Development of Albumin-bound closoDodecaborate and its Promising Boron
Delivery Efficacy to Tumor
Daisuke Kanoh,1 Shoji Tachikawa,1 Shinichi Sato,2
Hiroyuki Nakamura*,2
1) Department of Chemistry, Faculty of Science,
Gakushuin University, Japan
2) Synthetic Organic Division, Chemical Resources Laboratory
Tokyo Institute of Technology, Japan
Requirements for Efficient BNCT
>25 ppm 10B in Tumor, T/B and T/N >2.5
In the clinical study: L-BPA (500 mg/kg) 1 hr before irradiation
⇒ Total dose: 30 g /person (60 kg body weight)
Large amount administration is required! ⇒ Significantly low toxic
Different approach is needed
in comparison with conventional antitumor drug design
How can we deliver a large amount of 10B to tumor?
Use of Nanocarriers
(Micelles, Liposomes…)
Nano Carrier Drug Delivery System (DDS)
-Enhanced permeability and retention (EPR) effect
→ particle size of 100 nm
- Escape from Reticulo-endothelial-system (RES)
→ PEG-conjugation
Liposome
Tumor tissue
Abnormal
architectures
without tight
junction
Tumor
cells
Tumor blood vessel
Liposomal Boron Agents for BNCT
Boron lipids
Na
H
O
H
O
O
10B
Total lipid dose
743 mg/kg
Total lipid dose
147 mg/kg
Ligand
2013
H. Nakamura, et al
Ligand
10
2009Ligand
High Boron Content Liposome
PEG
B
K, Tomizawa, H. Matsui et al
2004Anti-EGFR-antibody-Liposome
H. Nakamura, et al
2004Boron Lipid Liposome
O
O
Ligand
K. Maruyama, et al
S
10B
O
O
2003TF-PEG-Liposome
O
2
E. B. Kullberg, et al
O
2002EGF-PEG-Liposome
R. J. Lee, et al
DSBL
1992FA-PEG-Liposome
M. F. Hawthorne, et al
Boronated DSPC Liposome
1991H. Yanagie, et al
Boron conc. in tumor
100 nm
Boronated anti-CEA
immunoliposome
143 ppm (50 mgB/kg)
82 ppm (30 mgB/kg)
30 ppm (15 mgB/kg)
H. Koganei, et. al., Bioconjugate Chem. 2013, 24, 124.
Biopolymer-Based Boron Delivery
Antibody-conjugates
BSD
MoAb IB16-6
(B16-BL6 melanoma)
4.8 x 104 ~ 2.5 x 105 / cell
~1100 boron atoms/BSD
BSD
Cetuximab
F98 glioma-bearing rats
R. F. Barth, et al, Bioconjugate Chem. 1994, 5, 58-66
Growth factor-conjugates
EGF (53 aminoacid residues)
BSD
BNCT effect
BSD-EGF
R. F. Barth, et al, Bioconjugate Chem. 2004, 15, 185-194
5
Rolf. F. Barth, et al, Cancer Res. 2002, 62, 3159.
Biopolymer-Based Boron Delivery
12,000
17,000
68,000 (8 nm)
29,000
150,000 (15 nm)
EPR Effect was observed in the
proteins with the MW over 68,000
blood
tumor
Accumulation of Evans blue-stained
albumin complex in tumor
skin
6
Albumin as a Biopolymer DDS Vehicle
Albumin, a major plasma protein constituent, is composed approximately 55% of the
human plasma protein, and has been extensively investigated as a versatile carrier for
therapeutic and diagnostic agents.
Abraxane® (an albumin-paclitaxel nanoparticle)
-
FDA approval in 2005
Treatment of Breast cancer,
non-small cell lung cancer,
stomach cancer,
and adenocarcinoma of the pancreas
Structure of Abraxane
A 130 nm albumin-bound particle form of paclitaxel
Uptake of abraxane is presumably mediated by
the EPR effect and the gp60 transcytosis.
