Synthesis and biological evaluation of RGD peptides with the 99mTc/188Re chelated iminodiacetate core: Highly enhanced uptake and excretion kinetics of theranostics against tumor angiogenesis

Article abstract of DOI:10.1039/c2ra22460g

To develop a companion set of RGD-based agents for diagnostic and radiotherapeutic purposes, facile incorporation of ^99mTc(CO) ₃ or ¹⁸⁸Re(CO)₃ into the same precursor produced, respectively, a structurally and functionally matched radiodiagnostic and radiotherapeutic - or theranostic - pair. This work presents the synthesis of two ^99mTc-labeled RGD monomers (4 and 5) along with a ^99mTc-labeled RGD dimer (6) and an investigation of the influence of the small-sized and negatively charged ^99mTc-iminodiacetate (IDA) core on the in vitro and in vivo behavior of these three different RGD analogs for imaging integrin α_vβ₃ expression. Among the three ^99mTc-IDA-RGD analogs, 6 exhibited the highest integrin binding affinity with an IC₅₀ value of 0.5 nM and a tumor uptake with an ID/g value of 12.3 ± 5.15% at 60 min post-injection, whereas liver and intestinal levels remained relatively low with good metabolic stability (>97%), presumably because of the overall negative charge of the radiometal chelating system. Both ^99mTc/¹⁸⁸Re-labeled compounds (6 and 7), which were prepared from the precursor (18), provided a good tumor accumulation and a clearly visible image of the tumor with high contrast, as compared to the contralateral background in the U87-MG xenograft model. These data support the use of ^99mTc- and ¹⁸⁸Re-IDA-D-[c(RGDfK)] ₂ as a matched radio-theragnostic pair that can be used to individualize radiotherapy for angiogenesis-dependent cancer.

Full text of DOI:10.1039/c2ra22460g

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Synthesis and biological evaluation of RGD peptides
with the 99mTc/188Re chelated iminodiacetate core:
highly enhanced uptake and excretion kinetics of
theranostics against tumor angiogenesis3
Cite this: RSC Advances, 2013, 3, 782
Byung Chul Lee,a Byung Seok Moon,a Ji Sun Kim,a Jae Ho Jung,a Hyun Soo Park,a John
A. Katzenellenbogenb and Sang Eun Kim*ac
To develop a companion set of RGD-based agents for diagnostic and radiotherapeutic purposes, facile
incorporation of 99mTc(CO)3 or 188Re(CO)3 into the same precursor produced, respectively, a structurally
and functionally matched radiodiagnostic and radiotherapeutic—or theranostic—pair. This work presents
the synthesis of two 99mTc-labeled RGD monomers (4 and 5) along with a 99mTc-labeled RGD dimer (6) and
an investigation of the influence of the small-sized and negatively charged 99mTc-iminodiacetate (IDA)
core on the in vitro and in vivo behavior of these three different RGD analogs for imaging integrin avb3
expression. Among the three 99mTc-IDA-RGD analogs, 6 exhibited the highest integrin binding affinity
with an IC50 value of 0.5 nM and a tumor uptake with an ID/g value of 12.3 ¡ 5.15% at 60 min post-
injection, whereas liver and intestinal levels remained relatively low with good metabolic stability (.97%),
presumably because of the overall negative charge of the radiometal chelating system. Both 99mTc/188Re-
labeled compounds (6 and 7), which were prepared from the precursor (18), provided a good tumor
accumulation and a clearly visible image of the tumor with high contrast, as compared to the contralateral
background in the U87-MG xenograft model. These data support the use of 99mTc- and 188Re-IDA-D-
[c(RGDfK)]2 as a matched radio-theragnostic pair that can be used to individualize radiotherapy for
angiogenesis-dependent cancer.
Received 9th October 2012,
Accepted 7th November 2012
DOI: 10.1039/c2ra22460g
www.rsc.org/advances
‘‘RGD’’.4,5 RGD analogs have been widely investigated and
shown to be valuable tools for targeted tumor angiogenesis
Introduction
imaging.6,7 Single photon emission computed tomography
(SPECT)/computed tomography (CT) is a non-invasive imaging
modality that can longitudinally diagnose the target environ-
ment in the same animal across different time-points, and
RGD analogs can be potentially combined with SPECT/CT for
diagnostic purposes. We focused on the radionuclides 99mTc
and 188Re, respectively, to develop a companion set of RGD-
based agents for diagnostic and radiotherapeutic purposes.
99mTc is desirable for diagnostic imaging because of its ideal
nuclear properties (t1/2 = 6 h, 141 keV), while 188Re, with a half-
life of 17 h and a maximum beta energy of 2.12 MeV, is
favorable for tumor radiotherapy. Moreover, both 99mTc and
188Re are readily obtained by daily elution from 99Mo/99mTc-
and 188W/188Re-generator, respectively, and are thus very
convenient and suitable for routine clinical use. Over the past
decade, other research groups have tried to synthesize 99mTc-
labeled RGD analogs containing various 99mTc chelating
moieties, such as 2-mercaptoacetyl-glycylglycyl (MAG2),
6-hydrazinonicotinamide (HYNIC), nitrido-core and the 4 + 1
mixed ligand system.8,9 However, these 99mTc chelating
Targeted tumor-angiogenesis imaging can provide early
diagnosis, aid in treatment planning and monitoring of anti-
angiogenic therapies in cancer.1,2 Integrin avb3 is a suitable
target for both tumor-angiogenesis imaging and anti-angio-
genic therapy because it is highly expressed on activated
endothelial cells and new blood vessels found in tumors and
surrounding tissues but absent in most organs.3 Interest in
tumor-induced angiogenesis has increased enormously over
the past decade, and it is known that the binding region in
integrin avb3 contains the tripeptide sequence of arginine-
glycine-aspartic acid derivatives, commonly known as
aDepartment of Nuclear Medicine, Seoul National University Bundang Hospital,
Seoul National University College of Medicine, 300 Gumidong, Bundanggu,
Seongnam 463-707, Republic of Korea. E-mail: kse@snu.ac.kr; Fax: 82-31-787-4018;
Tel: 82-31-787-7671
bDepartment of Chemistry, University of Illinois, Urbana, Illinois 61801, USA
cProgram in Biomedical Radiation Sciences, Department of Transdisciplinary
Studies, Graduate School of Convergence Science and Technology, Seoul National
University, Seoul 151-742, Korea
Electronic supplementary information (ESI) available. See DOI: 10.1039/
3
c2ra22460g
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terms of their tumor accumulation and pharmacokinetic
properties.
moieties of a targeted compound have significant short-
comings, i.e., a bulky core size, low metabolic stability and
slow clearance from normal tissue. Therefore, certain RGD
analogs have been formulated to include polyethylene glycol
(PEG4) or glycine (G3) linkers to increase the integrin avb3
binding affinity in a ‘‘bivalent’’ fashion and to improve
radiotracer excretion kinetics from normal organs.10
Synthesis of 185/187Re-IDA-RGD analogs (1–3) and
radiochemical synthesis of 99mTc/188Re-IDA-RGD analogs (4–7)
Compound 8 was prepared from the Boc protected PEG acid
and benzyl chloride in the presence of cesium carbonate,
followed by acidic hydrolysis in a trifluoroacetic acid (TFA)–
methylene chloride mixture, as summarized in Scheme 1. To
introduce the 99mTc-(CO)3-IDA core and PEG chain onto the
lysine residue, treatment of glycine methyl ester 9 with tert-butyl
bromoacetate in CH3CN in the presence of DIEA, followed by
hydrolysis with LiOH, resulted in the formation of the acid
compound 10. The amino group of compound 8 was linked with
the acid compound 10 using an amide bond condensation and
then, the IDA moiety linked PEG acid 11 was prepared in the
presence of 10% palladium on charcoal within a hydrogen
atmosphere. Compound 14 was synthesized using the same
procedures described above as the introduction of an IDA
moiety and deprotection of two benzyl groups at acid residues.
