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16th ICNCT - Helsinki, 16th June 2014
Gadolinium Neutron Capture Therapy Agents
Targeting Mitochondria
[email protected]
Presenter: Dr. Madleen Busse
1
Gadolinium Neutron Capture Therapy (GdNCT)
•
- Total energy = 7.94 MeV
- ~ 5 ACK electrons / NC event
- path-length of ACK electrons = a few nm
•- damage is highly localized
(1) Martin, R. F.; D'Cunha, G.; Pardee, M.; Allen, B. J. Int. J. Radiat. Biol. 1988, 54, 205; (2) Martin, R. F.; D'Cunha, G.;
Pardee, M.; Allen, B. J. Pigm. Cell Res. 1989, 2, 330; (3) Crossley, E.L.; Ching, H. Y. V.; Ioppolo, J. A.; Rendina, L. M.
"Boron and Gadolinium in the Neutron Capture Therapy of Cancer" in Bioinorganic Medicinal Chemistry, Wiley-VCH, 2011,
Alessio, E. ed., p. 283.
Why targeting the mitochondria?
(1) J. S. Modicanapolitano and J. R. Aprille, Cancer Res., 1987, 47(16), 4361-4365; (2) V. Gogvadze, S. Orrenius and B.
Zhivotovsky, Trends Cell. Biol., 2008, 18(4), 165-173; (3) M. P. Murphy and R. A. J. Smith, Adv. Drug Delivery Rev., 2000,
41(2), 235-250.
3
Why targeting the mitochondria?
Disrupted in
Cancer Cells
(1) D. Lichtshtein, H. R. Kaback and A. J. Blume, Proc. Natl. Acad. Sci. U. S. A., 1979, 76(2), 650-654; (2) I. Madar, L. Weiss
and G. Izbicki, J. Nucl. Med., 2002, 43(2), 234-238; (3) I. Madar, J. H. Anderson, Z. Szabo, U. Scheffel, P. F. Kao, H. T. Ravert
and R. F. Dannals, J. Nucl. Med., 1999, 40(7), 1180-1185.
4
Why targeting the mitochondria?
TPP – tetraphenylphosphonium
In Vivo Selectivity ratioT:N = 40:1
Disrupted in
Cancer Cells
(1) D. Lichtshtein, H. R. Kaback and A. J. Blume, Proc. Natl. Acad. Sci. U. S. A., 1979, 76(2), 650-654; (2) I. Madar, L. Weiss
and G. Izbicki, J. Nucl. Med., 2002, 43(2), 234-238; (3) I. Madar, J. H. Anderson, Z. Szabo, U. Scheffel, P. F. Kao, H. T. Ravert
and R. F. Dannals, J. Nucl. Med., 1999, 40(7), 1180-1185.
5
GdNCT Agents Targeting Mitochondria
p-Xylene
Triphenylphosphonium triflate
Prototype: PPh3-Gd-DO3A
DO3A = 1,4,7,10-Tetraazacyclodedecane-N,N’,N”-triacetic acid
6
Introduction of cell lines
SVG p12 (ATCC CRL-8621TM)
T98G (ATCC CRL-1690TM)
Organism: Homo sapiens, human
Tissue: brain
Cell type: astroglial; SV40 transforme
Morphology: fibroblast
Culture properties: adherent
Biosafety Level: 2 [Cells contain
polyomavirus DNA sequences]
Age: fetus, first trimester
Storage condition: liquid nitrogen
vapour phase
Organism: Homo sapiens, human
Tissue: brain
Morphology: fibroblast
Culture properties: adherent
Biosafety Level: 1
Disease: glioblastoma multiforme
