Comparison of radioiodine- or radiobromine-labeled rgd peptides between direct and indirect labeling methods

Article abstract of DOI:10.1248/cpb.c18-00081

Radiolabeled cyclic peptides containing the (Arg-Gly-Asp) RGD sequence for use in positron emission tomography (PET) imaging, single-photon emission computed tomography (SPECT) imaging, and targeted radionuclide therapy of cancer have been reported. In this study, RGD was used as a model carrier peptide for diagnosis and therapy of cancer. To evaluate the characteristics of radiohalogen-labeled peptides, several kinds of labeled RGD peptides [¹²⁵I-c(RGDyK), ⁷⁷Br-c(RGDyK), [¹²⁵I]SIB-c(RGDfK), [⁷⁷Br]SBrB-c(RGDfK), [¹²⁵I]SIB-EG₂-c(RGDfK), and [⁷⁷Br]SBrB-EG₂-c(RGDfK)] were designed, prepared, and evaluated. In these initial studies, ⁷⁷Br (t_1/2=57.0h) and ¹²⁵I (t_1/2=59.4d) were used because of their longer half-lives. Precursor peptides were synthesized using a standard 9-fluorenylmethyloxycarbonyl (Fmoc)-based solid-phase methodology. Radiolabeled peptides were prepared by chloramine-T method or conjugation of RGD peptides with [¹²⁵I]N-succinimidyl 3-iodobenzoate ([¹²⁵I]SIB) or [⁷⁷Br]N-succinimidyl 3-bromobenzoate ([⁷⁷Br]SBrB). Measurement of the partition coefficients, integrin binding assay, and biodistribution experiments in tumor-bearing mice were performed. ¹²⁵I and ⁷⁷Br labeling were successfully performed using similar methods, and in vitro characteristics and biodistributions were similar between the ¹²⁵I-labeled and corresponding ⁷⁷Br-labeled peptides. [¹²⁵I]SIB- and [⁷⁷Br]SBrB-conjugated RGD peptides showed higher partition coefficients, lower tumor uptakes, and higher intestinal uptake than ¹²⁵I-c(RGDyK) and ⁷⁷Br-c(RGDyK). [¹²⁵I]SIB-EG₂-c(RGDfK) and [⁷⁷Br]SBrB-EG₂-c(RGDfK), which possess an ethylene glycol linker, decreased lipophilicity and uptake in intestine compared with [¹²⁵I]SIB-c(RGDfK) and [⁷⁷Br]SBrB-c(RGDfK), which possess no linker. However, the improvement in biodistribution of [¹²⁵I]SIB-EG₂-c(RGDfK) and [⁷⁷Br]SBrB-EG₂-c(RGDfK)] was insufficient. In conclusion, directly radiohalogenated c(RGDyK) peptides are potentially more useful for tumor imaging and therapy than indirectly radiohalogenated ones.

Full text of DOI:10.1248/cpb.c18-00081

Vol. 66, No. 6
Chem. Pharm. Bull. 66, 651–659 (2018)
651
Regular Article
Comparison of Radioiodine- or Radiobromine-Labeled RGD Peptides
between Direct and Indirect Labeling Methods
,a,b
Kazuma Ogawa,* Takuya Takeda,b Masaru Yokokawa,b Jing Yu,b Akira Makino,c
Yasushi Kiyono,c Kazuhiro Shiba,d Seigo Kinuya,b and Akira Odanib
b
a Institute for Frontier Science Initiative, Kanazawa University; Kanazawa, Ishikawa 920–1192, Japan: Graduate
c
School of Medical Sciences, Kanazawa University; Kanazawa, Ishikawa 920–1192, Japan: Biomedical Imaging
d
Research Center, University of Fukui; Yoshida, Fukui 910–1193, Japan: and Advanced Science Research Center,
Kanazawa University; Kanazawa, Ishikawa 920–8640, Japan.
Received February 2, 2018; accepted March 22, 2018
Radiolabeled cyclic peptides containing the (Arg-Gly-Asp) RGD sequence for use in positron emission
tomography (PET) imaging, single-photon emission computed tomography (SPECT) imaging, and targeted
radionuclide therapy of cancer have been reported. In this study, RGD was used as a model carrier peptide
for diagnosis and therapy of cancer. To evaluate the characteristics of radiohalogen-labeled peptides, several
kinds of labeled RGD peptides [125I-c(RGDyK), 77Br-c(RGDyK), [125I]SIB-c(RGDfK), [77Br]SBrB-c(RGDfK),
[
125I]SIB-EG2-c(RGDfK), and [77Br]SBrB-EG2-c(RGDfK)] were designed, prepared, and evaluated. In these
initial studies, 77Br (t1/2=57.0h) and 125I (t1/2=59.4d) were used because of their longer half-lives. Precursor pep-
tides were synthesized using a standard 9-fluorenylmethyloxycarbonyl (Fmoc)-based solid-phase methodology.
Radiolabeled peptides were prepared by chloramine-T method or conjugation of RGD peptides with [125I]N-
succinimidyl 3-iodobenzoate ([125I]SIB) or [77Br]N-succinimidyl 3-bromobenzoate ([77Br]SBrB). Measurement
of the partition coefficients, integrin binding assay, and biodistribution experiments in tumor-bearing mice
were performed. 125I and 77Br labeling were successfully performed using similar methods, and in vitro char-
acteristics and biodistributions were similar between the 125I-labeled and corresponding 77Br-labeled pep-
tides. [125I]SIB- and [77Br]SBrB-conjugated RGD peptides showed higher partition coefficients, lower tumor
uptakes, and higher intestinal uptake than 125I-c(RGDyK) and 77Br-c(RGDyK). [125I]SIB-EG2-c(RGDfK) and
[77Br]SBrB-EG2-c(RGDfK), which possess an ethylene glycol linker, decreased lipophilicity and uptake in in-
testine compared with [125I]SIB-c(RGDfK) and [77Br]SBrB-c(RGDfK), which possess no linker. However, the
improvement in biodistribution of [125I]SIB-EG2-c(RGDfK) and [77Br]SBrB-EG2-c(RGDfK)] was insufficient.
In conclusion, directly radiohalogenated c(RGDyK) peptides are potentially more useful for tumor imaging
and therapy than indirectly radiohalogenated ones.
