Near-Infrared-Emitting BODIPY-trisDOTA111In as a Monomolecular Multifunctional Imaging Probe: From Synthesis to In Vivo Investigations

Article abstract of DOI:10.1002/chem.201602886

A new generation of monomolecular imaging probes (MOMIP) based on a distyryl-BODIPY (BODIPY=boron-dipyrromethene) coupled with three DOTA macrocycles has been prepared (DOTA=1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid). The MOMIP presents good fluorescence properties and is very stable in serum. The bimodal probe was conjugated to trastuzumab, and an optical in vivo study showed high accumulation of the imaging agent at the tumor site.¹¹¹In radiometallation of the bioconjugate was performed in high radiochemical yield, highlighting the potential of this new BODIPY-chelators derivative as a bimodal imaging probe.

Full text of DOI:10.1002/chem.201602886

A Journal of
Accepted Article
Title: NIR emitting BODIPY-based MOnomolecular Multifunctional Imaging Probes:
from synthesis to in vivo investigations
Authors: Nicolas Maindron; Martin Ipuy; Claire Bernhard; Damien Lhenry; Mathieu
Moreau; Sabin Carme; Alexandra Oudot; Bertrand Collin; Jean-Marc Vringe-
aud; Peggy Provent; Francois Bruntte; Franck Denat; Christine Goze
This manuscript has been accepted after peer review and the authors have elected
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Object Identifier (DOI) given below. The VoR will be published online in Early View as
soon as possible and may be different to this Accepted Article as a result of editing.
Readers should obtain the VoR from the journal website shown below when it is pu-
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of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.201602886
Link to VoR: http://dx.doi.org/10.1002/chem.201602886
Supported by

