Synthesis of Gd and 68Ga Complexes in Conjugation with a Conformationally Optimized RGD Sequence as Potential MRI and PET Tumor-Imaging Probes

Article abstract of DOI:10.1002/cmdc.201200043

We report the synthesis of novel chelates of Gd and ⁶⁸Ga with DPTA, DOTA, HP-DOA3, as well as with AAZTA, a novel chelating agent developed by our research group. These chelating agents were appropriately conjugated, prior to metal complexation, with DB58, an RGD peptidomimetic, conformationally constrained on an azabicycloalkane scaffold and endowed with high affinity for integrin α_νβ₃. Because α_νβ₃ is involved in neo-angiogenesis in solid tumors and is also directly expressed in cancer cells (e.g. glioblastomas, melanomas) and ovarian, breast, and prostate cancers, these constructs could prove useful as molecular imaging probes in cancer diagnosis by MRI or PET techniques. Molecular modeling, integrin binding assays, and relaxivity assessments allowed the selection of compounds suitable for multiple expression on dendrimeric or nanoparticulate structures. These results also led us to an exploratory investigation of ⁶⁸Ga complexation for the promising ⁶⁸Ga-PET technique; the AAZTA complex 15(⁶⁸Ga) exhibited uptake in a xenograft model of glioblastoma, suggesting potentially useful developments with new probes with improved affinity.

Full text of DOI:10.1002/cmdc.201200043

DOI: 10.1002/cmdc.201200043
68
Synthesis of Gd and Ga Complexes in Conjugation with
a Conformationally Optimized RGD Sequence as Potential
MRI and PET Tumor-Imaging Probes
Leonardo Manzoni,*[a] Laura Belvisi,*[b, c] Daniela Arosio,[a] Maria Paola Bartolomeo,[d]
Aldo Bianchi,[c] Chiara Brioschi,[d] Federica Buonsanti,[d] Claudia Cabella,[d]
Cesare Casagrande,[c] Monica Civera,[c] Marilenia De Matteo,[c] Lorenza Fugazza,[e]
Luciano Lattuada,[d] Federico Maisano,[d] Luigi Miragoli,[d] Cristina Neira,[d] Michael Pilkington-
Miksa,[c] and Carlo Scolastico[b, c]
We report the synthesis of novel chelates of Gd and 68Ga with
DPTA, DOTA, HP-DOA3, as well as with AAZTA, a novel chelat-
ing agent developed by our research group. These chelating
agents were appropriately conjugated, prior to metal complex-
ation, with DB58, an RGD peptidomimetic, conformationally
constrained on an azabicycloalkane scaffold and endowed with
high affinity for integrin anb3. Because anb3 is involved in neo-
angiogenesis in solid tumors and is also directly expressed in
cancer cells (e.g. glioblastomas, melanomas) and ovarian,
breast, and prostate cancers, these constructs could prove
useful as molecular imaging probes in cancer diagnosis by MRI
or PET techniques. Molecular modeling, integrin binding
assays, and relaxivity assessments allowed the selection of
compounds suitable for multiple expression on dendrimeric or
nanoparticulate structures. These results also led us to an ex-
ploratory investigation of 68Ga complexation for the promising
68Ga-PET technique; the AAZTA complex 15(68Ga) exhibited
uptake in a xenograft model of glioblastoma, suggesting po-
tentially useful developments with new probes with improved
affinity.
Introduction
Structural imaging is a powerful tool for detecting the differen-
tial features of pathological tissues, particularly of neoplastic
formation with respect to the anatomical features of normal
tissues. Computed tomography (CT), magnetic resonance
imaging (MRI), and ultrasound (US) techniques have thus
reached high diagnostic relevance,[1] especially when rein-
forced with appropriate contrast agents. The need for early
and specific assessment of the manifestation and progression
of cancer has prompted the development of functional imag-
ing by techniques that can highlight the metabolic differences
of cancer cells. Among these, positron emission tomography
(PET) imaging of [18F]2-fluoro-2-deoxy-d-glucose as a marker of
high glucose uptake[2] is the most widely employed. The next
conceptual advancement, and now the most promising, has
been the targeting of biomolecules that are specifically ex-
pressed in tumor cells[3] such as receptors, adhesion molecules,
and enzymes, using high-affinity ligands covalently linked with
suitable imaging moieties for radioactive, MRI, or US imaging.
This approach has an appealing therapeutic potential, because
when proving the specific delivery of imaging moieties to
tumor tissues, it opens the way to the design of molecular
constructs suitable to concentrate radio- or chemotherapeutic
agents in cancer tissues, sparing healthy tissues and organs.
