Examination of the usefulness as
the new boron compound of ACBC-BSH
2014
Osaka Medical College, Neurosurgery
Gen Futamura1 Shinji Kawabata1 Shin-Ichi Miyatake1 Toshihiko Kuroiwa1
Yoshihide Hattori2 Mitsunori Kirihata2
Hiroki Tanaka3 Yoshinori Sakurai3 Shinichiro Masunaga3 Koji Ono3
1Department
of Neurosurgery, Osaka Medical College
2Osaka Prefecture University
3Kyoto university research reactor institute
16th International Congress on Neutron Capture Therapy
June 16, 2014 Helsinki, Finland
Introduction
What is ACBC ?
2014
Osaka Medical College, Neurosurgery
1)1-amino-3-fluorocyclobutane-1-carboxylic acid (ACBC), unnatural
amino acid was reported as the agent which showed intense uptake in
glioblastoma1)-4)
*ACBC has high affinity to amino acid transporter.
*ACBC is not metabolized in cells.
*ACBC is not used for protein synthesis.
1)Washburn LC, et al, Cancer Res 1978;38:2271-2273
2)Washburn LC, et al, J Nucl Med 1979;20:1051-1055
3)Goodman MM, et al, J Labelled Compd Radiopharm 1994
4)Hubner K, et al, Mosby-Year Book, Inc 1992-10
Introduction
2) In a clinical study, [18F]-ACBC showed usefulness as a tracer
of PET imaging 5)
2014
Osaka Medical College, Neurosurgery
MRI(Gd+）
18F-ACBC
PET
FDG PET
T/N ratio 5.0
5) Shoup, et al, J NucI Med 1999; 40: 331
This PET imaging becomes useful as well as F-BPA
PET imaging in clinical BNCT
Introduction
We conjugate BSH to ACBC for further BNCT experiments.
2014
Osaka Medical College, Neurosurgery
BSH
ACBC
+
ACBC-BSH
2
N H










S
C O 2H

2N a
+
2
Introduction
ACBC-BSH
2-
BPA
H2N
CO2H

Osaka Medical College, Neurosurgery
N H










S
C O

2N a
2
2
H
+
Molecular weighht:439.75
HO
10
B
OH
Molecular weighht:208
*ACBC-BSH is high molecular weight and low electrical charge, so
it is difficult for this agent to reach regions that tumor cells invade
microscopically where the BBB seems to be intact.
2014
*We have adopted Convection Enhanced Delivery(CED) for
drug administration to solve this problem 6)-9)
6)Kawabata, et al, J Neurooncol 2011 June 1
7)Miyata et al, Neurosurgery 2011 68:1380-1387
8)Hiramatsu et al, Lasers Surg Med. 2011 January
9)Saito et al, Cancer Res 2004; 64(7):2572-2579
Introduction
Convection Enhanced Delivery (CED)
*CED is one of the methods for local drug infusion into the brain.
Osaka Medical College, Neurosurgery
*Continuous slow infusion into a local site of the brain under a small constant
positive pressure makes a ‘bulk flow’ in the interstitial space.
*Bulk flow enables to distribute high molecular weight drugs to a large area in
interstitial space , keeping high concentration without through the disrupted BBB.
CED enables to distribute ACBC-BSH to tumor cells invade microscopically
where the BBB seems to be intact.
2014
Convection Enhanced Delivery (CED)
2014
Osaka Medical College, Neurosurgery
Hamilton syringe
Hamilton syringe
F98 glioma
Brain
Brain
Objectives
1) in vivo biodistribution experiment
Osaka Medical College, Neurosurgery
To determine the biodistribution of ACBC-BSH following intracerebral
(i.c.) administration by CED to F98 glioma bearing rats.
Administration rate: 0.33 µl/min, 30 min (CED30min)
0.13 µl/min, 24 h
(CED24h)
2) in vivo BNCT therapy experiment
To determine the efficacy of ACBC-BSH as boron delivery agents for
BNCT in F98 glioma bearing rats.
