Gadolinium complex of 125I/127I-RGD-DOTA conjugate as a tumor-targeting SPECT/MR bimodal imaging probe

Article abstract of DOI:10.1021/ml3003499

The work describes the synthesis and in vivo application of [Gd(L)(H ₂O)]·xH₂O, where L is a (¹²⁵I/ ¹²⁷I-RGD)- DOTA conjugate, as a tumor-targeting SPECT/MR bimodal imaging probe. Here, (¹²⁵I/¹²⁷I-RGD)-DOTA signifies a cocktail mixture of radioisotopic (1a, L = ¹²⁵I-RGD-DOTA) and natural (1b, L = ¹²⁷I-RGD-DOTA) Gd complexes. The two complexes are chemically equivalent as revealed by HPLC, and their cocktail mixture exhibits the integrin-specific tumor enhancement, demonstrating that they constitute essentially a single bimodal imaging probe. Employment of a cocktail mixture thus proves to be a sole and practical approach to overcome the sensitivity difference problem between MRI and SPECT.

Full text of DOI:10.1021/ml3003499

Letter
pubs.acs.org/acsmedchemlett
125 127
Gadolinium Complex of I/ I‑RGD-DOTA Conjugate as a Tumor-
Targeting SPECT/MR Bimodal Imaging Probe
Ji-Ae Park,† Jung Young Kim,† Yong Jin Lee,† Wonho Lee,† Sang Moo Lim,‡ Tae-Jeong Kim,§
Jeongsoo Yoo,∥ Yongmin Chang,*,∥ and Kyeong Min Kim*,†
‡
†Molecular Imaging Research Center and Department of Nuclear Medicine, Korea Institute of Radiological and Medical Sciences,
Seoul 139-706, Republic of Korea
§Department of Applied Chemistry, Kyungpook National University, Daegu 702-701, Republic of Korea
∥Department of Molecular Medicine, Kyungpook National University, Daegu 700-422, Republic of Korea
S
* Supporting Information
ABSTRACT: The work describes the synthesis and in vivo
application of [Gd(L)(H2O)]·xH2O, where L is a (125I/127I-
RGD)- DOTA conjugate, as a tumor-targeting SPECT/MR
bimodal imaging probe. Here, (125I/127I-RGD)-DOTA signifies
a “cocktail mixture” of radioisotopic (1a, L = 125I-RGD-
DOTA) and natural (1b, L = 127I-RGD-DOTA) Gd
complexes. The two complexes are chemically equivalent as
revealed by HPLC, and their cocktail mixture exhibits the
integrin-specific tumor enhancement, demonstrating that they
constitute essentially a single bimodal imaging probe. Employ-
ment of a cocktail mixture thus proves to be a sole and
practical approach to overcome the sensitivity difference problem between MRI and SPECT.
