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 Universitynagasaki@bioa.eng.osaka-cu.ac.jp

16th International Congress on Neutron Capture Therapy@Pörssitalo, Helsinki, Finland (2014.14-19) 　

Clinically Used BNCT Agents

NH2

OH

O

BHO

OH

BHHBHB

BH

BH

BHBH

BHHB

HB

B

BH

SH

2Na

2-

L-BPALow water-solubility

BSH

Low tumor selectivity

Disadvantage

Development of novel boron agents possessing high water-solubility and tumor-selectivity is

required.

o-Carborane-Kojic acid Conjugate (CKA)

CKA

•Glucose metabolite produced by Aspergillus oryzae•Ferric chelator•Antifungal effect

Disadvantage: Poor water solubility

Kojic acid o-Carborane

•Boron cluster

Ligand candidate to melanocyte

The enhancement of boron neutron capture reaction

by high boron occupancy

Skin lightening effect (Inhibitor of tyrosinase) = Affinity toward melanocyte cells

Cyclodextrins

b-Cyclodextrin (b-CD)

n=1, 2, 3, 4 R2=R3=R6=H, CH3 , CH2CH3 ,

CO2H , OSO3Na ,

CH2CH(OH)CH3 ,

CH2CH2CH2CH2OSO3Na

SolubilityStabilityBioavailability

Solubilization of CKA with Hydroxypropyl-β-Cyclodextrin

CKA/HP-β-CD was used as a novel boron agent possessing melanoma selectivity in this

study.BHHB

HB

BH

BH

BHCH

BHHB

HB

C

BH

O

O

OH

HO

O

OH

CKA/HP-β-CD solution

Boron

Cluster

Milli-Q

Vortex Mixing

60 min

Sonication in bath

60 min

Supernatant

Precipitate

B ConcentrationICP-AES

9000 ppmB 90%

Cyclodextrin25℃

3000 rpm(10 min)

Centrifugation

Accumulation of CKA/HP-β-CD in vitro

0.5 1 3 6 12 240

200400600800

100012001400160018002000

time (hr)

boro

n c

on

cen

trati

on

(ng

/8.0

×1

05

ce

lls)

[Boron Agent] 10 ppm of BCKA/HP-β-CD

[Cell line] 8.0×105 cells ■ ： B16/BL6 (murine melanoma) ■ ： C2C12 (murine myoblast) 　 ■ ： colon26 (murine rectal cancer)

Cellular Interanalization B16/BL6 > C2C12, colon26

Boron concentration in cells were measured by ICP-AES.

Melanoma selectivity

Contribution of Kojic Acid moiety for internalization into melanoma cells

0.5 1 3 6 12 240

500

1000

1500

2000

2500

time (hr)

bo

ron

co

ncen

tra-

tio

n(n

g/8

.0×

10

5 c

ells)

[Cell line] B16/BL6 　 8.0×105 cells

[Boron Agent]■ ： CKA/HP-β-CD 　　　　 10 ppmB■ ： o-carborane/HP-β-CD 10 ppmB

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

100 10 1 00

100

200

300

400

500

600

700

KA/CKA ratio

Con

cen

trati

on

(pp

b/8

.0x1

08

cells)

[Cell line] B16/BL6 　 8.0×105 cells[Boron Agent] CKA/HP-β-CD 10 ppm B[Exposure time] 3 hours

Accumulation of CKA/HP-β-CD were inhibited dose-dependently with free Kojic Acid (KA).

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

0.5 1 2 30

2

4

6

8

10

12

14

16

18

muscle tumorliver bloodkidbey spleenspleen lung

time(hr)

con

cen

trati

on

(pp

m)

0 1 2 3 41.0

10.0

100.0

1000.0

10000.0

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

time(hr)

T/N

rati

o

T/B

rati

o

(N=6)

kidney

High tumor-selectivityPeak of accumulation in “1 hour” after administration

Intraperitoneal administration of CKA complex was carried out with melanoma-bearing mice.