Key words Arg-Gly-Asp (RGD) peptide; integrin; tumor; radiobromine; positron emission tomography (PET)
Some pathological processes such as tumor development are (for SPECT), 124I (for PET), 125I (for basic research or Auger
related to angiogenesis. Integrin molecules are heterodimeric therapy), 131I (for β− therapy), and 211At (for α therapy).14)
A
transmembrane receptors for cell adhesion, and it has been labeling method for proteins that uses radioiodine and an oxi-
reported that αvβ3 integrin is a representative subtype char- dation agent, such as chloramine-T, has been used classically.
acterized as a regulator of angiogenesis.1) Therefore, the αvβ3 The chloramine-T method can effectively and easily intro-
integrin has been identified as a marker of angiogenic blood duce radioiodine into tyrosine residue in proteins. Moreover,
vessels.2) Moreover, it is known that αvβ3 integrin expression the chloramine-T method can be adapted for peptides with a
is upregulated on endothelial cells during neovascularization tyrosine residue and small molecules containing a phenolic
associated with solid tumors,3) and αvβ3 integrin is overex- group.15,16) Another representative labeling approach that uses
pressed in some kinds of cancer cells, but less expressed in radioiodine is a method using an iododestannylation reaction
normal organs.4) Therefore, αvβ3 integrin has garnered much from a corresponding precursor with a trialkylstannyl group.
interest as an important target for imaging and therapy of can- This method has been widely used for radioiodination of
cer in nuclear medicine.5) Ligands that have a high affinity for small molecules17) or indirect labeling. One of the reagents for
αvβ3 integrin have been investigated and developed.6) As the indirect labeling with radioiodine is N-succinimidyl 3-(tri-n-
most typical ligand for the αvβ3 integrin, cyclic peptides con- butylstannyl)benzoate (ATE), which has been used for radiola-
taining Arg-Gly-Asp (RGD) sequence have been investigated beling of proteins, such as antibodies.18,19) The tri-n-butylstan-
because of their high affinity for αvβ3 integrin.7) A number nyl group in ATE could be easily substituted by radioiodine.
of radiolabeled RGD peptide derivatives for use in positron In the case of preparing radiolabeled proteins or peptides,
emission tomography (PET) imaging, single-photon emission
[
125I]N-succinimidyl 3-iodobenzoate ([125I]SIB) is prepared by
computed tomography (SPECT) imaging, and targeted radio- the iododestannylation reaction following conjugation with
nuclide therapy have been reported.8–13)
proteins or peptides.
Among the halogens, many useful radioisotopes for nuclear
Labeling with radiofluorine normally requires different
medicine and life sciences exist, such as 18F (for PET), 76Br methods of radioiodine labeling due to distinct physical prop-
(for PET), 77Br (for basic research or Auger therapy), 123I erties of fluorine.20) The same methodology for radioiodine
*To whom correspondence should be addressed. e-mail: kogawa@p.kanazawa-u.ac.jp
© 2018 The Pharmaceutical Society of Japan

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Chem. Pharm. Bull.
Vol. 66, No. 6 (2018)
Fig. 1. Structures of Iodinated and Brominated RGD Peptides (a) I-c(RGDyK) and Br-c(RGDyK), (b) SIB-c(RGDfK) and SBrB-c(RGDfK), (c) SIB-
EG2-c(RGDfK) and SBrB-EG2-c(RGDfK)
labeling could be used for labeling with radiobromine21): in- tides were performed using the same precursors and the simi-
troduction of a radiobromine molecule at the next position of a lar procedure. Namely, 125I-c(RGDyK) (2) and 77Br-c(RGDyK)
hydroxyl group in a phenolic group using an oxidation agent, (4) were prepared by the introduction of radioisotopes into
such as chloramine-T, and introduction of a radiobromine the tyrosine residue of c(RGDyK), which was prepared using
molecule at any position of the benzene ring using a bromod- standard 9-fluorenylmethyloxycarbonyl (Fmoc)-based solid-
estannylation reaction from corresponding precursor with a phase methodology, using the chloramine-T method under no-
trialkylstannyl group, such as [77Br]N-succinimidyl 3-bromo- carrier-added conditions with high radiochemical yields (89
benzoate ([77Br]SBrB) from ATE.
and 73%, respectively). However, radiochemical yields of in-
In this study, RGD peptide was used as a model peptide and direct labeling using standard electrophilic halogenation of the
a carrier for diagnosis and therapy of cancer. We designed, tri-n-butylstannyl group of ATE and following conjugation of
prepared, and evaluated in vitro and in vivo several types RGD peptides with [125I]SIB or [77Br]SBrB were low as 30%
of directly and indirectly labeled RGD peptides for evaluat- for [125I]SIB-c(RGDfK) (6), 25% for [77Br]SBrB-c(RGDfK)
ing characteristics between radioiodine- and radiobromine- (8), 34% for [125I]SIB-EG2-c(RGDfK) (10), and 22% for
labeled peptides (Fig. 1; 125I-c(RGDyK) (2), 77Br-c(RGDyK) [77Br]SBrB-EG2-c(RGDfK) (12). These results reflected the
(4),
125I]SIB-EG2-c(RGDfK) (10), and [77Br]SBrB-EG2-c(RGDfK)
(12)) and for applying to astatine-labeled peptides in the fu-
[
125I]SIB-c(RGDfK) (6), [77Br]SBrB-c(RGDfK) (8), low yields of the conjugation of c(RGDfK) peptides with
125I]SIB or [77Br]SBrB because, in part, the active ester in
125I]SIB or [77Br]SBrB could be hydrolyzed to the carboxyl
[
[
[
ture regarding labeling methods, physical characteristics, and group, and a twice purification HPLC procedure was neces-
radionuclides. Although we are interested in developing 76Br- sary. The radiochemical yields of [125I]SIB and [77Br]SBrB
(t1/2=16.2h), 123I-(t1/2=13.2h), 124I-(t1/2=4.2d), or 131I-(t1/2=8.0d) were 65 and 53%, respectively. These results suggested that
labeled RGD peptides for αvβ3 integrin receptor SPECT (123I) the radiochemical yields of the radiobrominations were a little
or PET (76Br and 124I) imaging and targeted radionuclide ther- lower than those of radioiodinations, which is consistent with
apy (131I), in this study, 77Br (t1/2=57.0h) and 125I (t1/2=59.4d) the results from our previous studies of radiobromination and
were used as alternative radionuclides because of their longer radioiodination performed using the same precursor.17,22)
half-lives.