The small size of the 99mTc-(CO)3-IDA core led us to design
and synthesize three 99mTc-labeled RGD analogs as radiotracers
for tumor angiogenesis. The 99mTc-labeled RGD monomer
precursor 16, IDA-c(RGDfK), for [99mTc(H2O)3(CO)3]+ labeling,
was synthesized by reacting tert-butyl bromoacetate with the
free amine of lysine in the protected cyclic-R(Pbf)-G-D(OtBu)-f-
K-NH2 15, and the subsequent removal of tert-butyl and
Recently, the tricarbonyl technetium-99m (99mTc-(CO)3)
unit has been shown to be useful for introducing 99mTc into
biomolecules because of its high chemical stability and small
size. The fac-[99mTc(H2O)3(CO)3]+ synthon can be readily
2
generated from 99mTcO4 and CO gas in the presence of
NaBH4.11,12 Additionally, therapeutic radionuclides such as
b-emitting rhenium-188 [188Re(H2O)3(CO)3]+ also could be
prepared from the same class of ligands used for chelation
of the [99mTc(H2O)3(CO)3]+ moiety.13
Many small 99mTc-(CO)3 cores
(
99mTc(I)-complex) with
tridentate ligands have been developed and used for the
preparation of 99mTc-(CO)3-labeled radiotracers.14–19 We have
also previously reported the use of 99mTc-labeled glucosamino-
D-c(RGDfK) that includes a 99mTc(CO)3-(iminodiacetate, IDA)
core as a new radiotracer for tumor angiogenesis imaging.20,21
Although 99mTc-labeled glucosamino-D-c(RGDfK) had a high
integrin binding affinity in vitro, this radiotracer was not
useful as a diagnostic agent because of its low tumor uptake in
vivo. We hypothesized that the overall negative charge of the
radiometal interacted with the nearby positive charge of
arginine in the RGD peptide, thus interfering with the binding
of the peptide unit to the integrin target in vivo. However,
including a negative charge in the 99mTc core could lead to the
development of new analogs having improved biodistribution
as well as desirable pharmacokinetic properties.22
As a continuation of our interest in 99mTc-(CO)3-labeled
RGD peptides with a negative charge on the 99mTc-(CO)3 core,
we designed two 99mTc-labeled RGD monomers with or
without a PEG chain (4 and 5), along with a 99mTc-labeled
RGD dimer (6), and evaluated in vitro and in vivo their capacity
to image integrin avb3 expression. Accordingly, a 188Re-labeled
RGD analog (7) was prepared for a radiotherapeutic purpose
from the selected precursor. Our goal was to develop a
companion set of RGD-based agents (radio-theranostics),
which incorporated tricarbonyl 99mTc or 188Re complexes into
the same precursor, with the choice of radionuclide determin-
ing the potential diagnostic and therapeutic utility.
2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl
(Pbf)
groups on the side chains of the IDA moiety, aspartic acid
and arginine. The 99mTc-labeled RGD monomer precursor with
PEG chain 17 and 99mTc-labeled RGD dimer precursor 18 were
prepared from 15 using an amide bond condensation with
compound 11 and 14, respectively, as shown in Scheme 2.
Subsequently, the protecting groups, three tert-butyl and Pbf
groups, were removed by the treatment of a mixture of TFA–
HSCH2CH2SH–H2O (95 : 2.5 : 2.5) to obtain the desired pre-
cursors 16, 17 and 18. The ‘‘cold’’ rhenium coordination
reaction with the precursors (16, 17, or 18) was performed
in a water–methanol mixture (1 : 1) at 65 uC using (NEt4)2
[
185/187ReCl3(CO)3] (Scheme 2). The desired 185/187Re(CO)3
coordinated RGD-peptides were synthesized from each pre-
cursor (16, 17, and 18) in 60–78% yields and revealed HPLC
retention times of 21.8 min for 1, 22.1 min for 2 and 19.2 min
for 3. The reaction was monitored by HPLC until the precursor
peak disappeared. Incorporation of 99mTc/188Re-(CO)3 into the
IDA moiety in the precursors was performed in aqueous media.
The overall radiochemical yields were 72–79% for 4, 5 and 6 and
50–55% for 7, and all compounds were obtained with high
radiochemical purity (.95%).
Results and discussion
In vitro integrin receptor-binding assay
Chemical synthesis
As shown in Chart 1, the IC50 values of the compounds suggested
that the monomeric RGD analogs (1 and 2) had similar binding
affinity values, while the dimeric RGD analog 3 possessed higher
affinity than either of the monomeric RGD analogs, possibly
because of the bivalency effect. Thus, it appears that the small
size of the 185/187Re-(CO)3-IDA core introduced into the RGD
analogs (1, 2, and 3) resulted in conjugates having high binding
affinity for integrin-positive cells.
We have focused on the development of a diagnostic imaging
agent containing an RGD peptide for tumor angiogenesis,
aiming to introduce the small-sized 99mTc-(CO)3-IDA core into
an RGD peptide in order to increase the hydrophilicity of the
targeted molecule, and to do this, we have made a comparative
evaluation of three 99mTc-IDA-RGD analogs (4, 5, and 6) in
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Scheme 1
the three 99mTc-IDA-RGD analogs (4, 5, and 6) were adminis-
tered intravenously, and at various time points thereafter,
mice were sacrificed to perform tissue distribution studies
(Table 1).
Biodistribution studies
In ex vivo distribution studies, we used human glioblastoma
U87-MG cells, an integrin avb3-positive tumor cell line.23,24
Tumor cells were engrafted on the flanks of Balb/c nude mice.
After tumor volume reached approximately 62.8 ¡ 16.8 mm3,
Scheme 2
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Chart 1
The blood levels of both peptides 5 and 6 were around 0.6%
ID/g (30 min post-injection), whereas that of 4 is even lower with
rapid clearance in the blood. The tumor radioactivity levels of
the three 99mTc-labeled RGD analogs at 60 min post-injection
were 0.40 ¡ 0.14% ID/g (4), 3.35 ¡ 1.23% ID/g (5), and 12.3 ¡
5.15% ID/g (6), respectively. Although 4 has a relatively high
receptor binding affinity, the additional negative charge in the
RGD analog 4 led to an unfavorable biodistribution in vivo. This
lack of correlation between receptor binding affinity and tumor
accumulation may be due to the polarity-dependent tumor
penetration and the binding affinity for the receptors, as
reported for other radiolabeled peptides.22 We also had
anticipated that the negatively charged 99mTc-(CO)3-IDA core
might induce unwanted interactions with the positive charge of
the arginine residue, thus reducing the affinity of the integrin-
binding in vivo. Our previously reported RGD analogs, 99mTc-
labeled glucosamino-D-c(RGDfK) containing the 99mTc-(CO)3-
IDA core, also showed similar poor biodistribution profiles.20
Among other reported 99mTc-(CO)3-labeled RGD analogs, the
99mTc-(CO)3-pyrazolyl conjugate of c(RGDyK), which simply
linked the positive-charge core through the side arm of the
lysine residue, showed relatively high tumor and liver uptake
compared to 4, which contained the negative charge on the
99mTc core.16
Finally, we selected the dimeric cRGD precursor 18 and
introduced the 188Re-(CO)3-IDA core as the therapeutic agent.
The radiochemical synthesis of [188Re(CO)3(H2O)3]+ was per-
formed as previously described.13 The highest tumor radio-
activity level of 7 was 12.3 ¡ 1.7% ID/g at 30 min post-injection.
The liver uptake of both peptides was similar at 30 min post-
injection, whereas liver uptake of 7 gradually became lower than
that of 6 by 2 h. Over time, tumor-to-liver ratios of 7 increased
continuously to 11.2 until 8 h. Thus, ex vivo biodistribution
demonstrated that the matched pair of 99mTc and 188Re-labeled
peptides (6 and 7) have both similar and very high integrin
binding affinity in a tumor xenograft.