Age: 61 years
Gender: male
Ethnicity: Caucasian
Storage condition: liquid nitrogen
vapour phase
7
Cytotoxicity Studies of PPh3-Gd-DO3A
8
Uptake and In-Vitro Selectivity Studies
of PPh3-Gd-DO3A
Incubation
time [h]
In-Vitro
Selectivity
of
T98G/SVG
p12
Dosing
Concentration
[µM]
SVG p12
ng Gd / mg
Protein
T98G
ng Gd / mg
Protein
SVG p12
Gd atoms /
cell
[x 108]
T98G
Gd atoms /
cell
[x 108]
0
2.69 ± 0.58
2.84 ± 0.97
0.03 ± 0.09
0.04 ± 0.12
10
19.00 ± 1.07
168.83 ± 43.25
0.22 ± 0.09
4.18 ± 4.13
9:1
100
259.58 ± 45.35
963.42 ± 52.34
3.21 ± 0.60
23.96 ± 5.40
4:1
1000
3828.60 ± 326.60
2474.08 ± 404.64
61.33 ± 31.70
53.00 ± 3.30
~ 1:1
0
9.13 ± 2.00
18.52 ± 3.22
0.74 ± 0.17
0.32 ± 0.20
10
31.79 ± 5.02
420.14 ± 5.42
0.30 ± 0.03
0.95 ± 0.21
13:1
100
308.67 ± 18.42
1486.96 ± 146.12
2.98 ± 0.20
30.32 ± 6.50
5:1
1000
6440.50 ± 177.47
3000.29 ± 457.66
62.16 ± 2.97
68.60 ± 26.36
~ 1:1
24
48
9
Uptake and In-Vitro Selectivity Studies
of PPh3-Gd-DO3A
Incubation
time [h]
In-Vitro
Selectivity
of
T98G/SVG
p12
Dosing
Concentration
[µM]
SVG p12
ng Gd / mg
Protein
T98G
ng Gd / mg
Protein
SVG p12
Gd atoms /
cell
[x 108]
T98G
Gd atoms /
cell
[x 108]
0
2.69 ± 0.58
2.84 ± 0.97
0.03 ± 0.09
0.04 ± 0.12
10
19.00 ± 1.07
168.83 ± 43.25
0.22 ± 0.09
4.18 ± 4.13
9:1
100
259.58 ± 45.35
963.42 ± 52.34
3.21 ± 0.60
23.96 ± 5.40
4:1
1000
3828.60 ± 326.60
2474.08 ± 404.64
61.33 ± 31.70
53.00 ± 3.30
~ 1:1
0
9.13 ± 2.00
18.52 ± 3.22
0.74 ± 0.17
0.32 ± 0.20
10
31.79 ± 5.02
420.14 ± 5.42
0.30 ± 0.03
0.95 ± 0.21
13:1
100
308.67 ± 18.42
1486.96 ± 146.12
2.98 ± 0.20
30.32 ± 6.50
5:1
1000
6440.50 ± 177.47
3000.29 ± 457.66
62.16 ± 2.97
68.60 ± 26.36
~ 1:1
24
48
10
Uptake and In-Vitro Selectivity Studies
of PPh3-Gd-DO3A
Incubation
time [h]
In-Vitro
Selectivity
of
T98G/SVG
p12
Dosing
Concentration
[µM]
SVG p12
ng Gd / mg
Protein
T98G
ng Gd / mg
Protein
SVG p12
Gd atoms /
cell
[x 108]
T98G
Gd atoms /
cell
[x 108]
0
2.69 ± 0.58
2.84 ± 0.97
0.03 ± 0.09
0.04 ± 0.12
10
19.00 ± 1.07
168.83 ± 43.25
0.22 ± 0.09
4.18 ± 4.13
9:1
100
259.58 ± 45.35
963.42 ± 52.34
3.21 ± 0.60
23.96 ± 5.40
4:1
1000
3828.60 ± 326.60
2474.08 ± 404.64
61.33 ± 31.70
53.00 ± 3.30
~ 1:1
0
9.13 ± 2.00
18.52 ± 3.22
0.74 ± 0.17
0.32 ± 0.20
10
31.79 ± 5.02
420.14 ± 5.42
0.30 ± 0.03
0.95 ± 0.21
13:1
100
308.67 ± 18.42
1486.96 ± 146.12
2.98 ± 0.20
30.32 ± 6.50
5:1
1000
6440.50 ± 177.47
3000.29 ± 457.66
62.16 ± 2.97
68.60 ± 26.36
~ 1:1
24
48
11
Uptake and In-Vitro Selectivity Studies
of PPh3-Gd-DO3A
Incubation
time [h]
In-Vitro
Selectivity
of
T98G/SVG
p12
Dosing
Concentration
[µM]
SVG p12
ng Gd / mg