We became particularly interested in targeting avb3 integrin,
a member of the superfamily of integrin adhesion molecules.[4]
This integrin governs the adhesion of endothelial cells to the
extracellular matrix (ECM) in the process of angiogenesis under
normal and pathological conditions. In the latter, in tissues
claiming increased oxygen and nutrient supplies, such as ische-
mic and tumor tissues, avb3 is markedly overexpressed and
supports neo-angiogenesis.[5] In recent years, it has also been
observed that avb3 not only plays a role in cancer progression
via angiogenesis, but is also significantly expressed on the
[a] Dr. L. Manzoni, Dr. D. Arosio
Consiglio Nazionale delle Ricerche
Istituto di Scienze e Tecnologie Molecolari
Via Fantoli 16/15, 20138, Milano (Italy)
E-mail: leonardo.manzoni@istm.cnr.it
[b] Dr. L. Belvisi, Prof. C. Scolastico
Universitꢀ degli Studi di Milano
Dipartimento di Chimica Organica e Industriale
Via Venezian 21, 20133 Milano (Italy)
[c] Dr. L. Belvisi, Dr. A. Bianchi, Prof. C. Casagrande, Dr. M. Civera,
Dr. M. De Matteo, Dr. M. Pilkington-Miksa, Prof. C. Scolastico
Universitꢀ degli Studi di Milano
Centro Interdipartimentale Studi biomolecolari e applicazioni Industriali
Via Fantoli 16/15, 20138, Milano (Italy)
[d] Dr. M. P. Bartolomeo, Dr. C. Brioschi, Dr. F. Buonsanti, Dr. C. Cabella,
Dr. L. Lattuada, Dr. F. Maisano, L. Miragoli, Dr. C. Neira
Bracco Imaging SpA, Bracco Research Centre
Via Ribes 5, 10010 Colleretto Giacosa (TO) (Italy)
[e] Dr. L. Fugazza
Advanced Accelerator Applications
Via Ribes 5, 10010 Colleretto Giacosa (TO) (Italy)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201200043.
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outer membranes of certain cancer cells such as glioblastomas,
melanomas, and ovarian, breast, and prostate cancers, and is
involved in metastasis and proliferation.[6] Being correlated
with disease progression, the expression of avb3 integrin plays
a role as a marker of angiogenesis, tumor development, and
metastasis. Therefore, noninvasive visualization of this integrin
may provide new means for tumor detection and staging.[7]
Because avb3 integrin interacts with ECM and cellular pro-
teins by recognizing and binding at an Arg-Gly-Asp (RGD) se-
quence,[8] many research groups have devoted efforts to the
development of integrin ligands structurally based on linear
and cyclic peptides containing the RGD sequence.[9] We fo-
cused on inserting the RGD motif into rigid peptidomimetic
azabicycloalkane scaffolds with varying structural and stereo-
chemical features suitable for imposing specific conformations
onto the RGD sequence, in order to select the optimal spatial
disposition of the RGD pharmacophoric groups for interaction
with the integrin, while minimizing the peptidic features prone
to metabolic degradation in vivo.[10] Accordingly, we synthe-
sized small libraries of cyclic pentapeptide mimics, and in one
of these we identified the high-affinity and specific avb3/avb5
integrin ligand ST1646 (Figure 1). Biochemical and pharmaco-
logical studies revealed ST1646 as a potent and selective an-
tagonist endowed with significant in vitro and in vivo anti-an-
giogenic activity,[10b] suggesting the possibility of therapeutic
development of ST1646 for cancer applications.
pressing cell lines, and, in the case of the NIR probe, of in vivo
tumor uptake and persistence in mice xeno-implanted with
human glioblastoma U-87 MG cells expressing the integrin. We
therefore considered DB58 as an optimal starting point in the
development of novel avb3-targeted probes in clinically appli-
cable imaging techniques; herein we report the conjugation
and complexation of the amino derivative of DB58 with several
GdIII chelating moieties for MRI imaging, along with extension
to the synthesis of 68Ga complexes for PET imaging. Moreover,
with the aim of improving the signal-to-noise ratio, a divalent
compound featuring two gadolinium complex moieties was
synthesized.
MRI contrast agents based on gadolinium complexes are
currently employed as diagnostic agents in medicinal imag-
ing.[13] We therefore decided to exploit the most efficient and
clinically validated ligands for gadolinium coordination, based
on polyamino polycarboxylic structures such as DOTA, HP-
DO3A, and DTPA (Figure 2), which guarantee high thermody-
Figure 2. Ligands for gadolinium coordination.
namic and kinetic stability: very important properties for clini-
cal safety.[14] Gadolinium complexes of DOTA (Dotarem, Guer-
bet), HP-DO3A (Prohance, Bracco Imaging) and DTPA (Magnev-
ist, Bayer Schering Pharma AG) are examples of stable, extra-
cellular, nonspecific MRI contrast agents with wide clinical ap-
plication.[15] We also used AAZTA (Figure 2), a recently reported
ligand[16] that can form a stable gadolinium complex with high
relaxivity.
