Coordination-Mediated Synthesis of 67Ga-Labeled Purification-Free Trivalent Probes for in Vivo Imaging of Saturable Systems

Article abstract of DOI:10.1021/acs.bioconjchem.8b00337

A large excess of unlabeled ligands over gallium-67 (⁶⁷Ga) provides ⁶⁷Ga-labeled probes with high radiochemical yields in a short reaction time. However, the unlabeled ligands hinder target accumulation of radiolabeled probes by comp

Full text of DOI:10.1021/acs.bioconjchem.8b00337

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Article
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Coordination-Mediated Synthesis of Ga-Labeled Purification-
Free Trivalent Probes for In Vivo Imaging of Saturable Systems
Holis Abdul Holik, Tomoya Uehara, Soki Nemoto, Takemi Rokugawa, Yuumi
Tomizawa, Ayako Sakuma, Yuki Mizuno, Hiroyuki Suzuki, and Yasushi Arano
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00337 • Publication Date (Web): 10 Aug 2018
Downloaded from http://pubs.acs.org on August 13, 2018
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Coordination-Mediated Synthesis of 67Ga-Labeled
Purification-Free Trivalent Probes for In Vivo
Imaging of Saturable Systems
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Holis A. Holik†,‡, Tomoya Uehara†*, Soki Nemoto†, Takemi Rokugawa†, Yuumi Tomizawa†,
Ayako Sakuma†, Yuki Mizuno†,§, Hiroyuki Suzuki†, and Yasushi Arano†
† Laboratory of Molecular Imaging and Radiotherapy, Graduate School of Pharmaceutical
Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan
‡ Department of Pharmaceutical Analysis and Medicinal Chemistry, Faculty of Pharmacy,
Universitas Padjadjaran, Jl. Raya Bandung-Sumedang KM 21, Sumedang 46363, Indonesia
§ Laboratory of Physical Chemistry, Showa Pharmaceutical University, 3-3165 Higashi-
Tamagawagakuen, Machida, Tokyo 194-8543, Japan.
Keywords: gallium-67, SPECT, PET, schiff base
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ABSTRACT
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A large excess of unlabeled ligands over gallium-67 (67Ga) provide 67Ga-labeled probes with high
radiochemical yields in short reaction time. However, the unlabeled ligand hinders target
accumulation of radiolabeled probes by competing for target molecules. To circumvent the
problem, we investigated the way to prepare 67Ga-labeled multivalent probes from monovalent
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ligands. The reaction of a bi- or tri-dentate ligand with [67Ga]Ga-citrate resulted in Ga-labeled
probes of insufficient stability. However, the reaction of [67Ga]Ga-citrate with a mixture of RGD-
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conjugated salicylaldehyde and triamine provided a Ga-labeled trivalent probe with stability
sufficient for in vivo applications. Since the free Schiff base ligand decomposed rapidly upon
injection, the 67Ga-labeled trivalent probe visualized murine tumor without post-labeling
purification, which was not achieved with a 67Ga-labeled trivalent probe from a trivalent ligand.
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These findings indicate the availability of Schiff base ligands to prepare Ga-labeled trivalent
probes by a simple radiolabeling procedure.
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INTRODUCTION
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Bioactive molecules labeled with metallic radionuclides such as gallium-67/68 (67/68Ga),
technetium-99m (99mTc) and indium-111 have been developed and used in preclinical and clinical
studies.1-4 These radiolabeled probes are generally prepared by conjugating an appropriate chelator
to a biomolecule of interest (referred to as “ligand”) and subsequent complexation reaction with
metallic radionuclides to ensure high in vivo stability. The radiolabeled ligands are synthesized in
the presence of a large excess of ligands over metallic radionuclides to ensure high radiochemical
yields in short reaction time, due to the low concentration of metallic radionuclides (67Ga: 37
MBq/mL corresponding to 2.5 × 10-8 M) (Figure 1C).1 The presence of a large excess of unlabeled
ligands in the injectate impairs target uptake of the radiolabeled ligands by competing for target
molecules, which results in poor images of the target molecules of low expression levels.5-9 The
free ligands can be removed from the radiolabeled ligands by HPLC or other purification methods.
However, such manipulation significantly wastes time and radioactivity and impairs the practical
utility and advantages of using metallic radionuclides.
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To circumvent the problem, we recently developed a new design concept of metal coordination-
mediated synthesis of 99mTc-labeled trivalent probes using [99mTc][Tc(CO)3(OH2)3]+ and isonitrile-
conjugated RGD peptide (CN-RGD).8 In this design, three water molecules in
[
99mTc][Tc(CO)3(OH2)3]+ are displaced with the three monovalent ligands (CN-RGD) to prepare a
trivalent [99mTc][Tc(CO)3(CN-RGD)3]+ probe. [99mTc][Tc(CO)3(CN-RGD)3]+ acquired higher
integrin αvβ3 binding affinity than its monovalent ligand (Figure 1A-1). In SPECT imaging studies,
[
99mTc][Tc(CO)3(CN-RGD)3]+ clearly visualized murine tumors without removing the unlabeled
ligands. However, a 99mTc-labeled monovalent probe prepared from a monovalent ligand failed to
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(A) In situ multivalent “1 to 3” or “1 to 2” designs
Reaction solution
A-1
1:3
reaction
M
M
M
Higher affinity
(Multivalent effect)
(<1 µM)
(≈100 µM)
(<1 µM)
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Reaction solution
A-2
1:2
reaction
M
M
M
Higher affinity
(Multivalent effect)
(<1 µM)
(≈100 µM)
(<1 µM)
(B) Modified in situ multivalent “1 to 3” design
Reaction solution
1:1
reaction
M
M
Equilibrium
M
Higher affinity
(Multivalent effect)
(<1 µM)
(≈100 µM)
(<1 µM)
(C) Conventional design (trivalent “3 to 3” design)
Reaction solution
1:1
reaction
M
M
M
(<1 µM)
Similar affinity
: metal radionuclide : aldehyde group
(≈100 µM)
(<1 µM)
: targeting molecule
M
: amine group
: triamine compound
: Schiff base
Figure 1. A conceptual illustration of the three molecular design of trivalent probes. (A) In situ
multivalent “1 to 3” design (A-1) and “1 to 2” design (A-2). The radiolabeled probes prepared
from the monovalent ligand acquired higher binding affinity than the monovalent ligand upon
complexation by the multivalent effect. (B) Modified in situ “1 to 3” design. The radiolabeled
probe was synthesized by reacting a radiometal ion with a trivalent Schiff base hexadentate
ligand prepared from a monovalent ligand with an aldehyde group and a triamine molecule at
the molar ratio of 3:1. While the Schiff base is stabilized upon metal coordination, the unlabeled
Schiff base ligands present as equilibrium in the injectate are decomposed immediately after
injection. As a result, the metal complex is an only trivalent compound and possess higher
binding affinity than its unlabeled monovalent ligand. (C) A conventional trivalent “3 to 3”
design. A trivalent ligand is reacted with a radiometal ion to form a radiolabeled trivalent probe.
The radiolabeled probe possesses target binding affinity similar to that of the unlabeled ligand.
depict the tumors in the presence of the unlabeled ligands. We also reported the applicability of
this design concept to 99mTc-labeled bivalent probes using D-penicillamine as a chelating molecule,
suggesting that the design concept would apply to other metallic radionuclides (Figure 1A-2).9
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Since recent interest has rather been directed toward the use of trivalent metallic radionuclides
such as 67/68Ga, yttrium-90, indium-111, lutetium-177 and actinium-225 for molecular imaging
and targeted radionuclide therapy, the application of the design concept to these metallic
radionuclides would provide a versatile molecular design of radiolabeled probes for molecular
imaging and targeted radionuclide therapy. We focused our present efforts on radiogallium, 67Ga.
