 671.1279. H NMR (400
MHz, CDCl3): δ 7.55 – 7.01 (m, 4H), 4.50 (s, 2H), 3.99 – 3.23 (m, 14H), 2.95 – 2.62
(m, 2H), 2.62 – 2.27 (m, 2H), 2.28 – 1.92 (m, 2H), 1.90 – 1.43 (m, 2H). 13C NMR (101
MHz, CDCl3): δ 158.07– 156.48 (m), 138.23 – 136.41 (m), 130.55 – 128.42 (m), 120.99
– 111.59 (m), 61.02 – 58.94 (m), 56.06 – 44.52 (m), 33.30 – 32.47 (m), 29.95 – 23.65
(m). 19F NMR (376 MHz, CDCl3): δ -68.31 – -69.57 (m).
1,1',1''-(11-(4-(((2-aminoethyl)amino)methyl)benzyl)-1,4,8,11-
tetraazacyclotetradecane-1,4,8-triyl)tris(2,2,2-trifluoroethan-1-one) (3)
To a stirred solution of ethylenediamine (134 mg, 2.2 mmol) and K2CO3 (93 mg,
0.675 mmol) in dry CH3CN (5 mL) at -10 oC (acetone/ice bath) was added dropwise a
solution
of
1-[1-methylene-4-(bromo-methylene)phenylene]-4,8,11-
tris(trifluoroacetyl)-1,4,8,11-tetra-azacyclotetradecane (2) (300 mg, 0.45 mmol) in
CH3CN (20 mL) over 15-20 min. After stirring for a further 30 min, the reaction mixture
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was filtered and concentrated to provide a residue which was purified by
chromatography on silica gel using 1% TEA in EtOAC:MeOH (7:3) to give the product
as a white solid (180 mg, 62%). HRMS (ESI) = 651.2708 (M + H)+. Calc. for C26 H36
N6 O3 F9: 651.2705. 1H NMR (400 MHz, DMSO-d6): δ 7.33 – 7.10 (m, 4H), 3.87 – 3.18
(m, 15H), 3.09 – 2.24 (m, 12H), 2.18 – 1.95 (m, 2H), 1.83 – 1.63 (m, 2H). 13C NMR
(101 MHz, DMSO-d6): δ 157.30– 154.95 (m), 141.15 – 139.76 (m), 137.61 – 136.04
(m), 130.23 – 127.12 (m), 121.54 – 111.81 (m), 59.71 – 58.30 (m), 54.43 – 43.32 (m),
41.65 (s), 29.51 – 22.06 (m). 19F NMR (376 MHz, DMSO-d6): δ -66.66 – -74.74 (m).
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1,1',1''-(11-(4-(((2-((4-fluorobenzyl)amino)ethyl)amino)methyl)benzyl)-
1,4,8,11-tetraazacyclotetradecane-1,4,8-triyl)tris(2,2,2-trifluoroethan-1-one) (4)
To a stirred solution of the amine 3 (100 mg, 0.153 mmol) in dry CH3CN (10 mL),
at room temperature, were added 4-fluorobenzaldehyde (12.6 μL, 0.118 mmol), and
catalytical amount of glacial acetic acid. The resulting solution was stirred at room
temperature for 1 h and NaBH(OAc)3 (75 mg, 0.354 mmol) was added. The mixture
was left to stir overnight. Acetonitrile was removed under vacuum. The residue was
dissolved in EtOAc, washed with saturated aqueous NaHCO3, dried (Na2SO4),
concentrated, and purified by chromatography on silica gel using 0.5% TEA in
EtOAC:MeOH (8:2). The desired product was obtained as a white solid (40 mg, 34%).
MS (ESI): m/z 759 [MH+]. 1H NMR (400 MHz, DMSO-d6): δ 7.64 – 6.76 (m, 8H), 3.89
– 3.23 (m, 19H), 3.19 – 2.53 (m, 6H), 2.49 – 2.21 (m, 2H), 2.22 – 1.93 (m, 3H), 1.88-
1.60 (m, 2H). 13C NMR (101 MHz, DMSO-d6): δ 162.68 (s), 160.28 (s), 158.23– 154.29
(m), 140.47 – 139.58 (m), 138.16 – 135.95 (m), 130.84 – 127.03 (m), 121.41 – 111.86
(m), 115.25-115.04 (d, J=21.1 Hz), 59.86 – 58.02 (m), 54.38 – 43.14 (m), 52.46 (s),
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48.49 (s), 31.53 – 22.35 (m). 19F NMR (376 MHz, DMSO-d6): δ -67.85 – -68.75 (m), -
116.76 (s).