All compounds showed radiochemical purities >95% after
purification by reversed phase (RP)-HPLC. The identities of
radiolabeled peptides were verified by comparing the retention
Results and Discussion
Preparation of Radiolabeled RGD Peptides In this study, times of the corresponding peptides with those of nonradioac-
the preparation of radioiodinated and radiobrominated pep- tive iodine or bromine.

Vol. 66, No. 6 (2018)
Chem. Pharm. Bull.
653
Table 1. Partition Coefficients of 125I-c(RGDyK), 77Br-c(RGDyK), [125I]
SIB-c(RGDfK), [77Br]SBrB-c(RGDfK),
[77Br]SBrB-EG2-c(RGDfK)
[
125I]SIB-EG2-c(RGDfK), and
ClogP
LogP value
125I-c(RGDyK)
77Br-c(RGDyK)
−3.19
−3.38
−0.74
−1.00
−1.71
−1.97
−2.98 0.03
−3.19 0.04
−0.14 0.04
−0.32 0.00
−0.91 0.01
−1.15 0.02
[
125I]SIB-c(RGDfK)
[77Br]SBrB-c(RGDfK)
125I]SIB-EG2-c(RGDfK)
[77Br]SBrB-EG2-c(RGDfK)
[
Each logP value is log [radioactivity in 1-octanol/radioactivity in 0.1M phosphate
buffer (pH 7.4)]. Mean standard deviation (S.D.) for three or four experiments.
Table 2. IC50 Values of I-c(RGDyK), Br-c(RGDyK), SIB-c(RGDfK),
SBrB-c(RGDfK), SIB-EG2-c(RGDfK), and SBrB-EG2-c(RGDfK)
Fig. 2. Typical Displacement Curves of Competition Binding Assay
to the αvβ3 Integrin of 125I-c(RGDyK) with I-c(RGDyK), Br-c(RGDyK),
SIB-c(RGDfK), SBrB-c(RGDfK), SIB-EG2-c(RGDfK), and SBrB-EG2-
c(RGDfK)
IC50 (nM)
I-c(RGDyK)
16.6 9.9
33.5 8.8
22.1 13.3
54.1 13.3
41.2 14.2
50.5 17.4
Br-c(RGDyK)
and 77Br-c(RGDyK), [125I]SIB-c(RGDfK) and [77Br]SBrB-
c(RGDfK), or [125I]SIB-EG2-c(RGDfK) and [77Br]SBrB-EG2-
c(RGDfK) were co-injected into tumor-bearing mice to mini-
mize the number of mice and experimental errors. The results
of biodistribution experiments of radiolabeled RGD peptides
in U87MG tumor-bearing mice are shown in Tables 3–5. It has
SIB-c(RGDfK)
SBrB-c(RGDfK)
SIB-EG2-c(RGDfK)
SBrB-EG2-c(RGDfK)
Each IC50 value is shown as mean S.D. for three experiments.
Determination of Partition Coefficient The results been known that iodine anion accumulates in the thyroid and
of measuring the partition coefficients as the logP values stomach. Therefore, the uptakes in thyroid and stomach have
of 125I-c(RGDyK), 77Br-c(RGDyK),
[77Br]SBrB-c(RGDfK),
[
125I]SIB-c(RGDfK), been used as indexes of deiodination of radioiodine-labeled
[
125I]SIB-EG2-c(RGDfK),
and tracers in vivo. In this study, the accumulation in the stomach
[77Br]SBrB-EG2-c(RGDfK) are listed in Table 1. ClogP was not high for all radioiodine-labeled RGD peptides, which
values calculated using ChemBioDraw Ultra software ver- suggested that significant deiodination did not occur in vivo.
sion 14.0 (Cambridge Soft Corporation, Cambridge, MA, The biodistribution of 125I-c(RGDyK) and 77Br-c(RGDyK)
U.S.A.) are also listed in Table 1. The tendencies of these showed similar patterns, such as high tumor uptake based on
values were consistent between the measured logP val- the affinity of RGD motif for the αvβ3 integrin and low uptake
ues and ClogP values. The directly labeled RGD peptides, in non-target tissues except kidney as a tissue for excretion.
125I-c(RGDyK) and 77Br-c(RGDyK), showed much higher
hydrophilicity than
125I]SIB or [77Br]SBrB conjugated [77Br]SBrB-c(RGDfK) demonstrated similar patterns; par-
RGD peptides, [125I]SIB-c(RGDfK), [77Br]SBrB-c(RGDfK), ticularly, extremely higher accumulation in the small intestine
125I]SIB-EG2-c(RGDfK), and [77Br]SBrB-EG2-c(RGDfK). and lower accumulation in the kidney and tumor than those of
As expected,
125I]SIB-EG2-c(RGDfK) and [77Br]SBrB- 125I-c(RGDyK) and 77Br-c(RGDyK). The extremely higher ac-
In addition, the biodistribution of [125I]SIB-c(RGDfK) and
[
[
[
EG2-c(RGDfK) with an ethylene glycol chain as a hydrophilic cumulation in the intestine and lower accumulation in tumors
linker showed higher hydrophilicity than [125I]SIB-c(RGDfK) of [125I]SIB-c(RGDfK) and [77Br]SBrB-c(RGDfK) were not
and [77Br]SBrB-c(RGDfK) without a linker. Bromine-labeled predicted. Especially, the same degree of tumor accumula-
peptides showed a little lower logP values than iodine labeled tion was predicted because the affinity of SIB-c(RGDfK) and
peptides. HPLC analyses also supported these results, because SBrB-c(RGDfK) for αvβ3 integrin was maintained. Although
the retention times of the bromine-labeled peptides were a the reason for this finding was not elucidated, it might be be-
little shorter than those of the corresponding iodine-labeled cause of the increased lipophilicity due to the introduction of
peptides.