67Ga is a gamma emitter (T1/2=3.3 d) is a commercially available γ-emitting radionuclide and used
in the diagnosis of infection and inflammatory processes as well as tumor imaging by SPECT.1
In this study, we investigated an appropriate procedure to apply the chemical design developed
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with 99mTc-labeled probes to Ga-labeled ones. One approach involved the use of bidentate or
tridentate chelators based on the procedure achieved with 99mTc-labeled trivalent or bivalent
probes.8, 9 Among the bidentate chelators, we selected 3-hydroxy-1,2-dimethyl-4-pyridone (Hdpp),
due to a formation of a thermodynamically stable complex with Ga3+ ion (Figure 2A-1).10, 11 As
for the tridentate chelators, 2-acetylpyridine N,N-disubstituted thiosemicarbazone (PTSC)
derivatives were selected based on the recent studies (Figure 2A-2).12, 13 The other approach
involves the use of a Schiff base chelator,14 since free Schiff base chelators are present as
equilibrium and are rapidly decomposed upon dilution while the Schiff base remains stable through
metal coordination (Figures 1B and 2B)15 as well exemplified by 99mTc-labeled pyridoxal
isoleucine for hepatobiliary tract imaging.16, 17 The applicability of the latter approach was further
evaluated after conjugation of a cyclic RGDfK (-Arg-Gly-Asp-D-Phe-Lys-: c(RGDfK)) peptide,
a specific antagonist for integrin αvβ3 overexpressed during tumor angiogenesis,18 as a model of a
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targeting specific biomolecule. A Ga-labeled 1,4,7-triazacyclononane-1,4,7-tris-(glutaric acid)
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(NOTGA) conjugated c(RGDfK) ([67Ga]Ga-NOTGA(RGD)3) was synthesized and used as a
reference of the conventional “3 to 3” approach (Figure 2C).
Results and Discussion
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(A) in situ multivalent “1 to 3” or “1 to 2” designs
A -1
O
O
Ga
HO
O
N
N
Ga
3
Hdpp
1 Ga-(Hdpp)3
A -2
N
Ga
N
S
R2N
S
N
N
S
N
Ga
N
N
N
N
H
NR2
NR2
2 R=CH2-CH2-O-CH3: PTSC-1
3 R=CH2-CH(CH3)2: PTSC-2
4 R=CH2-CH2-O-CH3: Ga-(PTSC-1)2
5 R=CH2-CH(CH3)2: Ga-(PTSC-2)2
(B) Modified in situ trivalent “1 to 3” design
OH
H2N
OH
OH
NH2 NH2
N
N
N
N
Ga
TAMEol
OH
HO
Ga
O
N
N
OH
HO
O
O
R
R
O
R
R
R
R
R
6 R=H: Sal
7 R=H: (Sal)3TAMEol
8 R=H: Ga-(Sal)3TAMEol
17 R= Ahx-RGD
: Ga-(Sal-RGD)3TAMEol
16 R=Ahx-RGD
: Sal-RGD
(C) Conventional design (trivalent “3 to 3” design)
O
RGD
O
O
RGD
O
O
O
OH
N
RGD
RGD
O
N
N
Ga
N
Ga
N
O
O
OH
O
O
N
HO
O
O
O
O
RGD
RGD
18: NOTGA(RGD)3
19: Ga-NOTGA(RGD)3
Figure 2. (A-1) In situ trivalent “1 to 3” design using bidentate Hdpp and 67Ga. (A-2) In
situ bivalent “1 to 2” design using tridentate PTSC ligands and 67Ga. (B) Modified in situ
trivalent “1 to 3” design using hexadentate Schiff base (R-Sal)3TAMEol with 67Ga. The
Schiff base ligand ((R-Sal)3TAMEol) is stabilized upon 67Ga coordination, while the free
Schiff base ligand is decomposed to RGD-Sal and TAMEol after the change in the
equilibrium state such as dilution. (C) A conventional “3 to 3” design of [67Ga]Ga-
NOTGA(RGD)3 using NOTGA(RGD)3.
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Bidentate Hdpp chelator. When a solution of Hdpp in 0.1 M acetate buffer (pH 5.0) was reacted
with [67Ga]Ga-citrate at 80 °C for 30 min, the major radioactivity was observed at the origin in
cellulose acetate electrophoresis (CAE) analysis. Under these conditions, [67Ga]Ga-citrate
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migrated 4 cm towards the anode (Figures 3A and B), suggesting a formation of a Ga-labeled
(A) [67Ga]Ga-(Hdpp)3
(B) [67Ga]Ga-citrate
-5
0
Distance from origin
5
-5
0
5
Distance from origin
10
10
(cm)
(C) [67Ga]Ga-(Hdpp)3
(cm)
(D) [67Ga]Ga-(Hdpp)3
0
0.5
Rf value
1
0
0.5
Rf value
1
Figure 3. Analysis of [67Ga]Ga-(Hdpp)3. CAE radioactivity traces of (A) [67Ga]Ga-(Hdpp)3 and
(B) [67Ga]Ga-citrate. TLC radioactivity traces of [67Ga]Ga-(Hdpp)3 developed with MeOH/water
(C) without and (D) with 10 mM Hdpp.
Hdpp. The TLC analysis displayed a broad radioactivity peak from the Rf values of 0 to 0.2 (Figure
3C). [67Ga]Ga-citrate remained at the origin under the analytical conditions. When Hdpp (10 mM)
was added to the developing solvent, a sharp radioactivity peak was observed at an Rf value of 0.3
(Figure 3D) similar to that of non-radioactive Ga-(Hdpp)3 (1) characterized and verified by ESI-
MS. Similar results were observed by RP-HPLC analysis where the majority of the radioactivity
was adsorbed on the column and was not eluted. (data not shown). These results suggested that
while Hdpp would provide an objective [67Ga]Ga-(Hdpp)3 complex, the complex underwent rapid
decomposition under low Hdpp concentrations, which was not appropriate for in vivo applications.
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Tridentate PTSC derivatives. The PTSC derivatives were synthesized by reacting 2-
acetylpyridine with S-methyl dithiocarbazate19 in aqueous ethanol at 80 ºC, followed by the
alkylation with secondary amines, as shown in Scheme S1. The 67Ga-complexes were prepared by
reacting the PTSC derivatives (1 mM) with [67Ga]GaCl3 in 10% ethanolic solution of 100 mM
ammonium acetate (pH 4.5) at 80 ºC for 15 min. The RP-HPLC analyses of the reaction solution
showed a single radioactivity peak at retention times similar to those of the corresponding non-
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radioactive Ga-(PTSC)2 derivatives verified by ESI-MS (Figure 4). Both [67Ga]Ga-(PTSC-1)2 and
(A) [67Ga]Ga-(PTSC-1)2 (B) [67Ga]Ga-(PTSC-2)2
0
10
20
30
0
10
20
30
40
retention time (min)
retention time (min)
(C) Ga-(PTSC-1)2
(D) Ga-(PTSC-2)2
0
10
20
30
0
10
20
30
40
retention time (min)
retention time (min)
Figure 4. RP-HPLC radioactivity traces of [67Ga]Ga-(PTSC-1)2 (A) and [67Ga]Ga-(PTSC-2)2
(B) and UV (254 nm) traces of Ga-(PTSC-1)2 (C) and Ga-(PTSC-2)2 (D).
