7. Neutron Capture Therapy

概要

CO7-1

Development of Alubmin Binding Gadolinium Complexes for MRI-Guided BNCT

S. Okada, K. Nishimura1, Q. Ainaya1, I. B. Sivaev2, K.
Miura, T. Takada3, M. Suzuki3, H. Nakamura
Laboratory for Chemistry and Life Science, Institute of
Innovative Research, Tokyo Institute of Technology
1
School of Life Science and Technology, Tokyo Institute of
Technology
2
Russian Academy of Sciences, Russia
3
Institute for Integrated Radiation and Nuclear Science,
Kyoto University
INTRODUCTION: Boron Neutron Capture Therapy
(BNCT) is a promising cancer treatment of harsh and
un-operatable malignant tumors. In order to obtain effective BNCT treatment of patients, it is important to control
the biodistribution of the boron-containing drug and its
accumulation in tumors. The conventional method to
estimate biodistribution is positron emission tomography
(PET) based on drugs labeled by radioactive isotopes[1],
although 10B isotope is non-radioactive.
We focused on Gd contrast agents for magnetic resonance imaging (MRI), which is a non-invasive method
and one of the most widely used medical diagnostics. The
157
Gd isotope has the highest thermal neutron capture
cross section of all stable nuclides in the periodic table.
The thermal neutron capture cross section of the 157Gd
isotope exceeds that of the 10B isotope by more than 60
times. The neutron capture reaction by 157Gd causes
complex inner shell transitions that produce prompt
γ-emission displacing an inner-core electron, resulting in
internal-conversion electron emission, and finally in the
Auger electron emission, together with soft X-ray and
photon emission. Therefore, the synthesis of compounds
containing both boron and gadolinium can be useful not
only to estimate biodistribution of boron drugs under
MRI guide but also to develop efficient neutron capture
cancer therapy. We have developed maleimide-functionalized closo-dodecaborate (MID) albumin
conjugates that demonstrated high and selective accumulation in tumor tissue with no toxicity in the absence of
thermal neutrons, hence a promising boron delivery system [2].
In this study, we synthesized Gd complexes that functionalized MID albumin conjugates aiming MRI-guided
BNCT.
EXPERIMENTS: To a solution of bovine serum albumin (BSA) in 10 mM HEPES buffer (pH 7.4) was
added Gd-complex ligand. The reaction solution was
shaken at 800 rpm for 12 h at room temperature. The
reaction solution was subjected to six cycles of ultracentrifugation with a 30 kDa filter to remove excess ligand
before the addition of MID. The reaction solution was
shaken for another 12 h at 37oC. The final BSA-Gd-MID
conjugate was obtained after filtration of excess MID via
ultracentrifugation. The concentrations of 10B and Gd per
BSA were estimated by using ICP-OES. The conjugate
solution was diluted with PBS for the biodistribution

study.
CT26 tumor bearing mice (Balb/cCrSlc nu/nu female,
5−6 weeks old, 16−20 g) were injected via the tail vein
with 200 μL of MID-BSA or Gd-MID-BSA (7.5 mg
[10B]/kg). The plasma, liver, lung, kidney, spleen, muscle,
brain, and tumor were extracted 6, 12, and 24 hours after
the injection and proceeded to ashing process, followed
by quantification of B and Gd via ICP-OES.
RESULTS: Previously, we demonstrated that tumor
accumulation of MID-BSA was the largest 12 h after the
injection[3]. Therefore, we compared the B and Gd concentration in the organs at 6, 12, 24 hours after injection.
The results are shown in Fig. 1. The B concentration of
Gd-MID-BSA in tumor was comparable to that of
MID-BSA in all the time points, although the Gd concentration of Gd-MID-BSA was the largest in liver, followed
in order by spleen and tumor. Therefore, the concentration
of B was not consistent with that of Gd, indicating the
release of Gd from Gd-MID-BSA due to non-specific
binding of free Gd to BSA. These results indicate it is
necessary to synthesize more stable Gd complexes and
remove free Gd ions in reaction solution thoroughly to
proceed thermal neutron irradiation experiment.

Fig. 1. Biodistribution of boron and gadolinium in colon
26 (CT26) tumor bearing mice. Gd-MID-BSA was intravenously injected at a dose of 7.5 mg [10B]/kg via the tail
vein, and the organs were extracted 6, 12, and 24 hours
after injection. The concentration of B and Gd in organs
was quantified using ICP-OES after ashing.
REFERENCES:
[1] V. Tolmachev et al., Bioconjugate Chem., 10 (1999)
338-345.
[2] K. Kawai et al., Mol. Pharm., 17 (2020) 3740–3747.
[3] S. Kikuchi et al., J. Control. Release, 237 (2016).