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N1-(4-((1,4,8,11-tetraazacyclotetradecan-1-yl)methyl)benzyl)-N2-(4-
fluorobenzyl)ethane-1,2-diamine (5)
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To a stirred solution of the trifluoroacetyl-protected amine 4 (70 mg, 0.09 mmol) in
MeOH (2.0 mL), at room temperature was added 5N NaOH (200 μL). The resulting
solution was stirred at room temperature overnight. The solvent was removed under
reduced pressure, and CH2Cl2 (5 mL) and water (2.0 mL) were added to the residue.
The phases were separated, and the aqueous phase was extracted with CH2Cl2 (3 × 5
mL). The combined organic phases were washed with 1N NaOH, dried (Na2SO4) and
concentrated to give a free base (40 mg, 93%). HRMS (ESI) = 471.3609 (M + H)+.
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Calc. for C27 H44 N6 F: 471.3611. H NMR (400 MHz, CDCl3): δ 7.31-7.05 (m, 6H),
7.04-6.82 (m, 2H), 3.67 (s, 4H), 3.48 (s, 2H), 2.85-2.31 (m, 23H), 1.87-1.70 (m, 2H),
1.67-1.51 (m, 2H). 13C NMR (101 MHz, CDCl3): δ 163.07 (s), 160.64 (s), 139.04 (s),
137.47 (s), 136.23 (d, J=3.2 Hz), 129.62- 127.86 (m), 115.34-114.84 (d, J=21.2 Hz),
57.45 (s), 54.53 (s), 53.67 (s), 53.27 (s), 53.19 (s), 50.83 (s), 49.36 (s), 49.17 (s), 48.97
(s), 48.78 (s), 48.02 (s), 47.41 (s), 28.66 (s), 26.25 (s). 19F NMR (376 MHz, CDCl3): δ
-116.25 (s).
Radiosynthesis of [18F]MCFB
The no-carrier-added aqueous [18F]fluoride solution, typically 1.5 mL, 1.4 GBq,
was transferred to a FASTlabTM automated synthesis module (GE Healthcare Life
Sciences, Amersham, UK). The activity was trapped on a Waters QMA-carbonate Sep-
Pak SPE cartridge and eluted into a reaction vessel with 1000 μL of an eluent solution
(800 μL of Kryptofix 2.2.2 (6mg/mL in acetonitrile), 200 μL of KHCO3 (3.5mg/mL in
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H2O)). The eluate was evaporated to dryness by a combination of vacuum and nitrogen
flow at 120°C for 12 min. After drying, anhydrous acetonitrile (400 μL) was added to
a dried residue in the reaction vessel, and the remaining of the radiosynthesis was
carried out manually.
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To a Wheaton vial containing 2.0 mg of 4-trimethylammonium-benzaldehyde
trifluoromethanesulfonate precursor dissolved in acetonitrile (100 μL) dried
[18F]fluoride (370-555 MBq in 300 μL acetonitrile) was added at ambient temperature.
The labeling reaction was conducted at 90°C for 15 min before allowing it to reach
room temperature. A solution of cyclam-amine (6.0 mg) in acetonitrile (100 μL) was
then added followed by heating at 50°C for 15 min. Afterwards, reaction mixture was
cooled again to ambient temperature and neat sodium triacetoxyborohydride (4.5 mg)
was added and heating resumed for 30 min at 50°C. The reaction was quenched by
addition of 0.1%TFA in water (10 mL) and purified by semi-preparative radio-HPLC.
The fraction of eluent containing [18F]-4 was collected and diluted to a final volume of
40 mL with water, then immobilized on a Sep Pak tC18 light cartridge (Waters) [pre-
conditioned with acetonitrile (5 mL) and water (10 mL)]. The cartridge was then
washed with water (5 mL) followed by elution of the radioligand using ethanol (300
μL). To a solution of ethanol was added 1M NaOH (200 μL) and the mixture was left
at room temperature for 15 min. Formulation was achieved by addition of 1M HCl to
provide [18F]MCFB.
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Radiochemical purity was confirmed by co-elution with a reference compound
(Supplementary Figure 1).