[67Ga]Ga-(PTSC-2)2 remained stable after isolation from the free ligand by RP-HPLC. However,
both [67Ga]Ga-(PTSC-1)2 and [67Ga]Ga-(PTSC-2)2 rapidly decomposed after incubation in 0.1 M
bicarbonate buffer (pH 7.4) at 37 ºC (Table 1). Although the N-substitution group significantly
affected the stability of the resulting [67Ga]Ga-(PTSC)2 complexes, these results suggested that
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further modifications of PTSC structure would be needed to develop a chelator that forms a
kinetically inert 67Ga complex for in vivo applications.
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Table 1. Stability of [67Ga]Ga-(PTSC-1)2 and [67Ga]Ga-(PTSC-2)2 in 0.1 M bicarbonate
buffer (pH 7.4)a
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percent of intact radiolabeled complex (%)
time (min)
[67Ga]Ga-(PTSC-1)2
70.4 ± 9.67
[67Ga]Ga-(PTSC-2)2
31.0 ± 3.24
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30
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47.1 ± 4.83
5.60 ± 0.38
5.73 ± 1.55
0.71 ± 0.32
aExpressed as mean ± s.d. for three experiments.
Schiff base ligand. The Schiff base chelator (7) was synthesized by reacting salicylaldehyde (1,
Sal) with triamine compound, 2,2’,2”-tri(aminomethyl)ethanol (TAMEol, Scheme 1) in MeOH
(Figure 2B). [67Ga]Ga-(Sal)3TAMEol was prepared by reacting an EtOH solution of
(Sal)3TAMEol (7) (1 mM) and [67Ga]Ga-citrate, followed by heating the reaction mixture at 80 ˚C
for 15 min in 95% radiochemical yield. Since the solubility of the non-radioactive Ga-
(Sal)3TAMEol (8) was too low to be detected by UV detector, ICP-MS was used to determine the
retention time of compound 8. The RP-HPLC retention time of the [67Ga]Ga-(Sal)3TAMEol was
similar to that of the corresponding non-radioactive Ga-complex characterized by ESI-MS (Figure
5). [67Ga]Ga-(Sal)3TAMEol remained stable during RP-HPLC analysis and after isolation from
Scheme 1. Synthesis of TAMEol
Boc
OH N3
OH NH2
NH2 NH2
OH NH2
3 HCl
NH
OH Br
Br Br
OH
(a)
(b)
(c)
(d)
N3 N3
NH2 NH2
NH NH
Boc Boc
TAMEol
Reagents : (a) NaN3, (b) 10%Pd/C, H2, (c) (Boc)2O, (d) 4 M HCl/EtOAc.
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the excess Sal and TAMEol. Furthermore, [67Ga]Ga-(Sal)3TAMEol remained stable in the apo-
transferrin challenge. These results indicated that the use of a Schiff base compound would provide
67Ga complex possessing kinetic stability worth for further evaluation.
(A)
(B)
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0
10
retention time (min)
20
0
10
retention time (min)
20
Figure 5. RP-HPLC radioactivity profile of [67Ga]Ga-(Sal)3TAMEol (A) and 69Ga-ion count
profile of Ga-(Sal)3TAMEol (B).
Synthesis of RGD-conjugated Sal. In the design of multivalent probes for targeting integrin
αvβ3, the distance between each RGD motif plays a crucial role in eliciting the multivalent effect.20-
22 We introduced a hexanoate linker between RGD and Sal to adjust the recommended distance.8,
9, 22, 23
The synthesis procedure for RGD-Sal (16) is illustrated in Scheme 2. p-Hydroxybenzoic
Scheme 2. Synthesis of RGD-Sal
F
Fmoc
O
F
F
OH
Fmoc
OH
(a)
(b)
N
H
H2N
N
H
O
O
O
11
12
F
cR(Pbf)GD(OtBu)fK
(c)
(d)
Fmoc
cR(Pbf)GD(OtBu)fK
H2N
N
H
O
O
13
14
OH
OH
OH
OH
(f)
(e)
(g)
N
O
N
OH
OH
cR(Pbf)GD(OtBu)fK
O
O
OH
O
OH
N
H
O
OH
O
9
10
15
OH
O
(h)
c(RGDfK)
O
N
H
O
16: RGD-Sal
Reagents : (a) Fmoc-NHS, (b) TFP, DCC, (c) c(R(Pbf)GD(OtBu)fK) (d) 20% piperidine/DMF,
(e) TFA, hexamethylenetetramine, (f) o-aminophenol (g) DCC, HOAt, (h) TFA/water 9:1.
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acid was reacted with hexamethylenetetramine, and the resulting aldehyde 9 was protected with o-
amino-phenol to prepare 10. Compound 10 was conjugated with an aminohexanoic acid-
conjugated RGD derivative 14 to prepare 15. RGD-Sal (16) was obtained after removing the
protecting groups and subsequent RP-HPLC purification (Figure S2).
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Synthesis of [67Ga]Ga-(RGD-Sal)3TAMEol. [67Ga]Ga-(RGD-Sal)3TAMEol was prepared in
95% radiochemical yield by heating a mixed solution of RGD-Sal (16), TAMEol and [67Ga]Ga-
citrate at 70 ºC for 1 h. The final concentration of RGD-Sal was 2.5 × 10-4 M. The RP-HPLC
analysis of [67Ga]Ga-(RGD-Sal)3TAMEol showed a single radioactive peak at a retention time
similar to that of the non-radioactive Ga-(RGD-Sal)3TAMEol (17) characterized and verified by
ESI-MS (Figures 6). The RP-HPLC analysis of [67Ga]Ga-(RGD-Sal)3TAMEol by UV trace
showed two peaks corresponding to RGD-Sal (16) and benzyl alcohol, a preservative in [67Ga]Ga-
citrate, and the intact (RGD-Sal)3TAMEol was not observed (Figure 6B). These results indicated
that the Schiff base ligand (RGD-Sal)3TAMEol was rapidly decomposed to its constituents by the
(A)
(B)
BnOH
RGD-sal
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20
30
0
10
30
0
10
Retention time (min)
(C)
20
Retention time (min)
0
10
30
Figure 6. RP-HPLC profiles of [67Ga]Ga-(RGD-Sal)3TAMEol. (A) The radiotrace of [67Ga]Ga-
(RGD-Sal)3TAMEol and (B) the UV (254 nm) trace of the same sample. A single radioactivity
peak was observed at a retention time similar to that of non-radioactive Ga-(RDG-Sal)3TAMEol
(C) verified by ESI-MS. The UV trace showed two peaks, corresponding to RGD-Sal and
benzyl alcohol (BnOH).
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changes in the equilibrium during RP-HPLC analysis while the Schiff base was stabilized by 67Ga
coordination to provide stable [67Ga]Ga-(RGD-Sal)3TAMEol. [67Ga]Ga-NOTGA(RGD)3 used as
a representative compound by the conventional “3 to 3” design was prepared in 95% radiochemical
yield as described previously (Figure S3).24
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Lipophilicity and stability estimation of [67Ga]Ga-(RGD-Sal)3TAMEol. The lipophilicity
and the stability of [67Ga]Ga-(RGD-Sal)3TAMEol were evaluated after removing RGD-Sal and
TAMEol by RP-HPLC, eliminating organic solvents and reconstituted in an appropriate buffer
solution. [67Ga]Ga-(RGD-Sal)3TAMEol remained stable after the process (data not shown). The
lipophilicity of [67Ga]Ga-(RGD-Sal)3TAMEol was determined by measuring the Log D7.4 value
using [67Ga]Ga-NOTGA(RGD)3 as a reference. The Log D7.4 values of [67Ga]Ga-(RGD-
Sal)3TAMEol and [67Ga]Ga-NOTGA(RGD)3 were -3.16 ± 0.03 and -4.10 ± 0.07, respectively.