KEYWORDS: dual modality, SPECT/MR, RGD peptide, small molecule, tumor targeting
Several methods have been proposed to overcome such
ccomplishment of multimodality molecular imaging is one
Aof the ultimate goals in preclinical and clinical medicine. difficulties.5,6 One such attempt would involve incorporation
of gadolinium dendrimers or nanomaterials with heavy loading
capability of MRI agent.7 Yet, even the nanobased MRI
platform exhibits a sensitivity difference greater than 3 orders of
magnitude. In addition, a serious drawback in the use of such
nanoplatforms lies in the fact that they exhibit a limited in vivo
biodistribution. Namely, one study shows that less than 5% of a
dual modal nanoplatform reaches the target site after
intravenous injection, while the rest is taken up by
reticuloendothelial system (RES).8 Further practical limitations
of nanoplatforms may come from their inherent in vivo
toxicity.9,10
An alternative approach for the design of a dual modality
imaging agent is to use a low molecular weight bifunctional
chelating agent (BFCA).11 An advantage of Gd-BFCA as
compared to gadolinium nanoparticles is that the former
exhibits good in vivo biodistribution and as a result high target
specificity without a significant RES uptake. For example,
Dirksen has recently reported the synthesis of a gadolinium
complex of DTPA-RGD conjugate incorporating a fluorescent
dye for use as a tumor-specific MRI/optical dual modal imaging
probe.12 A major drawback of this probe is, as expected, the
The combination of radioimaging and magnetic resonance
imaging (MRI), among others, is the most plausible to visualize
the molecular events in vivo because the combination can
provide simultaneously high sensitivity and high resolution,
which are prerequisites for effective molecular imaging.1,2
Furthermore, contrary to optical imaging, neither radioimaging
nor MRI does not suffer from the penetration depth problem,
thus making it feasible to image deep organs. Single photon
emission-computed tomography (SPECT) has widely been
used in clinical radioimaging. SPECT, although it provides
lower sensitivity and resolution than PET, possesses a unique
advantage of clinical availability of many SPECT radioisotopes
such as 99mTc, 123I, 201Tl, and 111In. Besides, it is economic and
convenient to use them in clinics as compared with PET
isotopes.3,4 In this work, we have used 125I as a source of 123I
since 125I (T1/2 = 59.4 days, 35 KeV) can readily turn into 123I
(T1/2 = 13.2 h, 159 KeV) for better SPECT imaging.
For the development of bimodal SPECT/MRI probes, one
of the most important issues is how to overcome the sensitivity
difference between SPECT and MRI, not to mention their
chemical and structural differences. In general, the imaging
sensitivity of SPECT is 6 orders of magnitude higher than that
of MRI.4 Thus, for example, the use of a 1:1 stoichiometry
between SPECT and MRI probes leads the amount of the
SPECT radioisotope far out of the safety limit for clinical usage.
Received: October 15, 2012
Accepted: December 17, 2012
Published: December 17, 2012
© 2012 American Chemical Society
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Letter
huge sensitivity difference between MR and optical images as a
result of 1:1 stoichiometry of Gd-DTPA and the fluorescent
dye within the same molecule.
The chemical equivalence of 1a and 1b can be established by
analytical HPLC chromatography as demonstrated in Figure 1.
We have recently demonstrated that the use of a “cocktail
mixture” of two different probes would be an only alternative to
overcome such a sensitivity difference and at the same time
preserve the same in vivo biodistribution.13 The BFCA is a
binucleating DTPA-bis(histidylamide), and the cocktail mixture
consists of Gd{DTPA-bis(histidylamide)} labeled with rhe-
nium (Re)/technetium-99m (99mTc). We have shown that both
complexes are chemically equivalent and exhibit the same ex/in
vivo biodistribution.13 In this proof-of-concept study, radio-
active 99mTc was substituted by Re in Gd{DTPA-bis-
(histidylamide)} to minimize the sensitivity difference between
SPECT and MRI. One minor problem inherent to this cocktail
mixture of Re/99mTc has yet to be recognized, since the two
isotopes are not identical (although similar). It would be
therefore beneficial to use the same isotopes in a cocktail
mixture.
Figure 1. Analytical HPLC chromatograms of 1a (blue, 254 nm) and
corresponding 1b (red, γ trace).
The figure shows the same retention times (Rt) of 19.5 and
20.0 min for 1a and 1b, respectively. A slight difference in the
retention times may be rationalized by different serial
arrangement of the detectors with radiometric and UV
detector.15
Both 1a and 1b reveal high stability in mouse serum for 3
days at 37 °C, demonstrating kinetic inertness toward
degradation by endogenous enzymes. Figure 2a shows the in
For example, two iodine isotopes such as radioactive 125I and
nonradioactive 127I form an identical pair and thus meet the
requirement for the cocktail mixture. In our continued effort to
develop a more sophisticated bimodal MR/SPECT imaging
probe with a tumor-targeting nature, we have prepared a BFCA
comprising DOTA and cyclic RGD (Arg-Gly-Asp), where
cRGD is derivatized by 125I/127I. cRGD is well-known to
possess high and specific affinity for ανβ3-integrin and has been
widely used as a molecular marker for tumor.14 Yet, few studies
are available on the synthesis and the application of 125I/127I-
RGD-DOTA as a BFCA.