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LogD determination
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Briefly, 7 MBq of the ligand in 20 μL of phosphate buffered saline (PBS) was added
to a mixture of 2 mL of n-octanol and 2 mL of PBS, pH 7.4; the n-octanol was washed
with PBS before use. The mixture was vigorously shaken for 1 h at room temperature
and then centrifuged at 5000 rpm for 5 min; 100 μL aliquots from each of the two phases
were drawn and their radioactivity content was measured in a γ-counter (LKB Wallac
1282 Compugamma laboratory gamma counter, PerkinElmer, Massachusetts, USA).
The n-octanol/PBS partition coefficient was determined by dividing the radioactivity
found in the n-octanol layer by that found in the PBS layer. The LogDOctanol/PBS value is
reported as the average of three independent experiments.
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Cell culture
The human triple-negative metastatic breast cancer cell lines MDA-MB-231-luc,
MDA-MB-231 shSC, and MDA-MB-231 shCXCR4, the CXCR4-overexpressing
glioma cell line U87.CD4.CXCR4, as well as the diffuse large B-cell lymphoma cell
lines U2932 and SuDHL8 were cultured in Roswell Park Memorial Institute (RPMI)
Medium (Sigma-Aldrich). MDA-MB-231-luc (hitherto referred to as MDA-MB-231)
is a luciferase-expressing wild-type MDA-MB-231 cell line obtained from PerkinElmer
(Waltham, MA, USA). CXCR4 expression was silenced by doxycycline-inducible
shRNA targeting CXCR4 (pTRIPZ CXCR4 shRNA clone V3THS_346208;
Dharmacon, Lafayette, USA) to obtain MDA-MB-231 shCXCR4, or non-targeting
shRNA (Dharmacon) to obtain MDA-MB-231 shSC. Viruses were packaged using
Trans-Lentiviral shRNA Packaging System (Dharmacon). Stable clones were FACS
sorted based on the expression of a doxycycline-inducible TurboRFP reporter gene
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within the same construct. CXCR4 knockdown was induced by incubation with
doxycycline-hyclate (Sigma-Aldrich) for indicated times. All cell lines were cultured
in the presence of 10% Fetal Bovine Serum (FBS) (Sigma-Aldrich), 2mM L-glutamine
(Invitrogen, Carlsbad, USA) and 100 U/mL penicillin-streptomycin (Invitrogen) in a
humidified atmosphere of 5% CO2 at 37°C.
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[125I]SDF-1 ligand binding assay
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The [125I]SDF-1 ligand binding to CXCR4 on U87.CD4.CXCR4 cells and
antagonism by [19F]MCFB was conducted as previously described by us.28 AMD3465
was used as positive control.
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In vitro radioligand uptake
Cells were seeded in 6-well plates at a density of 400,000-600,000 cells/well 24h
prior to the experiment. For blocking studies, U2932 and SuDHL8 cells were treated
with vehicle or 500 µg/mL AMD3465 for 5 min before addition of radioactivity. For
knockdown studies, MDA-MB-231 shCXCR4 or relevant shSC control cells were
treated with doxycycline at a concentration of 0.5 μg/mL for 24 h prior to uptake
studies. In hypoxia studies, cells were placed within a filtered hypoxia chamber (Oxoid
Anaerojar, Thermo Fisher Scientific) that achieves < 1% O2, and incubated at 37°C,
5% CO2 (balance N2) for 16 h. Cells evaluated under normoxic conditions were
incubated in 21% O2, 5% CO2 and balance N2 for 16 h at 37°C.
Cells were then incubated with radioligand at 0.74 MBq/well for 60 min at 37°C,
under 5% CO2. Following incubation, cells were placed on ice, and washed 3 times
with ice-cold PBS. After washing, cells were lysed with RIPA buffer (Sigma-Aldrich)
for 5 min. The radioactivity of the lysate was measured by γ-counting. Counts/min data
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were expressed as a percentage of the incubated dose (ID) of radioactivity in each well
and total cellular protein concentration of the sample, determined using Pierce® BCA
assay (Thermo Fisher Scientific), i.e., %ID/mg protein.