The stability of [67Ga]Ga-(RGD-Sal)3TAMEol was evaluated in the presence of apo-transferrin at
37 ˚C. As shown in Table 2, more than 95% of the radioactivity was present as an intact [67Ga]Ga-
(RGD-Sal)3TAMEol after 6 h incubation, demonstrating that [67Ga]Ga-(RGD-Sal)3TAMEol
possessed stability sufficient for in vivo applications.
Table 2. Stability of [67Ga]Ga-(RGD-Sal)3TAMEol in Apo-transferrin solutiona
time (h)
percent of intact radiolabeled complex (%)
1
3
6
98.0 ± 0.4
97.3 ± 0.6
95.7 ± 0. 3
aExpressed as mean ± s.d. for three experiments.
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Binding Affinity to Integrin αvβ3. The binding of Ga-(RGD-Sal)3TAMEol (17) to integrin αvβ3
on human glioma U87MG cells was evaluated with the competitive binding assay using [125I]I-
c(RGDyV). The IC50 values are summarized in Table 3, and the displacement curves of [125I]I-
c(RGDyV) are presented in Figure S4. The binding affinities of Ga-(RGD-Sal)3TAMEol (17), Ga-
NOTGA(RGD)3 (19), and NOTGA(RGD)3 (18) were similar to one another (1.01, 2.31, and 2.26
nM, respectively) and these values were also similar to that of a 99mTc-labeled trivalent RGD
peptide reported previously.8 These results indicated that the metal coordination-mediated
stabilization of Schiff base chelator constitutes a useful procedure to synthesize 67Ga-labeled
trivalent probes. The higher binding affinity of the monovalent RGD-Sal (16) (6.14 nM) compared
to c(RGDyV) (32.27 nM) would be attributable to the aldehyde-mediated non-specific binding to
protein molecules on the cells, since the binding affinity decreased when the aldehyde group of
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RGD-Sal (16) was reduced to an alcohol group (data not shown).
Table 3. IC50 Values of Each Compound
design
compound
RGD-Sal
IC50, nM
6.14
95% C.I. a
4.92-7.67
0.74-1.37
1.81-2.82
1.80-2.97
27.1-39.5
“1 to 3”
Ga-(RGD-Sal)3TAMEol
NOTGA(RGD)3
Ga-NOTGA(RGD)3
c(RGDyV)
1.01
2.26
“3 to 3”
2.31
reference
32.27
aC.I. = confidential interval.
Biodistribution and small animal SPECT/CT imaging studies. The biodistribution of
radioactivity at 1 h postinjection in nude mice bearing U87MG xenografts was determined in the
following 4 samples: (1) RP-HPLC-purified [67Ga]Ga-(RGD-Sal)3TAMEol, (2) unpurified
[67Ga]Ga-(RGD-Sal)3TAMEol (containing 10 nmol of RGD-Sal (16)), (3) RP-HPLC-purified
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[67Ga]Ga-NOTGA(RGD)3 (without NOTGA(RGD)3 (18)), and (4) unpurified [67Ga]Ga-
NOTGA(RGD)3 (containing 10 nmol of NOTGA(RGD)3 (18)). The results are summarized in
Table 4. The RP-HPLC-purified [67Ga]Ga-(RGD-Sal)3TAMEol showed the radioactivity levels in
the bone, a known tissue of free 67Ga accumulation,25, 26 similar to those of 67Ga-labeled
compounds that exhibit high in vivo stability26. These results along with the in vitro studies
confirmed that [67Ga]Ga-(RGD-Sal)3TAMEol possesses stability sufficient for in vivo
applications. The lower accumulation of unpurified [67Ga]Ga-(RGD-Sal)3TAMEol in the bone and
other tissues compared with purified [67Ga]Ga-(RGD-Sal)3TAMEol would be attributable to the
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inhibition of non-specific accumulation of the Ga-labeled compounds by the free unlabeled
Table 4. Biodistribution at 1 h Postinjection in Tumor-Bearing Micea
[67Ga]Ga-(RGD-Sal)3TAMEol
(“1 to 3” design)
[67Ga]Ga-NOTGA(RGD)3
(“3 to 3” design)
purified
0
unpurified
10
purified
0
unpurified
10
ligand (nmol)
blood
0.95 ± 0.01
4.17 ± 0.57c
11.85 ± 0.34
5.01 ± 0.51c
7.98 ± 1.02c
1.09 ± 0.27
2.48 ± 0.24c
4.74 ± 0.40c
1.07 ± 0.18
2.46 ± 0.52
11.46 ± 0.99
2.19 ± 0.34
4.87 ± 0.71
0.72 ± 0.24
1.26 ± 0.17
3.59 ± 0.16
0.38 ± 0.14
3.02 ± 0.52d
7.90 ± 0.69
3.95 ± 0.44d
9.91 ± 0.78d
1.05 ± 0.40
1.38 ± 0.23
4.19 ± 0.24d
0.43 ± 0.12
0.73 ± 0.13
6.24 ± 1.16
0.60 ± 0.26
7.95 ± 0.64
0.62 ± 0.12
n.d.
liver
kidney
spleen
intestine b
muscle
bone
tumor
1.62 ± 0.35
aThe results were expressed as percent injected dose per gram (%ID/g) ± SD (n=4-5).
bExpressed as %ID. Statistical analysis was performed using one-way analysis of variance
c
followed by Tukey’s multiple-comparison test; (different from unpurified [67Ga]Ga-(RGD-
d
Sal)3TAMEol, p<0.05), (different from unpurified [67Ga]Ga-NOTGA(RGD)3, p<0.05). n.d.;
not determined.
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Tumor
Tumor
1%ID/g
30%ID/g
Figure 7. SPECT/CT images of U87MG tumor-bearing nude mice over 45−75 min post i.v.
injection of (A) 1.8 MBq of the unpurified [67Ga]Ga-(RGD-Sal)3TAMEol and (B) 2.2 MBq of
the unpurified [67Ga]Ga-NOTGA(RGD)3. Images are shown at the same signal intensity scale.
Red circle indicates the U87MG tumor.
ligands, RGD-Sal (16) and TAMEol. Similar results were observed with [67Ga]Ga-
NOTGA(RGD)3 where the non-specific accumulation of [67Ga]Ga-NOTGA(RGD)3 were reduced
by unlabeled NOTGA(RGD)3 (18) (Table 4), as also observed in our previous studies of 99mTc-
labeled probes.8,
The RP-HPLC purified [67Ga]Ga-(RGD-Sal)3TAMEol and [67Ga]Ga-
9
NOTGA(RGD)3 showed similar tumor uptakes (4.74 ± 0.40 and 4.19 ± 0.24 %ID/g, respectively),
reflecting the similar binding affinities to U87MG cells (Table 3) and the high in vivo stability of
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the two Ga-labeled probes. When the Ga-labeled probes were injected without post-labeling
purification, the tumor accumulation of [67Ga]Ga-NOTGA(RGD)3 was inhibited by 60% in the
presence of 10 nmol of NOTGA(RGD)3 (18). On the other hand, only 25% reduction of tumor
accumulation was observed after injection of [67Ga]Ga-(RGD-Sal)3TAMEol in the presence of 10
nmol of RGD-Sal (16) (and 2.7 nmol of TAMEol), which indicates that the free (RGD-
Sal)3TAMEol present as an equilibrium in the injectate was rapidly decomposed to RGD-Sal (16)
and TAMEol upon injection, as also observed in the RP-HPLC analysis (Figure 6). Some RGD-
Sal (16) may have bound to plasma proteins through the aldehyde group, which may also reduce
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the inhibitory action of the monovalent RGD-Sal (16) on tumor cells. Such characteristics were
well reflected in the SPECT/CT images where unpurified [67Ga]Ga-(RGD-Sal)3TAMEol depicted
a clear image of the tumor whereas unpurified [67Ga]Ga-NOTGA(RGD)3 showed a poor image of
the tumor (Figure 7). These results demonstrated that the coordination-mediated stabilization of
Schiff base chelator constitutes a useful procedure to apply the chemical design achieved with
99mTc-labeled trivalent probes to 67Ga-labeled ones.