SIB and SBrB molecules. Actually, the logP values, an index
αvβ3 Integrin Binding Assay The affinities of Br- of lipophilicity, were much different between the directly la-
c(RGDyK), I-c(RGDyK), SBrB-c(RGDfK), SIB-c(RGDfK) beled RGD peptides and indirectly labeled RGD peptides via
SBrB-c(RGDfK), and SIB-EG2-c(RGDfK) for the αvβ3 in- SIB or SBrB (Table 1). The higher lipophilicity might cause
tegrin were determined by performing competitive binding rapid liver uptake and fast transition to the small intestine,
assays. The typical results of these assays are shown in Fig. 2 which also could explain the low accumulation in the kidney
and are summarized in Table 2. Binding of 125I-c(RGDyK) to and tumor.
αvβ3 integrin was competed by each RGD peptide in a concen-
We designed and prepared [125I]SIB-EG2-c(RGDfK) and
tration-dependent manner. Because these values were similar [77Br]SBrB-EG2-c(RGDfK), which possess an ethylene glycol
(statistically not significant using Tukey–Kramer test), the SIB chain as a linker between the RGD peptide and SIB or SBrB
unit or SBrB unit and ethylene glycol linker did not signifi- to increase hydrophilicity, to improve the biodistribution
cantly impede the affinity of RGD peptide for αvβ3 integrin.
of the indirectly labeled RGD peptides. LogP values were
Animal Experiments In this study, 125I-c(RGDyK) significantly decreased, and the affinities for αvβ3 integrin

654
Chem. Pharm. Bull.
Vol. 66, No. 6 (2018)
Table 3. Biodistribution of Radioactivity after Concomitant Intravenous Injection of 125I-c(RGDyK) and 77Br-c(RGDyK) in U87MG Tumor Bearing
Mice
125I-c(RGDyK)
77Br-c(RGDyK)
Tissue
1h
4h
1h
4h
Blood
0.37 (0.26)
2.88 (0.54)
9.99 (2.78)
2.25 (0.50)
0.81 (0.22)
2.19 (0.49)
0.44 (0.22)
1.39 (0.40)
0.68 (0.25)
0.68 (0.12)
1.93 (0.62)
0.93 (0.62)
0.08 (0.02)
4.37 (0.10)
0.08 (0.05)
1.29 (0.22)
5.33 (1.08)
1.22 (0.09)
1.95 (1.27)
1.60 (0.40)
0.34 (0.08)
1.02 (0.48)
0.33 (0.02)
0.29 (0.06)
0.56 (0.18)
0.09 (0.10)
0.04 (0.00)
3.58 (0.71)
0.37 (0.25)
2.57 (0.48)
0.28 (0.05)*
1.48 (0.34)
5.99 (0.54)
1.24 (0.13)
1.76 (1.16)
1.61 (0.43)
0.55 (0.08)*
0.94 (0.21)
0.43 (0.07)
0.33 (0.08)
0.82 (0.39)
0.11 (0.11)
0.12 (0.00)**
3.44 (0.40)
Liver
Kidney
Small-intestine
Large-intestine
Spleen
10.46 (2.82)**
1.98 (0.49)**
0.73 (0.19)*
1.97 (0.53)**
0.54 (0.33)
Pancreas
Lung
1.17 (0.34)**
0.54 (0.25)**
0.27 (0.10)**
1.67 (0.63)
Heart
Stomach‡
Bone
Muscle
Brain
1.33 (0.57)
0.07 (0.01)
Tumor
4.22 (0.32)
Expressed as % injected dose per gram. Each value represents the mean (S.D.) for four animals. ‡ Expressed as % injected dose. Significance was determined using paired
Student’s t-test (*p<0.05, **p<0.01 vs. 125I-c(RGDyK)).
Table 4. Biodistribution of Radioactivity after Concomitant Intravenous Injection of [125I]SIB-c(RGDfK) and [77Br]SBrB-c(RGDfK) in U87MG Tumor
Bearing Mice
[
125I]SIB-c(RGDfK)
[77Br]SBrB-c(RGDfK)
Tissue
1h
4h
1h
4h
Blood
0.67 (0.14)
2.85 (0.80)
2.16 (0.07)
46.54 (4.46)
2.90 (1.64)
1.22 (0.44)
0.93 (0.15)
0.34 (0.08)
1.03 (0.13)
1.03 (0.10)
1.19 (0.14)
0.69 (0.29)
0.13 (0.01)
1.50 (0.40)
0.57 (0.11)
1.55 (0.45)
1.40 (0.11)
2.43 (0.59)
0.70 (0.17)
3.93 (0.64)
1.74 (0.26)*
49.80 (2.75)
1.91 (0.67)
1.42 (0.37)
1.04 (0.46)**
0.59 (0.20)
0.80 (0.14)
0.67 (0.09)
1.19 (0.56)
0.50 (0.15)
0.56 (0.04)**
1.51 (0.36)
0.27 (0.08)
2.58 (0.32)**
1.22 (0.11)**
3.16 (0.52)**
17.08 (0.84)*
1.08 (0.05)
0.89 (0.13)
0.38 (0.02)
0.68 (0.09)
0.63 (0.05)
1.01 (0.33)
0.43 (0.10)
0.21 (0.05)
1.45 (0.30)*
Liver
Kidney
Small-intestine
Large-intestine
Spleen
13.03 (1.69)
0.95 (0.07)
0.74 (0.22)
0.32 (0.03)
0.80 (0.24)
0.71 (0.14)
0.82 (0.30)
0.31 (0.07)
0.14 (0.03)
1.24 (0.27)
Pancreas
Lung
Heart
Stomach‡
Bone
Muscle
Brain
Tumor
Expressed as % injected dose per gram. Each value represents the mean (S.D.) for four animals. ‡ Expressed as % injected dose. Significance was determined using paired
Student’s t-test (*p<0.05, **p<0.01 vs. [125I]SIB-c(RGDfK)).
were not attenuated by the introduction of an ethylene gly- experiments and can be labeled indirectly using ATE.23) Un-
col chain, as expected. In addition, [125I]SIB-EG2-c(RGDfK) fortunately, directly labeled RGD peptides are much better for
and [77Br]SBrB-EG2-c(RGDfK) showed similar biodistribu- imaging and therapy of cancer than indirectly radiohalogen-
tion patterns. Contrary to our expectation, radioactivity labeled RGD peptides prepared in this study. The unfavorable
in the liver after injection of [125I]SIB-EG2-c(RGDfK) and biodistribution of the indirectly radiohalogen-labeled RGD
[77Br]SBrB-EG2-c(RGDfK) was not decreased relative to that peptides may be because of their insufficient hydrophilicity.