SPECT/CT imaging studies in U87-MG tumor-bearing mice
As shown in Fig. 1, comparison of the SPECT/CT images of the
three 99mTc-IDA-RGD analogs and 188Re-IDA-D-[c(RGDfK)]2
visually reflected the ex vivo biodistribution of 99mTc/188Re
uptake in the animal model. All the 99mTc-IDA-RGD analogs
showed rapid clearance from the blood after injection, and the
major radioactivity was excreted in the urine (Fig. 1). The
introduction of the spacer in 5 significantly influenced tumor
accumulation. Among the four analogs examined here, 6 and 7
possessed high tumor uptake as well as low liver and intestinal
uptake. The higher kidney uptake is probably caused by the
increased arginine residues. This influence of the positive
charge on the renal uptake has been reported for radiolabeled
multimeric cyclic RGD peptides and other peptides.10,25 On
the basis of both high tumor uptake and low uptake in
receptor-negative tissues of 6 and 7, we believe that low uptake
and rapid reduction in hepatic levels might be the result of
rapid renal clearance engendered by the negative charge on
the 99mTc/188Re-(CO)3-IDA core attached to the RGD peptide
dimer. These results indicate that modification of the polarity
with a single negative charge can change the pharmacoki-
netics and have a desirable effect on the biodistribution of
biomolecules. The diminutive and hydrophilic 99mTc/188Re-
(CO)3-IDA core is expected to only minimally perturb the
biological activity of RGD, with subsequently increased tumor
targeting and improved in vivo kinetics.
To avoid the undesirable interaction between the 99mTc-
(CO)3-IDA core and the arginine, we inserted a PEG linker on the
side arm of the lysine in the RGD analog. The uptake of 99mTc-
IDA-PEG-c(RGDfK) (5) in integrin-positive tumor cells was
similar to other monomeric RGD analogs, with slightly faster
clearance from normal organs than the positive charge-contain-
ing 99mTc-(CO)3-pyrazolyl conjugate of c(RGDyK). The liver
uptake of both peptides 5 and 6 was similar at 30 min post-
injection; however, the intestinal uptake of 6 was gradually
reduced to significantly lower levels than that of 5 at 240 min
post-injection. Remarkably, the tumor uptake of 99mTc-IDA-D-
[c(RGDfK)]2 (6) was rapid and high, whereas its liver and
intestinal levels remained relatively low. Thus, over time, the
tumor-to-liver ratios of 6 increased from 4.0 at 10 min post-
injection to a peak of 5.2 at 1 h, which was maintained until 4 h.
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Table 1 Ex vivo biodistribution studies (% ID/g ¡ SD)a of 99mTc-IDA-RGD analogsb (4–7) in U87-MG xenografts
99mTc-IDA-c(RGDfK), 4
Tissue/organ
10 min
30 min
60 min
120 min
240 min
Blood
Liver
Lung
Spleen
Kidneys
Small intestine
Large intestine
Thyroid
Muscle
Brain
Tumor
0.37 ¡ 0.04
5.12 ¡ 0.76
1.72 ¡ 0.86
0.56 ¡ 0.32
1.65 ¡ 0.48
4.41 ¡ 1.00
0.55 ¡ 0.37
0.05 ¡ 0.02
0.56 ¡ 0.27
0.04 ¡ 0.01
1.43 ¡ 0.15
0.10 ¡ 0.01
0.92 ¡ 0.10
0.54 ¡ 0.14
0.19 ¡ 0.10
0.50 ¡ 0.09
5.59 ¡ 0.86
0.31 ¡ 0.06
0.01 ¡ 0.00
0.16 ¡ 0.04
0.02 ¡ 0.00
0.78 ¡ 0.40
0.03 ¡ 0.00
0.45 ¡ 0.09
0.27 ¡ 0.07
0.12 ¡ 0.03
0.23 ¡ 0.02
3.95 ¡ 0.22
0.53 ¡ 0.81
0.01 ¡ 0.00
0.08 ¡ 0.07
0.01 ¡ 0.01
0.40 ¡ 0.14
0.02 ¡ 0.00
0.30 ¡ 0.09
0.40 ¡ 0.46
0.08 ¡ 0.03
0.13 ¡ 0.01
4.00 ¡ 0.41
0.11 ¡ 0.01
0.03 ¡ 0.03
0.05 ¡ 0.02
0.03 ¡ 0.02
0.33 ¡ 0.06
—
—
—
—
—
—
—
—
—
—
—
99mTc-IDA-PEG-c(RGDfK), 5
Tissue/organ
10 min
30 min
60 min
120 min
240 min
Blood
Liver
Lung
Spleen
Kidneys
Small intestine
Large intestine
Thyroid
Muscle
Brain
Tumor
1.43 ¡ 0.23
5.30 ¡ 0.95
3.50 ¡ 0.18
2.31 ¡ 0.36
7.53 ¡ 0.46
17.6 ¡ 2.20
2.06 ¡ 0.25
0.20 ¡ 0.04
1.31 ¡ 0.02
0.10 ¡ 0.01
5.43 ¡ 0.50
0.66 ¡ 0.09
2.62 ¡ 0.28
2.87 ¡ 0.56
1.45 ¡ 0.18
4.33 ¡ 1.60
15.6 ¡ 2.89
1.21 ¡ 0.20
0.12 ¡ 0.03
0.97 ¡ 0.19
0.07 ¡ 0.01
4.27 ¡ 1.15
0.35 ¡ 0.06
1.41 ¡ 0.21
1.52 ¡ 0.19
0.77 ¡ 0.07
2.80 ¡ 0.89
4.79 ¡ 2.13
0.98 ¡ 0.30
0.08 ¡ 0.02
0.66 ¡ 0.13
0.06 ¡ 0.01
3.35 ¡ 1.23
0.17 ¡ 0.02
0.98 ¡ 0.15
0.65 ¡ 0.08
0.47 ¡ 0.10
1.13 ¡ 0.11
2.77 ¡ 0.96
26.8 ¡ 2.80
0.03 ¡ 0.00
0.52 ¡ 0.47
0.04 ¡ 0.02
2.63 ¡ 0.74
0.11 ¡ 0.01
0.64 ¡ 0.08
0.32 ¡ 0.04
0.20 ¡ 0.02
0.61 ¡ 0.14
0.37 ¡ 0.17
28.1 ¡ 5.82
0.01 ¡ 0.01
0.07 ¡ 0.01
0.02 ¡ 0.00
1.41 ¡ 0.24
99mTc-IDA-D-[c(RGDfK)]2, 6
Tissue/organ
10 min
30 min
60 min
120 min
240 min
Blood
Liver
Lung
Spleen
Kidneys
Small intestine
Large intestine
Thyroid
Muscle
Brain
Tumor
1.16 ¡ 0.08
3.10 ¡ 0.58
4.34 ¡ 0.69
3.33 ¡ 0.89
11.5 ¡ 1.36
5.82 ¡ 0.08
4.42 ¡ 0.85
0.19 ¡ 0.03
1.48 ¡ 0.21
0.13 ¡ 0.02
12.4 ¡ 3.89
0.62 ¡ 0.11
2.24 ¡ 0.33
3.34 ¡ 0.80
2.67 ¡ 0.63
10.9 ¡ 1.71
4.10 ¡ 1.09
3.93 ¡ 0.57
0.15 ¡ 0.02
1.49 ¡ 0.44
0.10 ¡ 0.02
11.5 ¡ 1.53
0.42 ¡ 0.01
2.36 ¡ 0.21
3.55 ¡ 0.26
2.92 ¡ 0.29
12.1 ¡ 1.62
3.90 ¡ 0.48
4.08 ¡ 0.53
0.14 ¡ 0.03
2.23 ¡ 1.44
0.10 ¡ 0.01
12.3 ¡ 5.15
0.26 ¡ 0.05
2.06 ¡ 0.54
2.19 ¡ 0.35
2.12 ¡ 0.32
10.1 ¡ 2.82
2.85 ¡ 0.99
2.63 ¡ 0.84
0.12 ¡ 0.04
1.11 ¡ 0.43
0.08 ¡ 0.01
9.81 ¡ 1.92
0.16 ¡ 0.02
1.24 ¡ 0.19
1.33 ¡ 0.09
1.29 ¡ 0.21
7.14 ¡ 0.85
1.62 ¡ 0.18
1.54 ¡ 0.20
0.10 ¡ 0.02
0.64 ¡ 0.07
0.06 ¡ 0.01
6.15 ¡ 0.84
188Re-IDA-D-[c(RGDfK)]2, 7
Tissue/organ
10 min
30 min
60 min
120 min
480 min
Blood
Liver
Lung
Spleen
Kidneys
Small intestine
Large intestine
Thyroid
Muscle
Brain
Tumor
1.13 ¡ 0.18
3.77 ¡ 0.13
4.52 ¡ 0.76
3.30 ¡ 0.48
11.8 ¡ 0.74
3.68 ¡ 0.36
2.14 ¡ 0.41
0.22 ¡ 0.10
1.58 ¡ 0.14
0.19 ¡ 0.04
10.5 ¡ 1.19
0.58 ¡ 0.02
2.58 ¡ 0.26
3.56 ¡ 0.37
2.81 ¡ 0.67
9.25 ¡ 1.24