F. Kratz, B. Elsadek, J. Control. Release 161 (2012) 429–445
Maleimide Doxorubicin Conjugate in situ Binding to Albumin
+
Chemical structure of INNO-206,
the (6-maleimidocaproyl)hydrazone derivative of doxorubicin
Tumor targeting based on in situ binding to the
circulating albumin after intravenous injection.
C. Unger, B. Haring, M. Medinger, J. Drevs, S. Steinbild, F. Kratz, K.
Mross, Clin. Cancer Res. 13 (16) (2007) 4858.
8
Design of Maleimide Boron Cluster Conjugates
2
-
O
H
O
N
O
N
O
O
2
N
a
BSA
Maleimide-closo-dodecaborate conjugate（B12H12, MID）
(bovine serum albumin)
Easy to synthesize and low toxic
(COCl)2
68%
F(ab’)2
O
2-
O
N
H
O
O
O
O
N
H
N
O
2 Et3NH
antibody
Maleimide-decaborate conjugate（B10H10）
D.S. Wilbur, M. F. Hawthorne, Bioconjugate Chem. 2007, 18, 1226−1240
9
Synthesis of Maleimide-closo-Dodecaborate Conjugate (MID)
-
2-
O
2-
O
N
a
B
F
,
H
C
l
4
O
N3
O
T
B
A
N
P
h
P
,
H
O
3
3
2
1
,
4
d
i
o
x
a
n
e
C
H
C
l
T
H
F
2
2
2 TBA
TBA
1
2 TBA
87%
2 steps
2
3
quant
V. I. Bregadze, Appl.Organometal. Chem., 2007, 21, 98-100
2O
O
O
H
O
N
O
N
4
M
a
l
e
i
m
i
d
o
b
u
t
y
r
i
c
A
c
i
d
,
B
O
P
,
E
t
N
3
2-
NH2
O
T
H
F
2 TBA
4
2 TBA
77%
O
5 (MID-TBA)
O
H
O
N
O
N
1
)
T
M
A
C
l
/
E
t
O
H
O

2
)
N
a
A
m
b
e
r
l
i
t
e
/
H
O
2
O
2-
2 Na
89%
2 steps
MID
10
Cytotoxicity of closo-Dodecaborates
MTT assay (CT26 and B16 cell、72h)
IC50
(mM)
2SH
>1
BSH
2 Na
2O
2 Na
>1
4
2O
NH2
O
O
H
N
O
N
O
2 Na
O
>1
MID
MID is low toxic!
11
Reaction of MID with Cysteines
2O
O
O
H
N
N
O
+
HS
OH
RHN
O
2 TMA
O
7a: R = H
7b: R = Ac
6 (MID-TMA)
HO
2O
N
S
NHR
O
PBS (pH 7.4)
O
2 TMA
MID-2TMA
ESI-negative MS
m/z 205.1
O
O
O
H
N
8a: R = H
8b: R = Ac
The reaction process was monitored by ESI-TOF MS.
The reaction proceeded quantitatively in an hour at rt.
8a
m/z 265.6
8b
m/z 286.4
12
Conjugation of MID to BSA in vitro
antibody*
BSH was detected by the anti-BSH
antibody in Western blot analysis.
The intensity of BSH-conjugated BSA
bands increased until the mixture of an
1:1 ratio and then became plateau.
* anti-BSH antibody was kindly donated by Prof. Kirihata (Osaka Prefecture University)
13
Structure of BSA
Cys34
Total number of Cys: 33
14
Analysis MID-BSA Conjugation by MALDI-TOF-MS
BSA was digested with trypsin and the peptide fragments were analyzed by MALDI-TOF-MS.
2-
The peptide including Cys34
O
O
O
H
N
N
O
2 TMA
O
MID: MW 410
+410
MID : BSA = 100 : 1
MID : BSA = 10 : 1
MID : BSA = 1 : 1
BSA only
MW 2,835(+48)
MW 2,845
We could detect the conjugation of MID and the
peptide fragment including Cys34 in BSA.
15
Biodistribution of MID-BSA Conjugates in tumor-bearing mice
• Tumor boron conc. reached 60 ppm in the mice treated with MID-BSA conjugates
(10:1, 30 mgB/kg) 12 hours after injection from tail vein.