Figure 1. Peptidomimetic integrin ligands ST1646 and DB58 and their IC50
values in the inhibition of biotinylated vitronectin binding to purified recep-
tors.
On the basis of these results, we progressed to new synthet-
ic developments aimed at making azabicycloalkane scaffolds
suitable for conjugation with therapeutic or diagnostic molecu-
lar moieties. We recently reported a convenient synthesis of
conformationally constrained homoSer-Pro dipeptide mimics
and their use in the preparation of cyclic RGD compounds.[11]
These cyclic RGD pentapeptide mimics have an etheroalkyl
side chain terminated by a hydroxy group that can be easily
transformed into various reactive functionalities (amino, azido,
Specificity of targeting of gadolinium complexes can be ach-
ieved by conjugating bifunctional derivatives of the chelating
agents (see examples in Figure 3) to a moiety, such as a pep-
tide, that is suitable for inducing delivery and accumulation of
the MRI probes into specific normal or pathological tissues;[17]
for this study an integrin ligand was used.
thio, etc.), enabling us to conjugate diverse functional units for Results and Discussion
application in medical diagnosis and therapy.
Chemistry
Among the new compounds, we selected DB58 (Figure 1),
which showed the highest affinity for avb3 integrin in an in vi-
tro binding assay,[11b] and we conjugated this active ligand to
probes for optical imaging (i.e., fluorescein and the near-infra-
red (NIR) probe Cy5.5).[12] These conjugates provided us with
interesting indications of specific binding on various avb3-ex-
We first investigated the influence of the distance between the
Gd cage and the cyclic RGD ligand on the affinity of the conju-
gated compounds toward avb3 integrin. Next, we examined
the influence of various chelating agents toward the ability of
the Gd complex to lower T1 values. Finally, we explored the
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Gd and 68Ga Complex–RGD Conjugates as MRI and PET Probes
(Scheme 1). In the case of compounds 10–12, after
reduction of the azide, the orthogonally protected
spacers 6–8 were conjugated, respectively, and, after
nitrogen protecting group removal, coupled with 3
to afford the desired protected compounds. Finally,
the side chain protecting groups of the RGD se-
quence and the five tert-butyl esters of the DTPA che-
lator were removed by standard methods used in
peptide chemistry (TIPS, 1,2-ethanedithiol, water, thi-
oanisole, TFA) to furnish the desired compounds 9–
12 (52–85% overall yields).
Figure 3. Bifunctional chelating agents.
The second aspect we evaluated is the influence of
the type of chelating agent on the ability of the GdIII
complex to lower the T1 values. Therefore, we considered
a DOTA derivative,[19] a HP-DO3A derivative,[20] and an AAZTA
derivative (the synthesis of the bifunctional chelating agent 4
is described in the Supporting Information) as gadolinium che-
lators, and we conjugated them to the RGD mimic. The syn-
thetic route for their preparation is identical to that reported
for DTPA derivatives (Scheme 2 and Scheme 3). All final conju-
gated compounds 9–16 were purified by semi-preparative RP
HPLC followed by lyophilization before the complexation with
gadolinium.
possibility of increasing the signal-to-noise ratio through a diva-
lent presentation of the Gd cage.
For the first aspect, we selected DTPA as the lanthanide che-
lator and prepared four compounds differing in the distance
between the chelating and targeting groups. In compound 9
the chelating cage is conjugated directly to the targeting RGD
moiety, whereas in compounds 10–12 three linkers, constitut-
ed by varying numbers of ethylene glycol units, are intro-
duced. The synthetic path proceeds from the bifunctional che-
lating agent 3, where on a total of six carboxylic groups, five
are protected with tert-butyl ester, and one is free.[18] To syn-
thesize compound 9, the known azide 5a, derived from
DB58,[11b] was hydrogenated with palladium on carbon, and
the corresponding amine was coupled to 3 with standard pro-
cedures for amide formation (HBTU, HOBT, DIPEA, DMF)
As anticipated, with the aim of improving the signal-to-noise
ratio in MRI analysis, we designed a divalent compound featur-
ing two Gd complex moieties. Our approach to the synthesis
of divalent systems is based on glutamic acid dendrimers.
Compound 17 was easily prepared using glutamic acid as the
Scheme 1. a) H2, 10% Pd/C, EtOH, RT, 24 h; b) 3, HOBT, HBTU, DIPEA, DMF, RT, 4–8 h, 58–99% over two steps; c) TIPS/1,2-etanedithiol/H2O/thioanisole
(2.5:2.5:2.5:5), TFA, RT, 5–6 h, 80–85%; d) 6, HOAT, HATU, DIPEA, DMF, RT, 24 h, 72%; e) 7, HOAT, HATU, DIPEA, DMF, RT, 24 h, 95%; f) 8, HOAT, HATU, DIPEA,
DMF, RT, 24 h, 99%.