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CONCLUSIONS
We investigated the way to apply the coordination-mediated synthesis of multivalent probes
67
developed with 99mTc-labeled probes to Ga-labeled ones. Hdpp, a bidentate chelator, failed to
67
prepare Ga-labeled probes since high Hdpp concentration was needed to maintain the intact
structure of the radiometal chelate. PTSC derivatives, tridentate chelators, provided [67Ga]Ga-
labeled probes of much higher stability than that from Hdpp. However, the radiolabeled probes
still suffered from insufficient stability for in vivo applications, and further structural modification
was needed. Thus, the approaches to prepare 99mTc-labeled multivalent probes were not suitable
to prepare 67Ga-labeled multivalent probes. Contrary to these approaches, the Schiff base chelator
provided 67Ga-labeled trivalent probes with stability sufficient for in vivo applications.
Furthermore, (RGD-Sal)3TAMEol exhibited the much less inhibitory effect on the [67Ga]Ga-
(RGD-Sal)3TAMEol accumulation to the target molecules compared with [67Ga]Ga-
NOTGA(RGD)3 prepared under the conventional “3 to 3” approach, due to rapid decomposition
of (RGD-Sal)3TAMEol to RGD-Sal and TAMEol upon injection. As a result, [67Ga]Ga-(RGD-
Sal)3TAMEol visualized the murine tumor without post-labeling purification. Although further
studies are required such as the reduction of Schiff base ligand concentration and the improvement
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of pharmacokinetic behavior, the present study indicates that the use of the Schiff base ligand
would constitute a useful procedure to prepare 67Ga-labeled purification-free trivalent probes.
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EXPERIMENTAL SECTION
General. [67Ga]Ga-citrate and [67Ga]GaCl3 was purchased from FUJIFILM RI Pharma Co., Ltd.
(Tokyo, Japan). Mass spectrometry was carried out using an Agilent 6130 Series Quadrupole
LC/MS electrospray system (Agilent Technologies, Tokyo) or JMS-T100LP (JEOL Ltd., Tokyo).
1H-NMR spectra were recorded on a JEOL JNM-ECS-400 (400 MHz) spectrometer (JEOL Ltd.).
The melting points were measured using a MP-500D (Yanaco, Kyoto, Japan) and were reported
uncorrected. c(-Arg(Pbf)-Gly-Asp(tBu)-D-Phe-Lys-) (c(R(Pbf)GD(tBu)fK)), NOTGA(tBu)3 and
methyl 3-[1-(2-pyridyl)ethylidene]hydrazinecarbothione were synthesized according to the
procedures of reported previously.18, 19, 24 All commercially available chemicals were of analytical
grade and used without further purification. All compounds purity used in affinity assays,
biodistribution studies and SPECT studies were greater than 95%. Analytical and preparative RP-
HPLC were performed in different system (system 1-9) and are detailed in supporting information.
TLC analyses were performed with silica plates (Silica gel 60F254, Merck Ltd., Tokyo). Cellulose
acetate electrophoresis (CAE) strips were run in a veronal buffer (pH 8.5, I=0.06) at constant
current of 1 mA/cm for 30 min. Radioactivity was measured using a MiniGita Star Gamma TLC
Scanner (raytest) and an auto well gamma counter.
Synthesis of 2,2’,2”-tri(aminomethyl)ethanol/3HCl (TAMEol). Pentaerythritol tribromide
(2.08 g, 6.40 mmol) and sodium azide (4.8 g, 73.8 mmol) were dissolved in dimethylformamide
(DMF, 35 mL) and mixed at 80 ˚C for 24 h under N2 atmosphere. After the solvent was removed
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in vacuo, the residue was resolved in H2O (20 mL) and then extracted with Et2O (30 mL). The
organic layer was dried over anhydrous MgSO4. After removing the solvent, pentaerithritol
triazide was obtained as a colorless oil. This product was used without further purification.
Pentaerithritol triazide was dissolved in MeOH (10 mL), and then 10% Pd/C (1.03 g) was added
portion-wise. After the mixture was stirred for 7 h, Pd/C was removed by filtration. After the
solvent was evaporated in vacuo, a colorless transparent oil was obtained. The resulting oil was
dissolved in the solution (20 mL, sat. NaHCO3 : acetonitrile/1 : 1) and then di-tert-butyl
dicarbonate (Boc2O, 5.79 g, 26.5 mmol) dissolved in acetonitrile (20 mL) was added dropwise on
ice. After mixing for 15 h, the solvent was evaporated in vacuo and then the residue was purified
with open column chromatography using silica gel and subsequently eluted with CHCl3 : Et2O/20 :
1 to obtain N,N,N-(tert-butoxycarbonyl)-2,2’,2”-tri(aminomethyl)ethanol (TAMEol-Boc3).
TAMEol-Boc3 was dissolved in 4 M HCl/EtOAc (5 mL) and mixed for 150 min. After removing
the solvent in vacuo, HCl salt of compound TAMEol was obtained as a white solid (190 mg, 12%).
1H-NMR (D2O), (ppm): 3.18 (t, 6H, CH2), 3.75 (s, 2H, CH2) (Figure S1). ESI-MS, m/z: 134
[M+H]+, Found 134. M.p. 315-316 ˚C.
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Synthesis of Ga-(3-hydroxy-1,2-dimethyl-4-pyridinone)3 (Ga-(Hdpp)3, 1). 3-Hydroxy-1,2-
dimethyl-4-pyridinone (Hdpp, 30 mg, 0.22 mmol) was suspended in water (5 mL). GaCl3 (12.9
mg, 0.073 mmol) dissolved in water (50 µL) was added to the solution and then the solution
became clear. After the pH of the solution was adjusted to 9 by 2 N NaOH, the reaction mixture
was heated at 70 ˚C until the reaction volume was <2.5 mL. After cooling room temperature, the
precipitate was collected by filtration and then compound 1 was obtained as an orange solid (20.6
mg, 58%). ESI-MS, m/z: 484 [M+H]+, Found 484.
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Synthesis of 2-acethylpyridine thiosemicarbazones. (PTSC-1 (2) and PTSC-2 (3)). To 200
mg (0.89 mmol) of methyl 3-[1-(2-pyridyl)ethylidene]hydrazinecarbothioate dissolved in 2.2 mL
of EtOH was added 0.89 mmol of bis(2-methoxyethyl)amine for PTSC-1 or diisobutylamine for
PTSC-2. The solution was heated under reflux for 24 h. After cooling to room temperature, the
resulting crystals were collected, washed with EtOH (30 mL) to afford PTSC-1(2) and PTSC-2
(3). PTSC-1: (82.2 mg, 29.9%) 1H-NMR (CD3OD), ꢀ: 2.44 (m, 3H, CH3), 3.37 (m, 6H, OCH3),
3.75 (m, 4H, CH2O), 4.01 (s, 4H, NCH2), 7.47 (m, 1H, Pyr), 8.03 (m, 2H, Pyr), 8.62 (m, 1H, Pyr).
ESI-MS, m/z: 310 [M+H]+, Found 310. PTSC-2: (101 mg, 37.5%) 1H-NMR (CD3OD), ꢀ: 0.95 (m,
12H, CH3), 2.46 (s, 3H, CH3), 3.69 (m, 4H, CH2), 7.47 (m, 1H, Pyr), 7.94 (m, 2H, Pyr), 8.65 (m,
1H, Pyr). ESI-MS, m/z: 307 [M+H]+, Found 307.