3.14 ¡ 0.90
1.16 ¡ 0.20
0.21 ¡ 0.16
1.17 ¡ 0.36
0.14 ¡ 0.06
12.3 ¡ 1.73
0.33 ¡ 0.04
1.78 ¡ 0.34
2.68 ¡ 0.66
1.66 ¡ 1.01
7.88 ¡ 1.40
2.95 ¡ 0.63
1.10 ¡ 0.12
0.13 ¡ 0.03
1.02 ¡ 0.24
0.10 ¡ 0.02
11.5 ¡ 1.41
0.10 ¡ 0.02
1.07 ¡ 0.18
1.41 ¡ 0.12
1.39 ¡ 0.24
6.11 ¡ 0.76
1.81 ¡ 0.16
1.77 ¡ 0.27
0.07 ¡ 0.01
0.71 ¡ 0.18
0.06 ¡ 0.00
10.6 ¡ 0.31
0.03 ¡ 0.00
0.35 ¡ 0.05
0.67 ¡ 0.07
0.70 ¡ 0.15
3.30 ¡ 0.42
0.86 ¡ 0.07
4.49 ¡ 0.58
0.05 ¡ 0.01
0.33 ¡ 0.03
0.04 ¡ 0.01
3.91 ¡ 0.27
a
b
Percent of injected dose per gram of tissue; mean ¡ SD (n = 4). Tracer (4, 5, 6, or 7) was dissolved in saline and injected into U87-MG-
bearing nude mice. The dose per mouse was 0.74 MBq via the tail vein.
To confirm the receptor-binding specificity of 6, a blocking
experiment was performed. In vivo blocking with excess
cRGDyV (18 mg kg21) resulted in significantly reduced
radioactivity uptake in the tumor. In addition, the use of an
integrin non-binding, negative control peptide, 99mTc-IDA-D-
[c(RADfK)]2, indicated that the uptake of the major fraction of
6 in the tumor was selectively integrin-mediated, as shown in
Fig. 2. This RAD peptide does not bind to integrin avb3, due to
the addition of a single methyl group that results from
changing glycine to alanine.26
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Fig. 1 Comparison of SPECT/CT images of 99mTc-IDA-RGD analogs (4, 5, and 6) and 188Re-IDA-D-[c(RGDfK)]2 (7).
blood (Fig. 3). The metabolic stability of 6 was evaluated in
human U87-MG glioma tumor-bearing nude mice and
determined in blood and urine samples and in liver, kidneys,
and tumor homogenates at 30 min, and 2 h each after
intravenous injection. The extraction efficiency of all organs
Metabolic stability
In metabolic stability studies, the radioactivity in the tumor
remained stable from 30 min until 2 h, with retained activity
being over 98% for non-metabolized species; furthermore, the
labeled peptides also remained stable in the other organs and
Fig. 2 SPECT/CT imaging of the tumor region with or without cRGDyV and SPECT/CT imaging of 99mTc-IDA-D-[c(RADfK)]2 for a negative control peptide. Yellow
circles indicate the tumor location. Color bars indicate the range of radio-uptake as a percentage of the injected dose per gram of tissue.
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high tumor accumulation and desirable tumor to liver ratios
for a radiotherapeutic purpose. Optimization of integrin avb3
expression therapy for human tumors will ultimately require
further xenografts; however, the pharmacokinetic behavior of
7 described here holds considerable promise. The results of
our ex vivo biodistribution and in vivo SPECT imaging studies,
including a sensitive visibility in tumor xenograft presented
here, suggest that the two agents that we have developed,
99mTc- and 188Re-IDA-D-[c(RGDfK)]2, hold promise as radio-
theranostics in the field of tumor-induced angiogenesis.
Experimental
General
All commercial reagents and solvents were used without
further purification unless otherwise specified. Reagents and
solvents were commercially purchased from Sigma-Aldrich
(Seoul, Korea). Flash column chromatography was performed
with silica gel (230–400 mesh, ASTM; Merck). All reactions
were monitored on pre-coated plates (silica gel 60F254; Merck).
NMR spectra were recorded on a Varian 400-MR (Palo Alto, CA,
USA) spectrometer at ambient temperature. Chemical shifts
were reported in parts per million (ppm, d units). Electrospray
ionization (ESI) was obtained on a Varian 500-MS ion trap
mass spectrometer (Varian, Palo Alto, CA, USA) and MALDI-
TOF mass spectra were performed on a Voyager-DE STR
MALDI-TOF mass spectrometer (San Francisco, CA, USA).
HPLC was carried out on a Thermo Separation Products
System (Fremont, CA, USA) equipped with a semi-preparative
column (Eclipse XDB-C18 column 5 mm, 9.4 6 250 mm;
Agilent Co., Palo Alto, CA, USA). Chromatography systems were
fitted with a UV detector (SectraSystem UV3000 set at 214 nm;
Thermo, Fremont, USA) and a gamma-ray detector (Flow-
Count fitted with a NaI(Tl) detector; Bioscan, Washington DC,
USA). Thin Layer Chromatography (TLC) of radiosynthesis was
performed using Merck F254 silica plates and analyzed on a
Bioscan radio-TLC scanner (Washington DC, USA). All radio-
activities were measured using a VDC-505 activity calibrator
from Veenstra Instruments (Joure, The Netherlands). The
cyclic pentapeptides, i.e., cyclic-Arg(Pbf)-Gly-Asp(OtBu)-D-Phe-
Lys-NH2, cyclic-Arg(Pbf)-Ala-Asp(OtBu)-D-Phe-Lys-NH2 (for
99mTc-IDA-D-[c(RADfK)]2, details of observation and analysis
Fig. 3 Identification of parent and metabolites of 6 in a mouse xenograft
model. HPLC profiles of each sample at 30 and 120 min post-injection: blood
samples (a), urine samples (b), liver samples (c), kidney samples (d), and tumor
samples (e).
was over 87% for 30 min post-injection, except for blood
(Online Supplementary Information Table 2 ). The retention
3
time for 6 was 19.5 min. The HPLC profiles showed two
metabolites having retention times of 9 and 19 min in the
blood, liver, and kidneys. 99mTc-IDA-D-[c(RGDfK)]2 reached
stable levels in all the samples by 2 h after injection; however,
some degradation was observed at later time points in the
blood (19%), liver (18%), and kidneys (13%). The amount of
intact tracer in the tumor was greater than 98% by 2 h.