Chemistry - A European Journal
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COMMUNICATION
NIR
emitting
BODIPY-trisDOTA111In
as
MOnomolecular
Multifunctional Imaging Probe: from synthesis to in vivo
investigations
Nicolas Maindrona§, Martin Ipuya§, Claire Bernharda, Damien Lhenrya, Mathieu Moreaua, Sabin Carmea,
Alexandra Oudotc, Bertrand Collina,c, Jean-Marc Vrigneaudc, Peggy Proventb, François Brunottec,
Franck Denat*a, Christine Goze*a
Abstract: A new generation of MOnomolecular Imaging Probes
(MOMIP) based on a distyryl-BODIPY coupled with three DOTA
macrocycles has been prepared. The MOMIP presents good
fluorescence properties and is very stable in serum. The
bimodal probe was conjugated to trastuzumab, and optical in-
vivo study showed high accumulation of the imaging agent at
the tumor site. 111In radiometallation of the bioconjugate was
performed in high radiochemical yield, highlighting the
potential of this new BODIPY-chelators derivative as a bimodal
imaging probe.
Imaging Probe (MOMIP). This MOMIP can be conjugated on
one single attachment point, eventually in a site specific manner
using bioorthogonal reactions. This allows a better control of the
pharmacokinetic properties and specificity of the resulting
“chemically defined” bioconjugate. The elaboration of such type
of MOMIP platforms represents a synthetic challenge and the
different building blocks (fluorescent dye, chelator, linker) need
to be carefully chosen to meet the criteria and specifications
required for in vivo imaging: high water solubility and stability,
good fluorescence properties in the Near Infrared Region (also
called therapeutic window, between 650 and 900 nm) and good
radiolabeling efficiency. Several MOMIP systems have been
reported with promising in vivo behavior.4 However, the choice of
the fluorescent dye is often limited to classically used rhodamine
and cyanines. More intriguingly, despite all the advantages and
the versatility of the BODIPY derivatives5 (i.e. very good
photophysical properties, good stability), only a very few
examples of BODIPY-based bimodal probes are described in
the literature, and none of them are suited for antibody labeling.6-
8 We wished to design new BODIPY-polyazamacrocycle MOMIP
optimized for in vivo optical/SPECT or PET bimodal imaging of
antibodies. To this aim, we decided to elaborate a platform
In molecular imaging, combining fluorescence with nuclear
imaging techniques (PET or SPECT) can be really
advantageous due to their complementarity, both for preclinical
and clinical use.1 This dual-modality may provide crucial
information in preclinical studies, fluorescence microscopy and
ex vivo optical investigations completing in vivo PET or SPECT
data. In the clinical field, an emerging and promising application
is the use of such bimodal probes for intraoperative image-
guided surgery, with PET or SPECT images allowing
preoperative staging of cancer patients, while intraoperative
fluorescence imaging could help the surgeon to precisely
operate and remove tumor merging, thus improving therapeutic
outcomes.2 In this context, the use of radiometals is of particular
interest because of the wide range of nuclear properties (half-
life) suitable for the labeling of various targeting biovectors.
Designing bimodal tracers represents therefore a challenging
research field. In order to elaborate an efficient hybrid imaging
agent, the vector of interest (antibody, antibody fragment or
peptide) has to be labeled with both the appropriate radiometal
and the fluorescent dye. This is often performed using a
sequential approach, i.e. two successive bioconjugation
reactions to attach the imaging tags to the biovector, without any
control of the balance chelator/NIR dye.1b,3 A more elegant and
efficient approach consists in combining the two different probes
in one single molecule called MOnomolecular Multimodal
containing
a 3,5-distyryl BODIPY moiety, emitting in the
therapeutic window, and three DOTA moieties in order to i)
compensate the high lipophilicity of the distyryl-BODIPY core, ii)
minimize aggregation phenomena, iii) facilitate the radiolabeling
step with long half-life radioisotopes such as 111In. This new
compound was called TBK-686 for TrisDOTA-BODIPY-Lysine-
686 nm.
We report herein the synthesis and the stepwise validation of
this novel MOMIP platform, including a study of the stability in
physiological medium and the evaluation of 111In radiolabeling
efficiency after conjugation to trastuzumab,
a monoclonal
antibody targeting HER2. We also present the first in vivo
fluorescence images of an antibody conjugated to a NIR
emitting-BODIPY/chelator-based MOMIP, highlighting the
potential of this strategy for the design of dual-labeled targeting
imaging agents.
[a]
ICMUB UMR CNRS 6302, CNRS, Univ. Bourgogne Franche-Comté,
F-21000 Dijon, France
The synthetic route to construct TBK-686 is based on the use of
two key building blocks, 5 and 6, containing appropriate and
carefully selected orthogonal amine protective groups (Scheme
1) in order that the cleavage steps proceed gently enough to
keep intact the di-styryl BODIPY structure. For that purpose,
Fmoc and Dde turned out to be suitable moieties. Di-styryl
BODIPY core was obtained through an optimized Knoevenagel
condensation between the methyl groups at the 3,5 positions of
1 and 4-hydroxybenzaldehyde (see ESI section for experimental
details). After alkylation of the phenols with 2-azidoethyl tosylate,
9, avenue Alain Savary, 21078 Dijon, France
Fax: (+) 33 3 80 39 61 17
E-mail: franck.denat@u-bourgogne.fr; christine.goze@u-
bourgogne.fr
Oncodesign, 20 rue Jean Mazen, BP27627, 21076 Dijon CEDEX,
France
Centre Georges François Leclerc, Service de médecine nucléaire, 1
rue Professeur Marion, BP77980, 21079 Dijon Cedex
The two authors contributed equally to this work
[b]
[c]
§
Supporting information for this article is given via a link at the end of the
document.