127I-RGD, 127I-RGD-DOTA, and the corresponding gadoli-
nium complex (1a) were obtained in high yields (>95%) after
HPLC purification. Their formation was confirmed by MALDI,
and the purity of each compound was confirmed by the
appearance of a single peak in the reverse-phase HPLC. Chart 1
Chart 1. Structure of Complexes 1
Figure 2. In vitro serum stability of 1a (a) by UV detector at 254 nm
and 1b (b) by γ detector. The compounds were incubated in human
serum at 37 °C and analyzed by HPLC at different time intervals.
vitro serum stability of 1a as a function of time as measured by
UV absorption. HPLC chromatograms show dissociations of
RGD (Rt, 8.2 min), I-RGD (Rt, 12.8 min), I-RGD-DOTA (Rt,
12.6 min), and/or 1a. Assignment of 1a eluted at 13.1 min was
made by comparison with a standard. The radiochromato-
graphic profiles for serum samples with 1b at different
incubation times are shown in Figure 2b. The figure shows a
single major peak (Rt, 20 min) assignable to 1b regardless of
the incubation time. No peak corresponding to free 125I (Rt, 5
min) separated from 1b was observed in 72 h of incubation.
The cytotoxicity of 1a remains very low when incubated for 5
days (Figure S1 in the Supporting Information) and
comparable with that of Dotarem. Table S1 in the Supporting
Information summarizes the proton relaxivity r1 and r2 for 1a,
Dotarem, and Omniscan. The r1 and r2 of 1a are the highest to
exhibit 6.17 0.13 and 6.37 0.05 mM−1 s−1 at 298 K and 128
MHz, respectively. It is worth noting that the r1 value of 1a is
twice as high as that Dotarem. A slower tumbling motion in 1a
shows the structures of 1a,b. Corresponding radioactive 1b was
obtained with a moderate radiochemical yield (≥40%) and
purified by HPLC. Although radiochemical purity reaches as
high as 99%, its use in in vivo assays and imaging is
inappropriate due to the presence of acetonitrile (5%), an
inner HPLC eluent. Thus, further purification can be achieved
by purging a solution of 1b with N2 after which the specific
activity of solid 1b reaches up to 2200 Ci/mmol after drying. A
solution of 1b solution sterilized by a sterile filter (0.22 μm)
was used for in vitro and in vivo studies.
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as a result of an increase in molecular weight achieved through
conjugation with RGD may partially explain such increases in
relaxivities as compared with Dotarem and Omniscan.16
Moreover the radioactivity did not have any effect on magnetic
relaxation. Taken together, if measured at the same
concentration, three complexes (1a, 1b, and 1a/1b) are
expected to show the same relaxation behavior.
0.59% ID/g) 30 min after injection. The high uptake by
stomach may suggest partial deiodination of 125I. Sodium iodide
symporter (NIS), which is an integral membrane protein, is
known to transport two sodium ions and one iodide ion into
cells in thyroid tissue, salivary glands, stomach, and breast.17,18
Therefore, specific uptake by stomach, a NIS-expressing organ,
suggests the possible decomposition of the 125I-labeled complex
into iodine ion and ligand. That is, iodide is dissociated from 1b
to be taken up by stomach. The tumor also shows high uptake
Figure 3 shows in vivo dual SPECT/MR images of the
integrin ανβ3 positive U87MG tumor visualized with a mixture
of 1b (1.12
0.09% ID/g). Here again, the behavioral
difference between 1b and a mixture of 1a/1b can be
recognized. For instance, for the reason unknown, the
biodistribution profiles with a mixture of 1a/1b exhibit
radioactivity twice as high as that exhibited by 1b alone (Figure
S2 in the Supporting Information).
