Transcript 16th International Congress on Neutron Capture Therapy

Carborane-kojic Acid Conjugate for
Melanoma-Targeting Boron Neutron Capture
Therapy
T. Nagasaki1, R. Kawasaki1, Y. Sakurai2,
S. Masunaga2, K. Ono2, M. Kirihata3
1Graduate
School of Engineering, Osaka City University
2Research Reactor Institute, Kyoto University
3Research Center for BNCT, Osaka Prefecture University
[email protected]
16th International Congress on Neutron Capture Therapy
@Pörssitalo, Helsinki, Finland (2014.14-19)
Clinically Used BNCT Agents
L-BPA
Low water-solubility
Disadvantage
BSH
Low tumor selectivity
Development of novel boron agents possessing high
water-solubility and tumor-selectivity is required.
o-Carborane-Kojic acid Conjugate (CKA)
Kojic acid
•Glucose metabolite produced
by Aspergillus oryzae
•Ferric chelator
•Antifungal effect
Skin lightening effect (Inhibitor of tyrosinase)
= Affinity toward melanocyte cells
•Boron cluster
o-Carborane
The enhancement of
boron neutron capture reaction
by high boron occupancy
Ligand candidate to melanocyte
CKA
Disadvantage: Poor water solubility
Cyclodextrins
Solubility
Stability
Bioavailability
b-Cyclodextrin (b-CD)
n=1, 2, 3, 4
R2=R3=R6=H, CH3 , CH2CH3 ,
CO2H , OSO3Na ,
CH2CH(OH)CH3 ,
CH2CH2CH2CH2OSO3Na
Solubilization of CKA with Hydroxypropyl-β-Cyclodextrin
Cyclodextrin
Milli-Q
Boron
Cluster
Vortex
Mixing
Precipitate
25℃
3000 rpm
(10 min)
60 min
B Concentration
ICP-AES
9000 ppmB 90%
Centrifugation
Sonication
in bath
60 min
Supernatant
CKA/HP-β-CD solution
CKA/HP-β-CD was used as a novel
boron agent possessing melanoma
selectivity in this study.
OH
O
boron concentration(ng/8.0×105 cells)
Accumulation of CKA/HP-β-CD in vitro
2000
[Boron Agent] 10 ppm of B
CKA/HP-β-CD
[Cell line] 8.0×105 cells
■：B16/BL6 (murine melanoma)
■：C2C12 (murine myoblast)
■：colon26 (murine rectal cancer)
1500
1000
500
Boron concentration in cells
were measured by ICP-AES.
0
0.5
1
3
6
12
24
time (hr)
Cellular Interanalization
B16/BL6 > C2C12, colon26
Melanoma selectivity
2500
(ng/8.0×105 cells)
boron concentration
Contribution of Kojic Acid moiety for
internalization into melanoma cells
2000
[Cell line] B16/BL6 8.0×105 cells
1500
[Boron Agent]
■：CKA/HP-β-CD
10 ppmB
■：o-carborane/HP-β-CD 10 ppmB
1000
500
0
0.5
1
3
6
12
24
time (hr)
Accumulation of boron agents: CKA/HP-β-CD >> o-carborane/HP-β-CD
Accumulation of CKA/HP-β-CD towards B16BL6
was enhanced by Kojic Acid-moiety.
Competitive inhibition with free Kojic Acid
[Cell line]
B16/BL6 8.0×105 cells
[Boron Agent] CKA/HP-β-CD 10 ppm B
[Exposure time] 3 hours
(ppb/8.0x108 cells)
Concentration
800
600
400
200
0
100
10
1
0
Accumulation of CKA/HP-β-CD were
inhibited dose-dependently with free
Kojic Acid (KA).
KA/CKA ratio
CKA/HP-β-CD possesses melanoma-selectivity by ligand effect.
Enhanced uptake mechanism mediated by Kojic Acid-receptor is suggested.
Intracellular-distribution of CKA/HP-β-CD
in melanoma cells
[Cell line]
B16/BL6 8.0×105 cells
[Boron Agent]
CKA/HP-β-CD 40 ppmB
[Exposure time] For 1 hour
[Affinity Constant] Kd= 1.9 x 10-6 M
(A) Anti-BSH (green)
(B) DAPI (blue)
(C) Phase contrast
Internalized CKA was rapidly localized into nuclei.
Pharmacokinetics of CKA/HP-β-CD
(N=6)
Intraperitoneal administration of CKA complex was carried out with melanoma-bearing mice.