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branching unit, and multiple
copies of the chelating agent
were bound to this inert branch-
ing core (Figure 4).
Commercially available l-Boc
glutamic acid was activated with
N-hydroxysuccinimide (NHS) in
the presence of N,N-diisopropyl-
carbodiimide (DIC). The activated
ester was coupled with propar-
gylamine to give compound 18.
Removal of the Boc group with
trifluoroacetic acid yielded the
deprotected amine 19, ready for
the coupling reaction. The azido
group of 5b was hydrogenated
with 10% Pd/C to give the corre-
sponding amine, which was
treated with succinic anhydride
in the presence of DIPEA to give
the acid 20. Activation of the
carboxylic group with TBTU and
HOBT, followed by coupling with
the previously synthesized amine
19 gave the alkynyl derivative 21
(Scheme 4).
Scheme 2. a) H2, 10% Pd/C, EtOH, RT, 24 h; b) 1, HOBT, HBTU, DIPEA, DMF, RT, 18 h, 75%; c) TIPS/1,2-etanedithiol/
H2O/thioanisole (2.5:2.5:2.5:5), TFA, RT, 5–7 h, 68–70%; d) 2, HOAT, HATU, DIPEA, DMF, RT, 24 h, 99%.
Functionalization of the dendri-
mer ends with DTPA derivative is
based on the 1,3-dipolar cycload-
dition between terminal triple
bonds and azides. The copper(I)-
catalyzed reaction is very rapid
and completely regioselective.
The catalyst is prepared by in situ
reduction of Cu(OAc)2 by sodium
l-ascorbate. Thus, azide 22,
which was obtained by coupling
compound 3 with the commer-
cially available azido-PEG-amine,
Scheme 3. a) H2, 10% Pd/C, EtOH, RT, 24 h; b) 4, HOBT, HBTU, DIPEA, DMF, RT, 6–24 h, 99%; c) TIPS/1,2-etanedi-
Figure 4. Divalent compound 17 featuring two gadolinium complex moieties.
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thÝiol/HÝO/thioanisole (2.5:2.5:2.5:5), TFA, RT, 6–7 h, 75–77%; d) 6, HOAT, HATU, DIPEA, DMF, RT, 24 h, 97%.
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Gd and 68Ga Complex–RGD Conjugates as MRI and PET Probes
Molecular modeling
Monte Carlo conformational
searches[21]
(AMBER*
force
field,[22] implicit water GB/SA sol-
vation model)[23] were performed
on ligands 9(Gd) and 10(Gd)
within the X-ray crystal structure
of the integrin avb3 binding
site[24] to evaluate, at the molec-
ular level, the effect of the Gd–
chelant unit spacer on the bind-
ing of the RGD moiety.
Considering the X-ray struc-
ture of cilengitide bound to the
avb3 headpiece (PDB ID: 1L5G)
as representative of the optimal
RGD interaction mode, conform-
ers of 9(Gd) and 10(Gd) were
evaluated for their ability to re-
produce the experimental bind-
ing mode. In the X-ray structure,
the acidic and basic moieties of
the RGD sequence point in op-
posite directions, acting like an
electrostatic clamp and attach-
Scheme 4. a) NHS, DIC, propargylamine, RT, 15 h; b) TFA, CH2Cl2, RT, 2 h; c) H2, 10% Pd/C, MeOH, RT, 24 h, 94%;
d) succinic anhydride, DIPEA, THF, RT, 18 h 88%; e) 19, HOBT, TBTU, DIPEA, DMF, RT, 3 h, 77%; f) 22, Cu(OAc)2,
sodium l-ascorbate, tert-butanol/H2O, RT, 42%; g) phenol/thioanisole/1,2-etanedithiol/H2O, (5:5:2.5:5), TFA, RT.
was subjected to the cycloaddition reaction conditions in the
presence of the protected alkynyl derivative 21 to give diva-
lent protected DTPA dendrimer. Finally, deprotection using
a mixture of trifluoroacetic acid, phenol, thioanisole, 1,2-etha-
nedithiol, and water gave compound 17 (Scheme 4). Complex-
ation of 9–17 with gadolinium afforded Gd-chelated com-
pounds 9(Gd)–17(Gd) (see Supporting Information).
ing to charged regions of the integrin binding site. The posi-
tively charged guanidinium group of the Arg residue interacts
with the negatively charged side chains of Asp218 and
Asp150 in the a unit. One carboxylate oxygen atom of the
ligand Asp side chain is coordinated to MIDAS metal cation
region of the b unit, while the second carboxylate oxygen
atom forms hydrogen bonds with the backbone amides of
Asn215 and Tyr122 in the b unit. A hydrogen bond between
the ligand Asp NH group and the C=O function of b3-Arg216
provides further stabilization to the complex.