The molecular specific tumor imaging can be further
validated by MRI imaging and MRI blocking experiments.
Although MR images are acquired 90 min after administration
of a mixture of 1a and 1b, the integrin ανβ3 positive tumor can
be still enhanced by MR because the signal for 1a remains
almost steady for 3 h (Figure 5). The CNR profile of a mixture
Figure 3. In vivo MR and SPECT/MR images of U87MG tumor-
bearing mice obtained with a mixture of 1a and 1b (a−c) and 1b (d−
f). Quantitative analyses of SPECT imaging of tumors were 0.56
0.05 and 0.24 0.09 at c and f, respectively.
of 1a and 1b. To overcome the sensitivity difference, the
relative amounts of 1a and 1b were adjusted as follows: [1a] for
MRI = 2.7 mg (0.1 mmol Gd/kg); [1b] for SPECT = 1.5 ×
10−5 mg (200 μCi). It is worth noting that the U87MG tumor
is more clearly visualized with high contrast in SPECT by a
mixture of 1a and 1b. Similar observations were also made with
MRI as well: that is, a strong signal enhancement with a mixture
of 1a and 1b, while it remains minimal with 1b alone.
Figure 4 shows the tissue distribution profiles of 1b in nude
mice bearing the U87MG glioblastoma tumors. The weighted
size of the dissected tumors ranges from 5 to 7 mm. Rapid
clearance of radioactivity from the blood circulation is observed.
Among the organs evaluated, stomach and kidney show the
highest uptake (stomach, 3.69 0.29% ID/g; kidney, 2.79
Figure 5. Contrast-to-noise ratio (CNR) as a function of time
measured from the targeting, blocking, and Dotarem experiments by
MRI.
of 1a and 1b shows a significant increase in CNR for as long as
up to 1 h after injection, and the signal persists for 3 h. In
contrast, however, the CNR profile of Dotarem shows a rapid
increase within 30 min followed by a sharp drop since then.
Here, steady signal enhancement by a mixture of 1a and 1b
indicates molecular specific binding to integrin ανβ3 in the
U87MG tumor. The integrin-specific nature of a mixture of 1a
and 1b can be further confirmed by blocking experiments as
shown in Figure 5.
In summary, we successfully synthesized small molecule-
based dual modal MR/SPECT imaging agents 1a and 1b. The
BFCA consists of DOTA conjugate of cyclic RGD, where RGD
incorporates a 125I/127I moiety to the side chain amino group of
the peptide to truly accomplish the chemical equivalence in the
cocktail mixture of 1a and 1b.
ASSOCIATED CONTENT
* Supporting Information
■
S
Synthesis of 1a and 1b and other detailed procedures: in vitro
serum stability assay, in vitro cell toxicity, relaxivity measure-
ments, biodistribution, and in vivo imaging studies. This
material is available free of charge via the Internet at http://
pubs.acs.org.
Figure 4. Organ uptake (% ID/g) of (1b) by mice (n = 4) in 30 min
and 1, 3, and 24 h.
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Letter
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AUTHOR INFORMATION
Corresponding Author
*Tel: +82-53-420-5471. Fax: +82-53-422-2677. E-mail:
ychang@knu.ac.kr. (Y.C.). Tel: +82-2-970-1387. Fax: +82-2-
970-2416. E-mail: kmkim@kirams.re.kr (K.M.K.).
■
Funding
This work was partially supported through The Programs of
Development Research Center of PET Application Technology
& The Programs of Clinical Application Research of
Radiopharmaceuticals in the National Nuclear Technology
Program, ROK. The work was also partially supported by
Nuclear R&D Program (Grant Nos. 2009-0081817 to Y.C. and
2012013722 to K.M.K.) through the National Research
Foundation of Korea (NRF) funded by MEST, ROK.
Kyungpook National University is also acknowledged for
partial support of financial support through KNU Research
Fund, 2012.
Notes
The authors declare no competing financial interest.
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