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In vitro internalization studies
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For internalization experiments, MDA-MB-231 cells were plated in 6-well plates at
a density of 600 000 cells/well and allowed to recover for 24 h at 5% CO2, at 37°C. On
the day of the assay, cells were incubated with approximately 0.74 MBq/well of
[18F]MCFB for 30 min at 4°C (assuming inhibition of receptor internalization at this
temperature) or at 37°C. In conditions incubated at 4°C, the media was replaced with
fresh 37°C media and incubated for 5, 15, 30 and 60 min at 37°C. After incubation,
surface-bound radioactivity was removed by washing the cells with 400 µL of ice-cold
50 mM glycine in 150 mM NaCl (pH=3) for 5 min on ice, followed by two washes with
ice-cold PBS. To obtain the internalized fraction, cells were lysed with 400 µL of 1M
of NaOH. The radioactivity of the internalized fraction was measured by γ-counting.
Counts/min data were expressed as a percentage of the incubated dose (ID) of
radioactivity in each well and total cellular protein concentration of the sample,
determined using Pierce® BCA assay, i.e., %ID/mg protein. The percentage of
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internalization was calculated as:
internalized fraction
Internalization(%)=
x 100
surface bound fraction + internalized fraction
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Immunoblotting
For western blot analysis, cells (80-90% confluency) were placed on ice, the medium
was removed, and cells were washed 3 times with ice-cold PBS and lysed with RIPA
buffer supplemented with 100X Pierce™ protease and phosphatase inhibitor cocktail
(Thermo Fisher Scientific) for 10 min on ice. Whole lysates were sonicated, and total
protein concentration was quantified using the Pierce™ BCA protein assay kit (Thermo
Fisher Scientific). Lysates were mixed with NuPage® LDS loading buffer and reducing
agent (Invitrogen), and denatured at 70°C for 10 min. Equal amounts of protein (30
μg) were resolved on 4-15% Mini-PROTEAN® TGX™ gels (Bio-Rad, Hemel
Hempstead, UK) and separated by gel electrophoresis at 290V for 15 min. The gels
were then transferred to PVDF membranes (Trans-Blot Turbo Transfer Packs, Bio-
Rad) using the Trans-Blot® Turbo™ System (Bio-rad). Membranes were blocked for
1 h in 5% milk in PBS containing 0.1% v/v Tween® 20 (Sigma-Aldrich) (PBST) and
incubated with rabbit anti-human CXCR4 clone UMB2 (1:1000; ab124824, Abcam),
Danvers, USA), and mouse anti-HIF-1α clone 54 (1:1000; BD Biosciences, USA)
overnight at 4°C. After washing with PBST, goat anti-rabbit Immunoglobin G (IgG)
horseradish peroxidase (IgG-HRP) (1:2000, SC-2004, Santa Cruz Biotechnology,
Dallas, USA) or goat anti-mouse lgG-HRP (1:2000, SC-2005, Santa Cruz
Biotechnology) were incubated in 5% milk-TBST for 1 hour at room temperature.
Signals were detected using Amersham enhanced chemiluminescence (ECL) Plus
Western Blotting Detection Reagent kit (GE Healthcare Life Sciences) and Amersham
Hyper-film (GE Healthcare Life Sciences). Intensity of protein bands was normalized
to β-actin (mouse anti-β –actin antibody (1:10,000, ab6276, Abcam)) and analyzed
using ImageJ version 1.44h (National Institutes of Health).
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Immunofluorescence
For immunofluorescence staining of tumor CXCR4 expression, formalin-fixed
paraffin embedded tumor slices (of 5 µm thickness) were stained with rabbit anti-
CXCR4 UMB2 clone antibody (1:200) in blocking buffer overnight at 4°C. The slides
were subsequently washed with PBST thrice and incubated with Alexa Fluor® 488 goat
anti-rabbit IgG secondary antibody (1:400; Molecular Probes™, Thermo Fisher
Scientific) in blocking buffer for 1 h at room temperature, protected from light.
Following another washing step, the slides were mounted using ProLong® gold
antifade reagent with 4’-6-diamidino-2-phenylindole (DAPI) (Life Technologies Ltd.,
Thermo Fisher Scientific). Immunofluorescence imaging was performed using a 40X
or 60X UPlanAPO objective lens on an Olympus BX-51 wide-field microscopy UIS2
optical system (Olympus Life Science Europa GMBH, Hamburg, Germany) equipped
with a DP70 digital camera. Images were acquired using an Olympus U-RFL-T
epifluorescence source and DPController 1.2.1.108 imaging software (Olympus
Optical Co. Ltd, Tokyo, Japan) in the red, blue and green channels. Image processing
was performed using ImageJ version 1.44h.