2014
1)
Materials and Methods
*Animal: Fischer 344 male rat
Osaka Medical College, Neurosurgery
*Cell line: F98 rat glioma cells（bearing 1×105cells）
(provided by Rolf F. Barth, Department of Pathology, Ohio State University)
*Way of administration: CED or iv
*CED Administration rate:
0.33 µl/min, 30 min (total 10 µl)
0.13 µl/min, 24h (total 200 µl)
*Evaluation of boron concentration: ICP-AES
(Tumor, Blood, brain, skin, muscle, heart, lung, liver, spleen, kidney)
2014
1)
Materials and Methods
Osaka Medical College, Neurosurgery
Boron compound
*ACBC-BSH 1000 µg10B/ml
*BPA
1000 µg10B/ml
Injection methods
*ACBC-BSH CED30min
*ACBC-BSH CED24h
*ACBC-BSH iv
*BPA
iv
dose 10µl
dose 200µl
dose 2000µl
dose 2000µl
=250 mg10B/kg b.w.
2014
1)
Materials and Methods
Timing of boron concentration measurement
2014
Osaka Medical College, Neurosurgery
CED30min
i.c. tumor implantation
CED
over 30 min
1h
6h
24 h
i.c. tumor implantation
CED
over 24 h
1h
6h
24 h
iv
1h
6h
CED24h
iv
i.c. tumor implantation
1)
Result
Boron concentrations (Blood, Brain, Tumor)
Osaka Medical College, Neurosurgery
Boron concentrations ± SD（µg10B/g）
Timea(h) n
Agent
CEDc
30min
d
CED
ACBC-BSH
BPA
24h
ive
ivf
1
6
24
1
6
24
1
6
1
6
4
3
3
3
3
3
4
3
3
3
Blood
0.8 ± 0.3
0.9 ± 0.6
0.8 ± 0.1
3.3 ± 2.2
1.3 ± 0.0
0.5 ± 0.1
6.3 ± 1.7
7.0 ± 3.4
8.5 ± 0.3
4.1 ± 1.3
Brain
Tumor
0.1±0.0
0.2±0.1
0.1±0.0
1.5±1.3
0.4±0.3
0.0±0.0
0.2±0.0
0.3±0.1
3.0±0.6
3.1±1.3
10.7 ± 3.0
0.5 ± 0.5
0.4 ± 0.3
21.1 ± 3.3
17.8 ± 10.1
0.0 ± 0.1
2.7 ± 0.5
6.0 ± 1.7
19.7 ± 1.4
14.4 ± 3.4
Ratiosb
T/Blood T/Brain
13.3
0.5
0.4
6.4
13.7
0.1
0.4
0.9
2.3
3.6
147.2
2.8
6.2
14.2
42.4
1.4
14.9
18.6
6.7
4.7
2014
1)
Result
（µg10B/g）
Osaka Medical College, Neurosurgery
Boron concentration (Tumor)
Tumor Boron concentrations
30.0
CED30min
CED24h
25.0
ACBC-BSH iv
BPA iv
20.0
15.0
10.0
5.0
2014
0.0
1h
6h
1)
Result
Boron biodistribution in normal tissue
（1hour after intracerebral drug administration）
2014
Osaka Medical College, Neurosurgery
Boron concentrations ± SD（µg10B/g）
ACBC-BSH
CED
i.v.
30min
BPA
Organ
i.v.