Conformers found within 3 kcalmolÀ1 of the global mini-
mum of ligand 9(Gd) are positioned within the integrin bind-
ing site according to two binding modes. The lowest-energy
conformation represents 45% of the Boltzmann-weighted con-
former population and displays the Gd cage and the RGD
motif both interacting with the b subunit. Relative to the cilen-
gitide RGD interaction mode, the Arg guanidinium group loses
the electrostatic interaction with Asp218 and Asp150 side
chains of the a subunit. The remaining 55% of conformers
form all the key RGD crystallographic interactions with the
charged Gd cage directed toward the ADMIDAS region of the
b subunit.
In contrast, all the structures found within 3 kcalmolÀ1 of
the global minimum of compound 10(Gd) form the RGD refer-
ence system interactions (Figure 5). The chelant unit is directed
toward the outside of the integrin binding site, growing unhin-
dered. In agreement with binding data (Table 1), ligand 10(Gd)
appears to have the optimal linker length to properly fit the
RGD sequence and the Gd cage within the avb3 binding site.
Synthesis of 68Ga-labeled compounds
Labeling of DTPA and AAZTA derivatives 10 and 15, respective-
ly, with 68Ga was performed at pH 3.8; the pH was then cor-
rected to the physiological value, and the final radiochemical
purity was assessed. Evaluation of the stability at physiological
pH (7.4) of the 68Ga complexes 10(68Ga) and 15(68Ga) was per-
formed by HPLC analysis with radiometric detection. Com-
pound 15(68Ga) with the AAZTA coordination cage was ob-
served to be stable under physiological conditions, as the peak
of the complex remains clearly detectable (figure S1, Support-
ing Information) while no free 68Ga is observed. For compound
10(68Ga), which differs from 15(68Ga) only in the gallium coordi-
nation cage (DTPA instead of AAZTA), HPLC analysis showed
that the complex is completely dissociated at pH 7.4 and only
the peak of free gallium is present in the chromatogram (fig-
ure S2, Supporting Information). Therefore, the complex with
the AAZTA coordination cage was chosen for in vivo experi-
ments.
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binding affinity. The presence of a mid-length linker (in 10(Gd)
and 11(Gd)) maintains affinity at the same level of the unconju-
gated compound (DB58), possibly because it keeps the ligand
cage distal from the RGD binding site. At the other extreme,
a spacer that is too long, as in 12(Gd), may also be detrimental
to binding because the high spacer flexibility allows interfer-
ence of the Gd complex with the RGD side of the molecule.
Rather unexpectedly, the presence of a cyclic ligand, i.e.,
DOTA, HP-DO3A, or AAZTA, markedly decreased binding affini-
ty, resulting in IC50 values consistently >100 nm. Moreover, fur-
ther increases to the bulkiness of the derivatives deteriorated
binding properties, as can be observed by comparing the
mono-DTPA derivatives with 17(Gd), which carries two Gd-
DTPA units.
Relaxivity data
The Gd-chelated RGD derivatives were characterized by relax-
ometry. Table 2 shows the millimolar relaxivity at 20 MHz, 258C
for each tested compound. Fittings of the NMRD profiles (fig-
Figure 5. Global minimum of the complex 10(Gd): avb3 headpiece forming
the X-ray interactions. Cilengitide is in black wire and 10(Gd) is in tube rep-
resentation (O, N, Gd in black; C in gray and polar hydrogen atoms in light
gray). Receptor is in gray wire representation, with interacting key residues
highlighted as tubes and metal ions shown as black spheres. Nonpolar hy-
drogen atoms are removed for clarity.
Table 2. Relaxometric characterization of Gd-chelated RGD derivatives.
[a]
Compound
r1p [mmÀ1 sÀ1
]
Gd-DTPA
9(Gd)
4.45Æ0.18
7.55Æ0.12
7.63Æ0.12
7.49Æ0.10
7.91Æ0.09
6.20Æ0.04
12.94Æ0.48
13.32Æ0.16
8.37Æ0.29
Table 1. IC50 values of Gd-chelated RGD derivatives.
10(Gd)
11(Gd)
12(Gd)
14(Gd)
15(Gd)
16(Gd)
17(Gd)
Compound
IC50 [nm][a]
DB58
9(Gd)
26Æ4
270Æ140
30Æ11
10(Gd)
11(Gd)
12(Gd)
13(Gd)
14(Gd)
15(Gd)
16(Gd)
17(Gd)
54Æ24
130Æ64
>1000
[a] Measurements were acquired at 20 MHz, 258C; values represent the
mean ÆSD from three experiments.
830Æ120
290Æ65
570Æ110
450Æ22
ure S3, Supporting Information) and comparison with the re-
laxivity behavior as a function of temperature (figure S4, Sup-
porting Information) demonstrated that with respect to Gd-
DTPA, the increase in relaxivity observed at all examined fields
is a consequence of the increase in molecular reorientation
time due to conjugation with the peptide.