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Synthesis of Ga-(PTSC-1)2 (4) and Ga-(PTSC-2)2 (5). PTSC-1 (2) or PTSC-2 (3) (20 mg, 65
µmol) and Ga(NO3)3/nH2O (ca. 17 mg) was dissolved in EtOH (0.5 mL) and mixed at 40 ˚C for 1
h. A solution of ammonium hexafluorophosphate (13 mg, 0.08 mmol) in EtOH (200 µL) was added
to the solution. After mixing for 10 min, the precipitates were obtained by filtration to afford Ga-
(PTSC-1)2 (4) or Ga-(PTSC-2)2 (5). compound 4: 7.5 mg (16.8%) ESI-MS, m/z: 687 [M]+, Found
687. compound 5: 4.3 mg (9.7%) ESI-MS, m/z: 679 [M]+, Found 679.
Synthesis of Trisalicyldiminomethyl Ethanol ((Sal)3TAMEol, 7). TAMEol/3HCl (170 mg,
0.70 mmol) and salicylaldehyde (6) (489 mg, 4.00 mmol) was dissolved in MeOH (10 mL). After
standing overnight, the solvent was removed in vacuo. The residue was recrystallized from MeOH
to afford (Sal)3TAMEol (7) as a yellow solid (83 mg, 27%).1H-NMR (CD3OD), (ppm): 3.72 (s,
6H, CH2), 3.75 (s, 2H, CH2), 6.89 (t, 3H, CH), 8.41 (s, 3H, CH=N). ESI-MS, m/z: 446 [M+H]+,
Found 446.
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Synthesis of Ga-(Sal)3TAMEol (8). Compound 7 (12.8 mg, 28.8 µmol) and gallium-
acetylacetonate (10.5 mg, 28.6 µmol) was dissolved in EtOH (8.5 mL) and then refluxed at 80 ˚C
for 12 h. The precipitate was collected by filtration and then compound 8 was obtained as a pale
yellow solid (8 mg, 54%). ESI-MS, m/z: 512 [M+H]+, Found 512.
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Synthesis of 3-Formyl-4-Hydroxybenzoic Acid (9). p-Hydroxybenzoic acid (1.97 g, 14.3
mmol) was dissolved in trifluoroacetic acid (TFA, 6 mL). Hexamethylenetetramine (2.0 g, 14.3
mmol) dissolved in TFA (8 mL) was added to the solution dropwise and mixed at 70 ˚C for 22 h.
After cooling to room temperature, 4 N HCl aq. (45 mL) was added to the solution and mixed for
6 h. The precipitate was collected and washed with H2O (400 mL) to obtain compound 9 as a pale
yellow solid (1.3 g, 55%). 1H-NMR (CD3OD), ꢀ: 7.02 (d, 1H, Aryl), 8.15 (dd, 1H, Aryl), 8.40 (s,
1H, Aryl), 10.11 (s, 1H, CHO). ESI-MS, m/z: 167 [M+H]+, Found 167. M.p. 225-227 ˚C.
Synthesis of 3-[(2-Hydroxyphenylimino)-Methyl]-4-Hydroxybenzoic Acid (10). Compound
9 (503 mg, 3.03 mmol) and o-aminophenol (330 mg, 3.02 mmol) were dissolved in MeOH (30
mL) and refluxed at 70˚C for 10 min. Compound 10 was crystallized from MeOH (543 mg, 70%).
1H-NMR (CD3OD), ꢀ: 6.94 (m, 3H, Aryl), 7.18 (m, 1H, Aryl), 7.47 (m, 1H, Aryl), 8.11 (t, 1H,
Aryl), 8.24 (s, 1H, Aryl), 9.02 (s, 1H, CHN). ESI-MS, m/z: 258 [M+H]+, Found 258. M.p. 259-
260 ˚C.
Synthesis of Fmoc-6-Aminohexanoic Acid (11). 6-Aminohexanoic acid (1.25 g, 9.53 mmol)
was dissolved in H2O (20 mL) and then 10% NaHCO3 solution (20 mL) was added. N-(9-
fluorenylmethoxycarbonyloxy) succinimide (Fmoc-OSu, 3.56 g, 10.6 mmol) dissolved in dioxane
(20 mL) was added to the solution at 0 ˚C and mixed overnight. After removing the solvent in
vacuo, 1 N HCl aq. (10 mL) was added and then extracted by CH2Cl2 (20 mL × 3). The organic
solvent was dried over MgSO4. Compound 11 was recrystallized from CH2Cl2 and obtained as a
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white solid (2.4 g, 71%). 1H-NMR (CDCl3), ꢀ: 1.51 (m, 6H, CH2), 2.34 (m, 2H, CH2CO), 3.17 (m,
2H, CH2NH), 4.19 (t, 1H, CH), 4.41 (m, 2H, CH2), 4.77 (br, 1H, NH), 7.33 (m, 4H, Fmoc), 7.57
(d, 2H, Fmoc), 7.74 (d, 2H, Fmoc). ESI-MS, m/z: 376 [M+Na]+, Found 376. M.p. 117-118˚C.
Synthesis of Fmoc-6-Aminohexanoic Acid 2,3,5,6-Tetrafluorophenyl Ester (12). Compound
11 (1.01 g, 2.86 mmol), 2,3,5,6-tetrafluorophenol (TFP, 540 mg, 3.25 mmol) was dissolved in 2
mL of DMF. N, N'-dicyclohexylcarbodiimide (DCC, 670 mg, 3.25 mmol) in 0.7 mL of DMF was
added dropwise at 0 ˚C under argon atmosphere. After stirring for 24 h, DCurea was filtered off
and the solvent removed in vacuo. The residue was purified by preparative TLC (CHCl3 :
AcOEt/10:1) to obtain compound 12 as a white solid (0.99 g, 69%). 1H-NMR (CDCl3), ꢀ: 1.49 (m,
6H, CH2), 2.67 (t, 2H, CH2CO), 3.21 (m, 2H, CH2NH), 4.20 (m, 1H, Fmoc), 4.39 (d, 2H, CH2),
4.76 (br, 1H, NH), 6.97 (m, 1H, TFP), 7.34 (dd, 4H, Fmoc), 7.57 (d, 2H, Fmoc), 7.75 (d, 2H,
Fmoc). ESI-MS, m/z: 524 [M+Na]+, Found 524. M.p. 102 ˚C.
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Synthesis of Fmoc-6-aminohexanoate-c[R(Pbf)GD(tBu)fK] (13). c(R(Pbf)GD(tBu)fK) (230
mg, 0.252 mmol) was dissolved in 300 µL of DMF. After compound 12 (161 mg, 0.321
mmol) was added slowly, and then the solution was stirred at room temperature for 2 h under
argon atmosphere. After removing the solvent in vacuo, the residue was purified by preparative
TLC using CHCl3 : MeOH/10 : 1 as a solvent. Compound 13 was obtained as a white solid (206
mg, 66%). ESI-MS, m/z: 1269 [M+Na]+, Found 1269. M.p. 201-202 ˚C.
Synthesis of 6-aminohexanoate-c[R(Pbf)GD(tBu)fK] (14). Compound 13 (156 mg, 0.125
mmol) was dissolved in 5 mL of 20% piperidine/DMF and stirred at room temperature for 25 min.
After the solvent was removed in vacuo, the residue was dissolved in a small amount of MeOH.
An excess of Et2O was added to the solution and then the resulting precipitate was collected by
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filtration. The precipitate was washed with CH2Cl2 (5 mL) and hexane (5 mL) to obtain compound
14 as a white solid (118 mg, 92%). ESI-MS, m/z: 1025 [M+H]+, Found 1025. M.p. 208-209 ˚C.