Anti tumor efficacy of BNCT at KUR -1

Fluence dependencyPe

rcen

t st

ill a

live(%

)

Time in days(day)

Neutron Fluence Full: 4 x 1012 neutron/cm2

Half: 2 x 1012 neutron/cm2

Quarter: 1 x 1012 neutron/cm2

Anti tumor efficacy of BNCT at KUR -1CKA Dose dependency

BPA hot CKA/HP-β-CD hot

Perc

en

t st

ill a

live(%

)

Time in days(day)

Boron-containing phenoxyacetanilide derivatives as hypoxia-inducible factor (HIF)-1α inhibitors

H. Nakamura et al., Bioorg. Med. Chem. Lett., 20, 1453 (2010)

HIF-1?

16

16

16 16

HIF-1Suppression

CKA complex act as hypoxia-inducible factor (HIF)-1α inhibitors

BPA(1500 ppmB)

After 14 days

CKA/HP-β-CD(4500 ppm B)After 21 days

Fig. Inhibition effect of CKA complex on hypoxia-induced accumulation of HIF-1 in HeLa cells.

Fig. Inhibition effect of CKA complex on metastasis to lung after BNCT.

Conclusions

CKA/HP-β-CD is promising for melanoma-BNCT

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　 　

BHHBHB

BH

BH

BHCH

BHHB

HB

C

BH

O

O

OH

HO

O

OH

Thank you for your attention!

Kiitos!!

Cytotoxicity of CKA/HP-b-CD (WST assay)

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

boron concentration (ppm)Rela

tive c

ell v

iavilit

y

[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

No serious toxicity was observed

0 1 2 315

16

17

18

19

20

days after treatment (day)

we

igh

t (g

)

6000 ppmB 4500 ppmB3000 ppmB

Methasesis

CKA hot 群では比較的抑制された

コウジ酸構造の優位性

細胞内取り込み量CKA > o-carborane

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

100 10 1 00

100

200

300

400

500

600

700

KA/CKA ratioco

nce

ntr

ati

on

(pp

b/

8.0

x1

08

ce

lls)

CKA carborane0

100

200

300

400

500

600

700

800

900

co

nce

ntr

ati

on

(pp

b/

8.0

x1

05

ce

lls)

コウジ酸の濃度依存的に取り込み量が低下

o-carborane との比較 コウジ酸による競合阻害

ホウ素中性子捕捉療法 (BNCT)

10Btumor tissue

thermal neutron

normal tissue

thermal neutron10B

α particles

Li nuclei

< 10 μm

Boron Neutron Capture Reaction

腫瘍細胞特異的に殺傷可能な副作用の小さな治療法

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

正常細胞を傷付けず、腫瘍組織を選択的に殺傷可能

原理と特徴

AgateBalls

Solubilizer Vibration Milling25 Hz, 20 min

Extraction with Milli-Q

Centrifugation25 ,3000 rpm,10 min℃

Boron

Cluster

Supernatant

Precipitate

１． High Speed Vibration Milling (HSVM)

２． Vortex Mixing (VM)

B ConcentrationICP-AES

Milli-Q

Vortex Mixing

60 min

Sonication in bath60 min

25℃3000 rpm,10 min

Supernatant

Precipitate

B ConcentrationICP-AES

Solubilizer

Boron

Cluster

Centrifugation

HB BHBH

BH

HB

HBHC

BHBH

BH

C

BH

O

H

O

OH

H

HO

コウジ酸修飾カルボラン (CKA)コウジ酸

O

H

O

OH

H

HO

HB BHBH

BH

HB

HBHC

BHBH

BH

C

BH

O

H

O

OH

H

HO

CKA

メラノサイト親和性

o-carborane

高いホウ素集積能

メラノーマ選択性ホウ素キャリア

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

細胞内取り込み量CKA > o-carborane

100 10 1 00

100

200

300

400

500

600

700

KA/CKA ratioco

nce

ntr

ati

on

(pp

b/

8.0

x1

08

ce

lls)

コウジ酸の濃度依存的に取り込み量が低下

o-carborane との比較 コウジ酸による競合阻害

0.5 1 3 6 12 240

500

1000

1500

2000

2500

time (hr)

bo

ron

co

nce

ntr

ati

on

(ng

/8.0

×1

05

ce

lls)