Skin
Muscle
Heart
Liver
Spleen
Kidney
Lung
Blood
Brain
Tumor
5.2 ±
6.7 ±
7.8 ±
9.6 ±
14.6 ±
45.7 ±
8.5 ±
8.5 ±
3.0 ±
19.7 ±
1.6
1.6
0.5
1.4
5.3
12.3
2.1
0.3
0.6
1.4
5.1 ± 2.1
2.6 ± 1.9
1.4 ± 0.5
20.7 ± 5.4
6.1 ± 3.1
106.4 ± 15.7
11.7 ± 8.2
6.3 ± 1.7
0.2 ± 0.0
2.7 ± 0.5
1.0
0.2
0.1
0.3
3.0
0.6
0.3
0.8
0.1
10.7
±
±
±
±
±
±
±
±
±
±
1.9
0.4
0.1
0.1
1.9
0.1
0.1
0.3
0.0
3.0
CED
24h
0.5 ±
2.1 ±
1.3 ±
1.9 ±
4.4 ±
1.5 ±
1.2 ±
3.3 ±
1.5 ±
21.1 ±
0.6
1.3
0.9
1.8
2.0
0.4
0.8
2.2
1.3
3.3
1)
Result
Immunostaining of F98 glioma cell（in vitro）
Osaka Medical College, Neurosurgery
microscope
immunostaining
overlay
BPA
diffusely distuributed in the cytoplasm and cell nuclei
ACBC-BSH
2014
aggregated on the fringe of the cell nuclei
1)
Snmmary of biodistribution experiment
Osaka Medical College, Neurosurgery
*The tumor boron concentrations of ACBC-BSH CED24h
(21.1µg/g) was highest and was similar to BPA iv (19.7µg/g).
*Immunostaining showed that ACBC-BSH was incorporated into
the cytoplasm of the F98 cells and aggregated on the fringe of
the cell nuclei compared with BPA.
2014
We expected that the BNCT using ACBC-BSH
delivered by CED24h therapeutic effect would show
greater therapeutic effect than BPA iv administration .
2)
Materials and Methods
*Animal: Fischer 344 male rat
Osaka Medical College, Neurosurgery
*Cell line: F98 rat glioma cells（bearing 1×103cells）
(provided by Rolf F. Barth, Department of Pathology, Ohio State University)
*Boron delivery system: ACBC-BSH(1000 µg10B/ml)
*Studies were initiated at KUR.
*Way of administration for BNCT study
2014
a) untreated
b) Irradiated（neutron irradiation...1MW 1hour）
c) BPA iv + neutron irradiation
d) ACBC CED24h + neutron irradiation
e) ACBC CED24h + BPA iv + neutron irradiation
2)
Result
Radiation doses
Osaka Medical College, Neurosurgery
Agent
Boron concentrations±SD
（µg10B/g）
Timea
Route
(h)
2014
Without
compound
s
Untreated
Controls
Tumor
Biological radiation dose
c (Gy-eq)
Brain
Tumor
Brain
Tumor
1
1.5 ± 1.3 21.1±3.3
1.1
3.7
2.1d
12.3d
iv
1
3.0 ± 0.6 19.7±1.4
1.3
3.5
2.3
11.6
-
-
0±0
0±0
0.9
0.9
-
-
-
-
0±0
0±0
0
0
0
0
ACBC-BSH CDE24h
BPA
Brain
Physical radiation
doseb (Gy)
2014
Osaka Medical College, Neurosurgery
Survival rate
2)
Result
Kaplan-Meier survival curves
Discussion
1)The CBE factor is specific for different tissues and for different
Osaka Medical College, Neurosurgery
boron compounds and depends on the microdistribution of 10B in
the cells. Therefore, therapeutic effect by the same boron
concentration varies according to the localization.10)-12)
Conceptual diagram of CBE
alpha particle
B
cytoplasm
B B B
nucleus
B
B B BB
B
B
B
B
B
B
B
As much as a boron
compound accumulates
it near the nucleus,
the therapeutic effect
becomes higher.
=
2014
high CBE
10) Fox JC, et al, BJR supplement / BIR. 1992;24:48-52.
11) Santa Cruz GA, et al, Radiation research. 2004;162:702-10.
12) Dagrosa MA, et al, International journal of radiation oncology. 2011;79:262-8.
Discussion
Osaka Medical College, Neurosurgery
1)The therapeutic effect of ACBC-BSH CED24 and BPA iv was
approximately same. The reason is that I think extracellular
accumulations of ACBC-BSH was high.