• Boron concentration in other organs was low compared to boron liposomes.
Tumor
60 ppm
38 ppm
CT26-bearing mice (Balb/c/♀; 5 weeks) i.v.
16
Biodistribution of BSH-Boron Lipid Liposomes
BSH-Liposome
180
BSH 3000ppm
Tumor
10%DSBL 3000ppm
160
10%DSBL 5000ppm
140
Boron (ppm)
120
100
B/P = 2.7
80
10% DSBL
60
30 and 50 mg B/kg
40
20
24
500
Boron (ppm)
Blood
400
36
80
BSH 3000ppm
10%DSBL 3000ppm
10%DSBL 5000ppm 60
time (h)
48
Kidney
72
BSH 3000ppm
10%DSBL 3000ppm
10%DSBL 5000ppm
250
Spleen
200
BSH 3000ppm
10%DSBL 3000ppm
10%DSBL 5000ppm
150
300
40
100
200
20
100
0
50
0
24
36time (h)48
72
0
24
36 time (h) 48
72
24
36 time (h) 48
H. Koganei, M. Ueno, S. Tachikawa, L. Tasaki, H. S. Ban, M. Suzuki, K. Shiraishi, K. Kawano, M.
Yokoyama, Y. Maitani, K. Ono, H. Nakamura, Bioconjugate Chem. 2013, 24, 124.
72
Biodistribution of MID-BSA Conjugates in tumor-bearing mice
• Tumor boron conc. reached 60 ppm in the mice treated with MID-BSA conjugates
(10:1, 30 mgB/kg) 12 hours after injection from tail vein.
• Boron concentration in other organs was low compared to boron liposomes.
• EPR effect was also observed even in the case of the injection of MID itself, suggesting
that the conjugation of MID to serum albumin in circulating blood.
Tumor
60 ppm
38 ppm
CT26-bearing mice (Balb/c/♀; 5 weeks) i.v.
18
MID + blood
Control
(blood only)
MID + blood
Control
(blood only)
Conjugation of MID to Serum Albumin in Blood
~60 kDa
(serum albumin)
MID was treated with the blood of mice for 1 h at 37oC and the MID-conjugated
protein was determined by Western blotting analysis using anti-BSH antibody.
The higher accumulation of the MID-BSA (10：1) conjugate is probably due
to in situ binding of MID to circulating albumin after intravenous injection.
19
BNCT Effect of Mice Injected with MID-BSA (10:1) Conjugates
1400
MID(10:1) 3000
ppm
30 mg[B]/kg
Tumor volume (mm3)
1200
MID(10:1) 1500
ppm
15 mg[B]/kg
1000
MID(10:1) 750
7.5ppm
mg[B]/kg
Hot Control
800
600
thermal
neutron
400
200
0
0
3
6
10
14
17
Neutron flux 1.5-2.2 x 1012 n/cm2
Kyoto University Reactor (KUR)
Days after Irradiation
Tumor growth of mice injected with MID-BSA conjugates at a dose range of 7.5-30
mg[B]/kg was suppressed after BNCT, and the tumor of some mice completely
disappeared 14 days after neutron irradiation.
Collaborated with Prof. M. Suzuki’s group at KUR
20
Conclusion
1. We succeeded in the synthesis of maleimide closo-dodecaborate (MID) for
conjugation of boron sources to biopolymers.
2. MID is low toxic.
3. MID readily reacted with cysteines including Cys34 in BSA.
4. MID also reacted with serum albumin in blood of mice.
5. Tumor boron conc. reached 60 ppm in the mice treated with MID-BSA (30 mg[B]/kg)
conjugates 12 hours after injection.
6. Tumor growth of mice injected with MID-BSA conjugates was suppressed after BNCT.
Acknowledgment
We thank Professor Mitsunori Kirihata (Osaka Prefecture University) for kindly
donating anti-BSH antibody and Stella Pharma Co. Ltd. for the supply of 10B12H12.
We also thank Professors Suzuki, Kondo, Sakurai, and Tanaka for their kind
supports to proceed thermal neutron irradiation experiments at KUR.
21
Thank you very much for
your kind attention!
7F
9F