[a] Values represent the mean ÆSE from 2–3 replicate assays carried out
in triplicate.
Binding affinity evaluation
As expected from their molecular weights, the r1p values of
the Gd-chelated RGD compounds measured at 20 MHz and
All synthesized products were evaluated after complexation
with GdIII. Affinity for the receptor was measured by the ability
to compete with vitronectin, a natural target in the extracellu-
lar matrix, for the binding to immobilized purified human avb3
integrin.[9a] Accordingly, we calculated the IC50 values, which
represent the peptide concentrations required to inhibit 50%
of vitronectin binding to the receptor.
258C were higher than that of the precursor Gd-DTPA (r1p
=
4.45 mmÀ1 sÀ1). However, the r1p values for the avb3 integrin li-
gands were slightly lower than what was expected from the
extrapolation of the linear correlation law occurring between
r1p and molecular weight for lower-molecular-weight com-
pounds (figure S5, Supporting Information). This is probably
due to the presence of intramolecular motions of the peptide
moiety that make the molecular reorientation time shorter
than what would be expected simply on the basis of molecular
weight. For the two Gd-AAZTA conjugates, the relaxivity is
higher, as expected for these complexes, which coordinate two
water molecules.
The peptides were able to partially or completely suppress
binding of the natural ligand to the isolated receptor, and the
binding curves followed the expected sigmoidal pattern. Com-
paring the IC50 data (Table 1) with structural features of the
linear chelating compounds, it is apparent that the presence of
a linker that is too short (9(Gd)) or too long (12(Gd)) decreases
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Gd and 68Ga Complex–RGD Conjugates as MRI and PET Probes
PET
time the coronal section is indicated with a yellow arrow show-
ing the tumor mass. The clearance behavior of the two radio-
tracers is quite different, with the untargeted molecule being
eliminated from the bloodstream more quickly than the target-
ed one, with a rapid accumulation in the bladder. The 68Ga-
AAZTA signal in the tumor is detectable 20 min after adminis-
tration; it then decreases rapidly, being undetectable ~50 min
later. On the other hand, 15(68Ga) has slower kinetics and accu-
mulates in the tumor. In fact, ~40 min following injection, the
tumor is highlighted and readily detectable due to improved
contrast between the tumor mass and the surrounding tissues.
The signal remains appreciable in the tumor until 70 min post-
injection.
In recent studies we achieved good evidence for the value of
our RGD-carrying scaffolds in vivo by NIR imaging of the Cy5.5-
coupled amine derived from compound 5a,[12b] showing specif-
ic uptake and persistence in the tumor tissue of mice bearing
U-87 MG glioblastoma. Glioblastoma, the most frequent sub-
type of human primary brain tumors, is the target of a phase III
trial of cilengitide, the only RGD-based agent in advanced clini-
cal investigations.[5b] Therefore, we decided to seek, in the
same experimental tumor, a proof-of-concept for the present
metal-chelated conjugates using 68Ga-PET, which is orders of
magnitude more sensitive than Gd-MRI. In our preliminary ex-
periment, the 68Ga-AAZTA RGD conjugate 15(68Ga) was intrave-
nously injected in mice (n=3) and compared with 68Ga-AAZTA
(n=2). Figure 6 shows representative images of 15(68Ga)
(panel a) and 68Ga-AAZTA (panel b) uptake over time. At each
Figure 7 shows the time course of tumor uptake of the two
compounds. The RGD conjugate appears to concentrate in
tumor tissue and to persist at higher concentrations than the
non-targeted molecule up to 150 min. Moreover, by simultane-
ous injection of the integrin ligand c(RGDfV), the expected
competitive antagonism in integrin binding was observed as
a decreased uptake of 15(68Ga), similar to that of the nonspe-
cific compound (Figure 7).
68
^
Figure 7. Time course uptake of 15( Ga) alone ( ), in co-administration with
68
~
&
c(RGDfV) ( ), and Ga(AAZTA) ( ) in U-87 MG xenograft mice.
Conclusions
Our exploration of chemical diversity in Gd chelates with a con-
strained RGD ligand showed, in the case of DPTA derivatives,
a modulation of the affinity for integrin avb3 by changes in
length of the spacer setting the distance between the chelated
Gd ion and the RGD moiety, with good affinity at intermediate
length, preserved at the level of the parent ligand DB58 in
compound 10(Gd). The extension of the exploration to DOTA,
HP-DO3A, or AAZTA with various linkers, and also with a gemi-
nal presentation of two Gd chelates, did not produce com-
pounds with adequate affinity.
Relaxivity, another relevant property of the chelated com-
pounds, definitely increased with respect to DPTA-Gd chelate
for all compounds, although less than proportionally with mo-
lecular weight, at variance from the correlation reported for
a set of non-targeted Gd chelates. It is conceivable that the
conformational freedom added by the linker–ligand moiety
may allow molecular motions that oppose the increase in re-
laxivity.