Synthesis of Sal-Ahx-cR(Pbf)GD(tBu)fK (15). Compound 14 (13 mg, 12.7 µmol), compound
10 (5 mg, 19 µmol), 1-hydroxy-7-azabenzotriazole (HOAt, 10 mg, 73 µmol) was dissolved in
DMF (200 µL) and stirred. Then, DCC (6 mg, 29 µmol) dissolved in DMF (300 µL) was added
dropwise at 0 ˚C under argon atmosphere. After stirred for 48 h at room temperature, a resulting
DC urea was filtered off and the solvent was removed in vacuo. The residue was purified by
preparative TLC with CHCl3 : MeOH/10 : 1 as a solvent to obtained compound 15 as a yellow-red
solid (13 mg, 81 %). ESI-MS, m/z: 1286 [M+Na]+, Found 1286. M.p. 198-199˚C.
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Synthesis of Sal-Ahx-cRGDfK (16, RGD-Sal). Compound 15 (14 mg, 0.011 mmol) was
dissolved in 5 mL of TFA : H2O/9 : 1 solution. The solution was stirred for 3 h at room temperature.
After the solvent was removed in vacuo, the residue was dissolved in water (5 mL) and washed
with CHCl3 (3 × 5 mL). The aqueous layer was purified by preparative RP-HPLC (system 7) to
obtain compound 16 (8.0 mg, 84%). ESI-MS, m/z: 887 [M+Na]+, Found 887. HRMS, m/z:
865.42083 [M+H]+ calculated for C41H57N10O11, Found 865.42484 . M.p. 217 ˚C (dec).
Synthesis of Ga-(RGD-Sal)3TAMEol (17). Compound 16 (5.6 mg, 6.5 µmol) and
TAMEol/3HCl (266 µg, 1.1 µmol) was dissolved in 1 M ammonium acetate solution (400 µL) and
then the pH of the solution was adjusted to 8 with 25% aqueous ammonia (solution 1). GaCl3 (5
mg/mL) in 50 mM HCl aq was mixed with 1 M ammonium acetate solution and then the pH of
the solution was adjusted to 8 with 25% aqueous ammonia (solution 2, final GaCl3-concentration:
0.45 mg/mL). Solution 2 (440 µL) was added to solution 1 and stirred for 8 h at 70 ˚C while the
pH of the solution was maintained at 8 with 25% aqueous ammonia. The solution was purified by
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preparative RP-HPLC (system 9) to obtain compound 17 (2.5 mg, 83%). ESI-MS, m/z: 914
[M+3H]3+, Found 914.
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Synthesis of NOTGA-(tBu)3(Ahx-R(pbf)GD(tBu)fK)3. NOTGA(tBu)3 (12 mg, 17.5 µmol)
and TFP (28 mg, 169 µmol) were dissolved in CH2Cl2 (350 µL) and then DCC (17 mg, 82 µmol)
dissolved in CH2Cl2 (100 µL) was added to the solution at 0 ˚C under N2 atmosphere. After mixing
for 12 h, the white precipitates were removed by filtration. The solvent was evaporated and then
the residue was dissolved in DMF (400 µL). After compound 14 (54 mg, 52.7 µmol) was added
to the solution, the solution was mixed for 24 h. The white precipitate was obtained after excess
of Et2O was added to the solution. The white precipitate was purified by preparative TLC (CHCl3 :
MeOH/5 : 1) to obtain NOTGA-(tBu)3(Ahx-R(pbf)GD(tBu)fK)3 (46.6 mg, 72%). ESI-MS, m/z:
1877 [M+2Na]2+, Found 1877.
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Synthesis of NOTGA(RGD)3 (18). NOTGA-(tBu)3(Ahx-R(pbf)GD(tBu)fK)3 (46 mg, 12.4
µmol) were dissolved in 5 mL of TFA : triethylsilane : H2O/9.5 : 0.25 : 0.25 and then stirred at
room temperature for 16 h. After removing the solvent in vacuo, the residue was dissolved in
MeOH : H2O/3 : 7 (10 mL) and then washed by CHCl3 (3 × 10 mL). Aqueous phase was purified
with preparative RP-HPLC (system 7) to obtain compound 18 (15.3 mg, 47.1%). ESI-MS, m/z:
1308 [M+2H]2+, Found 1308.
Synthesis of Ga-NOTGA(RGD)3 (19). The solution of Ga(NO3)3 (0.1 M, 26 µL, H2O) was
added to the solution of compound 18 (5 mg, 1.91 µmol, 800 µL, H2O) and then mixed at 70 ˚C.
After 20 min, sodium acetate (0.1 M, 20 µL) was added to adjust pH 4 and then the mixture stirred
for another 1 h at 70 ˚C. The solution was purified by preparative RP-HPLC (system 8) to obtain
compound 20 (3.85 mg, 75%). ESI-MS, m/z: 895 [M+3H]3+, Found 895.
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Preparation of [67Ga]Ga-(Hdpp)3. [67Ga]Ga-citrate (74 MBq/mL) was diluted 10 times with
saline. The solution of Hdpp (50 µL, 10 mM, saline) was mixed with the diluted [67Ga]Ga-citrate
(50 µL) and heated at 80 ˚C for 30 min. The reaction solution was analyzed by TLC (MeOH :
water/1 : 1 with or without 10 mM Hdpp), CAE, and RP-HPLC (system 3).
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Preparation of [67Ga]Ga-(Sal)3TAMEol. [67Ga]Ga-citrate (74 MBq/mL) was diluted 10 times
with water. The diluted [67Ga]Ga-citrate (20 µL) was added to an ethanolic solution of compound
2 (0.5 mg, 1.12 µmol, 1.12 mL) and then heated at 80 ˚C for 15 min. The reaction solution was
analyzed by RP-HPLC (system 4).
Preparation of [67Ga]Ga-(PTSC-1)2 and [67Ga]Ga-(PTSC-2)2. [67Ga]GaCl3 (5 µL, 37
MBq/mL) was mixed with 0.1 M ammonium acetate (pH 4.5, 30 µL) and left for 5 min. A 10 µL
of this solution was mixed with PTSC derivatives (1.1 mM, 90 µL) dissolved in a mixture of EtOH
and 0.1 M ammonium acetate pH 4.5 (1:8), and the reaction mixture was heated at 80 ˚C for 15
min. RP-HPLC analysis was conducted with the system 5 or 6.
Preparation of [67Ga]Ga-(RGD-Sal)3TAMEol. Compound 16 (3.75 × 10-4 M) and
TAMEol/3HCl (1.07 × 10-4 M) were dissolved in ammonium acetate (1.0 M, pH 8, 30 µL).
[67Ga]Ga-citrate (74 MBq/mL) was diluted 10 times with ammonium acetate (2.0 M, pH 8) and
the diluted [67Ga]Ga-citrate (15 µL) was added to the above mixture and incubated at 70 ˚C for 1
h (final concentration of compound 16: 2.5 × 10-4 M). For SPECT/CT studies, [67Ga]GaCl3 was
used. [67Ga]GaCl3 was mixed with the ammonium acetate (1.0 M, pH 7) to prepare [67Ga]Ga-
acetate (7.4 MBq/mL). [67Ga]Ga-acetate (15 µL) was added to the above mixture (30 µL) and then
incubated at 70˚C for 1 h. The radiochemical yield and purity were determined by TLC
(ammonium acetate : MeOH/1 : 1) and RP-HPLC (systems 1 or 2). Furthermore, RP-HPLC
analysis of the labeling solution using UV detector was also performed.