Chemistry - A European Journal
10.1002/chem.201602886
COMMUNICATION
free amine BODIPY 4 was obtained by reduction of the azide
into amine via a Staudinger reaction.
Figure 1 shows absorption and emission spectra of TBK-686.
Absorption and emission profiles of TBK-686 present typical
features of “distyryl” BODIPY derivatives, i.e. two absorption
bands at 667 and 375 nm (corresponding to the S0-S1 and S0-
S2 transitions respectively), and an emission band at 686 nm
(Figure 1).The quantum yield of TBK-686 is high (30% in
DMSO), and it is worthy to note that In(III) labelling had no
impact at all on the photophysical properties of the compound
(i.e. absorption maximum at 667 nm, emission maximum at 685
nm and a quantum yield of 30% in DMSO). In PBS, this
fluorescence is not affected until 100 nM, which is considerably
higher than the dose injected in vivo.
To make the CuAAC reaction with 6 successful, primary amines
on BODIPY 4 had to be protected, probably due to solubility
issues. Afterwards, selective deprotection enabled us to obtain
building block 8 containing three reactive amines for the
introduction of the chelators, and a Dde-protected amine
function for adding the bioconjugatable group in the last step.
Three DOTA moieties were introduced by reaction of 8 with an
excess
of
DOTA-NHS.
Further
treatment
with
hydroxylamine/imidazole lead to the desired MOMIP TBK-686.
Cold complex (In)3-TBK-686 was also prepared as a model to
mimic the system after Indium 111 radiolabeling.
O
N
NH
O
NH
O
N
NH
O
NH
O
H
OH
N3
PPh3, THF/H2O
OTs
N
N
N
N
R.T., 12h
B
B
DMF, K2CO3
60°C, 4h
B
piperidine, APTS
Toluene, 100°C, 4h
F
F
94%
N
N
F
F
F F
B
72%
F
F
53%
O
O
HO
OH
O
O
1
2
3
4
N3
N3
H2N
NH2
Fmoc-Cl, DIEA R.T., 4h
O
O
THF
O
HN
HN
86%
N
N
HN
N
NHFmoc
NH2
N
N
N
NHFmoc
N3
O
NH
O
N
NH
O
NH
HN
HN
HN
O
O
O
O
O
O
N
N
Et2NH, CH2Cl2
6
B
N
N
N
B
F
F
B
R.T., 24 h
F
F
F
F
CuI, DIPEA, CH2Cl2, R.T., 4h
98%
95%
7
O
O
8
O
O
O
O
5
H2N
NH2
FmocHN
NHFmoc
NHFmoc
FmocHN
1) DOTA-NHS, NEt3, DMF
50 °C, 7h
then R.T., overnight
2) NH2OH.HCl/imidazole
N-Methyl-2-pyrrolidone
R.T., 9h
S
HN
NH
O
O
HN
HN
HN
N
N
N
N
N
N
NH
HN
NH
CO2H
CO2H
CO2H
S
N
N
N
O
O
O
N
NH
O
NH
InCl3
MeOH, NEt3
N
N
N
HO2
C
HO2C
H2N
(In)3TBK-686
N
N
50 °C, 2h
CO2H
N
B
N
N
B
F
F
F
F
Trastuzumab
PBS
SCN
NCS
TBK-686
O
O
O
O
TBK-686-NCS
NEt3, DMSO, R.T., 1h
37°C, 12h
NH
NH
HN
O
HN
O
HO2
C
HO2C
COOH
COOH
CO2H
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
O
O
N
HO2C
HO2C
TBK-686 trastuzumab
CO2H
CO2H
CO2H
HO2C
HO2C
Scheme 1. Synthetic Scheme of TBK-686 and the imaging agent TBK-686-Trastuzumab