Figure 6. Representative U-87 MG tumor mass images at various time points
after administration of a) 15(68Ga) and b) 68Ga(AAZTA); arrows show the
tumor position.
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L. Manzoni, L. Belvisi, et al.
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head group of integrin has been identified in the X-ray structure as
the ligand binding region. The protein was truncated to residue se-
quences 41–342 for chain a and 114–347 for chain b. Due to a lack
of parameters, the Mn2+ ions in the experimental protein structure
were modeled by replacing them with Ca2+ ions. The resulting
structure was prepared using the Protein Preparation Wizard of the
graphical user interface Maestro. All nonpolar hydrogen atoms of
the protein were removed except for the hydrogen atoms of aro-
matic residues within 3 ꢁ of the X-ray structure ligand. The coordi-
nation shell of metal cation belonging to MIDAS (metal-ion-depen-
dent adhesion site) and ADMIDAS (adjacent to MIDAS) was com-
pleted on the basis of the integrin aIIbb3 X-ray structures[26] by
adding one and two water molecules, respectively.
Comparative modeling of compounds 9(Gd) and 10(Gd)
showed that the fitting of the latter in the integrin avb3 bind-
ing site is in good agreement with the interactions observed in
the crystallographic study of cilengitide, whereas the fitting of
9(Gd) does not completely reproduce the X-ray structure bind-
ing mode. The degree of integrin affinity of compound 10(Gd)
is similar to, or somewhat better than, that of the fluorescein-
linked analogues we previously reported.[12a] These had shown
selective uptake and internalization in avb3-expressing cells.
The current trend for novel targeted Gd contrast agents is
toward multiple presentation of Gd atoms, aiming at increased
sensitivity of the MRI method.[25] Thus, we are considering the
transfer of the structural features and the Gd–RGD distance of
compound 10(Gd) in multiple moieties bound on dendrimeric
or nanoparticulate structures.
Ligand setup
The X-ray structure of the Gd cage coordinated to one water mole-
cule[27] was linked to the bound-like conformation Invg(Asp)+
b[Gly,Asp] of the RGD cyclopeptide.[11b] The distances of nitrogen
and oxygen atoms coordinated to the Gd3+ ion were kept fixed by
a force constant of 500 kJmolÀ1 ꢁ2.
Our efforts to transfer the favorable binding properties of
10(Gd) directly to the complex 10(68Ga), as a tool for PET with
68Ga probes (a promising new sensitive and easily accessible
PET technique), resulted in a complex unstable under physio-
logical conditions. In contrast, the complexation of 68Ga in
compound 15, resulting in the stable complex 15(68Ga), afford-
ed the first targeted AAZTA derivative suitable for in vivo ex-
periments. Notwithstanding the modest binding affinity of the
corresponding 15(Gd) compound, we decided to run a prelimi-
nary in vivo experiment in a murine model of human glioblas-
toma, expressing avb3, on which we recently reported interest-
ing observations with our Cy5.5-linked DB58-derived NIR
probe. The present preliminary observations, within their ex-
perimental limitations, are in accordance with the NIR results
and indicate a better uptake and persistence in the tumor with
respect to the non-targeted AAZTA(68Ga) complex. Specificity
of targeting was pointed out by the antagonism produced by
administration of the avb3 integrin ligand c(RGDfV). The results
suggest additional chemical and biological investigations for
improved high-affinity ligands, aiming at the development of
clinically applicable integrin-targeted 68Ga-PET probes.
Monte Carlo conformational searches
Monte Carlo conformational searches of compounds 9(Gd) and
10(Gd) (MCMM,[28] AMBER* force field,[22] implicit water GB/SA sol-
vation model[23]) were performed as implemented in the frame-
work of MacroModel[21] version 9.6 within the integrin avb3 binding
site prepared as described in the protein setup. For each search, at
least 1000 starting structures for each variable torsion angle were
generated and minimized until the gradient was <0.05 kJꢁmolÀ1
using the truncated Newton–Raphson algorithm.[29] Duplicate con-
formations of ligands and those with an energy >6 kcalmolÀ1
above the global minimum were discarded. Default parameters for
ligand rotation and translation were used.
The substructure utility was used to model the receptor atoms.
Residue atoms within 5 ꢁ of X-ray ligand were constrained to crys-
tallographic coordinates by a harmonic potential (with a force con-
stant K=100 kJmolÀ1 ꢁ2 for atoms up to 3 ꢁ and with K=
300 kJmolÀ1 ꢁ2 for atoms between 3 and 5 ꢁ) allowing the polar
and aromatic hydrogen atoms free movement. Residue atoms be-
tween 5 and 10 ꢁ were frozen to their X-ray position (K=À1). The
remaining receptor atoms were ignored during the calculations.