of [125I]SIB-c(RGDfK) and [77Br]SBrB-c(RGDfK). Radioac- In fact, the introduction of the ethylene glycol chain was in-
tivity in the small intestine was prominently decreased by sufficient for increasing the hydrophilicity to the same level
their higher hydrophilicities; we speculate that intact tracers as that of the directly labeled RGD peptides. In our previous
or radioactive metabolites moved from the liver to the intes- study, a 67Ga-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraace-
tine earlier time than 1h postinjection, but tumor uptake did tic Acid (DOTA) complex conjugated c(RGDfK), 67Ga-DOTA-
not improve, and the decrease of radioactivity in the intestine c(RGDfK), was evaluated.24) Although 67Ga-DOTA-c(RGDfK)
was insufficient. Considering the application to astatine label- is not a radiohalogen-labeled RGD peptide, but rather a
ing for α-targeted therapy, which has attracted much attention radiometal-labeled RGD peptide, 67Ga-DOTA-c(RGDfK) is a
because of its prominent therapeutic effects, indirect label- compound in which the [125I]SIB moiety as a radiolabeling site
ing methods could be useful because astatine element was of [125I]SIB-c(RGDfK) is substituted with 67Ga-DOTA, which
not introduced into the tyrosine residue in our preliminary has a much higher hydrophilic moiety than that of [125I]SIB.

Vol. 66, No. 6 (2018)
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655
Table 5. Biodistribution of Radioactivity after Concomitant Intravenous Injection of [125I]SIB-EG2-c(RGDfK) and [77Br]SBrB-EG2-c(RGDfK) in
U87MG Tumor-Bearing Mice
[
125I]SIB-EG2-c(RGDfK)
[77Br]SBrB-EG2-c(RGDfK)
Tissue
1h
4h
1h
4h
Blood
1.97 (0.52)
3.64 (1.50)
2.95 (0.50)
17.27 (1.79)
1.22 (0.55)
1.11 (0.17)
1.83 (0.95)
1.89 (0.65)
0.81 (0.13)
1.20 (0.27)
1.24 (0.44)
1.15 (0.82)
0.19 (0.11)
1.65 (0.48)
1.52 (0.27)
1.45 (0.23)
1.35 (0.17)
2.93 (0.63)
9.28 (1.20)
0.95 (0.10)
1.05 (0.20)
1.38 (0.17)
0.73 (0.19)
0.95 (0.09)
1.05 (0.21)
0.76 (0.40)
0.15 (0.04)
1.54 (0.19)
1.66 (0.08)
4.16 (1.85)
3.10 (0.43)
18.80 (2.13)**
1.04 (0.40)
0.91 (0.09)
1.43 (0.66)
1.49 (0.09)
0.60 (0.09)*
0.70 (0.16)*
0.84 (0.22)
1.33 (0.72)
0.28 (0.03)
1.31 (0.20)
1.61 (0.40)
1.66 (0.21)**
1.88 (0.12)**
3.21 (0.63)*
13.08 (1.61)**
1.12 (0.07)**
0.97 (0.08)
Liver
Kidney
Small-intestine
Large-intestine
Spleen
Pancreas
Lung
1.81 (0.11)*
0.96 (0.08)
Heart
Stomach‡
0.40 (0.05)**
1.02 (0.17)
Bone
Muscle
Brain
0.62 (0.20)
0.48 (0.03)**
1.16 (0.06)*
Tumor
Expressed as % injected dose per gram. Each value represents the mean (S.D.) for four animals. ‡ Expressed as % injected dose. Significance was determined using paired
Student’s t-test (*p<0.05, **p<0.01 vs. [125I]SIB-EG2-c(RGDfK)).
The radioactivities (%Dose/g) in blood, liver, kidney, small CHEMICAL Co., Ltd. (Tokyo, Japan). 1,1,1,3,3,3-Hexafluoro-
intestine, and tumor at 1h postinjection of 67Ga-DOTA- 2-propanol (HFIP) was purchased from Tokyo Chemical
c(RGDfK) were 0.24 0.06, 1.31 0.18, 2.77 0.57, 1.76 0.45, Industry Co., Ltd. (Tokyo, Japan). Other reagents were of
and 3.45 0.33, respectively, in U87MG tumor-bearing mice. reagent grade and used as received.
The biodistribution of 67Ga-DOTA-c(RGDfK) was comparable
Production of Bromine-77 77Br was produced in Univer-
or better to those of directly labeled RGD peptides. These re- sity of Fukui. Radiosynthetic isolation and purification of 77Br
sults and the higher hydrophilicity of 67Ga-DOTA-c(RGDfK) were performed according to a previously reported method.22)
(LogP value: −3.50 0.01) indicate that high hydrophilicity Namely, 77Br was obtained from a 77Se(p,n)77Br reaction on
may be important for biodistribution of RGD peptides. There- an isotopically enriched Cu277Se-coated tungsten target with
fore, a novel labeling method using compounds with high 8µA/ 11 MeV proton beam on a RDS Eclipse HP/RD cyclo-
hydrophilicity must be developed for astatine labeling of RGD tron (Siemens, Knoxville, TN, U.S.A.).
peptides in the future.
Preparation of c(RGDfK) and c(RGDyK) The peptide
synthesis was performed manually using a standard Fmoc-
based solid-phase methodology. Namely, at first, Fmoc-Gly-
Conclusion
The biodistributions of RGD peptides labeled with ATE OH (4 equiv.) was dissolved in dichloromethane. 2-Chlorot-
were inadequate for tumor imaging and radionuclide therapy, rityl chloride resin and DIEA (3.5 equiv.) were added. The
even if ethylene glycol was introduced, compared with that reaction mixture was rotated for 1h, and 1mL of methanol
of RGD peptides with direct labeling in tyrosine residue. For was added to react for another 30min. Next, the peptide chain
application to alpha targeted therapy using astatine labeling was constructed in cycles of (I) 15min of deprotection with
RGD peptide, novel labeling method must be developed in the 20% piperidine in dimethylformamide (DMF) and (II) 2h of
future.