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In vivo radioligand uptake
All animal experiments were done by licensed investigators in accordance with the
UK Home Office Guidance on the Operation of the Animal (Scientific Procedures) Act
1986 (HMSO, London, UK, 1990) and within guidelines set out by the UK National
Cancer Research Institute Committee on Welfare of Animals in Cancer Research.29
The in vivo models were established in female athymic nude mice (MDA-MB-231
tumor model) or in NOD/SCID mice (U2932 and SuDHL8 tumor models) aged 6 to 8
weeks (Harlan, Bicester, UK Ltd). Xenografts were established under 2.5% isoflurane
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anaesthesia by subcutaneous injection of MDA-MB-231 cell line (5x106 cells in 100
µL PBS) or U2932 and SuDHL8 (5x106 cells in 100 µL of 50% PBS and 50% Matrigel
(Corning, Amsterdam, The Netherlands)) in the back of the neck of the mice. Tumor
dimensions were measured by caliper and volumes calculated using the ellipsoid
formula that is best for estimating tumor mass: Volume (mm3) = (π/6) x a x b x c, where
a, b and c represent 3 orthogonal axes of the tumor. Mice were used for imaging when
tumor volume reached approximately 100 mm3 (at approximately 4-6 weeks post-
induction).
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For imaging, mice were anesthetized with 2.5% isoflurane and placed in a
thermostatically controlled rig in a dedicated small animal Genisys4 PET scanner
(SOFIE Biosciences, Culver City, USA). Following injection of 0.925 MBq of
[18F]MCFB via lateral tail vein cannula, PET scans were acquired in a list-mode format
over either 0-60 min (dynamic scan) or 40-60 min after injection (static scan) to give
decay-corrected values of radioactivity accumulation in tissues. The collected data were
ordered into 6 (dynamic scan) or 2 (static scan) time frames of 600 seconds and
reconstructed with a 3-dimensional maximum likelihood estimation method (3D ML-
EM). Volumes of interest (VOIs) for tumors and different organs were defined using
Siemens Inveon Research Workplace software (Siemens Molecular Imaging Inc.,
Knoxville, USA) and the count densities (MBq/mL) of these VOIs were averaged for
the time points corresponding to 40-60 min (where equilibrium was observed). Tissue
radioactivity uptake values were normalized to whole-body uptake, a method used in
previous publications.30–32
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Metabolism Studies
Non-tumor-bearing were injected intravenously with 3.7 MBq of [18F]MCFB.
Plasma, urine and liver were collected at the indicated time and were snap-frozen in
liquid nitrogen for subsequent HPLC analysis. For extraction, ice cold CH3CN (1.0 mL)
was added to plasma. The mixture was centrifuged (15,493 x g, 4°C, 3 min) and the
resulting supernatant was evaporated to dryness under vacuum at 40°C using a rotary
evaporator. Liver samples were diced and homogenized with ice cold CH3CN (1.0 mL)
using Precellys® 24 homogenizer prior to centrifugation. The supernatant was then
decanted and evaporated to dryness. The samples were resuspended in HPLC mobile
phase (1.2 mL) and filtered through a 13 mm Pall Acrodisc syringe filter (0.2 μm). The
samples were analyzed by radio-HPLC.
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Biodistribution studies were carried out 60 min after injection of 0.925 MBq of
[18F]MCFB via lateral tail vein cannula. Tissue samples were quickly collected, and
radioactivity content was determined by γ-counting and normalized to sample weight.
Treatment with metformin hydrochloride (Sigma-Aldrich) was carried out by injection
of 50 mg/kg of metformin intravenously in U2932-bearing mice 30 min prior injection
of radioactivity. The dose of metformin selected was based on the work by Wilcock
and Bailey33 that indicates that a 50 mg/kg bolus is equivalent to the maximum
recommended daily dosage in humans.
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Statistics
Data were expressed as mean ± SEM. Statistical analysis was done using unpaired 2-
tailed Student’s t-test or two-way ANOVA with Bonferroni correction as appropriate
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and defined as significant (*, 0.01<p<0.05), very significant (**, 0.001<p<0.01) and
extremely significant (***, p<0.001).