These results indicate 6 to be a metabolically stable peptide
that can monitor overexpressed integrin avb3 on newly formed
blood vessels near and within tumor tissue. This agent can
thus be potentially useful for monitoring the therapeutic anti-
angiogenesis effect of a corresponding anti-cancer treatment
in an in vivo tumor model.
appear in the online Supplementary Information ), and cyclic-
3
Arg-Gly-Asp-D-Tyr-Val (for RGD[125I]yV) were prepared using a
solid support coupling protocol according to previously
described methods.21,27 We purchased 99Mo/99mTc- and
188W/188Re-generator from Samyoung Unitech (Seoul, Korea)
and Enviro Korea (Daejeon, Korea), respectively.
Benzyl 1-amino-3,6,9,12-tetraoxapentadecan-15-oate (8)
A
stirred solution of 1-[N-(tert-butyloxycarbonyl)-amino]-
3,6,9,12-tetraoxapentadecanoic acid (224 mg, 0.61 mmol) and
cesium carbonate (596 mg, 1.83 mmol) in acetonitrile (CH3CN,
1 mL) at room temperature was treated with benzyl chloride
(0.21 mL, 1.83 mmol) under argon atmosphere. The reaction
mixture was warmed to 80 uC for 30 min. After cooling to room
temperature, the reaction solvent was evaporated and the
Conclusions
The tumor uptake of 6 was clearly visible; it was significantly
higher than that in the contralateral background at all time
points, and it demonstrated integrin-targeting specificity and
high metabolic stability. In addition, 7 showed significantly
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crude mixture was purified by silica gel flash column
chromatography using 5% MeOH–CH2Cl2 as the eluant, and
combined benzylate in dichloromethane (CH2Cl2, 10 mL) was
added in trifluoroacetic acid (1 mL) at 0 uC. The reaction
mixture was stirred for 1 h at room temperature and basified
by saturated sodium bicarbonate (25 mL). After extraction with
CH2Cl2 (100 mL 6 3), the combined organic layers were
concentrated, and purified by silica gel flash column chroma-
tography using MeOH–CH2Cl2–triethylamine (5 : 93 : 2) as the
eluant, affording 194 mg (81%) of the title compound 8 as a
pale yellow oil: 1H NMR (400 MHz, CDCl3) d 7.38–7.33 (m, 5H),
5.14 (s, 2H), 3.78 (t, J = 6.2 Hz, 2H), 3.65–3.62 (m, 12H), 3.58 (t,
J = 4.8 Hz, 2H), 2.93 (brs, 2H), 2.66 (t, J = 6.2 Hz, 2H), 2.51 (brs,
2H); 13C NMR (100 MHz, CDCl3) d 172.2, 135.6, 128.6, 128.4,
128.2, 70.3, 70.0 (63), 69.85, 69.79, 66.9, 66.7, 66.5, 39.8, 34.7;
MS (ESI) m/z 356 (M + H)+; HRMS calcd for C18H30NO6
356.2068, found 356.2052.
desired compound 11 (120 mg, 76%) was obtained as a colorless
oil: 1H NMR (400 MHz, CDCl3) d 9.02 (brs), 8.20 (brs), 3.75–3.70
(m, 2H), 3.63–3.56 (m, 14H), 3.52–3.47 (m, 2H), 3.38–3.36 (m,
6H), 2.57 (t, J = 6.0 Hz, 2H), 1.44 (s, 18H); 13C NMR (100 MHz,
CDCl3) d 175.0, 171.8, 170.5, 81.7, 70.34, 70.32, 70.29, 70.2,
70.10, 70.06, 69.8, 67.0, 58.8, 56.6, 38.8, 35.5, 28.1; MS (ESI) m/z
551 (M + H)+; HRMS calcd for C25H47N2O11 551.3174, found
551.3158.
N-a-Bis(tert-butoxycarbonylmethyl)-aspartic acid dibenzyl
ester (13)
To a solution of L-aspartic acid dibenzyl ester (12, 1 g, 2.85
mmol) and DIEA (1.5 mL 8.57 mmol) in CH3CN (15 mL), tert-
butylbromoacetate (1.27 mL, 8.57 mmol) was added at 90 uC
under nitrogen gas. After the reaction mixture was refluxed for
12 h, the solvent was removed in vacuo, and the residue was
dissolved in EtOAc (100 mL), the organic solution was washed
with saturated sodium chloride (30 mL), followed by water (30
mL) and dried over anhydrous sodium sulfate. The organic
solution was concentrated, and purified by silica gel flash
column chromatography using 20% EtOAc–hexane as the
eluant, affording 770 mg (50%) of the title compound 13 as a
2-[Bis(2-tert-butoxy-2-oxoethyl)amino]acetic acid (10)
A stirred solution of glycine methyl ester hydrochloride (9, 500
mg, 3.98 mmol) and N,N9-diisopropylethylamine (DIEA, 2.10
mL, 11.9 mmol) in CH3CN (5 mL) at room temperature was
treated with tert-butyl bromoacetate (1.76 mL, 11.9 mmol).
After the mixture was stirred at 60 uC for 12 h, the cooled
solution was concentrated in vacuo and extracted with ethyl
acetate (EtOAc, 100 mL 6 3). After the combined organic
layers were dried and concentrated, a solution of the crude
product in methanol (MeOH, 10 mL) was treated with a 1 M
LiOH solution (2.0 equiv.) and stirred at 50 uC for 1 h. After the
cooled solution was concentrated in vacuo, the residue was
dissolved in water and acidified to pH 5 with 1 M HCl and
extracted with EtOAc (100 mL 6 3). The combined organic
layers were concentrated, and purified by silica gel flash
column chromatography using 5% MeOH–CH2Cl2 as the
eluant, affording 603 mg (50%) of the title compound 10 as
a white solid: mp 117–124 uC; 1H NMR (400 MHz, CDCl3) d
3.49 (s, 2H), 3.47 (s, 4H), 1.48 (s, 18H); 13C NMR (100 MHz,
DMSO-d6) d 172.3, 169.9, 80.3, 55.2, 54.5, 27.8; MS (ESI) m/z
304 (M + H)+; HRMS calcd for C14H26NO6 304.1755, found
304.1750.
1
colorless oil: H NMR (400 MHz, CDCl3) d 7.36–7.26 (m, 10H),
5.14–5.06 (m, 4H), 3.99 (dd, J = 9.2, 5.2 Hz, 1H), 3.61–3.50 (m,
4H), 2.94 (dd, J = 16.8, 9.6 Hz, 1H), 2.82 (dd, J = 16.4, 5.6 Hz,
1H), 1.42 (s, 18H); 13C NMR (100 MHz, CDCl3) d 171.2, 170.6,
170.5, 135.74, 135.66, 128.48, 128.46, 128.24, 128.22, 128.18,
128.13, 81.0, 66.7, 66.5, 61.0, 53.5, 35.9, 28.1; MS (FAB) m/z 542
(M+H)+; HRMS calcd for C30H40NO8 542.2754, found 542.2751.
N-a-Bis(tert-butoxycarbonylmethyl)-aspartic acid (14)
A solution of compound 13 (460 mg, 0.85 mmol) in methanol
(10 mL) was treated with 10% palladium on charcoal (2.0
equiv.) under nitrogen atmosphere. After gas atmosphere was
exchanged from N2 to H2 gas, the reaction mixture was stirred
for 12 h at room temperature. The reaction mixture was
filtered through a Celite layer and then the solvent was
removed in vacuo. The desired compound 14 (278 mg, 91%)
1
was obtained as a colorless oil: H NMR (400 MHz, CDCl3) d
9.60 (brs, 2H), 3.90 (t, J = 6.6 Hz, 1H), 3.55–3.40 (m, 4H), 2.98
(dd, J = 16.4, 5.2 Hz, 1H), 2.66 (dd, J = 16.8, 7.6 Hz, 1H), 1.46 (s,
18H); 13C NMR (100 MHz, CDCl3) d 175.3, 174.6, 172.3, 82.7,
62.7, 54.2, 34.4, 28.0; MS (EI) m/z 361 (M+H)+; HRMS calcd for
6-(2-tert-Butoxy-2-oxoethyl)-2,2-dimethyl-4,8-dioxo-
3,12,15,18,21-pentaoxa-9-azatetracosan-24-oic acid (11)
C
16H27NO8 361.1736, found 361.1738.