Experimental Section
Chemistry
Receptor binding assays
The synthesis of the bifunctional chelating agent 4, the synthesis
and the characterization of the conjugated compounds 9–17, the
general procedure for the preparation of Gd complexes using
GdCl3, and the preparation of the 68Ga-labeled compounds are re-
ported in the Supporting Information.
A 96-well microtiter plate was coated overnight at 48C with avb3
integrin (Chemicon; 0.5 mgmLÀ1, 100 mLwellÀ1) in coating buffer
(20 mm Tris·HCl pH 7.4, 150 mm NaCl, 1 mm MnCl2, 0.5 mm MgCl2,
2 mm CaCl2). The plate was washed once with blocking buffer (1%
BSA in coating buffer, 200 mLwellÀ1) and then incubated in the
same buffer (200 mLwellÀ1) for 2 h at room temperature. Afterward,
the plate was rinsed twice with blocking buffer and incubated
(100 mLwellÀ1, 3 h at room temperature) with various concentra-
tions of test compounds (0.01–10000 nm) in the presence of bio-
tinylated vitronectin (1 mgmLÀ1). The plate was then washed three
times with blocking buffer and incubated with streptavidin–HRP
(Strep-HRP, GE Healthcare; 1:10000 in PBS, 100 mLwellÀ1) for 1 h at
room temperature. After additional four washes in PBS, the plate
was incubated with TMB solution (Sigma–Aldrich; 100 mLwellÀ1) for
3–5 min, and the colorimetric reaction was stopped by adding
50 mL 2n H2SO4. To quantify competitive binding, we determined
the remaining bound biotinylated vitronectin by reading the ab-
Molecular modeling
All calculations were run using the Schrçdinger suite of programs
(http://www.schrodinger.com) through the Maestro graphical inter-
face.
Protein setup
The crystal structure of the extracellular domain of the integrin
avb3 receptor in complex with cilengitide (PDB ID: 1L5G)[24b] was
used for Monte Carlo conformational searches. Calculations were
performed only on the globular head of the integrin because the
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Gd and 68Ga Complex–RGD Conjugates as MRI and PET Probes
sorbance values at l 450 nm. All assays were carried out in tripli-
cate and repeated 2–3 times. IC50 values were determined by non-
linear regression analysis using GOSA-fit software (Global Optimisa-
tion by Simulated Annealing; http://www.bio-log-software.com/).
Keywords: chelates · imaging agents · integrins · RGD · tumor
targeting
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Human glioblastoma cells (U-87 MG) were supplied by the ATCC.
Cells were grown in EMEM supplemented with 10% fetal bovine
serum, 2 mm glutamine, 100 IUmLÀ1 penicillin, and 100 mgmLÀ1
streptomycin. CD-1 nude mice (six-week-old females) were sup-
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ducted according to national and international laws on animal ex-
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PET experiments
The aim of this study was to evaluate the uptake of 15(68Ga) ad-
ministered in a xenograft mouse model of human glioblastoma (U-
87 MG) cells in CD1 nude mice. U-87 MG cells were collected and
washed two times with PBS; cells (5ꢂ106) were resuspended in
0.1 mL PBS and injected subcutaneously in the right flank of each
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as a function of breathing rate. All the animals were warmed with
a heating pad (308C) for 30 min before injection up to the end of
the PET experiment. 15(68Ga) or 68Ga(AAZTA) were administered in-
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Acknowledgements
We gratefully acknowledge the Ministero dell’Universitꢀ e della
Ricerca, MIUR (PRIN prot. 2008J4YNJY, FIRB RBNE03LF7X) for fi-
nancial support. Expert assistance in relaxivity measurements by
Luisa Poggi (Bracco Imaging SpA, Bracco Research Centre, Via
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L. Manzoni, L. Belvisi, et al.
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Published online on && &&, 0000
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FULL PAPERS
Persistence pays: Novel Gd and 68Ga
chelates conjugated with a cyclic RGD
peptidomimetic integrin ligand were
prepared. Preliminary in vivo evaluation
of the first integrin-targeted AAZTA-
(68Ga) complex showed good uptake
and persistence in a xenograft mouse
model of human glioblastoma (U-87
MG) cells.
L. Manzoni,* L. Belvisi,* D. Arosio,
M. P. Bartolomeo, A. Bianchi, C. Brioschi,
F. Buonsanti, C. Cabella, C. Casagrande,
M. Civera, M. De Matteo, L. Fugazza,
L. Lattuada, F. Maisano, L. Miragoli,
C. Neira, M. Pilkington-Miksa,
C. Scolastico
&& – &&
Synthesis of Gd and 68Ga Complexes
in Conjugation with
a Conformationally Optimized RGD
Sequence as Potential MRI and PET
Tumor-Imaging Probes
ChemMedChem 0000, 00, 1 – 11
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www.chemmedchem.org
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