coupling with 2.5eq of Fmoc-amino acid [Fmoc-Arg(Pbf)-
OH, Fmoc-Lys(Boc)-OH, Fmoc-D-Phe-OH, Fmoc-D-Tyr (tBu)-
OH, or Fmoc-Asp(OtBu)-OH], DIPC (2.5 equiv.) and HOBt
Experimental
Materials 1H-NMR spectra were recorded on a JEOL (2.5 equiv.) in DMF. The coupling reaction was then repeated
JNM-ECS400 spectrometer (JEOL Ltd., Tokyo, Japan), and after Kaiser test25) was positive for the resin to obtain Resin-
the chemical shifts were reported in ppm downfield from an Gly-Arg(Pbf)-Lys(Boc)-D-Tyr(tBu)-Asp(OtBu)-OH or Resin-
internal tetramethylsilane standard. Electrospray ionization Gly-Arg(Pbf)-Lys(Boc)-D-Phe-Asp(OtBu)-OH. To cleave pep-
(ESI)-MS were obtained with JEOL JMS-T100TD (JEOL tides from the resin, the resin-bound peptides were treated
Ltd.). [125I]Sodium iodide (644GBq/mg) were purchased with 30% HFIP in dichloromethane for 5min. After filtration,
from PerkinElmer, Inc. (Waltham, MA, U.S.A.). 67GaCl3 was the solvent in the filtrate was removed in vacuo. The residue
kindly provided by Nihon Medi-physics (Tokyo, Japan). Fmoc- was used in the next reaction without further purification.
Lys(Boc)-OH was purchased from Merck (Darmstadt, Germa- For cyclization of the peptides, NaHCO3 (5 equiv.) and diphe-
ny). 2-Chlorotrityl chloride resin, Fmoc-Arg(Pbf)-OH, Fmoc- nylphosphoryl azide (DPPA, 3 equiv.) were added to 5mM of
Asp(OtBu)-OH, Fmoc-Gly-OH, Fmoc-D-Tyr(tBu)-OH, and peptides solution in DMF. After 24h stirring at room tempera-
Fmoc-D-Phe-OH were purchased from Watanabe Chemical In- ture, the solid NaHCO3 was removed by filtration, and DMF
dustries, Ltd. (Hiroshima, Japan). N,N-Diisopropylethylamine was removed in vacuo. The residue was used in the next reac-
(DIEA) was purchased from Nacalai Tesque (Kyoto, Japan). tion without further purification.
N,N′-Diisopropylcarbodiimide (DIPC) and 1-hydroxyben-
For deprotection, the cyclic peptides were treated with 95%
zotriazole hydrate (HOBt) were purchased from KOKUSAN trifluoroacetic acid (TFA), 2.5% water, and 2.5% triisopropyl-

656
Chem. Pharm. Bull.
Vol. 66, No. 6 (2018)
Fig. 3. Syntheses Scheme of SIB-EG2-c(RGDfK) and SBrB-EG2-c(RGDfK)
(a) HOBt, DIPC; (b) TFA:triisopropylsilane:H2O (38:1:1).
silane for 24h at room temperature. The crude peptide was aqueous solution (10mg/mL) and then purified by RP-HPLC
purified by RP-HPLC performed with a Cosmosil 5C18-AR-II on Cosmosil 5C18-AR-II (10×150mm; Nacalai Tesque) at a
column (10×150mm; Nacalai Tesque, Kyoto, Japan) at a flow flow rate of 4mL/min, the isocratic mobile phase was 28%
rate of 4.0mL/min with a gradient mobile phase of 40% meth- acetonitrile in water with 0.1% TFA. Chromatograms were
anol in water with 0.1% TFA to 60% methanol in water with obtained by monitoring the UV absorption at a wavelength
0.1% TFA for 20min in the case of c(RGDfK), and with a gra- of 220nm. The fractions containing Br-c(RGDyK) was deter-
dient mobile phase of 15% methanol in water with 0.1% TFA mined by mass spectrometry, and collected. The solvent was
to 25% methanol in water with 0.1% TFA for 20min in the removed by lyophilization to provide Br-c(RGDyK) (3) as a
case of c(RGDyK). The column temperature was maintained white powder (820µg, 30%). ESI-MS m/z: 698 ([M+H]+), 700
at 40°C. Chromatograms were obtained by monitoring the ([M+H]+)
UV adsorption at a wavelength of 220nm. The fractions con-
Preparation of Nonradioactive SIB-c(RGDfK) (5)
taining c(RGDfK) and c(RGDyK) were determined by mass and SBrB-c(RGDfK) (7) SIB-c(RGDfK) (5) and SBrB-
spectrometry, and collected. The solvents were removed by ly- c(RGDfK) (7) were prepared by conjugation of c(RGDfK)
ophilization to provide c(RGDfK) (40%) and c(RGDyK) (47%) peptide with N-succinimidyl 3-iodobenzoate (SIB) or N-suc-
as white powders. c(RGDfK): ESI-MS m/z: 604 ([M+H]+). cinimidyl 3-bromobenzoate (SBrB).
c(RGDyK): ESI-MS m/z: 620 ([M+H]+)
SIB was synthesized according to a previous report with
Preparation of Nonradioactive I-c(RGDyK) (1) and Br- a slight modification.26) Namely, 3-iodobenzoic acid (719mg,
c(RGDyK) (3) NaNO2 (69mg, 1.0mmol) and I2 (254mg, 2.90mmol) was dissolved in 10mL of acetonitrile. N-
1.0mmol) were dissolved in 10mL of 50% methanol in water. Hydroxysuccinimide (NHS) (401mg, 3.48mmol) was added
After being stirred for 30min at room temperature, 130µL of to the solution, and 50mL of acetonitrile containing N,N′-
this solution was taken to a microtube. c(RGDyK) (6.2mg, dicyclohexylcarbodiimide (DCC) (718mg, 3.48mmol) was
10 µmol) was added to the solution. The reaction mixture was added dropwise at 0°C. The reaction mixture was stirred at
shaken for 2h at room temperature, and purified by RP-HPLC room temperature for 5h. After filtration, the solvent in filtrate
with a Cosmosil 5C18-AR-300 (10×150mm) at a flow rate of was removed in vacuo. The residue was purified by chroma-
4mL/min, the gradient mobile phase was 20% methanol in tography on silica gel using ethyl acetate–hexane (1:2) as the
1
water with 0.1% TFA to 30% methanol in water with 0.1% eluent to obtain SIB (742mg, 74%) as a white solid. H-NMR
TFA for 20min. The solvent was removed by lyophilization to (CDCl3) δ: 2.92 (4H, s), 7.27 (1H, t, J=8.0Hz), 8.01 (1H, d,
provide I-c(RGDyK) (1) as a white powder (2.6mg, 35%). ESI- J=7.6Hz), 8.11 (1H, d, J=7.6Hz), 8.47 (1H, s).