A stirred solution of compound 10 (60 mg, 0.19 mmol),
1-hydroxybenzotriazole (HOBT, 51 mg, 0.38 mmol) and
O-benzotriazole-N,N,N9,N9-tetramethyl-uronium-hexafluoropho-
sphate (HBTU, 144 mg, 0.38 mmol) in N,N9-dimethylformamide
(DMF, 5 mL) at room temperature was treated with the mixture
of compound 8 (47 mg, 0.13 mmol) and DIEA (133 mL, 0.76
mmol) via dropwise addition. The resulting solution was stirred
for 16 h at room temperature, and then the solvent was removed
in vacuo. The crude reaction mixture was purified by silica gel
flash column chromatography using 5% MeOH–CH2Cl2 as the
eluant. A solution of the crude product in methanol (5 mL) was
treated with 10% palladium on charcoal (2.0 equiv.) under
nitrogen atmosphere. After gas atmosphere was exchanged
from N2 to H2 gas, the reaction mixture was stirred for 16 h at
room temperature. The reaction mixture was filtered through a
Celite layer and then the solvent was removed in vacuo. The
N-a-Bis(hydroxycarbonylmethyl)-cyclic(Arg-Gly-Asp-D-Phe-Lys),
IDA-c(RGDfK) (16)
A stirred solution of cyclic[Arg(Pbf)-Gly-Asp(OtBu)-D-Phy-Lys-
NH2] (15, 240 mg, 0.26 mmol) and DIEA (130 mL, 0.77 mmol) in
CH3CN (5 mL) was treated with tert-butyl bromoacetate (115
mL, 0.77 mL) in CH3CN (1 mL). The resulting solution was
stirred at 50 uC for 12 h and then the cooled solution was
evaporated under reduced pressure. The crude reaction
mixture was purified by silica gel flash column chromatogra-
phy using 10% MeOH–CH2Cl2 as the eluant, affording the
protected
peptide,
N-a-bis(tert-butyloxycarbonylmethyl)-
cyclic[Arg(Pbf)-Gly-Asp(OtBu)-D-Phy-Lys], as a colorless oil;
MS (MALDI) m/z 1163 (M + Na)+. Subsequently, the obtained
peptide was treated with a solution of trifluoroacetic acid–
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HSCH2CH2SH–water (5 mL, 95 : 2.5 : 2.5). The reaction
mixture was stirred for 24 h at room temperature and then
the solution was evaporated in vacuo. To get the solid, excess
diethyl ether was added to the residue mixture. The resulting
solid was filtered and washed with diethyl ether. After the solid
dried under reduced pressure, it gave compound 16 (134 mg,
74%) as a white solid: MS (MALDI) m/z 720.8 (M + Na)+.
from the column was monitored with a 214 nm UV detector.
The chemically pure 185/187Re-IDA-RGD analogs eluted with a
retention time of 21.8 min for 1, 22.1 min for 2 and 19.2 min
for 3. The product isolated from semi-preparative HPLC was
diluted with excess water, passed through a tC18 Sep-Pak
cartridge, washed with water (5 mL) and eluted with methanol
(2 mL). After methanol was removed under reduced pressure,
it gave 185/187Re-IDA-RGD analogs (1, 2, and 3, 60–78%) as a
white solid: MS (ESI) m/z 990.85 (M + H)+ for 1, MS (MALDI) m/
z 1293.4 (M + H)+ for 2 and MS (ESI) m/z 1688.5 (M + H)+ for 3.
N-a-Bis(hydroxycarbonylmethyl)-PEG-cyclic(Arg-Gly-Asp-D-Phe-
Lys), IDA-PEG-c(RGDfK) (17)
To a solution of compound 11 (50 mg, 0.089 mmol), HOBT (36
mg, 0.27 mmol), and HBTU (100 mg, 0.27 mmol) in DMF (7
mL) was added a mixture of compound 15 (51 mg, 0.089
mmol) and DIEA (93 mL, 0.534 mmol) in DMF (3 mL) at room
temperature under nitrogen gas. After the reaction mixture
was stirred for 12 h, the solvent was removed under reduced
pressure. The crude reaction mixture was purified by silica gel
flash column chromatography using 10% MeOH/CH2Cl2 as
eluant, affording the protected peptide, N-a-bis(tert-butylox-
ycarbonylmethyl)-PEG-cyclic[Arg(Pbf)-Gly-Asp(OtBu)-D-Phy-
Lys], as a white solid; MS (ESI) m/z 1444.7 (M + H)+.
Subsequently, deprotection and purification of the protected
peptide were followed using the same conditions as described
above in the method for compound 16. After the solid dried
under reduced pressure, it gave compound 17 (62 mg, 69%) as
a white solid: MS (ESI) m/z 1024.5 (M + H)+.
General method for 99mTc-IDA-RGD analogs (4–6)
The fac-[99mTc(H2O)3(CO)3]+ synthon was prepared according
to Alberto et al.11,12 A solution of [99mTc(H2O)3(CO)3]+ (370–740
MBq) in saline (200 mL) was added to the precursor (16, 17 or
18, 0.3 mg) in water (300 mL). The reaction mixture was stirred
at 75 uC for 30 min and then cooled in an ice bath. The
radiotracer was purified by semi-preparative HPLC (Agilent Co.
Eclipse XDB-C18 column; 5 mm, 9.4 6 250 mm) and used the
same gradient condition as described in the general method
for 185/187Re-IDA-RGD analogs. The effluent from the column
was monitored with a 214 nm UV detector followed by a
gamma radioactive detector. The radiochemically pure 1, 2, or
3 eluted off with a retention time of 20.6 min for 1, 21.4 min
for 2 and 18.4 min for 3, and the radiochemical yield ranged
between 72–79%. The product isolated from semi-preparative
HPLC was diluted with excess water, passed through a tC18
Sep-Pak cartridge and washed with water (5 mL). The desired
product was eluted by 80% ethanol–saline (1.5 mL) and
evaporated under a stream of nitrogen gas. The product was
made with saline and sterilized by passing through a sterile
filter (0.22 mm).
N-a-Bis(hydroxycarbonylmethyl)-D-[cyclic(Arg-Gly-Asp-D-Phe-
Lys)]2, IDA-D-[c(RGDfK)]2 (18)
To a solution of compound 14 (39.5 mg, 0.11 mmol), HOBT
(59.4 mg, 0.44 mmol), and HBTU (167 mg, 0.44 mmol) in DMF
(7 mL) was added a mixture of compound 15 (350 mg, 0.38
mmol) and DIEA (153 mL, 0.88 mmol) in DMF (3 mL) at room
temperature under nitrogen gas. After the reaction mixture
was stirred for 12 h, the solvent was removed under reduced
pressure. The crude reaction mixture was purified by silica gel
flash column chromatography using 10% MeOH–CH2Cl2 as
the eluant, affording the protected peptide, N-a-bis(tert-
butyloxycarbonylmethyl)-D-{cyclic[Arg(Pbf)-Gly-Asp(OtBu)-D-
Phy-Lys]}2, as a white solid; MS (MALDI) m/z 2171.06 (M + Na)+.
Subsequently, deprotection and purification of the protected
peptide were followed using the same conditions as described
above in the method for compound 16. After the solid dried
under reduced pressure, it gave compound 18 (97 mg, 62%) as
a white solid: MS (ESI) m/z 1420.5 (M + H)+.