10000.0
muscle
liver
kidney
kidbey
spleen
15
10
tumor
blood
spleen
lung
5
1000.0
3.00
2.50
100.0
2.00
1.50
10.0
1.00
0.50
1.0
0
0.5
1
2
time(hr)
3
0.00
0
1
2
time(hr)
3
High tumor-selectivity
Peak of accumulation in “1 hour” after administration
4
T/B ratio
20
4.00
3.50
T/N ratio
concentration(ppm)
25
Percent still alive(%)
Anti tumor efficacy of BNCT at KUR -1
Fluence dependency
Time in days(day)
Neutron Fluence
Full: 4 x 1012 neutron/cm2
Half: 2 x 1012 neutron/cm2
Quarter: 1 x 1012 neutron/cm2
Percent still alive(%)
Anti tumor efficacy of BNCT at KUR -1
CKA Dose dependency
Time in days(day)
BPA hot
CKA/HP-β-CD hot
Boron-containing phenoxyacetanilide derivatives
as hypoxia-inducible factor (HIF)-1α inhibitors
H. Nakamura et al., Bioorg. Med. Chem. Lett., 20, 1453 (2010)
16
16
16
16
?
HIF-1
CKA complex act as hypoxia-inducible
factor (HIF)-1α inhibitors
Fig. Inhibition effect of CKA complex
on hypoxia-induced accumulation of
HIF-1 in HeLa cells.
BPA
(1500 ppmB)
After 14 days
CKA/HP-β-CD
(4500 ppm B)
After 21 days
Fig. Inhibition effect of CKA complex
on metastasis to lung after BNCT.
Suppression
HIF-1
Conclusions
OH
CKA/HP-β-CD possesses
・Melanoma selectivity
・Nuclear localization ability
・Highly tumor-selectivity in vivo
・Strong anti tumor effect with BNCT
similar to BPA
・Inhibition effect on HIF-1 accumulation
CKA/HP-β-CD is promising for melanoma-BNCT
O
Thank you for your attention!
Kiitos!!
Relative cell viavility
Cytotoxicity of CKA/HP-b-CD (WST assay)
1.60
1.40
1.20
1.00
0.80
0.60
0.40
0.20
0.00
0
0.625 1.25
2.5
5
10
20
40
80
160
boron concentration (ppm)
[Cell line]
■：B16/BL6(murine melanoma cell)
■：C2C12(murine myoblast cell)
■：colon26(murine colon cancer cell)
Sensitivity of cells: B16/BL6 > C2C12, colon26
Acute toxicity of CKA/HP-b-CD
6000 ppmB
4500 ppmB
3000 ppmB
20
weight (g)
19
18
17
16
15
0
1
2
3
days after treatment…
No serious toxicity was observed
Methasesis
CKA hot群では比較的抑制された
コウジ酸構造の優位性
o-carboraneとの比較
concentration(ppb/8.0x108 cells)
concentration(ppb/8.0x105 cells)
1000
コウジ酸による競合阻害
800
600
400
200
0
CKA
carborane
細胞内取り込み量
CKA > o-carborane
800
700
600
500
400
300
200
100
0
100
10
1
0
KA/CKA ratio
コウジ酸の
濃度依存的に取り込み量が低下
コウジ酸構造によって、
細胞内への取り込みが促進される
ホウ素中性子捕捉療法(BNCT)
Boron Neutron Capture Reaction
α particles
thermal neutron
10B
Li nuclei
< 10 μm
thermal neutron
原理と特徴
ホウ素薬剤を腫瘍に集積
熱・熱外中性子照射
細胞障害性のある粒子線が発生
粒子線の飛程が細胞一つ分
10B
tumor tissue
normal tissue
正常細胞を傷付けず、
腫瘍組織を選択的に殺傷可能
腫瘍細胞特異的に殺傷可能な副作用の小さな治療法
１．High Speed Vibration Milling (HSVM)
Solubilizer
Agate
Balls
Vibration Milling
25 Hz, 20 min
Boron
Cluster
Supernatant
B Concentration
ICP-AES
Extraction
with Milli-Q
Centrifugation
25℃,3000 rpm,10 min
２．Vortex Mixing (VM)
Solubilizer
Boron
Cluster
Vortex Mixing
Milli-Q
60 min
Precipitate
Supernatant
25℃
3000 rpm,10 min
Centrifugation
Sonication in bath
60 min
Precipitate
B Concentration
ICP-AES
コウジ酸修飾カルボラン(CKA)
コウジ酸
O
H
OH
CKA
HO
O
H
メラノサイト親和性
o-carborane
メラノーマ選択性ホウ素キャリア
高いホウ素集積能
メラノーマ選択性を有する
BNCT用ホウ素薬剤としての評価を行った
Contribution of Kojic Acid moiety for
accumulation toward melanoma
o-carboraneとの比較
コウジ酸による競合阻害
concentration(ppb/8.0x108 cells)
2000
(ng/8.0×105 cells)
boron concentration
2500
1500
1000
500
0
0.5 1
3
6
time (hr)
細胞内取り込み量
CKA > o-carborane
12 24
800
700
600
500
400
300
200
100
0
100
10
1
KA/CKA ratio
0
コウジ酸の
濃度依存的に取り込み量が低下