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Article 67
Coordination-Mediated Synthesis of Ga-Labeled PurificationFree 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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Bioconjugate Chemistry
Coordination-Mediated Synthesis of 67Ga-Labeled Purification-Free Trivalent Probes for In Vivo Imaging of Saturable Systems 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 HigashiTamagawagakuen, Machida, Tokyo 194-8543, Japan.
Keywords: gallium-67, SPECT, PET, schiff base
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ABSTRACT
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 ligands. The reaction of a bi- or tri-dentate ligand with [67Ga]Ga-citrate resulted in 67Ga-labeled probes of insufficient stability. However, the reaction of [67Ga]Ga-citrate with a mixture of RGDconjugated salicylaldehyde and triamine provided a
67
Ga-labeled trivalent probe with stability
sufficient for in vivo applications. Since the free Schiff base ligand decomposed rapidly upon injection, the
67
Ga-labeled trivalent probe visualized murine tumor without post-labeling
purification, which was not achieved with a 67Ga-labeled trivalent probe from a trivalent ligand. These findings indicate the availability of Schiff base ligands to prepare
67
Ga-labeled trivalent
probes by a simple radiolabeling procedure.
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Bioconjugate Chemistry
INTRODUCTION 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. To circumvent the problem, we recently developed a new design concept of metal coordinationmediated synthesis of 99mTc-labeled trivalent probes using [99mTc][Tc(CO)3(OH2)3]+ and isonitrileconjugated
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 (