General method for 188Re-IDA-D-[c(RGDfK)]2 (7)
The fac-[188Re(H2O)3(CO)3]+ was prepared according to the
literature.13 To a solution of [188Re(H2O)3(CO)3]+ (50–100 mCi)
in saline (200 mL) was added the precursor 18 (0.5 mg, 0.35
mmol) in water (300 mL). The reaction mixture was stirred at 75
uC for 60 min and then cooled in an ice bath. The radiotracer
was purified by semi-preparative HPLC (Agilent Co. Eclipse
XDB-C18 column; 5 mm, 9.4 6 250 mm) with the same
gradient condition as described above. The effluent from the
column was monitored with a 214 nm UV detector followed by
a gamma radioactive detector. The radiochemically pure 188Re-
IDA-D-[c(RGDfK)]2 eluted with a retention time of 19.2 min
and the radiochemical yield ranged 50–55%. The product
isolated from semi-preparative HPLC was diluted with excess
water, passed through a tC18 Sep-Pak cartridge and washed
with water (5 mL). The desired product was eluted by 80%
ethanol–saline (1.5 mL) and evaporated under a stream of
nitrogen gas. The product was made isotonic with sodium
chloride and sterilized by passing through a sterile filter (0.22
mm). Specific radioactivity of 188Re-IDA-D-[c(RGDfK)]2; 3.3 Ci/
mmol was obtained after purification in an HPLC column.
185/187Re-IDA-RGD analogs (1–3)
To a solution of compounds 16, 17 or 18 (10 mmol) in a
solution of water (0.5 mL) and MeOH (0.5 mL) was added
(NEt4)2[185/187ReCl3(CO)3] (9.1 mg, 15 mmol). The reaction
mixture was stirred at 60 uC for 3 h. The solution was removed
under reduced pressure and then the product was purified by
semi-preparative HPLC (Agilent Co. Eclipse XDB-C18 column;
5 mm, 9.4 6 250 mm). The column was eluted with a solvent
mixture of CH3CN–water–0.1% trifluoroacetic acid using a
gradient condition. The HPLC eluent started with 20%
CH3CN–water (0.1% trifluoroacetic acid) from 0 to 5 min and
the ratio was increased with a solvent mixture of 60% CH3CN–
water (0.1% trifluoroacetic acid) over 40 min. The effluent
Cell lines and animal model
For the in vitro competitive binding assay, U87-MG cells
(ATCC) were cultured in Dulbecco’s modified Eagle’s medium
supplemented with 4 mM L-glutamine, 4500 mg L21 glucose, 1
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mM sodium pyruvate, 1.5 g mL21 sodium bicarbonate, 10%
FBS and antibiotic-antimycotic (Gibco, Grand Island, NY, USA)
at 37 uC in a humidified atmosphere of 5% CO2. Human
glioblastoma U87-MG cells were cultured for 2–3 days, without
changing the media, and harvested with trypsin-EDTA in PBS.
We resuspended the U87-MG cells (5 6 106) in 100 mL with 100
mM PBS (pH 7.4) and injected them subcutaneously into the
right shoulder of 6–7 week-old male Balb/c nu/nu mice (Orient
Bio Inc., Seongnam, Korea). Animal protocols were approved
by the Seoul National University Bundang Hospital Animal
Care and Use Committee. We also performed caliper measure-
ments of the longest perpendicular tumor diameters.
3500 rpm at 4 uC. After removal of the supernatant, the pellets
were washed twice with cold PBS (2 mL). For each sample, the
supernatants of both centrifugation steps for the blood, liver,
kidneys, and tumor were combined and passed through tC-18
Sep-Pak cartridges. The urine samples were diluted with 2 mL
of cold PBS and passed through a tC-18 Sep-Pak cartridge, and
the cartridge was washed with 2 mL of water and eluted with
CH3CN (3 mL). Then, organic fractions (400 mL) were diluted
with water (1.6 mL), and the aliquots were analyzed by HPLC.
The identification of the percentage of the intact tracer was
performed using RP-HPLC (YMC C18, 5 mm, 7.9 6 250 mm)
with a 25% CH3CN–water–0.1% trifluoroacetic acid gradient (2
mL min21, tR = 19.5 min).
In vitro integrin receptor-binding assay
The receptor binding affinity studies of 185/187Re-IDA-RGD
analogs (1, 2, and 3) were performed using 125I-c(RGDyV) as
the integrin-specific radio-ligand in integrin avb3-positive
human glioblastoma U87-MG cells. One hundred microliters
of a cell suspension (105 glioma cells) in Eppendorf tubes were
incubated with 125I-c(RGDyV) (0.037 MBq/tube) in the presence
of increasing concentrations of cold (185/187Re) RGD analogs.
Tubes were centrifuged for 5 min at 2000 rpm at 4 uC. After
removing the unbound 125I-c(RGDyV), centrifuged cell pellets
were washed three times with cold PBS (1 mL). The radio-
activity was determined using a gamma counter (Perkin Elmer,
wizard 1480, Turku, Finland). The IC50 values were calculated
by fitting the data by nonlinear regression using GraphPad
PrismTM (GradPad Software, Inc., San Diego, CA). Experiments
were carried out three times with triplicate samples. IC50
values are reported as an average plus/minus the standard
deviation in Chart 1.
CT procedure
An animal CT scanner system (NanoSPECT/CT, Bioscan Inc.,
Washington DC) consisting of a low-energy X-ray tube (X-ray
energy, 45 kVp, 177 mA; 180 projections) and a precision-
motion translation stage was used. The detector and X-ray
source rotate about a fixed bed, allowing the mouse to be kept
in the same horizontal position in the CT scanner as in the
SPECT scanner. The images were acquired with the X-ray
source set at 45 kVp and 177 mA. Two-dimensional slices of the
bed position were reconstructed using an Exact Cone Beam
Filter Back Projection (FBP) algorithm with a Shepp-Logan
filter. The CT images were reconstructed on a voxel–pixel size
of 0.20 : 0.192 mm, providing image sizes (x, y, z) of 176 6 176
6 136 with an image resolution of 48 mm. For reconstruction
in Hounsfield units, the system was calibrated using a 50 mL
polypropylene tube filled with water.
SPECT/CT imaging
Ex vivo distribution
We performed SPECT imaging studies when the tumor volume
reached 81.8 ¡ 13.5 mm3. SPECT images and X-ray CT images
were acquired using an animal SPECT/CT scanner system
(NanoSPECT/CT, Bioscan Inc., USA). SPECT imaging was
performed using a low-energy and high-resolution pyramid
collimator. We obtained high-resolution scans (18 frames) of
each mouse from 0 min to 180 min after the intravenous
injection of radiotracers (4–6 and 99mTc-IDA-[c(RADfK)]2, 18.5
MBq, n = 3). Mice were placed in a prone position on the bed
under anesthesia with 2% isoflurane. On the anesthetized
mouse, whole body images were obtained in 24 projections
over a 10 min period using a 4-head scanner with 4 6 9 (1.4
mm) pinhole collimators in helical scanning mode. The X-ray
source and detectors are mounted on a circular gantry
allowing it to rotate 360u around the mouse positioned on a
stationary bed. The energy window was set at 140 KeV ¡ 15%.
SPECT imaging was followed by CT image acquisition with the
animal in exactly the same position. The software programs
HiSPECT version 1.0 (Bioscan Inc.) and InVivoScope version
1.43 (Bioscan Inc.) were used for image reconstruction and
quantification, respectively. The SPECT images were recon-
structed to produce an image size (x, y, z) of 176 6 176 6 136
with a voxel size (x, y, z) of 0.2 6 0.2 6 0.2 mm. Images of
99mTc-labeled compounds in Fig. 1 and 2were obtained at a
mid-scan time of 25 min, for a scan duration of 10 min in
tumor-bearing nude mice.