R4017
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Iodophenyl-Conjugated closo-Dodecaborate as a Promising Small Boron Agent for BNCT

(A) BC-IP (15 μgB/g)
Boron concentration [μgB/g]

INTRODUCTION: Boron Neutron Capture Therapy
(BNCT) is attracting attention as a non-invasive radiotherapy in the treatment of cancer. The efficiency of boron agent depends highly on tumor selectivity, sufficient
amount of boron agent in tumor site, non-toxicity, tumor/normal tissues ratio (>3) and absorption of thermal
neutrons by boron. In March 2020, accelerator-based
BNCT
for
head
and
neck
cancer
using
4-borono-L-phenylalanine (L-BPA) was approved by the
Pharmaceuticals and Medical Devices Agency in Japan,
making BNCT more accessible treatment [1]. L-BPA is
known to actively accumulate into tumor cells thorough
L-type amino acid transporter 1 (LAT-1). However, there
are still many patients for whom L-BPA is not applicable.
Therefore, the development of novel boron carriers applicable to various cancers including BPA-negative tumors is required for further expansion of BNCT.
We recently developed maleimide-functionalized closo-dodecaborate sodium form (MID), which conjugates
not only the free SH group of a cysteine residue (Cys34)
but also several lysine residues in serum albumin under
physiological conditions [2,3]. The MID-conjugated albumin selectively accumulated in tumors due to the enhanced permeability and retention effect and significantly
inhibited tumor growth in colon 26 tumor-bearing mice
subjected to thermal neutron irradiation [2].
In this study, we focused on small molecule albumin
ligands that bind noncovalently to albumin.
4-Iodophenylbutanamide (IP) was chosen as the albumin
ligand and conjugated with closo-dodecaborate to
demonstrate in vivo biodistribution.
EXPERIMENTS:
Boron-conjugated
4-iodophenylbutanamide (BC-IP) was designed and synthesized from closo-dodecaborate. Tumor-bearing mice
(female, 5-6 weeks old) were prepared by injecting subcutaneously (s.c.) a suspension of U87MG human glioblastoma cells in PBS. The mice were kept on a regular
chow diet and water for a week. The tumor-bearing mice
were injected i.v. with 200 μL of PC-IP or MID dissolved
in ultrapure (Milli-Q) water at the final dose of 15µgB/g.
At 3, 12, and 24 h after injection, the mice were lightly
anesthetized and blood samples were collected from heart.
The mice were then sacrificed by cervical dislocation and
dissected. Liver, spleen, kidney, brain, and tumor were
excised, washed with saline, and weighted. Each tissue
was digested with 1 mL of HNO₃ at 90 ℃ for 3 h, and
then the digested samples were diluted with distilled wa-

20
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School of Life Science and Technology, Tokyo Institute of
Technology
1
Laboratory for Chemistry and Life Science, Institute of
Innovative Research, Tokyo Institute of Technology
2
Institute for Integrated Radiation and Nuclear Science,
Kyoto University

ter. After filtering through a membrane filter (0.5 µmφ,
13JP050AN, ADVANTEC, Japan), boron concentrations
were measured by ICP-OES. All protocols for in vivo
studies involving the use of mice were approved by the
Institutional Animal Care and Use Committee of Tokyo
Institute of Technology.
RESULTS: The boron concentration in the tumor
showed the highest value of 11 μgB/g at 3 h and gradually decreased to 2.4 and 2.3 μgB/g at 12 and 24 h respectively (Fig. 1A). In contrast, boron concentrations in other tissues remained below 5.0 μgB/g at all time periods,
demonstrating the highest BNCT effect can be achieved
with neutron irradiation at 3 h post injection. For comparison, we investigated the biodistribution of MID at 12
h post injection, the time point at which MID exhibited
the highest accumulation in tumors. MID tended to accumulate in plasma, followed by lung, tumor, and kidney
(Fig. 4B). The boron concentration in tumor was about 5
μgB/g and thus much lower than was observed with
BC-IP. These results suggest that IP, a non-covalent albumin ligand, can enhance tumor accumulation over the
maleimide, covalent ligand. Therefore, BC-IP has a potential to act as a more efficient boron carrier than MID
in BNCT.

■3h

■ 12 h ■ 24 h

(B) MID (15 μgB/g)
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K. Nishimura, K. Kawai, T. Morita1, S. Okada1, K.
Miura1, T. Takada,2 M. Suzuki2, H. Nakamura1

Boron concentration [μgB/g]

CO7-2

■ 12 h

Fig. 1. Biodistribution of (A) BC-IP and (B) MID in
U87MG tumor mouse models with i.v. injection. Data are
expressed as mean ± SD (n = 3).
REFERENCES:
[1] H. Kanno et al., The Oncologist, 26 (2021)
e1250-e1255.
[2] S. Kikuchi et al., J. Control. Release, 237 (2016)
160-167.
[3] K. Kawai et al., Mol. Pharm, 17 (2020) 3740-3747.

R4026
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CO7-3

Effects of overexpression of LAT1 in cancer stem cell-like cells on suppression of
tumor growth by boron neutron capture therapy

K. Ohnishi1, T. Tani2 and M. Suzuki3
Department of 1Biology, Ibaraki Prefectural University of
Health Sciences
2
QST Hospital, National Institutes for Quantum and Radiological Science and Technology
3
Institute for Integrated Radiation and Nuclear Science,
Kyoto University
INTRODUCTION: Outcome from BNCT largely depends on amount of intracellular accumulation of boron
compound. ...