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Preparation of [67Ga]Ga-NOTGA(RGD)3. Compound 18 (2.0 × 10-4 M) was dissolved in
ammonium acetate (1.0 M, pH 5, 15 µL). [67Ga]Ga-acetate (7.4 MBq/mL) prepared by the above
described method was added to the solution (15 µL) and then incubated at 25 ˚C for 20 min. The
radiochemical yield and purity were determined by RP-HPLC (systems 1 or 2). Furthermore, RP-
HPLC analysis of the labeling solution using UV detector was also performed.
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Cell Line. U87MG glioma cell was purchased from the American Type Culture Collection
(ATCC, Manassas, VA). These cells were cultured according to protocols prescribed by the ATCC.
Binding Affinity to Integrin αvβ3. This study was performed according to the procedures
described in our previous studies.8, 9 The half-maximal inhibitory concentration (IC50) was
determined in competition against [125I]I-c(RGDyV).18 Multiscreen DV filter plates (Merck
Millipore, MA) were seeded with 2 × 105 cells in the binding buffer (20 mM Tris, 150 mM NaCl,
2 mM CaCl2, 1 mM MgCl2, 1 mM MnCl2, 0.1% BSA, pH 7.4), and then [125I]I-c(RGDyV) in the
presence of c(RGDyV),18 RGD-Sal, Ga-(RGD-Sal)3TAMEol, NOTGA(RGD)3 or Ga-
NOTGA(RGD)3 were added. The total incubation volume was adjusted to 200 µL. After
incubation for 1 h at 37 ˚C, the plates were filtered and washed twice with 200 µL of the ice-cold
binding buffer. The polyvinylidene fluoride filters were collected and the radioactivity was
determined using an autowell γ-counter. The IC50 values were calculated by fitting the data by
nonlinear regression using GraphPad Prism (GraphPad Software, Inc., San Diego, CA). All the
binding experiments were carried out with quadruplet samples.
Determination of Log D7.4 Values. [67Ga]Ga-(RGD-Sal)3TAMEol and [67Ga]Ga-
NOTGA(RGD)3 were purified by RP-HPLC (system 1), and the eluents of the two compounds
were evaporated in vacuo. Then, the residues were dissolved in 25 mM phosphate buffer (pH 7.4)
to prepare 3.7 MBq/mL of sample solutions. Ten µL (37 kBq) of each sample solution was added
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to an equal volume mixture (3 mL : 3 mL) of n-octanol and 25 mM phosphate buffer (pH 7.4)
preincubated for 24 h to reach equilibrium. The mixtures were vortexed for 1 min, followed by
being left to stand for 1 min. This procedure was repeated 3 times. The mixtures were then
centrifuged at 2000 rpm for 10 min. The samples for both n-octanol and aqueous layers were taken
and counted in an auto well γ-counter. The log D7.4 values were reported as mean ± SD of 3
experiments.
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Stability Study. Ga-labeled complexes were purified by RP-HPLC to remove unlabeled
ligands. The radioactive peak was collected and the solvent was removed in vacuo. The residue
was reconstituted in carbonate buffer (0.1 M, pH 7.4). To 200 µL of this solution (74 MBq), a
solution of apo-transferrin (2 mg/mL, 300 µL, 0.1 M carbonate buffer pH 7.4) was added and
incubated at 37 ˚C. Aliquots of samples were collected after being incubated for 1, 3 and 6 h, and
the radioactivity was analyzed by TLC (ammonium acetate : MeOH/1 : 1).
Animal Model. Animal studies were conducted in accordance with the institutional guidelines
approved by the Chiba University Animal Care Committee. Five-week-old male BALB/c nu/nu
mice (body weight: ca. 22 g, Japan SLC, Inc., Hamamatsu, Japan) were xenografted by
subcutaneous injection of U87MG human glioblastoma cells (5 × 106 cells/50 µL of culture
medium) into their right hind legs. The mice were subjected to biodistibution and SPECT/CT
imaging studies when the tumor weight reached 0.1−0.5 g (ca. 3-4 weeks after xenograft).
Biodistribution study. Male nude mice bearing U87MG tumor xenografts were injected via the
tail vein with 100 µL (11.1 kBq) of [67Ga]Ga-(RGD-Sal)3TAMEol in the presence of RGD-Sal
(10 nmol) as the trivalent “1 to 3” design. For comparison, [67Ga]Ga-NOTGA(RGD)3 in the
presence of NOTGA(RGD)3 (10 nmol) was administered to the mice. Moreover, RP-HPLC-
purified [67Ga]Ga-(RGD-Sal)3TAMEol or [67Ga]Ga-NOTGA(RGD)3 were also evaluated using
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similar methods. The animals were sacrificed and dissected at 1 h after administration. The tissues
of interest were excised and weighed, and the radioactivity was determined with an auto-well γ-
counter. The results are presented as the percentage injected dose per gram (%ID/g) or percentage
injected dose per tissues (%ID). Values were expressed as mean ± SD for a group of 4-5 mice.
Urine Analysis. Six hours after the injection of [67Ga]Ga-(RGD-Sal)3TAMEol (74 kBq) into 6-
week-old ddy-mice,27 urine samples were collected. For RP-HPLC analysis of the urine sample,
200 µL of acetonitrile was added to 200 µL of urine to precipitate the proteins. The supernatants
were filtered through a polycarbonate membrane (0.45 µm) before being analyzed by RP-HPLC
(system 2).
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Small Animal SPECT/CT Imaging Studies. SPECT images were taken 45 min after
administration of [67Ga]Ga-(RGD-Sal)3TAMEol (100 µL, 1.8 MBq, RGD-sal=10 nmol) or
[67Ga]Ga-NOTGA(RGD)3 (100 µL, 2.2 MBq, NOTGA(RGD)3=10 nmol) to male BALB/c nu/nu
mice bearing U87MG xenografts from the tail vein. CT scans were performed before SPECT scans
for anatomic reference. The mice were anaesthetized with 1−1.5% (v/v) isoflurane (DS Pharma
Animal Health, Osaka, Japan) and positioned on the animal bed where anesthesia was continuously
delivered via a nose cone system. SPECT imaging and X-ray CT imaging were performed by small
animal SPECT/CT system (Triumph LabSPECT4/CT, TriFoil Imaging Inc., Chatsworth, CA)
equipped with a five pinhole (1.0 mm) collimator. Data acquisition was performed for 34 min at
120 s per projection with stepwise rotation of 16 projections over 360 ˚. All SPECT data were
reconstructed by a 3D-ordered subset expectation maximization (3D-OSEM) algorithm method
with 2 subset and 5 iterations in FLEX-RECON software (TriFoil Imaging Inc.).
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Statistical Analysis. Data are expressed as the means ± SD where appropriate. Results were
statistically analyzed using the unpaired Student’s t-test or Tukey’s multiple-comparison test.
Differences were considered statistically significant when p was <0.05.
ASSOCIATED CONTENTS
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Supporting Information
HPLC methods, RP-HPLC radio- and UV-chromatograms of [67Ga]Ga-NOTGA(RGD)3, In
vitro inhibition curves of [125I]I-(RGDyV) bound to U87MG glioma cells, 1H-NMR spectrum of
TAMEol in D2O, synthesis of PTSC derivatives.
AUTHOR INFORMATION
Corresponding author
Phone: +81-43-226-2898; Fax: +81-43-226-2897; E-mail: tuehara@chiba-u.jp.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGEMENTS
We thank Dr. Noriyuki Suzuki for ICP-MS analysis. This work was supported in part by a Grant-
in-Aid for Young Scientists (A) (No. 25713045) (to T.U.) from the Ministry of Education, Culture,
Sports, Science and Technology of Japan and the Development of Systems and Technology for
Advanced Measurement and Analysis Program (Development of SPECT probe for molecular
imaging) from Japan Science and Technology Agency, JST and JSPS Research Fellows Program
(No. 264896) (to Y.A.).
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