presenter: dr. madleen busse 1 gadolinium neutron capture therapy agents targeting mitochondria...
TRANSCRIPT
Presenter: Dr. Madleen Busse
1
Gadolinium Neutron Capture Therapy Agents Targeting Mitochondria
16th ICNCT - Helsinki, 16th June 2014
(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 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
Why targeting the mitochondria?
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(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.
Why targeting the mitochondria?
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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.
Why targeting the mitochondria?
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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.
TPP – tetraphenylphosphonium
In Vivo Selectivity ratioT:N = 40:1
P
GdNCT Agents Targeting Mitochondria
6
O
O
F
F
F
P
N N
NNO
O
OO
O
OGd
DLC
Linker
Gd-chelating macrocycle
OH2
H2O
DO3A = 1,4,7,10-Tetraazacyclodedecane-N,N’,N”-triacetic acid
Triphenylphosphonium triflate
p-Xylene
Prototype: PPh3-Gd-DO3A
Introduction of cell lines
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T98G (ATCC CRL-1690TM) Organism: Homo sapiens, humanTissue: brainMorphology: fibroblastCulture properties: adherentBiosafety Level: 1Disease: glioblastoma multiformeAge: 61 yearsGender: maleEthnicity: CaucasianStorage condition: liquid nitrogen vapour phase
SVG p12 (ATCC CRL-8621TM) Organism: Homo sapiens, humanTissue: brainCell type: astroglial; SV40 transformeMorphology: fibroblastCulture properties: adherentBiosafety Level: 2 [Cells contain polyomavirus DNA sequences]Age: fetus, first trimesterStorage condition: liquid nitrogen vapour phase
Cytotoxicity Studies of PPh3-Gd-DO3A
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Uptake and In-Vitro Selectivity Studies of PPh3-Gd-DO3A
9
Incubation time [h]
Dosing Concentration
[µM]
SVG p12ng Gd / mg
Protein
T98Gng Gd / mg
Protein
SVG p12Gd atoms /
cell[x 108]
T98GGd atoms /
cell[x 108]
In-VitroSelectivity
of T98G/SVG
p12
24
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
48
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
Uptake and In-Vitro Selectivity Studies of PPh3-Gd-DO3A
10
Incubation time [h]
Dosing Concentration
[µM]
SVG p12ng Gd / mg
Protein
T98Gng Gd / mg
Protein
SVG p12Gd atoms /
cell[x 108]
T98GGd atoms /
cell[x 108]
In-VitroSelectivity
of T98G/SVG
p12
24
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
48
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
Uptake and In-Vitro Selectivity Studies of PPh3-Gd-DO3A
11
Incubation time [h]
Dosing Concentration
[µM]
SVG p12ng Gd / mg
Protein
T98Gng Gd / mg
Protein
SVG p12Gd atoms /
cell[x 108]
T98GGd atoms /
cell[x 108]
In-VitroSelectivity
of T98G/SVG
p12
24
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
48
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
Uptake and In-Vitro Selectivity Studies of PPh3-Gd-DO3A
12
Incubation time [h]
Dosing Concentration
[µM]
SVG p12ng Gd / mg
Protein
T98Gng Gd / mg
Protein
SVG p12Gd atoms /
cell[x 108]
T98GGd atoms /
cell[x 108]
In-VitroSelectivity
of T98G/SVG
p12
24
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
48
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
(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)
- K-edge of Gd = 50.2 keV- irradiation occurred at 51 keV
• - no PAT effect found
Gadolinium Photon Activation Therapy (GdPAT)
Efficient mass absorption coefficient
(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.
- K-edge of Gd = 50.2 keV- irradiation occurred at 51 keV
• - no PAT effect found
GdPAT Experiment
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Control
Gd-PPh3-pXY-DO3A Control agent: Gd-DTPA Vehicle Control Milli-Q
Repeats
C-Min
Serial Dilution C-Max
C-Min
Serial Dilution C-Max
16.88
37.75 67.50 125.00
250.00
16.88
37.75 67.50 125.00
250.00
A
B
C
D
E
F
G
1 2 3 4 5 6 7 8 9 10 11 12 H
How to Quantify the PAT Effect?
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Drug
absolute Control Drug [µM]
Binary Effect:Drug and Irradiation
Irradiation
Irradiation [Gy]
X component
Y component
Z component
GdPAT Experiment
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SVG p12 - control T98G - GBMMean SEM Mean SEM
absolute control 100.00 1.53 100.00 3.16
drug control (17 uM) - no radiation 96.62 0.70 100.00 2.31
radiation control - no drug 98.49 0.96 95.84 0.72
Gd-DTPA - 17 uM 92.05 3.23 80.58 2.48
Gd-DO3A - 17 uM 99.24 2.74 51.94 5.74
Conditions:- BE = 64 keV- IT = 48 h- PT = 12 h- No media change, i.e. same Gd conc. in all wells
Gd-DTPA