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Non-radioactive synthesis of precursor 3 and reference compound 5
O
O
Br
Br
F3C
N
N
N
F3C
N
HN
N
NH HN
EtOTFA, TEA
MeOH, rt
O
O
K2CO3, CH3CN, reflux
Br
N
N
HN
NH
CF3
CF3
O
O
F3C
F3C
2
(67%)
H2N
1
(93%)
NH2
K2CO3,
CH3CN,
-10 o
O
O
O
F3C
N
N
N
N
C
F
F3C
H
N
N
N
H
N
O
H
N
H
O
NaBH(OAC)3, CH3CN, rt
N
O
N
NH2
O
F3C
F
CF3
F3C
CF3
3
(62%)
4
(34%)
5M NaOH
MeOH, rt
NH
NH
N
H
N
HN
N
H
F
5
(93%)
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Scheme 1. Synthesis of compound 5 from cyclam.
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The synthesis of precursor 3 and reference compound 5 is shown in (Scheme 1). The
protection of cyclam with EtOTFA was done using previously optimized conditions
with triethylamine.34 The subsequent N-alkylation of the tri-protected cyclam 1 with α,
α’-dibromo-p-xylene using K2CO3 in acetonitrile gave the desired bromomethyl
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intermediate 2 in 67% isolated yield. Initial attempts to alkylate ethylenediamine with
intermediate 2 at elevated temperature unfortunately increased formation of by-
products, which were subsequently minimized by performing the reaction at -10°C and
using an excess of the diamine to give a 62% yield. Additionally, the precursor 3 was
further used for the preparation of reference compound 5. First, the compound 4 was
prepared by the reductive amination of 4-fluorobenzaldehyde with 3 using
NaBH(OAc)3 in acetonitrile. Straight-forward mild deprotection of the amides via
MeOH in aqueous NaOH afforded free base within 16 hours in a yield of 93%.
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Radiochemistry and Partition Coefficient
The labeled variant of 5 was prepared via a three-step procedure: a) radiosynthesis of
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the F-labeling prosthetic group 4-[18F]fluorobenzaldehyde ([18F]FBA), followed by
b) reductive amination and c) global deprotection. [18F]FBA was synthesized from 4-
N,N,N-trimethylammonium benzaldehyde (triflate salt) via a standard fluorination route
(K[18F]F/Kryptofix 222) in acetonitrile at 90°C for 15 min and was used for the next
step without further purification (Scheme 2A). Substitution of potassium carbonate for
the less basic potassium hydrogen carbonate improved reproducibility of the reaction
significantly, most likely due to the higher stability of the precursor under these
conditions (as seen by the UV trace from analytical HPLC of the incorporation). The
average radiochemical incorporation as measured by analytical radio-HPLC was >95%.
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Scheme 2. Radiosynthesis of [18F]MCFB. A. Synthesis of [18F]FBA. B. Synthesis of
[18F]MCFB from precursor 3 using [18F]FBA.
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The subsequent step was the reductive alkylation of amine 3 with the intermediate
[18F]FBA using NaBH(OAc)3 as the reducing agent (Scheme 2B). The reaction was
performed as one-pot two-step synthesis. Parameters such as temperature, solvent,
reaction time and sequence of reagent addition were investigated. The optimal
conditions were found to be 15 min reaction time for the initial formation of the imine
(which was not isolated) and a further 30 min for subsequent reduction with
NaBH(OAc)3. The solvent of choice for both steps was acetonitrile, with the reaction
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carried out at 50°C to produce the alkylated amine [18F]4.
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Finally, deprotection of [18F]4 was carried out using 1M NaOH at room temperature
affording the desired molecule [18F]MCFB with complete conversion within 10 min.
As the procedure was carried out manually with low amounts of radioactivity, only a
modest specific activity (5.7 GBq/μmol) was achieved. The radiosynthetic procedure
from 18F fluoride including formulation of [18F]MCFB took 2.5 h in total. Overall, the
decay-corrected yield of 14.1 ± 2.5 % (n=5) was achieved.
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The LogDOctanol/PBS of [18F]MCFB was found to be -1.64 ± 0.06.
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Competitive Binding, Receptor Expression and Internalization of [18F]MCFB
Binding of [19F]MCFB to CXCR4 was estimated from its inhibitory effect on binding
of the native ligand CXCL12 to CXCR4 in cells. [19F]MCFB or AMD3465 (positive
control) inhibited CXCL12-CXCR4 interaction in a concentration-dependent manner
(Supplementary Figure 2). The 50% inhibitory values (IC50) for [19F]MCFB and
AMD3465 were 111.3 ± 4.0 and 89.8 ± 3.5 nM, respectively.