MS m/z: 746 ([M+H]+)
SBrB (68mg, 76%) was obtained as a white solid from
c(RGDyK) (2.4mg, 3.9µmol) was dissolved in 100µL of 5% 3-bromobenzoic acid (60mg, 300µmol) as a starting material
acetic acid aqueous solution, and Br2 in acetic acid and 24µL instead of 3-iodobenzoic acid in the above-mentioned method.
of chloramine-T aqueous solution (2mg/mL) was added to 1H-NMR (CDCl3) δ: 2.92 (4H, s), 7.41 (1H, t, J=8.0Hz), 7.81
c(RGDyK) solution. After 3h of stirring at room temperature, (1H, d, J=8.0Hz), 8.07 (1H, d, J=7.6Hz), 8.28 (1H, s).
the reaction mixture was quenched with 24µL of NaHSO3
SIB (5.4mg, 16.0µmol) and c(RGDfK) (2.7mg, 4.5µmol)

Vol. 66, No. 6 (2018)
Chem. Pharm. Bull.
657
were dissolved in 400µL of DMF, then 1µL of triethylamine nitrile in water with 0.1% TFA to 60% acetonitrile in water
(TEA). The mixture was shaken for 3h and purified by RP- with 0.1% TFA for 20min. The fraction containing SIB-EG2-
HPLC on Cosmosil 5C18-AR-300 (10×150mm) at a flow rate c(RGDfK) (9) was determined by mass spectrometry, and col-
of 4mL/min, the gradient mobile phase was 40% methanol lected. The solvent was removed by lyophilization to provide
in water with 0.1% TFA to 90% methanol in water with 0.1% compound 9 (170 µg, 64%) as a white powder. ESI-MS m/z:
TFA for 20min. Chromatograms were obtained by monitor- 979 ([M+H]+)
ing the UV absorption at a wavelength of 220nm. The frac-
SBrB-EG2-c(RGDfK) (63%) (11) was obtained as a white
tions containing SIB-c(RGDfK) (5) was determined by mass powder using SBrB (19) instead of SIB (17) in the above-men-
spectrometry, and collected. The solvent was removed by tioned method. ESI-MS m/z: 931 ([M+H]+), 933 ([M+H]+)
lyophilization to provide SIB-c(RGDfK) (5) as a white powder
(2.2mg, 59%). ESI-MS m/z: 834 ([M+H]+)
Preparation of 125I-c(RGDyK) (2) and 77Br-c(RGDyK)
(4) c(RGDyK) was labeled with 125I using the chloramine-T
SBrB (4.7mg, 16.0µmol) and c(RGDfK) (2.4mg, 4.0µmol) method.28) Briefly, [125I]sodium iodide solution (3.7MBq/1µL)
were reacted and purified at the above-mentioned method to was added to c(RGDyK) in 100µL of 0.1M phosphate buffered
obtain SBrB-c(RGDfK) (7) (1.1mg, 35%) as a white powder. saline (PBS) pH 7.4 (1mg/mL). Following mixing, 10µL of
ESI-MS m/z: 786 ([M+H]+), 788 ([M+H]+)
chloramine-T aqueous solution (1mg/mL) was added. After
Preparation of Nonradioacitve SIB-EG2-c(RGDfK) (9) 5min of standing at room temperature, the reaction mixture
and SBrB-EG2-c(RGDfK) (11) SIB-EG2-c(RGDfK) (9) and was quenched with 10µL of Na2H2SO5 solution (10mg/mL) in
SBrB-EG2-c(RGDfK) (11) were synthesized according Fig. 3.
0.1 M acetate buffer pH 6.8 and then purified by RP-HPLC on
[2-[2-(tert-Butyloxycarbonyl)aminoethoxy]ethoxy]acetic Cosmosil 5C18-AR-II (4.6×150mm) at a flow rate of 1mL/min.
acid (Boc-NH-EG2-COOH) (14) was prepared in 4 steps (5%) The gradient mobile phase was 20% methanol in water with
from 2-[2-(2-chloroethoxy)ethoxy]ethanol using a method 0.1% TFA to 30% methanol in water with 0.1% TFA for
described previously.27) 1H-NMR (CDCl3) δ: 1.44 (9H, s), 3.33 20min.
(2H, m), 3.53-3.73 (6H, m), 4.14 (2H, s). Direct analysis in real
77Br-c(RGDyK) was prepared by a similar method as prepa-
ration of 125I-c(RGDyK). 77Br− (3.7MBq) in water was added
time (DART)-MS m/z: 264 ([M+H]+)
c[Arg(Pbf)-Gly-Asp(OtBu)-D-Phe-Lys] (2.0mg, 2.1µmol) to 100µL of 0.1M acetate buffer pH 6.8 containing 100µg of
(13), which was synthesized using a method described pre- c(RGDyK), and then 10µL of chloramine-T aqueous solution
viously,24) was dissolved in 100µL of DMF. Boc-NH-EG2- (1mg/mL) was added. After 5min standing at room tem-
COOH (14) (2.3mg, 8.6µmol), HOBt (1.2mg, 4 equiv.), and perature, the reaction mixture was quenched with 10µL of
DIPC (1.3µL, 4 equiv.) were added to the solution. After 11h Na2S2O5 solution (10mg/mL) and then purified by RP-HPLC
stirring at room temperature, the crude peptide was purified using the above-mentioned method.