Chemistry - A European Journal
10.1002/chem.201602886
COMMUNICATION
basic buffer. The degree of Label-Antibody Ratio (LAR) was
assessed by spectrophotometry (absorbance measurement at
280 and 660 nm) and confirmed by HRMS analysis. Using a 20-
fold excess of TBK-686-NCS, the resulting bimodal imaging
agent TBK-686-trastuzumab was obtained with 2.5 MOMIP per
antibody. With the bioconjugate in hand, we worked on the
radiolabeling process, which is a crucial step for the validation of
the BODIPY MOMIP as an efficient platform for the design of
nuclear/optical bimodal agents. Indium 111 was chosen because
its half life (67h) is adapted for labeling slowly clearing
biomolecules such as antibodies and this radioisotope enabled
us to follow the uptake of trastuzumab in HER2 expressing
tumors by SPECT.9 The results indicate that i) the BODIPY core
did not interfere with the incorporation of 111In in the macrocyclic
moieties and thus did not affect the radiolabeling efficiency of
the antibody conjugate (specific activity = 490 MBq/mg), ii) the
1,20
1,00
0,80
0,60
0,40
0,20
0,00
300
400
500
600
700
800
wavelength(nm)
Figure 1. Absorption and emission spectra of TBK-686 in DMSO.
standard
protocol
commonly
used
to
purify
radioimmunoconjugates could be applied to obtain the pure
111In-TBK-686-trastuzumab with a radiochemical purity > 95%
(see ESI section), iii) The 111In-conjugate remains fluorescent
after radiolabeling. A challenge test was performed to evaluate
the labeling stability of the conjugate 111In-TBK-686-
trastuzumab in the presence of 2000-fold molar excess of
EDTA. Radio-ITLC controls performed after 12h, 24h, 48h and
72h demonstrated that no demetallation occurs (radiochemical
purity >92%), underlying the high stability of the DOTA-based
conjugate (see ESI section).
In order to further validate the compounds before in vivo
evaluation, in vitro stability of the cold complex In(III)3-TBK-686
was investigated in FBS (Fetal Bovine Serum, Figure 2). HPLC
chromatograms recorded at different time clearly highlight the
very high stability of In(III)-MOMIP platform (no degradation
occurring after 48h incubation).
100
90
80
70
60
50
40
30
20
10
The ability of TBK-686-trastuzumab to target HER2+
expressing tumor was investigated in vivo in a model of BT474
tumor bearing mice. Whole-body fluorescence images were
recorded at 24h and 48h post injection, revealing an intense and
focal fluorescent signal at the tumor site (Figure 3A). These
exciting preliminary results were confirmed by ex-vivo imaging
and organ uptake, which showed a much stronger fluorescence
signal in the tumor rather than in other organs (Figures 3B and
3C).
mAU
0
0
10
20
30
40
50
20
10
Incubation time (h)
t = 48h
0.0
-10
-20
t = 0
-30
-40
In conclusion, a rational synthetic pathway has been devised for
the preparation of a versatile, stable and highly fluorescent
MOnomolecular Multimodal Imaging Probe TBK-686 for
optical/SPECT imaging, containing a BODIPY dye and three
radiometal chelators. Very originally, the presence of three
DOTA chelators around the BODIPY core is highly beneficial to
the properties of the MOMIP platform in terms of hydrosolubility
and radiometalation efficiency. The MOMIP TBK-686 was
successfully conjugated to trastuzumab antibody, and the
radiolabeling of the resulting bioconjugate with 111In was
performed in high radiochemical yield. The accumulation of the
bioconjugate in HER2 expressing tumors in mice was clearly
evidenced by both in vivo and ex vivo optical imaging, thus
highlighting the potential of this bimodal imaging agent based
0.47
1.00
2.00
3.00
4.00
5.00
6.00 t (min)
Figure 2. Stability study of the In(III)3-TBK-686 complex in Fetal Bovin Serum
(incubation at 37°C). Evolution of the HPLC chromatograms recorded after
45min, 2h15, 4h45, 7h, 22h and 48h. Compound retention time: 3.56 min, =
650 nm. Percentage of In(III)3-TBK-686 was determined relative to the
reference at t = 0 min (100%).
TBK-686 was activated by introducing an isothiocyanate
function suited for efficient bioconjugation to lysine residues of
antibodies (Scheme 1). The resulting TBK-686-NCS was
conjugated to trastuzumab through peptidic coupling in a slightly

Chemistry - A European Journal
10.1002/chem.201602886
COMMUNICATION
A
48h
24h
B
C
3,00E+06
2,50E+06
2,00E+06
1,50E+06
1,00E+06
5,00E+05
0,00E+00
L
S
B
TUMOR
LIVER
KIDNEYS SPLEEN BLOOD
K
T
Figure 3. A/ Direct comparison of NIR fluorescence images of a subcutaneous BT474 tumor-bearing mouse at 24 and 48h post injection (Ex/Em: 660/710 nm).
Mouse received a single injection of TBK-686-trastuzumab (0.3 nmol). The same scale is used for both images. The tumor site is indicated with a white arrow. B/
Ex vivo fluorescence imaging on the tumor, blood and selected organs. C/ Quantification of fluorescence uptake (ROI 26%, Average radiant efficiency); K =
Kidneys, L = Liver, T = Tumor, B = Blood, S = Spleen (Ex/Em: 660/710 nm).
Zhang, G. W. Severin, Y. Yang, J. W. Engle, G. Niu, R. J. Nickles, X.
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Acknowledgements
This study is a part of Pharm’image® project. Support was
provided by the CNRS, the University of Burgundy, the Conseil
Régional de Bourgogne and the FEDER program through the
PARI research program. This work was supported by a French
Government grant managed by the French National Research
Agency (ANR) under the program “Investissements d’Avenir”
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Keywords: BODIPY • bimodal probes • MOMIP • optical
imaging • DOTA
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Chemistry - A European Journal
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COMMUNICATION
Entry for the Table of Contents
COMMUNICATION
A
new generation of BODIPY-
Nicolas Maindron, Martin Ipuy, Claire
trisDOTA MOnomolecular Multimodal
Imaging Probes for optical /SPECT
imaging was developed and validated
in vivo, highlighting the potential of
Bernhard, Damien Lhenry, Mathieu
Moreau, Sabin Carme, Alexandra
Oudot, Bertrand Collin, Jean-Marc
Vrigneaud, Peggy Provent, François
Brunotte, Franck Denat*, Christine
Goze*
BODIPY
fluorophore for
future
imaging developments.
Page No. – Page No.
Title