For in vitro binding studies, we used a highly-expressing native CXCR4 B-cell
lymphoma cell line, U2932, and compared it against another B-cell lymphoma cell line
with lower CXCR4 expression, SuDHL8.35 The [18F]MCFB uptake was higher in
U2932 cell line (0.86 ± 0.15%ID/mg) than in SuDHL8 (0.52 ± 0.05%ID/mg) and co-
incubation with AMD3465 resulted in partial inhibition of tracer binding (37% for
U2932 and 45% for SuDHL8). The difference in tracer uptake between cell lines was
smaller than the variation of CXCR4 protein expression (96% difference of CXCR4/β-
actin) (Figure 2A), and together with the partial blocking of tracer uptake after
AMD3465 treatment, suggests some level of non-specific binding.
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Figure 2. In vitro characterization of [18F]MCFB. A. Uptake of [18F]MCFB normalized
to cellular protein in U2932 and SuDHL8 cell lines, with or without blocking for 5 min
with AMD3465. B. Effect of CXCR4 knockdown on [18F]MCFB radioactive uptake.
MDA-MB-231 cells transduced with a doxycycline-inducible CXCR4-targeted
(shCXCR4) short hairpin RNA construct were incubated under normoxia or hypoxia
(< 1% O2 for 16 h) in the presence or absence of doxycycline (0.5 mg/ml for 24 h).
Typical western blots of CXCR4 expression (and β-actin for loading control) in U2932,
SuDHL8 and MDA-MB-231 shCXCR4 cell lines are shown below. C. Internalization
of [18F]MCFB in MDA-MB-231 cell line. All data are mean ± SEM, n=6.
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Specific uptake of [18F]MCFB was further confirmed by the use of isogenic MDA-MB-
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(shCXCR4). Tracer accumulation was 2-fold higher in hypoxia-induced cells compared
to normoxic conditions, in accordance with the higher CXCR4 expression (Figure 2B).
Hypoxia induction was confirmed by HIF-1α expression (Supplementary Figure 3).
Receptor knockdown in the MDA-MB-231 shCXCR4 cell line by treatment with
doxycycline resulted in reduced tracer uptake, again demonstrating [18F]MCFB
sensitivity for CXCR4 expression. In the control MDA-MB-231 shSC cell line, cellular
uptake of [18F]MCFB was unperturbed by the doxycycline treatment, as expected
(Supplementary Figure 4). To determine uptake mechanism, we incubated MDA-
MB-231 cells with [18F]MCFB at 4°C, subsequently transitioning them to 37°C.
[18F]MCFB was rapidly internalized, with the internal fraction reaching 40% of the total
uptake within the first 15 min (Figure 2C). Indeed, at 37°C, the internalized fraction
was 9.8 ± 0.9%ID/mg, whereas at 4°C, only 2.9 ± 0.3%ID/mg was found intracellularly
(Supplementary Figure 5), pointing to the role of internalization as part of tracer
uptake mechanism.
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PET Imaging
Subsequently, we investigated tracer accumulation in mice bearing U2932 and
SuDHL8 tumors, together with a non-transgenic variant of MDA-MB-231.
Representative PET images show high [18F]MCFB accumulation in the tumor models
at 60 min post-radioligand injection (Figure 3A). Uptake was 2-fold higher in U2932
(T/WB ratio 4 ± 0.8) than in SuDHL8 (T/WB ratio 2.1 ± 0.3) and MDA-MB-231 (T/WB
ratio 1.6 ± 0.1) (Figure 3B). Interestingly, the signal uptake (U2932 > SuDHL8 >
MDA-MB-231) was not in accordance with expression of CXCR4 (U2932 > MDA-
MB-231 > SuDHL8).
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Figure 3. [18F]MCFB discriminates differential CXCR4 expression in vivo. A.
Representative sagittal [18F]MCFB-PET images derived from summed 60 min dynamic
scans in U2932, SuDHL8 and MDA-MB-231 xenograft-bearing mice. CXCR4
expression in excised tumors was determined by western blot (results shown for 3
independent samples per tumor model). B. Quantification of [18F]MCFB uptake in the
different tumor models by region of interest analysis normalized to whole-body
radioactivity. Data are mean ± SEM, n=4. C. Representative axial [18F]MCFB-PET
image of a MDA-MB-231 xenograft-bearing mouse where necrosis is visible. White
arrows indicate tumors.