by RP-HPLC performed with a Cosmosil 5C18-AR-II column
Preparation of [125I]SIB-c(RGDfK) (6) N-Succinimidyl
(10×150mm) at a flow rate of 4.0mL/min with a gradient mo- ester of 3-(tri-n-butylstannyl)benzoate (ATE) (0.5mg), which
bile phase of 71% methanol in water with 0.1% TFA to 97% was synthesized using a method reported previously with
methanol in water with 0.1% TFA for 18min. The column a slight modification from 3-broobenzoic acid via a four-
temperature was maintained at 40°C. Chromatograms were step,18,29) was dissolved in 79µL of ethanol. Fifteen microliter
obtained by monitoring the UV adsorption at a wavelength of chloramine-T (1mg/mL) in ethanol, 21µL of 1% acetic
of 220nm. The fraction containing Boc-NH-EG2-c[Arg(Pbf)- acid in ethanol, and [125I]NaI (3.7MBq) were added to the
Gly-Asp(OtBu)-D-Phe-Lys] (15) was determined by mass ATE solution. After standing at room temperature for 10min,
spectrometry, and collected. The solvent was removed by lyo-
[
125I]SIB (18) was purified by RP-HPLC on Cosmosil 5C18-
philization to provide compound 15 (29%) as a white powder. AR-II (4.6×150mm) at a flow rate of 1mL/min. The gradient
Boc-NH-EG2-c[Arg(Pbf)-Gly-Asp(OtBu)-D-Phe-Lys] (3): ESI- mobile phase was 50% methanol in water with 0.1% TFA to
MS m/z: 1158 ([M+H]+)
70% methanol in water with 0.1% TFA for 20min. Fractions
For deprotection of functional groups, compound 15 was containing [125I]SIB (18) was collected, and the solvent was
treated with a mixture of 95% TFA, 2.5% water, and 2.5% tri- dried by N2 gas. [125I]SIB (18) was dissolved in 50µL of DMF,
isopropylsilane for 2h at room temperature. The crude peptide and 50µg of c(RGDfK) and 0.5µL of TEA were added to the
was purified by RP-HPLC performed with a Cosmosil 5C18-
[
125I]SIB (18) solution. The mixture solution was incubated
AR-II column (10×150mm) at a flow rate of 4.0mL/min with for 40min at room temperature, and purified by RP-HPLC on
a gradient mobile phase of 20% methanol in water with 0.1% Cosmosil 5C18-AR-II (4.6×150mm) at a flow rate of 1mL/min.
TFA to 56% methanol in water with 0.1% TFA for 18min. The gradient mobile phase was 40% methanol in water with
The fraction containing NH2-EG2-c(RGDfK) (16) was deter- 0.1% TFA to 90% methanol in water with 0.1% TFA for
mined by mass spectrometry, and collected. The solvent was 20min.
removed by lyophilization to provide compound 16 (50%) as
a white powder. NH2-EG2-c(RGDfK) (16): ESI-MS m/z: 749 was dissolved in 55µL of ethanol. Fifteen microliter of chlora-
mine-T (1mg/mL) in ethanol, 21µL of 1% acetic acid in etha-
Preparation of [77Br]SBrB-c(RGDfK) (8) ATE (0.5mg)
([M+H]+)
Compound 16 (200µg, 0.41µmol) was dissolved in 100µL nol, and 77Br− (3.7MBq) was added to the ATE solution. The
of DMF. SIB (370µg, 1.09µmol) (17), which was synthesized reaction mixture stood at room temperature for 10min. After
as above-mentioned, and 1.0µL of TEA were added to the purification of [77Br]SBrB (20) by RP-HPLC, conjugation of
reaction mixture. After stirring for 2h at room temperature, [77Br]SBrB (20) with c(RGDfK) were performed by the same
the crude compound 9 was purified by RP-HPLC performed manner as above-mentioned to obtain [77Br]SBrB-c(RGDfK)
with a Cosmosil 5C18-AR-II column (4.6×150mm) at a flow (8).
rate of 1.0mL/min with a gradient mobile phase of 20% aceto-
Preparation of [125I]SIB-EG2-c(RGDfK) (10) and [77Br]-

658
Chem. Pharm. Bull.
Vol. 66, No. 6 (2018)
SBrB-EG2-c(RGDfK) (12)
[
125I]SIB-EG2-c(RGDfK) (10) ments, 125I activity was determined because the radioactivity
and [77Br]SBrB-EG2-c(RGDfK) (12) were prepared by con- of 77Br was negligible at that time. The radioactivity counts of
jugation of [125I]SIB (18) and [77Br]SBrB (20), respectively, 125I were measured after attenuation of 77Br.
with NH2-EG2-c(RGDfK) (16) using same method as above-
Statistical Evaluation IC50 values in integrin binding
mentioned.
assays among I-c(RGDyK), Br-c(RGDyK), SIB-c(RGDfK),
Determination of Partition Coefficient The parti- SBrB-c(RGDfK), SIB-EG2-c(RGDfK), and SBrB-EG2-
tion coefficients of 125I-c(RGDyK) (2), 77Br-c(RGDyK) c(RGDfK) was determined using a one-way ANOVA followed
(4),
[
[
125I]SIB-c(RGDfK) (6), [77Br]SBrB-c(RGDfK) (8), by the Tukey–Kramer post hoc test. Significance between
125I]SIB-EG2-c(RGDfK) (10), [77Br]SBrB-EG2-c(RGDfK) the biodistribution data of 125I-labeled peptides and the cor-
(12), and 67Ga-DOTA-c(RGDfK), which was prepared ac- responding 77Br-labeled peptides was determined using paired
cording to the method reported previously, as a reference Student’s t-test. Results were considered statistically signifi-
compound,24) were measured as described previously.30) The cant at p<0.05.
partition coefficient was determined by calculating the ratio of
cpm/mL in 1-octanol to that in the 0.1M phosphate buffer pH
7.4, and expressed as a common logarithm (logP).
Conflict of Interest The authors declare no conflict of
interest.
αvβ3 Integrin Binding Assay The affinities of I-
c(RGDyK) (1), Br-c(RGDyK) (3), SIB-c(RGDfK) (5),
SBrB-c(RGDfK) (7), SIB-EG2-c(RGDfK) (9), and SBrB-
EG2-c(RGDfK) (11) for αvβ3 integrin were evaluated by
competitive inhibition between the each peptide and 125I-
c(RGDyK) to αvβ3 integrin according to procedures reported
previously.24)
References
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SBrB-EG2-c(RGDfK) (12) in Tumor-Bearing Mice Ex-
periments with animals were conducted in strict accordance
with the Guidelines for the Care and Use of Laboratory
Animals of Kanazawa University. The animal experimental
protocols used were approved by the Committee on Animal
Experimentation of Kanazawa University. The animals were
housed with free access to food and water at 23°C with a 12-h
alternating light/dark schedule. U87MG glioblastoma cell line
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(40kBq) and 77Br-c(RGDyK) (4) (56kBq), a mixed solution of
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