Tissue biodistribution studies were performed in 6 week-old
male nude mice, who were injected subcutaneously with 5 6
106 U87-MG cells into the right shoulder. After tumor volume
reached 62.8 ¡ 16.8 mm3 at 2 weeks post cell inoculation,
mice were injected with 99mTc or 188Re-IDA-RGD analogs (0.74
MBq, 200 mL in saline) into the tail vein, sacrificed at the
indicated time points (n = 4, 10, 30, 60, 120, and 240 min post-
injection for 4–6; 10, 30, 60, 120, and 480 min post-injection
for 7). Blood, tumor, normal organs (liver, lung, spleen,
kidneys, small intestine, large intestine, thyroid, brain), and
muscle were promptly extracted and measured for radio-
activity using a gamma counter (PerkinElmer, Wellesley, MA,
USA). Radioactivity in the organs was presented as the
percentage of the injected dose per gram of tissue in
comparison with samples of a standard dilution of the initial
dose (% ID/g).
Metabolic stability studies
The metabolic stability of 99mTc-IDA-D-[c(RGDfK)]2 (6) was
evaluated in the tumor-bearing mice. U87-MG-bearing mice
injected via the tail vein with 99mTc-IDA-D-[c(RGDfK)]2 (37
MBq) were subsequently sacrificed and dissected at 0.5 and 2
h post-injection. Blood was immediately centrifuged for 5 min
at 3500 rpm at 4 uC and the serum supernatant was collected.
The liver, kidneys, and tumors were each homogenized with
cold PBS (2 mL) in an ice chamber and centrifuged for 5 min at
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In the SPECT/CT image of 7 (37 MBq), a whole body image
RSC Advances
10 J. Shi, Y.-S. Kim, S. Zhai, Z. Liu, X. Chen and S. Liu,
Bioconjugate Chem., 2009, 20, 750.
was obtained according to the similar acquisition protocol of
4–6 except for 12 projections over a 30 min period with 155
KeV ¡ 15% of the energy window. The image of 7 in Fig. 1 was
obtained at a mid-scan time of 45 min, for a scan duration of
30 min in tumor-bearing nude mice. The accumulated
radioactivity of 99mTc/188Re-IDA-RGD analogs in the tumor at
the specified time-points was extracted from the images by
drawing regions of interest (ROI) using the 37–55.5 MBq
radioactivity of 99mTc and 188Re as a reference source. The
percentage of the injected dose per gram of weight (% ID/g)
was determined from the radioactivity in the ROI on the tumor
after intravenous injection of 99mTc/188Re-compounds, with
the location of the edge of the ROI being the contour for 70%
of the maximum intensity.
11 R. Alberto, R. Schibli, A. Egli, A. P. Schubiger, U. Abram and
T. A. Kaden, J. Am. Chem. Soc., 1998, 120, 7987.
12 R. Alberto, R. Schibli, R. Waibel, U. Abram and A.
P. Schubiger, Coord. Chem. Rev., 190–192, 901.
13 R. Schibli, R. Schwarzbach, R. Alberto, K. Ortner,
H. Schmalle, C. Dumas, A. Eqli and P. A. Schubiger,
Bioconjugate Chem., 13, 750.
14 R. Schibli, M. Netter, L. Scapozza, M. Birringer,
P. Schelling, C. Dumas, J. Schoch and P. A. Schubiger, J.
Organomet. Chem., 2003, 668, 67.
15 B. C. Lee, D. H. Kim, J. H. Lee, H. J. Sung, Y. S. Choe, D.
Y. Chi, K.-H. Lee, Y. Choi and B. T. Kim, Bioconjugate
Chem., 2007, 18, 1332.
16 S. Alves, J. D. G. Correia, L. Gano, T. L. Rold,
A. Prasanphanich, R. Haubner, M. Rupprich, R. Alberto,
C. Decristoforo, I. Santos and C. J. Smith, Bioconjugate
Chem., 2007, 18, 530.
Acknowledgements
17 B. L. Oliveira, P. D. Raposinho, F. Mendes, F. Figueira,
I. Santos, A. Ferreira, C. Cordeiro, A. P. Freire and J.
D. Correia, Bioconjugate Chem., 2010, 21, 2168.
18 E.-M. Kim, M.-H. Joung, C.-M. Lee, H.-J. Jeong, S. T. Lim,
M.-H. Sohn and D. W. Kim, Bioorg. Med. Chem. Lett., 2010,
20, 4240.
This work was supported by the National Research Foundation
of Korea (NRF) grant funded by the Korea government (MEST)
(20110018410, 20110030953, 20110006321) and the grant of
the Korea Healthcare Technology R&D Project, Ministry of
Health and Welfare, Republic of Korea (A102158).
19 L. Huang, H. Zhu, Y. Zhang, X. Xu, W. Cui, G. Yang and Y.
M. Shen, Steroids, 2010, 75, 905.
20 K.-H. Jung, K.-H. Lee, J.-Y. Paik, B.-H. Ko, J.-S. Bae, B.
C. Lee, H. J. Sung, D. H. Kim, Y. S. Chi and D. Y. Chi, J.
Nucl. Med., 2006, 47, 2000.
21 B. C. Lee, H. J. Sung, J. S. Kim, K.-H. Jung, Y. S. Choe, K-
H. Lee and D. Y. Chi, Bioorg. Med. Chem., 2007, 15, 7755.
22 E. G. Garayoa, C. Schweinsberg, V. Maes, L. Brans,
References
1 M. H. Michalski and X. Chen, Eur. J. Nucl. Med. Mol.
Imaging, 2011, 38, 385.
2 L. Fass, Mol. Oncol., 2008, 2, 115.
3 P. C. Brooks, A. M. Montgomery, M. Rosenfeld, R.
A. Reisfeld, T. Hu, G. Klier and D. A. Cheresh, Cell, 1994,
79, 1157.
4 M. Gurrath, G. Muller, H. Kessler, M. Aumailley and
R. Timpl, Eur. J. Biochem., 1992, 210, 911.
5 E. Ruoslahti and M. D. Pierschbacher, Cell, 1986, 44, 517.
6 R. Haubner, H. J. Wester, W. Weber, C. Mang, S. L. Ziegler
and S. L. Goodman, Cancer Res., 2001, 61, 1781.
7 M. L. Janssen, W. I. Oyen, I. Dijkgraaf, L. F. Massuger,
C. Frielink, S. Edwards, M. Rajopadhye, H. Boonstra, F.
H. Corstens and O. C. Boerman, Cancer Res., 2002, 62,
6146.
8 J. Shi, Y.-S. Kim, S. Chakraborty, B. Jia, F. Wang and S. Liu,
Bioconjugate Chem., 2009, 20, 1559.
9 C. Decristoforo, I. Santos, H. J. Pietzsch, J. U. Kuenstler,
A. Duatti, C. J. Smith, A. Rey, R. Alberto, E. Von Guggenberg
and R. Haubner, Q. J. Nucl. Med. Mol. Imaging, 2007, 51, 33.
¨
´
P. Blauenstein, D. A. Tourwe, R. Schibli and P.
A. Schubiger, Bioconjugate Chem., 2008, 19, 2409.
23 Z. Liu, B. Jia, H. Zhao, X. Chen and F. Wang, Mol. Imaging
Biol., 2011, 13, 112.
24 H. Wang, K. Chen, W. Cai, Z. Li, L. He, A. Kashefi and
X. Chen, Mol. Cancer Ther., 2008, 7, 1044.
25 H. Akizawa, Y. Arano, M. Mifune, A. Iwado, Y. Saito,
T. Mukai, T. Uehara, M. Ono, Y. Fujioka, K. Ogawa, Y. Kiso
and H. Saji, Nucl. Med. Biol., 2001, 28, 761.
26 L. Sancey, E. Garanger, S. Foillard, G. Schoehn, A. Hurbin,
C. Albiges-Rizo, D. Boturyn, C. Souchier, A. Grichine,
P. Dumy and J. L. Coll, Mol. Ther., 2009, 17, 837.
27 R. Haubner, H. J. Wester, U. Reuning, R. Senekowitsch-
¨
Schmidtke, B. Diefenbach, H. Kessler, G. Stocklin and
M. Schwaiger, J. Nucl. Med., 1999, 40, 1061.
792 | RSC Adv., 2013, 3, 782–792
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