Protein
T98G
ng Gd / mg
Protein
SVG p12
Gd atoms /
cell
[x 108]
T98G
Gd atoms /
cell
[x 108]
0
2.69 ± 0.58
2.84 ± 0.97
0.03 ± 0.09
0.04 ± 0.12
10
19.00 ± 1.07
168.83 ± 43.25
0.22 ± 0.09
4.18 ± 4.13
9:1
100
259.58 ± 45.35
963.42 ± 52.34
3.21 ± 0.60
23.96 ± 5.40
4:1
1000
3828.60 ± 326.60
2474.08 ± 404.64
61.33 ± 31.70
53.00 ± 3.30
~ 1:1
0
9.13 ± 2.00
18.52 ± 3.22
0.74 ± 0.17
0.32 ± 0.20
10
31.79 ± 5.02
420.14 ± 5.42
0.30 ± 0.03
0.95 ± 0.21
13:1
100
308.67 ± 18.42
1486.96 ± 146.12
2.98 ± 0.20
30.32 ± 6.50
5:1
1000
6440.50 ± 177.47
3000.29 ± 457.66
62.16 ± 2.97
68.60 ± 26.36
~ 1:1
24
48
12
Gadolinium Photon Activation Therapy (GdPAT)
•
- K-edge of Gd = 50.2 keV
- irradiation occurred at 51 keV
- no PAT effect found
(1) Martin, R. F.; D'Cunha, G.; Pardee, M.; Allen, B. J. Int. J. Radiat. Biol. 1988, 54, 205; (2) Martin, R. F.; D'Cunha, G.;
Pardee, M.; Allen, B. J. Pigm. Cell Res. 1989, 2, 330; (3) Crossley, E.L.; Ching, H. Y. V.; Ioppolo, J. A.; Rendina, L. M.
"Boron and Gadolinium in the Neutron Capture Therapy of Cancer" in Bioinorganic Medicinal Chemistry, Wiley-VCH, 2011,
Alessio, E. ed., p. 283.
Gadolinium Photon Activation Therapy (GdPAT)
Efficient mass absorption coefficient
•
- K-edge of Gd = 50.2 keV
- irradiation occurred at 51 keV
- no PAT effect found
(1) De Stasio, G.; Rajesh, D.; Ford, J. M.; Daniels, M. J.; Erhardt, R. J.; Frazer, B. H.; Tyliszczak, T.; Gilles, M. K.; Conhaim, R.
L.; Howard, S. P.; Fowler, J. F.; Esteve, F.; Mehta, M. P. Clin. Cancer Res. 2006, 12, 206; (2) Biston, M.-C.; Joubert, A.; Adam, J.F.; Elleaume, H.; Bohic, S.; Charvet, A.-M.; Esteve, F.; Foray, N.; Balosso, J. Cancer Res., 2004, 64, 2317.
CMin
16.8
8
Serial Dilution
37.75
67.50
125.0
0
Control agent: Gd-DTPA
CMax
CMin
250.0
0
16.8
8
Serial Dilution
37.75
67.50
125.0
0
CMax
250.0
0
Repeats
Control
Gd-PPh3-pXY-DO3A
Vehicle Control
Milli-Q
GdPAT Experiment
A
B
C
D
E
F
G
1
2
3
4
5
6
7
8
9
10
11
12
H
15
How to Quantify the PAT Effect?
Drug
absolute
Control
Drug [µM]
Z component
X component
Irradiation
Binary Effect:
Drug and Irradiation
Irradiation [Gy]
Y component
16
GdPAT Experiment
Gd-DTPA
Conditions:
- BE = 64 keV
- IT = 48 h
- PT = 12 h
- No media change,
i.e. same Gd conc. in all wells
SVG p12 - control
Mean
SEM
absolute control
drug control (17 uM) - no radiation
radiation control - no drug
Gd-DTPA - 17 uM
Gd-DO3A - 17 uM
T98G - GBM
Mean
SEM
100.00
1.53
100.00
3.16
96.62
0.70
100.00
2.31
98.49
0.96
95.84
0.72
92.05
3.23
80.58
2.48
99.24
2.74
51.94
5.74
17
Acknowledgements
[email protected]
Kiitos
[email protected]