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This mismatch may be due to the central necrotic areas identified in the MDA-MB-
231 tumors (Figure 3C). This phenotype has been described for this tumor model36 and
the compromised vasculature can significantly reduce tracer delivery, not only to the
necrotic core but also to the surrounding tissue. Thus, receptor binding in the whole
tumor is likely to be affected, hindering differences in uptake between treatment groups
as was evident in our work. High tracer uptake was also seen in liver, bladder and the
snout region. Immunofluorescent staining of the excised tumors showed expression of
CXCR4 in both cell lymphoma lines (Figure 4A), although significantly higher in the
U2932 compared to the SuDHL8 (p<0.001) (Figure 4B).
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Figure 4. A. Representative images of excised tumor tissues stained by
immunofluorescence for CXCR4 (green) and DAPI (blue). B. Quantification of
CXCR4-fluorescence intensity normalized to DAPI.
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[18F]MCFB Biodistribution
Biodistribution was carried out in U2932 and SuDHL8 tumor-bearing mice 60 min
after injection of [18F]MCFB (Figure 5A). Tumor uptake was 3.3 ± 0.9%ID/g for
U2932 tumors, and 1.81 ± 0.04%ID/g for SuDHL8 tumors. The low levels of bone
uptake for both SuDHL8 and U2932-bearing mice (1.6 ± 0.3%ID/g and 1.4 ± 0.2%ID/g,
respectively) suggest that tracer defluorination did not ensue upon intravenous
injection, and renal elimination occurred as indicated by the high tracer localization in
kidneys (42.1 ± 1.8%ID/g and 43.1 ± 7.3%ID/g, respectively) and urine (521.7 ±
178.5%ID/g and 688.8 ± 50.4%ID/g, respectively). High [18F]MCFB accumulation in
the liver was also seen (51.9 ± 2.9%ID/g for SuDHL8 and 63 ± 4.9%ID/g for U2932-
bearing mice).
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Figure 5. Biodistribution of [18F]MCFB. A. Mice bearing U2932 and SuDHL8
xenografts B. Mice bearing U2932 treated with 50 mg/kg of metformin for 30 min were
injected with 1.48 MBq of [18F]MCFB and tissues were excised 60 minutes after
injection. Radioactivity in the tissues was counted and is expressed as fraction of
injected dose normalized to tissue weight. Data are mean ± SEM, n=4.
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Radio-chromatographic analysis of plasma and liver extracts obtained at 15 or 60
min after [18F]MCFB injection showed only parent compound indicating good in vivo
metabolic stability (Supplementary Figure 6) suggesting that the high liver uptake
was unlikely due to metabolism. Similarly to AMD3465, [18F]MCFB has a positive
charge at physiological pH. We hypothesized that polyspecific transporters, such as the
organic cation transporter 1 (OCT1), 2 (OCT2) and 3 (OCT3), may be involved in the
transport of the tracer into the metabolic organs. To test this hypothesis, we treated
U2932 tumor-bearing mice with 50 mg/kg of metformin, an oral insulin-sensitizing
drug that is a substrate for OCT1, OCT2 and OCT3, and imaged the mice with
[18F]MCFB (Figure 5B and Supplementary Figure 7). Biodistribution studies
showed a marked decrease in urinary excretion of [18F]MCFB after metformin
treatment (688.8 ± 50.4% to 2.8 ± 0.4%ID/mg), and moderate decrease in the kidneys
and liver; all the other organs showed small increases in [18F]MCFB uptake.
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DISCUSSION
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We report the development of the 18F cyclam-based radioligand, [18F]MCFB,
designed for sensitive detection of CXCR4. Several PET-dedicated tracers have been
developed for the imaging of CXCR4 by exploiting modifications of therapeutically
relevant antagonists to accommodate different radioisotopes. Cyclam-based tracers,
such as AMD3100 and AMD3465, have shown promising characteristics for CXCR4
imaging; however, they have also been plagued with high hepatic accumulation. Given
that CXCR4 is expressed in the liver, some degree of specific liver uptake is expected.
Most strategies employ 64Cu for the radiolabeling of cyclams, whose structures
accommodate metals without the need of a chelator. However, 64Cu is known to
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