Conceptual diagram of CBE in this case
ACBC-BSH
B
B
B
cytoplasm
B
B
nucleus
B
B
B
BPA
alpha particle
B
B
B
B
B
B
B
B
B
BB
B
2014
Discussion
2014
Osaka Medical College, Neurosurgery
2)We did not compete for the therapeutic effect by using ACBCBSH together in BPA. Difference in drug distribution at the cell
level was regarded as the cause.
microscope
BPA
ACBC-BSH
immunostaining
overlay
Conclusion
2014
Osaka Medical College, Neurosurgery
*This study suggested the possibility that
ACBC-BSH became the drug to add therapeutic
effect to.
2014
Osaka Medical College, Neurosurgery
Thank you for your attention
Discussion
3)The F98 brain tumor model that we used is the animal model that is
resistant to standard treatment using temozolomide 13).
Osaka Medical College, Neurosurgery
Rolf F. BARTH et al 14) reported that the following survival data.
*untreated.....................................................................Rat survival 24days (mean)
*irradiation (Physical dose 4.42Gy)…………….........Rat survival 29days (mean)
*BPA iv(250mg/kg) +BSH iv(30mg/kg)+BNCT..……Rat survival 41days (mean)
+17days duration of survival
This time our study result
*untreated……….........................................................Rat survival 26days (mean)
*irradiation (Physical dose 0.9Gy)...............................Rat survival 30days (mean)
*BPA iv+ BNCT..........................................................+10days duration of survival
*ACBC-BSH CED24h+BNCT....................................+11days duration of survival
*ACBC-BSH CED24h+BPA iv+ BNCT.....................+16days duration of survival
Other time our study result
*untreated …………………………………................Rat survival 30days (mean)
*irradiation (Physical dose 1.0Gy)……………….......Rat survival 31days (mean)
*BPA iv(250mg/kg) +BSH iv(100mg/kg)+BNCT……+10days duration of survival
％ILS
16
68
13
40
43
59
3
32
2014
BNCT using ACBC-BSH CED24 and BPA iv expect higher therapeutic
effect than existing treatment of BNCT.
13) Yang W, et al, Journal of neuro-oncology. 2011;101:379-90
14) R.F.Barth, et al, Int.J.Radiation Oncology Biol.Phys.2000;209-218
in vivo BNCT efficacy study
Summary of BNCT efficacy study
Osaka Medical College, Neurosurgery
*The therapeutic effect of both ACBC-BSH CED24h
and BPA iv was approximately same.
*The therapeutic effects by ACBC-BSH and BPA did
not compete.
2014
in vivo BNCT efficacy study
Result
survival times of F98 glioma bearing rats
Osaka Medical College, Neurosurgery
Agent/Route
Group
na
Survival Time
Mean±SD Median Range
%ILSb
Median
Untreated Controls 6
27.2±2.4
26.5
25-31
-
Irradiated Controls 5
29.8±1.9
30.0
27-32
13.2
BPA/i.v.
5
ACBC-BSH/CED24h 7
*
p<0.0382
ACBC-BSH/CED24h
6
+ BPA/i.v.
2014
%ILS =
37.4±2.6
37.0
34-40
39.6
37.0±5.2
38.0
31-44
43.4
44.3±8.0
42.0
38-60
58.5
MeST(each groups) - MeST(untreated controls)
MeST(untreated controls)
*log-rank test p<0.05
*p<0.0013
*p<0.0015
*
p<0.0005
BSH
ACBC
+
Figure4
F98 in vitro Boron up take
14
□ centrifugation(-)
■ centrifugation(+)
12
10
8
6
4
2
0
tACBC 1mM
BSH 1mM
BPA 1mM
Kyoto university research reactor
institute
Discussion
F98 glioma cellを用いた免疫染色
BPA
顕微鏡写真
重ね合わせ
免疫染色
細胞核を含む
細胞内全体に分布
ACBC-BSH
細胞核周辺に凝集
α粒子
B
細胞質
B B B
B核 B
B B
B
B
B
B
B
B
B
B
核のより近くにホウ素が
集積した方が、殺腫瘍
細胞効果が高い
F98 in vitro Boron up take
各化合物のモル濃度をそろえて24時間暴露
14
12
10
8
6
4
2
0
tACBC 1mM
cACBC 1mM
BPA 1mM